Abstract: BACKGROUND: Diabetic nephropathy is closely related to ferroptosis. Previous studies have found that Huidouba has a significant hypoglycemic effect, protects against diabetic nephropathy-induced renal oxidative stress injury and fibrosis, and also reduces the level of fibrosis in high glucose-induced HK-2 cells. 
OBJECTIVE: To investigate the role of Huidouba in regulating glutathione peroxidase 4 (GPX4) to inhibit ferroptosis and attenuate cellular fibrosis in human renal cortical proximal tubular epithelial cells (HK-2) cultured in high glucose conditions.
METHODS: (1) In order to investigate the interventional effect of Huidouba on ferroptosis and fibrosis in high glucose-cultured HK-2 cells, the cells were divided into six groups: control group, model group, low-, medium-, high-dose Huidouba groups, and rosiglitazone group. The morphology of HK-2 cells was observed. Cell viability, levels of oxidative stress-related indicators (malondialdehyde and glutathione), and protein and mRNA expression levels of ferroptosis-related indicators (GPX4, ferritin heavy chain 1, nuclear factor-E2-associated factor 2, transferrin receptor) and fibrosis-related indicators (transformed growth factor-β1 and kidney injury molecule 1), were detected. (2) To observe the effect of siRNA interfering with GPX4 expression in HK-2 cells, the cells were divided into negative control group and si-GPX4 group. The expression of GPX4 at protein and mRNA levels was detected. (3) In order to observe the effect of siRNA interference with GPX4 on the effect of Huidouba on the inhibition of ferroptosis and mitigation of cellular fibrosis in high glucose-cultured HK-2 cells, the cells were divided into negative control group, high glucose group (treated with 30 mmol/L glucose), high glucose+Huidouba group (treated with 30 mmol/L glucose+400 μg/mL Huidouba water extract), and high glucose+Huidouba+si-GPX4 group (treated with GPX4-siRNA followed by intervention with 30 mmol/L glucose+400 μg/mL Huidouba water extract). The levels of malondialdehyde and glutathione, and the protein and mRNA expression of GPX4, ferritin heavy chain 1, nuclear factor-E2-associated factor 2, transferrin receptor, transformed growth factor-β1, and kidney injury molecule 1 were detected. 
RESULTS AND CONCLUSION: (1) Compared with the control group, the cell viability, glutathione level, the protein and mRNA expression of GPX4, ferritin heavy chain 1, and nuclear factor-E2-associated factor 2 were significantly reduced in the model group, while the malondialdehyde level and protein and mRNA expression of transferrin receptor, transformed growth factor-β1, and kidney injury molecule 1 were significantly increased. Compared with the model group, the above indicators were improved to varying degrees in different dose groups of Huidouba and rosiglitazone group. (2) The protein and mRNA expression of GPX4 was significantly lower in the si-GPX4 group compared with the negative control group. Compared with the high glucose+Huidouba group, the glutathione level, the protein and mRNA expression of GPX4, ferritin heavy chain 1, and nuclear factor-E2-associated factor 2 were significantly reduced in the high glucose+Huidouba+si-GPX4 group, while the malondialdehyde level and protein and mRNA expression of transferrin receptor, transformed growth factor-β1, and kidney injury molecule 1 were significantly increased. To conclude, Huidouba attenuates cell fibrosis by inhibiting ferroptosis in high glucose-cultured HK-2 cells through the upregulation of GPX4.

Key words: diabetic nephropathy, fibrosis, HK-2 cells, ferroptosis, Huidouba, GPX4