Protective Effect Of GYY4137 On Chondrocytes Via Inhibiting The NF-κB Pathway And Matrix Metalloproteinase 13 In Vivo And In Vitro

Objective:Osteoarthritis is a chronic joint disease that can lead to progressive cartilage destruction and subsequent joint dysfunction.Over the years,many scholars have devoted themselves to the etiology and mechanism of Osteoarthritis,which are mostly related to metabolism,heredity,local factors,pathophysiology,etc.,inflammatory factors such as interleukin,Tumor necrosis factors and some inflammatory pathways have been shown to play an important role in the development of osteoarthritis.But there is no cure for the disease,and conservative treatment can only slow its progress.Knee replacement can remove diseased joint tissue,but only if the joint is damaged enough to meet the standard of surgery,this process can take decades or even decades,and patients often endure long periods of pain and psychological torture.And the operation cost is high,the postoperative pain is intense,needs the long time recovery and the function exercise,causes the patient to bear the huge material and the mental pressure.Therefore,to find an effective way to reduce the inflammatory reaction and even reverse the destruction of cartilage in osteoarthritis has become the goal of many scholars.It has been demonstrated that endogenous hydrogen sulfide（H₂S）exerts antioxidant and anti-inflammatory properties in Vivo during different inflammatory diseases,which may be due to the inhibition of H₂S on the production of lymphocyte and inflammatory mediators by immune cells.When H₂S is used as an inflammatory regulator,its proinflammatory and anti-inflammatory effects on immune cells depend on its concentration,physiological concentration of H₂S can produce better anti-inflammatory effects,and high concentration of H₂S May aggravate the progress of inflammation.It has been shown that the NF-κB pathway is one of the key targets for its anti-inflammatory effect.In the past few years,the effects of H₂S on the pathophysiology of organisms exposed to inflammatory responses and reperfusion injury in Vivo have been studied,including the effects of H₂S on Arthritis:At low concentrations,H₂S has anti-inflammatory effects on tissues and cells,these results suggest that H₂S has anti-inflammatory effect on arthritis,and high concentration of H₂S has destructive effect on cartilage.Therefore,a number of studies have confirmed that hydrogen sulfide can effectively protect the articular cartilage of knee arthritis,reduce the inflammatory response to cartilage damage.In the past,the release of H₂S from Nash has been used in Cell and animal studies.However,the release of H₂S from Nash is extremely unstable and can not simulate the release process and concentration change of H₂S in organism,and toxic to operators.Therefore,we have found a new H₂S sustained release agent:GYY4137,which can be released slowly in animals or solution,and can simulate the concentration and change of H₂S in animals well.GYY4137,alias p-4-methoxyphenyl-P-4-morpholinyl-dithiophosphoric acid,has the following molecular structure.H₂S Is a H₂S sustained release agent,the rate of H₂S generation is 1lM/hr,has multiple effects on neuroregulation,hypertension,inflammation and vascular tension,and has been used to study various biological effects of H2s.GYY4137 has been shown in several studies to be safe for other uses in animal studies,such as in mice with endotoxic shock,where it has been shown to have an anti inflammatory effect that inhibits the secretion of IL-8 and the proliferation of Primary Human Airway smooth muscle cells,and stimulate the synthesis of anti-inflammatory chemokine IL-10 in rat plasma during sepsis.However,its application in osteoarthritic cartilage protection has not been reported.The authors studied whether GYY4137 also had a protective effect on osteoarthritis.GYY4137 has been used safely in animal experiments,but its application in osteoarthritic cartilage protection has not been reported.The authors studied whether GYY4137 also had a protective effect on osteoarthritis.In this study,we investigated the effects of GYY4137 on osteoarthritic cartilage destruction induced by complete freund’s adjuvant in rats and the expression of inflammatory factors in serum,the expressio n of MMP-13 and its effect on NF-κB signaling pathway in chondroitin inflammatory cells induced by IL-1 were also studied.The completion of this study will lead to more suitable experimental drugs for H₂S research,open GYY4137 for osteoarthritis research blank,further for H₂S for osteoarthritis protection provides a better directionMethods:One:Effects of GYY4317 on cartilage protection and inflammatory factors in CFA induced knee arthritis in rats.Rat Arthritis model was established by complete CFA and right hind limb knee joint.GYY4137 was used in experimental group.After successful establishment of the model,the blood of abdominal aorta was taken from rats,and the knee joint tissue was collected,and the subsequent indexes were measured.SD Rats were divided into three groups:Blank Group,Control Group and Experimental Group.The rats in the control group were injected with CFA and 0.1 ml GYY4137 for 4 weeks.The rats in the experimental group were injected with CFA and0.1 ml GYY4137.1.The swelling degree of knee joint was compared by measuring and comparing the joint diameter of rats.2.Observe the destruction degree of cartilage layer of knee joint in rats.3.The levels of serum-associated inflammatory factors（TNF-αand IL-1β）in rat abdominal aorta were measured by Elisa.4.The cartilage layer thickness of each experimental group was measured and compared by CaseViewer software.5.Hematoxylin and eosin stain and safraninO-Fsat Green staining were used to observe the thickness of cartilage layer,the degree of inflammatory cell infiltration and the destruction of Chondrocytes in the knee joint of ratsTwo:Different concentrations of GYY4137 protect chondrocytes induced by IL-1Human Chondrocytes were cultured and added GYY4137 with different gradients in the medium.The Cultured Human Chondrocytes were divided into three groups:Blank Group,Control Group and Experimental Group.The culture medium was DMEM containing 10%fetal bovine serum and 1%Penicillin（10,000IU）and Streptomycin（10,0000μg/mL）.The chondrocytes in the blank group were cultured in normal medium without any special treatment.IL-1（1ng/ml）medium was added to the control group for induction,and IL-1（1ng/ml）in the experimental group for induction,the medium containing 200,500and 1000 mmo L/L GYY4137 was used.1.The survival rate and inhibition rate of chondrocytes in different concentration GYY4137 medium were determined by CCK-8 method.2.The expression of NF-κB65,p-NF-κB65 and MMP-13 in chondrocytes was determined by Western blot.Results:One:GYY4317 has a protective effect on articular cartilage of knee osteoarthritis induced by CFA and has a good inhibitory effect on inflammation of OA.1:COMPARED WITH CAF induction group,GYY4137 treatment group significantly reduced the mean knee diameter.2:Compared with the Control Group,the mean thickness of cartilage layer decreased in CAF induction group and GYY4137 treatment group,but increased in Gyy4137Treatment Group compared with CAF induction group.3:Staining results:The inflammatory cells in the complete freund’s adjuvant-induced group had more infiltration,and the safranine-solid Green staining showed less blue-green staining,while the GYY4137 group had less infiltration and lighter blue-green staining,but still retained layers.3:ELISA results:The levels of TNF-and IL-1 in CAF induction group and GYY4137treatment group were higher than that in control group.Two:The protective effect of GYY4137 on chondrocytes induced by IL-11:CCK-8 test showed that there was no difference in the survival rate of chondrocytes after 24 h treatment.2:Compared with the blank group,the levels of MMP-13,NF-Bp65,p-NF-b P65 in CAF-induced group were significantly higher.Compared with CAF induction group,GYY4137 treatment group had significantly lower protein level,and different concentration of GYY4137 also affected the results.Conclusion:GYY4137 can effectively reduce the swelling of knee joint and the destruction of cartilage layer,and inhibit the production of inflammatory factors in Vivo.GYY4137 can participate in the regulation of MMP-13,NF-Bp65,p-NF-b P65 expression through NF-b pathway,thus reducing the destruction of cartilage matrix and effectively protecting the cartilage of knee joint.