Mechanism Research On A Potential Role Of TRPV6 As A Regulator In Rat Intra-articular Chondrocyte

Background and Objective:Osteoarthritis is a common chronic degenerative diseases,synovitis and cartilage reconstruction is the main features.Associated with obesity and aging problem becoming seriously,The incidence of the disease has been improved.Nowadays,how to prevention and treatment of osteoarthritis has become a huge work for medical worker.As a uniqueness cell in cartilago articularis,chondrocyte secrete and regulate EMC.Promote chondrocyte proliferation and inhibit apoptosis,is one of the most important aspects of the prevention and treatment of osteoarthritis.TRPV6(and its closest relative,TRPV5)are the only highly Ca2+-selective channels of the entire TRP superfamily.It can influence cell proliferation by changing the intracellular calcium ion concentration.The goal of this study was to determine the potential role of TRPV6 in regulating intra-articular chondrocyte function.Such as proliferation?apoptosis and secretion of ECM.Providing new theory for the pathogenesis of articular cartilage degeneration and a new way for prevention and treatment of the disease.Methods:Chondrocytes were isolated from articular cartilage in the joints of rats as eight-week-old male,Alcian blue staining was performed to authenticate it..Chondrocytes were infected with TRPV6 shRNA lentiviral particles and TRPV6 lentiviral activation particles.Testing each group RPV6 expression level By Western Bolt experiment to verify the interference effect.Using ELIASA testing The absorbance at 600 nm each group.Testing each group COL-2 and COL-1 expression level By Western Bolt experiment.Using Cell counting kit(CCK-8)assay,EdU incorporation assay and CFSE assay to test Chondrocytes proliferation in each group.Chondrocyte apoptosis was measured by flow cytometry.Measurement of caspase-3 activity by fluorescent quantitation and BCL-2,Bax expression level by Western Bolt experiment.Results :Chondrocytes were isolated from rat joints and cells were identified using chondrocyte-specific Alcian blue staining.Western blot analysis of TRPV6 expression confirmed that the induced deletion and overexpression of TRPV6 were effective,Down-regulation of TRPV6 significantly decreased the staining intensity of extracellular proteoglycans,whereas overexpression of TRPV6 markedly attenuated this inhibitory effect.In addition,Western blot analysis showed reduced expression of COL-2 in chondrocytes infected with Lenti-shRNA-TRPV6.However,co-application of Lenti-shRNA-TRPV6 and Lenti-TRPV6 almost completely eliminated the effect of Lenti-shRNA-TRPV6,In contrast,the level of COL-1 expression was up-regulated in chondrocytes infected with Lenti-TRPV6,which was alleviated in chondrocytes co-transfected with Lenti-shRNA-TRPV6 and Lenti-TRPV6.CCK-8 assay,EdU incorporation assay and CFSE assay results show that down-regulation of TRPV6 expression led to decreased chondrocyte viability in a time-dependent manner,whereas overexpression of TRPV6 markedly attenuated this inhibitory effect.Apoptotic progression was monitored in chondrocytes via flow cytometric analysis of PS exposure and plasma membrane integrity.After silencing TRPV6 expression,a population of apoptotic chondrocytes was observed.However,these changes were significantly relieved when chondrocytes were infected with Lenti-TRPV6.Overexpression of TRPV6 significantly attenuated the increased caspase-3 activity caused by TRPV6 gene silencing in chondrocytes.Levels of BCL2 and BAX in chondrocytes were analyzed by Western blot.Silencing of TRPV6 caused a reduction in the BCL2/BAX ratio in chondrocytes.However,this decrease was relieved in chondrocytes infected with Lenti-TRPV6.Conclusions :TRPV6 promotes the secretion of ECM by chondrocytes.TRPV6 promotes chondrocyte proliferation and inhibits chondrocyte apoptosis.