维胃方对乙酸致大鼠胃溃疡的治疗作用及与血浆NT相关性研究
|
摘要
课题背景:
消化性溃疡病是消化系统中常见的疾病,呈世界性分布,约有5%～10%的人一生中患过此病,其发病机制较为复杂,多为胃、十二指肠黏膜攻击因子和黏膜自身防御-修复因子之间失衡所致。其临床特点为病程长,易复发,且常伴发一些严重的并发症。
临床治疗GU主要采用抑制胃酸和胃黏膜保护剂。目前H2受体阻滞剂及质子泵抑制剂在临床上广泛应用,这两种药物的使用,使GU的愈合率显著提高,但由于费用、副作用、耐药性、患者依从性等问题的存在,最终使该病的复发率仍居高不下。因此,研究GU理想的治疗方案仍是一件急迫而重要的任务,近几年来人们对于GU药物的开发,常从天然药物寻找突破口。
维胃方是导师的经验方,通过临床实践证明对GU有较好疗效,七及方是维胃方中的组成部分。本实验采用适合药物疗效观察的乙酸致大鼠GU模型,以中医药基础理论为指导,临床疗效为依据,通过GU的大体愈合情况、溃疡面积、GU周围黏膜病理组织切片光学显微镜下观察,从不同层次阐述维胃方治疗GU的效果。并检测胃液PH值、测定血清GaS、血浆NT水平,其中以整体性调节作为切入点,选用NT作为主要指标,旨在阐述维胃方治疗GU的相关机制,为维胃方的临床运用和开发提供实验依据。
实验方法:
1、制备冰醋酸致大鼠GU模型
参照Takagi等乙酸致大鼠GU造模方法,并加以改造。大鼠造模前24h,禁食不禁水。经腹腔注射10%水合氯醛溶液,按3.5ml/kg注射麻醉大鼠。腹部剃毛、消毒,于剑突下腹中线打开腹腔,手术切口长约2cm,暴露出胃,在胃前壁浆膜面接近窦体交界处的胃窦同一部分(避开血管)用浸有100%乙酸,直径为5mm的圆形滤纸接触60s,然后还纳胃体,逐层缝合切口,酒精消毒,保护切口。
2、实验动物分组和相关指标检测
雄性SPF级Wistar大鼠72只,根据完全随机法分为9组,每组8只:A正常组,系非造模大鼠,自由饮水,进食。B模型组,乙酸致大鼠GU,自由饮水,进食。C自愈组,乙酸致大鼠GU,自由饮水,进食。D雷尼替丁组,乙酸致大鼠GU,第4天开始灌胃雷尼替丁水悬液2ml,共7天,每天1次。E1维胃方低剂量组,乙酸致大鼠GU,第4天灌胃低剂量维胃方2ml,共7天,每天1次;E2维胃方中剂量组,乙酸致大鼠GU,第4天开始灌胃中剂量维胃方2ml,共7天,每天1次;E3维胃方高剂量组,乙酸致大鼠GU,第4天,灌胃高剂量维胃方2ml,共7天,每天1次。F1七及方中剂量组,乙酸致大鼠GU,第4天开始灌胃中剂量七及方2ml,共7天,每天1次;F2七及方高剂量组,乙酸致大鼠胃溃疡,第4天,灌胃高剂量七及方2ml,共7天,每天1次。除模型组在第4天,其余各组均在第11天麻醉后取材,并观察胃溃疡的大体愈合情况,检测胃液PH值、溃疡面积,光学显微镜下观察胃溃疡周围黏膜病理组织切片,测定血清Gas、血浆NT水平。
统计学处理:
实验数据用SPSS11.5统计软件分析,数据以χ±S表示,采用单因素方差分析方法(One-Way-ANOVA)进行多样本均数比较。大鼠溃疡面积的比较,方差不齐,采用Welch检验,各组间存在差异,采用Dunnett's T3法检验;大鼠胃液pH值的比较,方差齐,各组间存在差异,采用LSD检验;大鼠血清Gas水平的比较,方差齐,但各组差别无统计学意义,无需做两两比较;大鼠血浆NT水平的比较,方差不齐,采用Welch检验,各组间存在差异,采用Dunnett's T3检验。
主要结果:
1、胃黏膜大体标本肉眼观察
维胃方中剂量组溃疡绝大部分愈合,新生组织与周围正常组织无明显差别,仅见个别小溃疡,且溃疡表浅,个别可见炎症分泌物。
2、用药组与自愈组大鼠GU面积比较
方差不齐,用Welch检验,各组间存在差异,采用Dunnett's T3法检验。雷尼替丁组,维胃方低、中、高剂量组,七及方中、高剂量组与自愈组比较差别有统计学意义(P=0.005,P=0.028,P=0.001,P=0.007,P=0.001,P=0.001)。维胃方中剂量组与雷尼替丁组相比差别有统计学意义(P=0.013)。维胃方中剂量组、七及中剂量组、七及高剂量组与维胃方高剂量组相比差别有统计学意义(P=0.006,P=0.043,P=0.036)。维胃方对溃疡面积大小的影响并不成量效依赖。
3、光学显微镜下观察胃黏膜病理组织切片
维胃方中剂量组黏膜基本完整,仅有极个别溃疡深及上皮与胃腺区交界处,面积小,上皮细胞形态,腺体结构良好,腺体细胞增生数量较多,腺体间隙较小,排列较整齐,黏膜下层仅见少量炎症细胞浸润,黏膜下微循环未见明显病理改变,肌层变薄,肉芽组织增生,排列较整齐,小部分浆膜断裂。
4、大鼠胃液PH值的比较
方差齐,各组间存在差异,采用LSD检验,模型组、七及方中剂量组、七及方高剂量组与正常组比较差别有统计学意义(P=0.006,P=0.000,P=O.034)。维胃方中剂量组与正常组比较差别无统计学意义(P=0.746)。各组可以分为三个水平:pH值处于较低水平的是正常组、雷尼替丁组、维胃方各组,pH值处于中等水平的是自愈组,pH处于较高水平的是模型组、七及方各组。
5、大鼠血清GAS水平的比较
方差齐,但各组差别无统计学意义,无需做两两比较。
6、大鼠血浆NT水平的比较
方差不齐,采用Welch检验,各组间存在差异,采用Dunnett's T3检验,模型组、雷尼替丁组、维胃方低剂量组、七及方中剂量组与自愈组相比差别有统计学意义(P=0.021,P=0.020,P=0.032,P=0.016)。自愈组、维胃方高剂量组与模型组相比差别有统计学意义(P=0.021,P=0.003)。各组与正常组相比差别均无统计学意义(均P>0.05),其中维胃方中剂量组接近正常组(P=1)。各组大体可以分为三个层次:NT值处于较低水平的是模型组、雷尼替丁组、维胃方低剂量组、七及方中剂量组,NT值处于中等水平的是正常组、维胃方中剂量组、七及方高剂量组,NT值处于较高水平的是自愈组、维胃方高剂量组。维胃方对血浆NT水平的影响呈量效正相关(r=1,P=0.002)。
结论:
1、从溃疡面积中看,造模成功率高达到100%,自愈组大鼠对GU的自愈效果差,用药组起到一定治疗作用,维胃方中剂量组的疗效最佳。
2、维胃方和七及方由于配伍及剂量的关系出现了复杂的量效关系,说明在相同煎煮等技术条件下,中药配伍、剂量对疗效影响极为重要。
3、从胃黏模大体标本观察中发现,在用药各组中维胃方中剂量组效果最好。
4、光学显微高倍镜下观察胃黏膜病理组织切片,维胃方中剂量组效果最佳,部分机制是通过改善微循环、抗炎和细胞再生这些方面发挥作用。
5、模型组血浆NT水平低于正常组,说明溃疡形成时,体内合成和释放NT明显减少。
6、模型组胃液pH值高于正常组,表明手术造模对动物本身就是一种应激,为防止乙酸的损害作用,大鼠机体应激性的调动体内的平衡机制,迅速中和酸性,以保护其受损伤的胃黏膜,这可能是模型组胃液pH值升高的原因。
7、维胃方各剂量组血浆NT存在一种剂量依赖的正相关关系(r=1,P=0.002),但是血清Gas并不成量效依赖关系,说明在脱离乙酸致大鼠GU的手术应激后的治疗过程中,推测NT对胃黏膜的影响不是直接通过G细胞分泌Gas而发挥作用,很可能是通过合成生长抑素等。
8、根据pH的统计结果,七及方各组pH值高于维胃方各组,说明三七、白及二者单独合用,在提高pH值,抗胃酸方面能够起着重要作用。七及方中剂量组和维胃方低剂量组NT处于同等水平,但低于正常水平时,七及方中剂量组因为pH值高于维胃方低剂量组,所以能够发挥更好的疗效。
9、维胃方中剂量组在胃液pH值、血清Gas、血浆NT水平均接近正常组,从GU病理组织切片上看,维胃方中剂量组修复受损胃黏膜效果更强,部分机制可能是通过NT的神经机制,从抗炎、改善微循环、细胞再生等方面,对机体起着整体性调节,发挥其整体治疗GU的作用。
10、维胃方中剂量组血浆NT水平接近正常组(P=1),推测血浆NT发挥其生理学效应,修复和保护胃黏膜存在一定阈值,生理浓度或接近生理浓度的血浆NT,对胃黏膜有更重要的意义,能调节机体尽可能达到常态。
综上实验结果表明:通过GU的大体愈合情况、溃疡面积以及GU周围黏膜病理组织切片光学显微镜下观察,发现维胃方中剂量组的疗效最佳,其相关机制可能是通过NT,直接或间接使胃液pH值接近正常水平,并从抗炎、改善微循环、细胞再生等方面,对机体起着整体性调节,发挥其整体治疗GU的作用。
Background:
Peptic ulcer(PU) disease is one large kind of digestive system diseases, which spreads cosmopolitism. It has been estimated that 5%～10% people have suffered this disease in their lives. The pathogenesy is complicit. It has been summarized that the balance is destroyed between aggression factor of gastric mucosa intestinal mucosa and mucosa self defend- reparative factor. PU has the characteristics of long course of disease, ease palindromia and some severe complication.
Gastric acid inhibition and gastric mucosa protectant are two main methods of clinical therapeutics. At present, histamine-2-receptor antagonists and proton pump antagonists which widely use in clinic make GU accrete rate predominance raise. However, the problem of cost, side-effect, drug resistance, patient dependence makes the GU palindromia high. Therefore, GU therapeutics is still a urge and important task. Recently, the exploitation of GU drug turns to naturally occurring drugs.
Weiweifang(WWF) is Liu Xiaowei professor's empirical formula. It has been proved that it has a good effect to GU in clinic. Qijifang(QJF) is one part of WWF .The experiment which bases the Traditional Chinese Medicine(TCM) theory and clinical effect adopts acetic acid—inducted gastric ulcer mode. We detected gastric mucosa, ulcer size and gastric ulcer circa histopathologic slide to find out WWF's Therapeutical effect. The gastric fluid pH, serum gastrin(Gas) and plasma neurotensin(NT) concentration had been detected. The purport is to find out the correlate treatment mechanism of WWF. NT which has hol-modulation function is the enter-point in our experiment. We hope that we can support experiment evidences for WWF's clinical apply and exploitation.
Methods:
1. Acetic Acid-Induced Gastric ulcer
The experiments were performed according to the method described by Takagi et al., with some modifications. Eight groups of male Wistar rats that had fasted for 24 h were used in this experiment. Under anesthesia, a laparotomy was performed on all animals through a midline epigastric incision. After exposing the stomach, filter pape in 5 mm area with 100% acetic acid solution was contacted with the subserosal layer in the glandular part of the anterior wall. The abdomen was then closed and all animals were fed normally.
2. Rats subgroup and relative indicatrix detection
Seventy two male SPF Wistar rats were randomly divided into nine groups of 8 each, respectively marked with normal group, model group, autotherapy group, Ranitidine group, WWF groups and QJF groups. Normal group is untreated and freely drink and eat. Model group is inducted gastric ulcer by acetic acid and freely drink and eat. When acetic acid—inducted gastric ulcer in rats of autotherapy group, Ranitidine group, WWF groups and QJF groups , the groups which drink and eat freely are respectively treated with nothing, but Ranitidine, three different doses of WWF (low ,medium , high ) and two different doses of QJF (medium , high )7 days. Except model group in the forth days, other groups are killed in the eleventh day. Afer they are killed, We detected the gastric fluid pH, gastric mucosa, ulcer size, gastric ulcer circa histopathologic slide, serum gastric acid secretion(Gas) and plasma NT concentration.
Stastical analysis:
The data result of study is analysis with SPSS11.5 statistics software. The data is expressed with (x|-)±S. One-Way-ANOVA is used to multiple comparisons. Comparison in gastric ulcer area, when the test of Homogeneity of vriances is not existent we use Welch test and that the group's disparation is existent, we use Dunnett's T3 test. Comparison in gastric fluid pH in rats, when the test of Homogeneity of vriances and the group's disparation is existent, we use LSD test. Comparison in serum Gas in rats, the test of Homogeneity of vriances is existent but the group's disparation is not existent, so we do nothing. Comparison in plasma NT concentration in rats, when the test of Homogeneity of vriances is not existent we use Welch test and that the group's disparation is existent, we use Dunnett's T3 test.
Results:
1. Gastric mucosa detected with eyes , for no equipment
The ulcer of WWF medium dose group (WWFMG) is almost healing, neogenesis tissue has nothing different with normal tissue. There are only light aphthae and little inflammatory exudate seen.
2. comparision in drug groups and autotherapy group in gastric ulcer area in rats
The test of Homogeneity of vriances is not existent. It is used Welch test and that the group's disparation is existent, we use Dunnett's T3 test. Drug groups includes Ranitidine group, Weiweifang low dose group (WWFLG),WWFMG, Weiweifang high dose group(WWFHG), Qijifang medium group (QJFMG), Qijifang high group (QJFHG) have discrepancy significantly compared with autotherapy group (P=0.005, P=0.028,P=0.001,P=0.007, P=0.001, P=0.001) .WWFMG has discrepancy significantly compared with Ranitidine group (P=0.013) . WWFMG, QJFMG, QJFHG have discrepancy significantly compared with WWFHG (P=0.006, P=0.043, P=0.036) .The effective of WWF is not rely quantity in gastric ulcer area.
3. To detect gastric ulcer circa histopathologic slide in light microscope.
WWFMG's mucosa is almost integrity. Only a few of ulcer which is small in area reach the juncture of endepidermis and gastric area glandularis. The recovery of cellula epithelialis and glandular organ are fine, glandular organ Cellular proliferation is nice, include quantity , space, alignment. There are a few of inflammatory cell infiltration in mucosa underlayer. Microcirculation has not significant patho-change. Muscular layer turn to slide. Granulation tissue grow in regularity. A few of serosa membrane are abruptly.
4. Comparison in gastric fluid pH in rats.
When the test of Homogeneity of variances and the group's disparation are existent, we use LSD test. Model group, QJFMG,QJFHG have discrepancy significantly compared with normal group (P=0.006, P=0.000, P=0.034) . WWFMG has not discrepancy significantly compared with normal group (P=0.746) .All the groups can divide in three levers of pH. Normal group, Ranitidine group and WWF groups are in the low lever, autotherapy group is in the middle lever. Model group and QJF groups are in the high lever.
5. Comparison in serum Gas in rats
The test of the Homogeneity of vriances is existent but the group's disparation is not existent, so we do nothing.
6. Comparison in plasma NT concentration in rats
The test of the Homogeneity of vriances is not existent. We use Welch test and that the group's disparation is existent, we use Dunnett's T3 test. Model group, Ranitidine group, WWFLG, QJFMG and QJFHG have discrepancy significantly compared with autotherapy group (P=0.021, P=0.020, P=0.032, P=0.016) . Autotherapy group and WWFHG have discrepancy significantly compared with model group (P=0.021, P=0.003) . All groups have not discrepancy significantly compared with normal group. WWFMG is close to normal group (P=1) .All the groups can divide in three levers of NT. Model group, Ranitidine group, WWFLG and QJF groups are in the low lever. Normal group and WWFMG are in the middle lever. Autotherapy group and WWFHG are in the high lever. The effective of WWF has the tendency to rely quantity in plasma NT concentration in rats (r=1, P= 0.002) .
Conclusion:
1. The mode achievement ratio is reach 100% in gastric ulcer area in rats. Autotherapy group has bad effect. All the drug groups have more or less therapeutical effect. WWFMG has the best effect.
2. Because of compatibility and dose, WWF groups and QJF groups have complicate dose-effect relationship. It manifests that compatibility and dose of Tradition Chinese drags in same specification are important to therapeutical effect.
3. WWFMG has the best therapeutical effect by detecting the gastric mucosa with eyes, for nothing equipment.
4. WWFMG has the best therapeutical effect by detecting gastric ulcer circa histopathologic slide in light microscope. It has good effect in microcirculation, anti-inflammatory and cell regeneration.
5. In plasma NT concentration, model group is lower than normal group. It manifests that the synthesis and release of NT is in lower lever in elcosis.
6. Model group is higher in gastric fluid pH than normal group. It manifests that acetic Acid-Induced Gastric ulcer is a stress for rats. For self-protect, rats irritably remove the equilibrium mechanism to counteract Acetic Acid. It may be the reason why the model group is in higher lever in gastric fluid pH.
7.The effective of WWF has the tendency to rely quantity in plasma NT concentration in rats (r=1, P=0.002) , but not in serum Gas . It manifests that NT can effect the gastric mucosa during the treatment period, but not through Gas derectly. It may be through growth hormone release inhibiting hormone (GIH) .
8. QJF groups have discrepancy significantly compared with WWF groups in gastric fluid pH. It proves that solitude use with notoginseng and bletillae rhizome can elevate pH to inhibit the gastric acid secretion. When QJFMG and WWFLG are in the same lever, but lower than normal lever in plasma NT concentration, QJFMG have better effect through the higher pH.
9. WWFMG is close to normal group in gastric fluid pH, serum Gas and plasma NT concentration. From the evidence of gastric ulcer circa histopathologic slide, WWFMG has the best effect in recovering damaged gastric mucosa, Partial mechanism is that plasma NT may be playing a hol-regulation role by anti-inflammatory, microcirculation improvement and cell regeneration.
10. WWFMG is close to normal group in plasma NT concentration(P=l). It has been supposed that plasma NT which exist leak point close to normal concentration play an important role for gastric mucosa. Plasma NT in this concentration can regulate the body as to normal.
In above, it has been find out that WWFMG has the best effect by detecting gastric mucosa, ulcer size, gastric ulcer circa histopathologic slide. Partial mechanism is that plasma NT may be playing a hol-regulation role by adjusting pH to normal lever and by anti-inflammatory, microcirculation improvement and cell regeneration.
消化性溃疡病是消化系统中常见的疾病,呈世界性分布,约有5%～10%的人一生中患过此病,其发病机制较为复杂,多为胃、十二指肠黏膜攻击因子和黏膜自身防御-修复因子之间失衡所致。其临床特点为病程长,易复发,且常伴发一些严重的并发症。
临床治疗GU主要采用抑制胃酸和胃黏膜保护剂。目前H2受体阻滞剂及质子泵抑制剂在临床上广泛应用,这两种药物的使用,使GU的愈合率显著提高,但由于费用、副作用、耐药性、患者依从性等问题的存在,最终使该病的复发率仍居高不下。因此,研究GU理想的治疗方案仍是一件急迫而重要的任务,近几年来人们对于GU药物的开发,常从天然药物寻找突破口。
维胃方是导师的经验方,通过临床实践证明对GU有较好疗效,七及方是维胃方中的组成部分。本实验采用适合药物疗效观察的乙酸致大鼠GU模型,以中医药基础理论为指导,临床疗效为依据,通过GU的大体愈合情况、溃疡面积、GU周围黏膜病理组织切片光学显微镜下观察,从不同层次阐述维胃方治疗GU的效果。并检测胃液PH值、测定血清GaS、血浆NT水平,其中以整体性调节作为切入点,选用NT作为主要指标,旨在阐述维胃方治疗GU的相关机制,为维胃方的临床运用和开发提供实验依据。
实验方法:
1、制备冰醋酸致大鼠GU模型
参照Takagi等乙酸致大鼠GU造模方法,并加以改造。大鼠造模前24h,禁食不禁水。经腹腔注射10%水合氯醛溶液,按3.5ml/kg注射麻醉大鼠。腹部剃毛、消毒,于剑突下腹中线打开腹腔,手术切口长约2cm,暴露出胃,在胃前壁浆膜面接近窦体交界处的胃窦同一部分(避开血管)用浸有100%乙酸,直径为5mm的圆形滤纸接触60s,然后还纳胃体,逐层缝合切口,酒精消毒,保护切口。
2、实验动物分组和相关指标检测
雄性SPF级Wistar大鼠72只,根据完全随机法分为9组,每组8只:A正常组,系非造模大鼠,自由饮水,进食。B模型组,乙酸致大鼠GU,自由饮水,进食。C自愈组,乙酸致大鼠GU,自由饮水,进食。D雷尼替丁组,乙酸致大鼠GU,第4天开始灌胃雷尼替丁水悬液2ml,共7天,每天1次。E1维胃方低剂量组,乙酸致大鼠GU,第4天灌胃低剂量维胃方2ml,共7天,每天1次;E2维胃方中剂量组,乙酸致大鼠GU,第4天开始灌胃中剂量维胃方2ml,共7天,每天1次;E3维胃方高剂量组,乙酸致大鼠GU,第4天,灌胃高剂量维胃方2ml,共7天,每天1次。F1七及方中剂量组,乙酸致大鼠GU,第4天开始灌胃中剂量七及方2ml,共7天,每天1次;F2七及方高剂量组,乙酸致大鼠胃溃疡,第4天,灌胃高剂量七及方2ml,共7天,每天1次。除模型组在第4天,其余各组均在第11天麻醉后取材,并观察胃溃疡的大体愈合情况,检测胃液PH值、溃疡面积,光学显微镜下观察胃溃疡周围黏膜病理组织切片,测定血清Gas、血浆NT水平。
统计学处理:
实验数据用SPSS11.5统计软件分析,数据以χ±S表示,采用单因素方差分析方法(One-Way-ANOVA)进行多样本均数比较。大鼠溃疡面积的比较,方差不齐,采用Welch检验,各组间存在差异,采用Dunnett's T3法检验;大鼠胃液pH值的比较,方差齐,各组间存在差异,采用LSD检验;大鼠血清Gas水平的比较,方差齐,但各组差别无统计学意义,无需做两两比较;大鼠血浆NT水平的比较,方差不齐,采用Welch检验,各组间存在差异,采用Dunnett's T3检验。
主要结果:
1、胃黏膜大体标本肉眼观察
维胃方中剂量组溃疡绝大部分愈合,新生组织与周围正常组织无明显差别,仅见个别小溃疡,且溃疡表浅,个别可见炎症分泌物。
2、用药组与自愈组大鼠GU面积比较
方差不齐,用Welch检验,各组间存在差异,采用Dunnett's T3法检验。雷尼替丁组,维胃方低、中、高剂量组,七及方中、高剂量组与自愈组比较差别有统计学意义(P=0.005,P=0.028,P=0.001,P=0.007,P=0.001,P=0.001)。维胃方中剂量组与雷尼替丁组相比差别有统计学意义(P=0.013)。维胃方中剂量组、七及中剂量组、七及高剂量组与维胃方高剂量组相比差别有统计学意义(P=0.006,P=0.043,P=0.036)。维胃方对溃疡面积大小的影响并不成量效依赖。
3、光学显微镜下观察胃黏膜病理组织切片
维胃方中剂量组黏膜基本完整,仅有极个别溃疡深及上皮与胃腺区交界处,面积小,上皮细胞形态,腺体结构良好,腺体细胞增生数量较多,腺体间隙较小,排列较整齐,黏膜下层仅见少量炎症细胞浸润,黏膜下微循环未见明显病理改变,肌层变薄,肉芽组织增生,排列较整齐,小部分浆膜断裂。
4、大鼠胃液PH值的比较
方差齐,各组间存在差异,采用LSD检验,模型组、七及方中剂量组、七及方高剂量组与正常组比较差别有统计学意义(P=0.006,P=0.000,P=O.034)。维胃方中剂量组与正常组比较差别无统计学意义(P=0.746)。各组可以分为三个水平:pH值处于较低水平的是正常组、雷尼替丁组、维胃方各组,pH值处于中等水平的是自愈组,pH处于较高水平的是模型组、七及方各组。
5、大鼠血清GAS水平的比较
方差齐,但各组差别无统计学意义,无需做两两比较。
6、大鼠血浆NT水平的比较
方差不齐,采用Welch检验,各组间存在差异,采用Dunnett's T3检验,模型组、雷尼替丁组、维胃方低剂量组、七及方中剂量组与自愈组相比差别有统计学意义(P=0.021,P=0.020,P=0.032,P=0.016)。自愈组、维胃方高剂量组与模型组相比差别有统计学意义(P=0.021,P=0.003)。各组与正常组相比差别均无统计学意义(均P>0.05),其中维胃方中剂量组接近正常组(P=1)。各组大体可以分为三个层次:NT值处于较低水平的是模型组、雷尼替丁组、维胃方低剂量组、七及方中剂量组,NT值处于中等水平的是正常组、维胃方中剂量组、七及方高剂量组,NT值处于较高水平的是自愈组、维胃方高剂量组。维胃方对血浆NT水平的影响呈量效正相关(r=1,P=0.002)。
结论:
1、从溃疡面积中看,造模成功率高达到100%,自愈组大鼠对GU的自愈效果差,用药组起到一定治疗作用,维胃方中剂量组的疗效最佳。
2、维胃方和七及方由于配伍及剂量的关系出现了复杂的量效关系,说明在相同煎煮等技术条件下,中药配伍、剂量对疗效影响极为重要。
3、从胃黏模大体标本观察中发现,在用药各组中维胃方中剂量组效果最好。
4、光学显微高倍镜下观察胃黏膜病理组织切片,维胃方中剂量组效果最佳,部分机制是通过改善微循环、抗炎和细胞再生这些方面发挥作用。
5、模型组血浆NT水平低于正常组,说明溃疡形成时,体内合成和释放NT明显减少。
6、模型组胃液pH值高于正常组,表明手术造模对动物本身就是一种应激,为防止乙酸的损害作用,大鼠机体应激性的调动体内的平衡机制,迅速中和酸性,以保护其受损伤的胃黏膜,这可能是模型组胃液pH值升高的原因。
7、维胃方各剂量组血浆NT存在一种剂量依赖的正相关关系(r=1,P=0.002),但是血清Gas并不成量效依赖关系,说明在脱离乙酸致大鼠GU的手术应激后的治疗过程中,推测NT对胃黏膜的影响不是直接通过G细胞分泌Gas而发挥作用,很可能是通过合成生长抑素等。
8、根据pH的统计结果,七及方各组pH值高于维胃方各组,说明三七、白及二者单独合用,在提高pH值,抗胃酸方面能够起着重要作用。七及方中剂量组和维胃方低剂量组NT处于同等水平,但低于正常水平时,七及方中剂量组因为pH值高于维胃方低剂量组,所以能够发挥更好的疗效。
9、维胃方中剂量组在胃液pH值、血清Gas、血浆NT水平均接近正常组,从GU病理组织切片上看,维胃方中剂量组修复受损胃黏膜效果更强,部分机制可能是通过NT的神经机制,从抗炎、改善微循环、细胞再生等方面,对机体起着整体性调节,发挥其整体治疗GU的作用。
10、维胃方中剂量组血浆NT水平接近正常组(P=1),推测血浆NT发挥其生理学效应,修复和保护胃黏膜存在一定阈值,生理浓度或接近生理浓度的血浆NT,对胃黏膜有更重要的意义,能调节机体尽可能达到常态。
综上实验结果表明:通过GU的大体愈合情况、溃疡面积以及GU周围黏膜病理组织切片光学显微镜下观察,发现维胃方中剂量组的疗效最佳,其相关机制可能是通过NT,直接或间接使胃液pH值接近正常水平,并从抗炎、改善微循环、细胞再生等方面,对机体起着整体性调节,发挥其整体治疗GU的作用。
Background:
Peptic ulcer(PU) disease is one large kind of digestive system diseases, which spreads cosmopolitism. It has been estimated that 5%～10% people have suffered this disease in their lives. The pathogenesy is complicit. It has been summarized that the balance is destroyed between aggression factor of gastric mucosa intestinal mucosa and mucosa self defend- reparative factor. PU has the characteristics of long course of disease, ease palindromia and some severe complication.
Gastric acid inhibition and gastric mucosa protectant are two main methods of clinical therapeutics. At present, histamine-2-receptor antagonists and proton pump antagonists which widely use in clinic make GU accrete rate predominance raise. However, the problem of cost, side-effect, drug resistance, patient dependence makes the GU palindromia high. Therefore, GU therapeutics is still a urge and important task. Recently, the exploitation of GU drug turns to naturally occurring drugs.
Weiweifang(WWF) is Liu Xiaowei professor's empirical formula. It has been proved that it has a good effect to GU in clinic. Qijifang(QJF) is one part of WWF .The experiment which bases the Traditional Chinese Medicine(TCM) theory and clinical effect adopts acetic acid—inducted gastric ulcer mode. We detected gastric mucosa, ulcer size and gastric ulcer circa histopathologic slide to find out WWF's Therapeutical effect. The gastric fluid pH, serum gastrin(Gas) and plasma neurotensin(NT) concentration had been detected. The purport is to find out the correlate treatment mechanism of WWF. NT which has hol-modulation function is the enter-point in our experiment. We hope that we can support experiment evidences for WWF's clinical apply and exploitation.
Methods:
1. Acetic Acid-Induced Gastric ulcer
The experiments were performed according to the method described by Takagi et al., with some modifications. Eight groups of male Wistar rats that had fasted for 24 h were used in this experiment. Under anesthesia, a laparotomy was performed on all animals through a midline epigastric incision. After exposing the stomach, filter pape in 5 mm area with 100% acetic acid solution was contacted with the subserosal layer in the glandular part of the anterior wall. The abdomen was then closed and all animals were fed normally.
2. Rats subgroup and relative indicatrix detection
Seventy two male SPF Wistar rats were randomly divided into nine groups of 8 each, respectively marked with normal group, model group, autotherapy group, Ranitidine group, WWF groups and QJF groups. Normal group is untreated and freely drink and eat. Model group is inducted gastric ulcer by acetic acid and freely drink and eat. When acetic acid—inducted gastric ulcer in rats of autotherapy group, Ranitidine group, WWF groups and QJF groups , the groups which drink and eat freely are respectively treated with nothing, but Ranitidine, three different doses of WWF (low ,medium , high ) and two different doses of QJF (medium , high )7 days. Except model group in the forth days, other groups are killed in the eleventh day. Afer they are killed, We detected the gastric fluid pH, gastric mucosa, ulcer size, gastric ulcer circa histopathologic slide, serum gastric acid secretion(Gas) and plasma NT concentration.
Stastical analysis:
The data result of study is analysis with SPSS11.5 statistics software. The data is expressed with (x|-)±S. One-Way-ANOVA is used to multiple comparisons. Comparison in gastric ulcer area, when the test of Homogeneity of vriances is not existent we use Welch test and that the group's disparation is existent, we use Dunnett's T3 test. Comparison in gastric fluid pH in rats, when the test of Homogeneity of vriances and the group's disparation is existent, we use LSD test. Comparison in serum Gas in rats, the test of Homogeneity of vriances is existent but the group's disparation is not existent, so we do nothing. Comparison in plasma NT concentration in rats, when the test of Homogeneity of vriances is not existent we use Welch test and that the group's disparation is existent, we use Dunnett's T3 test.
Results:
1. Gastric mucosa detected with eyes , for no equipment
The ulcer of WWF medium dose group (WWFMG) is almost healing, neogenesis tissue has nothing different with normal tissue. There are only light aphthae and little inflammatory exudate seen.
2. comparision in drug groups and autotherapy group in gastric ulcer area in rats
The test of Homogeneity of vriances is not existent. It is used Welch test and that the group's disparation is existent, we use Dunnett's T3 test. Drug groups includes Ranitidine group, Weiweifang low dose group (WWFLG),WWFMG, Weiweifang high dose group(WWFHG), Qijifang medium group (QJFMG), Qijifang high group (QJFHG) have discrepancy significantly compared with autotherapy group (P=0.005, P=0.028,P=0.001,P=0.007, P=0.001, P=0.001) .WWFMG has discrepancy significantly compared with Ranitidine group (P=0.013) . WWFMG, QJFMG, QJFHG have discrepancy significantly compared with WWFHG (P=0.006, P=0.043, P=0.036) .The effective of WWF is not rely quantity in gastric ulcer area.
3. To detect gastric ulcer circa histopathologic slide in light microscope.
WWFMG's mucosa is almost integrity. Only a few of ulcer which is small in area reach the juncture of endepidermis and gastric area glandularis. The recovery of cellula epithelialis and glandular organ are fine, glandular organ Cellular proliferation is nice, include quantity , space, alignment. There are a few of inflammatory cell infiltration in mucosa underlayer. Microcirculation has not significant patho-change. Muscular layer turn to slide. Granulation tissue grow in regularity. A few of serosa membrane are abruptly.
4. Comparison in gastric fluid pH in rats.
When the test of Homogeneity of variances and the group's disparation are existent, we use LSD test. Model group, QJFMG,QJFHG have discrepancy significantly compared with normal group (P=0.006, P=0.000, P=0.034) . WWFMG has not discrepancy significantly compared with normal group (P=0.746) .All the groups can divide in three levers of pH. Normal group, Ranitidine group and WWF groups are in the low lever, autotherapy group is in the middle lever. Model group and QJF groups are in the high lever.
5. Comparison in serum Gas in rats
The test of the Homogeneity of vriances is existent but the group's disparation is not existent, so we do nothing.
6. Comparison in plasma NT concentration in rats
The test of the Homogeneity of vriances is not existent. We use Welch test and that the group's disparation is existent, we use Dunnett's T3 test. Model group, Ranitidine group, WWFLG, QJFMG and QJFHG have discrepancy significantly compared with autotherapy group (P=0.021, P=0.020, P=0.032, P=0.016) . Autotherapy group and WWFHG have discrepancy significantly compared with model group (P=0.021, P=0.003) . All groups have not discrepancy significantly compared with normal group. WWFMG is close to normal group (P=1) .All the groups can divide in three levers of NT. Model group, Ranitidine group, WWFLG and QJF groups are in the low lever. Normal group and WWFMG are in the middle lever. Autotherapy group and WWFHG are in the high lever. The effective of WWF has the tendency to rely quantity in plasma NT concentration in rats (r=1, P= 0.002) .
Conclusion:
1. The mode achievement ratio is reach 100% in gastric ulcer area in rats. Autotherapy group has bad effect. All the drug groups have more or less therapeutical effect. WWFMG has the best effect.
2. Because of compatibility and dose, WWF groups and QJF groups have complicate dose-effect relationship. It manifests that compatibility and dose of Tradition Chinese drags in same specification are important to therapeutical effect.
3. WWFMG has the best therapeutical effect by detecting the gastric mucosa with eyes, for nothing equipment.
4. WWFMG has the best therapeutical effect by detecting gastric ulcer circa histopathologic slide in light microscope. It has good effect in microcirculation, anti-inflammatory and cell regeneration.
5. In plasma NT concentration, model group is lower than normal group. It manifests that the synthesis and release of NT is in lower lever in elcosis.
6. Model group is higher in gastric fluid pH than normal group. It manifests that acetic Acid-Induced Gastric ulcer is a stress for rats. For self-protect, rats irritably remove the equilibrium mechanism to counteract Acetic Acid. It may be the reason why the model group is in higher lever in gastric fluid pH.
7.The effective of WWF has the tendency to rely quantity in plasma NT concentration in rats (r=1, P=0.002) , but not in serum Gas . It manifests that NT can effect the gastric mucosa during the treatment period, but not through Gas derectly. It may be through growth hormone release inhibiting hormone (GIH) .
8. QJF groups have discrepancy significantly compared with WWF groups in gastric fluid pH. It proves that solitude use with notoginseng and bletillae rhizome can elevate pH to inhibit the gastric acid secretion. When QJFMG and WWFLG are in the same lever, but lower than normal lever in plasma NT concentration, QJFMG have better effect through the higher pH.
9. WWFMG is close to normal group in gastric fluid pH, serum Gas and plasma NT concentration. From the evidence of gastric ulcer circa histopathologic slide, WWFMG has the best effect in recovering damaged gastric mucosa, Partial mechanism is that plasma NT may be playing a hol-regulation role by anti-inflammatory, microcirculation improvement and cell regeneration.
10. WWFMG is close to normal group in plasma NT concentration(P=l). It has been supposed that plasma NT which exist leak point close to normal concentration play an important role for gastric mucosa. Plasma NT in this concentration can regulate the body as to normal.
In above, it has been find out that WWFMG has the best effect by detecting gastric mucosa, ulcer size, gastric ulcer circa histopathologic slide. Partial mechanism is that plasma NT may be playing a hol-regulation role by adjusting pH to normal lever and by anti-inflammatory, microcirculation improvement and cell regeneration.
引文
[1]Katz J.Multicenter study of Helicobacter pylori infection prevalence in patients with chronic gastroduodenal disease.Various epidemiologic features.Acta Gastroenterol Latinoam[J],1997,27:253-2571.
[2]姒健敏,王杭勇,蔡建庭,吕宾,俞方,黄怀德,钱可大,项柏康.中国人溃疡病流行状况及其治疗依从性.中华内科杂志[J],2000,39(3):153-155.
[3]Adeyemi EO,Bastaki SA,Chandranath IS,Hasan MY,Fahim M,Adem A.Mechanisms of action of leptin in preventing gastric ulcer[J].World J Gastroenterol,2005,11(27):4154-4160.
[4]Gyires K.Neuropeptides and gastric mucosal homeostasis.Curr Top Med Chem [J],2004,4:63-73.
[5]Carraway R.Characterization of radioimmunoassay neurotensin in the rat,its differental distribution in the central nervous system,small intestine and stomach.J Bio Chem[J],1976,251(22):7045-7052.
[6]Shaw C,Stockman F,Conlon J M.Xenopsin and neurotensin like peptides in gastric juice from patients with duodenal ulcers.Eur J Chin Invest[J],1987,17(4):306-312.
[7]梅懋华.消化道生理学与临床.北京:人民卫生出版社,1990,24.
[8]ZhangL,XingL,Demers L,et al.Central neurotensin inhibits gastric acid secretion.an adrenergic mechanism in rats.Gastroenterology[J],1989,97(5):1130-1134.
[9]周颖斌,徐珞,陶尚敏.神经降压素对大鼠应激性溃疡细胞保护作用的研究.胃肠道学[J],2006,11(1):34-36.
[10]徐珞,陶尚敏.神经降压素抗实验性胃溃疡作用初步探讨.中国行为医学科学[J],2005,9,14(9):775-776.
[11]徐珞,郭云良,于业军,刘晓萍,陶尚敏.神经降压素对大鼠应激性胃溃疡的影响及与表皮生长因子的相关性研究.中国行为医学科学[J],1998,7(1):17-18.
[12]孙瑞卿,王述琦,陶尚敏,陈家津.中枢神经降压素对大鼠胃应激性溃疡的作用及多巴胺的关系.中国病理生理杂志[J],1999,15(4):309-311.
[13]Sipponen P,Harkonen M,Allanko A,et al.Diagnosis of atrophic gastritis from a serum samp le.Clin Lab[J].2002,48:505.
[14]温怀凯,刘素霞.胃癌患者血清Gastrin含量分析及临床意义.放射免疫学杂志[J],2006,19(1):9-11.
[15]钟英强,曾志勇,黄志清,等1胃溃疡并出血患者血清胃泌素水平变化和临床意义.中国实验诊断学[J].2001,5(1):451.
[16]张惠芳,侯敏,高志刚.神经降压素和胃泌素在消化性溃疡中的水平变化及其相关性探讨.胃肠病学[J],2004,9(2):117-118.
[17]陈志东.消化性胃溃疡模型在研究新药中的应用.现代中西医结合杂志[J],2006,15(15):2137-2138.
[18]卢文丽,方肇勤,潘志强,侯俐,刘小美.6种胃溃疡小鼠模型的证候特征及比较.上海中医药杂志[J].2007,41(8):63-67.
[19]郑芝田主编.消化性溃疡.北京:人民卫生出版社,1998:830.
[20]Birdane FM,Cemek M,Birdane YO,Gulcin I,Buyukokuroglu ME.Beneficial effects of Foeniculum vulgare on ethanol-induced acute gastric mucosal injury in rats[J].World J Gastroenterol,2007,13(4):607-611.
[21]TAKAGI K,OKABE S,SAZIKI R.A new method for the production of chronic gastric ulcer in rats and the effect of several drugs on its healing[J].Jpn J Pharmacol,1969,19:418-426.
[22]王军,王春宏,任晓玲,王少臣.白芨胶的临床应用.中国医院药学杂志[J],2004,24(9):556-557.
[23]杨志刚,陈阿琴,俞颂东.三七药理研究新进展.上海中医药杂志[J],2005,39(4):59-62.
[24]张璐,刘强.茯苓多糖制备工艺及药理作用研究进展.中国实验方剂学杂志 [J],2006,12(4):61-64.
[25]吴艳玲,朴慧善.蒲公英的药理研究进展.时珍国医国药[J],2004,15(8):519-520.
[26]贺德辉.中药量效关系刍议[J],时珍国医国药,2000,11(10),913.
[27]Dai XP,Li JB,Liu ZQ,Ding X,Huang CH,Zhou B.Effect of Jianweiyuyang granule on gastric ulcer recurrence and expression of VEGF mRNA in the healing process of gatric ulcer in rats[J].World J Gastroenterol,2005,11(35):5480-5484.
[28]Szabo S,Deng X,Khomenko T.Gene expression and gene therapy in experimental duodenal ulceration[J].J Physiol Paris,2001,95:325-335.
[29]Milani S,Calabro A.Role of growth factors and their receptorsin gastric ulcer healing[J].Microsc Res Tech,2001,53:360-371.
[30]袁禧先,王凤荣,薛鸿鹏,姜威.幽门螺杆菌相关性胃溃疡胃癌组织中VEGF 的表达及意义[J].黑龙江医药科学,2006,29(4):32-33.
[31]凌江红,陈业强,李家邦,潘宇政,曹阳,申定珠.健胃愈疡颗粒对愈合和复发胃溃疡大鼠胃黏膜炎症反应及NF-κB表达的影响[J].中国中药杂志,2006,31(17):1454-1457.
[32]Nishida T,Tsuji S,Kimura A,Tsujii M,Ishii S,Yoshio T,Shinzaki S,Egawa S,Irie T,Yasumaru M,Iijima H,Murata H,Kawano S,Hayashi N.Endothelin-1,an ulcer inducer,promotes gastric ulcer healing via mobilizing gastric myofibroblasts and stimulates production of stroma-derived factors[J].Am J Physiol Gastrointest Liver Physiol.2006,290(5):G1041-1050.
[2]姒健敏,王杭勇,蔡建庭,吕宾,俞方,黄怀德,钱可大,项柏康.中国人溃疡病流行状况及其治疗依从性.中华内科杂志[J],2000,39(3):153-155.
[3]Adeyemi EO,Bastaki SA,Chandranath IS,Hasan MY,Fahim M,Adem A.Mechanisms of action of leptin in preventing gastric ulcer[J].World J Gastroenterol,2005,11(27):4154-4160.
[4]Gyires K.Neuropeptides and gastric mucosal homeostasis.Curr Top Med Chem [J],2004,4:63-73.
[5]Carraway R.Characterization of radioimmunoassay neurotensin in the rat,its differental distribution in the central nervous system,small intestine and stomach.J Bio Chem[J],1976,251(22):7045-7052.
[6]Shaw C,Stockman F,Conlon J M.Xenopsin and neurotensin like peptides in gastric juice from patients with duodenal ulcers.Eur J Chin Invest[J],1987,17(4):306-312.
[7]梅懋华.消化道生理学与临床.北京:人民卫生出版社,1990,24.
[8]ZhangL,XingL,Demers L,et al.Central neurotensin inhibits gastric acid secretion.an adrenergic mechanism in rats.Gastroenterology[J],1989,97(5):1130-1134.
[9]周颖斌,徐珞,陶尚敏.神经降压素对大鼠应激性溃疡细胞保护作用的研究.胃肠道学[J],2006,11(1):34-36.
[10]徐珞,陶尚敏.神经降压素抗实验性胃溃疡作用初步探讨.中国行为医学科学[J],2005,9,14(9):775-776.
[11]徐珞,郭云良,于业军,刘晓萍,陶尚敏.神经降压素对大鼠应激性胃溃疡的影响及与表皮生长因子的相关性研究.中国行为医学科学[J],1998,7(1):17-18.
[12]孙瑞卿,王述琦,陶尚敏,陈家津.中枢神经降压素对大鼠胃应激性溃疡的作用及多巴胺的关系.中国病理生理杂志[J],1999,15(4):309-311.
[13]Sipponen P,Harkonen M,Allanko A,et al.Diagnosis of atrophic gastritis from a serum samp le.Clin Lab[J].2002,48:505.
[14]温怀凯,刘素霞.胃癌患者血清Gastrin含量分析及临床意义.放射免疫学杂志[J],2006,19(1):9-11.
[15]钟英强,曾志勇,黄志清,等1胃溃疡并出血患者血清胃泌素水平变化和临床意义.中国实验诊断学[J].2001,5(1):451.
[16]张惠芳,侯敏,高志刚.神经降压素和胃泌素在消化性溃疡中的水平变化及其相关性探讨.胃肠病学[J],2004,9(2):117-118.
[17]陈志东.消化性胃溃疡模型在研究新药中的应用.现代中西医结合杂志[J],2006,15(15):2137-2138.
[18]卢文丽,方肇勤,潘志强,侯俐,刘小美.6种胃溃疡小鼠模型的证候特征及比较.上海中医药杂志[J].2007,41(8):63-67.
[19]郑芝田主编.消化性溃疡.北京:人民卫生出版社,1998:830.
[20]Birdane FM,Cemek M,Birdane YO,Gulcin I,Buyukokuroglu ME.Beneficial effects of Foeniculum vulgare on ethanol-induced acute gastric mucosal injury in rats[J].World J Gastroenterol,2007,13(4):607-611.
[21]TAKAGI K,OKABE S,SAZIKI R.A new method for the production of chronic gastric ulcer in rats and the effect of several drugs on its healing[J].Jpn J Pharmacol,1969,19:418-426.
[22]王军,王春宏,任晓玲,王少臣.白芨胶的临床应用.中国医院药学杂志[J],2004,24(9):556-557.
[23]杨志刚,陈阿琴,俞颂东.三七药理研究新进展.上海中医药杂志[J],2005,39(4):59-62.
[24]张璐,刘强.茯苓多糖制备工艺及药理作用研究进展.中国实验方剂学杂志 [J],2006,12(4):61-64.
[25]吴艳玲,朴慧善.蒲公英的药理研究进展.时珍国医国药[J],2004,15(8):519-520.
[26]贺德辉.中药量效关系刍议[J],时珍国医国药,2000,11(10),913.
[27]Dai XP,Li JB,Liu ZQ,Ding X,Huang CH,Zhou B.Effect of Jianweiyuyang granule on gastric ulcer recurrence and expression of VEGF mRNA in the healing process of gatric ulcer in rats[J].World J Gastroenterol,2005,11(35):5480-5484.
[28]Szabo S,Deng X,Khomenko T.Gene expression and gene therapy in experimental duodenal ulceration[J].J Physiol Paris,2001,95:325-335.
[29]Milani S,Calabro A.Role of growth factors and their receptorsin gastric ulcer healing[J].Microsc Res Tech,2001,53:360-371.
[30]袁禧先,王凤荣,薛鸿鹏,姜威.幽门螺杆菌相关性胃溃疡胃癌组织中VEGF 的表达及意义[J].黑龙江医药科学,2006,29(4):32-33.
[31]凌江红,陈业强,李家邦,潘宇政,曹阳,申定珠.健胃愈疡颗粒对愈合和复发胃溃疡大鼠胃黏膜炎症反应及NF-κB表达的影响[J].中国中药杂志,2006,31(17):1454-1457.
[32]Nishida T,Tsuji S,Kimura A,Tsujii M,Ishii S,Yoshio T,Shinzaki S,Egawa S,Irie T,Yasumaru M,Iijima H,Murata H,Kawano S,Hayashi N.Endothelin-1,an ulcer inducer,promotes gastric ulcer healing via mobilizing gastric myofibroblasts and stimulates production of stroma-derived factors[J].Am J Physiol Gastrointest Liver Physiol.2006,290(5):G1041-1050.