Pravastatin Alleviates Interleukin 1?-induced Cartilage Degradation By Restoring Impaired Autophagy Associated With MAPK Pathway Inhibition

Part ? Construction of in vitro osteoarthritic cartilage degeneration model and effect of pravastatin on it Objective: To establish an in vitro model of osteoarthritic cartilage degeneration in rat chondrocytes and to examine the protective effect of pravastatin on the degradation of chondrocyte matrix in inflammatory environment.Methods: The chondrocytes were isolated from rat knee articular cartilage and the cells were cultured and passaged.Chondrocytes were treated with pravastatin at different concentrations(0,5,20 and 60 ?M)for 24 hours,and their viability was evaluated by CCK8.Chondrocytes pretreated with increasing concentrations of pravastatin(0,5,10,and 20 ?M)for 2 hours were stimulated by IL-1?(10 ng/mL)for 24 hours.Then,the expression levels of osteoarthritis-related effectors such as MMP3,MMP13 and ADAMTS5,at the mRNA and protein levels,were examined by quantitative real-time polymerase chain reaction(RT-PCR)and Western blotting,respectively.Rat articular chondrocytes were pretreated with pravastatin(0 or 20 ?M)and subsequently stimulated with IL-1?(0 or 10 ng / mL)for 24 hours,then stained with the toluidine blue to evaluate extracellular matrix degradation,as well as collected for RNA and protein samples to assess,the expression levels of osteoarthritis-related effectors such as MMP3,MMP13 and ADAMTS5,at the mRNA and protein levels by quantitative RT-PCR and Western blotting,respectively.Results: The isolated rat chondrocytes contained high homogeneity and stable traits.Treatment with increasing concentrations of pravastatin in rat chondrocytes resulted in no significant alteration in cell viability compared with control values.Pravastatin exerted dose-dependent therapeutic effects on inflammatory chondrocytes.Pravastatin can significantly alleviate chondrocyte degeneration and matrix destruction induced by IL-1? and downregulate matrix degradation-related effector genes and protein expression.Conclusion: The in vitro model of osteoarthritic cartilage degeneration in rat chondrocytes is stable and reliable.Pravastatin exerts protective effects on the degradation of chondrocyte matrix in inflammatory environment.Part ? Effect of pravastatin on the impaired autophagy activity in rat osteoarthritic chondrocyte Objective: To investigate the physiological mechanism involved in the chondroprotective effect of pravastatin against osteoarthritic matrix degradation.Methods: Rat articular chondrocytes were pretreated with pravastatin(0 or 20 ?M)for 2 hours and subsequently stimulated with IL-1?(0 or 10 ng / m L)for 24 hours,then their viability was evaluated by CCK8 as well as their cell apoptosis rate was evaluated by Annexin V and 7-AAD stained flow cytometry.Chondrocytes pretreated with pravastatin(0 or 20 ?M)for 2 hours were stimulated by IL-1?(10 ng/m L)for 24 hours.Then,the expression levels of autophagy-related effectors including atg7,atg12,Beclin1 and LC3,at the m RNA and protein levels,were examined by quantitative real-time polymerase chain reaction(RT-PCR)and Western blotting,respectively.Autophagic flux in chondrocytes in different treatment groups was monitored via GFP-m RFP-LC3 adenovirus transfection and confocal microscopy.Results: Compared with the blank control,pravastatin treatment and IL-1 ? exposure resulted in no significant alteration on the proliferation viability and apoptosis rate of articular chondrocytes.Pravastatin remarkably reversed the downregulation of autophagy related genes induced by IL-1? and enhanced autophagosome conversion to autophagolysosomes inhibited by IL-1? stimulation,while no change was observed in unstimulated chondrocytes after pravastatin treatment.Conclusion: In the early stage of osteoarthritis pathogenesis,pravastatin do not regulate cell proliferation or apoptosis.Pravastatin alleviates IL-1? induced cartilage degeneration by restoring impaired autophagy activity in inflammatory response.Part ? The mechanisms of pravastatin on impaired autophagy activity in osteoarthritic chondrocytes Objective: To identify the mechanisms of the restorement of autophagy activity by pravastatin in osteoarthritic chondrocytes.Methods: After pretreatment of chondrocytes with the specific autophagy inducer rapamycin or specific autophagy inhibitor 3-MA for 2 hours,the chondrocytes were stimulated with IL-1?(0 or 10 ng / m L)for 24 hours and then extracted fore protein and RNA.Their expression levels of osteoarthritis and autophagy-related effectors such as MMP3,MMP13 and ADAMTS5,at the m RNA and protein levels,were examined by quantitative real-time polymerase chain reaction(RT-PCR)and Western blotting,respectively.Activation of diverse MAPK,PI3K/AKT/m TOR and NF-?B pathways in chondrocytes with or without pravastatin treatment during IL-1? stimulation was examined by Western blotting.Furthermore,chondrocytes pretreated with p38 phosphorylation inhibitor SB203580 or p65 translocation inhibitor JSH-23 for 2 hours were stimulated by IL-1? for 24 hours.Then,the expression levels of specific pathways activation and autophagy-related proteins including Beclin1 and LC3 were examined by Western blotting.Results: Rapamycin treatment significantly downregulated the expression of cartilage degradation related metalloproteinases induced by IL-1? in rat articular chondrocytes,while 3-MA treatment resulted in the contrary effect.IL-1? stimulation can significantly increase the phosphorylation level of MAPK pathway,PI3 K / AKT / m TOR pathway and NF-?B pathway-related protein in chondrocytes,while pravastatin pretreatment mainly inhibits the phosphorylation of MAPK pathway and NF-?B pathway.Further experiments using selective inhibitors of specific pathway showed that p38 phosphorylation inhibition reduced autophagy impairment induced by IL-1? stimulation,whereas p65 activation inhibition did not significantly affect this autophagy impairment.Conclusion: The regulation on impaired autophagy activity during inflammatory response in chondrocytes effectively affect the severity of cartilage matrix degradation.The effect that pravastatin alleviates interleukin 1?-induced cartilage degradation by restoring impaired autophagy is mainly associated with MAPK pathway inhibition.