Abstract:

Objective To investigate the improvement effect of ginsenoside Rg3 （G-Rg3） on high glucose-induced injury in human renal tubular epithelial HK2 cells， and to elucidate its mechanism. Methods A high glucose-induced injury model of human renal tubular epithelial HK2 cells was established using high glucose（HG） medium （containing 25 mmol·L^-1 glucose）. Cells were divided into negative control （NC） group （medium containing 5.5 mmol·L^-1 glucose）， HG group （HG medium）， nuclear factor erythroid 2-related factor 2 （Nrf2） inhibitor ML385 group （ML385 group） （HG medium+5 μmol·L^-1 ML385）， G-Rg3 intervention group （Rg3 group） （HG medium+40 μmol·L^-1 G-Rg3）， and ML385+Rg3 group （HG medium + 5 μmol·L^-1 ML385 + 40 μmol·L^-1 G-Rg3）. Cell counting kit-8 （CCK-8） method was used to detect the cell activities in cells in various groups. 2'，7'-dichlorodihydrofluorescein diacetate（DCFH-DA） fluorescence method was used to detect the levels of reactive oxygen species （ROS） in the cells in various groups. Commercial kits were used to detect the activities of superoxide dismutase （SOD） and lactate dehydrogenase （LDH） and the levels of malondialdehyde （MDA） and ferrous ion（Fe²⁺） in the cells in various groups. Western blotting method was used to detect the expression levels of Nrf2， heme oxygenase-1 （HO-1）， glutathione peroxidase 4 （GPX4）， and acyl-CoA synthetase long-chain family member 4 （ACSL4） proteins in the cells in various groups. Results The results of CCK-8 method， DCFH-DA fluorescence method and kit method showed that， after 48 h of culture， compared with NC group， the cell activity was significantly decreased in HG group （P<0.01），the level of ROS， the activity of LDH， and the levels of MDA and Fe²⁺ in the cells were significantly increased （P<0.01）， and the activity of SOD was significantly decreased （P<0.01）. Compared with HG group， the level of ROS， the activity of LDH， and the levels of MDA and Fe²⁺ in the cells in ML385 group were significantly increased （P<0.05）， and the activity of SOD was significantly decreased （P<0.05）； the cell activity in Rg3 group was significantly increased （P<0.01）， the level of ROS， the activity of LDH， and the levels of MDA and Fe²⁺ in the cells were significantly decreased （P<0.01）， and the activity of SOD was significantly increased （P<0.01）. Compared with Rg3 group， the cell activity in ML385+Rg3 group was significantly decreased （P<0.01）， the level of ROS， the activity of LDH， and the levels of MDA and Fe²⁺ in the cells were significantly increased （P<0.01）， and the activity of SOD was significantly decreased （P<0.01）. The results of Western blotting method showed that， compared with NC group， the expression levels of Nrf2， HO-1， and GPX4 proteins in HK2 cells were significantly decreased in HG group （P<0.01）， and the expression level of ACSL4 protein was significantly increased （P<0.01）. Compared with HG group， the expression level of GPX4 protein in the cells in ML385 group was significantly decreased （P<0.01）， and the expression level of ACSL4 protein was significantly increased （P<0.01）； the expression levels of Nrf2， HO-1， and GPX4 proteins in HK2 cells were significantly increased in Rg3 group （P<0.01）， and the expression level of ACSL4 protein was significantly decreased （P<0.05）. Compared with Rg3 group， the expression levels of Nrf2， HO-1， and GPX4 proteins in HK2 cells in ML385+Rg3 group were significantly decreased （P<0.01）， and the expression level of ACSL4 protein was significantly increased （P<0.05）. Conclusion ?G-Rg3 showed an ameliorative effect on HG-induced oxidative injury in human renal tubular epithelial cells， and its mechanism may be associated with upregulating Nrf2 expression， activating the Nrf2/HO-1/GPX4 signaling pathway， and inhibiting ferroptosis.

Key words: Ginsenoside Rg3, Renal tubular epithelial cell, High-glucose environment, Ferroptosis, Nrf2 signaling pathway