Research Of The Mechanism Of Necroptosis On The Pathological Changes In Rat Mandibular Condylar Cartilage

Part I The research of RIP1?RIP3 mediated necroptosis in primary rat mandibular condylar chondrocytes[Objective]To investigate the generation and molecular mechanism of necroptosis induced by treatment of drug in primary rat mandibular condylar chondrocytes,and to study the protective effects of Nec-1 on this progress.[Method]3 weeks SD rats were killed to take the temporomandibular joint condylar cartilage to get primary chondrocytes.Then the chondrocytes were treated with TNF-a(10 ng/L)and cycloheximide(0.2 mM)to induce necroptosis,and a combination of Nec-1 and(or)Z-VAD-FMK were used in this expreiment.The changes of cell size and shape were observed by optical microscope;the cell vitality detection kit was used to check the cell vitality;Using flow cytometry to detect the rate of apoptosis and necrosis;and JC-1 mitochondrial membrane potential detection kits was applied to detect the changes of mitochondrial membrane potential;and cells ROS levels were examined with ROS kits.And qRT-PCR was used to test the expression changes of RIP1,RIP3 and caspase8.[Results]There were no significant changes among the TNF-a alone,the CHX alone and the control group,but the combination treatment of TNF-a and CHX could induce the cell death,the decrease of mitochondrial membrane potential,and the increase of cellular ROS content.And the expression of RIP1,RIP3 and caspase8 also increased.However,after the combination treatment of Z-VAD-FMK,cell death did not changed significantly.Whereas,when Nec-1 was also treated,the number of death cells decreased significantly,and mitochondrial membrane potential and ROS levels restorated,what's more,the expression of RIP 1 and RIP3 also decreased.[Conclusion]The combination treatment of TNF-a,CHX and Z-VAD-FMK could induce the RIP1 and RIP3 dependent necroptosis of primary rat mandibular condylar chondrocytes,and Nec-1 could suppressed it.Part ? The generation and mechanism of necroptosis on the pathological changes in rat mandibular condylar cartilage induced by compressive mechanical stress[Objective]As the only linkage joints in the human body,temporo mandibular joints are in complex mechanical environment.A moderate amount of force is vital for maintaining intra-articular environment steady,while too much physiological stimulation stress can cause its pathological damage and bone cartilage degenerative changes,such as the thinning of the condylar cartilage,the degradation of cartilage matrix and the death of chondrocyte.However,its etiology and pathogenesis is still unclear.The purpose of this study was to explore whether the newly discovered programmed cell death mode-necroptosis participated in the process,as well as its specific molecular mechanism and pathological significance,to provide new train of thought and scientific basis for the prevention and treatment of temporomandibular joint disease caused by abnormal mechanical force.[Method]Male Sprague-Dawley(SD)rats aged 6 weeks were used in this study.According to the animal model of rat temporomandibular joint compressive loading device(patent N.O.:201120210396.4),which was designed by our research group,an upward and backward compressive stress(80 g)was exerted on rat condylar cartilage of the force-applying groups.For the nonforce-applying groups,the same operation was performed,but force was not applied.A combination of necroptosis inhibitor necrostatin-1(Nec-1)and(or)van-caspase inhibitor Z-VAD-FMK were used in this experiment.Hematoxylin-eosin(HE)staining was used to observe the thickness and morphological changes of rat condylar cartilage after applying strength and(or)drug;use transmission electron microscope to observe the apoptosis and necrosis of the chonstocytes;And Polymerase Real-time Chain Reaction(qRT-PCR)and immunohistochemistry(IHC)were applied to detect the expression changes of inflammation relevant factors such as TN-a,IL-1 and MAC-1,and the markers of necroptosis like RIP1,RIP3,and the marker of apoptosis,Caspase 8.[Results]1.HE staining results showed that there were no significant changes between control group and the drug-only groups about the condylar cartilage thickness,but the cartilage thickness of the force-applied group decreased obviously,as the time extened,the degree increased.However,when drug and force were both used,the thinning cartilage recovered,and the effect of Nec-1 was more significant than Z-VAD-FMK.2?The results of ransmission electron microscope showed that a large number of chondrocytes died after condylar cartilage getting force.There were organelles changes such as swelling,rupture in condylar chondrocytes,and there were more necroptosis,other than apoptosis.However,drug treatment can alleviate part of cell death.3?The results of qRT-PCR and IHC showed that the expression of TNF-a,IL-1,MAC-1,RIP1,RIP3 and caspase8 of rat condylar chondrocytes increased significantly during the early force-applied period.And after the articular cavity got injection of Z-VAD-FMK or Nec-1,above situation could be inhibited,and the effect of the combined group was the most obvious,in turn,better than Nec-1 and Z-VAD-FMK.However,the expression of the above proteins did not increase significantly during the later force-applied period,and there were no significant differences among all the groups.[Conclusion]RIP1 and RIP3 mediated necroptosis plays an important role in the pathological changes of the thinning of condylar cartilage caused by compressive stress.And Nec-1,the specific inhibitors of necroptosis,can relieve the thinning effect of the condylar cartilage,and inhibit the cell death of rat condylar cartilage.Nec-1 can reduce the expression of logo molecular of necroptosis to reduce cell death,and reduce the related inflammation,plays a protective effect on rat condylar cartilage.We consider that this approach may have significant potential for translation into a useful anti TMJ OA therapy.