Experimental Study On The Effect Of Protein Tyrosine Kinase Inhibitor PP1 On Knee Osteoarthritis

Objective: The increased expression of matrix metalloproteinase and polyproteoglycan in Knee Osteoarthritis(KOA)leads to degradation of collagen and proteoglycan and degeneration and necrosis of articular chondrocytes.However,the pathogenesis of osteoarthritis is not yet clear.Currently,commonly used clinical drugs can only relieve OA symptoms,and there is still a lack of drugs to improve OA condition.Therefore,it is of great significance to find new effective drugs to treat OA.To investigate and verify the expression of protein tyrosine kinase inhibitor PP1 in chondrocytes of osteoarthritis,and to explore the effect of intraperitoneal injection of PP1 on joint degeneration and progression of osteoarthritis in mice.Methods:(1)In vitro cell experiment: Normal chondrocytes were extracted from rat Suckling mice and cultured.The chondrocytes were divided into four groups.Interlcukin-1beta,IL-1β)to construct chondrocyte inflammation model,control group(F12basal medium culture),IL-1β group(10ng/ml IL-1β+F12 co-culture),PP1 group(5μM,50μM PP1+ IL-1β10 ng/ml co-culture),respectively.Real-time polymerase chain reaction(RT-PCR),Western blot and Cellular immunofluorescence were used technique)to detect the anti-inflammatory effect and regulatory mechanism of PP1 on IL-1β-induced chondroinflammatory cells in Suckling mice.(2)In vivo experiment: The mice were divided into 4 groups: sham operation group,OA group,low dose PP1 group and high dose PP1 group,with 6 mice in each group.Destabilization of the Medial meniscus(DMM)has destabilization of the knee osteoarthritis model in mice.The sham-operation group and group 0A have been injected with normal saline 10 m L/kg/ time every 2 days.Low and high dose PP1 groups were intraperitoneally injected with 0.5mg PP1 and 5mg/kg PP1 once every 2 days,respectively,and the mice were sacrificed after continuous intervention for 4 weeks.Micro-CT scanning,three-dimensional reconstruction and statistical analysis of mouse knee joint were performed,hematoxylin-Eosinstin staining(HE),muscored O-fast green staining,Alsin blue staining,immunofluorescence detection and analysis of hematoxylin-Eosinstin staining(HE),to investigate the anti-joint degeneration of mouse KOA by PP1 The role of osteoarthritis progression.Methods:(1)In vitro experiment: Normal rat chondrocytes were extracted and cultured and divided into four groups.Interlcukin-1beta,IL-1β)to construct chondrocyte inflammation model,control group(F12 basal medium culture),IL-1β group(10ng/ml IL-1β+F12co-culture),PP1 group(5μM,50μM PP1+ IL-1β10 ng/ml co-culture),respectively.Real-time polymerase chain reaction(RT-PCR)and Western blot were used to investigate the anti-inflammatory effects of PP1 on IL-1β-induced chondroinflammatory cells in Suckling mice and its regulatory mechanism.(2)In vivo experiment: The mice were divided into 4groups: sham operation group,OA group,low dose PP1 group and high dose PP1 group,with6 mice in each group.Since the Destabilization of the Medial meniscus(DMM)has been destabilization of the knee osteoarthritis animal models of mice.The sham-operation group and group 0A have been injected with normal saline 10 m L/kg/ time intradiscally 2 days/time.PP1 low dose and high dose groups were intraperitoneally injected with 10 mg/kg PP1 and100mg/kg PP1 every 2 days,respectively.The mice were sacrificed after continuous intervention for 4 weeks.Micro-CT scanning of knee joint,three-dimensional reconstruction and statistical analysis were performed,hematoxylin-Eosinstin staining(HE),Alcin blue staining,and immunofluorescence detection were performed to investigate the effect of PP1 on the anti-joint degeneration of KOA in mice.Results:(1)In the cell experiment,compared with IL-1β group,the proportion of cell apoptosis in PP1 group was less.RT-PCR,Western blot and immunofluorescence results showed that PP1 reduced the expression of inflammatory factors,and PP1 had a protective effect on chondrocytes,which was proportional to the increase of the concentration of PP1.(2)In the animal experiments of knee osteoarthritis in mice,the staining results of HE,Alcian blue,crosin O-solid green and tissue fluorescence,as well as the results of Micro-CT scan showed that intraperitoneal injection of PP1 could inhibit the cartilage destruction and osteophyte formation induced by DMM surgery,and PP1 could effectively delay the pathological changes of the bone microstructure of knee osteoarthritis.Conclusions:(1)In vitro experiments,IL-1β was used to construct inflammatory chondrocytes,and it was found that PP1 could inhibit the expression of inflammatory mediators in chondrocytes and reduce the inflammatory response.(2)In this study,by constructing a mouse KOA model and using PP1 intraperitoneal injection intervention,it was found that PP1 could delay the degenerative changes of knee osteoarthritis in mice.