Mechanism of Rapamycin on the Proliferation, Migration and Epithelial Mesenchymal Transformation of Podocytes Induced by High Glucose
ZHAO Xuehui1*, WANG Huaihuai1, LI Weiwei2, LI Yingjie1, JIA Junli1
The aim of this study was to investigate the effects of rapamycin on D-glucose-induced proliferation, migration, EMT (epithelial-mesenchymal transition) and PI3K/AKT (phosphatidylinositol-3-kinase/serine-threonine kinase) signaling pathway of human glomerular podocytes. The human glomerular podocyte HGPC cell lines were cultured in vitro and divided into control group (5 mmol/L D-glucose), high glucose group (30 mmol/L D-glucose), and low/ medium/high concentration group (30 mmol/L D-glucose+2.5, 5, 10 μmol/L rapamycin). The optimum concentration of rapamycin was selected by the expression levels of inflammatory factor IL-8 (interleukin-8), TNF-α (tumor necrosis factor-α) detected by ELISA (enzyme-linked immunosorbent assay) and cell viability measured by CCK-8 (cell counting kit-8). The supplementation groups were further divided into LY294002 group (30 mmol/L D-glucose+10 μmol/L PI3K/ AKT pathway inhibitor LY294002), rapamycin+LY294002 group (30 mmol/L D-glucose+10.0 μmol/L rapamycin+10 μmol/L PI3K/AKT pathway inhibitor LY294002) and rapamycin+SC79 group (30 mmol/L D-glucose +10.0 μmol/L rapamycin+10 μmol/L PI3K/AKT pathway agonist SC79), intervened for 24 h. Cell proliferation was detected by EdU (5-ethynyl-2’deoxyuridine); the migration ability of cells was determined by wound healing; the expression levels of EMT and PI3K/AKT pathway-related proteins were determined by WB (Western blot). The results showed that compared with the control group, the expression levels of the inflammatory factor IL-8 and TNF-α in the high glucose group were significantly increased, and the cell viability was significantly decreased. Compared with the high glucose group, the expression level of inflammatory factor IL-8 and TNF-α in high concentration group were decreased, while the cell viability was increased (P<0.05). Therefore, 10.0 μmol/L rapamycin was determined to the optimal concentration for further experiments. Compared with the control group, the cell proliferation rate and E-cadherin protein expression level in the high glucose group were significantly decreased, while the cell migration rate and the proteins expression levels of N-cadherin, Vimentin, FN, p-PI3K/ PI3K and p-AKT/AKT were significantly increased in the high glucose group (P<0.05). Compared with the high glucose group, the change trend of above indexes was reversed by rapamycin group and inhibitor group (P<0.05). Compared with the rapamycin group, LY294002 in the rapamycin+LY294002 group enhanced the trend of these indexes, while SC79 in the rapamycin+SC79 group weakened the trend of these indexes (P<0.05). In conclusion, rapamycin could promote the proliferation and inhibit the migration and EMT process of human glomerular podocyte HGPC cells induced by D-glucose, and the mechanism may be related to blocking PI3K/AKT pathway signal transduction.
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