THE MTORC1 SIGNALING PATHWAY INHIBITOR RAPAMYCIN INHIBITS LEPTIN INDUCED OSTEOGENIC DIFFERENTIATION OF TDSCS AND ECTOPIC OSSIFICATION OF ACHILLES TENDON

Li Yao¹, Bo Sun², Bing Wang¹, Jian Liu^{1, *} 

¹Department of Orthopaedics, The First People's Hospital of Lianyungang, Lianyungang 222061, PR China - ²Department of Obstetrics, Lianyungang Maternal and Child Heath Care Hospital, Lianyungang 222003, PR China

Objective: To investigate the inhibitory effect of mammalian rapamycin target protein complex 1(mTORC1) signaling pathway inhibitor rapamycin on leptin induced osteogenic differentiation of tendon stem cells (TDSCs) and the formation of heterotopic Achilles tendon ossification (HO). 

Methods: Seventy-six healthy male Sprague-Dawley rats were selected for in vitro and in vivo experiments. In vitro experiment; rat TDSCs were extracted for osteogenic induction culture. TDSCs were treated with different concentrations of leptin (1, 10, 100ng/ml) or 100ng/ml leptin +10nM rapamycin for four days. The expressions of alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2), osteogenic transcription factor (OSX) and osteocalcin (OCN) were detected by qRT-PCR. The expressions of the phosphorylated ribosome S6 protein kinase (PS6K1) and phosphorylated ribosome S6 protein (PS6) downstream of Runx2, OSX and mTORC1 signaling pathways were detected by Western Blot. In vivo experiment; by using a random numbers table, 76 rats were divided into the normal group, heterotopic ossification (HO), heterotopic ossification + rapamycin group (HO+RA), heterotopic ossification + leptin group (HO+LEP), and the heterotopic ossification + leptin + rapamycin group (HO+LEP+RA) using the immunohistochemical method to detect the expression of Runx2, leptin and OSX and mTORCl downstream marker PS6. 

Results: The in vitro experiments, with the increase of leptin concentration, the expressions of osteogenic factors ALP, Runx2, OSX, OCN mRNA and proteins of Runx2, OSX and downstream factors PS6K1 and PS6 of mTORCl signaling pathway were significantly increased (P<0.05). The expressions of PS6K1, PS6, Runx2 and OSX in the leptin group were significantly higher than those in the control group and the leptin + rapamycin group (P<0.05). The in vivo experiments showed that the expression of leptin in the HO group was significantly higher than that in the normal group (P<0.05). The expressions of Runx2 and OSX in the HO+RA group were significantly lower than those in the HO group (P<0.05). The expressions of Runx2 and OSX in the HO+LEP group were significantly higher than those in the HO group and the HO+LEP+RA group (P<0.05). There was no significant difference in the expression of Runx2 and OSX between the HO group and the HO+LEP+RA group (P>0.05). The expression of PS6 in the HO+RA group was significantly lower than that in the HO group (P<0.05). PS6 expression in the HO+LEP group was significantly higher than that in the HO group (P<0.05). The expression of PS6 in the HO+LEP+RA group was significantly lower than that in the HO+LEP group (P<0.05). 

Conclusion: Rapamycin, a mTORC1 signaling pathway inhibitor, can effectively inhibit the osteogenic differentiation and ectopic ossification of TDSCs in tendinous tissues induced by leptin.