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随着我国人口老龄化的到来,心脑血管疾病已经成为我国城乡居民致死的首要因素,糖尿病作为冠状动脉疾病、高血压的独立危险因素,加重了冠心病、心力衰竭等心血管疾病的发病进程。其中,糖尿病心肌病(DCM)是糖尿病病人致死的主要原因之一。目前研究发现DCM主要病理生理机制包括心肌细胞凋亡、心肌纤维化、左心室功能障碍和代谢紊乱等[1]。但具体发病机制并未完全阐明。因此,探究DCM发病机制及病理过程,被认为是治疗DCM的关键因素。MAPKs信号通路是目前发现的最主要的生长信号调节蛋白之一,广泛存在于细胞质内, 对细胞的增殖、分化与凋亡过程起着重要的作用。既往研究[2]发现,MAPKs信号通路及其下游因子参与高糖诱导的心肌损伤中炎性反应、细胞凋亡等病理过程。
厄贝沙坦是一种血管紧张素Ⅱ受体抑制剂,被认为是改善心肌细胞结构及功能、降低心力衰竭远期死亡风险的临床常用药物。既往研究[3]也发现厄贝沙坦可以减轻糖尿病心肌细胞炎性反应等,达到保护心肌损伤的作用。但厄贝沙坦是否通过影响MAPKs信号通路发挥其抗糖尿病心肌损伤作用目前尚未明确。本研究拟通过复制大鼠糖尿病心肌损伤,运用Western blotting技术检测糖尿病心肌组织中MAPKs通路下游因子的蛋白表达,明确厄贝沙坦是否通过调控MAPKs通路改善糖尿病心肌损伤,为后期探讨糖尿病心肌损伤的分子机制提供理论依据。现作报道。
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HC组大鼠FBG、BW、H/B、LVWI与Con组差异均无统计学意义(P>0.05);与Con、HC组比较,各糖尿病组大鼠FBG均明显升高,BW均降低,H/B及LVWI均增高(P < 0.05~P < 0.01);与DM4W、DM8W组比较,DM8W+Ir组H/B及LVWI均降低(P < 0.05~P < 0.01)(见表 1)。
分组 FBG/(mmol/L) BW/(mg/g) (H/B)/(mg/g) LVWI/(mg/g) Con组 5.13±0.27 252.44±9.73 2.70±0.03 1.47±0.04 HC组 6.12±0.54 317.04±11.76 2.58±0.03 1.60±0.05 DM4W组 21.11±3.07**## 223.17±7.73*## 3.99±0.07*# 2.79±0.04*# DM8W组 23.48±3.45**## 219.05±9.81*## 4.48±0.06*# 2.90±0.04*# DM8W+Ir组 22.04±3.11**## 246.01±8.89*##▲■ 3.06±0.04*#▲■■ 2.01±0.04*#▲■ F 132.90 165.10 2 926.00 2 467.00 P < 0.01 < 0.01 < 0.01 < 0.01 MS组内 6.273 93.598 0.002 0.002 q检验:与Con组比较*P < 0.05,**P < 0.01;与HC组比较#P < 0.05,##P < 0.01;与DM4W组比较▲P < 0.05;与DM8W组比较■P < 0.05,■■P < 0.01 表 1 各组大鼠FBG、BW、H/B及LVWI比较(x±s; ni=10)
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Masson染色后,显微镜下观察各组大鼠心肌组织,心肌细胞呈红色,胶原纤维呈蓝绿色,Con组和HC组心肌细胞排列规则,胶原纤维呈条索状散在分布;DM4W组、DM8W组大鼠心肌组织纤维粗大,堆积成片状,分布不均,沉积增多,DM8W+Ir组大鼠心肌组织心肌形态则有明显改善(见图 1)。
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HC组大鼠IL-1β、IL-6、IL-1水平与Con组差异均无统计学意义(P>0.05);与Con组和HC组比较,各糖尿病组大鼠IL-1β、IL-6和IL-1水平均升高(P < 0.05);与DM4W组和DM8W组比较,DM8W+Ir组IL-1β、IL-6和IL-1水平均降低(P < 0.05)(见表 2)。
分组 IL-1β IL-6 IL-1 Con组 111.26±9.78 79.29±13.68 145.29± 9.26 HC组 103.68±10.32 85.32±10.03 139.87±13.02 DM4W组 166.30±13.57#* 149.95±9.02#* 221.53±8.77#* DM8W组 181.72±12.03#* 186.78±11.87#* 252.63±11.98#* DM8W+Ir组 140.04±11.22#*▲■ 117.52±10.73#*▲■ 198.04±9.57#*▲■ F 87.07 163.00 208.90 P P < 0.01 P < 0.01 P < 0.01 MS组内 131.381 125.027 113.457 q检验:与Con组比较*P < 0.05;与HC组比较#P < 0.05;与DM4W组比较▲P < 0.05;与DM8W组比较■P < 0.05 表 2 各组大鼠心肌细胞IL-1β、IL-6、IL-1水平比较(x±s; ni=10;pg/mL)
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HC组MKP-1、P38MAPK、P-ERK1/2蛋白表达与Con组差异均无统计学意义(P>0.05);与HC组比较,各糖尿病组大鼠P38MAPK、P-ERK1/2蛋白表达明显增加,MKP-1表达降低(P < 0.05);与DM8W组相比,DM8W+Ir组P38MAPK、P-ERK1/2蛋白表达均明显降低,而MKP-1表达增加(P < 0.05)(见图 2、表 3)。
分组 MKP-1 P38MAPK p-ERK1/2 Con组 0.63±0.14 0.52±0.33 0.49±0.34 HC组 0.61±0.07 0.50±0.52 0.501±0.46 DM4W组 0.52±0.24# 0.59±0.69# 0.55±0.69# DM8W组 0.49±0.06# 0.70±0.34# 0.63±0.54# DM8W+Ir组 0.57±0.14#*▲■ 0.60±0.74#*▲■ 0.52±0.38#*▲■ F 1.65 0.20 0.12 P >0.05 >0.05 >0.05 MS组内 0.021 0.304 0.248 q检验:与Con组比较*P < 0.05;与HC组比较#P < 0.05;与DM4W组比较▲P < 0.05;与DM8W组比较■P < 0.05 表 3 各组大鼠心肌组织MKP-1、P38MAPK、P-ERK1/2蛋白表达水平比较(x±s; ni=10)
厄贝沙坦对糖尿病大鼠心肌损伤中MAPKs信号通路及相关因子的影响
Effect of irbesartan on the MAPKs signaling pathway and related factors in myocardial injury of diabetic rats
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摘要:
目的观察厄贝沙坦在糖尿病大鼠心肌损伤中发挥的作用,并探讨其对丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPKs)信号通路及其相关因子表达影响。 方法雄性SD大鼠50只,随机分为糖尿病4周(DM4W)组、糖尿病8周(DM8W)组、糖尿病8周+厄贝沙坦(DM8W+Ir)组、对照(Con)组、高糖高胆固醇饮食(HC)组,各10只。复制2型糖尿病大鼠模型成功后,DM8W+Ir组给予厄贝沙坦溶液灌胃干预;饲喂第4周时,处死DM4W组大鼠并留取心脏组织标本;饲喂8周后,处死剩余组大鼠。比较各组大鼠空腹血糖、体质量、心体比和左心室质量指数;取大鼠离体心肌行Masson染色观察心肌细胞纤维化改变;ELISA法检测心肌细胞内炎症细胞因子白细胞介素(interleukin,IL)-1β、IL-6、IL-1表达水平;Western blotting法检测心肌组织丝裂原活化蛋白激酶磷酸酶-1(mitogen-activated protein kinase phosphatase-1,MKP-1)、P38丝裂原激活蛋白激酶(P38 mitogen-activated protein kinase,P38MAPK)、细胞外信号调节激酶(extracellular signal-related kinases,P-ERK1/2)蛋白表达水平。 结果HC组大鼠空腹血糖、体质量、心体比、左心室质量指数与Con组差异均无统计学意义(P>0.05),IL-1β、IL-6、IL-1表达水平和MKP-1、P38MAPK、P-ERK1/2蛋白表达与Con组差异亦均无统计学意义(P>0.05)。与Con组、HC组相比,各糖尿病组大鼠Masson染色心肌细胞明显纤维化改变;体质量减轻,心肌细胞内炎症细胞因子IL-1β、IL-6、IL-1表达水平升高,心肌组织MKP-1蛋白表达降低,P38MAPK、P-ERK1/2蛋白表达升高(P < 0.05~P < 0.01);与DM4W、DM8W组比较,DM8W+Ir组大鼠IL-1β、IL-6、IL-1表达水平降低,MKP-1蛋白表达升高,P38MAPK、P-ERK1/2表达蛋白降低(P < 0.05~P < 0.01)。 结论厄贝沙坦通过调控MAPKs信号通路发挥改善糖尿病心肌损伤的作用。 Abstract:ObjectiveTo observe the effects of irbesartan on myocardial injury in diabetic rats, and to explore the effect of irbsartan on MAPKs signaling pathway and related factors expression. MethodsFifty male SD rats were randomly divided into the four weeks of diabetes (DM4W) group, eight weeks of diabetes(DM8W) group, eight weeks of diabetes + irbesartan(DM8W+Ir) group, control(Con) group and high sugar and cholesterol diet(HC) group(10 rats in each group).After the model of type 2 diabetic rats were successfully established, the DM8W+Ir group was administered using irbesartan solution by gavage.At the fourth week of feeding, the DM4W group was sacrificed, and heart tissue specimens were collected.After 8 weeks of feeding, the remaining rats were sacrificed.The fasting blood glucose(FBG), body weight(BW), heart to body ratio(H/B) and left ventricular mass index(LVWI) were compared among five groups.The Masson staining was used to observe the changes of myocardial fibrosis in isolated rat myocardium.The levels of IL-1β, IL-6, IL-1 and IL-1 were detected using ELISA.Western blotting was used to detect the expression levels of mitogen-activated protein kinase-1(MKP-1), P38 mitogen-activated protein kinase(P38MAPK) and extracellular signal-related kinases(P-ERK1/2) in myocardium. ResultsThere was no statistical significance in the levels FBG, BW, H/B, LVWI, IL-1β, IL-6 and IL-1, MKP-1, and protein levels of P38MAPK, P-ERK1/2 between HC group and Con group(P>0.05).Compared with the Con group and HC group, the fibrotic changes of Masson staining myocardial cells were observed, the body weight lightened, the expression levels of inflammatory cytokines IL-1, IL-6 and IL-1 in myocardial cells increased, the protein expression levels of MKP-1 in myocardial tissues decreased, and the protein expression levels of P38MAPK and P-ERK1/2 increased in diabetic rats(P < 0.05 to P < 0.01).Compared with the DM4W and DM8W groups, the expression levels of IL-1 β, IL-6 and IL-1 decreased, the expression level of MKP-1 protein increased, and the expression levels of P38MAPK and P-ERK1/2 decreased in DM8W+Ir group(P < 0.05 to P < 0.01). ConclusionsIrbesartan can improve the diabetic myocardial injury by regulating the MAPKs signaling pathway. -
表 1 各组大鼠FBG、BW、H/B及LVWI比较(x±s; ni=10)
分组 FBG/(mmol/L) BW/(mg/g) (H/B)/(mg/g) LVWI/(mg/g) Con组 5.13±0.27 252.44±9.73 2.70±0.03 1.47±0.04 HC组 6.12±0.54 317.04±11.76 2.58±0.03 1.60±0.05 DM4W组 21.11±3.07**## 223.17±7.73*## 3.99±0.07*# 2.79±0.04*# DM8W组 23.48±3.45**## 219.05±9.81*## 4.48±0.06*# 2.90±0.04*# DM8W+Ir组 22.04±3.11**## 246.01±8.89*##▲■ 3.06±0.04*#▲■■ 2.01±0.04*#▲■ F 132.90 165.10 2 926.00 2 467.00 P < 0.01 < 0.01 < 0.01 < 0.01 MS组内 6.273 93.598 0.002 0.002 q检验:与Con组比较*P < 0.05,**P < 0.01;与HC组比较#P < 0.05,##P < 0.01;与DM4W组比较▲P < 0.05;与DM8W组比较■P < 0.05,■■P < 0.01 表 2 各组大鼠心肌细胞IL-1β、IL-6、IL-1水平比较(x±s; ni=10;pg/mL)
分组 IL-1β IL-6 IL-1 Con组 111.26±9.78 79.29±13.68 145.29± 9.26 HC组 103.68±10.32 85.32±10.03 139.87±13.02 DM4W组 166.30±13.57#* 149.95±9.02#* 221.53±8.77#* DM8W组 181.72±12.03#* 186.78±11.87#* 252.63±11.98#* DM8W+Ir组 140.04±11.22#*▲■ 117.52±10.73#*▲■ 198.04±9.57#*▲■ F 87.07 163.00 208.90 P P < 0.01 P < 0.01 P < 0.01 MS组内 131.381 125.027 113.457 q检验:与Con组比较*P < 0.05;与HC组比较#P < 0.05;与DM4W组比较▲P < 0.05;与DM8W组比较■P < 0.05 表 3 各组大鼠心肌组织MKP-1、P38MAPK、P-ERK1/2蛋白表达水平比较(x±s; ni=10)
分组 MKP-1 P38MAPK p-ERK1/2 Con组 0.63±0.14 0.52±0.33 0.49±0.34 HC组 0.61±0.07 0.50±0.52 0.501±0.46 DM4W组 0.52±0.24# 0.59±0.69# 0.55±0.69# DM8W组 0.49±0.06# 0.70±0.34# 0.63±0.54# DM8W+Ir组 0.57±0.14#*▲■ 0.60±0.74#*▲■ 0.52±0.38#*▲■ F 1.65 0.20 0.12 P >0.05 >0.05 >0.05 MS组内 0.021 0.304 0.248 q检验:与Con组比较*P < 0.05;与HC组比较#P < 0.05;与DM4W组比较▲P < 0.05;与DM8W组比较■P < 0.05 -
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