The Effects Of Inhibiting MD2 Or HSP90 On The Activities Of Celastrol-regulated Transcript Factors

Objective Celastrol is an important biological active component of Tripterygium wilfordii Hook.f which plays varieties of pharmacological roles and has a broad application potential in clinic.To clarify the action mechanisms of celastrol is the basis for its clinical transformation.At present,it has been found that celastrol can act on multiple molecular targets,but there are two views on how it can affect many molecules:one notion is that celastrol acts on a few of common upstream targets and then affects multiple down-streaming targets;the second notion is that there is no common upstream targeted celastrol directly affects multiple targets.In this study,we investigate whether the four transcription factors?TFs?affected by celastrol have a common upstream target,to test whether the first notion above be reasonable.The four TFs in this study are NF-?B,HSF-1?and HSP70 regulated by it?,Nrf2?and HO-1 regulated by it?and HIF-1,because these four factors have been extensively studied before and the affections on these factorscan fully explain the basic pharmacological effects of celastrol.The upstream target molecules chosen in this study are MD2 and HSP90,because it has been proved that celastrol could bind these two molecules,and both of them have reportedly regulated the aforementioned four TFs.This study is to further reveal the molecular mechanism underlying the therapeutic effects of celastrol and provide guidance for future work.Methods The in vitro culture HepG2 were used as study model in this study.First,we screened the safe and effective dose for celastrol to be used.Second,we observed the effect of celastrol on NF-?B,HSF-1?and its regulated HSP70?,Nrf2?demonstrated as HO-1expression?,and HIF-1.Third,we observed the effects of NF-?B inhibitor PDTC or siRNA down-regulating MD2 on the four TFs,both of which was compared with celastrol.Finally,the effect of HSP90 inhibitor 17-AAG or direct interference with HSP90 by siRNA on the four TFs was explored and compared with those of celastrol.Results We found that the IC₅₀0 for celastrol on HepG2 cells was 3.32?M.Celastrol can inhibit NF-?B phosphorylation and induce the expression of HSF-1?HSP70?,HO-1 and HIF-1.The effect of PDTC on NF-?B,HSF-1?HSP70?and HO-1,was the same as that of celastrol,but the effect on HIF-1 was different.After MD2 was down-regulated,only NF-?B change was consistent with celastrol's action,indicating that MD2 was not the common upstream target for celastrol's regulation of the four TFs.Moreover,the effect of HSP90inhibitor 17-AAG on NF-?B and HSF-1?HSP70?was the same as that of celastrol,while the effects on another two other TFs were different.Moreover,after HSP90 was down-regulated,only the alteration in NF-?B was the same as celastrol treatment,indicating that HSP90 was not the common upstream target of celastrol to regulate the four TFs.In addition,we found that there may be a positive feedback relationship between inhibition of NF-?B and inhibition of HSP90,but the exact mechanism is unclear.Conclusion In this study,we added new evidence to the reported effects of celastrol in regulating NF-?B,HSF-1?and HSP70?,Nrf2?and HO-1?and HIF-1.Our findings supported the notion that the pharmacological effects of celastrol is achieved through targeting multiple molecules and pathways,however did not support the notion that there is a few of common upstream target.Therefore,the mechanism of celastrol regulating the four TFs should be studied separately.In addition,there may be a positive feedback between the two main effects of celastrol,namely,inhibition of NF-?B and inhibition of HSP90,the underling mechanisms of which need further exploration.