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
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摘要
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-124-3 p 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-124-3 p in each groups of INS-1 cells was measured. The potential targets of miR-124-3 p were predicted by bioinformatics methods. The expression of miR-124-3 p's target gene PLC-B1 and IP3 R-1 were measured. miR-124-3 p target gene was verified by double fluorescent reporter gene assay. The expression of PLC-B1 and IP3 R-1 in ISD-INS-1 cells transfected with miR-124-3 p mimic and miR-124-3 p inhibitor was measured by RT-PCR and Westernblot. 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), YD-hi group BIS and GSIS were significantly higher than model group(P<0.05); The expression of miR-124-3 p in model group was lower than control group(P<0.05). Bioinformatics analysis results showed that PLC-B1 was the potential target gene of miR-124-3 p, the relative expression of PLC-B1 and IP3 R-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 miRNA transfection showed that compared with the control group, the expression of PLC-B1 and IP3 R-1 were significantly decreased after transfection of miR-124-3 p mimics(P<0.05). The expression of PLC-B1 and IP3 R-1 did not change after transfected with miR-124-3 p inhibitor(P>0.05). YD could reduce the expression of PLC-B1 and IP3 R-1 mRNA and protein in ISD-INS-1 cells transfected with miR-124-3 p mimics(P<0.05). Conclusion: Yiqi Huazhuo Decoction(YD) can promote the insulin secretion of ISD-INS-1 cells. Overexpression of miR-124-3 p 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-124-3 p, increasing the expression of PLC-B1, decreasing the inhibition of PLC-B1/IP3 R-1 pathway in ISD-INS-1 cells.
Objective: The aim of this study was to investigate the mechanism of Yiqi Huazhuo Decoction regulating miR-124-3 p 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-124-3 p in each groups of INS-1 cells was measured. The potential targets of miR-124-3 p were predicted by bioinformatics methods. The expression of miR-124-3 p's target gene PLC-B1 and IP3 R-1 were measured. miR-124-3 p target gene was verified by double fluorescent reporter gene assay. The expression of PLC-B1 and IP3 R-1 in ISD-INS-1 cells transfected with miR-124-3 p mimic and miR-124-3 p inhibitor was measured by RT-PCR and Westernblot. 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), YD-hi group BIS and GSIS were significantly higher than model group(P<0.05); The expression of miR-124-3 p in model group was lower than control group(P<0.05). Bioinformatics analysis results showed that PLC-B1 was the potential target gene of miR-124-3 p, the relative expression of PLC-B1 and IP3 R-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 miRNA transfection showed that compared with the control group, the expression of PLC-B1 and IP3 R-1 were significantly decreased after transfection of miR-124-3 p mimics(P<0.05). The expression of PLC-B1 and IP3 R-1 did not change after transfected with miR-124-3 p inhibitor(P>0.05). YD could reduce the expression of PLC-B1 and IP3 R-1 mRNA and protein in ISD-INS-1 cells transfected with miR-124-3 p mimics(P<0.05). Conclusion: Yiqi Huazhuo Decoction(YD) can promote the insulin secretion of ISD-INS-1 cells. Overexpression of miR-124-3 p 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-124-3 p, increasing the expression of PLC-B1, decreasing the inhibition of PLC-B1/IP3 R-1 pathway in ISD-INS-1 cells.
引文
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[20] 谷英敏,柴可夫.糖耐量低减治疗的中医组药举隅[J].中华中医药杂志,2016,31(4):1161-1164.
[2] 王晖,周建扬,陈霞波,等.降浊合剂治疗气虚痰浊型2型糖尿病66例临床研究[J].中医杂志,2007,48(9):803-805.
[3] 陈霞波,龚文波,张业,等.降浊合剂治疗糖尿病前期气虚痰浊证临床研究[J].中华中医药学刊,2013,31(6):1385-1387.
[4] 龚文波,陈霞波,翁思颖.益气化浊法与养阴清热法对GK大鼠糖耐量及胰岛素抵抗的影响[J].中华中医药杂志,2013,28(12):3660-3662.
[5] 龚文波,陈霞波,周开,等.益气化浊法与养阴清热法对GK大鼠胰岛素分泌及血清GLP-1的影响[J].中华中医药学刊,2013,31(10):2249-2251.
[6] 刘丽娟,合湫,苏恒,等.细胞外调节蛋白激酶1/2在小鼠胰岛瘤βTC-6细胞胰岛素分泌功能中的作用探讨[J].中国糖尿病杂志,2015,23(2):165-168.
[7] Ebert MS,Sharp PA.Roles for microRNAs in conferring robustness to biological processes[J]. Cell,2012,149(3):515-524.
[8] Rossbach O, Bindereif A.Eukaryotic non-coding RNAs: new targets for diagnostics and therapeutics? [J].Pharmazie,2016,71(1):3-7.
[9] Guay C,Regazzi R.Role of islet microRNAs in diabetes:which model for which question?[J]. Diabetologia,2015,58(3):456-463.
[10] Fuchsberger C, Flannick J, Teslovich TM, et al. The genetic architecture of type 2 diabetes[J]. Nature, 2016, 536: 41-47.
[11] Fu X, Dong B, Tian Y, et al. MicroRNA-26a regulates insulin sensitivity and metabolism of glucose and lipids[J].J Clin Invest, 2015,125:2497-2509.
[12] 曹海明,武哲丽.从系统生物学探讨microRNA在肝癌证候中的研究[J].中华中医药学刊,2014,32(3):486-488.
[13] 徐丽,贾连群,张哲,等.基于MicroRNAs探讨冠心病痰浊血瘀证候及从脾论治冠心病的科学内涵[J].中华中医药学刊,2017,35(9):2249-2252.
[14] 罗蔚,郑景辉,陈建军,等.急性心肌缺血血瘀证大鼠模型microRNAs差异表达的研究[J].中华中医药学刊,2018,36(5):1142-1146.
[15] 杨泽民,洪敏,陈滢宇,等.2型糖尿病脾虚证患者血清microRNA表达谱和生物信息学分析[J].中华中医药杂志,2017,32(8):3677-3683.
[16] Thore S, Dyachok O, Gylfe E,et al. Feedback activation of phospholipase C via intracellular mobilization and store-operated influx of Ca2+ in insulin-secreting β-cells[J].J Cell Sci,2005,118(19): 4463-4471.
[17] Zapata-Martínez J, Medina MF, Gramajo-Bühler MC,et al. Participation of PLA2 and PLC in DhL-induced activation of Rhinella arenarum oocytes[J].Zygote,2016,24(4):495-501.
[18] Lovis P,Gattesco S,Regazzi R.Regulation of the expression of components of the exocytotic machinery of insulin-secreting cells by micro RNAs[J]. Biol Chem, 2008,389(3):305-312.
[19] 柴可夫,张曾亮.糖尿病脾胰相关论[J].浙江中医药大学学报,2007(6):678-679.
[20] 谷英敏,柴可夫.糖耐量低减治疗的中医组药举隅[J].中华中医药杂志,2016,31(4):1161-1164.