半夏泻心汤对RIN-m5F胰岛细胞凋亡的影响及其机制研究

发布时间：2018-06-27 18:53

本文选题：半夏泻心汤 + 胰岛β细胞凋亡　；参考：《中国中医科学院》2017年硕士论文

【摘要】：2型糖尿病是一种常见的具有遗传易感性并受环境等多种因素影响的慢性非传染性疾病。胰岛β细胞凋亡是2型糖尿病的关键发病机制之一,如何减少胰岛β细胞的凋亡成为防治糖尿病及其并发症的重要方法之一。许多中医学者认为脾虚胃热、痰瘀互结,胃肠失调是导致糖尿病发生发展的重要病机。中医调和胃肠法能够改善胃肠道功能、健脾清热、祛瘀化痰,可用于糖尿病及其相关疾病的治疗。本研究为证实半夏泻心汤(中医调和胃肠法的经典代表方)可用于糖尿病的治疗,并探索其分子机制,分别从综述和实验研究两部分进行阐释。第一部分文献综述2型糖尿病的发病率近年来呈现不断攀升的趋势,严重影响居民的生活质量和生命安全,并带来巨大的经济负担。本病可由多种原因引起,诸如遗传因素、环境因素,目前对于2型糖尿病病因研究较为关注的是胰岛β细胞凋亡对2型糖尿病发生发展的关键作用。越来越多的研究证实高糖、高脂、高浓度游离脂肪酸以及多种炎症因子等因素可加重胰岛β细胞氧化应激反应,进而导致胰岛β细胞的凋亡,诱发或加重2型糖尿病病情和病程。随着对糖尿病这一病因研究的深入,许多药物的相关治疗机制正在研究过程中,诸如GLP-1类似物(如利拉鲁肽、艾塞那肽)和DPP-IW抑制剂(如沙格列汀),许多研究表明这些药物可以抑制胰岛β细胞的凋亡并促进GLP-1活性的发挥,有利于2型糖尿病的改善。然而这些药物可能存在未纳入医保范围,价格昂贵,不方便携带和保存等缺点。许多中医学者认为脾虚胃热、痰瘀互结,胃肠失调是导致糖尿病发生发展的重要病机。调和胃肠法能够改善胃肠道功能、健脾清热、祛瘀化痰。而且“胃-肠-胰岛轴”的观点与半夏泻心汤从调和胃肠方面治疗糖尿病不谋而合。因此,可以用于糖尿病及其相关疾病的防治。半夏泻心汤作为调和胃肠法的经典方,现代临床及实验研究表明半夏泻心汤复方及其单味药可改善糖、脂代谢,降低血糖,改善胃肠道激素的分泌,提高机体的抗氧化能力和免疫功能,抑制胰岛细胞的凋亡,用于治疗糖尿病及其相关疾病。但半夏泻心汤的作用机制尚不明确,有待验证。第二部分实验研究目的:体外培养RIN-m5F胰岛细胞,建立RTN-m5F细胞凋亡模型并加以药物干预,通过CCK8、流式细胞术以及蛋白印迹的方法,证实半夏泻心汤抗RIN-m5F细胞凋亡的作用及其作用机制,从分子水平证明半夏泻心汤治疗2型糖尿病的有效性和科学性。为2型糖尿病的防治提供新思路和深入研究的技术平台。为临床治疗2型糖尿病提供新的思路和方法。方法:33只雄性SD大鼠随机分组灌胃,取血收集含药血清(正常血清、含半夏泻心汤药物血清、含利拉鲁肽药物血清),H_2O_2处理RIN-m5F细胞建立氧化应激细胞凋亡模型,造模成功后探索半夏泻心汤最佳浓度后随机分组,分别为:空白对照组、模型组、半夏泻心汤+H_2O_2组、利拉鲁肽+H202组。流式细胞术检测各组细胞凋亡率、放免法测定胰岛素含量、蛋白印迹(Western blot)法测定Bcl-2,Bax,Caspase-3,PI3K,AKT,p-AKT的蛋白表达量。所得数据用Microsoft EXCEL 2010进行管理,以均数土标准差(x一±s)表示,运用SPSS20.0统计软件,各组数据进行正态性检验和方差齐性检验后,采用单因素方差分析的方法进行统计学分析,组间比较采用LSD-t检验,P0.05认为有统计学意义。结果:通过上述实验得出:(1)建立了 RIN-m5F细胞的凋亡模型。H_2O_2的适宜造模浓度为300μM;(2)确立了半夏泻心汤含药血清的最佳浓度。运用CCK8检测不同浓度的半夏泻心汤含药血清对RIN-m5F细胞的保护作用,发现半夏泻心汤含药血清最佳浓度为10%,差异有统计学意义(P0.05);(3)证实了半夏泻心汤含药血清能够抑制RIN-m5F细胞的凋亡。流式细胞术结果显示与模型组相比,药物干预后,细胞活力显著增强,细胞凋亡率显著下降,差异有统计学意义(P0.05);(4)证实了半夏泻心汤含药血清能够促进胰岛素的分泌。放免法检测胰岛素分泌结果显示与模型组相比,药物干预后,葡萄糖刺激前后的胰岛素分泌均显著增加,差异有统计学意义(P0.05);(5)验证了半夏泻心汤含药血清能够降低Bax,Caspase-3的表达,升高Bcl-2、p-AKT的表达。Westernblot检测相关蛋白表达结果显示与模型组相比,药物干预后各组细胞Bax,Caspase-3的表达量明显降低、Bcl-2的表达量明显升高,p-AKT的表达明显升高,差异均具有统计学意义(P0.05)。结论:半夏泻心汤可改善RIN-m5F细胞凋亡,促进胰岛素的分泌,其可能是通过激活PI3K/AKT通路,从而调节下游因子表达有关。
[Abstract]:Type 2 diabetes is a common chronic non communicable disease with genetic susceptibility and environmental factors. The apoptosis of islet beta cells is one of the key pathogenesis of type 2 diabetes. How to reduce the apoptosis of islet beta cells is one of the important methods to prevent diabetes and its complications. Deficiency of stomach heat, phlegm and stasis, gastrointestinal disorders are important pathogenesis for the development of diabetes. Traditional Chinese medicine harmonization gastrointestinal function can improve gastrointestinal function, invigorate the spleen and heat, remove stasis and phlegm, and can be used in the treatment of diabetes and related diseases. This study confirms that the Banxia Xiexin Decoction (classical representative of Chinese medicine and gastrointestinal method) can be used in diabetes Treatment, and explore its molecular mechanism, from the two parts of the review and experimental research respectively. Part 1 literature review the incidence of type 2 diabetes in recent years has been rising trend, seriously affecting the quality of life and life safety of residents, and bring huge economic burden. This disease can be caused by a variety of reasons, such as genetic factors. Environmental factors are concerned about the key role of islet beta cell apoptosis to the development of type 2 diabetes in the study of the etiology of type 2 diabetes. More and more studies have confirmed that high glucose, high fat, high concentration of free fatty acids and many inflammatory factors can aggravate the oxidative stress reaction of islet beta cells, and then lead to islet beta cells. Apoptosis, inducing or aggravating the disease and course of type 2 diabetes. With the in-depth study of this cause of diabetes, many related mechanisms are being studied, such as GLP-1 analogues (such as lyalalu, iserin) and DPP-IW inhibitors (such as Shah Glenn Dean), and many studies have shown that these drugs can inhibit islet beta cells The apoptosis and the promotion of GLP-1 activity are beneficial to the improvement of type 2 diabetes. However, these drugs may not be included in medical insurance coverage, expensive, inconvenient to carry and preserve. Many Chinese scholars believe that spleen deficiency stomach heat, phlegm and stasis are interconnected, gastrointestinal disorders are important pathogenesis for the development of glycuria. Improving the function of gastrointestinal tract, invigorating the spleen and clearing heat, removing stasis and eliminating phlegm, and the idea of "stomach bowel islet axis" and Banxia Xiexin Decoction in the treatment of diabetes from the harmonization of stomach and intestines. Therefore, it can be used for the prevention and treatment of diabetes and its related diseases. Banxia Xiexin soup is a classic recipe for harmonization of gastrointestinal methods. Modern clinical and experimental studies show that Pinellia Xiexin Decoction compound and its single flavor medicine can improve sugar, lipid metabolism, reduce blood sugar, improve the secretion of gastrointestinal hormones, improve the body's antioxidant capacity and immune function, inhibit the apoptosis of islet cells, and use it for the treatment of diabetes and related diseases. However, the mechanism of Banxia Xiexin soup is not clear and needs to be verified. Second parts of experimental research items are still needed. The RIN-m5F islet cells were cultured in vitro, the RTN-m5F cell apoptosis model was established and drug intervention was established. The effect and mechanism of Banxia Xiexin Decoction on the apoptosis of RIN-m5F cells were confirmed by CCK8, flow cytometry and Western blot. The effectiveness and scientificity of Banxia Xiexin Decoction in the treatment of type 2 diabetes was proved at the molecular level. The prevention and control of type 2 diabetes provides a new idea and a technical platform for deep study. Method: 33 male SD rats were randomly divided into two groups, and collected blood to collect serum containing medicine (normal serum, serum containing Banxia Xiexin Decoction, serum containing relagin drug), and H_2O_2 treatment of RIN-m5F cells After the optimum concentration of the oxidative stress cell apoptosis model, the optimal concentration of Banxia Xiexin Decoction was randomly divided into two groups: blank control group, model group, group +H_2O_2 of Banxia Xiexin Decoction and +H202 group of liararu. Flow cytometry was used to detect the apoptosis rate of each group, radioimmunoassay was used to determine islet content, and Western blot method was used to determine Bcl-2, Bax, Ca. The protein expression of spase-3, PI3K, AKT, p-AKT. The data were managed with Microsoft EXCEL 2010, and the average soil standard deviation (x one + s) was expressed. The SPSS20.0 statistical software was used. The data of each group were tested by the normal test and the variance homogeneity test. The statistical analysis was carried out by the single factor variance analysis method, and the LSD-t test was used among the groups. The results of P0.05 were statistically significant. Results: (1) the optimum model concentration of RIN-m5F cell apoptosis model.H_2O_2 was established. (2) the optimum concentration of Banxia Xiexin Decoction serum was established. The protective effect of serum containing Banxia Xiexin soup with different concentrations on RIN-m5F cells was detected by CCK8, and the Pinellia ternata was found. The best concentration of the serum of Xiexin Decoction was 10%, the difference was statistically significant (P0.05); (3) it was confirmed that the serum of Banxia Xiexin Decoction could inhibit the apoptosis of RIN-m5F cells. The results of flow cytometry showed that compared with the model group, the cell viability was significantly enhanced and the apoptosis rate decreased significantly (P0.05); (4) (4) The results of radioimmunoassay showed that the insulin secretion was significantly increased after the treatment of the model group compared with the model group, and the difference was statistically significant (P0.05). (5) it was proved that the serum of Banxia Xiexin Decoction can reduce the Bax, Caspase-3 table. The expression of.Westernblot detected by the expression of.Westernblot in p-AKT showed that the expression of Bax, Caspase-3 was significantly decreased, the expression of Bcl-2 increased obviously, the expression of p-AKT increased obviously, and the difference of p-AKT was significantly increased, and the difference was statistically significant (P0.05). Conclusion: Banxia Xiexin Decoction can improve RIN-m5F fine. Apoptosis promotes insulin secretion, which may be mediated by activating the PI3K/AKT pathway, thereby regulating the expression of downstream factors.
【学位授予单位】：中国中医科学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R259

【参考文献】