Leptin Accelerates The Pathogenesis Of Heterotopic Ossification In Rat Tendon Tissues Via Upregulation Of MTORC1 Signaling

Objective:To investigate the effect and underlying action mechanism of leptin on the osteogenic differentiation of tendon-derived stem cells(TDSCs)in vitro and the HO formation in rat tendons.Methods:Isolated rat TDSCs were treated with various concentrations of leptin(1、10、100 n/ml)in the presence or absence of mTORC1 signaling specific inhibitor rapamycin(10nM)in vitro.After treatment of 14 days,alkaline phosphatase(ALP)activity/staining assays,Alizarin red staining,quantitative PCR,and Western blot assays were performed to measured the expression of osteogenic markers,such as ALP,runt-related transcription factor(Runx2),osterix(OSX),osteocalcin(OCN),and mTORC1 markers phosphorylation of p70 ribosomal S6 kinase1(PS6K1)and p70 ribosomal S6(PS6).Additionally,effect of leptin on TDSCs proliferation was performed by CCK-8 experiments for 3 days culture.A rat model with Achilles tenotomy was employed to evaluate the effect of leptin on HO formation together with or without rapamycin treatment.HE staining,micro-CT scan,immunohistochemistry staining of Runx2,OSX,and PS6 were performed.Results:In vitro studies with TDSCs showed that leptin increased the expression of osteogenic biomarkers(ALP,Runx2,OSX,OCN)and enhanced mineralization of TDSCs via activating the mTORC1 signal pathway(as indicated by PS6K1 and PS6).However,mTORC1 signaling blockade with rapamycin treatment suppressed leptin-induced osteogenic differentiation and mineralization.In vivo studies showed that leptin promoted HO formation in the Achilles tendon after tenotomy,and rapamycin treatment blocked leptin-induced HO formation.Conclusion:Leptin can promote TDSCs osteogenic differentiation and HO formation via mTORCl signaling in both vitro and vivo models,which provides a new potential therapeutic target for HO prevention.