Therapeutic Effect And Mechanism Of Protectin DX On Osteoarthritis

Objectives: Osteoarthritis is the most common age-related chronic joint disease.At present,the treatment of osteoarthritis is mainly about pharmaceutical treatment,which is mostly limited to the relief of symptoms.The non-steroidal anti-inflammatory drugs have achieved good clinical effects in the clinical treatment and thus are most commonly used.However the side effects(cardiovascular and gastrointestinal tract)cannot be ignored.Therefore,the development of novel anti-inflammatory drugs is imminent.In recent years,it has been discovered that a novel serial of drugs called "specialized pro-resolving mediators" have played a powerful and extensive anti-inflammatory role in many disease models.Protectin DX,the representative one,has been reflected in previous research.Other members of the same family have demonstrated inhibition of arthritis progression in vitro,so the anti-inflammatory effect of protectin DX in arthritis models remains to be further explored.NF-κB,a classic inflammatory pathway,has been proven to be the therapeutic mechanism of many anti-osteoarticular drugs.AMPK,a classic protein widely present in tissues,has been shown to be closely related to the protector DX in previous studies.Whether the AMPK / NF-κB pathway is a protective mechanism of protectin DX needs further investigation.The research above will provide a theoretical basis for new drugs to treat osteoarthritis.Methods: The first part of the study is to use rat primary chondrocytes for research.First,cells were cultured and identificated,and then different concentrations and different time of Protectin DX(PDX)were tested to select the most suitable concentration.Then ELISA,Western blot and RT-qPCR were used to detect the expression of inflammation-related proteins and changes in gene transcription levels.The second part was selected from rat primary synovial fibroblasts for research.First,the culture was identified,and then PDX with different concentrations and different times was selected to select the most suitable concentration gradient.Then the same methods as in the first part were used to test the inflammation-related proteins and genes Changes in transcription levels were detected by Western blot and cellular immunofluorescence to determine whether NF-κB p65 nuclear translocation was found in synovial fibroblasts.In the third part,the knee joint osteoarthritis model was established by injecting iodoacetic acid into the knee joint cavity,and then divided into blank group,OA group,OA + PDX group for drug administration,material extraction,histology,imaging,immunoblot analysis,detection.The therapeutic effect of PDX on osteoarthritis in vivo.In the fourth part,the primary chondrocytes of rats were used for research.According to the results in the first part,the concentration with the greatest anti-inflammatory effect was selected.Using ELISA,Western blot and cellular immunofluorescence methods,PDX or NF-κB was applied on the basis of IL-1β.Inhibitor PDTC,explore the link between NF-κB and downstream proteins,and then PDX and or AMPK inhibitor compound C were added under the same conditions,exploring the link between AMPK activation and NF-κB and downstream inflammation-related proteins.Results:Part 1: Therapeutic effect of protectin DX on primary chondrocytes in inflammatory model rats1.After 24 h and 48 h intervention,the cell viability of PDX at 0.5,1,2 and 4 μM concentrations was not different from that of the blank group,while at 8 μM concentrations,cell viability was significantly reduced.So PDX concentrations of 0.5,1,2 and 4 μM can be used as the working concentration for subsequent experiments.2.After applying IL-1β(10ng / ml)in chondrocytes,it was found that the cell viability was significantly reduced,but the chondrocyte activity was improved after increasing the concentration of PDX.3.IL-1β significantly stimulated the chondrocytes of rats to secrete NO,but NO was gradually decreased after increasing the concentration of PDX.4.IL-1β obviously stimulated the chondrocytes of rats to secrete PGE2,but PGE2 was gradually decreased after increasing the concentration of PDX.5.Under the inflammatory stimulation of IL-1β(10 ng / ml)in each group except the blank group,the intervention group was given an increasing concentration of PDX(1,2,and 4 μM),and the mRNA of inflammation-related genes was detected by RT-qPCR.It was found that IL-1β significantly stimulated the increase of chondrocyte inflammationrelated mRNA expression,but on the basis of increasing the concentration of PDX,the expression of inflammation-related mRNA gradually decreased,which was statistically significant.6.Under the inflammatory stimulation of IL-1β(10 ng / ml)in each group except the blank group,the intervention group was given an increasing concentration of PDX(1,2,and 4 μM).Western blotting was used to detect the levels of inflammation-related proteins.IL-1β significantly stimulated the expression of chondrocyte inflammation-related proteins(iNOS,COX-2,MMP3,MMP13,ADAMTS4,COLLAGEN II),but the expression of inflammation-related proteins gradually decreased after PDX was given an increasing concentration gradient.Is statistically significant.Part 2: Therapeutic effect of protectin DX on primary synovial fibroblasts in inflammatory model rats1.After 24 h and 48 h,the cell viability of PDX in 0.25,0.5,1,2 μM concentration gradients was not different from that of the blank group,but at 4 μM concentration,the cell viability was significantly reduced.So PDX concentrations of 0.5,1 and 2 μM can be used as the working concentration for subsequent experiments.2.IL-1β stimulates the synovial fibroblasts of rats to secrete NO.On the basis of this,the expression of NO is gradually reduced after increasing the concentration of PDX.3.IL-1β significantly stimulated the synovial fibroblasts of rats to secrete PGE2,but PGE2 expression gradually decreased after PDX was given in a dose-dependent manner.4.RT-qPCR detection of mRNA levels found that IL-1β significantly increased the expression of inflammation-related mRNA in rat synovial fibroblasts,but the expression of inflammation-related mRNA gradually decreased after PDX was given in a dosedependent manner.5.Western blotting detected inflammation-related protein levels and found that IL-1β significantly stimulated the expression of inflammation-related proteins(iNOS,COX-2 and MMP3,MMP13)in synovial fibroblasts of rats,but an increasing concentration gradient was given on this basis After PDX,the expression of inflammation-related proteins gradually decreased,which was statistically significant.6.After IL-1β(5 ng / ml)stimulation,NF-κB nuclear translocation was founded in synovial fibroblasts.Under PDX stimulation,synovial fibroblast nuclear translocation phenomenon is reversed and redistributed outside the cell nucleus.Compared with IL-1β,the differences were significant.Part III: Therapeutic effect of protectin DX on the knee joint of osteoarthritis model rats1.The cartilage and surrounding tissues were significantly damaged in the OA group,while the tissue conditions were significantly improved in the abdominal cavity group given PDX.2.The results of HE,Safranin O staining and OARSI score showed that joint destruction was more severe in the OA group than in the blank group,and PDX significantly improved the inflammatory damage.3.Detection of TNF-α in serum and joint cavity lavage fluid by ELISA showed that the level of TNF-α in OA group was the highest in serum and joint cavity lavage fluid,and decreased in the OA + PDX group.4.Western blot experiments showed that type II collagen was most expressed in the blank group,and the OA group was significantly reduced.The addition of PDX improved cartilage destruction.The WB results of MMP13 also confirmed the anti-inflammatory effect of PDX in vivo.Part 4: Mechanism of Protectin DX on Primary Chondrocytes in Inflammatory Rats1.The phosphorylation levels of NF-κB p65 and IκBα increased significantly after IL-1β treatment,and IL-1β caused significant degradation of IκBα.However,PDX can dose-dependently inhibit IL-1β-mediated increase in NF-κB p65 and IκBα phosphorylation.2.Adding PDTC,a selective inhibitor of NF-κB,can partially inhibit the inflammatory response mediated by IL-1β,and the indicators of iNOS,MMP13 and NO are significantly reduced after the application of PDX.3.The phosphorylation level of AMPK is reduced by IL-1β,and then its level is increased by PDX.At the same time,Compound C reduces the degree of AMPK activation caused by PDX.Under the same conditions,the phosphorylation level of p65 was stimulated by IL-1β and then inhibited by PDX.On this basis,the activation level of NF-κB p65 was reduced after Compound C was added.Similarly,the change of this important inflammatory protein,MMP13,is the same as that of P65.4.Most of P65 existed in the cytoplasm of the blank group,and the p65 nuclear shift was manifested after IL-1β stimulation,and PDX could block this expression and retransfer p65 to the cytoplasm.Based on this,compound C reversed the previous nuclear shift phenomenon.Conclusion:1.Rat primary chondrocytes have no cytotoxic response to PDX at concentrations of 1,2,and 4 μM.IL-1β(10ng / ml)has a significant inhibitory effect on rat primary chondrocyte proliferation,and PDX can improve it.Inhibition of chondrocyte viability.2.In the case of IL-1β-induced inflammatory response,PDX can improve its function of causing the synthesis and secretion of cartilage matrix components and degradation.3.In the case of IL-1β-induced inflammatory response,PDX can improve the phenotype change of synovial fibroblasts,especially the synthesis and secretion of cartilage matrix components and the degradation of inflammatory mediators and mechanism degradation enzymes.4.PDX can exert anti-inflammatory effects by inhibiting the translocation of NF-κB from the synovial fibroblasts into the nucleus.5.Iodoacetic acid injection in the knee joint cavity can cause changes in knee arthritis,and intraperitoneal injection of PDX can reduce the local and systemic inflammation of the knee joint.6.In the case of IL-1β-mediated inflammatory response,PDX can block the activation of the NF-κB signaling pathway.7.PDX can exert its anti-inflammatory effect through the AMPK / NF-κB signaling pathway.