The Role Of Autophagy In The Regulatory Effects Of Polyphenolic Compounds On The Osteoclastogenesis

BackgroundThe incidence of osteoporosis is increasing year by year.The therapeutic drugs are expensive,and have many side effects.It is imperative to choose a safer and more effective treatment for osteoporosis.As plant polyphenols,curcumin and puerarin have been reported for their effects on osteoporosis.However,the underlying mechanisms regarding their treatment on osteoporosis have not yet been clarified.Therefore,it is necessary to explore the detailed mechanisms related to curcumin and puerarin in the treatment of osteoporosis.ObjectiveThe purpose of this study is to elucidate the specific role of autophagy in the osteoclastogenesis regulated by curcumin and puerarin.Materials and Methods1.The role of autophagy in curcumin-treated osteoclastogenesisIn vitro experiments:under the intervention of curcumin,the autophagy protein expression levels and autophagy activity of osteoclast precursors were detected;the effect of curcumin on autophagy activity of osteoclast precursors was observed under the intervention of RANKL;under the use of chloroquine,the effect of autophagy inhibition on the proliferation of osteoclast precursors treated by curcumin was observed;under the joint intervention of curcumin and RANKL,combined with the application of chloroquine,the differentiation ability of osteoclasts was observed;after Beclinl,ATG5 and Atg7 were silenced,the effects of autophagy proteins downregulation on the autophagy of osteoclast precursors jointly controlled by curcumin and RANKL and the differentiation ability of osteoclasts regulated by curcumin were observed;After overexpression of TRAF3,the difference of osteoclast formation induced by curcumin was analyzed.In vivo experiment:OVX rats were treated with curcumin and chloroquine to observe the bone loss and osteoclast formation.2.The role of autophagy in puerarin-treated osteoclast formationIn vitro experiment:after Beclinl,ATG5 and Atg7 were downregulated,the effect of puerarin on autophagic proteins and autophagic activity of OCP were observed;after RANKL intervention,the effect of puerarin on autophagic activity of OCP was observed;after Beclinl,ATG5 and Atg7 overexpression,the effect of puerarin on the proliferation level of OCP was observed.Results1.The role of autophagy in curcumin-treated osteoclastogenesisThe expression of ATG7 or Beclinl in OCPs was increased in a concentration-dependent manner under curcumin intervention;The LC3 conversion rate of OCPs was increased under curcumin intervention;The number of LC3 puncta in OCPs was increased significantly under curcumin intervention.Curcumin,like RANKL,can enhance LC3 transformation,LC3 puncta and autophagosome formation in OCPs.Curcumin reduced LC3 transformation,LC3 puncta and autophagosome formation of OCPs in the presence of RANKL.Overexpression of Beclinl can reverse the formation of LC3 puncta in OCPs inhibited by curcumin in the presence of RANKL.Curcumin can directly promote the number of EdU-positive cells in OCPs.The addition of chloroquine reversed the number of EdU-positive cells in OCPs promoted by curcumin on the basis of autophagy inhibition.Curcumin significantly decreased the number of mature osteoclasts and the levels of three osteoclast marker enzymes(CTSK,TRAP and MMP-9).Chloroquine further enhanced the role of curcumin in promoting osteoclastogenesis.Silence of Atg7 or Beclinl further enhanced the inhibition of curcumin on osteoclastogenesis.Micro-CT results showed that curcumin rescued the reduction in bone mineral density and trabecular bone loss in OVX rats,which could be further enhanced by chloroquine.H&E staining showed that the reduced trabecular areas in OVX rats were improved by curcumin,and further ameliorated by co-administration of curcumin and chloroquine.The increasing serum level of TRAP-5b(the in vivo osteoclast activity marker)in OVX rats was significantly reduced after curcumin administration,and decreased more markedly following the injection of chloroquine.Curcumin and RANKL could both reduce the expression of TRAF3 protein.The addition of curcumin alleviated RANKL-reduced TRAF3 proteins.Under the co-administration of curcumin and RANKL plus M-CSF,the mature osteoclasts were further decreased following TRAF3 overexpression.2.The role of autophagy in puerarin-treated osteoclast formationUnder directly intervened of puerarin,the expression of autophagic proteins Atg5,Atg7 and Beclinl in OCPs decreased in a concentration-dependent manner.Puerarin could significantly inhibit LC3 conversion rate and autophagosome formation in osteoclast precursors.Overexpression of Atg5,Atg7 and Beclin1 significantly reversed the inhibition of puerarin on LC3 conversion and autophagosome formation in OCPs.Overexpression of Atg7,Atg5 or BECN1 can reverse the number of EdU-positive cells in OCPs inhibited by puerarin Overexpression of Atg7,Atg5 or BECN1 can reverse the relative number of osteoclast precursors inhibited by puerarin.Puerarin can reverse the expression of autophagic protein in osteoclast precursor enhanced by RANKL.Puerarin can reverse the LC3 transformation level of OCPs promoted by RANKL.Puerarin can reverse the formation of LC3 puncta in OCPs promoted by RANKL.The LC3 transformation level of OCPs inhibited by puerarin was restored by overexpression of BECN1.The level of osteoclast differentiation inhibited by puerarin was reversed by overexpression of BECN1.ConclusionsCurcumin and puerarin,as plant polyphenols,have therapeutic effects on osteoporosis,which are related to autophagy.