【作者】 陈俊；

【导师】 赵映前；

【作者基本信息】 湖北中医药大学 ， 中医临床基础， 2010， 博士

【摘要】 目的在系统整理研究古代文献的基础上,结合现今消渴及其并发症的发病特点,对消渴胃病(糖尿病胃轻瘫)的理法方药进行理论探讨,丰富中医学对消渴胃病的认识；探讨糖尿病胃轻瘫大鼠胃窦SCF-Kit信号途径在Cajal间质细胞受损中的作用,进一步阐明中药糖胃康治疗糖尿病胃轻瘫的作用部位、环节及分子机制,为临床开发防治糖尿病胃轻瘫的有效中药制剂奠定基础。方法理论研究：对消渴胃病的历史源流、病机特点及防治方法等进行分析研究,讨论糖胃康的组方原则及药效分析。实验研究：采用链脲佐菌素及高糖高脂饲料不规则喂养(即单日上午进食,双日下午进食)诱导糖尿病胃轻瘫大鼠模型,随机分为7组(正常对照组、糖尿病模型组、糖尿病胃轻瘫模型组、糖胃康高剂量组、糖胃康中剂量组、糖胃康低剂量组和西沙比利治疗组)进行实验研究。观察糖胃康对糖尿病胃轻瘫模型组大鼠糖、脂代谢及胰岛β细胞功能的影响；通过透射电镜观察各组大鼠胃窦组织ICC超微结构的变化；采用Western blot检测各组大鼠胃窦平滑肌组织c-kit蛋白、SCF蛋白表达水平,RT-PCR测定各组大鼠胃窦平滑肌组织SCF mRNA的表达。结果1.消渴胃病具有“本虚标实”的病机特点,且“本虚之中,脾虚为要”、“标实之中,毒邪为甚”,糖胃康针对此病机而设,具有健脾和胃,理气排毒之功效。2.糖尿病及糖尿病胃轻瘫造模研究中：在造模后前4周糖尿病模型组及糖尿病胃轻瘫模型组大鼠与正常对照组大鼠相比较,日进食量均明显增加,有显著性差异(P<0.05或P<0.01),且以糖尿病模型组尤为明显；糖尿病胃轻瘫模型组大鼠第6周后,日进食量减少,第8周与正常对照组相比较,有统计学意义(P<0.01)；与糖尿病模型组相比较,第4周后,糖尿病胃轻瘫模型组大鼠日进食量减少,有显著性差异(P<0.01)。造模后前2周,糖尿病模型组和糖尿病胃轻瘫模型组大鼠日饮水量均显著增加,与正常对照组比较,有显著性差异(P<0.01)；造模4周后,糖尿病胃轻瘫模型组大鼠日饮水量较糖尿病模型组减少,第6周后尤为明显,有统计学意义(P<0.05或P<0.01)。糖尿病模型组和糖尿病胃轻瘫模型组大鼠体重随着时间的延长(造模2周后)均呈现体重下降的趋势,与正常对照组相比较,有显著性差异(P<0.05或P<0.01),且以糖尿病胃轻瘫组大鼠下降尤为明显；与糖尿病模型组相比较,糖尿病胃轻瘫模型组大鼠体重下降有统计学意义(P<0.05或P<0.01)。同时,造模后糖尿病模型组及糖尿病胃轻瘫模型组大鼠均出现大便量少、色淡、质稀软、互相粘连、不成形等大便性状的改变,且以糖尿病胃轻瘫模型组大鼠尤为明显。在胃排空功能的测定方面,糖尿病模型组和糖尿病胃轻瘫模型组大鼠胃排空功能均有所下降,与正常对照组相比较,糖尿病模型组大鼠胃排空下降无统计学意义(P>0.05),而糖尿病胃轻瘫模型组大鼠有显著性差异(P<0.01)。3.糖胃康对糖尿病胃轻瘫模型大鼠糖脂代谢和胰岛β细胞功能影响的研究中：糖胃康高、中剂量组分别与糖尿病胃轻瘫模型组相比较,大鼠空腹血糖及胰岛素水平均明显下降(P<0.01),胰岛素敏感指数均升高(P<0.01)、抵抗指数均降低(P<0.01)；胰岛形状较规则,细胞数量增多,胞浆较丰富；胰岛β细胞团平均面积均明显增大(P<0.05)；且胃排空功能与西沙比利治疗组相比较无明显差异。说明高、中剂量的糖胃康可通过其有效的降糖作用来改善IR,又可以通过其改善IR的作用,增加胰岛素敏感性,恢复胰岛β细胞的分泌功能,缓解糖尿病胃轻瘫的临床症状。模型组(糖尿病模型组和糖尿病胃轻瘫模型组)大鼠胰岛内有明显的脂质沉积,可能是糖尿病及糖尿病胃轻瘫形成的重要病理因素。实验后期,模型组大鼠体重出现了明显的下降,且以糖尿病胃轻瘫模型组为甚,分析原因可能为血糖较高,细胞供能不足,机体分解脂肪供能而导致的消瘦,与临床上未经控制的血糖,糖尿病病程进展而出现“消瘦”的临床特征相符合；糖尿病胃轻瘫模型组大鼠因饮食减少,供能更为不足而致体重下降更加明显。药物干预后,糖胃康高、中剂量组大鼠体重均明显增加,内脏脂肪/体重均明显降低,说明其对血脂具有调节作用,能够延缓糖尿病病程进展。4.糖胃康对糖尿病胃轻瘫模型大鼠Cajal间质细胞的影响研究中：电镜观察结果显示,糖尿病胃轻瘫模型组大鼠胃窦ICC与其它的ICC之间、平滑肌细胞之间以及神经末梢之间的缝隙连接明显减少,尚存细胞间的连接结构不清,间隙增大。部分ICC的基膜与细胞膜分离,形成空洞；线粒体肿胀、空泡样变、甚至溶解；胞质广泛溶解,有大量的胞质内空泡形成；细胞器数量减少明显。药物干预后,糖胃康高、中剂量组大鼠胃窦ICC与其它的ICC之间、平滑肌细胞之间以及神经末梢之间的缝隙连接均明显增多,ICC的基膜结构趋于完整,部分线粒体轻度肿胀,细胞器数量较正常对照组无明显差异。Western blot检测显示：糖尿病胃轻瘫模型组大鼠胃窦平滑肌组织c-kit蛋白表达水平显著降低,与正常对照组相比,具有显著性差异(P<0.01)；药物干预后,糖胃康高、中剂量组大鼠胃窦平滑肌组织c-kit蛋白表达水平较糖尿病胃轻瘫模型组均显著升高(P<0.01),且与正常对照组比较差异均无统计学意义(P>0.05)。5.糖胃康对糖尿病胃轻瘫模型大鼠SCF蛋白和基因表达的影响研究中：糖尿病胃轻瘫模型组大鼠胃窦平滑肌组织SCF的m RNA及蛋白质相对表达水平均显著降低,与正常对照组相比,具有显著性差异(P<0.01)；药物干预后,糖胃康高、中剂量组大鼠胃窦平滑肌组织SCF的m RNA及蛋白质相对表达水平较糖尿病胃轻瘫模型组均显著升高,差异有统计学意义(P<0.05或P<0.01),且与正常对照组比较差异无统计学意义(P>0.05)。结论1.脾虚失运是消渴病形成的关键环节,热毒、湿毒、瘀毒、浊毒等毒邪既是消渴病的病理产物,又是消渴胃病形成的重要致病因素。消渴胃病具有“本虚标实”的病机特点,且“本虚之中,脾虚为要”、“标实之中,毒邪为甚”,治疗原则上应病证结合、标本兼顾。针对消渴胃病“脾虚毒盛”的主要病机,结合中医药理论及现代药理研究,精心组方而成的糖胃康具有健脾和胃,理气排毒的功效。2.在单次腹腔注射STZ的基础上,采用高糖高脂饲料不规则喂养(即单日上午进食,双日下午进食)的方法能成功复制糖尿病胃轻瘫模型。3.糖胃康对糖尿病胃轻瘫模型大鼠糖、脂代谢紊乱具有明显的改善作用,在一定程度上能改善IR,恢复胰岛β细胞的功能,增强DGP的胃排空功能,且呈一定的量效关系。糖胃康可通过改善糖尿病胃轻瘫模型大鼠糖、脂代谢紊乱,上调SCF表达水平,调控ICC的分化、发育和表型维持,恢复SCF/Kit的信号传递系统通路,从而使胃平滑肌收缩功能改变,增强DGP的胃排空功能。更多还原

【Abstract】 ObjectiveOn the basis of ancient literature, combined with today’s clinical peculiarity of diabetes and complications of diabetes,probe into diabetes gastroparesis treatment for theoretical approach, and enrich Chinese medicine on diabetes stomach; Study the role of diabetes gastroparesis (DGP) rats gastric antral SCF-Kit pathway in the damage of interstitial cells of cajal (ICC) and the mechanism of TangWeiKang in order to lay the foundation to develop an effective medicine to control DGP.MethodsTheoretical studies:Research the origin, the pathogenesis and treatment methods of gastric disease of diabetes, and discuss TangWeikang’s prescription principles and efficacy analysis.Experimental studies:At first,we have used Streptozotocin by cavitas abdominalis injection and high sugar high fat diet to irregularly feed (morning meal in odd-number days, afternoon eating in even-number days) rats to induce DGP model, and have divided rats into 7 Groups (normal comparison group, diabetes model group, diabetes gastroparesis model group,TangWeiKang high dose group, TangWeiKang medium dose group, TangWeiKang low dose group and XiShaBiLi treatment group) for experimental research. Then we have observed TangWeiKang’influence on improving carbohydrate and lipid metabolism and pancreaticβcell function of DGP model group rats; and observed each group of rats gastric antrum ICC ultrastructure through transmission electron microscopy; and taken the Western blot to detect gastric tissue c-kit and SCF protein expression level, and taken RT-PCR to determinate the expression of gastric tissue SCF mRNA.Results.1.Gastric disease of diabetes has a pathogenesis characteristic that Ben is deficient and Biao is excess. Furthermore, the spleen’s deficiency is the most important,and the toxic’exuberance is the most obvious. Aiming at these, we have established a prescription TangWeiKang that can make the spleen-stomach healthy and smooth Qi and eliminate toxins.2. In the research of making DM model and DGP model:Former 4 weeks after the model, quantities of food-intake of DM model group and DGP model group rats have obviously increased, compared with normal comparison group, and differences are significant (P<0.05 or P<0.01).Moreover, DM model group is more obvious. After 6 weeks, quantities of food-intake of DGP model group rats have decreased, compared with normal comparison group, and differences are significant in 8thweek(P<0.01). Former 2 weeks after the model, quantities of hydroposia of DM model group and DGP model group rats have obviously increased, compared with normal comparison group, and differences are significant (P<0.01). After 4 weeks, quantities of hydroposia of DGP model group rats have decreased more than that of DM model group.Besides, differences are more significant after 6 weeks, and are significant (P<0.05 or P<0.01). As an extension of time, the weights of DM model group and DGP model group rats have obviously reduced, compared with normal comparison group, and differences are signif icant(P<0.05 or P<0.01). Moreover, DGP model group is more obvious. and differences are significant (P<0.05 or P<0.01), compared with DM model group. At the same time, the stool of DM model group and DGP model group rats has appeared some changes, such as less, pale, softer, mutual adhesion, irregular shape and so on. Moreover, DGP model group is more obvious. The function of gastric emptying of DM model group and DGP model group rats has decreased, and the decline of gastric emptying of DM model group rats is non-obvious(P>0.05), compared with normal comparison group,but that of DGP model group rats is significant (P<0.01).3. TangWeiKang’ influence on improving carbohydrate and lipid metabolism and pancreaticβcell function of DGP model group rats: Fasting plasma glucose and fasting serum insulin and Homa-IR of TangWeiKang high dose group and medium dose group rats have obviously descended,but insulin sensitivity index of TangWeiKang high dose group and medium dose group rats have went up. Islet cell shape of TangWeiKang high dose group and medium dose group rats is more regular, and quantity of cell is more,and endochylema is more rich, compared with DGP model group rats. But the function of gastric emptying of TangWeiKang high dose group and medium dose group rats has no obvious difference with XiShaBiLi treatment group rats. These have explained TangWeiKang high dose and medium dose can improve IR by means of reducing glucose, and raise insulin sensibility,and renew pancreaticβcell function, and relieve clinical symptoms. There are obvious lipid accumulation in langerhans’islet in the DM model group and DGP model group rats, which can be important pathology factors that come into being DM and DGP. The weights of model group (DM model group and DGP model group)rats have dropped obviously in the experiment later stage, and DGP model group is most obvious. The reason can be high plasma glucose that lead to fat to resolve. The weights of TangWeiKang high dose group and medium dose group rats have obviously increased after invasion, and the ratio of fat in viscera and weight has obviously decreased,which have illustrated TangWeiKang can adjust lipid and put off the progress of DM.4. TangWeiKang’ influence on ICC of DGP model group rats:The gap joints between gastric antral ICC and smooth muscle and nerve endings have markedly decreased in the DGP model group rats through transmission electron microscopy, and the structures of intercellular connections are vague,and the gap has magnified. A part of ICC’ basement layer has been separated with cytomembrane, which has formed cavity.we can also find mitochondria swelling, vacuolar degeneration,even dissolution, and cytoplasm resolving, and the quantity of cell organ has decreased. The gap joints between gastric antral ICC and smooth muscle and nerve endings are markedly more in the TangWeiKang high dose group and medium dose group than DGP model group after invasion, with more developed slide endoplasmic reticulum, Golgi apparatus and mitochondria. We have found the expression levels of c-kit protein of DGP model group have obviously decreased by western blot analysis method, compared with normal comparison group, and differences are significant (P<0.01). The expression levels of c-kit protein in the TangWeiKang high dose group and medium dose group are obviously higher than DGP model group after invasion (P<0.01), but the differences are non-obvious (P>0.05), compared with normal comparison group.5. TangWeiKang’influence on SCF protein and gene expression of DGP model group rats:We have found the expression levels of SCF protein and gene expression of DGP model group have obviously decreased, compared with normal comparison group, and differences are significant (P<0.01). The expression levels of SCF protein and gene expression in the TangWeiKang high dose group and medium dose group are obviously higher than DGP model group after invasion (P<0.05 or P<0.01), but the differences are non-obvious (P>0.05), compared with normal comparison group.Conclusion1. Spleen-deficiency and losing in the transformation and transportation is a key link during diabetes mellitus formation. Toxins, such as toxic heat, damp toxin, ecchymosis toxin, turbid toxin and so on, are not only pathological products of diabetes mellitus,but also important pathogenic factors during gastric disease of diabetes formation. Gastric disease of diabetes have a pathogenesis characteristic that Ben is deficient and Biao is excess. Furthermore, the spleen’s deficiency is the most importance, and the toxic’exuberance is the most obvious. Treatment principle should combine Bing with Zheng,and treat Biao and Ben at the same time. Aiming at these, we have established a prescription TangWeiKang that can make the spleen-stomach healthy and smooth Qi and eliminate toxins.2. On the basis of single intraperitoneal injection STZ, we have fed model group rats on high sugar and high fat diet irregularly (morning meal in odd-number days, afternoon eating in even-number days).we have successfully copied diabetic gastroparesis model in this way.3.TangWeiKang have a positive influence on improving carbohydrate and lipid metabolism and pancreaticβcell function, to a certain extent, can improve the IR, restore pancreatic isletβcell functions, enhance gastric emptying of DGP, and these are at the dose-effect relationship. TangWeiKang may rebuild the SCF-Kit pathway for ICC phenotype reversion by upregulating the level of SCF, so as to maintain normal gastrointestinal motor function.更多还原