Endogenous H₂S Affects Cholesterol Efflux And Apoptosis Of Osteoclasts Through LXRα/ABCG1 Pathway

Background and objective:cholesterol as an important part of cell membrane lipid raft and cell metabolism of important members of the material,it’s contents in cells and cell membrane were closely related to apoptosis.Osteoclast cholesterol efflux is mainly mediated by ATP-binding cassette transporter G1（ABCG1）on its cell membrane.Liver X receptorα（LXRα）promotes the efflux of intracellular cholesterol by upregulating ABCG1 expression.Hydrogen sulfide（H₂S）,one of three kinds of gas signal molecules,which is catalyzed by Cystathionine-γ-lyase（CSE）in osteoclasts and has a regulatory role in many cell apoptosis.But it is not clear whether endogenous H₂S regulates osteoclast cholesterol efflux to regulate osteoclast apoptosis.In this study,we used RAW264.7 cell as preosteoclasts and added Receptor activator of nuclear factor kappa-B ligand（RANKL）to stimulate conversion of these cells into osteoclasts,thereby observing the effects of endogenous H₂S on cholesterol efflux and osteoclast apoptosis and exploring the underlying mechanism.Methods:The osteoclast precursor RAW264.7 cells were cultured in vitro and RANKL was added to induce osteoclast differentiation form mature osteoclasts.PcDNA 3.1/myc-His（-）-CSE+LXRαsiRNA and pcDNA3.1/myc-His（-）-CSE,CSE siRNA,pcDNA 3.1/myc-)-CSE+ABCG1 siRNA was used to treat mature osteoclasts of logarithmic phase and divided into control,LXRαinhibition,ABCG1 inhibition,CSE overexpression,CSE inhibition,CSE overexpression+LXRαinhibition,and CSE overexpression+ABCG1 inhibition,respectively.The numbers of mature osteoclasts and the nucleus pyknosis of each group were observed at different time points by using tartaric acid phosphatase staining to determine the number of osteoclasts in each group.Concentrations of H₂S were measured using a microplate reader with a spectrophotometer.The expression of CSE mRNA,LXRαmRNA,ABCG1 mRNA and caspase3 mRNA was detected by quantitative PCR.The protein expression of CSE,LXRα,ABCG1 and caspase 3 in each group were detected by Western blotting.The intracellular cholesterol efflux was detected by liquid scintillation counter.Results:Osteoclasts overexpressing CSE exhibited a marked increase in H₂S concentration,cholesterol efflux and apoptosis rate.Moreover,the expression of LXRαand ABCG1 were upregulated in CSE overexpression group.However,inhibition of the LXRα/ABCG1 by their siRNAs abrogated CSE-enhanced cholesterol efflux.Conclusions:Endogenous H₂S can promote cholesterol efflux from osteoclast and increase osteoclast apoptosis by activating the LXRα/ABCG1 pathway.