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

doi:10.12122/j.issn.1673-4254.2025.03.03

Abstract

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

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.

Figures/Tables 13

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

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

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

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

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

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

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

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.

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.

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

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.

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

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.

References 31

1	Raval N, Kumawat A, Kalyane D, et al. Understanding molecular upsets in diabetic nephropathy to identify novel targets and treatment opportunities[J]. Drug Discov Today, 2020, 25(5): 862-78.
2	Xu L, Shao FM. Sitagliptin protects renal glomerular endothelial cells against high glucose-induced dysfunction and injury[J]. Bioengineered, 2022, 13(1): 655-66.
3	Li QQ, Xin YL, Liu PL, et al. Deciphering the therapeutic mechanisms of Fructus Ligustri Lucidi on diabetic nephropathy via bioinformatics analysis[J]. J Chin Pharmaceut Sci, 2024, 33(11): 1025-39.
4	Huang JH, Wang JZ. Selective protein degradation through chaperone-mediated autophagy: Implications for cellular homeo-stasis and disease (Review)[J]. Mol Med Rep, 2024, 31: 13.
5	Yu L, Wei J, Liu PD. Attacking the PI3K/Akt/mTOR signaling pathway for targeted therapeutic treatment in human cancer[J]. Semin Cancer Biol, 2022, 85: 69-94.
6	杨继, 江宗岳, 周婧雅. 从微型癥瘕理论探讨糖尿病血管并发症的病机转变[J]. 中华中医药杂志, 2024, 39(3): 1270-4.
7	储全根, 王玲, 张凯, 等. 抵当汤调控NF-κB通路干预DM大鼠心肌炎症反应的机制[J]. 中国实验方剂学杂志, 2018, 24(1): 109-13.
8	王玲, 储全根, 张凯, 等. 抵当汤对糖尿病大鼠心肌微血管病变的影响[J]. 江西中医药, 2017, 48(12): 27-9.
9	许笑雯, 储全根, 储俊, 等. 痰瘀同治法对糖尿病大鼠心肌微血管病变AGEs/RAGE轴及氧化应激的影响[J]. 南方医科大学学报, 2021, 41(10): 1527-33.
10	张凯, 储全根, 毕华剑, 等. 抵当汤对糖尿病大鼠心肌组织JAK2/STAT3信号通路的影响[J]. 安徽中医药大学学报, 2016, 35(2): 65-9.
11	蔡正银, 储俊, 储全根, 等. 抵当陷胸汤对糖尿病大鼠心肌超微结构和CTGF及其受体LRP蛋白表达的影响[J]. 中国实验方剂学杂志, 2022, 28(22): 100-5.
12	陈静, 储全根, 储俊, 等. 基于JAK/STAT信号通路探讨痰瘀同治法对糖尿病大鼠心肌病变的影响[J]. 安徽中医药大学学报, 2023, 42(5): 88-93.
13	罗宝璐, 储全根, 储俊, 等. 抵当汤含药血清对高糖诱导大鼠肾小球内皮细胞损伤的影响[J]. 中成药, 2024, 46(9): 2930-5.
14	储全根, 蔡正银, 储俊, 等. 痰瘀同治法对糖尿病大鼠肾脏TGF-β1/Smad3信号通路的影响[J]. 南方医科大学学报, 2020, 40(5): 708-12.
15	国家药典委员会. 中华人民共和国药典-一部: 2020年版[M]. 北京: 中国医药科技出版社, 2020: 24-220.
16	王群, 唐甜甜, 韩伟, 等. 基于网络药理学和实验验证的黄芪六一汤药效组分促肾小管上皮细胞自噬抗糖尿病肾病的机制研究[J]. 药物评价研究, 2024, 47(6): 1187-98.
17	范永升, 姜德友. 金匮要略[M]. 5版. 北京: 中国中医药出版社, 2021: 95.
18	唐容川. 血证论[M]. 北京: 人民军医出版社, 2007: 113.
19	吴以岭, 魏聪, 贾振华, 等. 从络病学说探讨糖尿病肾病的病机[J]. 中国中医基础医学杂志, 2007, 13(9): 659-60.
20	王新苗, 杨浩宇, 顾成娟, 等. 黄芪、水蛭粉、大黄治疗糖尿病肾病经验: 仝小林三味小方撷萃[J]. 吉林中医药, 2020, 40(1): 5-7.
21	傅强, 王世东, 肖永华, 等. 吕仁和教授分期辨治糖尿病学术思想探微[J]. 世界中医药, 2017, 12(1): 21-4.
22	Lassén E, Daehn IS. Molecular mechanisms in early diabetic kidney disease: glomerular endothelial cell dysfunction[J]. Int J Mol Sci, 2020, 21(24): 9456.
23	Jennifer Weil E, Lemley KV, Mason CC, et al. Podocyte detachment and reduced glomerular capillary endothelial fenestration promote kidney disease in type 2 diabetic nephropathy[J]. Kidney Int, 2012, 82(9): 1010-7.
24	马华根, 刘海琴, 刘昭德, 等. 原代大鼠肾小球微血管内皮细胞的培养与鉴定[J]. 生理学报, 2021, 73(6): 926-30.
25	Rops AL, van der Vlag J, Jacobs CW, et al. Isolation and characterization of conditionally immortalized mouse glomerular endothelial cell lines[J]. Kidney Int, 2004, 66(6): 2193-201.
26	Dylewski JF, Wilson N, Lu SZ, et al. Isolation, purification, and conditional immortalization of murine glomerular endothelial cells of microvascular phenotype[J]. MethodsX, 2020, 7: 101048.
27	Yang DY, Livingston MJ, Liu ZW, et al. Autophagy in diabetic kidney disease: regulation, pathological role and therapeutic potential[J]. Cell Mol Life Sci, 2018, 75(4): 669-88.
28	Levine B, Kroemer G. Biological functions of autophagy genes: a disease perspective[J]. Cell, 2019, 176(1/2): 11-42.
29	Huang JD, Chen LY, Wu JX, et al. Targeting the PI3K/AKT/mTOR signaling pathway in the treatment of human diseases: current status, trends, and solutions[J]. J Med Chem, 2022, 65(24): 16033-61.
30	Kim YC, Guan KL. mTOR: a pharmacologic target for autophagy regulation[J]. J Clin Invest, 2015, 125(1): 25-32.
31	Dong RX, Zhang X, Liu YD, et al. Rutin alleviates EndMT by restoring autophagy through inhibiting HDAC1 via PI3K/AKT/mTOR pathway in diabetic kidney disease[J]. Phytomedicine, 2023, 112: 154700.

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