Targeted Inhibition Of TXNRD1 Prevents Cartilage Extracellular Matrix Degeneration By Activating Nrf2 Pathway In Osteoarthritis

Background and objectiveOsteoarthritis(OA)is a common degenerative joint condition that worsens with aging.By 2020,OA will surpass diabetes as the fourth most crippling disease in the world,imposing a tremendous financial pressure on families and society.The prevalence of those over 65 years old is over 50%,and the disease has a significant incidence in the middle-aged and older population.Chronic joint pain and restricted mobility are the predominant clinical symptoms of OA,which are primarily driven on by synovitis,articular cartilage deterioration,osteophyte development,and subchondral bone sclerosis.Although aging,obesity,and metabolic problems have all been identified as OA risk factors,there are presently no effective therapies to prevent the progression of OA.The only effective treatment for osteoarthritis(OA)currently is joint replacement.Early treatment of OA involves mainly joint cavity injection lubricant or use of non-steroidal anti-inflammatory drugs and steroid drugs to alleviate symptoms,reduce joint pain,and inhibit inflammation.However,the above method cannot fundamentally reverse the process of cartilage degeneration and inflammation,can only temporarily relieve partial pain,and disease progression.There are still limitations despite the enormous potential of contemporary medications and regenerative therapies.The seek for OA treatment methods that are safer,more efficient,and reliable has thus taken precedence.TXNRD1 is mainly found in the cytoplasm of tissues and is the most studied and understood isoenzyme.It is a selenoprotein that promotes thioredoxin 1(Trx1)reduction in an NADPH-dependent manner,and it involves cell proliferation,DNA replication,stress-induced premature senescence,and anti-oxidative stress.In our previous study,we found that TXNRD1 was significantly upregulated in IL-1β-stimulated mouse primary chondrocytes and in cartilage from OA patients,demonstrating that TXNRD1 has an important role in articular cartilage homeostasis and OA progression.However,no studies have reported the expression of related genes in chondrocytes after TXNRD1 inhibition.Our study revealed that inhibiting TXNRD1 has been shown to activate Nrf2 signaling,and several studies over the past few years showed that an active Trx system,and TXNRD1 in particular,is important for inhibiting Nrf2 activation.Therefore,we speculate that the inhibition of TXNRD1 is mainly activated through the activation of Nrf2,thus alleviating the degradation of the extracellular matrix of the articular cartilage and then inhibiting the development of OA.Given the critical biological functions of TXNRD1 in OA,it is imperative to elucidate the role of TXNRD1 in the development of OA and its molecular mechanisms.Research methods(1)Western blot,q PCR and immunohistochemistry were used to determine the expression level of TXNRD1 in the cartilage tissues of OA patients and normal individuals.IL-1β was used on mouse chondrocytes to establish vitro OA model,Chondrocytes from mouse were stimulated with IL-1β for various durations(0,2,4,6days),and the expression of TXNRD1,COL2a1,ADAMTS5,SOX9,MMP9 and MMP13 were assessed by q PCR and Western Blot.(2)Auranofin and small interfering RNA(si RNA)were utilized to investigate how inhibition of TXNRD1 affected the IL-1β-induced ECM degradation and the expression of ECM-related genes.q PCR was originally utilized to examine the m RNA expression levels of MMP9,MMP13,COL2a1 and SOX9.Western Blot was used to measure the expression of MMP13,ADAMTS5,COL2a1 and SOX9 following auranofin treatment or si TXNRD1.(3)The si-Nrf2 was transfected in mouse chondrocytes,followed by the detection of the changes in the expression levels of the chondrocyte phenotype and ECM-associated proteins,thus further clarifying the specific mechanism.(4)ML385(Nrf2 inhibitor)was used to explore the mechanism of si-TXNRD1 on cartilage ECM,and Western blot was used to detect the expression level changes of the chondrocyte phenotype and its ECM-associated proteins,so as to further clarify the specific mechanism.(5)Auranofin was injected into the DMM mouse model,and the degree of cartilage surface damage and protein expression level were measured by HE staining,Safranin O/fast green staining and immunohistochemistry.Results and Conclusions(1)The mRNA and protein levels of TXNRD1 were significantly upregulated in the articular cartilage of OA patients,and in vitro cytology experiments demonstrated that the m RNA and protein expression levels were upregulated under IL-1β treatment,and the protein expression level increased in a time gradient.(2)Auranofin promote the expression of Nrf2 and HO-1 and the synthesis of extracellular matrix and inhibit the degradation of extracellular matrix in the safe concentration range.Silencing TXNRD1 can up-regulate the expression levels of COL2A1 and SOX9 and down-regulate the expression levels of MMP13 and ADAMTS5,thus maintaining the homeostasis of chondrocyte extracellular matrix.(3)Silencing Nrf2 inhibited the protective effect of auranofin on cartilage ECM;similarly,inhibiting Nrf2 counteracts the protective effect of si TXNRD1,suggesting that TXNRD1 inhibition may exert the protective effect on cartilage extracellular matrix by activating Nrf2 signaling pathway.(4)Injection of auranofin into the knee joint of mice effectively alleviated the degree of articular cartilage destruction and reduced the expression of Nrf2,further supporting the role of TXNRD1 inhibition in OA and its mechanism in vivo.