Ganoderic acid A from Ganoderma lucidumretard ADPKD renal cyst development via down-regulating Ras/MAPK signaling pathway

摘要

OBJECTIVE Autosomal dominant polycystic kidney disease (ADPKD) is a common-monogenetic disease characterized by progressive development of renal cysts. Thereis still further need for effective therapy.Based on our precious study that Ganoderma triterpenes(GT),which is the major secondary metabolites of Ganoderma lucidum,is able to attenuate renal cyst development.The aim of this study was to investigate the effect of a monomer,Ganoderic acid A(GA-A)that was purified from the GT,which has been reported to exhibit antinociceptive,antioxidative,hepatoproctive and anti-cancer activities,to have a potent anti-cyst effect in ADPKD. METHODS We first evaluated the potential cytotoxicity of GA-A on MDCK cells using a CCK-8 assay.Then we used MDCK cyst model,cultivated MDCK cells in vitro to form fluid-filled cysts surrounded by monolayer cells.MDCK cells were co-incu-bated with 10 μmol·L-1FSK with or without GA-A(25 μg·mL-1)and equal concentration GT as positive control from day 0 to day 6 to investigate the inhibitory effect of GA-A on cyst formation.And to further investigate the inhibitory effect of GA-A on cyst enlargement, MDCK cysts were treated with different concentration of GA-A(6.25,25 and 100 μg·mL-1)from day 5 to day 12.Next we used an embryonic kidney cyst model, wile-type mice kidneys were taken out on embryonic day 13.5 to form renal cysts stimulated with 8-Br-cAMP to prove the renal cyst inhibition at organ level.Meanwhile,we explored the possible mechanisms underlying GA-A inhibition on renal cyst development using MDCK cells treated with 10 μmol·L-1FSK co-incubated with GA-A(25 μg·mL-1)and equal concentration GT.Several key components of Ras/MAPK pathway was evaluated by Western blot,the protein expression of H-ras,B-raf, p-ERK, Egr-1 and c-fos was evaluated. RESULTS MDCK cell viability was not affected by GA-A that were used ofincreasing concentrations up to 200 μg·mL-1. GA-A had no significant influence on cyst formation,but inhibited cyst enlargement dose-dependently and the inhibitory effect is significantly better than that of the same concentration of GT which proved that GA-A may be an effective monomer from GT.And after washing out GA-A on day 8,MDCK cysts regrew to a large size,suggesting that the inhibitory effect of GA-A on MDCK cyst enlargement was reversible. GA-A inhibited embryonic kidney cyst development significantly in a dose-dependent and reversible manner proving GA-A cyst inhibition at organ level,which is also more effective than equal concentration GT.Treatment of MDCK cells with FSK caused a significant elevation of H-ras,B-raf,p-ERK,Egr-1 and c-fos signaling molecules,while treatment with GA-A reduced the level of H-ras,B-raf,p-ERK,Egr-1 and c-fos expression.GA-A down-regulated Ras/MAPK signaling pathway could contribute to its inhibitory effect on cyst development. CONCLUSION Ganoderic acid A from Ganoderma lucidum retard ADPKD renal cyst development via down-regulating Ras/MAPK signaling pathway.