ABSTRACT
OBJECTIVE Chronic kidney disease (CKD) has become a global public health problem with 10％-15％incidence rate, and inhibiting the renal interstitial fibrosis is considered to be a potential strategy to delay the progression of CKD. Z-Guggulsterone (Z-GS), an active compound from derived from Commiphora mukul, has been proved to be effective in various diseases. The present study aimes to determine the protective effect and the molecular mechanism of Z-GS on renal fibrosis. METHODS Unilateral ureteral obstruction (UUO) mice and hypoxia-induced HK-2 cells were used to simulate renal fibrosis in vitro and in vivo, respectively. The mice and cells were treated with different doses of Z-GS to observe the pharmacological action. Renal function, including Scr, BUN, and UA, were detected by commercial kits. H&E and Masson staining were performed to observe histopathological changes of kidney. Cell viability and LDH release of HK-2 cells were detected by commercial kits. Cell cycle distribution and apoptosis rate were analyzed by flow cytometry. Fibrosis markers were detected by immunohistochemistry and immunofluorescence analysis. Cell cycle related proteins and Klotho/p53 signaling were analyzed by Western blotting. RESULTS The results showed that Z-GS decreased the rise of Scr, BUN, and UA and lightened renal histopathological injury, which were induced by UUO. Besides, Z-GS administration alleviated renal fibrosis in mice by inhibiting the expressions of α-SMA, TGF-β and colla?genⅣ, and delayed G2/M cell cycle arrest by promoting the expressions of CDK1 and cyclinD1/B1 rate. Experiments in vitro indicated that Z-GS treatment significantly increased the cell viability while decreased the LDH release in hypoxia-induced HK-2 cells. In addition, hypoxia induced fibrosis and G2/M cycle arrest in HK-2 cells were retarded by Z-GS. The study of its possible mechanism exhibited that Z-GS treatment increased the level of Klotho and inhibited P53 level. Nev?ertheless, the effect of Z-GS on Klotho/P53 signaling was reversed by siRNA-Klotho. Moreover, siRNA-Klotho treatment eliminated the effects of Z-GS on G2/M cell cycle arrest and fibrosis. CONCLUSION This study clarified that Z-GS allevi?ated renal fibrosis and G2/M cycle arrest through Klotho/P53 signaling pathway. People who have suffered CKD may potentially benefit from treatment with Z-GS.
ABSTRACT
Objective: To study the effect of rocaglaol from Aglaia odorata on HepG2 proliferation and to explore the potential anti-tumor mechanism. Methods: The MTT, colony formation, EdU incorporation, and CFDA-SE assays were used to determine the anti-proliferative activity of rocaglaol in HepG2 cells. Apoptosis and cell cycle distribution effect induced by rocaglaol were carried out by flow cytometry. The effect of rocaglaol on protein involved in the G2/M checkpoint and the MAPK pathway were performed by Western blotting analysis. Results: Rocaglaol significantly inhibited the viability of HepG2 cells in a dose-dependent and time-dependent manner. Rocaglaol was more effective than doxorubicin in the growth inhibition of HepG2 cells. However, rocaglaol-induced cytotoxicity in normal human hepatic cell line L02 was lower than that of doxorubicin. Treatment with different concentrations of rocaglaol at 48 h caused G2/M cell cycle progression inhibition, rather than apoptosis in HepG2 cells. Rocaglaol can significantly reduce the expression of G2/M cell cycle-regulating proteins cdc25C, cdc2, and cyclin B1 as well as increase the expression of ERK and JNK phosphorylation levels. Further study found that U0126 can partly abrogate the anti-proliferative activity in HepG2 cells, G2/M phase arrest and the reduction in the protein expression levels of cdc2 and cdc25C induced by rocaglaol. Conclusion: Our results demonstrated that rocaglaol was superior to doxorubicin in the inhibition of HepG2 cells proliferation and the selectivity of L02 cell activity. We provided evidence that the rocaglaol had the ability to continuously over-activate the ERK signaling in HepG2 cells, leading to the inhibition of cell proliferation through G2/M phase arrest.