益气化浊汤下调胰岛素分泌受损INS-1细胞miR-124-3p改善胰岛素分泌

Effects of Yiqi Huazhuo Decoction on Improvement of Insulin Secretion in ISD-INS-1 Cells by Down-regulating miR-124-3p

目的:研究益气化浊汤调控miR-124-3p改善胰岛素分泌受损INS-1细胞的作用机制。方法:四氧嘧啶诱导INS-1细胞建立胰岛素分泌受损(ISD)模型,分设model组、低剂量益气化浊汤干预组(YD-lo)、高剂量益气化浊汤组(YD-hi),正常INS-1细胞设为control组,测各组细胞胰岛素分泌水平及测miR-124-3p表达丰度;生物信息学法预测miR-124-3p潜在靶基因;测各组细胞miR-124-3p靶基因PLC-B1及其下游IP3R表达;双荧光报告基因实验行靶基因验证;测miR-124-3p mimic与miR-124-3p inhibitor转染后ISD-INS-1细胞PLC-B1及IP3R表达。结果:model组基础胰岛素分泌(BIS)、葡萄糖刺激后胰岛素分泌(GSIS)较control组均下降(P<0.05),YD-lo组BIS、GSIS较model组无差异(P>0.05),YD-hi组BIS、GSIS较model组均升高(P<0.05); miR-124-3p表达丰度,model组较control组下降(P<0.05),YD-lo组较model组无差异(P>0.05),YD-hi组较model组升高(P<0.05)。生物信息学法分析结果显示PLC-B1为miR-124-3p潜在靶基因;PLC-B1、IP3R-1 mRNA相对表达量,model组较control组均下降(P<0.05),YD-lo组较model组均无差异(P>0.05),YD-hi组较model组均升高(P<0.05);双荧光报告基因实验验证PLC-B1为miR-124-3p靶基因;相较control组,转染miR-124-3p mimics后,PLC-B1、IP3R的mRNA及蛋白表达量均明显降低(P<0.05),转染miR-124-3p inhibtor后,PLC-B1、IP3R的mRNA及蛋白表达量均无变化(P>0.05);益气化浊汤可减少转染miR-124-3p mimics后ISD-INS-1细胞PLC-B1、IP3R基因及蛋白表达量的降低(P<0.05)。结论:益气化浊汤能促进ISD-INS-1细胞胰岛素分泌;miR-124-3p过表达是INS-1细胞胰岛素分泌下降的机制之一;益气化浊汤可通过下调ISD-INS-1细胞miR-124-3p表达,增加PLC-B1表达,减少它对PLC-B1/IP3R通路的抑制,改善胰岛素分泌。

Objective: The aim of this study was to investigate the mechanism of Yiqi Huazhuo Decoction regulating miR - 124 -3p to improve insulin secretion of INS - 1 cells. Methods: ISD( insulin secretion dysfunction)- INS - 1 cells established by Alloxan were divided into model group, YD - lo group ( low does group by Yiqi Huazhuo Decoction), YD-hi group( high does group by Yiqihuazhuo Decoction), and normal INS-1 cells as control group. Insulin secretion level and expression of miR-124-3p in each groups of INS - 1 cells was measured. The potential targets of miR-124 - 3p were predicted by bioinformatics methods. The expression of miR-124-3p' s target gene PLC - Bl and IP3R - 1 were measured. miR - 124 - 3p target gene was verified by double fluorescent reporter gene assay. The expression of PLC - Bl and IP3R - 1 in ISD - INS -1 cells transfected with miR - 124 - 3p mimic and miR - 124 - 3p inhibitor was measured bv RT - PCR and Westembiot. Results: The levels of BIS and GSIS in model group were significantly lower than those in control group ( P <0. 05 ). There was no significant difference in BIS and GSIS between YD - lo group and model group( P >0. 05), YI)-hi group BIS and GSIS were significantly higher than model group ( P < 0. 05 );The expression of miR - 124 - 3p in model group was lower than control group ( P < 0. 05 ). Bioinformatics analysis results showed that PLC - Bl was the potential target gene of miR - 124 -3p, the relative expression of PLC - Bl and IP3R - 1 in model group ( P < 0. 05), YD-lo group had no difference compared with model group ( P > 0. 05 ), YD - hi group than model group ( P < 0. 05). The results of iniRNA transfection showed that compared with the control group, the expression of PLC-Bl and IP3R - 1 were significantly decreased after transfection of miR - 124 -3p mimics (P <0. 05). The expression of PLC - Bl and IP3R - 1 did not change after transfected with miR - 124-3p inhibitor ( P >0. 05). YD could reduce the expression of PLC-Bl and IP3R - 1 mRNA and protein in ISD - INS-1 cells transfected with miR-124 - 3p mimics ( P < 0. 05 ). Conclusion : Yiqi Huazhuo Decoction ( YD ) can promote the insulin secretion of ISD - INS-1 cells. Overexpression of miR - 124 - 3p is one of the mechanisms of insulin secretion decline in INS - 1 cells. YD may improve the insulin secretion by decreasing the expression of miR -124 - 3p, increasing the expression of PLC - Bl , decreasing the inhibition of PLC - B1/IP3R - 1 pathway in ISD - INS - 1 cells.