| Objectives:Heterotopic ossification(HO)refers to the ectopic formation of bone in soft tissue.Traumatic HO mostly occurs in clinic and the underlying mechanism is not completely understood.The objective of this study is to assess changes in Treg at early stages of HO and explore the role of metformin in regulation of Treg and its effect on HO.Methods:Mice were received with Achilles tenotomy and dorsal burns to induce HO.The experimental mice were treated with metformin or PBS for different period of time and the muscle tissue around the Achilles tendon was collected.q PCR and WB were used to detect changes in indicated parameters,micro-CT and HE staining to observe HO around the Achilles tendon,and flow cytometry to detect the ratio of Treg in the spleen,lymph nodes,injured muscle tissue and blood.Result:1.Compared with non-surgical control group,m RNA levels of Treg markers,Tigit,Lag3,Gltr,Clta-4,Foxp3 and Helios in the injured muscle tissue,were significantly decreased at day 1(p<0.01),There was no significant change at day 3,but increased significantly at day 7(p<0.05);the expressions of SMAD6 and SMURF1 showed a similar trend to changes in Treg(1 day: p<0.01;7 days: p<0.01);The level of AMPK(p-AMPK)decreased significantly after surgical injury(p<0.05).2.Treatment of mice with metformin at different time points,the m RNA levels of Treg markers in the injured muscle tissue were significantly increased(p<0.05),concurrently with increases in p-AMPK(p<0.05)and decreases in HO in soft tissues around the surgical spot.3.Treatment of mice with metformin at the early stages(1～7 days)increases in the m RNA levels of Treg markers in the injured muscle tissue(p<0.05),accompanied by increases in the expressions of SMAD6 and SMURF1(p<0.05),p-AMPK(p<0.05)and the ratio of Treg(p<0.05).However,Treg in blood was significantly decreased(p<0.01).Conclusion:1.Treg exerts an inhibitory role in the early stage of heterotopic ossification.2.Metformin may inhibit the early progression of heterotopic ossification by activating AMPK at the trauma site,increasing the proportion of Treg,and inhibiting the BMP signaling pathway. |
