黄芪苷对胃粘膜损伤的保护作用研究
|
摘要
[研究背景]
开展中药有效部位的研究,近年来成为中药、天然药新药开发的重要方向之一以中医药理论为依据,结合现代科学实验技术开展健脾益气中药对于胃粘膜保护作用的药理学研究是我们课题组多年来坚持不懈的研究方向。
在前期研究工作中,课题组成员探讨了黄芪总苷(AST)对脾虚大鼠胃壁细胞功能及形态的影响,发现AST作为黄芪的主要有效部位,在调节细胞信号传导功能的同时,具有保护胃粘膜屏障、维持组织结构完整性,调节整体胃酸分泌的重要作用。AST作为黄芪皂苷类化合物的混合体,是黄芪的有效部位之一；黄芪甲苷(AST-Ⅳ)作为黄芪中的单体皂苷类化合物,被中药药典规定为黄芪药材质量评价的指标性物质。我们知道,中药多靶点发挥药效作用的物质基础往往来源于有效部位中多个同类化合物群体的协同作用,单一化合物的活性均不强。因此,以药效为标准对中药有效部位与单体成分进行对比研究,能够揭示中药有效部位与单体的药理作用差异,阐明有效部位中多成分之间相互协同增效的科学意义。基于这种研究思路,本实验对基于胃粘膜保护的AST及AST-Ⅳ的作用及其差异开展进一步研究,目前尚未见相关研究报道。
[目的]
本文旨在以黄芪“健脾益气”作用的中医药理论为依据,结合现代科学实验技术研究黄芪皂苷类有效部位(AST)及单体成分(AST-Ⅳ)对胃粘膜保护作用的功效,并从多方面对比AST与AST-Ⅳ保护胃粘膜的作用差异。主要包括以下几个方面：
一、建立固相萃取-高效液相-蒸发光散射检测(SPE-HPLC-ELSD)法测定大鼠血清中AST-Ⅳ含量的方法,比较AST、AST-Ⅳ在大鼠腹腔注射给药后相同时间大鼠血清中AST-Ⅳ的含量,并找出AST及AST-Ⅳ血药浓度的达峰时间。为后期AST与AST-Ⅳ对大鼠急性胃粘膜损伤的实验研究中造模给药时间点的确立打下基础,并为两药抗急性胃粘膜损伤药效学作用的差异提供药代动力学参数的依据。
二、从胃粘膜病理形态学的角度,观察研究AST及AST-Ⅳ不同剂量对无水乙醇致大鼠急性胃粘膜损伤的保护作用。同时,为了观察药效的累积效应,分别观察给药1次及连续给药4次的药效作用,以期从多层次研究探讨AST与AST-Ⅳ作用的差异性。
三、从胃粘膜上皮细胞超微结构的改变,探讨AST与AST-Ⅳ预防及治疗给药后对慢性胃粘膜损伤的保护作用。
四、从细胞学的角度,观察AST及AST-Ⅳ对大鼠急性损伤的胃粘膜细胞凋亡及增殖的影响,以期明确这两种药物保护胃粘膜细胞的作用机制及差异。
[方法]
一、对AST、AST-Ⅳ在大鼠体内AST-Ⅳ的血药浓度及达峰时间考察,采用SPE法提取分离大鼠血清中的AST-Ⅳ, HPLC-ELSD法考察其线性关系及最低检测浓度、专属性、精密度、稳定性及回收率,建立AST-Ⅳ含测方法学。分别以AST、AST-Ⅳ(按AST-Ⅳ计20mg/kg相等剂量)两种药物予大鼠腹腔注射,给药后30,60,75,90,105,120min测定大鼠血清中AST-Ⅳ的含量,并对各时间点两种药物的血药浓度进行分析、比较,找出各自血药浓度的达峰时间。
二、在AST及AST-Ⅳ不同剂量对大鼠急性胃粘膜损伤的保护作用研究中,以AST-Ⅳ为控制指标,设定AST及AST-Ⅳ不同剂量,并保证二者对应的大、中、小剂量中AST-Ⅳ的含量一致。将SD大鼠随机分为模型组,AST大、中、小剂量组,AST-Ⅳ大、中、小剂量组,西药阳性对照组。各组分别给予溶剂注射液,AST大(200mg/kg)、中(100mg/kg)、小(50mg/kg)剂量注射液,AST-Ⅳ大(4.08mg/kg)、中(2.04mg/kg)、小(1.02mg/kg)剂量注射液,西米替丁(100mg/kg)注射液腹腔注射。于给药1次90min、75min(分别是AST与AST-Ⅳ各自血药浓度的达峰时间)后及给药4次于末次给药90min、75min后,用无水乙醇(1ml/100g)灌胃诱导大鼠急性胃粘膜损伤。1h后取腺胃区组织测量胃粘膜损伤分数,并制作石蜡切片做HE染色,在光学显微镜下观察胃粘膜组织形态的病理变化。
三、AST及AST-Ⅳ对慢性胃粘膜损伤大鼠胃粘膜上皮细胞超微结构的影响,仍以AST-Ⅳ为控制指标,设定AST及AST-Ⅳ高、低剂量,并保证二者对应的高、低剂量中AST-Ⅳ的含量一致。将SD大鼠随机分为正常组,模型组,AST高、低剂量预防组,AST-Ⅳ高、低剂量预防组,模型自然恢复组,AST高、低剂量治疗组,AST-Ⅳ高、低剂量治疗组。采用大黄灌胃造成大鼠慢性胃粘膜损伤模型,分别腹腔给予溶剂注射液、AST高(200mg/kg)、低(100mg/kg)剂量注射液,AST-Ⅳ高(4.08mg/kg)、低(2.04mg/kg)剂量注射液。预防组连续给药14d,治疗组造模结束后治疗6d。用扫描电镜观察慢性胃粘膜损伤大鼠胃粘膜表面上皮细胞超微结构的变化及AST与AST-Ⅳ预防给药和治疗给药对其的影响。
四、AST及AST-Ⅳ对大鼠急性损伤胃粘膜细胞凋亡及增殖的影响,采用免疫组织化学SP法,检测早期凋亡相关基因(Bcl-2/Bax)以及增殖性细胞核抗原(PCNA)在急性胃粘膜损伤大鼠胃粘膜的蛋白表达,并通过CMIASWIN图像分析系统对免疫组化结果做定量分析及统计学处理。
五、统计分析方法,采用单因素方差分析(One-Way ANOVA),组间比较采用LSD法。
[结果]
一、SPE-HPLC-ELSD法研究AST、AST-Ⅳ在大鼠体内AST-Ⅳ的血药浓度,结果表明用SPE法提取分离大鼠血清中的AST-IV,操作简便、去除蛋白效果好,且对AST-Ⅳ有很好的富集作用。用HPLC-ELSD法检测大鼠血清中的AST-Ⅳ含量,能解决成分微量、复杂的血清样品的定量分析问题。其线性关系良好,线性方程为：Y=1.272X+2.98,r=0.9995,最低检测浓度为1.536μg/mL,其专属性、精密度、稳定性、回收率及萃取回收率均符合要求,成功建立AST-Ⅳ含测方法学。
在大鼠腹腔注射等量AST-Ⅳ时,测得AST血药浓度达峰时间在90min左右,AST-Ⅳ血药浓度达峰时间在75min左右。两种药物各时点血清中AST-Ⅳ的浓度前者均是后者3倍以上,且达峰时的浓度前者是后者的3.5倍。AST达峰时血清中AST-Ⅳ的浓度为33.05±5.36μg/mL, AST-Ⅳ达峰时血清中AST-Ⅳ的浓度为9.29±3.63μg/mL。
二、在AST及AST-Ⅳ对大鼠急性胃粘膜损伤的保护作用研究中表明：给药1次及给药4次中除AST-Ⅳ小剂量组外,其余各组均有不同程度的降低损伤分数和光镜下损伤指数的作用(P<0.05),且AST各剂量组优于AST-Ⅳ各剂量组(P<0.05),AST大剂量组明显优于其他剂量组(P<0.05),与西药阳性对照组作用相当(P>0.05)；给药1次与给药4次相同的剂量组相比,无显著性差异(P>0.05);AST各组给药1次作用优于AST-Ⅳ各组给药4次(P<0.05)。
三、在AST及AST-Ⅳ对慢性胃粘膜损伤大鼠胃粘膜上皮细胞超微结构影响的实验中,我们发现慢性胃粘膜损伤大鼠胃粘膜上皮细胞广泛坏死、破碎、形态模糊不完整,排列紊乱,细胞肿胀、扁平,细胞间无界限,多个细胞挤压扭结成团；细胞表面微绒毛稀少、脱落,分泌颗粒稀少；胃小凹结构畸形的。AST高(200mg/kg)、低(100mg/kg)剂量及AST-Ⅳ高剂量(4.08mg/kg),预防给药和治疗给药,均能够扭转慢性胃粘膜损伤大鼠胃粘膜上皮的病变,且AST各剂量的作用均优于AST-Ⅳ各剂量,AST高剂量预防给药和治疗给药的作用均明显优于其余各剂量,与正常组的水平相当；AST低剂量预防给药和治疗给药的作用均优于AST-Ⅳ高剂量。
四、从AST及AST-Ⅳ对大鼠急性胃粘膜损伤细胞凋亡及增殖的影响中可以看出,实验结果与模型组比较,AST各个剂量组、AST-Ⅳ大、中剂量组及西药阳性对照组,Bax蛋白阳性表达减弱(P<0.05), Bcl-2蛋白表达增强(P<0.05)；而AST各个剂量组、AST-Ⅳ大剂量组及西药阳性对照组,胃粘膜PCNA蛋白阳性表达明显增强,与模型组比较,有显著性差异(P<0.05)。AST各剂量组减弱Bax表达,增强Bc1-2及PCNA表达的作用优于AST-Ⅳ各剂量组(P<0.05),AST大剂量组明显优于其他剂量组(P<0.05),接近于西药阳性对照组。
[结论]
一、SPE-HPLC-ELSD法检测AST、AST-IV在大鼠体内AST-Ⅳ的血药浓度,其方法学正确合理。以AST-Ⅳ为指标,可以认为AST较AST-Ⅳ更易于吸收。这一结果为黄芪皂苷类有效部位抵抗胃粘膜损伤作用优于单体成分,提供了有力的证据,也为后期的药效学研究打下了基础。
二、在AST及AST-Ⅳ对大鼠急性胃粘膜损伤的保护作用研究中证实：AST及AST-Ⅳ对无水乙醇所致的大鼠急性胃粘膜损伤有保护作用,AST的作用明显优于AST-Ⅳ,且随剂量的增加,AST的优越性更加明显,AST大剂量的保护作用已经接近阳性对照药西米替丁的水平；连续给药4次,AST和AST-Ⅳ均不存在药效叠加的现象；AST单次给药的保护作用优于AST-Ⅳ多次给药。
三、AST及AST-Ⅳ对慢性胃粘膜损伤大鼠胃粘膜上皮细胞超微结构影响的实验表明：慢性胃粘膜损伤大鼠的胃粘膜屏障受到破坏,AST及AST-Ⅳ均具有保护胃粘膜屏障的作用；AST的作用明显优于AST-Ⅳ,且随剂量的增加,AST的优越性更加明显,高剂量的作用接近正常组的水平；AST在较低剂量的情况下可以达到甚至超过AST-Ⅳ高剂量的作用效应。
四、AST及AST-Ⅳ对大鼠急性胃粘膜损伤细胞凋亡及增殖的影响结果说明：AST及AST-Ⅳ可通过下调Bax表达,上调Bc1-2以及PCNA表达水平,不同程度地抑制胃粘膜细胞凋亡,促进胃粘膜细胞增殖,从而发挥保护胃粘膜的作用；AST的抑制胃粘膜细胞凋亡、促进胃粘膜细胞增殖作用优于AST-IV,且随剂量的增加,AST的优越性更加明显,AST大剂量的保护作用已经接近阳性对照药西米替丁的水平。
通过上述的实验研究,说明黄芪皂苷类有效部位AST及单体AST-IV具有明确的胃粘膜保护作用,证明了中药黄芪“益气健脾、生肌”作用的科学含义。揭示了黄芪皂苷类有效部位的药效作用优于单体成分,有效部位在较低剂量的情况下可以达到甚至超过单体高剂量的作用效应。这说明由于中药有效部位中的多成分之间相互合理配伍,起到协同增效的作用,这种作用类似于中药复方,是多种同类成分之间相互影响、综合作用的结果。这一研究结果为胃粘膜损伤疾病的防治提供了新的思路,也为今后从药物组分水平上开展黄芪相关的药物开发提供理论依据。
Research Background
Carrying on the research of traditional Chinese medicine effective parts has become one of the most important directions in traditional Chinese medicine, crude drug and new drug development in recent years. It is our research group's persistent research direction to develop pharmacology study of nourishing spleen and reinforcing qi TCM for gastric mucosal protective effect basing on TCM theory and combining modern scientific experiment technology.
In earlier research work, members of our research group have studyed the effects of AstragaIosides (AST) on function and morphology of gastric parietal cells of spleen deficiency rats. We have found that AST as the main effective parts has an important role on regulating the functions of gastric parietal cells, protecting the gastric mucosal barrier, maintaining the integrity of structure and modulating gastric acid secretion. As the mixture of Astragalus saponins compounds, AST is one of the effective parts of Astragalus. As the monomer of Astragalus saponins compounds, AstragalosideⅣ(AST-Ⅳ) is formulated as the quality evaluation index of Astragalus by TCM Pharmacopeia. As we know, the physical base of TCM multiple targets effects often root in the synergistic effect of multiple similar compounds in effective parts. The activity of single compound is not strong. Therefore, Carry on contrast study of TCM effective parts and monomer using efficacy as standard, which can reveal the difference of TCM effective parts and monomer and clarify the science significance of effective parts synergistic effects. In view of this research ideas, this experiment carry on further study based on the gastric mucosal protective effects of AST and AST-Ⅳ. At present, the related research reports has not yet been seen. Objective
This paper is aimed at studying the gastric mucosal protective effects of Astragalus saponins effective part (AST) and the monomer (AST-Ⅳ) basing on TCM theory and combining with modern scientific experiment technology. It mainly includes the following aspects:
First, solid phase extraction-high performance liquid chromatography-evaporative light scattering detection (SPE-HPLC-ELSD) was established for determining the serum concentration of AST-Ⅳin rats after separately intraperitoneal injection of AST and AST-Ⅳat the same time and finding the peak time of AST and AST-Ⅳserum concentration. Thus to lay a foundation for establishing molding time in the experimental study of AST and AST-IV to acute gastric mucosal injury in rats in late, and also provide the basis of pharmacokinetic parameters for the pharmacodynamic difference of the two medicine to resisting acute gastric mucosa injury.
Second, study the protective effect of AST and AST-Ⅳdifferent doses on absolute alcohol induced acute gastric mucosal injury in rats from the degree of gastric mucosal pathological morphology. Meanwhile, we observe pharmacodynamics separately from medication one time and medication four times in order to observe the cumulative effect, and expect to study the difference of AST and AST-Ⅳfrom multilevel.
Third, probe the protective effects of AST and AST-IV on chronic gastric mucosal injury after prevention medicine and treatment medicine from the gastric mucosal epithelial cells ultrastructural changes.
Fourth, observe the effects of AST and AST-Ⅳon acute injury gastric mucosal cells' apoptosis and proliferation in rats from the degree of cytology and expect to formulate the gastric mucosal protective effects mechanisms and difference of these two drugs.
Methods
First, investigate AST-Ⅳserum concentration and it's peak time of AST and AST-IV in rats. Extract the AST-IV in serum by SPE. Investigate the linear relationship and minimum detection concentration, specificity, precision, stability and recovery by HPLC-ELSD to establish the AST-IV content test methodology. Serum AST-Ⅳcontent was detected and compared after separately intraperitoneal injection of AST and AST-Ⅳ(equivalent of AST-Ⅳ20 mg/kg) for 30,60,75,90,105 and 120min. Then the peak time of plasma concentration of each medicine was found.
Second, with the AST-Ⅳas the control index, we set AST and AST-Ⅳdifferent doses and ensure the AST-Ⅳcontent of their corresponding large, medium and small doses consistent in the experimental research of AST and AST-Ⅳdifferent doses protecting on acute gastric mucosal injury in rats. SD rats were randomly divided into model group, AST large, medium and small dose group, AST-Ⅳlarge, medium and small dose group and western medicine positive control group, these groups were respectively given intraperitoneal injection of solvent, AST large (200mg/kg), medium (100mg/kg), small (50mg/kg) doses injection and AST-Ⅳlarge (4.08mg/kg), medium (2.04mg/kg), small (1.02mg/kg) doses injection and cimetidine (200mg/kg) injection. Then we used absolute alcohol to induce acute gastric mucosal injury in rats after medication one time and last time of medicine four times 90 min and 75 min (which are the peak time of serum concentrion of AST and AST-Ⅳrespectively). The gland stomach tissues were taken to measure the gastric mucosal injury scores after one hour and paraffin sections were stained with HE and pathomorphological change of gastric mucosal was observed under an optical microscope.
Third, with the AST-IV as the control index, we still set AST and AST-Ⅳhigh and low doses and ensure the AST-Ⅳcontent of their corresponding high and low doses consistent in the experiment research of AST and AST-Ⅳinfluence on gastric mucosal epithelial cells ultrastructure of chronic gastric mucosal injury rats. SD rats were randomly divided into normal group, model group, AST high and low dose prevention group, AST-Ⅳhigh and low dose prevention group and model natural recovery group, AST high and low dose treatment group, AST-Ⅳhigh and low dose treatment group. The model of chronic gastric mucosa injury in rats was established by giving Rheum into stomach. These groups were respectively given intraperitoneal injection of solvent, AST high (200mg/kg) and low (100mg/kg) doses injection and AST-Ⅳhigh (4.08mg/kg) and low (2.04mg/kg) doses injection. Prevention groups were given 14 days dosing continuously and treatment groups were given 6 days cure after molding. Then we observed the ultrastructural changes of gastric mucosal surface epithelial cells and the effects of AST and AST-Ⅳprevention and treatment medication on these model rats by scanning electron microscope.
Fourth, in the experimental research of AST and AST-Ⅳon cell apoptosis and proliferation to acute injury gastric mucosa in rats, the SP method of immunohistochemistry was used to detect the proteins expressions of early apoptosis related gene (Bcl-2/Bax) and proliferating cell nuclear antigen (PCNA) in acute injury gastric mucosa of rats. CMIASWIN image analysis system was used to quantitatively analysis immunohistochemical results and then we do statistical processing.
Fifth, single factor analysis variance (One-Way ANOVA) was been adopted to statistic analysis and LSD test was been used to compare among groups.
Results
First, the SPE-HPLC-ELSD was used to detect serum AST-IV concentration in rats with AST and AST-Ⅳ. The results showed that:The method of SPE to extract and separate serum AST-Ⅳin rats was easy and simple to handle, the effects of which to remove protein and to enrich AST-IV were very well. HPLC-ELSD to detect serum AST-Ⅳconcentration in rats could solve the quantitative analysis problem of micro-componential and complicated serum sample. The linear relationship was good. The linear equation was Y=1.272X+2.98, r=0.9995. The minimum detection concentration was 1.536μg/mL. With the specificity, precision, stability, recovery and extraction recovery were all desirable, we established the AST-Ⅳcontent test methodology successfully.
We measured the peak time of AST serum concentration was about 90 min and AST-Ⅳwas about 75 min after equivalent of AST-Ⅳintraperitoneal Injection. At any time point, the serum concentration of AST-Ⅳin rats after intraperitoneal injection for AST was three times as much as that in rats after intraperitoneal injection for AST-Ⅳ, and even arrived 3.5 times in peak time. The serum concentration of AST-Ⅳwas 33.05±5.36μg/mL when AST reached it's peak time, while AST-Ⅳwas 9.29±3.63μg/mL when AST-IV reached it's peak time.
Second, the results of AST and AST-Ⅳprotective effects on acute gastric mucosa injury in rats indicated that:Each group except AST-Ⅳsmall dose group could take different degree effect on reducing gastric injury score and gastric injury index under the light microscope in medication one time and four times (P<0.05). AST each dose group was superior to AST-Ⅳ(P<0.05), AST large dose group which had the same effect with western medicine positive control group (P>0.05) was obviously superior to the rest groups (P<0.05). The groups of medication one time compare with medication four times had no significant difference (P>0.05). The effect of medication one time of AST each dose group was superior to medication four times of AST-Ⅳeach dose group (P<0.05)
Third, in the experiment research of AST and AST-IV influence on gastric mucosal epithelial cells ultrastructural of chronic gastric mucosal injury rats, We found that:The gastric mucosal epithelial cells of chronic gastric mucosal injury rats became generally necrosis, fragmentation, fuzzy and incomplete form, disorganized arrangement, swelling and applanation, no delimitation among cells, a number of cells squeeze and tangle into lump. Microvillus of cell surface became rare and detached with few secretory granules. The construction gastric pits became malformed. AST high (200mg/kg) and low(100mg/kg) doses as well as AST-IV high dose(4.08mg/kg), prevention and therapy medication, were able to reverse the gastric mucosal epithelial lesions in rats with chronic gastric mucosal injury, the effect of AST each dose was superior to all doses of AST-Ⅳ, and the effect of AST high dose of prevention and therapy medication was superior to the rest doses and equal to the normal group. The effect of AST low dose of prevention and therapy medication was superior to AST-Ⅳhigh dose.
Fourth, as we can see from effects of AST and AST-Ⅳon cell apoptosis and proliferation to acute injury gastric mucosa, Compared with model group, AST each dose group, AST large and medium dose group and western medicine positive control group, Bax protein positive expression were weaken (P<0.05), Bcl-2 protein positive expression were strengthen (P<0.05). While AST each dose group, AST large dose group and western medicine positive control group, gastric mucosal PCNA protein expression enhanced obviously, which had significant difference compared with model group (P<0.05).The effects of weakening Bax expression, enhancing Bcl-2 and PCNA expression of AST each dose group were superior to AST-IV each dose group (P<0.05). AST large dose group which had close to the western medicine positive control group was obviously superior to the rest groups (P<0.05)
Conclusion
First, the method of SPE-HPLC-ELSD which had been used to detect the AST-Ⅳserum concentration in rats with AST and AST-Ⅳwas correct and reasonable. With AST-Ⅳas the serum concentration index, we could consider that AST was easier to be absorbed than AST-Ⅳ. This result provided powerful evidence that the effects of Astragalus saponins effective parts were superior to the monomer and also laid a foundation for late pharmacodynamics study.
Second, the study of AST and AST-Ⅳprotective effected on acute gastric mucosa injury in rats confirmed that:AST and AST-IV had a protective effect on rats' alcohol-induced acute gastric mucosal injury, the effect of AST was obviously superior to AST-Ⅳ, and with the dosage's increasing, the superiority of AST was more obviously. The protective effect of AST large dose group had already access to positive control medicine cimetidine. There was no potency stacked phenomenon in AST and AST-Ⅳcontinuous medication four times. The protective effect of AST medication once was better than AST-IV multiple medication.
Third, the experiment research of AST and AST-Ⅳinfluenced on gastric mucosal epithelial cells ultrastructure of chronic gastric mucosal injury rats demonstrated:gastric mucosal barrier of chronic gastric mucosal injury rats got damaged, AST and AST-Ⅳhad a protective effect on gastric mucosal barrier. The effect of AST was obviously superior to AST-Ⅳ. With the dosage's increasing, the superiority of AST was more obviously, the protective effect of AST high dose had already access to the normal group. AST in the case of lower dose could achieve even more than AST-Ⅳhigh dose effect.
Fourth, the influence of AST and AST-Ⅳon cell apoptosis and proliferation to acute injury gastric mucosa illustrated:AST and AST-IV could descend the expression of Bax, and ascend the expression of Bcl-2 and PCNA. Thus restrained gastric mucosal cell apoptosis and promoted gastric mucosal cell proliferation from different degree for alleviating the injury of gastric mucosa. That was one of the mechanisms of these two medicines resisting acute gastric mucosa injury. The effect of AST was obviously superior to AST-Ⅳ. With the dosage's increasing, the superiority of AST was more obviously. The protective effect of AST large dose had already access to the positive control medicine Cimetidine.
We have illustrate the effective parts of Astragalus saponins AST and the monomer AST-Ⅳhave clearly protective effects on gastric mucosa via the above-mentioned experimental study. Thus have certified the scientific meaning of TCM Astragalus "nourishing spleen and reinforcing qi" and "promote new growth of tissues". We have also revealed the effects of Astragalus saponins effective parts are superior to the monomer composition, and the effective parts in the case of lower dose can achieve even more than the monomer high dose effect. Thus state that reasonable compatibility among multicomponent of TCM effective parts play the role of synergistic function. This function is similar to TCM compound which is the result of multiple similar compositions combined action by influencing each other. This research results provide a new thought on the prevention and treatment for gastric mucosal injury, and also provide theory basis for carrying on medicine development of Astragalus from medicine components level.
开展中药有效部位的研究,近年来成为中药、天然药新药开发的重要方向之一以中医药理论为依据,结合现代科学实验技术开展健脾益气中药对于胃粘膜保护作用的药理学研究是我们课题组多年来坚持不懈的研究方向。
在前期研究工作中,课题组成员探讨了黄芪总苷(AST)对脾虚大鼠胃壁细胞功能及形态的影响,发现AST作为黄芪的主要有效部位,在调节细胞信号传导功能的同时,具有保护胃粘膜屏障、维持组织结构完整性,调节整体胃酸分泌的重要作用。AST作为黄芪皂苷类化合物的混合体,是黄芪的有效部位之一；黄芪甲苷(AST-Ⅳ)作为黄芪中的单体皂苷类化合物,被中药药典规定为黄芪药材质量评价的指标性物质。我们知道,中药多靶点发挥药效作用的物质基础往往来源于有效部位中多个同类化合物群体的协同作用,单一化合物的活性均不强。因此,以药效为标准对中药有效部位与单体成分进行对比研究,能够揭示中药有效部位与单体的药理作用差异,阐明有效部位中多成分之间相互协同增效的科学意义。基于这种研究思路,本实验对基于胃粘膜保护的AST及AST-Ⅳ的作用及其差异开展进一步研究,目前尚未见相关研究报道。
[目的]
本文旨在以黄芪“健脾益气”作用的中医药理论为依据,结合现代科学实验技术研究黄芪皂苷类有效部位(AST)及单体成分(AST-Ⅳ)对胃粘膜保护作用的功效,并从多方面对比AST与AST-Ⅳ保护胃粘膜的作用差异。主要包括以下几个方面：
一、建立固相萃取-高效液相-蒸发光散射检测(SPE-HPLC-ELSD)法测定大鼠血清中AST-Ⅳ含量的方法,比较AST、AST-Ⅳ在大鼠腹腔注射给药后相同时间大鼠血清中AST-Ⅳ的含量,并找出AST及AST-Ⅳ血药浓度的达峰时间。为后期AST与AST-Ⅳ对大鼠急性胃粘膜损伤的实验研究中造模给药时间点的确立打下基础,并为两药抗急性胃粘膜损伤药效学作用的差异提供药代动力学参数的依据。
二、从胃粘膜病理形态学的角度,观察研究AST及AST-Ⅳ不同剂量对无水乙醇致大鼠急性胃粘膜损伤的保护作用。同时,为了观察药效的累积效应,分别观察给药1次及连续给药4次的药效作用,以期从多层次研究探讨AST与AST-Ⅳ作用的差异性。
三、从胃粘膜上皮细胞超微结构的改变,探讨AST与AST-Ⅳ预防及治疗给药后对慢性胃粘膜损伤的保护作用。
四、从细胞学的角度,观察AST及AST-Ⅳ对大鼠急性损伤的胃粘膜细胞凋亡及增殖的影响,以期明确这两种药物保护胃粘膜细胞的作用机制及差异。
[方法]
一、对AST、AST-Ⅳ在大鼠体内AST-Ⅳ的血药浓度及达峰时间考察,采用SPE法提取分离大鼠血清中的AST-Ⅳ, HPLC-ELSD法考察其线性关系及最低检测浓度、专属性、精密度、稳定性及回收率,建立AST-Ⅳ含测方法学。分别以AST、AST-Ⅳ(按AST-Ⅳ计20mg/kg相等剂量)两种药物予大鼠腹腔注射,给药后30,60,75,90,105,120min测定大鼠血清中AST-Ⅳ的含量,并对各时间点两种药物的血药浓度进行分析、比较,找出各自血药浓度的达峰时间。
二、在AST及AST-Ⅳ不同剂量对大鼠急性胃粘膜损伤的保护作用研究中,以AST-Ⅳ为控制指标,设定AST及AST-Ⅳ不同剂量,并保证二者对应的大、中、小剂量中AST-Ⅳ的含量一致。将SD大鼠随机分为模型组,AST大、中、小剂量组,AST-Ⅳ大、中、小剂量组,西药阳性对照组。各组分别给予溶剂注射液,AST大(200mg/kg)、中(100mg/kg)、小(50mg/kg)剂量注射液,AST-Ⅳ大(4.08mg/kg)、中(2.04mg/kg)、小(1.02mg/kg)剂量注射液,西米替丁(100mg/kg)注射液腹腔注射。于给药1次90min、75min(分别是AST与AST-Ⅳ各自血药浓度的达峰时间)后及给药4次于末次给药90min、75min后,用无水乙醇(1ml/100g)灌胃诱导大鼠急性胃粘膜损伤。1h后取腺胃区组织测量胃粘膜损伤分数,并制作石蜡切片做HE染色,在光学显微镜下观察胃粘膜组织形态的病理变化。
三、AST及AST-Ⅳ对慢性胃粘膜损伤大鼠胃粘膜上皮细胞超微结构的影响,仍以AST-Ⅳ为控制指标,设定AST及AST-Ⅳ高、低剂量,并保证二者对应的高、低剂量中AST-Ⅳ的含量一致。将SD大鼠随机分为正常组,模型组,AST高、低剂量预防组,AST-Ⅳ高、低剂量预防组,模型自然恢复组,AST高、低剂量治疗组,AST-Ⅳ高、低剂量治疗组。采用大黄灌胃造成大鼠慢性胃粘膜损伤模型,分别腹腔给予溶剂注射液、AST高(200mg/kg)、低(100mg/kg)剂量注射液,AST-Ⅳ高(4.08mg/kg)、低(2.04mg/kg)剂量注射液。预防组连续给药14d,治疗组造模结束后治疗6d。用扫描电镜观察慢性胃粘膜损伤大鼠胃粘膜表面上皮细胞超微结构的变化及AST与AST-Ⅳ预防给药和治疗给药对其的影响。
四、AST及AST-Ⅳ对大鼠急性损伤胃粘膜细胞凋亡及增殖的影响,采用免疫组织化学SP法,检测早期凋亡相关基因(Bcl-2/Bax)以及增殖性细胞核抗原(PCNA)在急性胃粘膜损伤大鼠胃粘膜的蛋白表达,并通过CMIASWIN图像分析系统对免疫组化结果做定量分析及统计学处理。
五、统计分析方法,采用单因素方差分析(One-Way ANOVA),组间比较采用LSD法。
[结果]
一、SPE-HPLC-ELSD法研究AST、AST-Ⅳ在大鼠体内AST-Ⅳ的血药浓度,结果表明用SPE法提取分离大鼠血清中的AST-IV,操作简便、去除蛋白效果好,且对AST-Ⅳ有很好的富集作用。用HPLC-ELSD法检测大鼠血清中的AST-Ⅳ含量,能解决成分微量、复杂的血清样品的定量分析问题。其线性关系良好,线性方程为：Y=1.272X+2.98,r=0.9995,最低检测浓度为1.536μg/mL,其专属性、精密度、稳定性、回收率及萃取回收率均符合要求,成功建立AST-Ⅳ含测方法学。
在大鼠腹腔注射等量AST-Ⅳ时,测得AST血药浓度达峰时间在90min左右,AST-Ⅳ血药浓度达峰时间在75min左右。两种药物各时点血清中AST-Ⅳ的浓度前者均是后者3倍以上,且达峰时的浓度前者是后者的3.5倍。AST达峰时血清中AST-Ⅳ的浓度为33.05±5.36μg/mL, AST-Ⅳ达峰时血清中AST-Ⅳ的浓度为9.29±3.63μg/mL。
二、在AST及AST-Ⅳ对大鼠急性胃粘膜损伤的保护作用研究中表明：给药1次及给药4次中除AST-Ⅳ小剂量组外,其余各组均有不同程度的降低损伤分数和光镜下损伤指数的作用(P<0.05),且AST各剂量组优于AST-Ⅳ各剂量组(P<0.05),AST大剂量组明显优于其他剂量组(P<0.05),与西药阳性对照组作用相当(P>0.05)；给药1次与给药4次相同的剂量组相比,无显著性差异(P>0.05);AST各组给药1次作用优于AST-Ⅳ各组给药4次(P<0.05)。
三、在AST及AST-Ⅳ对慢性胃粘膜损伤大鼠胃粘膜上皮细胞超微结构影响的实验中,我们发现慢性胃粘膜损伤大鼠胃粘膜上皮细胞广泛坏死、破碎、形态模糊不完整,排列紊乱,细胞肿胀、扁平,细胞间无界限,多个细胞挤压扭结成团；细胞表面微绒毛稀少、脱落,分泌颗粒稀少；胃小凹结构畸形的。AST高(200mg/kg)、低(100mg/kg)剂量及AST-Ⅳ高剂量(4.08mg/kg),预防给药和治疗给药,均能够扭转慢性胃粘膜损伤大鼠胃粘膜上皮的病变,且AST各剂量的作用均优于AST-Ⅳ各剂量,AST高剂量预防给药和治疗给药的作用均明显优于其余各剂量,与正常组的水平相当；AST低剂量预防给药和治疗给药的作用均优于AST-Ⅳ高剂量。
四、从AST及AST-Ⅳ对大鼠急性胃粘膜损伤细胞凋亡及增殖的影响中可以看出,实验结果与模型组比较,AST各个剂量组、AST-Ⅳ大、中剂量组及西药阳性对照组,Bax蛋白阳性表达减弱(P<0.05), Bcl-2蛋白表达增强(P<0.05)；而AST各个剂量组、AST-Ⅳ大剂量组及西药阳性对照组,胃粘膜PCNA蛋白阳性表达明显增强,与模型组比较,有显著性差异(P<0.05)。AST各剂量组减弱Bax表达,增强Bc1-2及PCNA表达的作用优于AST-Ⅳ各剂量组(P<0.05),AST大剂量组明显优于其他剂量组(P<0.05),接近于西药阳性对照组。
[结论]
一、SPE-HPLC-ELSD法检测AST、AST-IV在大鼠体内AST-Ⅳ的血药浓度,其方法学正确合理。以AST-Ⅳ为指标,可以认为AST较AST-Ⅳ更易于吸收。这一结果为黄芪皂苷类有效部位抵抗胃粘膜损伤作用优于单体成分,提供了有力的证据,也为后期的药效学研究打下了基础。
二、在AST及AST-Ⅳ对大鼠急性胃粘膜损伤的保护作用研究中证实：AST及AST-Ⅳ对无水乙醇所致的大鼠急性胃粘膜损伤有保护作用,AST的作用明显优于AST-Ⅳ,且随剂量的增加,AST的优越性更加明显,AST大剂量的保护作用已经接近阳性对照药西米替丁的水平；连续给药4次,AST和AST-Ⅳ均不存在药效叠加的现象；AST单次给药的保护作用优于AST-Ⅳ多次给药。
三、AST及AST-Ⅳ对慢性胃粘膜损伤大鼠胃粘膜上皮细胞超微结构影响的实验表明：慢性胃粘膜损伤大鼠的胃粘膜屏障受到破坏,AST及AST-Ⅳ均具有保护胃粘膜屏障的作用；AST的作用明显优于AST-Ⅳ,且随剂量的增加,AST的优越性更加明显,高剂量的作用接近正常组的水平；AST在较低剂量的情况下可以达到甚至超过AST-Ⅳ高剂量的作用效应。
四、AST及AST-Ⅳ对大鼠急性胃粘膜损伤细胞凋亡及增殖的影响结果说明：AST及AST-Ⅳ可通过下调Bax表达,上调Bc1-2以及PCNA表达水平,不同程度地抑制胃粘膜细胞凋亡,促进胃粘膜细胞增殖,从而发挥保护胃粘膜的作用；AST的抑制胃粘膜细胞凋亡、促进胃粘膜细胞增殖作用优于AST-IV,且随剂量的增加,AST的优越性更加明显,AST大剂量的保护作用已经接近阳性对照药西米替丁的水平。
通过上述的实验研究,说明黄芪皂苷类有效部位AST及单体AST-IV具有明确的胃粘膜保护作用,证明了中药黄芪“益气健脾、生肌”作用的科学含义。揭示了黄芪皂苷类有效部位的药效作用优于单体成分,有效部位在较低剂量的情况下可以达到甚至超过单体高剂量的作用效应。这说明由于中药有效部位中的多成分之间相互合理配伍,起到协同增效的作用,这种作用类似于中药复方,是多种同类成分之间相互影响、综合作用的结果。这一研究结果为胃粘膜损伤疾病的防治提供了新的思路,也为今后从药物组分水平上开展黄芪相关的药物开发提供理论依据。
Research Background
Carrying on the research of traditional Chinese medicine effective parts has become one of the most important directions in traditional Chinese medicine, crude drug and new drug development in recent years. It is our research group's persistent research direction to develop pharmacology study of nourishing spleen and reinforcing qi TCM for gastric mucosal protective effect basing on TCM theory and combining modern scientific experiment technology.
In earlier research work, members of our research group have studyed the effects of AstragaIosides (AST) on function and morphology of gastric parietal cells of spleen deficiency rats. We have found that AST as the main effective parts has an important role on regulating the functions of gastric parietal cells, protecting the gastric mucosal barrier, maintaining the integrity of structure and modulating gastric acid secretion. As the mixture of Astragalus saponins compounds, AST is one of the effective parts of Astragalus. As the monomer of Astragalus saponins compounds, AstragalosideⅣ(AST-Ⅳ) is formulated as the quality evaluation index of Astragalus by TCM Pharmacopeia. As we know, the physical base of TCM multiple targets effects often root in the synergistic effect of multiple similar compounds in effective parts. The activity of single compound is not strong. Therefore, Carry on contrast study of TCM effective parts and monomer using efficacy as standard, which can reveal the difference of TCM effective parts and monomer and clarify the science significance of effective parts synergistic effects. In view of this research ideas, this experiment carry on further study based on the gastric mucosal protective effects of AST and AST-Ⅳ. At present, the related research reports has not yet been seen. Objective
This paper is aimed at studying the gastric mucosal protective effects of Astragalus saponins effective part (AST) and the monomer (AST-Ⅳ) basing on TCM theory and combining with modern scientific experiment technology. It mainly includes the following aspects:
First, solid phase extraction-high performance liquid chromatography-evaporative light scattering detection (SPE-HPLC-ELSD) was established for determining the serum concentration of AST-Ⅳin rats after separately intraperitoneal injection of AST and AST-Ⅳat the same time and finding the peak time of AST and AST-Ⅳserum concentration. Thus to lay a foundation for establishing molding time in the experimental study of AST and AST-IV to acute gastric mucosal injury in rats in late, and also provide the basis of pharmacokinetic parameters for the pharmacodynamic difference of the two medicine to resisting acute gastric mucosa injury.
Second, study the protective effect of AST and AST-Ⅳdifferent doses on absolute alcohol induced acute gastric mucosal injury in rats from the degree of gastric mucosal pathological morphology. Meanwhile, we observe pharmacodynamics separately from medication one time and medication four times in order to observe the cumulative effect, and expect to study the difference of AST and AST-Ⅳfrom multilevel.
Third, probe the protective effects of AST and AST-IV on chronic gastric mucosal injury after prevention medicine and treatment medicine from the gastric mucosal epithelial cells ultrastructural changes.
Fourth, observe the effects of AST and AST-Ⅳon acute injury gastric mucosal cells' apoptosis and proliferation in rats from the degree of cytology and expect to formulate the gastric mucosal protective effects mechanisms and difference of these two drugs.
Methods
First, investigate AST-Ⅳserum concentration and it's peak time of AST and AST-IV in rats. Extract the AST-IV in serum by SPE. Investigate the linear relationship and minimum detection concentration, specificity, precision, stability and recovery by HPLC-ELSD to establish the AST-IV content test methodology. Serum AST-Ⅳcontent was detected and compared after separately intraperitoneal injection of AST and AST-Ⅳ(equivalent of AST-Ⅳ20 mg/kg) for 30,60,75,90,105 and 120min. Then the peak time of plasma concentration of each medicine was found.
Second, with the AST-Ⅳas the control index, we set AST and AST-Ⅳdifferent doses and ensure the AST-Ⅳcontent of their corresponding large, medium and small doses consistent in the experimental research of AST and AST-Ⅳdifferent doses protecting on acute gastric mucosal injury in rats. SD rats were randomly divided into model group, AST large, medium and small dose group, AST-Ⅳlarge, medium and small dose group and western medicine positive control group, these groups were respectively given intraperitoneal injection of solvent, AST large (200mg/kg), medium (100mg/kg), small (50mg/kg) doses injection and AST-Ⅳlarge (4.08mg/kg), medium (2.04mg/kg), small (1.02mg/kg) doses injection and cimetidine (200mg/kg) injection. Then we used absolute alcohol to induce acute gastric mucosal injury in rats after medication one time and last time of medicine four times 90 min and 75 min (which are the peak time of serum concentrion of AST and AST-Ⅳrespectively). The gland stomach tissues were taken to measure the gastric mucosal injury scores after one hour and paraffin sections were stained with HE and pathomorphological change of gastric mucosal was observed under an optical microscope.
Third, with the AST-IV as the control index, we still set AST and AST-Ⅳhigh and low doses and ensure the AST-Ⅳcontent of their corresponding high and low doses consistent in the experiment research of AST and AST-Ⅳinfluence on gastric mucosal epithelial cells ultrastructure of chronic gastric mucosal injury rats. SD rats were randomly divided into normal group, model group, AST high and low dose prevention group, AST-Ⅳhigh and low dose prevention group and model natural recovery group, AST high and low dose treatment group, AST-Ⅳhigh and low dose treatment group. The model of chronic gastric mucosa injury in rats was established by giving Rheum into stomach. These groups were respectively given intraperitoneal injection of solvent, AST high (200mg/kg) and low (100mg/kg) doses injection and AST-Ⅳhigh (4.08mg/kg) and low (2.04mg/kg) doses injection. Prevention groups were given 14 days dosing continuously and treatment groups were given 6 days cure after molding. Then we observed the ultrastructural changes of gastric mucosal surface epithelial cells and the effects of AST and AST-Ⅳprevention and treatment medication on these model rats by scanning electron microscope.
Fourth, in the experimental research of AST and AST-Ⅳon cell apoptosis and proliferation to acute injury gastric mucosa in rats, the SP method of immunohistochemistry was used to detect the proteins expressions of early apoptosis related gene (Bcl-2/Bax) and proliferating cell nuclear antigen (PCNA) in acute injury gastric mucosa of rats. CMIASWIN image analysis system was used to quantitatively analysis immunohistochemical results and then we do statistical processing.
Fifth, single factor analysis variance (One-Way ANOVA) was been adopted to statistic analysis and LSD test was been used to compare among groups.
Results
First, the SPE-HPLC-ELSD was used to detect serum AST-IV concentration in rats with AST and AST-Ⅳ. The results showed that:The method of SPE to extract and separate serum AST-Ⅳin rats was easy and simple to handle, the effects of which to remove protein and to enrich AST-IV were very well. HPLC-ELSD to detect serum AST-Ⅳconcentration in rats could solve the quantitative analysis problem of micro-componential and complicated serum sample. The linear relationship was good. The linear equation was Y=1.272X+2.98, r=0.9995. The minimum detection concentration was 1.536μg/mL. With the specificity, precision, stability, recovery and extraction recovery were all desirable, we established the AST-Ⅳcontent test methodology successfully.
We measured the peak time of AST serum concentration was about 90 min and AST-Ⅳwas about 75 min after equivalent of AST-Ⅳintraperitoneal Injection. At any time point, the serum concentration of AST-Ⅳin rats after intraperitoneal injection for AST was three times as much as that in rats after intraperitoneal injection for AST-Ⅳ, and even arrived 3.5 times in peak time. The serum concentration of AST-Ⅳwas 33.05±5.36μg/mL when AST reached it's peak time, while AST-Ⅳwas 9.29±3.63μg/mL when AST-IV reached it's peak time.
Second, the results of AST and AST-Ⅳprotective effects on acute gastric mucosa injury in rats indicated that:Each group except AST-Ⅳsmall dose group could take different degree effect on reducing gastric injury score and gastric injury index under the light microscope in medication one time and four times (P<0.05). AST each dose group was superior to AST-Ⅳ(P<0.05), AST large dose group which had the same effect with western medicine positive control group (P>0.05) was obviously superior to the rest groups (P<0.05). The groups of medication one time compare with medication four times had no significant difference (P>0.05). The effect of medication one time of AST each dose group was superior to medication four times of AST-Ⅳeach dose group (P<0.05)
Third, in the experiment research of AST and AST-IV influence on gastric mucosal epithelial cells ultrastructural of chronic gastric mucosal injury rats, We found that:The gastric mucosal epithelial cells of chronic gastric mucosal injury rats became generally necrosis, fragmentation, fuzzy and incomplete form, disorganized arrangement, swelling and applanation, no delimitation among cells, a number of cells squeeze and tangle into lump. Microvillus of cell surface became rare and detached with few secretory granules. The construction gastric pits became malformed. AST high (200mg/kg) and low(100mg/kg) doses as well as AST-IV high dose(4.08mg/kg), prevention and therapy medication, were able to reverse the gastric mucosal epithelial lesions in rats with chronic gastric mucosal injury, the effect of AST each dose was superior to all doses of AST-Ⅳ, and the effect of AST high dose of prevention and therapy medication was superior to the rest doses and equal to the normal group. The effect of AST low dose of prevention and therapy medication was superior to AST-Ⅳhigh dose.
Fourth, as we can see from effects of AST and AST-Ⅳon cell apoptosis and proliferation to acute injury gastric mucosa, Compared with model group, AST each dose group, AST large and medium dose group and western medicine positive control group, Bax protein positive expression were weaken (P<0.05), Bcl-2 protein positive expression were strengthen (P<0.05). While AST each dose group, AST large dose group and western medicine positive control group, gastric mucosal PCNA protein expression enhanced obviously, which had significant difference compared with model group (P<0.05).The effects of weakening Bax expression, enhancing Bcl-2 and PCNA expression of AST each dose group were superior to AST-IV each dose group (P<0.05). AST large dose group which had close to the western medicine positive control group was obviously superior to the rest groups (P<0.05)
Conclusion
First, the method of SPE-HPLC-ELSD which had been used to detect the AST-Ⅳserum concentration in rats with AST and AST-Ⅳwas correct and reasonable. With AST-Ⅳas the serum concentration index, we could consider that AST was easier to be absorbed than AST-Ⅳ. This result provided powerful evidence that the effects of Astragalus saponins effective parts were superior to the monomer and also laid a foundation for late pharmacodynamics study.
Second, the study of AST and AST-Ⅳprotective effected on acute gastric mucosa injury in rats confirmed that:AST and AST-IV had a protective effect on rats' alcohol-induced acute gastric mucosal injury, the effect of AST was obviously superior to AST-Ⅳ, and with the dosage's increasing, the superiority of AST was more obviously. The protective effect of AST large dose group had already access to positive control medicine cimetidine. There was no potency stacked phenomenon in AST and AST-Ⅳcontinuous medication four times. The protective effect of AST medication once was better than AST-IV multiple medication.
Third, the experiment research of AST and AST-Ⅳinfluenced on gastric mucosal epithelial cells ultrastructure of chronic gastric mucosal injury rats demonstrated:gastric mucosal barrier of chronic gastric mucosal injury rats got damaged, AST and AST-Ⅳhad a protective effect on gastric mucosal barrier. The effect of AST was obviously superior to AST-Ⅳ. With the dosage's increasing, the superiority of AST was more obviously, the protective effect of AST high dose had already access to the normal group. AST in the case of lower dose could achieve even more than AST-Ⅳhigh dose effect.
Fourth, the influence of AST and AST-Ⅳon cell apoptosis and proliferation to acute injury gastric mucosa illustrated:AST and AST-IV could descend the expression of Bax, and ascend the expression of Bcl-2 and PCNA. Thus restrained gastric mucosal cell apoptosis and promoted gastric mucosal cell proliferation from different degree for alleviating the injury of gastric mucosa. That was one of the mechanisms of these two medicines resisting acute gastric mucosa injury. The effect of AST was obviously superior to AST-Ⅳ. With the dosage's increasing, the superiority of AST was more obviously. The protective effect of AST large dose had already access to the positive control medicine Cimetidine.
We have illustrate the effective parts of Astragalus saponins AST and the monomer AST-Ⅳhave clearly protective effects on gastric mucosa via the above-mentioned experimental study. Thus have certified the scientific meaning of TCM Astragalus "nourishing spleen and reinforcing qi" and "promote new growth of tissues". We have also revealed the effects of Astragalus saponins effective parts are superior to the monomer composition, and the effective parts in the case of lower dose can achieve even more than the monomer high dose effect. Thus state that reasonable compatibility among multicomponent of TCM effective parts play the role of synergistic function. This function is similar to TCM compound which is the result of multiple similar compositions combined action by influencing each other. This research results provide a new thought on the prevention and treatment for gastric mucosal injury, and also provide theory basis for carrying on medicine development of Astragalus from medicine components level.
引文
[1]吕圭源,陈素红.关于中药有效部位新药研究的几点思考[J].世界科学技术-中医药现代化思路与方法,2004,6(5)：17-20
[2]冯寿章.重视中药二类新药(有效部位)研究,提高中药新药研制水平[J].中药新药与临床药理,1998,9(3)：134-137
[3]段亚丽,谢梅冬.黄芪化学成分及其有效成分黄芪甲苷含量测定的研究现状[J].中国兽药杂志,2005,39(3)：35-38
[4]黄可儿.黄芪总苷对脾虚大鼠胃壁细胞功能及形态影响的研究[D].广州：广州中医药大学,2006
[5]中国药典2005年版(一部)[S].北京：化学工业出版社,2005：212
[6]Luo Y, Qin Z, Hong Z, etal. Astragaloside IV pro tects against ischemic brain injury in a murine model of transient focal ischemia[J]. Neurosci Lett, 2004,363:218-223
[7]Wang Y P, Li X Y, Song C Q, et al. Effect of Astragaloside IV on T, Blymphocyte proliferation and peritoneal macrophage function in mice[J]. Acta Pharmacol Sin,2002,23:263-266
[8]WS3-B-3335-98,中华人民共和国卫生部药品标准中药成方制剂[S].卫生部药典委员会,1990.8
[9]冯祚臻,俸小平,官东秀.黄芪甲苷的提取及含量测定研究进展[J].中国药业,2005,14(11)：80-82
[10]高越,李翔,朱臻宇.大孔树脂吸附法对黄芪皂苷Ⅳ富集纯化工艺的研究[J].第二军医大学学报,2006,27(3)：334-336
[11]吕佳,侯利涛,李海滨.薄层扫描法测定补气养血颗粒黄芪甲苷含量[J].辽宁中医学院学报,2005,7(3)：271
[12]林朝展,祝晨陈,黄琳,等.薄层扫描法测定广州黄芪商品药材中黄芪甲苷的含量[J].广东药学,2002,12(5)：26-8
[13]杨克迪,李宏,龙云飞.固相萃取-高效液相色谱法测定黄芪中黄芪甲苷的含量[J].时珍国医国药,2007,18(1)：41-42
[14]顾泳川,王广基.HPLC-MS法测定大鼠尿中黄芪甲甙的含量及其尿药动力学研究[J].中国药科大学学报,2002,33(3)：222-225
[15]姚美村,齐莹,车镇涛,等.柱前衍生化HPLC测定黄芪中黄芪甲苷的含量[J].天然产物研究与开发,2000,12(4)：17-22
[16]崔颖,李灵芝,周欣.柱前衍生化法测定糖脂消胶囊中黄芪甲苷含量[J].武警医 学院学报,2007,16(3)：269-271
[17]王改香,朱智甲,林苗.柱前衍生反相高效液相色谱法测定黄芪中的黄芪甲苷[J].分析试验室,2009,28(7)：108-110
[18]谢红兵,常新耀,魏刚才,等.紫外分光光度法测定玉屏风散中黄芪甲苷的含量[J].光谱实验室,2008,25(6)：1092-1094
[19]梁瑞雪,张新军,贾元印.薄层扫描法测定尘肺清颗粒中黄芪甲苷的含量[J].时珍国医国药,2006,17(9)：1689-1690
[20]梁旭明,唐耀书,张小梅.薄层扫描法测定芪黄胶囊剂中黄芪甲苷的含量[J].时珍国医国药,2007,18(1)：22-23
[21]田丰,邓英杰.HPLC法测定黄芪中的黄芪甲苷含量[J].沈阳药科大学学报,2000,17(1)：43-45
[22]池玉梅,姜海英.HPLC示差折光检测测定黄芪精口服液中黄芪甲苷的含量[J].中国药学杂志,2001,36(1)：114-115
[23]吴巧凤,范英.HPLC法测定补血胶囊中黄芪甲苷的含量[J].中草药,2003,34(5)：425-426
[24]陈娟,师彦平.天然产物皂苷类化合物的高效液相色谱分析[J].药物分析杂志,2005,25(1)：123-128
[25]孙文基,谢世昌.天然药物成分定量分析[M].北京：中国医药科技出版社,2002：48
[26]赵陆华,吴孟华,单臻,等.高效液相色谱-蒸发光散射检测器法测定肾宝片及其中间体中黄芪甲苷的含量[J].药物分析杂志,2005,25(9)：1009-1011
[27]王鹏,叶利明,刘海峰,等.高效液相色谱-蒸发光散射检测法测定当归补血颗粒中黄芪甲苷的含量[J].药物分析杂志,2006,26(10)：1377-1379
[28]陈海明.HPLC-ELSD方法测定黄芪及其复方制剂中黄芪甲苷的含量[J].药物分析杂志,2007,27(3)：426-428
[29]胡斯,王中东,毕铭阳.HPLC-ELSD测定麦芪益肺合剂中黄芪甲苷的含量[J].中成药,2007,29(10)：1554-1555
[30]刘元媛,张振秋. HPLC-ELSD测定康艾扶正片中黄芪甲苷含量[J].中成药,2007,29(11)：1705-1706
[31]周洪波,葛晓明,房志坚.HPLC-ELSD法测定玉屏风颗粒中黄芪甲苷的含量[J],中药材,2007,30(6)：739-740
[32]贾晓斌,陈彦,蔡宝昌.HPLC-MS(TOF)法测定家兔血浆中黄芪甲苷的浓度[J].中成药,2005,27(3)：323-325
[33]陈宁,张琪,杜宇.黄芪甲苷在大鼠体内的药代动力学和组织分布研究[J].生物加工过程,2006,4(3)：67-72
[34]韩素琴,刘养清,任荣芳.香兰素荧光法测定黄芪口服液中的黄芪甲甙[J].山西农业大学学报,2002,22(1)：75-77
[35]游文玮,吴昭晖,佟丽,等.喂饲补阳还五汤大鼠血清中黄芪甲甙的固相萃取-化学衍生化-荧光分光光度法测定[J].第一军医大学学报,2003,23(4)：335-339
[36]陈娟,师彦平.天然产物皂苷类化合物的高效液相色谱分析[J].药物分析杂志,2005,25(1)：123-128
[37]Gu Y, Wang G, Fawcett JP. Determination of Astragaloside IV in rat plasma by liquid chromatography electrospray ionization mass spectrometry[J]. J Chromatogr B Analyt Technol Biomed Life Sci,2004,801(2):285-288
[38]DUY, ZHANG Q, CHENG G, etal. Pharmacokinetics of astragaloside Ⅳ in rats by liquid chromatography coupled with tandem mass spectrometry[J]. Eur J Drug Metab Pharmacokinet,2005,30(4):269-273
[39]ZHANG W D, ZHANG C, LIU RH, etal. Preclinical pharmacokinetics and tissue distribution of a natural card ioprotective agent astragaloside IV in rats and dogs[J]. Life Sci,2006,79(8):808-815
[40]Zhang Q, Zhu LL,Chen GG. Pharmacokinetics of astragaloside iv in beagle dogs[J]. Eur J Drug Metab Pharmacokinet,2007,32(2):75-79
[41]HUANG C R, WANG G F, WU X L, etal. Absorption enhancement study of astragaloside Ⅳ based on its transport mechanism in Caco-2 cells[J]. Eur J Drug Metab Pharmacokinet,2006,31(1):5-10
[42]Henke A, Launhardt H, KLement K, et al. Apoptosis in coxsackievirus B3-caused diseases:interaction between the capsid protein VP2 and the proapoptotic protein siva[J]. J Virol,2000,74(9):4284-4290
[43]钟飞,杜九中,于小华.黄芪甲甙对病毒性心肌炎细胞凋亡作用的研究[J].南华大学学报·医学版,2004,32(1)：17-19
[44]钟飞,李辉,周卫华.黄芪甲甙对大鼠阿霉素心肌细胞凋亡的影响[J].吉首大学学报(自然科学版),2008,29(6)：104-106
[45]Li L, Tao HY, Chen JB. Antiapoptosis effect of astragaloside on adriamycin induced rats' cardiotoxicity[J]. Zhongguo Zhong Xi Yi Jie He Za Zhi,2006, 26(11):1011-1024
[46]ZHANG Zhao-cai, YANG Ying-zhen, LI Shuang-jie, et al. Effect of astragal oside on my ocardial fibrosis in viral myocarditic mice[J]. Chin J New Drugs Clin Rem,2003,22 (9):515-519
[47]张召才,李双杰,杨英珍,等.黄芪甲甙对慢性心肌炎心肌纤维化的影响[J].中国中西医结合杂志,2007,27(8)：728-731
[48]李丽,陶辉宇,陈杰斌,等.黄芪甲甙抗阿霉素所致心肌凋亡作用及机制研究[C].第十二次全国中西医结合儿科学术会议论文汇编,2006：129-130
[49]Li ZP, Cao Q. Effects of astragaloside Ⅳ on myocardial calcium transport and cardiac function in ischemic rats[J].Acta Pharmacol Sin,2002,23(10): 898-904
[50]Gaasch WH, Delorey DE, Z ile MR, et al. Patterns of structural and functional remodeling of the left ventricle in chronic heart failure [J]. Am J Cardiol,2008,102(4):459-462
[51]许晓乐,季晖,谷舒怡,等.黄芪甲苷对异丙肾上腺素所致小鼠心肌肥厚的保护作用[J].中国药科大学学报,2008,38(5)：451-455
[52]石海莲,马春来,刘燕,等.黄芪皂苷甲抑制压力过载型心肌肥厚大鼠肾素血管紧张素的过度激活[J].中国中药杂志,2009,34(24)：3242-3245
[53]伍伟培.黄芪有效成分黄芪苷Ⅳ强心作用实验研究[J].中药材,2005,28(7)：591-592
[54]刘艳霞,刘在萍,焦建杰,等.黄芪苷Ⅳ对正常和心功能受抑制大鼠左心室心肌力学的影响[J].中草药,2001,32(4)：332-334
[55]郑培黎,戴建亚,陈红,等.黄芪甲苷对急性心衰犬心脏舒缩功能的影响[J].中国药理学通报,2005,21(12)：1534-1535
[56]Xu XL, Chen X J, JiH, et al. Astragaloside Ⅳ improved intracellular calcium handling in hypoxia-reoxygenated cardiomyocytes via the sarcoplasmic reticulum Ca-ATPase[J]. Pharmacology,2008,81(4):325-332
[57]Xu XL, Ji H, Gu SY, et al. Modification of alterations in cardiac function and sarcoplasmic reticulum by astragnaloside Ⅳ in myocardial in jury in vivo [J]. Eur J Pharmacol,2007,568(1-3):203-212
[58]Li ZP, Cao Q. Effects of astragaloside Ⅳ on myocardial calcium transport and cardiac function in ischemic rats [J]. Acta Pharmacol Sin,2002, 23(10):898-904
[59]Li HB, Ge YK, Zhang L, et al. Astragaloside Ⅳimproved barrier dysfunction induced by acute high glucose in human umbilical vein endothelial cells[J] Life Sci,2006,79(12):1186-1193
[60]杨富国,刘革新,王力.黄芪甲苷对缺氧/复氧损伤血管内皮细核转录因子-κB表达的影响[J].中国中西医结合急救杂志,2007,14(6)：367-369
[61]王旭慧,朱亮,陈红.黄芪甲苷对正常大鼠离体血管功能的影响[J].中国药理学通报,2006,22(11)：1319-1324
[62]Zhang W D, Zhang C, Wang XH, et al. Astragaloside Ⅳ dilates aortic vessels from normal and spontaneously hypertensive rats through endothelium-dependent and endothelium-independent ways[J]. Planta Med,2006,72(7):621-626
[63]Zhang C, Wang XH, Zhong MF, et al. Mechanisms underlying vasorelaxant action of astragaloside IV in isolated rat aortic rings [J]. Clin Exp Pharmacol Physiol,2007,34(5-6):387-392
[64]吴大正,胡之壁.黄芪皂苷甲对组胺引起的大鼠脑软膜微血管内皮增加的影响[J].中国中医基础医学杂志,2001,7(2)：36-38
[65]孙贝贝,文富强,王海霞.黄芪甲甙对吸烟大鼠气道β-defensin2表达的影响[J].西部医学,2008,20(3)：482-484
[66]张平,李杰平,于小华.黄芪甲甙对实验性肺纤维化大鼠Cathepsin B表达的影响[J].现代生物医学进展,2007,7(6)：860-862
[67]Navarrete A, Arrieta J, Terrones L, et al. Gastroprotective effect of Astragaloside IV:role of prostaglandins, sulfhydryls and nitric oxide[J].
J Pharm Pharmacol,2005,57 (8):1059-1064
[68]王畅,张艳军,冯英,等.黄芪甲苷对短暂性前脑缺血模型大鼠海马神经再生的影响[J].中草药,2009,40(5)：754-758
[69]柴丽娟,钟佩茹,周志焕,等.黄芪甲苷对体外神经干细胞增殖作用影响的研究[J].中国药理学通报,2010,26(5)：670-673
[70]Luo Y, Qin Z, Hong Z, et al. Astragaloside IV protects against ischemic brain injury in a murine model of transient focal ischemia[J]. Neurosci Lett, 2004,363(3):218-223
[71]于芹超,朱同玉,张永康.黄芪甲甙对大鼠肾缺血再灌注损伤的保护作用[J].中华实验外科学杂志,2004,21(3)：379
[72]廖秀清,熊平,肖晓山.黄芪甲甙对大鼠肺缺血再灌注损伤的保护作用[J].广东医学,2009,30(12)：1778-1780
[73]胡炯宇,黄跃生,宋华培.槲皮素和黄芪甲苷对大鼠缺氧心肌细胞的保护作用[J].中华烧伤杂志,2007,23(3)：175-177
[74]谢利霞,刘宏乐,夏志红.黄芪甲苷对帕金森病体外、体内模型的神经保护作用研究[J].中国药房,2010,21(23)：2132-2134
[75]王曦,石钰,C. Vienne,等.黄芪甲苷对人皮肤成纤维细胞增殖和凋亡的影响[J].中华医学美学美容杂志,2006,12(6)：93-97
[76]陈斌,康健,吕中明.黄芪甲甙对中波紫外线损伤皮肤角质形成细胞的保护作用[J].中国中西医结合皮肤性病学杂志,2009,8(1)：1-4
[77]Wang Y P, Li X Y, Song C Q, et al. Effect of astragaloside Ⅳ on T, B lymphocyte proliferation and peritoneal macrophage function in mice [J]. Acta Pharmacol Sin,2002,23(3):263-266
[78]张娟,陈建宗,张金平.黄芪甲甙体外抗乙型肝炎病毒的作用[J].第四军医大学学报,2007,28(24)：2291-2293
[79]ZHANG W J, HU FNAGL P, BINDER BR, et al. Anti-inflammatory activity of astragalosideⅣis mediated by inhibition of NF-kappaB activation and adhesion molecule expression[J]. Thromb Haemost,2003,90(5):904-914
[80]王琳,闫晨.玉屏风散中黄芪甲苷的制备及抗炎作用的研究[J].天津药学,2007,19(1)：31-32
[81]ZHANG W D, ZHANG C, WANG X H, et al. Astragaloside Ⅳ dilates aortic vessels from normal and spontaneously hypertensive rats through endothelium-dependent and endothelium-independent ways[J]. Planta Med,2006, 72(7):621-626
[82]卞俊,鲍蕾蕾,储智勇.黄芪甲苷对家兔血液流变学的影响[J].解放军药学学报,2005,21(6)：456-458
[83]刘维统,王跃,郝鹏.黄芪甲苷对关节软骨细胞功能影响的实验研究[J].实用医院临床杂志,2009,6(4)：44-47
[84]余俊先.黄芪甲苷和苄达赖氨酸防治糖尿病周围神经病变的药效学、药动学及PD-PK结合模型研究[D].南京：南京医科大学,2006
[85]王志洁,黄铁牛,刘焱文.黄芪总皂甙抗HSV2的实验研究[J].浙江中医学院学报,2001,25(5)：43-46
[86]董筠,刘建,万谟彬,等.黄芪静脉滴注对慢性乙型病毒性肝炎患者细胞免疫功能的影响[J].第二军医大学学报,2001,22(7)：664-666
[87]肖丽华,覃顺寿,覃昱,等.冬虫夏草、黄芪对乙肝表面抗原阳性转阴的研究[J].中国当代儿科杂志,2000,2(3)：231-232
[88]邹宇宏,杨雁,吴强,等.黄芪提取物的体外抗乙肝病毒作用[J].安徽医科大学学报,2003,38(4)：267-269
[89]古平,何民.黄芪总苷治疗小鼠柯萨奇B3病毒性心肌炎的实验研究[J].西南国防医药,2007,17(3)：281-284
[90]陈琦,孙云,邓杨梅,等.毛花黄芪总甙对小鼠抗衰老作用的研究[J].实用老年医学,2001,15(4)：189-191
[91]李维祖,李卫平,尹艳艳.黄芪总苷及黄芪甲苷对糖皮质激素诱导衰老大鼠氧自由基代谢的影响[J].中国中药杂志,2007,32(23)：2539-2542
[92]阿拉木斯,李爽,李燕舞,等.黄芪总苷对运动性疲劳大鼠胃粘膜胃泌素基因表达及氧自由基含量的影响[J].西安体育学院学报,2009,26(4)：461-463
[93]李爽,王蕾,冯云辉,等.黄芪总苷对一次性力竭游泳大鼠脑组织三磷酸腺苷酶活 性的影响[J].中国康复医学杂,2009,24(7)：641-643
[94]谭华,黄小平,邓常清.黄芪总苷和三七总皂苷配伍对小鼠缺血再灌注脑组织氧化应激的影响[J].中西医结合学报,2010,8(5)：448-452
f95]李燕舞,宋宁,王汝俊.黄芪总苷对应激大鼠胃黏膜氧自由基及褪黑素受体的影响[J].世界华人消化杂志,2008,16(29)：3321-3323
[96]王娜,朱科,岳华,等.黄芪总皂甙对心肌细胞氧化损伤的保护作用研究[J].河北中医药学报,2007,22(2)：8-9
[97]古平,何民.黄芪总苷对实验性猫急性心衰的治疗作用[J].西南国防医药,2008,18(3)：355-357
[98]孟丹,陈相健,杨笛,等.黄芪总皂甙对培养大鼠心肌细胞钙超载的影响[J].中国临床康复,2004,8(3)：462-463
[99]吴强,杨雁,薛绍礼,等.黄芪总苷对肝星状细胞增殖和合成胶原的抑制作用[J].中国药理学通报,2003,19(8)：892-895
100]宋少刚,杨雁,陈敏珠.黄芪总提物对大鼠肝星状细胞增殖及产生胶原的影响[J].中国临床药理学与治疗学,2001,6(2)：111-113
[101]朱虹,吴强,杨雁,等.黄芪总苷对血吸虫卵抗原活化的肝星状细胞增殖与胶原合成的影响[J].中国药理学通报,2006,22(5)：555-558
[102]丁向东,王红群,吴强,等.黄芪总苷对小鼠日本血吸虫病肝纤维化的影响[J].世界华人消化杂志,2008,16(2)：125-131
[103]杨雁,宋少刚,陈敏珠.黄芪总苷对肝癌细胞生长、甲胎蛋白分泌及-GT活性的影响[J].中国临床药理学与治疗学,2001,6(1)：18-20
[104]杨雁,陈敏珠.黄芪总苷对肝癌细胞凋亡及wtp53基因表达的影响[J].中国药理学通报,2001,17(4)：447-451
[105]尹艳艳,公惠玲,李维祖.黄芪总苷对血管性痴呆小鼠学习记忆功能的影响[J].安徽医药,2009,13(9)：1018-1020
[106]李维祖,李卫平,尹艳艳,等.黄芪总苷及黄芪甲苷对糖皮质激素诱导老前期小鼠记忆障碍的保护作用及其机制[J].安徽医药,2008,12(7)：592-594
[107]陈攻,朱洪斌.黄芪总苷预处理对小鼠脑缺血再灌注损伤的保护作用[J].铜陵职业技术学院学报,2008,2：53-54。
[108]卢炜卓.黄芪总苷对大鼠脑缺血机制与前列腺素的关系[J].安徽卫生职业技术学院学报,2007,6(4)：74-75
[109]尹艳艳,李卫平,李维祖,等.黄芪总苷对大鼠局灶性脑缺血/再灌注损伤后BDNF、TrkB和p75NTR mRNA表达的影响[J].中国药理学通报,2009,25(5)：672-676
[110]朱芬芳,李卫平,尹艳艳,等.黄芪总苷对缺氧/复氧诱导损伤细胞外信号调节蛋白激酶的影响[J].安徽医科大学学报,2009,44(4)：471-475
[111]黄迪,陈崇宏,余涓.黄芪总苷对小鼠吗啡条件性位置偏爱及中枢N0水平的影响[J].中草药,2008,39(11)：1704-1706
[112]牛艳,张羽飞.黄芪总苷对人肺腺癌A549细胞增殖及凋亡的影响[J].牡丹江医学院学报,2008,29(3)：16-18
[113]许杜娟,吴强,杨雁,等.黄芪总苷的抑瘤作用及其作用机制[J].中国药理学通报,2003,19(7)：823-826
[114]孙海燕,黄民,关溯.黄芪总苷对实验性急性胰腺炎的保护作用[J].时珍国医国药,2006,17(10)：1980-1982
[115]YANG Qin, LU Jing-Tao, ZHOU Ai-Wu, et al. Antinociceptive effect of astragalosides and its mechanism of action[J]. Acta Pharmacol Sin,2001,22(9): 809-812
[116]高建,徐先祥,徐先俊,等.黄芪总皂苷抗血栓形成作用实验研究[J].中成药,2002,24(2)：116-118
[117]徐先祥,夏伦祝,高家荣.黄芪总皂苷和赤芍总苷协同抗血小板作用研究[J].中药材,2002,25(9)：653-655
[118]姚余有,周爱武,陈敏珠.黄芪总苷对兔关节软骨和滑膜作用的体外实验[J].中国新药杂志,2004,13(4)：314-316
[119]黄可儿,李燕舞,王汝俊,等.AST对脾虚大鼠壁细胞胃泌素受体作用的研究[J].广州中医药大学学报,2006,23(1)：42-44
[120]Chen Y G, HaiL N, Zhang, Hu J. Study progress of compound prescription of Chinese crude drug[J]. J Yunnan Normal Univ,2002,22(3):70-74
[121]李维祖,李卫平,尹艳艳,等.黄芪总苷及黄芪甲苷对糖皮质激素诱导衰老大鼠氧自由基代谢的影响[J].中国中药杂志,2007,32(23)：2539-2542
[122]李维祖,李卫平,尹艳艳,等.黄芪总苷及黄芪甲苷对糖皮质激素诱导老前期大鼠记忆损伤的保护作用及机制[J].中国中药杂志,2009,34(2)：199-203
[123]扬雁,陈敏珠.黄芪总甙及其单体抗肝损伤作用的机理[J].中国药理学会通讯,2001,18(4)：10-11
[124]黄可儿,孙雪莲,杨小催,等.固相萃取HPLC法研究黄芪总苷、黄芪甲苷在大鼠体内黄芪甲苷血药浓度[J].中药新药与临床药理,2010,21(6)：630-633
[125]徐叔云,卞如濂,陈修.药理实验方法学[M].第3版.北京：人民卫生出版社,2002：1331
[126]陈奇.中药药理实验方法[M].北京：人民卫生出版社,1994：104
[127]陈艳芬,陈蔚文,李茹柳,等.“左金丸”与“反左金”的药效学反应比较研究[J].中国中医基础医学杂志,2002,8(10)：67-69
[128]Robet A, Nezamis JE, Lancaster C, et al. Cytoprotection by prostaglandins in rats. Prevention of gastric necrosis produced by alcohol, HCl, NaOH, hypertonic NaCl, and thermal injury[J]. Gastroenterology,1979,77(3):433-443
[129]黄可儿,赵敏,王建华.黄芪总苷的药理研究进展[J].中药新药与临床药理,2005,16(6)：461-463
[130]钟慈生.细胞和组织的超微结构[M].第一版.北京：人民卫生出版社,1984：124
[131]Wallace JL, Granger DN. The cellular and molecular basis of gastric mucosal defense[J]. The FASEB Journal,1996,10(7):731-740
[132]刘景丽.胃黏膜损伤的细胞增殖的相关研究[D].南京：南京医科大学,2008
[133]栾丽伟,董中校.临床医学综合研究[M].北京：中国科学技术出版社,2008：176
[134]Slomiany BL, Piotrowski J, Czajkowski A, et al. Gastric mucosal EGF and PDGF receptor expression with ulcer healing by ebrotidine[J]. Am J Gastroenterol,1994,89(6):894-897
[135]沈丽波,钱会南.脾虚模型实验研究方法概述[J].中国中医药信息杂志,2005,12(1)：93-94
[136]Karam S, Leblond CP. Dynamics of epithelial in the corpus of the mouse stomach Ⅲ. Inward migration of neck cells followed by progressive transformation into zymogenic cells. Anat Rec 1993,236:297-313
[137]Yang DH, Tsuyama S, Ge YB, et al. Proliferation and migration kinetics of stem cells in the rat fundic gland. Histol Histopathol 1997,12:717-728
[138]Que FG Gores GJ. Cell death by apoptosis:basic concepts and disease relevance for the gastroenterologist[J]. Gastroenterology,1996,110: 1238-1243
[139]Oltvai ZN, Milliman CL, etal. Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death[J]. Cell. 1993,74:609-619
[140]申洪,陆药丹.免疫组织化学染色的定量方法研究[J].生物医学工程学杂志,1993,10(3)：281-284
[141]周庚寅.组织病理学技术[M].北京：北京大学医学出版社,2006：223
[142]Maity P, Biswas K, etal. Smoking and the pathogenesis of gastroduodenal ulcer-recent mechanistic update [J]. Mol Cell Biochem,2003,253 (1-2):329-338
[143]杜长青,苏衍萍,高慧英.健胃益气汤对大鼠胃粘膜急性损伤的细胞保护作用[J].中国组织化学与细胞化学杂志,2007,16(5)：580-583
[144]Piofiowsld J, Slomiany A, etal. Activation of apoptosis caspase-3 and nitric oxide synthase-2 in gastric mucosal injury induced by indomethacin [J]. Scand J Gastroenterology,1999,34:129
[145]Haunstetter A, lzumo S. Apoptosis:basic mechanisms and implications for cardiovascular disease[J]. Circulation Res,1998,82:1111-1129
[146]Yin XN, Oltval ZN, etal. Bh1 and Bh2 domains of bcl2 are required for inhibition of apoptosis and heterodimerization with Bax[J]. Nature,1994, 369(2):321-323
[147]Tsujimoto Y, Shimizu S, etal. bcl-2 and bcl-Xl block apotosis as well as necrosis:possible involvement of common mediators in apoptotic necrotic singal transduction pathway[J]. Leukemia,1997,11:380-383
[148]Reed JC, Wong L, Hanby AM, et al. Bcl-2 and the regulation of programmed cell death[J]. J Cell Biol,1994,124:1-6
[149]彭黎明,王曾礼.细胞凋亡的基础与临床[M].第一版.人民卫生出版社,2000：246-247
[150]Yang E, Zha J, etal. Bad, a heterrodimeric partner for Bcl-xl and Bc1-2, displaces Bax and promotes cell death[J]. Cell.1995,80:285
[151]吴升华,周华辞.增殖性细胞核抗原的测定及应用[J].南京医科大学学报(中文版),1996,16(2)：129-131
[152]孙阳,杜军,陈敏,等.不同浓度酒精对慢性饮酒大鼠胃粘膜细胞增殖的影响[J].南京医科大学学报,2004,24(4)：348-350
[153]韩玲,朱家谷.中药与天然药物非临床药代动力学评价的思考[J].中国天然药物,2006,4(6)：473-476
[2]冯寿章.重视中药二类新药(有效部位)研究,提高中药新药研制水平[J].中药新药与临床药理,1998,9(3)：134-137
[3]段亚丽,谢梅冬.黄芪化学成分及其有效成分黄芪甲苷含量测定的研究现状[J].中国兽药杂志,2005,39(3)：35-38
[4]黄可儿.黄芪总苷对脾虚大鼠胃壁细胞功能及形态影响的研究[D].广州：广州中医药大学,2006
[5]中国药典2005年版(一部)[S].北京：化学工业出版社,2005：212
[6]Luo Y, Qin Z, Hong Z, etal. Astragaloside IV pro tects against ischemic brain injury in a murine model of transient focal ischemia[J]. Neurosci Lett, 2004,363:218-223
[7]Wang Y P, Li X Y, Song C Q, et al. Effect of Astragaloside IV on T, Blymphocyte proliferation and peritoneal macrophage function in mice[J]. Acta Pharmacol Sin,2002,23:263-266
[8]WS3-B-3335-98,中华人民共和国卫生部药品标准中药成方制剂[S].卫生部药典委员会,1990.8
[9]冯祚臻,俸小平,官东秀.黄芪甲苷的提取及含量测定研究进展[J].中国药业,2005,14(11)：80-82
[10]高越,李翔,朱臻宇.大孔树脂吸附法对黄芪皂苷Ⅳ富集纯化工艺的研究[J].第二军医大学学报,2006,27(3)：334-336
[11]吕佳,侯利涛,李海滨.薄层扫描法测定补气养血颗粒黄芪甲苷含量[J].辽宁中医学院学报,2005,7(3)：271
[12]林朝展,祝晨陈,黄琳,等.薄层扫描法测定广州黄芪商品药材中黄芪甲苷的含量[J].广东药学,2002,12(5)：26-8
[13]杨克迪,李宏,龙云飞.固相萃取-高效液相色谱法测定黄芪中黄芪甲苷的含量[J].时珍国医国药,2007,18(1)：41-42
[14]顾泳川,王广基.HPLC-MS法测定大鼠尿中黄芪甲甙的含量及其尿药动力学研究[J].中国药科大学学报,2002,33(3)：222-225
[15]姚美村,齐莹,车镇涛,等.柱前衍生化HPLC测定黄芪中黄芪甲苷的含量[J].天然产物研究与开发,2000,12(4)：17-22
[16]崔颖,李灵芝,周欣.柱前衍生化法测定糖脂消胶囊中黄芪甲苷含量[J].武警医 学院学报,2007,16(3)：269-271
[17]王改香,朱智甲,林苗.柱前衍生反相高效液相色谱法测定黄芪中的黄芪甲苷[J].分析试验室,2009,28(7)：108-110
[18]谢红兵,常新耀,魏刚才,等.紫外分光光度法测定玉屏风散中黄芪甲苷的含量[J].光谱实验室,2008,25(6)：1092-1094
[19]梁瑞雪,张新军,贾元印.薄层扫描法测定尘肺清颗粒中黄芪甲苷的含量[J].时珍国医国药,2006,17(9)：1689-1690
[20]梁旭明,唐耀书,张小梅.薄层扫描法测定芪黄胶囊剂中黄芪甲苷的含量[J].时珍国医国药,2007,18(1)：22-23
[21]田丰,邓英杰.HPLC法测定黄芪中的黄芪甲苷含量[J].沈阳药科大学学报,2000,17(1)：43-45
[22]池玉梅,姜海英.HPLC示差折光检测测定黄芪精口服液中黄芪甲苷的含量[J].中国药学杂志,2001,36(1)：114-115
[23]吴巧凤,范英.HPLC法测定补血胶囊中黄芪甲苷的含量[J].中草药,2003,34(5)：425-426
[24]陈娟,师彦平.天然产物皂苷类化合物的高效液相色谱分析[J].药物分析杂志,2005,25(1)：123-128
[25]孙文基,谢世昌.天然药物成分定量分析[M].北京：中国医药科技出版社,2002：48
[26]赵陆华,吴孟华,单臻,等.高效液相色谱-蒸发光散射检测器法测定肾宝片及其中间体中黄芪甲苷的含量[J].药物分析杂志,2005,25(9)：1009-1011
[27]王鹏,叶利明,刘海峰,等.高效液相色谱-蒸发光散射检测法测定当归补血颗粒中黄芪甲苷的含量[J].药物分析杂志,2006,26(10)：1377-1379
[28]陈海明.HPLC-ELSD方法测定黄芪及其复方制剂中黄芪甲苷的含量[J].药物分析杂志,2007,27(3)：426-428
[29]胡斯,王中东,毕铭阳.HPLC-ELSD测定麦芪益肺合剂中黄芪甲苷的含量[J].中成药,2007,29(10)：1554-1555
[30]刘元媛,张振秋. HPLC-ELSD测定康艾扶正片中黄芪甲苷含量[J].中成药,2007,29(11)：1705-1706
[31]周洪波,葛晓明,房志坚.HPLC-ELSD法测定玉屏风颗粒中黄芪甲苷的含量[J],中药材,2007,30(6)：739-740
[32]贾晓斌,陈彦,蔡宝昌.HPLC-MS(TOF)法测定家兔血浆中黄芪甲苷的浓度[J].中成药,2005,27(3)：323-325
[33]陈宁,张琪,杜宇.黄芪甲苷在大鼠体内的药代动力学和组织分布研究[J].生物加工过程,2006,4(3)：67-72
[34]韩素琴,刘养清,任荣芳.香兰素荧光法测定黄芪口服液中的黄芪甲甙[J].山西农业大学学报,2002,22(1)：75-77
[35]游文玮,吴昭晖,佟丽,等.喂饲补阳还五汤大鼠血清中黄芪甲甙的固相萃取-化学衍生化-荧光分光光度法测定[J].第一军医大学学报,2003,23(4)：335-339
[36]陈娟,师彦平.天然产物皂苷类化合物的高效液相色谱分析[J].药物分析杂志,2005,25(1)：123-128
[37]Gu Y, Wang G, Fawcett JP. Determination of Astragaloside IV in rat plasma by liquid chromatography electrospray ionization mass spectrometry[J]. J Chromatogr B Analyt Technol Biomed Life Sci,2004,801(2):285-288
[38]DUY, ZHANG Q, CHENG G, etal. Pharmacokinetics of astragaloside Ⅳ in rats by liquid chromatography coupled with tandem mass spectrometry[J]. Eur J Drug Metab Pharmacokinet,2005,30(4):269-273
[39]ZHANG W D, ZHANG C, LIU RH, etal. Preclinical pharmacokinetics and tissue distribution of a natural card ioprotective agent astragaloside IV in rats and dogs[J]. Life Sci,2006,79(8):808-815
[40]Zhang Q, Zhu LL,Chen GG. Pharmacokinetics of astragaloside iv in beagle dogs[J]. Eur J Drug Metab Pharmacokinet,2007,32(2):75-79
[41]HUANG C R, WANG G F, WU X L, etal. Absorption enhancement study of astragaloside Ⅳ based on its transport mechanism in Caco-2 cells[J]. Eur J Drug Metab Pharmacokinet,2006,31(1):5-10
[42]Henke A, Launhardt H, KLement K, et al. Apoptosis in coxsackievirus B3-caused diseases:interaction between the capsid protein VP2 and the proapoptotic protein siva[J]. J Virol,2000,74(9):4284-4290
[43]钟飞,杜九中,于小华.黄芪甲甙对病毒性心肌炎细胞凋亡作用的研究[J].南华大学学报·医学版,2004,32(1)：17-19
[44]钟飞,李辉,周卫华.黄芪甲甙对大鼠阿霉素心肌细胞凋亡的影响[J].吉首大学学报(自然科学版),2008,29(6)：104-106
[45]Li L, Tao HY, Chen JB. Antiapoptosis effect of astragaloside on adriamycin induced rats' cardiotoxicity[J]. Zhongguo Zhong Xi Yi Jie He Za Zhi,2006, 26(11):1011-1024
[46]ZHANG Zhao-cai, YANG Ying-zhen, LI Shuang-jie, et al. Effect of astragal oside on my ocardial fibrosis in viral myocarditic mice[J]. Chin J New Drugs Clin Rem,2003,22 (9):515-519
[47]张召才,李双杰,杨英珍,等.黄芪甲甙对慢性心肌炎心肌纤维化的影响[J].中国中西医结合杂志,2007,27(8)：728-731
[48]李丽,陶辉宇,陈杰斌,等.黄芪甲甙抗阿霉素所致心肌凋亡作用及机制研究[C].第十二次全国中西医结合儿科学术会议论文汇编,2006：129-130
[49]Li ZP, Cao Q. Effects of astragaloside Ⅳ on myocardial calcium transport and cardiac function in ischemic rats[J].Acta Pharmacol Sin,2002,23(10): 898-904
[50]Gaasch WH, Delorey DE, Z ile MR, et al. Patterns of structural and functional remodeling of the left ventricle in chronic heart failure [J]. Am J Cardiol,2008,102(4):459-462
[51]许晓乐,季晖,谷舒怡,等.黄芪甲苷对异丙肾上腺素所致小鼠心肌肥厚的保护作用[J].中国药科大学学报,2008,38(5)：451-455
[52]石海莲,马春来,刘燕,等.黄芪皂苷甲抑制压力过载型心肌肥厚大鼠肾素血管紧张素的过度激活[J].中国中药杂志,2009,34(24)：3242-3245
[53]伍伟培.黄芪有效成分黄芪苷Ⅳ强心作用实验研究[J].中药材,2005,28(7)：591-592
[54]刘艳霞,刘在萍,焦建杰,等.黄芪苷Ⅳ对正常和心功能受抑制大鼠左心室心肌力学的影响[J].中草药,2001,32(4)：332-334
[55]郑培黎,戴建亚,陈红,等.黄芪甲苷对急性心衰犬心脏舒缩功能的影响[J].中国药理学通报,2005,21(12)：1534-1535
[56]Xu XL, Chen X J, JiH, et al. Astragaloside Ⅳ improved intracellular calcium handling in hypoxia-reoxygenated cardiomyocytes via the sarcoplasmic reticulum Ca-ATPase[J]. Pharmacology,2008,81(4):325-332
[57]Xu XL, Ji H, Gu SY, et al. Modification of alterations in cardiac function and sarcoplasmic reticulum by astragnaloside Ⅳ in myocardial in jury in vivo [J]. Eur J Pharmacol,2007,568(1-3):203-212
[58]Li ZP, Cao Q. Effects of astragaloside Ⅳ on myocardial calcium transport and cardiac function in ischemic rats [J]. Acta Pharmacol Sin,2002, 23(10):898-904
[59]Li HB, Ge YK, Zhang L, et al. Astragaloside Ⅳimproved barrier dysfunction induced by acute high glucose in human umbilical vein endothelial cells[J] Life Sci,2006,79(12):1186-1193
[60]杨富国,刘革新,王力.黄芪甲苷对缺氧/复氧损伤血管内皮细核转录因子-κB表达的影响[J].中国中西医结合急救杂志,2007,14(6)：367-369
[61]王旭慧,朱亮,陈红.黄芪甲苷对正常大鼠离体血管功能的影响[J].中国药理学通报,2006,22(11)：1319-1324
[62]Zhang W D, Zhang C, Wang XH, et al. Astragaloside Ⅳ dilates aortic vessels from normal and spontaneously hypertensive rats through endothelium-dependent and endothelium-independent ways[J]. Planta Med,2006,72(7):621-626
[63]Zhang C, Wang XH, Zhong MF, et al. Mechanisms underlying vasorelaxant action of astragaloside IV in isolated rat aortic rings [J]. Clin Exp Pharmacol Physiol,2007,34(5-6):387-392
[64]吴大正,胡之壁.黄芪皂苷甲对组胺引起的大鼠脑软膜微血管内皮增加的影响[J].中国中医基础医学杂志,2001,7(2)：36-38
[65]孙贝贝,文富强,王海霞.黄芪甲甙对吸烟大鼠气道β-defensin2表达的影响[J].西部医学,2008,20(3)：482-484
[66]张平,李杰平,于小华.黄芪甲甙对实验性肺纤维化大鼠Cathepsin B表达的影响[J].现代生物医学进展,2007,7(6)：860-862
[67]Navarrete A, Arrieta J, Terrones L, et al. Gastroprotective effect of Astragaloside IV:role of prostaglandins, sulfhydryls and nitric oxide[J].
J Pharm Pharmacol,2005,57 (8):1059-1064
[68]王畅,张艳军,冯英,等.黄芪甲苷对短暂性前脑缺血模型大鼠海马神经再生的影响[J].中草药,2009,40(5)：754-758
[69]柴丽娟,钟佩茹,周志焕,等.黄芪甲苷对体外神经干细胞增殖作用影响的研究[J].中国药理学通报,2010,26(5)：670-673
[70]Luo Y, Qin Z, Hong Z, et al. Astragaloside IV protects against ischemic brain injury in a murine model of transient focal ischemia[J]. Neurosci Lett, 2004,363(3):218-223
[71]于芹超,朱同玉,张永康.黄芪甲甙对大鼠肾缺血再灌注损伤的保护作用[J].中华实验外科学杂志,2004,21(3)：379
[72]廖秀清,熊平,肖晓山.黄芪甲甙对大鼠肺缺血再灌注损伤的保护作用[J].广东医学,2009,30(12)：1778-1780
[73]胡炯宇,黄跃生,宋华培.槲皮素和黄芪甲苷对大鼠缺氧心肌细胞的保护作用[J].中华烧伤杂志,2007,23(3)：175-177
[74]谢利霞,刘宏乐,夏志红.黄芪甲苷对帕金森病体外、体内模型的神经保护作用研究[J].中国药房,2010,21(23)：2132-2134
[75]王曦,石钰,C. Vienne,等.黄芪甲苷对人皮肤成纤维细胞增殖和凋亡的影响[J].中华医学美学美容杂志,2006,12(6)：93-97
[76]陈斌,康健,吕中明.黄芪甲甙对中波紫外线损伤皮肤角质形成细胞的保护作用[J].中国中西医结合皮肤性病学杂志,2009,8(1)：1-4
[77]Wang Y P, Li X Y, Song C Q, et al. Effect of astragaloside Ⅳ on T, B lymphocyte proliferation and peritoneal macrophage function in mice [J]. Acta Pharmacol Sin,2002,23(3):263-266
[78]张娟,陈建宗,张金平.黄芪甲甙体外抗乙型肝炎病毒的作用[J].第四军医大学学报,2007,28(24)：2291-2293
[79]ZHANG W J, HU FNAGL P, BINDER BR, et al. Anti-inflammatory activity of astragalosideⅣis mediated by inhibition of NF-kappaB activation and adhesion molecule expression[J]. Thromb Haemost,2003,90(5):904-914
[80]王琳,闫晨.玉屏风散中黄芪甲苷的制备及抗炎作用的研究[J].天津药学,2007,19(1)：31-32
[81]ZHANG W D, ZHANG C, WANG X H, et al. Astragaloside Ⅳ dilates aortic vessels from normal and spontaneously hypertensive rats through endothelium-dependent and endothelium-independent ways[J]. Planta Med,2006, 72(7):621-626
[82]卞俊,鲍蕾蕾,储智勇.黄芪甲苷对家兔血液流变学的影响[J].解放军药学学报,2005,21(6)：456-458
[83]刘维统,王跃,郝鹏.黄芪甲苷对关节软骨细胞功能影响的实验研究[J].实用医院临床杂志,2009,6(4)：44-47
[84]余俊先.黄芪甲苷和苄达赖氨酸防治糖尿病周围神经病变的药效学、药动学及PD-PK结合模型研究[D].南京：南京医科大学,2006
[85]王志洁,黄铁牛,刘焱文.黄芪总皂甙抗HSV2的实验研究[J].浙江中医学院学报,2001,25(5)：43-46
[86]董筠,刘建,万谟彬,等.黄芪静脉滴注对慢性乙型病毒性肝炎患者细胞免疫功能的影响[J].第二军医大学学报,2001,22(7)：664-666
[87]肖丽华,覃顺寿,覃昱,等.冬虫夏草、黄芪对乙肝表面抗原阳性转阴的研究[J].中国当代儿科杂志,2000,2(3)：231-232
[88]邹宇宏,杨雁,吴强,等.黄芪提取物的体外抗乙肝病毒作用[J].安徽医科大学学报,2003,38(4)：267-269
[89]古平,何民.黄芪总苷治疗小鼠柯萨奇B3病毒性心肌炎的实验研究[J].西南国防医药,2007,17(3)：281-284
[90]陈琦,孙云,邓杨梅,等.毛花黄芪总甙对小鼠抗衰老作用的研究[J].实用老年医学,2001,15(4)：189-191
[91]李维祖,李卫平,尹艳艳.黄芪总苷及黄芪甲苷对糖皮质激素诱导衰老大鼠氧自由基代谢的影响[J].中国中药杂志,2007,32(23)：2539-2542
[92]阿拉木斯,李爽,李燕舞,等.黄芪总苷对运动性疲劳大鼠胃粘膜胃泌素基因表达及氧自由基含量的影响[J].西安体育学院学报,2009,26(4)：461-463
[93]李爽,王蕾,冯云辉,等.黄芪总苷对一次性力竭游泳大鼠脑组织三磷酸腺苷酶活 性的影响[J].中国康复医学杂,2009,24(7)：641-643
[94]谭华,黄小平,邓常清.黄芪总苷和三七总皂苷配伍对小鼠缺血再灌注脑组织氧化应激的影响[J].中西医结合学报,2010,8(5)：448-452
f95]李燕舞,宋宁,王汝俊.黄芪总苷对应激大鼠胃黏膜氧自由基及褪黑素受体的影响[J].世界华人消化杂志,2008,16(29)：3321-3323
[96]王娜,朱科,岳华,等.黄芪总皂甙对心肌细胞氧化损伤的保护作用研究[J].河北中医药学报,2007,22(2)：8-9
[97]古平,何民.黄芪总苷对实验性猫急性心衰的治疗作用[J].西南国防医药,2008,18(3)：355-357
[98]孟丹,陈相健,杨笛,等.黄芪总皂甙对培养大鼠心肌细胞钙超载的影响[J].中国临床康复,2004,8(3)：462-463
[99]吴强,杨雁,薛绍礼,等.黄芪总苷对肝星状细胞增殖和合成胶原的抑制作用[J].中国药理学通报,2003,19(8)：892-895
100]宋少刚,杨雁,陈敏珠.黄芪总提物对大鼠肝星状细胞增殖及产生胶原的影响[J].中国临床药理学与治疗学,2001,6(2)：111-113
[101]朱虹,吴强,杨雁,等.黄芪总苷对血吸虫卵抗原活化的肝星状细胞增殖与胶原合成的影响[J].中国药理学通报,2006,22(5)：555-558
[102]丁向东,王红群,吴强,等.黄芪总苷对小鼠日本血吸虫病肝纤维化的影响[J].世界华人消化杂志,2008,16(2)：125-131
[103]杨雁,宋少刚,陈敏珠.黄芪总苷对肝癌细胞生长、甲胎蛋白分泌及-GT活性的影响[J].中国临床药理学与治疗学,2001,6(1)：18-20
[104]杨雁,陈敏珠.黄芪总苷对肝癌细胞凋亡及wtp53基因表达的影响[J].中国药理学通报,2001,17(4)：447-451
[105]尹艳艳,公惠玲,李维祖.黄芪总苷对血管性痴呆小鼠学习记忆功能的影响[J].安徽医药,2009,13(9)：1018-1020
[106]李维祖,李卫平,尹艳艳,等.黄芪总苷及黄芪甲苷对糖皮质激素诱导老前期小鼠记忆障碍的保护作用及其机制[J].安徽医药,2008,12(7)：592-594
[107]陈攻,朱洪斌.黄芪总苷预处理对小鼠脑缺血再灌注损伤的保护作用[J].铜陵职业技术学院学报,2008,2：53-54。
[108]卢炜卓.黄芪总苷对大鼠脑缺血机制与前列腺素的关系[J].安徽卫生职业技术学院学报,2007,6(4)：74-75
[109]尹艳艳,李卫平,李维祖,等.黄芪总苷对大鼠局灶性脑缺血/再灌注损伤后BDNF、TrkB和p75NTR mRNA表达的影响[J].中国药理学通报,2009,25(5)：672-676
[110]朱芬芳,李卫平,尹艳艳,等.黄芪总苷对缺氧/复氧诱导损伤细胞外信号调节蛋白激酶的影响[J].安徽医科大学学报,2009,44(4)：471-475
[111]黄迪,陈崇宏,余涓.黄芪总苷对小鼠吗啡条件性位置偏爱及中枢N0水平的影响[J].中草药,2008,39(11)：1704-1706
[112]牛艳,张羽飞.黄芪总苷对人肺腺癌A549细胞增殖及凋亡的影响[J].牡丹江医学院学报,2008,29(3)：16-18
[113]许杜娟,吴强,杨雁,等.黄芪总苷的抑瘤作用及其作用机制[J].中国药理学通报,2003,19(7)：823-826
[114]孙海燕,黄民,关溯.黄芪总苷对实验性急性胰腺炎的保护作用[J].时珍国医国药,2006,17(10)：1980-1982
[115]YANG Qin, LU Jing-Tao, ZHOU Ai-Wu, et al. Antinociceptive effect of astragalosides and its mechanism of action[J]. Acta Pharmacol Sin,2001,22(9): 809-812
[116]高建,徐先祥,徐先俊,等.黄芪总皂苷抗血栓形成作用实验研究[J].中成药,2002,24(2)：116-118
[117]徐先祥,夏伦祝,高家荣.黄芪总皂苷和赤芍总苷协同抗血小板作用研究[J].中药材,2002,25(9)：653-655
[118]姚余有,周爱武,陈敏珠.黄芪总苷对兔关节软骨和滑膜作用的体外实验[J].中国新药杂志,2004,13(4)：314-316
[119]黄可儿,李燕舞,王汝俊,等.AST对脾虚大鼠壁细胞胃泌素受体作用的研究[J].广州中医药大学学报,2006,23(1)：42-44
[120]Chen Y G, HaiL N, Zhang, Hu J. Study progress of compound prescription of Chinese crude drug[J]. J Yunnan Normal Univ,2002,22(3):70-74
[121]李维祖,李卫平,尹艳艳,等.黄芪总苷及黄芪甲苷对糖皮质激素诱导衰老大鼠氧自由基代谢的影响[J].中国中药杂志,2007,32(23)：2539-2542
[122]李维祖,李卫平,尹艳艳,等.黄芪总苷及黄芪甲苷对糖皮质激素诱导老前期大鼠记忆损伤的保护作用及机制[J].中国中药杂志,2009,34(2)：199-203
[123]扬雁,陈敏珠.黄芪总甙及其单体抗肝损伤作用的机理[J].中国药理学会通讯,2001,18(4)：10-11
[124]黄可儿,孙雪莲,杨小催,等.固相萃取HPLC法研究黄芪总苷、黄芪甲苷在大鼠体内黄芪甲苷血药浓度[J].中药新药与临床药理,2010,21(6)：630-633
[125]徐叔云,卞如濂,陈修.药理实验方法学[M].第3版.北京：人民卫生出版社,2002：1331
[126]陈奇.中药药理实验方法[M].北京：人民卫生出版社,1994：104
[127]陈艳芬,陈蔚文,李茹柳,等.“左金丸”与“反左金”的药效学反应比较研究[J].中国中医基础医学杂志,2002,8(10)：67-69
[128]Robet A, Nezamis JE, Lancaster C, et al. Cytoprotection by prostaglandins in rats. Prevention of gastric necrosis produced by alcohol, HCl, NaOH, hypertonic NaCl, and thermal injury[J]. Gastroenterology,1979,77(3):433-443
[129]黄可儿,赵敏,王建华.黄芪总苷的药理研究进展[J].中药新药与临床药理,2005,16(6)：461-463
[130]钟慈生.细胞和组织的超微结构[M].第一版.北京：人民卫生出版社,1984：124
[131]Wallace JL, Granger DN. The cellular and molecular basis of gastric mucosal defense[J]. The FASEB Journal,1996,10(7):731-740
[132]刘景丽.胃黏膜损伤的细胞增殖的相关研究[D].南京：南京医科大学,2008
[133]栾丽伟,董中校.临床医学综合研究[M].北京：中国科学技术出版社,2008：176
[134]Slomiany BL, Piotrowski J, Czajkowski A, et al. Gastric mucosal EGF and PDGF receptor expression with ulcer healing by ebrotidine[J]. Am J Gastroenterol,1994,89(6):894-897
[135]沈丽波,钱会南.脾虚模型实验研究方法概述[J].中国中医药信息杂志,2005,12(1)：93-94
[136]Karam S, Leblond CP. Dynamics of epithelial in the corpus of the mouse stomach Ⅲ. Inward migration of neck cells followed by progressive transformation into zymogenic cells. Anat Rec 1993,236:297-313
[137]Yang DH, Tsuyama S, Ge YB, et al. Proliferation and migration kinetics of stem cells in the rat fundic gland. Histol Histopathol 1997,12:717-728
[138]Que FG Gores GJ. Cell death by apoptosis:basic concepts and disease relevance for the gastroenterologist[J]. Gastroenterology,1996,110: 1238-1243
[139]Oltvai ZN, Milliman CL, etal. Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death[J]. Cell. 1993,74:609-619
[140]申洪,陆药丹.免疫组织化学染色的定量方法研究[J].生物医学工程学杂志,1993,10(3)：281-284
[141]周庚寅.组织病理学技术[M].北京：北京大学医学出版社,2006：223
[142]Maity P, Biswas K, etal. Smoking and the pathogenesis of gastroduodenal ulcer-recent mechanistic update [J]. Mol Cell Biochem,2003,253 (1-2):329-338
[143]杜长青,苏衍萍,高慧英.健胃益气汤对大鼠胃粘膜急性损伤的细胞保护作用[J].中国组织化学与细胞化学杂志,2007,16(5)：580-583
[144]Piofiowsld J, Slomiany A, etal. Activation of apoptosis caspase-3 and nitric oxide synthase-2 in gastric mucosal injury induced by indomethacin [J]. Scand J Gastroenterology,1999,34:129
[145]Haunstetter A, lzumo S. Apoptosis:basic mechanisms and implications for cardiovascular disease[J]. Circulation Res,1998,82:1111-1129
[146]Yin XN, Oltval ZN, etal. Bh1 and Bh2 domains of bcl2 are required for inhibition of apoptosis and heterodimerization with Bax[J]. Nature,1994, 369(2):321-323
[147]Tsujimoto Y, Shimizu S, etal. bcl-2 and bcl-Xl block apotosis as well as necrosis:possible involvement of common mediators in apoptotic necrotic singal transduction pathway[J]. Leukemia,1997,11:380-383
[148]Reed JC, Wong L, Hanby AM, et al. Bcl-2 and the regulation of programmed cell death[J]. J Cell Biol,1994,124:1-6
[149]彭黎明,王曾礼.细胞凋亡的基础与临床[M].第一版.人民卫生出版社,2000：246-247
[150]Yang E, Zha J, etal. Bad, a heterrodimeric partner for Bcl-xl and Bc1-2, displaces Bax and promotes cell death[J]. Cell.1995,80:285
[151]吴升华,周华辞.增殖性细胞核抗原的测定及应用[J].南京医科大学学报(中文版),1996,16(2)：129-131
[152]孙阳,杜军,陈敏,等.不同浓度酒精对慢性饮酒大鼠胃粘膜细胞增殖的影响[J].南京医科大学学报,2004,24(4)：348-350
[153]韩玲,朱家谷.中药与天然药物非临床药代动力学评价的思考[J].中国天然药物,2006,4(6)：473-476