| Objective 1.To investigate whether PARP-1 involved in the progress of cartilage degradation of OA.2.To investigate the role and possible mechanisms of PARP-1 inhibition(including the use of specific inhibitor Olaparib and si RNA)in chondrocyte inflammation,cell senescence and autophagy in vitro.3.To investigate the effect of PARP-1 specific inhibitor Olaparib on the development of cartilage degradation of OA in vivo.Method 1.Detection of the expression of PARP-1 in OA cartilage chondrocytes.To investigate whether PARP-1 is involved in the development of cartilage degradation in OA,we detected the expression of PARP-1,collagen Ⅱ(Col 2)and p ADPr in interleukin-1β(IL-1β)stimulated rat chondrocytes.Moreover,anterior cruciate ligament resection surgery was used to build rat OA model in vivo,and we detected the change of the expression of PARP-1 and Col2 between normal cartilage and OA cartilage with immunohistochemical(IHC)staining.2.Detection of the inflammation related metabolism change in rat chondrocytes.First of all,we detected the expression of inflammatory cytokines and catabolic proteins(i NOS,MMP13 and COX2)in IL-1β stimulated rat chondrocytes.Then,we detected the effects of PARP-1 inhibition,including knocking down the expression of PARP-1 expression of rat chondrocytes with si RNA and the use of PARP-1 specific inhibitor Olaparib(Ola),on the expression of OA-related genes(MMP13,ADAMTS5,i NOS and COX2)in IL-1β induced rat chondrocytes.3.Detection of senescence-related marker in rat chondrocytes.We detected the expression of cell senescence-related marker p16INK4 a 、Acetyl-H3 and aging-related protein SIRT1 with western blot.Meanwhile,NAD+/NADH assay kit with WST-8 was used to determine intracellular NAD+.Investigation of the role of SIRT1 in the effect of PARP-1 inhibition on chondrocyte senescence.We detected the expression of p16INK4 a.SIRT1 specific inhibitor EX527 was used to co-treatment with PARP-1 inhibition in IL-1β-stimulated rat chondrocytes,and detected the change of expression of p16INK4 a.SA-β-gal staining kit was applied to measure the SA-β-gal activity in rat chondrocytes.Coimmunoprecipitation(co-IP)was applied to investigate the protein interaction between PARP-1 and SIRT1.4.Detection of autophagy impairment in chondrocytes.We measured the expression of autophagy-related proteins(Beclin1,p62 and LC3 Ⅰ/Ⅱ)in IL-1β-stimulated rat chondrocytes with western blot,and the change of autophagy flux was confirmed with the m RFP-GFP-LC3 adenovirus.5.Detection of OA-related signaling pathway in chondrocytes.Western blot was applied to measure the activity of NF-κB,MAPK and PI3K/Akt/m TOR signaling pathway in IL-1β-stimulated rat chondrocytes.Co-IP was used to investigate the protein interaction between PARP-1 and NF-κB signaling pathway.The nuclear translocation of p65 was detected with immunofluorescence(IF).To investigate the relationship of activity of SIRT1 and NF-κB signaling pathway,the expression of acetylated histone 3(Acetyl-H3)was detected with western blot in IL-1β-stimulated rat chondrocytes while treated with NF-κB specific inhibitor BAY 11-7085.6.Experiment of OA model in vivo.PARP-1 specific inhibitor Ola was injected into the knee joints of rat OA model to investigate the role of PARP-1 inhibition on the development of cartilage degradation in OA in vivo.H&E staining,Safranin O/Fast Green staining and Toluidine staining of rat cartilage were applied to assess the degree of progression of cartilage degeneration.And IHC staining of MMP13,Beclin1 and p16INK4 a in rat cartilage were applied to evaluate the effects of Ola in vivo.Result 1.In vitro,we found that IL-1β induced the increasing expression of PARP-1 and p ADPr in rat chondrocytes,while induced the decreasing expression of Col2.In vivo,we observed that the number of PARP-1 positive chondrocytes was obviously increased in rat OA model cartilage,while Col2 positive chondrocytes were significantly decreased.2.Ola inhibited the expression of p ADPr but not PARP-1 in IL-1β-stimulated rat chondrocytes while si-PARP-1 decreased both.PARP-1 inhibition included the use of inhibitor and si RNA.PARP-1 inhibition reversed IL-1β induced high expression of COX2,i NOS,MMP13 and ADAMTS5,indicating that PARP-1 inhibition attenuated IL-1β induced inflammatory responses and high expression of extracellular matrixdegrading enzymes.3.IL-1β induced the high expression of Acetyl-H3,p16INK4 a and high activity of SA-β-gal in rat chondrocytes,while decreased the expression of SIRT1 and the level of intracellular NAD+,indicating that IL-1β could promote cell senescence in chondrocytes.PARP-1 inhibition reversed the IL-1β induced the change of Acetyl-H3,p16INK4 a,intracellular NAD+ level and the activity of SA-β-gal,but not the expression of SIRT1.SIRT1 specific inhibitor EX527 reversed the effect of PARP-1 inhibition on IL-1β-induced high expression of p16INK4 a in chondrocytes,indicating that SIRT1 might be involved in the anti-senescence effect of PARP-1 on IL-1β-stimulated chondrocytes.The result of co-IP didn’t show there was direct protein interaction between PARP-1 and SIRT1 in rat chondrocytes.4.PARP-1 inhibition attenuated impaired autophagy in chondrocytes.PARP-1 inhibition increased the expression of autophagy-related proteins Beclin1 and promoted the conversion of LC3 Ⅰ to LC3 Ⅱ in IL-1β-stimulated rat chondrocytes,while decreased the accumulation of p62 and promoted the autophagy flux.5.The results of western blot showed that IL-1β stimulation led to the activation of NF-κB,PI3K/Akt/m TOR signaling pathway and MAPK signaling pathway which including p38,ERK and JNK.Concretely,the levels of phosphorylated p38,ERK and JNK peaked within 15 min after IL-1β exposure,while the levels of phosphorylated IκB,p65,PI3 K,Akt and m TOR peaked within 60 min.And PARP-1 inhibition could suppress the activation of IL-1β-induced NF-κB,PI3K/Akt/m TOR and JNK/MAPK,but not ERK/MAPK、p38/MAPK.The results of IF showed that PARP-1 inhibition could decrease the nuclear translocation of p65 in IL-1β-stimulated rat chondrocytes,and the results of co-IP showed there was protein interaction between PARP-1 and IκB.Besides,we found that the expression of Acetyl-H3 was increased with the activation of NF-κB signaling pathway,indicating that NF-κB activation might inhibited the activity of SIRT1 in chondrocytes.6.We found that the cartilage in OA model group showed severe superficial articular cartilage erosion and loss of compoents of extracellular matrix compared to the sham group.However,Ola treatment could significantly alleviate the cartilage destruction as well as loss of compoents of extracellular matrix.Additionally,the results of IHC showed that Ola treatment increased the expression of Beclin1 and decreased the expression of MMP13 and p16INK4 a,which consistent with the finding in vitro.Conclusion The expression of PARP-1 is increased in rat OA articular chondrocytes in vivo and in vitro.In vitro,PARP-1 inhibition attenuates IL-1β-induced inflammatory responses,high expression of extracellular matrix-degrading enzymes,impaired autophagy and cell senescence in rat chondrocytes.And the recovery of SIRT1 activity might be involved in the anti-senescence effect of PARP-1 inhibition in IL-1β-stimulated chondrocytes.Moreover,PARP-1 inhibition could suppress the activation of JNK/MAPK,PI3K/Akt/m TOR and NF-κB signaling pathway in IL-1β-stimulated chondrocytes,which indicates that these signaling pathway might be involved in the chondro-protective effects of PARP-1 inhibition.In vivo,PARP-1 inhibitor Ola relives cartilage destruction and alleviates cartilage degeneration. |
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