Astragaloside IV attenuated TGF-ß1- induced epithelial-mesenchymal transition of renal tubular epithelial cells via connexin 43 and Akt/mTOR signaling pathway.

INTRODUCTION: The objective of the study was to observe whether connexin 43 (Cx43) could regulate epithelial mesenchymal transformation (EMT) of renal tubular epithelial cells (RTECs) by influencing Akt/mTOR signaling pathway, and whether ASV could inhibit the development of renal interstitial fibrosis by regulating Cx43. METHODS: Lentivirus infection was transfected into RTECs with the final concentration of 50 ×PFU/ cell to regulate the expression of Cx43. And RTECs were intervened by different doses of Astragaloside IV (ASV). After synchronous culture of RTECs in each group，the expression levels of EMT-related indicators and Cx43 were detected by fluorescence microscope and Western-Blotting (WB), even the protein expressions and phosphorylation levels of AKT and mTOR in different groups were detected by WB. RESULTS: When the expression of Cx43 in RTECs was regulated by lentivirus infection, the degree of EMT induced by TGF­ß1 and the phosphorylation level of Akt and mTOR were changed accordingly, indicating that Akt/mTOR pathway might be a downstream molecular mechanism by which Cx43 could regulate EMT. After intervention with different doses of ASV, the expression level of Cx43 increased with obvious concentration dependence, and the expression levels of p-Akt and p- mTOR were significantly altered, suggesting that ASV could effectively increase the protein expressions of TGF­ß1-induced Cx43 in RTECs and inhibit the phosphorylation levels of Akt and mTOR. CONCLUSION: Cx43 were the main material basis of RTECs' injury, and ASV could inhibit TGF-ß1- induced RTECs' transdifferentiation. In-depth study of the mechanism might provide a broad application prospect for the treatment of renal interstitial fibrosis.

[Decursin reduces reactive oxygen species and inhibits cisplatin-induced apoptosis in rat renal tubular epithelial cells].

Objective To study the mechanism underlying the inhibitory effect of decursin on the apoptosis of rat renal tubular epithelial cells NRK-52E induced by cisplatin. Methods First, CCK-8 assay was used to detect the effects of 0, 10, 20, 40, 80, 100, 150, 200 µmol/L decursin and 0, 5, 10, 20, 30, 40, 50 µg/mL cispatin treatment for 24 hours on cell proliferation in NRK-52E cells via determining the half inhibitory concentration (IC50). Then, NRK-52E cells were stimulated with 20 µg/mL cisplatin combined with 10, 50, 100 µmol/L decursin, and cell activity was detected by CCK-8 assay. The cells were divided into normal control group, 20 µg/mL cisplatin stimulation group, and 10, 50, 100 µmol/L decursin treated groups. Cell morphological changes was observed under inverted microscope, morphological changes of nucleus was detected by DAPI staining, cell apoptosis was detected by flow cytometry, the level of intracellular ROS was detected by DCFH-DA staining, and the apoptosis marker proteins cleaved-caspase-3 and cleaved-PARP were examined by Western blot analysis. Results Compared with the normal control group, cisplatin significantly inhibited the activity of the cells, and IC50 was about 20 µg/mL; compared with the model group, in the decursin pretreatment groups, the level of intracellular ROS decreased remarkably, the expressions of cleaved-casspase-3 and cleaved-PARP proteins were reduced, and cell apoptosis was depressed. Conclusion Decursin can decrease the intracellular ROS level and inhibit the apoptosis of NRK-52E cells induced by cisplatin.