肾复康调控细胞衰老抑制D-Gal诱导的HK-2细胞纤维化作用及机制*

作者：黄 上1，胡 梁1，郑燕姣1，刘劲松1，龚雅琪2，黄 睿2，任佳乐2

单位：1.湖南省中西医结合医院/湖南省中医药研究院附属医院，湖南 长沙 410006； 2.湖南中医药大学，湖南 长沙 410208

引用：引用：黄上，胡梁，郑燕姣，刘劲松，龚雅琪，黄睿，任佳乐.肾复康调控细胞衰老抑制D-Gal诱导的HK-2细胞纤维化作用及机制[J].中医药导报,2025,31(4):42-47,52.

DOI：10.13862/j.cn43-1446/r.2025.04.007

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摘要：目的：探讨肾复康对D-半乳糖（D-Gal）诱导的HK-2细胞纤维化模型的干预作用及机制。方法：采用D-Gal处理构建HK-2细胞纤维化模型；采用噻唑蓝（MTT）检测肾复康对D-Gal培养条件下HK-2细胞活力的影响；将HK-2细胞分为对照组、D-Gal模型组、维生素E组（200 μg/mL）、肾复康低剂量组（200 μg/mL）和肾复康高剂量组（400 μg/mL），干预24 h。采用Western blotting法检测α-平滑肌肌动蛋白（α-SMA）、Ⅰ型胶原（Collagen Ⅰ）及Janus激酶2（JAK2）信号转导-转录活化蛋白3（STAT3）信号蛋白表达；免疫荧光检测核增殖抗原（Ki-67）表达；β-半乳糖苷酶染色检测细胞衰老情况；实时荧光定量PCR（RT-PCR）检测细胞白细胞介素-1β（IL-1β）mRNA、IL-6 mRNA及IL-8 mRNA表达水平；酶联免疫吸附试验（ELISA）检测细胞上清液中IL-1β、IL-6及IL-8水平。结果：D-Gal模型组细胞存活率低于对照组（P<0.01）；肾复康200、400 μg/mL组细胞存活率高于D-Gal模型组（P<0.01）。选用200 μg/mL、400 μg/mL分别作为低、高剂量。D-Gal模型组HK-2细胞α-SMA及Collagen Ⅰ蛋白相对表达量高于对照组（P<0.01）；维生素E组、肾复康低剂量组、肾复康高剂量组HK-2细胞α-SMA及Collagen Ⅰ蛋白相对表达量均低于D-Gal模型组（P<0.01）。D-Gal模型组细胞Ki-67蛋白荧光相对表达量低于对照组（P<0.01），β-半乳糖苷酶阳性率高于对照组（P<0.01）；维生素E组、肾复康低剂量组、肾复康高剂量组HK-2细胞Ki-67蛋白荧光相对表达量均高于模型组（P<0.05或P<0.01），β-半乳糖苷酶阳性率均低于D-Gal模型组（P<0.01）。D-Gal模型组HK-2细胞IL-1β mRNA、IL-6 mRNA及IL-8 mRNA相对表达量均高于对照组（P<0.01）；维生素E组、肾复康低剂量组、肾复康高剂量组HK-2细胞IL-1β mRNA、IL-6 mRNA及IL-8 mRNA相对表达量均低于D-Gal模型组（P<0.01）。D-Gal模型组HK-2细胞上清液中IL-1β、IL-6及IL-8水平均高于对照组（P<0.01）；维生素E组、肾复康低剂量组、肾复康高剂量组HK-2细胞上清液中IL-1β、IL-6及IL-8水平均低于D-Gal模型组（P<0.01）。D-Gal模型组HK-2细胞JAK2蛋白相对表达量及p-STAT3/ STAT3均高于对照组（P<0.01）；维生素E组、肾复康低剂量组、肾复康高剂量组HK-2细胞JAK2蛋白相对表达量及p-STAT3/ STAT3均低于D-Gal模型组（P<0.01）。结论：肾复康抑制D-Gal诱导的HK-2细胞纤维化的机制可能是调控细胞衰老。

关键词：肾纤维化；肾复康；细胞衰老；D-半乳糖；α-平滑肌肌动蛋白；β-半乳糖苷酶；Janus激酶2信号转导-转录活化蛋白3信号

Abstract：

Objective: To investigate the intervention effect and mechanisms of Shenfukang on D-Galactose (D-Gal)-induced fibrosis model in HK-2 cells. Methods: D-Gal was used to establish the fibrosis model in HK-2 cells. The MTT assay was employed to evaluate the influence of Shenfukang on the viability of HK-2 cells under D-Gal culture conditions. HK-2 cells were divided into control group, D-Gal model group, Vitamin E groups (200 μg/mL), Shenfukang low-dose group (200 μg/mL) and Shenfukang high-dose group (400 μg/mL) with the intervention time of 24 h. Western blotting was utilized to detect the expression of α-smooth muscle actin (α-SMA), Collagen I, Janus kinase 2 (JAK2), and signal transducers and activators of transcription 3 (STAT3). Immunofluorescence was conducted to examine Ki-67 expression. β-galactosidase staining was performed to assess cell senescence. Real-time quantitative PCR (RT-PCR) was used to measure the expression levels of interleukin-1β (IL-1β) mRNA, IL-6 mRNA and IL-8 mRNA in cells. Enzyme-linked immunosorbent assay (ELISA) was employed to measure the levels of IL-1β, IL-6 and IL-8 in cell supernatants. Results: The D-Gal model group showed lower cell viability than control group (P<0.01). The cell viability in the Shenfukang 200 and 400 μg/mL groups was higher than the D-Gal model group (P<0.01). Then doses of 200 μg/mL and 400 μg/mL were chosen as the Shenfukang low and high doses, respectively. The relative expression levels of α-SMA and Collagen I proteins in the D-Gal model group HK-2 cells were higher than the control group (P<0.01). The vitamin E group, Shenfukang low-dose group and Shenfukang high-dose group showed lower α-SMA and Collagen I protein relative expression levels than D-Gal model group (P<0.01). The D-Gal model group showed lower relative fluorescent expression level of Ki-67 protein than control group (P<0.01), while higher β-galactosidase positivity rate than control group (P<0.01). The vitamin E group, Shenfukang low-dose group and Shenfukang high-dose group showed higher relative fluorescent expression levels of Ki-67 protein than D-Gal model group (P<0.05 or P<0.01), while lower β-galactosidase positivity rate than D-Gal model group (P<0.01). The D-Gal model group showed higher relative expression levels of IL-1β mRNA, IL-6 mRNA and IL-8 mRNA than control group (P<0.01). The vitamin E group, Shenfukang low-dose group and Shenfukang high-dose group showed lower relative expression levels of IL-1β mRNA, IL-6 mRNA and IL-8 mRNA than D-Gal model group (P<0.01). The D-Gal model group showed higher levels of IL-1β, IL-6, and IL-8 in the cell supernatants, than control group (P<0.01). The vitamin E group, Shenfukang low-dose group and Shenfukang high-dose group showed lower levels of IL-1β, IL-6, and IL-8 in the cell supernatants, than D-Gal model group (P<0.01). The D-Gal model group showed higher relative expression levels of JAK2 protein and p-STAT3/STAT3 than control group (P<0.01). The vitamin E group, Shenfukang low-dose group and Shenfukang high-dose group showed lower relative expression levels of JAK2 protein and p-STAT3/STAT3 than D-Gal model group (P<0.01). Conclusion: Shenfukang may inhibit the D-Gal-induced fibrosis model in HK-2 cells by regulating cellular senescence.

Key words：renal fibrosis; Shenfukang; cellular senescence; D-Galactose; α-smooth muscle actin; β-galactosidase; JAK2-STAT3 signaling

发布时间：2025-12-20

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