Role And Mechanism Of PANoptosis With Pyroptosis As The Core During The Development Of Cartilage Injury In Osteoarthritis

Objective: Osteoarthritis(OA)is a chronic progressive disease,with articular cartilage destruction as the main pathological characteristics.With the globally inceased aging population and working years,the incidence of OA is higher year by year,leading to the huge economic burden to society and individuals.However,there is still no effective treatment to stop the development of OA.Recent years,mesenchymal stem cells(MSCs)have been widely used in the field of regenerative medicine,and make great progress in the treatment of knee OA,but the exact mechanism is still not clear.PANoptosis is the collective name for Pyroptosis,Apoptosis and Necroptosis,which display the most significant features of programmed cell death(PCD).PANoptosis has been reported to be associated with a variety of diseases,including autoimmune diseases,neurodegenerative diseases,cancer,microbial infections and metabolic diseases.Previously,our group has reported the role of pyroptosis,apotosis and necroptosis in OA respectively,but the relation between PANoptosis as a whole and OA is far to be demonstrated.In this study,we tried to predict the existence of PANoptosis as well as its key genes in OA by employing bioinformatics analysis.Thereafter,we investigated the activation of PANoptosis and its dynamic changes in monosodium iodoacetate(MIA)-induced OA model both in vivo and in vitro.Finally,PANoptosis-related atagonists and human mesenchymal stem cell(h MSC)were used to prove the possibility to treat OA by modulating PANoptosis.Methods:1.In the GEO database,gene expression files,including the control group and OA samples,were downloaded.Human osteoarthritis cartilage tissue data sets,GSE111357,GSE114007,GSE207881 were selected and the differentially expressed genes of OA patients were analyzed by R software.The related genes of Apoptosis,Pyroptosis and Necroptosis were downloaded from KEGG and other access websites to form PANoptosis related genes and key genes,then crossed with OA differential gene to obtain OA differential gene related to PANoptosis.GO,KEGG and GSEA analysis were used to further study the biological process and pathway of differentially expressed PANoptosis key genes in OA,and immune infiltration analysis was performed according to OA data set.2.Right knee joint cavity of rats were injected with 30 μ l MIA solution,concentration: 10mg/ml,to prepare rat OA model.According to the modeling time,rats were divided into four groups: control group(Sham),OA-2W,OA-4W and OA-6W.The weight,gravity distribution and Von Frey score of rats were measured during the whole observation period.The cartilage damage was evaluated by X-ray,gross pathology,H&E and toluidine blue staining.IHC staining,Western blot and q PCR were used to evaluate the protein and gen expression related to cartilage injury,inflammation and PANoptosis pathway.3.Human chondrocytes were isolated and cultured in vitro,and characterized by immunofluorescence staining.The in vitro OA model was established by MIA-injured human chondrocytes.The protein expressions for chondrocyte injury and PANoptosis were evaluated by immunofluorescence staining and Western Blot.MCC950 for NLRP3 inhibition and disulfiram(DSF)for pyropotosis inhibition were added to the in vitro model to investigate their influence on PANoptosis.4.Right knee joint cavity of rats were injected with 30 μ l MIA solution,concentration: 10mg/ml,to prepare rat OA model.After two weeks,h MSCs were injected into the injured knee joint cavity and rats were divided into four groups:Sham,OA,OA+h MSC and Sham+h MSC.The weight,gravity distribution and Von Frey score of rats were measured every week.Pathological samples were collected in two weeks after h MSC transplantation.The cartilage damage was evaluated by X-ray,gross pathology,H&E and toluidine blue staining.IHC staining,Western blot and q PCR were used to evaluate the expressions of proteins and genes related to cartilage injury,inflammation and PANoptosis.Result:1.Bioinformatics analysis found 343 genes related to PANoptosis pathway and23 key genes.The differentially expressed genes in OA cartilage tissue were up-regulated by 2641 and down-regulated by 691.After intersection of OA differential genes and key genes of PANoptosis pathway,there were 8 differentially expressed key genes obtained,among them,7 with up-regulation,and 4 genes derived from Pyroptosis pathway.2.MIA-induced rat OA model(1)The rats were divided into four groups according to the modeling time course:control group,OA-2W,OA-4W and OA-6W.(2)After injection of MIA into the rat knee joint,the weight of OA group decreased compared to the control rats.The weight distribution and Von Frey score of the affected limb decreased in OA group,with the lowest score at 2 weeks post-injury(WPI),and then gradually relieved.(3)X-ray,gross pathology,H&E and toluidine blue staining showed that the cartilage injury of knee joint aggravated during the OA development.At 2 WPI,the cartilage became thinner but still intact by gross observation and pathological staining,and the X-ray changes were not obvious.At 4 WPI,more severe damage was observed along with bone defects,and at 6 WPI,subchondral bone exposure was further visible.(4)IHC staining,Western blot and q PCR detection and scoring showed that the expressions of injury-related and inflammatory proteins and genes in the cartilage were the highest at 2 WPI,and then decreased gradually.At the same time,the expressions of pyroptosis pathway related proteins and genes increased at 2 WPI,and most reached the peak value,lasted until 4 WPI,and declined at 6 WPI.While the expressions of apoptosis and necroptosis pathway executive proteins increased at 2WPI and reached the peak at 4 WPI.3.The in vitro MIA-induced OA model with human chondrocytes(1)The second generation of primary cultured human chondrocytes were characterized by immunofluorescence staining for COL2A1-expressing cells.Different concentrations of MIA were added to the culture,and 0.75 n M was identified as the minimum concentration of MIA with significant difference in cartilage injury and inflammation according to the q PCR tests.(2)The in vitro MIA-induced OA model with human chondrocytes was set up with 0.75 n M MIA and the model was maintained for 24 hours.Then the cells were collected for immunofluorescence staining,and the expressions of Pyroptosis core pathway protein NLRP3 and GSDMD were detected.(3)GSDMD inhibitor DSF(1n M)and NLRP3 inhibitor MCC950(1μM)were added to the in vitro model as described previously.The protein expressions were evaluted by western-blot.After administration of MCC950 and DSF,the chondrocyte injury and inflammation were reduced;meanwhile the pyroptosis pathway,as well as apoptosis and necroptosis pathway,were inhibited.Moreover,MCC950 showed much stronger inhibition on apoptosis and necroptosis.4.h MSC transplantation in OA rats(1)Two WPI,h MSCs were injected into the injured knee joint cavity of rats.The rats were divided into 4 groups: Sham,Sham+h MSC,OA,OA+h MSC.(2)Compared with the control group,the weight of OA rats declined,whearas the weight of h MSC-treated rats increased.The weight distribution and Von Frey score of the affected limb in OA group decreased,and gradually relieved after h MSC treatment.(3)X-ray,gross observation,H&E and toluidine blue staining showed that the knee joint cartilage injury improved after h MSC treatment.(4)IHC staining,Western blot and q PCR detection and scoring showed that h MSC alleviated the injury and inflammatory responses of rat knee joint cartilage,restricted the occurrence of pyroptosis of cartilage tissue,and interrupted apoptosis and necroptosis.(5)IHC staining and Western blot evaluation showed that the expressions of several inflammatory proteins and genes in Sham+MSC group was higher than that in Sham group.Conclusion:1.Bioinformatics analysis revealed that there is the activation of PANoptosis pathway in OA,and the related genes and proteins of Pyroptosis pathway play a significant role in the occurrence and development of OA.2.In MIA-induced OA rats,the PANoptosis pathway was activated.3.The protein expressions of PANoptosis displayed dynamic changes in MIA-induced OA rats;the pyroptosis took place earlier than apoptosis and necroptosis,and was closely associated with OA-related pain and inflammation.4.Human chondrocytes could be employed to set up in vitro OA model.5.In in vitro human OA model,inhibition of NLRP3 inflammasomes and pyroptosis executive proteins could effectively modulate PANoptosis,which might be a novel and hopeful therapeutic target for OA.6.To modulate PANoptosis with pyroptosis as the core might be one possible mechanism for h MSC to treat OA.