溃结安康汤对溃疡性结肠炎大鼠模型影响及机制研究
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摘要
目的:
通过中药溃结安康汤对溃疡性结肠炎(Ulcerative colitis,UC)大鼠模型影响及机制研究,从溃疡性结肠炎大鼠模型的一般状态、肉眼观察大鼠结肠组织、显微镜观察(HE染色)大鼠结肠组织、大鼠肠电图(频率和波幅)、大鼠结肠组织氧自由基(MDA含量和SOD活性)、大鼠结肠组织炎性细胞因子(IL-1β、IL-10和TNF-α)的含量及蛋白表达、大鼠结肠组织表皮生长因子(EGF)的含量及mRNA表达等方面,探讨中药溃结安康汤治疗溃疡性结肠炎大鼠模型的治疗效果,并从多环节、多层次探讨其作用的可能靶点及机制,以期为中医药治疗溃疡性结肠炎提供实验依据。
材料与方法:
1.模型制作方法
健康Wistar大鼠60只,雌雄各半,雌雄分笼饲养,体重180-220g。适应性喂养1周,确定大鼠健康后,随机分为对照组(12只),造模组(48只)。造模组采用TNBS/乙醇法造模,对照组则等量生理盐水灌肠。3d后,随机抽取1只对照组大鼠和4只造模组大鼠,麻醉后,取结肠,进行组织形态学观察确认造模是否成功。
2.分组
对照组大鼠11只,为正常组;造模组大鼠随机分为4组:模型组、柳氮磺胺吡啶(SASP)组、补脾益肠丸组、溃结安康汤组,每组11只(各组大鼠均雌雄分笼饲养)。
3.给药
从造模第4d开始,根据人与大鼠之间等效剂量换算,补脾益肠丸组大鼠按1.62g/kg剂量,给予浓度为0.108g/ml补脾益肠丸3ml,日一次灌胃,连续灌胃21d:SASP组大鼠按0.36g/kg剂量,给予浓度0.024g/ml SASP 3ml,日一次灌胃,连续灌胃21d;溃结安康汤组大鼠按18g/kg剂量,给予浓度1.2g/ml溃结安康汤3ml,日一次灌胃,连续灌胃21d。正常组和模型组大鼠用生理盐水3ml,日一次灌胃,连续灌胃21d。
4.灌流与取材
4.1灌流
各组大鼠于灌胃第2ld晚,禁食不禁水,第22d,腹腔注射10%水合氯醛(0.2ml/100g)将各组大鼠麻醉,把大鼠固定于灌流台上,剖开大鼠胸腔,经左心室快速灌流37℃生理盐水30—50ml,继之灌注4℃的4%多聚甲醛溶液(PBS配制,PH7.2-7.4)30—50ml。
4.2取材
灌流后,切开腹腔,从肛门以上2cm处,向上截取大鼠结肠组织6-8cm,沿大鼠肠系膜缘纵向剖开肠腔,用生理盐水冲洗干净,然后将截取的大鼠结肠组织向上平铺,用大头针固定结肠组织,肉眼观察大鼠结肠粘膜组织损伤情况。然后在病变最严重处剪取大鼠结肠肠管1cm左右,用干冰速冻,放至-80℃冰箱冷冻保存。再留取大鼠结肠组织病变最明显处组织若干,置于10%甲醛溶液中后固定48小时以上,再将结肠组织修成1×0.5cm的小块,梯度酒精脱水,经石蜡包埋后,组织横切片,用涂有多聚赖氨酸的载玻片捞片后,送辽宁中医药大学教学实验中心检测。
结果:
1.大鼠一般状态
溃结安康汤组、补脾益肠丸组、SASP组:经溃结安康汤、补脾益肠丸、SASP灌胃给药10d,各组大鼠精神状态明显好转,毛发略显光泽,活动量增加,进食量明显增加,大便性状有好转,脓血便逐渐减少。
21d后治疗各组大鼠上述症状均有显著改善,粘液、脓血便明显减少,但与正常组比较,上述症状仍然略差,其中溃结安康汤组和SASP组恢复最好;模型组大鼠仍可见懒动、食量少、便稀、皮毛不泽等表现。
2.肉眼观察
模型组大鼠结肠粘膜组织损伤肉眼观积分与正常组比较,肉眼观积分明显增高,经统计学处理,具有统计学意义(P＜0.05);溃结安康汤组、补脾益肠丸组、SASP组与模型组比较,经统计学处理,肉眼观积分明显下降,具有统计学意义(P＜0.05);溃结安康汤组与补脾益肠丸组比较,肉眼观积分下降,经统计学处理,具有统计学意义(P＜0.05);溃结安康汤组与SASP组比较,肉眼观积分无明显差异,经统计学处理,无统计学意义(P＞0.05)。
3.光学显微镜观察
模型组大鼠结肠组织病理积分与正常组比较,病理积分明显升高,经统计学处理,具有统计学意义(P＜0.05);溃结安康汤组、补脾益肠丸组、SASP组与模型组比较,病理积分明显下降,经统计学处理,具有统计学意义(P＜0.05);溃结安康汤组与补脾益肠丸组比较,病理积分下降,经统计学处理,具有统计学意义(P＜0.05);溃结安康汤组与SASP组比较,病理积分无明显差异,经统计学处理,无统计学意义(P＞0.05)。
4.大鼠肠电图
模型组肠电图出现双向改变,与正常组比较,肠电图频率明显增快、幅值明显增大,经统计学处理,具有统计学意义(P＜0.05);溃结安康汤组、补脾益肠丸组、SASP组与模型组比较,肠电图频率明显下降、振幅明显下降,经统计学处理,具有统计学意义(P＜0.05);溃结安康汤组肠电图与补脾益肠丸组比较,肠电图频率下降、振幅下降,经统计学处理,具有统计学意义(P＜0.05);溃结安康汤组与SASP组比较,肠电图频率下降,经统计学处理,具有统计学意义(P＜0.05),而肠电图振幅下降无明显差异,经统计学处理,无统计学意义(P＞0.05)。
5.氧自由基
模型组结肠组织氧自由基与正常组比较,丙二醛(MDA)含量明显升高、超氧化物歧化酶(SOD)活性明显降低,经统计学处理,具有统计学意义(P＜0.05);溃结安康汤组、补脾益肠丸组、SASP组与模型组比较,MDA含量明显下降、SOD活性明显增强,经统计学处理,具有统计学意义(P＜0.05);溃结安康汤组与补脾益肠丸组比较,MDA含量下降、SOD活性增强,经统计学处理,具有统计学意义(P＜0.05);溃结安康汤组与SASP组比较,MDA含量下降,经统计学处理,具有统计学意义(P＜0.05),而SOD活性差异不大,经统计学处理,无统计学意义(P＞0.05)。
6.炎性细胞因子
模型组结肠组织炎性细胞因子与正常组比较,致炎细胞因子IL-1β、TNF-α含量均明显增高,IL-1β、TNF-α的蛋白表达也均明显增高,经统计学处理,具有统计学意义(P＜0.05),抗炎细胞因子IL-10含量明显下降,其蛋白表达也明显下降,经统计学处理,具有统计学意义(P＜0.05);溃结安康汤组、补脾益肠丸组、SASP组与模型组比较,IL-1β、TNF-α含量均明显下降,IL-1β、TNF-α的蛋白表达也均明显下降,经统计学处理,具有统计学意义(P＜0.05),IL-10含量明显升高,其蛋白表达也明显升高,经统计学处理,具有统计学意义(P＜0.05);溃结安康汤与补脾益肠丸组比较,IL-1β、TNF-α含量均下降,IL-1β、TNF-α的蛋白表达也均下降,经统计学处理,具有统计学意义(P＜0.05),IL-10含量升高,其蛋白表达也升高,经统计学处理,具有统计学意义(P＜0.05);溃结安康汤组与SASP组比较,IL-1β含量和IL-1β蛋白表达均差异不大,经统计学处理,无统计学意义(P＞0.05),TNF-α含量和TNF-α蛋白表达均下降,经统计学处理,具有统计学意义(P＜0.05),IL-10含量和IL-10蛋白表达均升高,经统计学处理,具有统计学意义(P＜0.05)。
7.表皮生长因子
模型组结肠组织表皮生长因子与正常组比较,EGF含量明显下降,EGFmRNA光密度明显下降、EGFmRNA强度/内参明显下降,经统计学处理,具有统计学意义(P＜0.05);溃结安康汤组、补脾益肠丸组、SASP组与模型组比较,EGF含量明显升高,EGFmRNA光密度明显升高、EGFmRNA强度/内参明显升高,经统计学处理,具有统计学意义(P＜0.05);溃结安康汤组与补脾益肠丸组,EGF含量升高,EGFmRNA光密度升高、EGFmRNA强度/内参升高,经统计学处理,具有统计学意义(P＜0.05)。溃结安康汤组与SASP组比较,EGF含量升高,EGFmRNA光密度升高、EGFmRNA强度/内参升高,经统计学处理,具有统计学意义(P＜0.05)。
结论:
1.肉眼观察溃疡性结肠炎大鼠模型有程度不同的粘膜的充血、水肿、糜烂和溃疡,光学显微镜下见结肠各层组织出现炎性细胞浸润,证明溃疡性结肠炎模型制作成功。
2.溃结安康汤对溃疡性结肠炎大鼠模型具有显著的减轻一般状态、减轻粘膜的充血、水肿、促进溃疡愈合,促进炎性细胞吸收的作用。
3.溃结安康汤对溃疡性结肠炎大鼠模型肠电图频率和振幅有明显降低的作用,从而减轻肠道运动,使腹泻减轻。
4.溃结安康汤对溃疡性结肠炎大鼠模型可以增加结肠组织SOD活性,清除氧自由基,抑制脂质过氧化反应,降低MDA含量,减轻结肠组织损伤。
5.溃结安康汤对溃疡性结肠炎大鼠模型通过下调致炎细胞因子和上调抗炎细胞因子使免疫功能恢复正常,控制炎症反应,提高了机体免疫力,降低结肠组织粘膜损伤。
6.溃结安康汤对溃疡性结肠炎大鼠模型通过上调结肠组织EGF的含量和EGFmRNA表达,提高了机体细胞的增殖化分能力和组织生长修复及保护肠粘膜等能力,促进溃疡愈合。
本实验研究从组织形态学、肌电生理、生物化学、免疫组织化学、分子生物学五个不同层次,说明中药溃结安康汤寒热并用,具有健脾益气、活血化瘀的功效。
Objective:
To research the treatment effect of Kuijieankang decoction on rats with ulcerative colitis,and to explore the role of the possible mechanism and target point with a number of links and multi-level,With a view to provide theoretical and experimental basis for the treatment of ulcerative colitis in Chinese medicine.All above would get help from experimental study of traditional Chinese medicine Kuijieankang decoction on rats with ulcerative colitis.The experiment will study the general state of model rat with ulcerative colitis,visual observation of the rats' colon tissue,optical microscopy of the rats' colon tissue(HE staining),the rats' electrointestinogram(frequency and amplitude), Oxygen free radicals of the rats' colon(MDA contents and SOD activity),contents and protein expression of the rats' colon tissue inflammatory cytokines(IL-1β, IL-10 and TNF-α),as well as contents epidermal growth factor(EGF) and mRNA expression of the rats' of colon tissue.
Materials and methods:
1.Method of model facture
60 healthy adult Wistar rats,weight 180-220g,were equally divided by male and female(male and female rear separately).After 1 week adapt feed in order to determine the health of rats.They were randomly divided into model group (48 rats),and the control group(12 rats).The UC model was established by TNBS/ethanol method and the control group was applied to normal saline enema. 3 days later,selected four model group rats and a control group rat randomly. Took the colon under anesthesia for morphological observation to confirm the success of model.
2.Grouping
The 11 rats in control group are look as the nomal rats.Model rats were divided into 4 groups by random method:model group,Bupiyichang pill group, SASP group,and Kuijieankang decoction group,11 rats in each group.We fed male and female rats in different cages.
3.Drugs given
From the fourth day after established model,Bupiyichang pill group's rats were given Bupiyichang pill 3ml intragastric administration daily,the concentration was 0.108g/ml,the amount was 1.62g/kg,converted as equivalent effect as health adult.SASP group's rats were given SASP 3ml intragastric administration daily,the concentration was 0.024g/ml,equivalent to 0.36g/kg dosage of health.And Kuijieankang decoction group's rats were given Kuijieankang decoction 3ml intragastric administration daily,the concentration was 1.2g/ml, equivalent to 18g/kg dosage of health,each rat of the normal group and the model group was given normal saline 3ml intragastric administration daily.All rats were given drugs at the same time every day for 21 days continuously.
4.Perfusion and drew the materials
4.1 Perfusion
In the night of the 21st day all rats were feed nothing but water.The rats were fixed after intraperitoneal anesthesia with 10%chloral hydrate,surgical incision of the chest cavity,perfus 30-50ml 37℃normal saline into the left ventricle quickly,and 30-50ml 4℃solution with 4%Paraformaldehyde(PBS confecte ph7.2-7.4) followed.
4.2 Drew the materials
After the perfusion,surgical incision of the abdomen.Took the department of 6-8cm colon from 2cm above the anus,then cut along the mesenteric edge mesocaval vertically.Then rinsed with saline,tiled and fixed them with pin. We observed colon mucosa with the naked eye to find intestinal lesions.Then Sheared the most obvious part about 1cm in severe lesions,quick-frozen with dry ice,transferred to -80℃cryopreservation refrigerator.Collected several colon lesions of the various groups' rats where most notably,Placed them into 10%formaldehyde solution for more than 48 hours.Every organization would be cut into a small piece of 0.5cm×1cm,dehydration with gradient alcohol.These organizations would be cut into cross-chip after paraffin-embedded.Then they would be posted on the slides coated with Poly-L-lysine.Finally,they would be sent to the laboratory center of Liaoning university of Traitional Chinese Medicine.
Results:
1.The general state of rats
10 days after intragastric administration,the rats of Kuijieankang decoction group,Bupiyichang pill group,and SASP group got marked improvement in mental state,more slightly shiny hair,activities increase,and an obvious increase in food intake.At the same time,traits of stool improved,pus and blood stool reduced gradually.
21 days later the symptoms of all rats in treatment groups were significantly improved,mucus and pus and blood stool reduced obviously.However,the above symptoms were still slightly worse compared with normal group.The rats of Kuijieankang decoction group and SASP group had got the best restoration.Model group rats still existed matte fur,less activities,thin stool,ate less,and so forth.
2.Visual observation
Compared with normal group,the general concept score of model rats' colon mucosa injury was increased significantly.Handled the data with the statisticmethod,it was statistically significant(P＜0.05).Compared with model group,the general concept score of Bupiyichang pill group,SASP group, and Kuijieankang decoction group dropped markedly.Handled the data with the statisticmethod,it was statistical significance(P＜0.05).Comparing Kuijieankang decoction group with SASP group,the general concept score had no significant difference.Handled the data with the statisticmethod,it wasn't statistical significance(P＞0.05).
3.Observation under light microscopy
Compared with normal group,the pathology score of model rats' colon was increased significantly.Handled the data with the statisticmethod,it was statistically significant(P＜0.05).Compared with model group,the pathology score of Bupiyichang pill group,SASP group,and Kuijieankang decoction group dropped markedly.Handled the data with the statisticmethod,it was statistical significance(P＜0.05).Comparing Kuijieankang decoction group with SASP group, the pathology score had no significant difference.Handled the data with the statisticmethod,it wasn't statistical significance(P＞0.05).
4.Electrointestinogram
Comparing the electrointestinogram of model rats with normal group,a two-way change emerged.Both the frequency and the amplitude increased obviously. Handled the data with the statisticmethod,it was statistically significant (P＜0.05).Compared with model group,both the frequency and the amplitude of Bupiyichang pill group,SASP group,and Kuijieankang decoction group dropped markedly.Handled the data with the statisticmethod,it was statistical significance(P＜0.05).And compared with Bupiyichang pill group,both indicators of Kuijieankang decoction group and SASP group dropped markedly.Handled the data with the statisticmethod,it was statistical significance(P＜0.05).Comparing Kuijieankang decoction group with SASP group,the frequency dropped.Handled the data with the statisticmethod,it was statistical significance(P＜0.05).While the amplitude had no statistical significance(P＞0.05).
5.Oxygen free radicals
Comparing Oxygen free radicals of the rats' colon in model group with normal group,the MDA contents of the former were much accentuated,and the SOD activity was much reduced.Handled the data with the statisticmethod,it was statistical significance(P＜0.05).Comparing Bupiyichang pill group,SASP group,and Kuijieankang decoction group with model group,the MDA contents were much reduced, and the SOD activity was much accentuated.Handled the data with the statisticmethod,it was statistical significance(P＜0.05).Comparing Kuijieankang decoction group with Bupiyichang pill group,the MDA contents were reduced,and the SOD activity was accentuated.Handled the data with the statisticmethod,it was statistical significance(P＜0.05).And comparing Kuijieankang decoction group with SASP group,the MDA content were reduced. Handled the data with the statisticmethod,it was statistical significance(P＜0.05).While the SOD activity was't statistical significance(P＞0.05).
6.Inflammatory cytokines
Comparing the rats' colon tissue inflammatory cytokines of model group with normal group,the contents and protein expression of IL-1βand INF-αwere much accentuated.Handled the data with the statisticmethod,it was statistical significance(P＜0.05).The contents and protein expression of IL-10 were much reduced.Handled the data with the statisticmethod,it was statistical significance(P＜0.05).Comparing Bupiyichang pill group,SASP group,and Kuijieankang decoction group with model group,the contents and protein expression of IL-1βand TNF-αwere much reduced.Handled the data with the statisticmethod,it was statistical significance(P＜0.05).The contents and protein expression of IL-10 were much accentuated.Handled the data with the statisticmethod,it was statistical significance(P＜0.05).Comparing Kuijieankang decoction group with Bupiyichang pill group,the contents and protein expression of IL-1βand TNF-αwere reduced.Handled the data with the statisticmethod,it was statistical significance(P＜0.05).The contents and protein expression of IL-10 were accentuated.Handled the data with the statisticmethod,it was statistical significance(P＜0.05).And comparing Kuijieankang decoction group with SASP group,the contents and protein expression of IL-1βwasn't statistical significance(P＞0.05).The contents and protein expression of TNF-αwere reduced.Handled the data with the statisticmethod,it was statistical significance(P＜0.05).The contents and protein expression of IL-10 were accentuated.Handled the data with the statisticmethod,it was statistical significance(P＜0.05).
7.Epidermal growth factor
Comparing model group with normal group,the contents of EGF,the optical density and the rate of EGF mRNA between the strength and internal reference were all reduced obviously.Handled the data with the statisticmethod,it was statistical significance(P＜0.05).Comparing Bupiyichang pill group,SASP group and Kuijieankang decoction group with model group,the contents of EGF,the optical density and the rate of EGF mRNA between the strength and internal reference were all accentuated obviously.Handled the data with the statisticmethod,it was statistical significance(P＜0.05).And comparing Kuijieankang decoction group with Bupiyichang pill group,the contents of EGF, the optical density and the rate of EGF mRNA between the strength and internal reference were all accentuated.Handled the data with the statisticmethod,it was statistical significance(P＜0.05).Compare Kuijieankang decoction group with SASP group,the contents of EGF,the optical density and the rate of EGF mRNA between the strength and internal reference were all accentuated.Handled the data with the statisticmethod,it was statistical significance(P＜0.05).
Conclusion:
1.The rat model of ulcerative colitis was successfully established through observation to the rats' general state,visual observation and light microscopy observation.
2.Kuijieankang decoction played a significant role of ulcer repair in the morphology to rats with ulcerative colitis.
3.Kuijieankang decoction could obviously reduce the frequency and the amplitude of ulcerative colitis rats' electrointestinogram.
4.To rats with ulcerative colitis,Kuijieankang decoction could increase the SOD activity of colon,clear Oxygen free radicals,inhibit lipid per oxidation, reduce MDA contents,and reduce colon mucosa injury.
5.Kuijieankang decoction could control inflammatory response,by reducing pro-inflammatory cytokines and increasing anti-inflammatory cytokines. Therefore it could make a recovery of ulcerative colitis rats' immune function, improve immunity,and reduce colon mucosa injury.
6.To rats with ulcerative colitis,Kuijieankang decoction could increase cellular proliferation capacity,tissue growth and repair capacity,protect colon mucosa,by increasing EGF contents and expression of EGF mRNA of colon. Then promote the healing of ulcer.
From the following five different levels including histomorphology, electromyography physiology,biochemistry,immunohistochemistry and molecular biology,the experimental study showed that Kuijieankang decoction used cooling and heat prescription together,and it could invigorate spleen and Qi as well as activate blood and resolve stasis.
通过中药溃结安康汤对溃疡性结肠炎(Ulcerative colitis,UC)大鼠模型影响及机制研究,从溃疡性结肠炎大鼠模型的一般状态、肉眼观察大鼠结肠组织、显微镜观察(HE染色)大鼠结肠组织、大鼠肠电图(频率和波幅)、大鼠结肠组织氧自由基(MDA含量和SOD活性)、大鼠结肠组织炎性细胞因子(IL-1β、IL-10和TNF-α)的含量及蛋白表达、大鼠结肠组织表皮生长因子(EGF)的含量及mRNA表达等方面,探讨中药溃结安康汤治疗溃疡性结肠炎大鼠模型的治疗效果,并从多环节、多层次探讨其作用的可能靶点及机制,以期为中医药治疗溃疡性结肠炎提供实验依据。
材料与方法:
1.模型制作方法
健康Wistar大鼠60只,雌雄各半,雌雄分笼饲养,体重180-220g。适应性喂养1周,确定大鼠健康后,随机分为对照组(12只),造模组(48只)。造模组采用TNBS/乙醇法造模,对照组则等量生理盐水灌肠。3d后,随机抽取1只对照组大鼠和4只造模组大鼠,麻醉后,取结肠,进行组织形态学观察确认造模是否成功。
2.分组
对照组大鼠11只,为正常组;造模组大鼠随机分为4组:模型组、柳氮磺胺吡啶(SASP)组、补脾益肠丸组、溃结安康汤组,每组11只(各组大鼠均雌雄分笼饲养)。
3.给药
从造模第4d开始,根据人与大鼠之间等效剂量换算,补脾益肠丸组大鼠按1.62g/kg剂量,给予浓度为0.108g/ml补脾益肠丸3ml,日一次灌胃,连续灌胃21d:SASP组大鼠按0.36g/kg剂量,给予浓度0.024g/ml SASP 3ml,日一次灌胃,连续灌胃21d;溃结安康汤组大鼠按18g/kg剂量,给予浓度1.2g/ml溃结安康汤3ml,日一次灌胃,连续灌胃21d。正常组和模型组大鼠用生理盐水3ml,日一次灌胃,连续灌胃21d。
4.灌流与取材
4.1灌流
各组大鼠于灌胃第2ld晚,禁食不禁水,第22d,腹腔注射10%水合氯醛(0.2ml/100g)将各组大鼠麻醉,把大鼠固定于灌流台上,剖开大鼠胸腔,经左心室快速灌流37℃生理盐水30—50ml,继之灌注4℃的4%多聚甲醛溶液(PBS配制,PH7.2-7.4)30—50ml。
4.2取材
灌流后,切开腹腔,从肛门以上2cm处,向上截取大鼠结肠组织6-8cm,沿大鼠肠系膜缘纵向剖开肠腔,用生理盐水冲洗干净,然后将截取的大鼠结肠组织向上平铺,用大头针固定结肠组织,肉眼观察大鼠结肠粘膜组织损伤情况。然后在病变最严重处剪取大鼠结肠肠管1cm左右,用干冰速冻,放至-80℃冰箱冷冻保存。再留取大鼠结肠组织病变最明显处组织若干,置于10%甲醛溶液中后固定48小时以上,再将结肠组织修成1×0.5cm的小块,梯度酒精脱水,经石蜡包埋后,组织横切片,用涂有多聚赖氨酸的载玻片捞片后,送辽宁中医药大学教学实验中心检测。
结果:
1.大鼠一般状态
溃结安康汤组、补脾益肠丸组、SASP组:经溃结安康汤、补脾益肠丸、SASP灌胃给药10d,各组大鼠精神状态明显好转,毛发略显光泽,活动量增加,进食量明显增加,大便性状有好转,脓血便逐渐减少。
21d后治疗各组大鼠上述症状均有显著改善,粘液、脓血便明显减少,但与正常组比较,上述症状仍然略差,其中溃结安康汤组和SASP组恢复最好;模型组大鼠仍可见懒动、食量少、便稀、皮毛不泽等表现。
2.肉眼观察
模型组大鼠结肠粘膜组织损伤肉眼观积分与正常组比较,肉眼观积分明显增高,经统计学处理,具有统计学意义(P＜0.05);溃结安康汤组、补脾益肠丸组、SASP组与模型组比较,经统计学处理,肉眼观积分明显下降,具有统计学意义(P＜0.05);溃结安康汤组与补脾益肠丸组比较,肉眼观积分下降,经统计学处理,具有统计学意义(P＜0.05);溃结安康汤组与SASP组比较,肉眼观积分无明显差异,经统计学处理,无统计学意义(P＞0.05)。
3.光学显微镜观察
模型组大鼠结肠组织病理积分与正常组比较,病理积分明显升高,经统计学处理,具有统计学意义(P＜0.05);溃结安康汤组、补脾益肠丸组、SASP组与模型组比较,病理积分明显下降,经统计学处理,具有统计学意义(P＜0.05);溃结安康汤组与补脾益肠丸组比较,病理积分下降,经统计学处理,具有统计学意义(P＜0.05);溃结安康汤组与SASP组比较,病理积分无明显差异,经统计学处理,无统计学意义(P＞0.05)。
4.大鼠肠电图
模型组肠电图出现双向改变,与正常组比较,肠电图频率明显增快、幅值明显增大,经统计学处理,具有统计学意义(P＜0.05);溃结安康汤组、补脾益肠丸组、SASP组与模型组比较,肠电图频率明显下降、振幅明显下降,经统计学处理,具有统计学意义(P＜0.05);溃结安康汤组肠电图与补脾益肠丸组比较,肠电图频率下降、振幅下降,经统计学处理,具有统计学意义(P＜0.05);溃结安康汤组与SASP组比较,肠电图频率下降,经统计学处理,具有统计学意义(P＜0.05),而肠电图振幅下降无明显差异,经统计学处理,无统计学意义(P＞0.05)。
5.氧自由基
模型组结肠组织氧自由基与正常组比较,丙二醛(MDA)含量明显升高、超氧化物歧化酶(SOD)活性明显降低,经统计学处理,具有统计学意义(P＜0.05);溃结安康汤组、补脾益肠丸组、SASP组与模型组比较,MDA含量明显下降、SOD活性明显增强,经统计学处理,具有统计学意义(P＜0.05);溃结安康汤组与补脾益肠丸组比较,MDA含量下降、SOD活性增强,经统计学处理,具有统计学意义(P＜0.05);溃结安康汤组与SASP组比较,MDA含量下降,经统计学处理,具有统计学意义(P＜0.05),而SOD活性差异不大,经统计学处理,无统计学意义(P＞0.05)。
6.炎性细胞因子
模型组结肠组织炎性细胞因子与正常组比较,致炎细胞因子IL-1β、TNF-α含量均明显增高,IL-1β、TNF-α的蛋白表达也均明显增高,经统计学处理,具有统计学意义(P＜0.05),抗炎细胞因子IL-10含量明显下降,其蛋白表达也明显下降,经统计学处理,具有统计学意义(P＜0.05);溃结安康汤组、补脾益肠丸组、SASP组与模型组比较,IL-1β、TNF-α含量均明显下降,IL-1β、TNF-α的蛋白表达也均明显下降,经统计学处理,具有统计学意义(P＜0.05),IL-10含量明显升高,其蛋白表达也明显升高,经统计学处理,具有统计学意义(P＜0.05);溃结安康汤与补脾益肠丸组比较,IL-1β、TNF-α含量均下降,IL-1β、TNF-α的蛋白表达也均下降,经统计学处理,具有统计学意义(P＜0.05),IL-10含量升高,其蛋白表达也升高,经统计学处理,具有统计学意义(P＜0.05);溃结安康汤组与SASP组比较,IL-1β含量和IL-1β蛋白表达均差异不大,经统计学处理,无统计学意义(P＞0.05),TNF-α含量和TNF-α蛋白表达均下降,经统计学处理,具有统计学意义(P＜0.05),IL-10含量和IL-10蛋白表达均升高,经统计学处理,具有统计学意义(P＜0.05)。
7.表皮生长因子
模型组结肠组织表皮生长因子与正常组比较,EGF含量明显下降,EGFmRNA光密度明显下降、EGFmRNA强度/内参明显下降,经统计学处理,具有统计学意义(P＜0.05);溃结安康汤组、补脾益肠丸组、SASP组与模型组比较,EGF含量明显升高,EGFmRNA光密度明显升高、EGFmRNA强度/内参明显升高,经统计学处理,具有统计学意义(P＜0.05);溃结安康汤组与补脾益肠丸组,EGF含量升高,EGFmRNA光密度升高、EGFmRNA强度/内参升高,经统计学处理,具有统计学意义(P＜0.05)。溃结安康汤组与SASP组比较,EGF含量升高,EGFmRNA光密度升高、EGFmRNA强度/内参升高,经统计学处理,具有统计学意义(P＜0.05)。
结论:
1.肉眼观察溃疡性结肠炎大鼠模型有程度不同的粘膜的充血、水肿、糜烂和溃疡,光学显微镜下见结肠各层组织出现炎性细胞浸润,证明溃疡性结肠炎模型制作成功。
2.溃结安康汤对溃疡性结肠炎大鼠模型具有显著的减轻一般状态、减轻粘膜的充血、水肿、促进溃疡愈合,促进炎性细胞吸收的作用。
3.溃结安康汤对溃疡性结肠炎大鼠模型肠电图频率和振幅有明显降低的作用,从而减轻肠道运动,使腹泻减轻。
4.溃结安康汤对溃疡性结肠炎大鼠模型可以增加结肠组织SOD活性,清除氧自由基,抑制脂质过氧化反应,降低MDA含量,减轻结肠组织损伤。
5.溃结安康汤对溃疡性结肠炎大鼠模型通过下调致炎细胞因子和上调抗炎细胞因子使免疫功能恢复正常,控制炎症反应,提高了机体免疫力,降低结肠组织粘膜损伤。
6.溃结安康汤对溃疡性结肠炎大鼠模型通过上调结肠组织EGF的含量和EGFmRNA表达,提高了机体细胞的增殖化分能力和组织生长修复及保护肠粘膜等能力,促进溃疡愈合。
本实验研究从组织形态学、肌电生理、生物化学、免疫组织化学、分子生物学五个不同层次,说明中药溃结安康汤寒热并用,具有健脾益气、活血化瘀的功效。
Objective:
To research the treatment effect of Kuijieankang decoction on rats with ulcerative colitis,and to explore the role of the possible mechanism and target point with a number of links and multi-level,With a view to provide theoretical and experimental basis for the treatment of ulcerative colitis in Chinese medicine.All above would get help from experimental study of traditional Chinese medicine Kuijieankang decoction on rats with ulcerative colitis.The experiment will study the general state of model rat with ulcerative colitis,visual observation of the rats' colon tissue,optical microscopy of the rats' colon tissue(HE staining),the rats' electrointestinogram(frequency and amplitude), Oxygen free radicals of the rats' colon(MDA contents and SOD activity),contents and protein expression of the rats' colon tissue inflammatory cytokines(IL-1β, IL-10 and TNF-α),as well as contents epidermal growth factor(EGF) and mRNA expression of the rats' of colon tissue.
Materials and methods:
1.Method of model facture
60 healthy adult Wistar rats,weight 180-220g,were equally divided by male and female(male and female rear separately).After 1 week adapt feed in order to determine the health of rats.They were randomly divided into model group (48 rats),and the control group(12 rats).The UC model was established by TNBS/ethanol method and the control group was applied to normal saline enema. 3 days later,selected four model group rats and a control group rat randomly. Took the colon under anesthesia for morphological observation to confirm the success of model.
2.Grouping
The 11 rats in control group are look as the nomal rats.Model rats were divided into 4 groups by random method:model group,Bupiyichang pill group, SASP group,and Kuijieankang decoction group,11 rats in each group.We fed male and female rats in different cages.
3.Drugs given
From the fourth day after established model,Bupiyichang pill group's rats were given Bupiyichang pill 3ml intragastric administration daily,the concentration was 0.108g/ml,the amount was 1.62g/kg,converted as equivalent effect as health adult.SASP group's rats were given SASP 3ml intragastric administration daily,the concentration was 0.024g/ml,equivalent to 0.36g/kg dosage of health.And Kuijieankang decoction group's rats were given Kuijieankang decoction 3ml intragastric administration daily,the concentration was 1.2g/ml, equivalent to 18g/kg dosage of health,each rat of the normal group and the model group was given normal saline 3ml intragastric administration daily.All rats were given drugs at the same time every day for 21 days continuously.
4.Perfusion and drew the materials
4.1 Perfusion
In the night of the 21st day all rats were feed nothing but water.The rats were fixed after intraperitoneal anesthesia with 10%chloral hydrate,surgical incision of the chest cavity,perfus 30-50ml 37℃normal saline into the left ventricle quickly,and 30-50ml 4℃solution with 4%Paraformaldehyde(PBS confecte ph7.2-7.4) followed.
4.2 Drew the materials
After the perfusion,surgical incision of the abdomen.Took the department of 6-8cm colon from 2cm above the anus,then cut along the mesenteric edge mesocaval vertically.Then rinsed with saline,tiled and fixed them with pin. We observed colon mucosa with the naked eye to find intestinal lesions.Then Sheared the most obvious part about 1cm in severe lesions,quick-frozen with dry ice,transferred to -80℃cryopreservation refrigerator.Collected several colon lesions of the various groups' rats where most notably,Placed them into 10%formaldehyde solution for more than 48 hours.Every organization would be cut into a small piece of 0.5cm×1cm,dehydration with gradient alcohol.These organizations would be cut into cross-chip after paraffin-embedded.Then they would be posted on the slides coated with Poly-L-lysine.Finally,they would be sent to the laboratory center of Liaoning university of Traitional Chinese Medicine.
Results:
1.The general state of rats
10 days after intragastric administration,the rats of Kuijieankang decoction group,Bupiyichang pill group,and SASP group got marked improvement in mental state,more slightly shiny hair,activities increase,and an obvious increase in food intake.At the same time,traits of stool improved,pus and blood stool reduced gradually.
21 days later the symptoms of all rats in treatment groups were significantly improved,mucus and pus and blood stool reduced obviously.However,the above symptoms were still slightly worse compared with normal group.The rats of Kuijieankang decoction group and SASP group had got the best restoration.Model group rats still existed matte fur,less activities,thin stool,ate less,and so forth.
2.Visual observation
Compared with normal group,the general concept score of model rats' colon mucosa injury was increased significantly.Handled the data with the statisticmethod,it was statistically significant(P＜0.05).Compared with model group,the general concept score of Bupiyichang pill group,SASP group, and Kuijieankang decoction group dropped markedly.Handled the data with the statisticmethod,it was statistical significance(P＜0.05).Comparing Kuijieankang decoction group with SASP group,the general concept score had no significant difference.Handled the data with the statisticmethod,it wasn't statistical significance(P＞0.05).
3.Observation under light microscopy
Compared with normal group,the pathology score of model rats' colon was increased significantly.Handled the data with the statisticmethod,it was statistically significant(P＜0.05).Compared with model group,the pathology score of Bupiyichang pill group,SASP group,and Kuijieankang decoction group dropped markedly.Handled the data with the statisticmethod,it was statistical significance(P＜0.05).Comparing Kuijieankang decoction group with SASP group, the pathology score had no significant difference.Handled the data with the statisticmethod,it wasn't statistical significance(P＞0.05).
4.Electrointestinogram
Comparing the electrointestinogram of model rats with normal group,a two-way change emerged.Both the frequency and the amplitude increased obviously. Handled the data with the statisticmethod,it was statistically significant (P＜0.05).Compared with model group,both the frequency and the amplitude of Bupiyichang pill group,SASP group,and Kuijieankang decoction group dropped markedly.Handled the data with the statisticmethod,it was statistical significance(P＜0.05).And compared with Bupiyichang pill group,both indicators of Kuijieankang decoction group and SASP group dropped markedly.Handled the data with the statisticmethod,it was statistical significance(P＜0.05).Comparing Kuijieankang decoction group with SASP group,the frequency dropped.Handled the data with the statisticmethod,it was statistical significance(P＜0.05).While the amplitude had no statistical significance(P＞0.05).
5.Oxygen free radicals
Comparing Oxygen free radicals of the rats' colon in model group with normal group,the MDA contents of the former were much accentuated,and the SOD activity was much reduced.Handled the data with the statisticmethod,it was statistical significance(P＜0.05).Comparing Bupiyichang pill group,SASP group,and Kuijieankang decoction group with model group,the MDA contents were much reduced, and the SOD activity was much accentuated.Handled the data with the statisticmethod,it was statistical significance(P＜0.05).Comparing Kuijieankang decoction group with Bupiyichang pill group,the MDA contents were reduced,and the SOD activity was accentuated.Handled the data with the statisticmethod,it was statistical significance(P＜0.05).And comparing Kuijieankang decoction group with SASP group,the MDA content were reduced. Handled the data with the statisticmethod,it was statistical significance(P＜0.05).While the SOD activity was't statistical significance(P＞0.05).
6.Inflammatory cytokines
Comparing the rats' colon tissue inflammatory cytokines of model group with normal group,the contents and protein expression of IL-1βand INF-αwere much accentuated.Handled the data with the statisticmethod,it was statistical significance(P＜0.05).The contents and protein expression of IL-10 were much reduced.Handled the data with the statisticmethod,it was statistical significance(P＜0.05).Comparing Bupiyichang pill group,SASP group,and Kuijieankang decoction group with model group,the contents and protein expression of IL-1βand TNF-αwere much reduced.Handled the data with the statisticmethod,it was statistical significance(P＜0.05).The contents and protein expression of IL-10 were much accentuated.Handled the data with the statisticmethod,it was statistical significance(P＜0.05).Comparing Kuijieankang decoction group with Bupiyichang pill group,the contents and protein expression of IL-1βand TNF-αwere reduced.Handled the data with the statisticmethod,it was statistical significance(P＜0.05).The contents and protein expression of IL-10 were accentuated.Handled the data with the statisticmethod,it was statistical significance(P＜0.05).And comparing Kuijieankang decoction group with SASP group,the contents and protein expression of IL-1βwasn't statistical significance(P＞0.05).The contents and protein expression of TNF-αwere reduced.Handled the data with the statisticmethod,it was statistical significance(P＜0.05).The contents and protein expression of IL-10 were accentuated.Handled the data with the statisticmethod,it was statistical significance(P＜0.05).
7.Epidermal growth factor
Comparing model group with normal group,the contents of EGF,the optical density and the rate of EGF mRNA between the strength and internal reference were all reduced obviously.Handled the data with the statisticmethod,it was statistical significance(P＜0.05).Comparing Bupiyichang pill group,SASP group and Kuijieankang decoction group with model group,the contents of EGF,the optical density and the rate of EGF mRNA between the strength and internal reference were all accentuated obviously.Handled the data with the statisticmethod,it was statistical significance(P＜0.05).And comparing Kuijieankang decoction group with Bupiyichang pill group,the contents of EGF, the optical density and the rate of EGF mRNA between the strength and internal reference were all accentuated.Handled the data with the statisticmethod,it was statistical significance(P＜0.05).Compare Kuijieankang decoction group with SASP group,the contents of EGF,the optical density and the rate of EGF mRNA between the strength and internal reference were all accentuated.Handled the data with the statisticmethod,it was statistical significance(P＜0.05).
Conclusion:
1.The rat model of ulcerative colitis was successfully established through observation to the rats' general state,visual observation and light microscopy observation.
2.Kuijieankang decoction played a significant role of ulcer repair in the morphology to rats with ulcerative colitis.
3.Kuijieankang decoction could obviously reduce the frequency and the amplitude of ulcerative colitis rats' electrointestinogram.
4.To rats with ulcerative colitis,Kuijieankang decoction could increase the SOD activity of colon,clear Oxygen free radicals,inhibit lipid per oxidation, reduce MDA contents,and reduce colon mucosa injury.
5.Kuijieankang decoction could control inflammatory response,by reducing pro-inflammatory cytokines and increasing anti-inflammatory cytokines. Therefore it could make a recovery of ulcerative colitis rats' immune function, improve immunity,and reduce colon mucosa injury.
6.To rats with ulcerative colitis,Kuijieankang decoction could increase cellular proliferation capacity,tissue growth and repair capacity,protect colon mucosa,by increasing EGF contents and expression of EGF mRNA of colon. Then promote the healing of ulcer.
From the following five different levels including histomorphology, electromyography physiology,biochemistry,immunohistochemistry and molecular biology,the experimental study showed that Kuijieankang decoction used cooling and heat prescription together,and it could invigorate spleen and Qi as well as activate blood and resolve stasis.
引文
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