甘草酸通过下调缺氧诱导因子-1α抑制小鼠肾小球系膜细胞炎症因子的表达

王珍; 曹雪; 侯绍章; 李媛

doi:10.3969/j.issn.2095-5227.2023.06.018

甘草酸通过下调缺氧诱导因子-1α抑制小鼠肾小球系膜细胞炎症因子的表达

Glycyrrhizin inhibited expression of inflammatory factors in mouse mesangial cells by down-regulating HIF-1α

摘要

摘要:
背景甘草酸(glycyrrhizic acid，GA)在糖尿病肾病保护领域取得了很大进展。缺氧诱导因子-1α(hypoxia-inducible factor-1α，HIF-1α)与糖尿病肾病的发生密切相关，甘草酸与HIF-1α在糖尿病肾病中的作用机制仍有待探索。
目的探讨甘草酸是否可通过影响HIF-1α的表达而抑制高糖条件下肾小球系膜细胞(SV40 MES13)炎症因子水平，为糖尿病肾病的治疗提供实验依据。
方法培养小鼠肾小球系膜细胞(SV40 MES13)，分为正常组(NG 5.6 mmol/L)、高糖组(HG 30 mmol/L)、高糖＋甘草酸组(HG 30 mmol/L＋GA 200 µmol/L)。Western blot方法检测各分组细胞HIF-1α、血管内皮生长因子(vascular endothelial growth factor，VEGF)蛋白因子的表达水平，免疫荧光方法检测各分组HIF-1α、VEGF在细胞内的表达。将细胞按照正常组(NG 5.6 mmol/L)、高糖组(HG 30 mmol/L)、高糖＋甘草酸组(HG 30 mmol/L＋GA 200 µmol/L)、高糖 + KC7F2组(HG 30 mmol/L＋KC7F2 7.5 µmol/L)、高糖 + 甘草酸 + KC7F2组(HG 30 mmol/L＋GA 200 µmol/L + KC7F2 7.5 µmol/L)、高糖 + DMOG组(HG 30 mmol/L＋DMOG 25 µmol/L)、高糖 + 甘草酸 + DMOG组(HG 30 mmol/L＋GA 200 µmol/L + DMOG 25 µmol/L)进行培养(48 h)，CCK-8检测细胞增殖水平，Western blot和酶联免疫吸附实验(ELISA)检测不同分组细胞白细胞介素(interleukin，IL)-1β、TNF-α、IL-6、IL-8的表达。
结果 Western blot、免疫荧光方法实验结果表明，甘草酸能够抑制高糖条件下小鼠肾小球系膜细胞(SV40 MES13) HIF-1α、VEGF蛋白因子的表达。CCK-8 实验表明，HIF-1α 的上调促进了小鼠肾小球细胞的增殖，甘草酸能够进一步抑制激活剂DMOG所诱导的细胞增殖水平，并且也能够协同抑制剂KC7F2进一步抑制肾小球系膜细胞的增殖。Western blot实验结果表明，甘草酸能显著抑制DMOG诱导的HIF-1α、VEGF、IL-1β、TNF-α、IL-6、IL-8蛋白因子的高表达(P＜0.05)，也能够联合KC7F2抑制HIF-1α、VEGF、IL-1β、TNF-α、IL-6、IL-8的表达(P＜0.05)，酶联免疫吸附实验(ELISA)得到同样的结果。
结论甘草酸通过下调HIF-1α的表达而抑制高糖条件下肾小球系膜细胞(SV40 MES13)炎症因子的表达。

Abstract:
Background Research on the protective effect of glycyrrhizic acid for diabetic nephropathy has achieved great progress. HIF-1α is closely related to the occurrence of diabetic nephropathy, but the mechanism of glycyrrhizic acid and HIF-1α in diabetic nephropathy remains to be explored.
Objective To investigate the effect of glycyrrhizic acid (GA) on inhibiting the inflammatory factors in glomerular mesangial cells SV40 MES13 under high glucose condition by affecting the expression of HIF-1α, so as to provide theoretical reference for the treatment of diabetic nephropathy.
Methods Mouse mesangial cells (SV40 MES13) were cultured and divided into normal group (NG 5.6mmol/L), high glucose group (HG 30mmol/L), and high glucose + glycyrrhizic acid group (HG 30mmol/L + GA 200µmol/L). Western blot was used to detect the expression levels of HIF-1α and VEGF protein factors in cells of different groups, and immunofluorescence was used to detect the expression levels of HIF-1α and VEGF in cells of different groups. The cells were divided into normal group (NG 5.6mmol/L), high glucose group (HG 30mmol/L), high glucose + glycyrrhiza group (HG 30mmol/L + GA 200µmol/L), high glucose + KC7F2 group (HG 30mmol/L + KC7F2 7.5µmol/L), high glucose + glycyrrhizic acid + KC7F2 group (HG 30mmol/L + GA 200µmol/L + KC7F2 7.5µmol/L), high glucose + DMOG group (HG 30mmol/L + DMOG 25µmol/L) and high glucose + glycyrrhizic acid + DMOG group (HG 30mmol/L + GA 200µmol/L + DMOG 25µmol/L) and cultured for 48h. Cell proliferation levels were detected by CCK-8. Western blot and enzyma-linked immunosorbent assay (ELISA) were used to analyze the expression levels of IL-1β, TNF-α, IL-6 and IL-8 in different groups.
Results The results suggested that glycyrrhizic acid could inhibit the expression of HIF-1α and VEGF protein factor in mouse mesangial cells (SV40 MES13) under the condition of high glucose. CCK8 experiment confirmed that HIF-1α up-regulation promoted the proliferation of mouse glomerular cells, glycyrrhizic acid could further inhibit the cell proliferation level induced by the activator DMOG, and could also cooperate with the inhibitor KC7F2 to further inhibit the proliferation level of glomerular mesangial cells. Western blot results showed that glycyrrhizic acid could significantly inhibit the high expression of HIF-1α, VEGF, IL-1β, TNF-α, IL-6, IL-8 protein factor induced by DMOG (P < 0.05), and could also inhibit the protein factor expression HIF-1α, VEGF, IL-1β, TNF-α, IL-6, IL-8 in combination with KC7F2 (P < 0.05). Enzyme-linked immunosorbent assay (ELISA) also showed the same results.
Conclusion GA can inhibit the expression of inflammatory factors in SV40 MES13 cells under high glucose condition by inhibiting expression of HIF-1α.

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