CTHRC1 Mediates IL-1β Induced Apoptosis In Chondrocytes Via JNK1/2 Signaling

Objective Osteoarthritis(OA),also known as degenerative joint disease or degenerative arthritis,is characterized by chondrocyte apoptosis.The aim of the present study was to investigate the effects of collagen triple helix repeat containing 1(CTHRC1)and the c?Jun N?terminal kinase(JNK)1/2 inhibitor SP600125 on rat chondrocytes cultured in vitro with interleukin(IL)?1β.Methods OA joint fluid samples(n=50)were collected from patients(67.9±7.2 years;male: female,11:39)with OA who underwent knee arthroplasty at the First Affiliated Hospital of Anhui Medical University(Hefei,China)between June 2012 and April2016.Human normal joint fluid samples were collected from 30 patients(62.8±11.2years old;male: female,1:4)with trauma and no history of OA or other joint diseases at The First Affiliated Hospital of Anhui Medical University.IL?1β and CTHRC1 expression in the joint fluid of patients with OA was determined using ELISA kit.Articular chondrocytes were harvested from 20 male 4?week?old Sprague Dawley rats.Chondrocytes were treated with different doses of IL?1β and cell viability and CTHRC1 expression were assessed using Cell Counting Kit?8 and western blot assays,respectively.In separate experiments,chondrocytes were treated with CTHRC1?expressing constructs(p LVX?Puro?CTHRC1)and/or SP600125,or IL?1βwith either CTHRC1 short hairpin(sh)RNA constructs(sh NRA?CTHRC1)or SP600125.The expression of CTHRC1,B?cell lymphoma Bcl-2,Bcl-2 associated X protein(Bax),cleaved caspase?3,poly ADP ribose polymerase(PARP)?1 and matrix metalloproteinase(MMP)?13 was measured using reverse transcription?quantitative polymerase chain reaction and western blotting assays.A Cell Counting Kit?8 assay was performed to examine cell viability.Annexin V/propidium iodide staining and flow cytometry assays were used to detect chondrocyte apoptosis.The expression of JNK1/2 and phosphorylated JNK1/2 was measured using western blotting.Results The levels of IL-1β and CTHRC1 were significantly higher in the joint fluid of patients with OA compared with the normal controls,with IL?1β and CTHRC1 expression 65.7 and 113.6% higher,respectively.Immunohistochemical staining demonstrated that Collagen II and SOX9 were highly expressed in normal primary cultured chondrocytes,which was indicative of a well?established chondrocyte system.CTHRC1 protein expression was increased in chondrocytes in a dose-dependent manner in response to IL-1β.Furthermore,chondrocyte proliferation was suppressed by IL-1β in a dose-dependent manner.The expression of CTHRC1 mRNA and protein was significantly increased in chondrocytes transfected with the p LVX-Puro-C THRC1 vector compared with chondrocytes transfected with the empty p LVX-Puro vector.CTHRC1 upregulation significantly activated JNK1/2,and this activation was markedly reduced by the inhibitor,SP600125.Following transfection with p LVX-Puro-C THRC1 for 24 h,the percentage of apoptotic cells and caspase-3activity were significantly increased in chondrocytes;however,apoptosis and caspase-3 activity were significantly decreased following SP600125 treatment.CTHRC1 upregulation significantly increased the expression of MMP-13,Bax,PARP-1 and cleaved caspase-3(P<0.01),whereas it significantly decreased Bcl-2expression(P<0.01)compared with cells transfected with the empty vector.CTHRC1 downregulation inhibits JNK1/2 activation in IL-1β-induced rat chondrocytes.When chondrocytes were transfected with p LKO.1-C THRC1-sh RNA or SP600125 was added prior to IL-1β,Bcl-2 expression was significantly higher compared with the IL-1β group(P<0.01);however,MMP-13,Bax,PARP-1 and cleaved caspase-3expression were significantly lower.Conclusion In conclusion,the results of the present study suggest that CTHRC1 downregulation promotes chondrocyte proliferation and inhibits apoptosis by directly regulating the JNK1/2 signaling pathway in IL-1β-induced primary rat chondrocytes.CTHRC1 upregulation may strengthen disease progression in an in vitro rat model of OA.CTHRC1 may serve as a novel therapeutictarget for the regeneration of cartilage in patients with OA.