The Anti-inflammatory Effect And Mechanism Study Of Dual Specificity Phosphatase 5(DUSP5) In Osteoarthritis

Osteoarthritis(OA)is a common degenerative joint disease manifested by articular cartilage destruction,osteophyte formation and narrow joint space.The main clinical manifestations of osteoarthritis are joint pain,joint swelling,restricted range of motion and joint deformities.And it seriously affects daily activities and quality of life in elderly people.At present,it is generally believed that risk factors such as age,gender,genetics,obesity and genes are closely related to the occurrence and development of osteoarthritis.Unfortunately,the exact pathophysiology and mechanism of OA are still not clear which result the effective therapy of OA is lacking.Numerous studies show that inflammatory response plays an important role in the development of OA.As two classic inflammatory signaling pathways,inhibiting the excessive activation of the NF-?B signaling pathway and the MAPK signaling pathway is believed to effectively reduce cartilage inflammation and cartilage degeneration,and can delay the development of OA.Therefore,finding genes that target these signaling pathways in chondrocytes may provide an effective therapeutic strategy for OA.A large number of studies have shown that Dual Specificity Phosphatase 5(DUSP5)can specifically anchor and dephosphorylate the ERK1/2 pathway in the MAPK signaling pathway.It has been proved that DUSP5 is widely involved in the occurrence and development of many diseases and plays an anti-inflammatory and protective role in them,including pulmonary fibrosis,autoimmune arthritis,cardiovascular disease and cancer,etc.However,the role and function of DUSP5 in OA chondrocytes has not yet been clarified.In this study,we aimed to reveal the anti-inflammatory effect of DUSP5 in osteoarthritis and its mechanism.First,we studied the expression difference of DUSP5 in cartilage tissue of OA patients and normal people.Then,we used small interfering RNA(si RNA)and lentivirus to construct knockdown or overexpression DUSP5 models in vivo and in vitro.Next,we used western-blot,QRT-PCR,Safranin O staining and immunofluorescence to detect characteristic inflammation indicators and related inflammation signal pathway indicators in osteoarthritis.In the end,we further improved the pathogenesis and mechanism of osteoarthritis,and clarified the anti-inflammatory effect and mechanism of DUSP5 in osteoarthritis,providing new ideas for the treatment of osteoarthritis.This research will be divided into the following 3 parts:1.The expression of DUSP5 in the OA cartilage tissue from patients and IL-1?-stimulated rat chondrocytes in vitro;2.The anti-inflammatory effect of DUSP5 in osteoarthritis in vitro and rat model of OA;3.The anti-inflammatory mechanism of DUSP5 in osteoarthritis.Chapter One: The expression of DUSP5 in the OA cartilage tissue from patients and IL-1?-stimulated rat chondrocytes in vitroObjective: To investigate the expression difference of DUSP5 between OA patients and normal people and the expression of DUSP5 in IL-1?-stimulated rat chondrocytes in vitro.Methods: We collected each 6 cases of articular cartilage tissue from patients with hip osteoarthritis and patients with femoral neck fracture who underwent total hip arthroplasty in the Second Affiliated Hospital of Zhejiang University.Then,we used Safranin O staining to study the difference of cartilage tissue between OA patients and normal people.Next,we used tissue immunofluorescence and western-blot technology to detect the expression of DUSP5 in human cartilage tissue.In addition,we applied IL-1? to stimulate rat chondrocytes cultured in vitro to induce an inflammatory response and construct an osteoarthritis model.Then,we used western-blot to explore the relationship between DUSP5 and IL-1?.Results: Safranin O staining shows that cartilage tissue wear and degeneration in OA patients is more significant than in normal people.Besides,tissue immunofluorescence and western-blot results show that the expression of DUSP5 in cartilage tissue of OA patients is significantly lower than that of normal human cartilage tissue(P<0.05).In vitro OA model,with the increase of IL-1? concentration,the expression level of DUSP5 also gradually increased.Under the stimulation of 10 ng/m L IL-1?,the expression of DUSP5 also increased with the extension of the stimulation time.Conclusion: DUSP5 expression was significantly lower in human OA cartilage tissue.While in rat chondrocyte OA model,the expression of DUSP5 increased with the increase of IL-1? concentration and showed a positive correlation with the extension of IL-1? stimulation in a short period of time.We speculate that inflammatory stimulation such as IL-1? will induce the expression of DUSP5 in a short period of time.But osteoarthritis is a chronic disease.Long-term inflammatory stimulation may inhibit the expression of DUSP5.And studies have found that DUSP5 is a short-lived protein that cannot be stably existed in the nucleus for a long time and is easily degraded by the ubiquitin-proteasome system(UPS).This may explain why DUSP5 expression is low in human OA cartilage tissue.Further research is needed.Chapter Two: The anti-inflammatory effect of DUSP5 in osteoarthritis in vitro and rat model of OAObjective: To investigate the regulatory effect of DUSP5 on inflammation in rat in vitro and in vivo osteoarthritis models.Methods: First,we used small interfering RNA(si RNA)or lentivirus to construct DUSP5 knockdown or overexpression rat chondrocytes.Then,in order to study the role of knockdown or overexpression of DUSP5 in IL-1?-induced rat chondrocyte inflammation model,we applied QRT-PCR and western-blot detection technology to detect the mRNA and protein expression of OA inflammatory indexes,MMP3,MMP9,MMP13,COX2 and i NOS.In vivo experiments,we constructed a knee joint OA model in rats by performing destabilization of the medial meniscus(DMM)on the knee joint of rats.Starting from 1 week after surgery,rats were injected with lentivirus into the articular cavity every 2 weeks for a total of 2 injections.50 rats were randomly divided into 5 groups of 10 rats each: The sham group was a control group,performed sham surgery,injected with sterile PBS;Len-OE was an overexpression group,injected with the same amount of DUSP5 overexpression lentivirus;Len-NC was an overexpression control group,injected with the same amount of overexpression control lentivirus;Sh-KD was a knockdown group,injected with the same amount of DUSP5 knockdown lentivirus;Sh-NC was a knockdown control group,injected with the same amount of DUSP5 knockdown control lentivirus.5 weeks after modeling,rats were sacrificed and knee cartilage tissue was taken for QRT-PCR to detect the expression of DUSP5 to verify the knockdown and overexpression efficiency.Finally,we used Safranin O staining and tissue immunofluorescence technology to detect cartilage wear,degeneration and expression of inflammation indicators in rat knee joint sections.Results: In vitro experiments,knockdown experiments showed that the use of si RNA can effectively reduce the m RNA and protein expression levels of DUSP5 in rat chondrocytes(P<0.05).QRT-PCR results showed that knockdown of DUSP5 significantly increased the m RNA expression of i NOS,COX2,MMP9,MMP13,and MMP3 in rat chondrocytes after IL-1? stimulation for 24 hours(P<0.05).Western-blot results also showed that knockdown of DUSP5 significantly increased the expression of iNOS,COX2,and MMP13 proteins in rat chondrocytes after 24 h of IL-1? stimulation(P<0.05).Overexpression experiments showed that the use of lentivirus can effectively increase the m RNA and protein expression levels of DUSP5 in rat chondrocytes(P<0.05).QRT-PCR results showed that overexpression of DUSP5 significantly reduced the m RNA expression of i NOS,COX2,MMP9,MMP13,and MMP3 genes in rat chondrocytes after IL-1? stimulation for 24 hours(P<0.05).Western-blot results showed that overexpression of DUSP5 significantly reduced the expression of i NOS,COX2,MMP13,and MMP9 proteins characteristic of inflammation in rat chondrocytes after 24 h of IL-1? stimulation(P<0.05).In vivo experiments,QRT-PCR results show that injection of lentivirus into the articular cavity can effectively knock down or overexpress DUSP5(P<0.05).Next,we used Safranin O staining and tissue immunofluorescence technology to detect the cartilage wear and degeneration.The results showed that the cartilage wear,degeneration degree and expression of COX2 in the overexpression group were significantly lower than those in the control group.Besides,the cartilage wear,degeneration degree and expression of COX2 in the knockdown group were significantly higher than those in the control group.The results of in vitro and in vivo experiments are consistent which effectively indicate that DUSP5 has an anti-inflammatory effect in osteoarthritis.Conclusion: DUSP5 acts as a protective effect on chondrocytes and an anti-inflammatory effect in osteoarthritis.Chapter Three: The anti-inflammatory mechanism of DUSP5 in osteoarthritisObjective: Explore the anti-inflammatory mechanism of DUSP5 in osteoarthritis.Methods: In isolated rat chondrocytes,we used small interfering RNA(si RNA)or lentivirus to knockdown or overexpress DUSP5.According to references,we used IL-1? for 10 min to induce the inflammation model needed for this part of the experiment.Then,we applied western-blot technology to detect the expression of p65 and p-p65 proteins in the NF-?B pathway and the expression of erk and p-erk proteins in the MAPK pathway.At the same time,we applied cellular immunofluorescence technology to detect the nuclear translocation of p65 to further study the anti-inflammatory mechanism of DUSP5 in osteoarthritis.In addition,in the DUSP5 knockdown experiment,we further designed a rescue experiment,using the specific inhibitor of the NF-?B pathway QNZ and the ERK1/2 pathway specific inhibitor GDC to specifically block the corresponding signaling pathways.Then,we applied western-blot technology to detect the expression of i NOS,COX2,and MMP9 in rat chondrocytes after IL-1? stimulation for 24 hours.Results: After knocking down DUSP5 in rat chondrocytes with si RNA,10 min after IL-1? stimulation,western-blot results showed that p-p65 and p-erk expression were significantly increased(P<0.05).In addition,cellular immunofluorescence showed a significant increase in p65 nuclear translocation compared to the control group.This fully shows that after knocking down DUSP5,IL-1? can induce higher activation of NF-?B and ERK1/2 pathways.And in DUSP5 overexpression group,10 min after IL-1? stimulation,western-blot results showed that p-p65 and p-erk expressions were significantly reduced(P<0.05).Cellular immunofluorescence showed a significant decrease in p65 nuclear translocation compared to the control group.This indicates that after overexpression of DUSP5,the activation of NF-?B and ERK1/2 pathways induced by IL-1? is effectively inhibited.Furthermore,in rescue experiments,after pretreatment of rat chondrocytes with NF-?B pathway specific inhibitors QNZ and ERK1/2 pathway specific inhibitors GDC,the high expression of i NOS,COX2 and MMP9 induced by IL-1? can be effectively inhibited(P<0.05).This further illustrates that DUSP5 does play an anti-inflammatory role in chondrocytes by inhibiting the NF-?B and ERK1/2 signaling pathways.Conclusion: In osteoarthritis,DUSP5 plays an anti-inflammatory role by inhibiting the activation of NF-?B and ERK1/2 signaling pathways.