Metformin Protects Osteoarthritis In Mice By Regulating The AMPK Signaling Pathways

Objective Osteoarthritis(Osteoarthritis,OA)is the most common degenerative joint disease,more than 25% of adults suffered from the disease.Pathological changes in OA including progressive articular cartilage injury,thickening of the subchondral bone,osteophyte formation,synovial variable inflammation and degeneration of the knee ligament and meniscus,hypertrophy of the joint capsule.OA include many factors such as joint damage,obesity,aging and genetic and so on.Although the etiology and pathogenesis of OA is not entirely clear,but the research has proved that Adenosine protein kinase(Adenosine 5 '-monophosphate-activated protein kinase,AMPK)signaling has close relationship with OA,in older mice and mice with OA cartilage,or the dynamic compression of bovine cartilage cells induced biomechanics injury,AMPK activity decreased significantly.The same result also observed people in OA cartilage cells and tissue of people.Then whether AMPK as targets in the treatment of OA,increasing AMPK activity could have protective effect on osteoarthritis progression? We did not see any related reports now.As we know that metformin as an AMPK agonist,which can increase the AMPK activity,therefore in this study we use of traumatic osteoarthritis model in mice,studying whether metformin plays a protective role for osteoarthritis in mice,and to explore its mechanism.We want to opens up new avenues that metformin can be used as targets of AMPK in the treatment of osteoarthritis.Methods(1)2 months old C57 BL / 6 mice(n = 36)were divided into control group and experimental group,each group of 18,anterior cruciate ligament and medial meniscus excision were done in right knee of both groups for arthritis model;(2)After the success of the building model,in the second day,experimental group was given metformin(2mg/kg/d)and control group was given the same saline lavage as intervention treatment for 8 weeks;(3)Through the Micro-CT and HE,sarranine O – fast green slice staining to observe structure change and give a mark according Mankin rating s;(4)We test the AMPK,mTOR activity and degree of autophagy of articular cartilage in two groups by Western Blot(5)Chondrocytes were cultivated in vitro and stimulated by metformin to observe the cell morphology,amount.The cell activity was determined by MTT method.The cell activity of AMPK,mTOR and autophagy were detected by Western Blot.Results(1)We succeeded in establishing the model of osteoarthritis in mice and found metformin could protect the osteoarthritis.General observation: the right knees in control group were slightly swollen,thickening of joint capsule and ligaments were foud,articular mobility was poor;And the metformin group showed no swollen joint and good joint mobility.MICRO-CT results showed: serious phenomenon of bone absorption,dissolution,coarse bone surface and a large number of osteophyte were seen in the control group;Metformin treatment group: no severe bone absorption,dissolution phenomena,some bone surface coarse,a few osteophyte were seen.HE and sarranine O – fast green staining results shows: the control group: joint face was serious erosion to bone and joint gap disappeared,a large number of fibroblasts filled;Metformin treatment group: joint space was slightly narrow,articular surface damage was lighter,pink,deep red bone cartilage were clearly visible.Compared with control group,the Mankin score of metformin treatment group significantly reduced.(2)Metformin activated AMPK and enhanced autophagy to protect osteoarthritis.Articular cartilage Western Blot results: compared with the control group,the p – AMPK rised,p-S6 reduced,LC3A/B rised in the articular cartilage of metformin treatment group.(3)Metformin inhibited Chondrocytes differentiation,and promoted chondrocytes autophagy.Chondrocytes culture in vitro showed that metformin treatment group: the number of cells decrease,differentiation decrease determined by MTT method.Western Blot results: compared with the control group,the p – AMPK rised,p-S6 reduced,LC3A/B rised in the articular cartilage of metformin treatment group.Discuss:(1)Metformin has a protective effect for traumatic osteoarthritis model in mice.(2)Metformin regulates AMPK-mTOR-autophagy pathways to protecte osteoarthritis.(3)Metformin might be as AMPK targets for the treatment of osteoarthritis.