抵当汤含药血清通过PI3K/Akt/mTOR信号通路增强高糖诱导的大鼠肾小球内皮细胞自噬

doi:10.12122/j.issn.1673-4254.2025.03.03

南方医科大学学报 ›› 2025, Vol. 45 ›› Issue (3): 461-469.doi: 10.12122/j.issn.1673-4254.2025.03.03

• • 上一篇

抵当汤含药血清通过PI3K/Akt/mTOR信号通路增强高糖诱导的大鼠肾小球内皮细胞自噬

董妍妍¹^,³(), 张可敬¹, 储俊¹^,²^,³, 储全根¹^,²^,³()

^1.安徽中医药大学中医学院，安徽合肥 230012
^2.合肥综合性国家科学中心大健康研究院新安医学与中医药现代化研究所，安徽合肥 230012
^3.安徽中医药大学新安医学教育部重点实验室，安徽合肥 230038

收稿日期:2024-12-04 出版日期:2025-03-20 发布日期:2025-03-28
通讯作者: 储全根 E-mail:yytung@ahtcm.edu.cn;286428483@qq.com
作者简介:董妍妍，在读博士研究生，E-mail: yytung@ahtcm.edu.cn
基金资助:
国家自然科学基金(81774189)

Didang Decoction-medicated serum enhances autophagy in high glucose-induced rat glomerular endothelial cells via the PI3K/Akt/mTOR signaling pathway

Yanyan DONG¹^,³(), Kejing ZHANG¹, Jun CHU¹^,²^,³, Quangen CHU¹^,²^,³()

^1.School of Traditional Chinese Medicine, Anhui University of Chinese Medicine, Hefei 230012, China
^2.Center for Xin'an Medicine and Modernization of Traditional Chinese Medicine, Institute of Health Research, Hefei Comprehensive National Science Center, Hefei 230012, China
^3.Key Laboratory of Xin'an Medicine, Ministry of Education, Anhui University of Chinese Medicine, Hefei 230038, China

Received:2024-12-04 Online:2025-03-20 Published:2025-03-28
Contact: Quangen CHU E-mail:yytung@ahtcm.edu.cn;286428483@qq.com
Supported by:
National Natural Science Foundation of China(81774189)

摘要/Abstract

摘要：

目的观察抵当汤含药血清通过磷脂酰肌醇3-激酶（PI3K）/蛋白激酶B（Akt）/哺乳动物雷帕霉素靶蛋白mTOR信号通路对高糖诱导的大鼠肾小球内皮细胞（RGECs）自噬的影响，以期为糖尿病肾病（DN）的治疗提供新的思路。方法连续过筛法结合胶原酶法提取与培养原代RGECs，使用第Ⅷ因子免疫荧光法对其鉴定。通过高糖培养基诱导RGECs模拟糖尿病环境下的细胞状态。将细胞分为5组：空白组（正常培养的RGECs）、高糖模型组（使用高糖培养基诱导的RGECs）、抵当汤组（在高糖诱导基础上加入抵当汤含药血清进行干预的RGECs）、3-MA组（在高糖诱导基础上加入自噬抑制剂3-MA进行干预的RGECs）和抵当汤+3-MA干预组（在高糖诱导基础上同时加入抵当汤含药血清和自噬抑制剂3-MA进行干预的RGECs）。CCK-8法筛选最佳造模条件和含药血清干预浓度，利用单丹磺酰卡巴胺（MDC）法观察自噬囊泡荧光强度，RT-qPCR法检测Beclin-1、p62mRNA表达，Western blotting法检测p-PI3K、p-Akt、p-mTOR、Beclin-1、p62、LC3B蛋白表达水平。结果与正常组比较，高糖模型组RGECs自噬荧光信号减少，荧光强度降低，Beclin-1 mRNA表达减少，p62 mRNA表达升高，Beclin-1蛋白表达和LC3Ⅱ/Ⅰ水平下降，P62、p-PI3K、p-Akt、p-mTOR蛋白表达上升（P<0.01）；与高糖模型组比较，抵当汤组RGECs自噬荧光面积与强度明显升高，Beclin-1 mRNA表达上升，p62 mRNA表达下降，Beclin-1蛋白表达上升，p62、p-PI3K、p-Akt、p-mTOR蛋白表达下降（P<0.01）。结论抵当汤含药血清可通过部分调节 PI3K/Akt/mTOR信号通路，增强 RGECs 的自噬，为糖尿病肾病的治疗提供新策略。

关键词: 糖尿病肾病, 抵当汤, 自噬, PI3K/Akt/mTOR信号通路, 肾小球内皮细胞

Abstract:

Objective To investigate the effect of Didang Decoction-medicated serum on autophagy in high glucose (HG)-induced rat glomerular endothelial cells (RGECs) and explore the pathway that mediates its effect. Methods Primary RGECs were isolated and cultured using sequential sieving combined with collagenase digestion, followed by identification using immunofluorescence assay for factor VIII. High glucose medium was used to induce RGECs to simulate a diabetic environment, and the effects of Didang Decoction-medicated serum and 3-MA (an autophagy inhibitor), either alone or in combination, on autophagy of HG-exposed cells were evaluated by observing autophagic vacuoles using monodansylcadaverine (MDC) staining. RT-qPCR and Western blotting were employed to measure mRNA and protein expression levels of Beclin-1, p62, LC3B, p-PI3K, p-Akt, and p-mTOR. Results Compared with the control cells, the HG-exposed RGECs showed significantly reduced autophagic fluorescence intensity, decreased Beclin-1 mRNA expression, increased p62 mRNA expression, downregulated Beclin-1 protein and LC3-II/I ratio, and upregulated p62, p-PI3K, p-Akt, and p-mTOR protein levels. Didang Decoction-medicated serum significantly enhanced autophagic fluorescence intensity in HG-exposed cells, increased Beclin-1 mRNA expression, decreased p62 mRNA expression, upregulated Beclin-1 protein, and downregulated p62, p-PI3K, p-Akt, and p-mTOR protein levels. Conclusion Didang Decoction-medicated serum enhances autophagy in HG-exposed RGECs by regulating the PI3K/Akt/mTOR signaling pathway, which sheds light on a new therapeutic strategy for diabetic nephropathy.

Key words: diabetic nephropathy, Didang decoction, autophagy, PI3K/Akt/mTOR signaling pathway, glomerular endothelial cells

董妍妍, 张可敬, 储俊, 储全根. 抵当汤含药血清通过PI3K/Akt/mTOR信号通路增强高糖诱导的大鼠肾小球内皮细胞自噬[J]. 南方医科大学学报, 2025, 45(3): 461-469.

Yanyan DONG, Kejing ZHANG, Jun CHU, Quangen CHU. Didang Decoction-medicated serum enhances autophagy in high glucose-induced rat glomerular endothelial cells via the PI3K/Akt/mTOR signaling pathway[J]. Journal of Southern Medical University, 2025, 45(3): 461-469.

图/表 13

表1 引物序列

Tab.1 Primer sequences for RT-qPCR

Primer	Length (bp)	Forward (5'→3')	Reverse (3'→5')
β-actin	150	CCCATCTATGAGGGTTACGC	TTTAATGTCACGCACGATTTC
Beclin-1	86	CAGCCTCTGAAACTGGACAC	TGGGCTGTGGTAAGTAATGG
P62	173	CGAAAGCTGACGCTGTTCAT	GGAGGACGGTACAAATCCATTA

图1 倒置荧光显微镜下观测大鼠肾小球微血管团

Fig.1 Observation of rat glomerular microvascular tufts under an inverted fluorescence microscope.

图2 接种后不同天数原代RGECs生长情况

Fig.2 Growth of primary RGECs at 3 (A), 4 (B), 6 (C) and 7 days (D) after seeding.

图3 第Ⅷ因子相关抗原抗体免疫荧光鉴定RGECs

Fig.3 Immunofluorescent identification of RGECs with factor VIII-related antigen antibody.

图4 不同浓度与时间葡萄糖干预下的RGECs相对活性

Fig.4 Changes of viability over time of the RGECs cultured in different glucose concentrations.

图5 不同浓度含药血清干预下的RGECs细胞相对活性

Fig.5 Changes of viability of RGECs treated with different concentrations of medicated serum. ***P<0.001, **P<0.01 vs 0% medicated serum.

图6 各组RGECs MDC自噬荧光染色结果

Fig.6 Fluorescent staining for analyzing autophagy in RGECs in each group.

图7 各组RGECs自噬荧光强度

Fig7 Autophagy fluorescent intensity of RGECs in each group. ###P<0.001 vs Control, ***P<0.001 vs High Glucose,▲▲▲P<0.001 vs Didang+3-Ma.

图8 各组Beclin-1、p62自噬相关基因表达

Fig.8 Transcriptional levels of autophagy-related genes (Beclin-1 and p62) in each group. ###P<0.001 vs Control. ***P<0.001,**P<0.01, *P<0.05 vs High Glucose.▲▲▲P<0.001, ▲▲P<0.01, ▲P<0.05 vs Didang+3-Ma.

图9 各组Beclin-1、LC3B、p62蛋白条带

Fig.9 Western blotting for detecting Beclin-1, LC3B and p62 protein expressions in each group.

图10 各组RGECs自噬相关蛋白表达

Fig.10 Expressions of autophagy-related proteins in each group of RGECs. ###P<0.001, ##P<0.01 vs Control. ***P<0.001,**P<0.01 vs High Glucose.▲▲▲P<0.001, ▲▲P<0.01, ▲P<0.05 vs Didang+3-Ma.

图11 各组p-PI3K、p-Akt、p-mTOR蛋白条带

Fig.11 Western blotting for detecting protein expressions of p-PI3K, p-Akt and p-mTOR in each group.

图12 各组RGECsp-PI3K、p-Akt、p-mTOR蛋白表达

Fig.12 Expression levels of p-PI3K, p-Akt and p-mTOR proteins in each group of RGECs. ###P<0.001 vs Control. ***P<0.001,*P<0.05 vs High Glucose.▲▲▲P<0.001, ▲P<0.05 vs Didang+3-Ma.

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抵当汤含药血清通过PI3K/Akt/mTOR信号通路增强高糖诱导的大鼠肾小球内皮细胞自噬

Didang Decoction-medicated serum enhances autophagy in high glucose-induced rat glomerular endothelial cells via the PI3K/Akt/mTOR signaling pathway

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