红车轴草总黄酮通过调控miR-99a-3p/CD36表达对高糖诱导的大鼠胰岛β细胞的保护作用

Protective effects of red clover flavonoids on rat pancreaticβcells induced by high glucose by regulating the expression of miR-99a-3p/CD36

目的研究红车轴草总黄酮对高糖诱导的大鼠胰岛β细胞损伤的影响,及其潜在的分子机制。方法本实验分为对照组,模型组,低、中、高剂量实验组,miR-NC组,miR-99a-3p组,anti-miR-NC,anti-miR-99a-3p组,模型+miR-NC组,模型+miR-99a-3p组,模型+si-NC组,模型+si-CD36组,高剂量实验+anti-miR-NC组和高剂量实验+anti-miR-99a-3p组。对照组给予5.5 mmol·L^(-1)葡萄糖处置;模型组给予25 mmol·L^(-1)葡萄糖处置;低、中、高剂量实验组分别给予12.5,25.0和50.0 mg·L^(-1)红车轴草总黄酮和25 mmol·L^(-1)葡萄糖处置;miR-NC组、miR-99a-3p组、anti-miR-NC组、anti-miR-99a-3p组分别转染miR-NC、miR-99a-3p、anti-miR-NC、anti-miR-99a-3p;模型+miR-NC组、模型+miR-99a-3p组、模型+si-NC组、模型+si-CD36组均给予25 mmol·L^(-1)葡萄糖处理,并分别转染miR-NC、miR-99a-3p、si-NC、si-CD36;高剂量实验+anti-miR-NC组、高剂量实验+anti-miR-99a-3p组均给予50.0 mg·L^(-1)红车轴草总黄酮+25 mmol·L^(-1)葡萄糖,并分别转染anti-miR-NC或anti-miR-99a-3p。用流式细胞术测定细胞凋亡率,用定量实时聚合酶链反应检测细胞中miR-99a-3p和CD36 mRNA的表达水平。结果模型组INS-1细胞中CD36 mRNA(2.74±0.27 vs 1.01±0.08)和细胞凋亡率[(27.63±2.71)%vs(5.69±0.58)%]均较对照组显著升高,miR-99a-3p(0.38±0.03 vs 1.00±0.09)较对照组显著降低,差异均有统计学意义(均P<0.05);与模型组比较,低、中、高剂量实验组的上述结果相反。模型+miR-99a-3p组的细胞凋亡率[(12.48±1.19)%vs(26.84±2.41)%]较模型+miR-NC组显著降低,miR-99a-3p表达量(0.79±0.07 vs 0.34±0.03)较模型+miR-NC组显著升高,差异均有统计学意义(均P<0.05)。模型+si-CD36组INS-1细胞中CD36 mRNA(1.35±0.12 vs 2.81±0.26)及细胞凋亡率[(13.54±1.32)%vs(25.87±2.52)%]均较模型+si-NC组显著降低,差异均有统计学意义(均P<0.05)。高剂量实验+anti-miR-99a-3p组INS-1细胞中miR-99a-3p(0.43±0.14 vs 0.88±0.06)较高剂量实验+anti-miR-NC组显著降低,CD36 mRNA(2.51±0.22 vs 1.41±0.13)及细胞凋亡率[(20.45±2.03)%vs(10.56±1.02)%]均较高剂量实验+anti-miR-NC组显著升高,差异均有统计学意义(均P<0.05)。结论红车轴草总黄酮可能通过调控miR-99a-3p/CD36以减轻高糖对大鼠胰岛β细胞INS-1的损伤,进而抑制细胞的凋亡。

Objective To investigate the effects of red clover flavonoids on the injury of rat pancreaticβcells induced by high glucose and its potential molecular mechanism.Methods This experiment was divided into control group,model group,experimental-L,-M,-H groups,miR-NC group,miR-99a-3p group,anti-miR-NC,anti-miR-99a-3p group,model+miR-NC group,model+miR-99a-3p group,model+si-NC group,model+si-CD36 group,experimental-H+anti-miR-NC group and experimental-H+anti-miR-99a-3p group.The pancreatic isletβcells in the control group were treated with 5.5 mmol·L^(-1)glucose with INS-1,the model group INS-1 cells were treated with 25 mmol·L^(-1)glucose,and the experimental-L,-M,-H groups INS-1 cells were treated with 12.5,25.0 and 50.0 mg·L^(-1)total flavonoids of red clover and 25 mmol·L^(-1)glucose treatment,miR-NC group,miR-99a-3p group,anti-miR-NC group,anti-miR-99a-3p Group INS-1 cells were treated with transfected miR-NC,miR-99a-3p,anti-miR-NC,anti-miR-99a-3p,model+miR-NC group,model+miR-99a-3p group,model+si-NC group,model+si-CD36 group INS-1 cells were treated with 25 mmol·L^(-1)glucose and transfected with miR-NC,miR-99a-3p,si-NC,siCD36,experimental-H+anti-miR-NC group,experimental-H+anti-miR-99a-3p group INS-1 cells were given 50.0 mg·L^(-1)total red clover flavonoids,25 mmol·L^(-1)glucose and transfected with anti-miR-NC or antimiR-99a-3p treatment.Flow cytometry was used to determine the rate of apoptosis,and quantitative real-time polymerase chain reaction was used to detect the levels of miR-99a-3p and CD36 mRNA in INS-1 cells.Results The levels of CD36 mRNA(2.74±0.27 vs 1.01±0.08)and apoptosis rate[(27.63±2.71)%vs(5.69±0.58)%]of INS-1 cells in the model group were significantly increased compared with the control group,while miR-99a-3p(0.38±0.03 vs 1.00±0.09)was significantly lower than that in control group(all P<0.05).Compared with the model group,the results of the experimental-L,-M,-H groups were opposite.Apoptosis rate[(12.48±1.19)%vs(26.84±2.41)%]in the model+miR-99a-3p group were significantly lower than that in the model+miR-NC group,and miR-99a-3p(0.79±0.07 vs 0.34±0.03)in the model+miR-99a-3p group was significantly increased compared with the model+miR-NC group(all P<0.05).The levels of CD36mRNA(1.35±0.12 vs 2.81±0.26)and apoptosis rate[(13.54±1.32)%vs(25.87±2.52)%]in INS-1 cells in the model+si-CD36 group were significantly lower than those in the model+si-NC group(all P<0.05).miR-99a-3p(0.43±0.14 vs 0.88±0.06)in the INS-1 cells of the experimental-H+anti-miR-99a-3p group was significantly lower than those of experimental-H+anti-miR-NC group,and the levels of CD36 mRNA(2.51±0.22 vs 1.41±0.13)and the apoptosis rate[(20.45±2.03)%vs(10.56±1.02)%]of cells in the experimental-H+anti-miR-99a-3p group were significantly increased compared with the experimental-H+anti-miR-NC group(P<0.05).Conclusion Red clover flavonoids may alleviate the injury of rat pancreaticβcells INS-1 induced by high glucose and inhibit the apoptosis by regulating miR-99a-3p/CD36.