Yiqi Yangyin Huazhuo Tongluo Formula alleviates diabetic podocyte injury by regulating miR-21a-5p/FoxO1/PINK1-mediated mitochondrial autophagy

doi:10.12122/j.issn.1673-4254.2025.01.04

Abstract

Abstract:

Objective To investigate the protective effect of Yiqi Yangyin Huazhuo Tongluo Formula (YYHT) against high glucose-induced injury in mouse renal podocytes (MPC5 cells) and the possible mechanism. Methods Adult Wistar rats were treated with 19, 38, and 76 g/kg YYHT or saline via gavage for 7 days to prepare YYHT-medicated or blank sera for treatment of MPC5 cells cultured in high glucose (30 mmol/L) prior to transfection with a miR-21a-5p inhibitor or a miR-21a-5p mimic. The changes in miR-21a-5p expressions and the mRNA levels of FoxO1, PINK1, and Parkin in the treated cells were detected with qRT-PCR, and the protein levels of nephrin, podocin, FoxO1, PINK1, and Parkin were detected with Western blotting. Autophagic activity in the cells were evaluated with MDC staining. The effect of miR-21a-5p mimic on FoxO1 transcription and the binding of miR-21a-5p to FoxO1 were examined with luciferase reporter gene assay and radioimmunoprecipitation assay. Results MPC5 cells exposed to high glucose showed significantly increased miR-21a-5p expression, lowered expressions of FoxO1, PINK1, and Parkin1 mRNAs, and reduced levels of FoxO1, PINK1, parkin, nephrin, and podocin proteins and autophagic activity. Treatment of the exposed cells with YYHT-medicated sera and miR-21a-5p inhibitor both significantly enhanced the protein expressions of nephrin and podocin, inhibited the expression of miR-21a-5p, increased the mRNA and protein expressions of FoxO1, PINK1 and Parkin, and upregulated autophagic activity of the cells. Transfection with miR-21a-5p mimic effectively inhibited the transcription of FoxO1 and promoted the binding of miR-21a-5p to FoxO1 in MPC5 cells, and these effects were obviously attenuated by treatment with YYHT-medicated sera. Conclusion YYHT-medicated sera alleviate high glucose-induced injury in MPC5 cells by regulating miR-21a-5p/FoxO1/PINK1-mediated mitochondrial autophagy.

Key words: diabetic nephropathy, Yiqi Yangyin Huazhuo Tongluo Formula, mitochondrial autophagy, podocyte injury, miR-21/FoxO1/PINK1

Kelei GUO, Yingli LI, Chenguang XUAN, Zijun HOU, Songshan YE, Linyun LI, Liping CHEN, Li HAN, Hua BIAN. Yiqi Yangyin Huazhuo Tongluo Formula alleviates diabetic podocyte injury by regulating miR-21a-5p/FoxO1/PINK1-mediated mitochondrial autophagy[J]. Journal of Southern Medical University, 2025, 45(1): 27-34.

Figures/Tables 8

Tab. 1 Primer sequences for RT-qPCR

Gene	Primer sequence (5'→3')	Length (bp)
FoxO1	Forward:CCCAGGCCGGAGTTTAACC Reverse:GTTGCTCATAAAGTCGGTGCT	132
PINK1	Forward:TTCTTCCGCCAGTCGGTAG Reverse:CTGCTTCTCCTCGATCAGCC	141
Parkin	Forward:TCTTCCAGTGTAACCACCGTC Reverse:GGCAGGGAGTAGCCAAGTT	115
β-actin	Forward:GGCTGTATTCCCCTCCATCG Reverse:CCAGTTGGTAACAATGCCATGT	154

Fig.1 Putative binding sites between miR-21a-5p and FoxO1 3'UTR and mutant sites in FoxO1-MUT reporter.

Fig.2 Effect of Yiqi Yangyin Huazhuo Tongluo Formula (YYHT)‑medicated rat sera on protein expressions of nephrin and podocin in MPC5 cells exposed to high glucose. A: Western blotting for nephrin and podocin expressions in MPC5 cells. B, C: Quantitative analysis of nephrin and podocin protein expressions. △△P<0.01 vs Control group; *P<0.05, **P<0.01 vs Model group.

Fig.3 Effect of YYHT-mediated sera on autophagosomes in MPC5 cells in each group. A: Autophagosomes in each group observed by MDC staining. B: Mean fluorescence intensity of each group. △△P<0.01 vs Control group; **P<0.01 vs Model group.

Fig.4 Effect of YYHT-medicated sera on expression levels of miR-21a-5p, FoxO1, PINK1 and Parkin mRNA in MPC5 cells in each group. △△P<0.01 vs Control group;*P<0.05, **P<0.01 vs Model group.

Fig.5 Effect of YYHT-medicated sera on protein expressions of FoxO1, PINK1 and Parkin in MPC5 cells in each group. A: Western blotting for FoxO1, PINK1 and Parkin expressions in MPC5 cells. B-D: Quantitative analysis of FoxO1, PINK1 and Parkin protein expressions. △△P<0.01 vs Control group; *P<0.05, **P<0.01 vs Model group.

Fig.6 Effect of YYHT-medicated sera on transcription of miR-21a-5p/FoxO1 detected by dual luciferase reporter gene assay. △△P<0.01 vs miR-21a-5p-mimic-NC group; *P<0.01,**P<0.01 vs miR-21a-5p-mimic group.

Fig.7 Effect of YYHT-medicated on binding of miR-21a-5p and FoxO1 in each group. △△P<0.01 vs miR-21a-5p-mimic-NC group; **P<0.01 vs miR-21a-5p-mimic group.

References 33

1	Wang N, Zhang C. Recent advances in the management of diabetic kidney disease: slowing progression[J]. Int J Mol Sci, 2024, 25(6): 3086.
2	Gupta S, Dominguez M, Golestaneh L. Diabetic kidney disease: an update[J]. Med Clin North Am, 2023, 107(4): 689-705.
3	DeFronzo RA, Reeves WB, Awad AS. Pathophysiology of diabetic kidney disease: impact of SGLT2 inhibitors[J]. Nat Rev Nephrol, 2021, 17(5):319-34.
4	Sugahara M, Pak WLW, Tanaka T, et al. Update on diagnosis, pathophysiology, and management of diabetic kidney disease[J]. Nephrology, 2021, 26(6): 491-500.
5	Hu SW, Hang X, Wei Y, et al. Crosstalk among podocytes, glomerular endothelial cells and mesangial cells in diabetic kidney disease: an updated review[J]. Cell Commun Signal, 2024, 22(1): 136.
6	Mohandes S, Doke T, Hu HL, et al. Molecular pathways that drive diabetic kidney disease[J]. J Clin Invest, 2023, 133(4): e165654.
7	Liu SM, Yuan YG, Xue Y, et al. Podocyte injury in diabetic kidney disease: a focus on mitochondrial dysfunction[J]. Front Cell Dev Biol, 2022, 10: 832887.
8	Wu QR, Yan RZ, Yang HW, et al. Qing-Re-Xiao-Zheng-Yi-Qi formula relieves kidney damage and activates mitophagy in diabetic kidney disease[J]. Front Pharmacol, 2022, 13: 992597.
9	Li W, Du MM, Wang QZ, et al. FoxO1 promotes mitophagy in the podocytes of diabetic male mice via the PINK1/parkin pathway[J]. Endocrinology, 2017, 158(7): 2155-67.
10	Mahtal N, Lenoir O, Tinel C, et al. MicroRNAs in kidney injury and disease[J]. Nat Rev Nephrol, 2022, 18(10): 643-62.
11	Wang J, Shen LY, Hong H, et al. Atrasentan alleviates high glucose-induced podocyte injury by the microRNA-21/forkhead box O1 axis[J]. Eur J Pharmacol, 2019, 852: 142-50.
12	Ma LL, Li J, Zhang XT, et al. Chinese botanical drugs targeting mitophagy to alleviate diabetic kidney disease, a comprehensive review[J]. Front Pharmacol, 2024, 15: 1360179.
13	Li XD, Zhang Y, Xing XD, et al. Podocyte injury of diabetic nephropathy: novel mechanism discovery and therapeutic prospects[J]. Biomedecine Pharmacother, 2023, 168: 115670.
14	郭东义, 张鹏, 卞华, 等. 益气养阴化浊通络方对早期糖尿病肾病患者NF‑κB、MCP-1及IL-6的影响[J]. 河南中医, 2016, 36(8): 1386-8.
15	张鹏, 吕芹, 郭东义, 等. 益气养阴化浊通络方对早期糖尿病肾脏疾病患者内皮细胞及血液流变学的影响[J]. 科学技术与工程, 2016, 16(17): 119-22.
16	韩立, 吕芹, 郭东义, 等. 益气养阴化浊通络方对早期糖尿病肾脏疾病患者氧化应激的影响[J]. 科学技术与工程, 2016, 16(14): 23-6.
17	郭克磊, 李颖利, 韩立, 等. 益气养阴化浊通络方对高糖诱导小鼠肾足细胞自噬ATG5、Beclin-1及BNIP3表达的影响[J]. 中国医院药学杂志, 2023, 43(16): 1808-13.
18	朱政洁, 马雷雷, 何立群. 从益气养阴、补肾活血论治糖尿病肾病[J]. 中华中医药杂志, 2022, 37(12):7170-3.
19	中华中医药学会, 北京中医药大学东直门医院, 北京中医药大学. 糖尿病肾脏疾病中西医结合诊疗指南[J]. 北京中医药大学学报, 2024, 47(4): 580-92.
20	Liu XQ, Jiang L, Li YY, et al. Wogonin protects glomerular podocytes by targeting Bcl-2-mediated autophagy and apoptosis in diabetic kidney disease[J]. Acta Pharmacol Sin, 2022, 43(1): 96-110.
21	Gong L, Wang R, Wang XY, et al. Research progress of natural active compounds on improving podocyte function to reduce proteinuria in diabetic kidney disease[J]. Ren Fail, 2023, 45(2): 2290930.
22	Wang SL, Long HJ, Hou LJ, et al. The mitophagy pathway and its implications in human diseases[J]. Signal Transduct Target Ther, 2023, 8(1): 304.
23	Zhang XF, Feng J, Li X, et al. Mitophagy in diabetic kidney disease[J]. Front Cell Dev Biol, 2021, 9: 778011.
24	Han R, Liu YT, Li SH, et al. PINK1-PRKN mediated mitophagy: differences between in vitro and in vivo models[J]. Autophagy, 2023, 19(5): 1396-405.
25	Han XD, Wang J, Li RL, et al. Placental mesenchymal stem cells alleviate podocyte injury in diabetic kidney disease by modulating mitophagy via the SIRT1-PGC-1alpha-TFAM pathway[J]. Int J Mol Sci, 2023, 24(5): 4696.
26	Dhas Y, Arshad N, Biswas N, et al. MicroRNA-21 silencing in diabetic nephropathy: insights on therapeutic strategies[J]. Biomedicines, 2023, 11(9): 2583.
27	Szostak J, Gor&#x; cy A, Durys D, et al. The role of microRNA in the pathogenesis of diabetic nephropathy[J]. Int J Mol Sci, 2023, 24(7): 6214.
28	Liu SJ, Wu WZ, Liao J, et al. MicroRNA-21: a critical pathogenic factor of diabetic nephropathy[J]. Front Endocrinol, 2022, 13: 895010.
29	Assmann TS, Recamonde-Mendoza M, Costa AR, et al. Circulating miRNAs in diabetic kidney disease: case-control study and in silico analyses[J]. Acta Diabetol, 2019, 56(1): 55-65.
30	向珈谊, 张会芳, 梁露群, 等. miR-21通过下调PPAR-α参与脂质代谢紊乱并促进糖尿病大鼠肾组织及肾小管上皮细胞纤维化病变[J]. 中国病理生理杂志, 2021, 37(10): 1858-67.
31	Orea-Soufi A, Paik J, Bragança J, et al. FOXO transcription factors as therapeutic targets in human diseases[J]. Trends Pharmacol Sci, 2022, 43(12): 1070-84.
32	Li XD, Wan TT, Li YB. Role of FoxO1 in regulating autophagy in type 2 diabetes mellitus (Review)[J]. Exp Ther Med, 2021, 22(1): 707.
33	Xu YM, Xu C, Huang J, et al. Astragalus polysaccharide attenuates diabetic nephropathy by reducing apoptosis and enhancing autophagy through activation of Sirt1/FoxO1 pathway[J]. Int Urol Nephrol, 2024, 56(9): 3067-78.

[1]	Yifan JIANG, Xiaorong LI, Jiayi GENG, Yongfeng CHEN, Bi TANG, Pinfang KANG. Quercetin ameliorates diabetic kidney injury in rats by inhibiting the HMGB1/RAGE/ NF-κB signaling pathway [J]. Journal of Southern Medical University, 2024, 44(9): 1769-1775.
[2]	Chengcheng JIANG, Yangyang LI, Kexin DUAN, Tingting ZHAN, Zilong CHEN, Yongxue WANG, Rui ZHAO, Caiyun MA, Yu GUO, Changqing LIU. Parkin deletion affects PINK1/Parkin-mediated mitochondrial autophagy to exacerbate neuroinflammation and accelerate progression of Parkinson's disease in mice [J]. Journal of Southern Medical University, 2024, 44(12): 2359-2366.
[3]	LI Ying, WANG Qian, CHEN Xiaoniao, XI Yue, YANG Jian, LIU Xiaomin, WANG Yuanda, ZHANG Li, CAI Guangyan, CHEN Xiangmei, DONG Zheyi. Validation and comparison of diabetic retinopathy-based diagnostic models for diabetic nephropathy [J]. Journal of Southern Medical University, 2023, 43(9): 1585-1590.
[4]	WANG Ying, ZHOU Mingjun, ZHU Qianwen, ZHANG Cui, WANG Lin, LI Shu, HU Zebo. HIF-1α activation induces cholesterol homeostasis dysfunction to accelerate progression of diabetic nephropathy in rats [J]. Journal of Southern Medical University, 2023, 43(10): 1782-1788.
[5]	WANG Huanlan, LIU Hong, ZHANG Yanmin, CHEN Weidong. MiR-34a alleviates podocyte injury in mice with diabetic nephropathy by targeted downregulation of Notch signaling pathway [J]. Journal of Southern Medical University, 2022, 42(12): 1839-1845.
[6]	HUANG Chao, WANG Hongting, WANG Cunqin. 27-P-CAUA induces apoptosis and mitochondrial autophagy in breast cancer cells by inhibiting HER2/PI3K/AKT signaling pathway [J]. Journal of Southern Medical University, 2022, 42(1): 63-70.
[7]	. Ginsenoside Rh2 inhibits renal fibrosis and renal cell apoptosis in rats with diabetic nephropathy by downregulating discoid domain receptor 1 [J]. Journal of Southern Medical University, 2021, 41(7): 1107-1113.
[8]	. Screening potential Chinese materia medica and their monomers for treatment of diabetic nephropathy based on caspase-1-mediated pyroptosis [J]. Journal of Southern Medical University, 2020, 40(09): 1280-1287.
[9]	. Intervention of phlegm and blood stasis inhibits TGF-β1/Smad3 signaling pathway in the kidney of diabetic rats [J]. Journal of Southern Medical University, 2020, 40(05): 708-712.
[10]	. Effect of traditional Chinese medicine for replenishing qi, nourishing yin and activating blood on renal Notch/Hes1 signaling in rats with diabetic nephropathy [J]. Journal of Southern Medical University, 2019, 39(07): 855-.
[11]	. Value of podocalyxin levels in urinary extracellular vesicles for diagnosis of diabetic nephropathy [J]. Journal of Southern Medical University, 2018, 38(09): 1126-.
[12]	. [J]. Journal of Southern Medical University, 2018, 38(03): 296-.
[13]	. Effect of Tripterygium glycosides on expression of hypoxia inducible factor-1α and endothelin-1 in kidney of diabetic rats [J]. Journal of Southern Medical University, 2015, 35(04): 499-.
[14]	. Effects of parthenolide on high glucose-induced cell proliferation, NF-κB activation and MCP-1 expression in rat mesangial cells [J]. Journal of Southern Medical University, 2013, 33(10): 1471-.
[15]	. Effect of exendin-4 on monocyte chemoattractant protein-1 expression in cultured rat glomerular mesangial cells induced by tumor necrosis factor-α in vitro [J]. Journal of Southern Medical University, 2013, 33(06): 930-.