The Effects And Mechanisms Of Transcription Factor KLF2 Regulating Chondrocyte Function By Modulating Oxidative Stress In Osteoarthritis

Objective: Osteoarthritis(OA)is one of the most common inflammatory and degenerative diseases in the musculoskeletal system,which can lead to significant joint function degeneration and decreased quality of life in patients.OA is predicted to be the greatest cause of disability among patients over 40 years old by 2040,which can cause serious living burden to patients and great economic burden to families and society.There have been a large number of studies on OA in recent years.However,due to the pathological mechanism of OA is very complicated,there is currently no efficient treatment that can arrest or reverse OA progression.In addition to physical therapy,regular exercise and weight loss,pharmacologic interventions can only alleviate some symptoms of OA.In the end-stage of OA,artificial joint replacement seems to be the only alternative strategy that can effectively improve joint function and quality of life.Hence,identifying an enhanced regulatory mechanism for cartilage homeostasis in OA will help in finding new specific therapeutic targets and therapeutic strategies.Kruppel-like factor 2(KLF2),also known as lung Kruppel-like factor(LKLF),is a member of the zinc finger family.KLF2 has been widely studied for its role in the activation of immune cells,cell proliferation,and the regulation of inflammation.Previous studies have shown that KLF2 is closely associated with a series of inflammatory diseases,however,the role of KLF2 in pathological process of OA have not been investigated in detail.Oxidative stress is increasingly recognized as a critical component of OA pathology.Studies have demonstrated that KLF2 can play an anti-oxidant role in some Inflammatory diseases,such as atherossclerosis.Nuclear factor-erythroid 2-related fator 2(NRF2)is a key transcription factor that play a crucial role in the protection against oxidative stress in OA.Despite these findings,there are no reports regarding direct link between KLF2 and NRF2 at present.By in-depth study of the potential role of KLF2 in the pathological progression of OA,this experimental research will provide new insights into OA pathogenesis regulated by KLF2 and a rationale for the development of effective OA intervention strategies.Methods: We first downloaded transcriptome data on human knee normal cartilage and OA cartilage from GEO database,and preliminarily observed the change of KLF2 expression between normal and OA cartilage.To further validate the change of KLF2 expression observed in transcriptome data,Rt-qPCR and immunohistochemistry were performed for normal and OA cartilage samples which collected from the clinic.Subsequently,the expression levels of KLF2 and COL2A1 were detected by Rt-qPCR in OA cartilage samples,and the correlation between KLF2 and COL2A1 expression levels were further analyzed.After determining the correlation between KLF2 expression level and OA,Rt-qPCR and Western blot analysis were performed to test the influence of different concentration and exposure time of IL-1? on the expression of KLF2 in SW1353 human chondrocytes.The expression of MMP13 and COL2A1 in chondrocytes treated with IL-1? was further detected by immunofluorescence assay.After that,western blot analysis was performed to measure the expression of MMP3,MMP9,MMP13 and COL2A1 in SW1353 human chondrocytes with and without KLF2 overexpression and stimulated with IL-1?.We next investigated the effects of KLF2 overexpression(pharmacological and lentiviral strategies)on IL-1?-induced cell viability decrease,reactive oxygen(ROS)increase and apoptosis in SW1353 cells by CCK8 and flow cytometry analysis.After identifying the above phenomenon,we then examined the effect of KLF2 overexpression on the expression of NRF2 downstream proteins(HO-1 and NQO1)in SW1353 cells by western blot analysis.Immunofluorescence analysis and western blot were performed to observe the nuclear translocation of NRF2 protein in SW1353 cells.To clarify the regulation mechanism of KLF2 on chondrocyte catabolism and apoptosis in OA pathological environment,Rt-qPCR and flow cytometry were performed to measure the expression of MMP13 and apoptosis in SW1353 human chondrocytes with and without KLF2 overexpression and stimulated with or without ML385 under IL-1? induced inflammatory environment.Finally,to further investigate the chondroprotective effect of KLF2,we used an OA animal model to assess the potential therapeutic effects of KLF2 on OA pathogenesis.Results:(1)Compared to that in human normal cartilage tissues,KLF2 expression in OA cartilage tissues was significantly decreased.(2)The expression levels of KLF2 in the damaged cartilage tissue were considerably decreased compared with that in the smooth cartilage tissue from the same patient.(3)The expression levels of KLF2 and COL2A1 in the knee cartilage of OA patients were positively correlated.(4)IL-1? caused dose-and time-dependent decreases in KLF2 m RNA and protein levels in SW1353 human chondrocytes.(5)Genetic or pharmacological overexpression of KLF2 in con-junction with IL-1? stimulation significantly reduced percentage of apoptosis,MMP3,MMP9,and MMP13 expression and increased COL2A1 expression in SW1353 cells.(6)IL-1? stimulation significantly increased the levels of ROS in SW1353 cells,whereas genetic or pharmacological overexpression of KLF2 in SW1353 cells suppressed the IL-1?-induced generation of ROS.(7)KLF2 overexpression inhibits IL-1?-induced MMP13 expression and cell apoptosis through the activation of the NRF2/ARE signaling pathway by increasing the nuclear translocation of NRF2 protein in SW1353 cells.(8)KLF2 protects against MIA-induced cartilage destruction and MMP13 expression in a rat OA model.Conclusion:(1)KLF2 expression level is correlated with the occurrence and development of OA,and may be a potential molecular target for clinical treatment of OA.(2)KLF2 overexpression can activate the NRF2/ARE anti-oxidative stress signaling pathway by increasing NRF2 nuclear translocation,thus regulating the catabolism and apoptosis of chondrocytes in OA.(3)Overexpression of KLF2 in rat knee cartilage has therapeutic effects on MIA-induced OA model and that KLF2 could be a potential therapeutic target for OA.