The Critical Role Of ASIC1a-mediated Necroptosis In AA Rat Articular Cartilage Injury And Its Mechanism

Rheumatoid arthritis(RA)is a polyarthritis featured with chronic and systemic inflammatory,resulting in synovitis,articular cartilage and bone erosion,and finally evolves into joint deformity.Cartilage destruction of affected joints is one of the important causes of RA.Necroptosis,a necrotic cell death pathway regulated by receptor interacting protein(RIP)1 and 3,plays many physiological roles in development,as well as additional roles in pathological processes,including degenerative,inflammatory and infectious diseases.However,whether necroptosis is involved in RA articular cartilage damage processes remain unclear.Acid-sensing ion channels(ASICs)belong to theepithelialsodium channel/degenerin family transiently activated by the extracellular H~+.Our previous work indicated that acid-sensing ion channel 1a(ASIC1a)expressed in rat articular chondrocytes and the expression significantly increased in adjuvant arthritis(AA)rat articular cartilage,which provide a novel physiological function of ASIC1a in rat articular cartilage injury.Of note,recent data has provided new ideas that ASIC1a-mediated RIP1 activation contributed to ischemic neuronal injury,and knockout of ASIC1a gene significantly prevented RIP1phosphorylation and brain injury simultaneously,but the exact mechanism is still unclear.Thus,we investigated whether activation of RIP1/RIP3/p-MLKL signaling was linked with ASIC1a-mediated necroptosis in AA rat articular cartilage injury in vitro and in vivo.We mainly carried out the following related experiments:1.Activation of necroptosis in articular cartilage from AA rats and its effects.140-160 g male Sprague-Dawley(SD)rats were used in this study.After 7 days of adaptation,the rats were randomly subdivided into the following groups non-arthritic group(n=8)and AA group(n=40).Then,rats in the AA group were divided respectively into 5 sub-groups by the time points:7 days,14 days,21days,28days,35 days(n=8).The body weight of the rats was weighed in the experiment,and the plasma,the ankle joints and the knee joints were collected for detection of the indicators.The results show that complete freund's adjuvant(CFA)successfully induced the AA rat model.The hind paw appeared obvious red and swelling accompanied with weight loss in AA rats compared with normal rats.Results of hematoxylin and eosin(H&E)staining showed normal rats had intact joints and smooth cartilage without damage while the AA rats were characterized by synovial hyperplasia cartilage destruction forming pannus inflammatory cells infiltrated into synovium bone erosion and cartilage serration.Immunohistochemical stained type II collagen the expression of articular chondrocyte marker was decreased sharply with time dependency.Furthermore,the levels of serum TNF-?and IL-1?in AA rats significantly increased after CFA inoculation.Necroptosis markers RIP1,RIP3,p-MLKL and PGAM5 protein expression increased sharply in the AA group compared to the normal group in rat articular cartilage by western blotting(WB).RIP1 and RIP3 immunohistochemistry results and WB results were consistent,suggesting that the RIP1/RIP3/p-MLKL pathway was activated.Moreover,proinflammatory cytokine TNF-?,IL-1?and IL-6 protein expression increased dramatically in the AA group than the normal group.These results suggest that necroptosis is involved in AA rat articular cartilage injury.2.Effect of ASIC1a-mediated necroptosis on the injury to articular cartilage in AA ratsImmunofluorescence double labeling was used to detect the co-expression of ASIC1a and RIP1 in articular cartilage tissue.In treatment groups,non-arthritic group(n=8)and AA group(n=24).The latter were divided into the following groups(each n=8):untreated AA group;amiloride-treated AA group;Nec-1-treated group.ASIC1a and RIP1 immunostaining signals were highly co-expressed in articular cartilage tissues,which are co-localization in the cytoplasm.With the treatment with Amiloride or Nec-1,the average weight of the rats and type II collagen were increased,the hind paw swelling was decreased and serum TNF-?and IL-1?levels were also decreased compared to the AA group.Additionally,H&E results showed that the pathological damage of articular cartilage was significantly reduced after administration of amiloride or Nec-1.Results of TEM showed that after CFA injection for 5 weeks,many chondrocytes showed markedly swollen,membrane lysis and organelles disappearing.In AA group treated with Nec-1 or amiloride,the integrity of chondrocytes was better preserved,and it was only a slightly swelling.Mechanism experiments demonstrated that Nec-1 or amiloride reduced necroptosis markers RIP1,RIP3,p-MLKL,PGAM5 and pro-inflammatory factors TNF-?,IL-1?and IL-6.The results suggest that blocking ASIC1a can inhibit AA rat articular cartilage necroptosis and relieve articular cartilage damage.3.Effect of extracellular acidification on necroptosis of primary rat articular chondrocytesIn vitro experimental cells are grouped into two groups:rat articular chondrocytes were treated with different pH extracellular acidification for 2 h(pH 7.4,pH 7.0,pH 6.5,pH 6.0,pH 5.5,pH 5.0)and treated with extracellular acid(pH 6.0)for different time periods(0,1,2,3,4 or 5h).Western bloting was used to detect the expression of necroptisis markers RIP1 and RIP3.The results showed that extracellular acidification significantly up-regulated the levels of RIP1 and RIP3 in rat articular chondrocytes in a pH-and time-dependent manner.The expression of RIP1and RIP3 was highest when acidified at pH 6.0 and pH 6.0 for 2 h.4.Effect of ASIC1a on necroptosis of primary rat articular chondrocytesIn vitro experimental cells are grouped into:pretreatment ASIC1a specific blocker PcTX-1 and RIP1 specific blocker Nec-1 group(normal group,pH6.0 group,pH6.0+PcTX-1 group and pH6.0+Nec-1 group);ASIC1a was silenced with ASIC1a short hairpin RNA(shRNA)(normal group,pH6.0 group,pH6.0+PcTX-1 group,pH6.0+ASIC1a-shRNA group and pH6.0+NC-shRNA group).The results showed that both PcTX-1 and Nec-1 could inhibit acid-reduced collagen type II expression and reduce acid-induced RIP1 and RIP3 expression in articular chondrocytes.The same result was verified by ASIC1a-short hairpin RNA.Taken together,our results suggest that activation of ASIC1a by extracellular acidification could induce articular chondrocyte necroptosis.In summary,ASIC1a mediates necroptosis in AA rat articular cartilage injury through RIP1/RIP3/p-MLKL Pathway.These findings indicated that pharmacological blockade of necroptosis could protect articular chondrocytes against acidosis-induced injury,which was associated with activation of ASIC1a.Our study enriches the function of ASIC1a and necroptosis in RA and provides a potential strategy for RA treatment.