MAPK信号通路在糖尿病性心肌病炎性反应中的作用及机制

发布时间：2018-05-04 22:18

本文选题：H9c2 + 心肌肥厚　；参考：《山西医科大学》2017年硕士论文

【摘要】：目的:本课题通过对大鼠胚胎心肌细胞(H9c2)进行高糖高脂干预,建造糖尿病心肌病肥厚模型,通过检测MAPK信号蛋白明确其对糖尿病心肌病中炎性反应的影响。方法:实验分为三部分:(1)采用随机数字表法,将体外培养的H9c2心肌细胞随机分组,每组设6个平行孔:对照组、高糖高脂A(葡萄糖33mmol/L+棕榈酸钠250μmol/L)、高糖高脂B(葡萄糖33mmol/L+棕榈酸钠500μmol/L)、高糖高脂C(葡萄糖33mmol/L+棕榈酸钠1000μmol/L)不同时间(12h、24h、36h、48h)处理组。采用荧光定量PCR(RT-PCR)技术分别检测心肌肥大相关指标心房钠尿肽(ANP)、α-肌动蛋白(α-SKA)的m RNA表达;利用苏木精-伊红染色法(HE染色)对各组细胞进行染色,观察高糖高脂对H9c2心肌细胞肥大程度的影响;检测细胞培养液中乳酸脱氢酶(LDH)活性,观察高糖高脂对H9c2心肌细胞损伤程度的影响。(2)结合第一部分实验,采用随机数字表法,将体外培养H9c2心肌细胞分组,每组设立6个平行对照孔:对照组、高糖高脂组(葡萄糖33mmol/L+棕榈酸钠500μmol/L)处理24小时。利用蛋白免疫印迹(Western blot)技术检测p-ERK/T-ERK、p-JNK/T-JNK、p-p38/T-p38的活化程度,观察高糖高脂与MAPK家族信号活化关系。(3)结合前两部分实验,采用随机数字表法,将体外培养H9c2心肌细胞分组,每组设立6个平行对照孔,对照组、高糖高脂组、抑制剂组(SB203580+高糖高脂组)。采用流式细胞术检测各组细胞中活性氧(ROS)的含量;通过化学检测法检测各组一氧化氮(NO)的水平;ELISA法检测各组细胞3-硝基酪氨酸(3-NT)、白介素-6(IL-6)、白介素-1β(IL-1β)的分泌程度,观察高糖高脂对H9c2心肌细胞炎症通路的影响及MAPK家族与炎性反应之间的关系。结果:1.高糖高脂处理导致H9c2细胞呈时间和浓度依赖性肥大和损伤。与对照组相比,高糖高脂处理后,ANP表达随着浓度和时间的逐渐增加而升高,且在36h高糖高脂B处理组ANP的表达量达到高峰(P0.01);相比对照组而言,高糖高脂促进α-SKA表达,但α-SKA的表达在36h高糖高脂B处理组达到最高(P0.05),与ANP的结果一致;与对照组相比,H9c2细胞的表面积变化与高糖高脂呈时间浓度依赖性,此外在36h高糖高脂B处理组,细胞的表面积变化最显著(P0.01),但36h高糖高脂C处理组的细胞形态改变异常;同时H9c2细胞上清中LDH的活性与高糖高脂处理间呈浓度及时间依赖性(P0.05)。据此,采用高糖高脂B(葡萄糖33mmol/L+棕榈酸钠500μmol/L)干预细胞36h为后续实验的干预浓度和时间。2.高糖高脂处理后H9c2细胞MAPK信号途径显著活化。与对照组相比,高糖高脂组中ERK、JNK、p38磷酸化水平明显提高,其中p-ERK和p-JNK的水平分别增加了1.39倍和1.47倍(P0.05),而p-p38的活化水平增加了3.34倍(P0.01)。3.高糖高脂显著诱导炎症反应变化并可被p38 MAPK抑制剂抑制。与高糖高脂组相比,抑制剂组(SB203580+高糖高脂组)可降低ROS含量、NO水平、3-NT含量、IL-6和IL-1β的分泌量(P0.05)。结论:1.高糖高脂可增加ANP及α-SKA的表达并使H9c2细胞表面积显著增加,且可使细胞上清中的LDH水平升高,这提示高糖高脂可显著诱导H9c2细胞肥大并使其发生损伤。2.高糖高脂可以增加MAPK家族活化程度,其中p38尤为明显,提示在糖尿病心肌病的发生过程中伴随有炎性信号通路活化。3.p38抑制剂可抑制高糖高脂诱导的ROS、NO、3-NT、IL-6、IL-1β水平的提高,提示p38 MAPK是介导高糖高脂导致糖尿病心肌病病理过程发生发展炎性反应的重要因素。
[Abstract]:Objective: to construct a hyperglycemic and hyperlipidemic model of diabetic cardiomyocytes (H9c2) to construct a hyperlipidemic model of diabetic cardiomyopathy, and to determine the effect of MAPK signal protein on the inflammatory response in diabetic cardiomyopathy. Methods: the experiment was divided into three parts: (1) the random digital table was used, and the H9c2 cardiomyocytes were randomly cultured in vitro. There were 6 parallel holes in each group: the control group, high sugar and high fat A (glucose 33mmol/L+ sodium palmitate 250 mol/L), high sugar and high fat B (glucose 33mmol/L+ palmitate sodium 500 u mol/L), high sugar and high fat C (dextrose 33mmol/L+ palmitate sodium 1000 mol/L) at different time (12h, 24h, 36h,) treatment group. Muscle hypertrophy related indexes of M RNA expression of atrial natriuretic peptide (ANP) and alpha actin (alpha -SKA); the effects of high glucose and high fat on the degree of hypertrophy of H9c2 cardiomyocytes by hematoxylin eosin staining (HE staining), and the activity of lactate dehydrogenase (LDH) in cell culture fluid, and the damage of high fat and high fat to H9c2 myocardial cells were observed. (2) combined with the first part of the experiment, the H9c2 myocardial cells were grouped in vitro with 6 parallel control holes in each group: control group, high glucose and high fat group (glucose 33mmol/L+ sodium palmitate 500 mol/L) for 24 hours. P-ERK/T-ERK, p-JNK/T-JNK, P were detected by protein immunoblotting (Western blot). The activation degree of -p38/T-p38 was observed. (3) combined with the first two parts of the experiment, the H9c2 myocardial cells were grouped in the first two parts, and 6 parallel control holes were set up in each group, the control group, the high sugar and high fat group and the inhibitor group (SB203580+ high fat group). The flow cytometry was used to detect each group. The content of active oxygen (ROS) in cells was detected by chemical detection, and the level of nitric oxide (NO) was detected by chemical detection. The secretory degree of 3- nitrotyrosine (3-NT), interleukin -6 (IL-6), and interleukin -1 beta (IL-1 beta) was detected by ELISA, and the relationship between high glucose and high fat on the inflammatory pathway of H9c2 cardiac myocytes and the relationship between MAPK family and inflammatory reaction was observed. Results: 1. high glucose and high fat treatment resulted in time and concentration dependent hypertrophy and injury of H9c2 cells. Compared with the control group, the expression of ANP increased with the increase of concentration and time, and the expression of ANP in the high glucose and high fat B treatment group of 36h reached the peak (P0.01). Compared with the control group, high sugar and high fat promoted the alpha -SKA table. But the expression of alpha -SKA was highest in 36h high fat B treatment group (P0.05), consistent with the result of ANP. Compared with the control group, the surface area changes of H9c2 cells were time dependent on high glucose and high fat, and the surface area of the cells in the 36h high fat and high fat B treatment group was the most significant (P0.01), but the cell morphology of the 36h high sugar and high fat C treatment group At the same time, the activity of LDH in H9c2 cell supernatant was dependent on the concentration and time dependence between high glucose and high fat treatment (P0.05). Accordingly, the intervention concentration and time of high fat and high fat B (glucose 33mmol/L+ palmitate 500 u mol/L) were used as the intervention concentration and time of.2. high glucose and high fat treatment, and the H9c2 cell MAPK signaling pathway was significantly activated. Compared with the control group, the level of ERK, JNK and p38 phosphorylation in the high glucose and high fat group increased significantly, in which the levels of p-ERK and p-JNK increased by 1.39 and 1.47 times respectively (P0.05), while the activation level of p-p38 increased by 3.34 times (P0.01).3. high sugar and high fat significantly induced the changes of the inflammatory reaction and could be inhibited by p38 MAPK inhibitor. Compared with the high glucose and high fat group, the inhibitor was inhibited. Group (SB203580+ high glucose group) could reduce the content of ROS, NO, 3-NT, IL-6 and IL-1 beta secretion (P0.05). Conclusion: 1. high sugar and high fat can increase the expression of ANP and alpha -SKA and increase the surface area of H9c2 cells significantly, and can increase the LDH level in the cell supernatant, which suggests that high sugar and high fat can significantly induce H9c2 cells hypertrophy and make it hair. .2. high glucose and high fat can increase the degree of activation of MAPK family, especially p38, suggesting that the activation of.3.p38 inhibitors with inflammatory signaling pathway in the pathogenesis of diabetic cardiomyopathy can inhibit high glucose and high lipid induced ROS, NO, 3-NT, IL-6, and IL-1 beta levels, suggesting that p38 MAPK is the mediating high glucose and high fat to lead to diabetic myocardium Pathological processes play an important role in the development of inflammatory response.

【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R587.2;R542.2

【相似文献】