HMS5552对糖尿病大鼠胰岛β细胞损伤的保护作用研究

Protective effects of HMS5552 on pancreaticβcell injury in diabetic rats

目的探究HMS5552对糖尿病大鼠胰岛β细胞损伤的保护作用及其机制研究。方法40只SD大鼠用高糖高脂饲料喂养8周联合腹腔注射链脲佐菌素(STZ)诱导2型糖尿病大鼠模型。将造模成功的大鼠分为模型组(灌胃给予0.9%NaCl)和实验组(灌胃给予30 mg·kg^(-1)HMS5552),另选20只正常大鼠为正常组(灌胃给予0.9%NaCl)。用苏木精-伊红(HE)染色法检测胰岛病理变化。将INS-1细胞分为空白组(正常培养)、模型组(高糖高脂培养液)和低、中、高剂量实验组(高糖高脂培养液+0.3、3.0、30.0μmol·L^(-1)HMS5552)。用细胞计数试剂盒-8(CCK-8)法检测INS-1细胞活力,用流式细胞术检测INS-1细胞凋亡率,用实时荧光定量聚合酶链反应法检测B淋巴细胞瘤2(Bcl-2)mRNA表达水平,用蛋白质印迹法检测脂酰肌醇3-激酶/蛋白激酶B(PI3K/AKT)通路及凋亡蛋白的表达水平。结果HE染色结果表明:实验组大鼠的胰腺病理损伤较模型组明显减轻。空白组、模型组和低、中、高剂量实验组的细胞活力分别为(99.67±2.40)%、(48.18±4.18)%、(61.74±4.78)%、(70.74±6.36)%和(78.30±4.68)%,细胞凋亡率分别为(10.40±4.66)%、(48.60±3.27)%、(35.90±4.45)%、(28.07±5.35)%和(18.30±3.04)%,Bcl-2 mRNA相对表达水平分别为1.00±0.03、0.32±0.04、0.59±0.05、0.62±0.04和0.72±0.12,磷酸化PI3K(p-PI3K)/PI3K比值分别为1.00±0.00、0.34±0.05、0.46±0.02、0.64±0.01和0.81±0.01,磷酸化AKT(p-AKT)/AKT比值分别为1.00±0.00、0.28±0.12、0.63±0.02、0.74±0.06和0.87±0.09。模型组的上述指标与空白组比较,低、中、高剂量实验组的上述指标与模型组比较,在统计学上差异均有统计学意义(P<0.05,P<0.01)。结论HMS5552可改善糖尿病大鼠糖代谢,并可能通过调控PI3K/AKT信号通路的表达来保护胰岛β细胞。

Objective To investigate the protective effect of HMS5552 on pancreatic isletβ-cell injury in diabetic rats and study the mechanism.Methods A total of 40 SD rats were fed with high-sugar and high-fat diet for 8 weeks combined with intraperitoneal injection of streptozotocin(STZ)to induce type 2 diabetic rat models.The successful model rats were divided into model group(0.9%NaCl by gavage)and experimental group(30 mg·kg^(-1)HMS5552 by gavage).The other 20 normal rats served as the normal group(0.9%NaCl was given by gavage).Hematoxylin-eosin(HE)staining was used to detect the pathological changes of pancreatic islets.The INS-1 cells were divided into blank group(normal cultured),model group(high-sugar and high-fat culture medium)and experimental-L,-M,-H groups(high-sugar and high-fat culture medium+0.3,3.0 and 30.0μmol·L^(-1)HMS5552).The viability of INS-1 cells was detected by cell counting kit-8(CCK-8)method,the apoptosis of INS-1 cells was detected by Annexin V/PI method,the mRNA expression level of B cell lymphocytoma 2(Bcl-2)was detected by real-time fluorescence quantitative polymerase chain reaction(RT-qPCR)method;the expression level of phosphoinositide 3-kinase/protein kinase B(PI3K/AKT)pathway and apoptosis protein was detected by western blotting.Results HE staining showed that the pathological damage of pancreas in the experimental group rats was significantly less than that in the model group.The cell viability of the blank group,model group and experimental-L,-M,-H groups were(99.67±2.40)%,(48.18±4.18)%,(61.74±4.78)%,(70.74±6.36)%and(78.30±4.68)%,respectively;apoptosis rates were(10.40±4.66)%,(48.60±3.27)%,(35.90±4.45)%,(28.07±5.35)%and(18.30±3.04)%,respectively;the relative expression levels of Bcl-2 mRNA were 1.00±0.03,0.32±0.04,0.59±0.05,0.62±0.04 and 0.72±0.12,respectively;phosphorylated PI3K(p-PI3K)/PI3K ratios were 1.00±0.00,0.34±0.05,0.46±0.02,0.64±0.01 and 0.81±0.01,respectively;phosphorylated AKT(p-AKT)/AKT ratios were 1.00±0.00,0.28±0.12,0.63±0.02,0.74±0.06 and 0.87±0.09,respectively.The above indexes in the model group compared with the blank group,the differences between the above indexes of the experimental-L,-M,-H groups and the model group were statistically significant(P<0.05,P<0.01).Conclusion HMS5552 can improve glucose metabolism in diabetic rats and protect pancreaticβ-cells by regulating the expression of PI3K/AKT signaling pathway.