lncRNA HAGLR对骨关节炎软骨细胞焦亡的影响及机制研究OA

Objective To investigate the effect of lncRNA HAGLR on pyroptosis of chondrocytes in osteoarthritis(OA)and its mecha-nism.Methods Animal experiment:Thirty-four SD rats were randomly divided into the Saline group(injected with 50 μL of normal saline into the left knee joint cavity)and the OA model group(injected with 50 μL of normal saline containing 5 mg monosodium iodoacetate into the left knee joint cavity),with 17 rats in each group.Two weeks after modeling,the degree of cartilage tissue injury was evaluated by HE staining and Safranin O/Fast green staining.The expressions of lncRNA HAGLR and miR-106b-5p were detected by qRT-PCR.The protein expressions of thioredoxin interacting protein(TXNIP),NOD-like receptor family pyrin domain containing 3(NLRP3),Caspase-1,and gasdermin D N-terminal(GSDMD-N)were detected by Western blot.Serum levels of interleukin-1β(IL-1β)and interleukin-18(IL-18)were determined by ELISA.In vitro experiments:The human chondrocyte cell line C28/I2 was divided into the following groups:the control group(cultured under normal conditions),OA group(stimulated with 20 ng/mL IL-1β for 48 hours to establish an in vitro OA model),si-NC group(transfected with si-NC for 24 hours followed by modeling),si-HAGLR group(transfected with si-HAGLR followed by modeling),mimic-NC group(transfected with mimic-NC followed by modeling),miR-106b-5p-mimic group(transfected with miR-106b-5p-mimic followed by modeling),and si-HAGLR+miR-106b-5p-mimic group(co-transfected with si-HAGLR and miR-106b-5p-mimic followed by modeling).Cell proliferation activity was detected by CCK-8 assay.The expressions of lncRNA HAGLR and miR-106b-5p were detected by q RT-PCR.The expressions of IL-1β and IL-18 were detected by immunofluorescence staining and ELISA.The protein expressions of TXNIP,NLRP3,Caspase-1,and GSDMD-N were detected by Western blot.The targeting relationship between lncRNA HAGLR and miR-106b-5p was verified by dual-luciferase reporter gene assay.Results Animal experiments:The cartilage injury in the OA model group was more serious than that in the Saline group.Compared with the Saline group,the OA model group exhibited high Mankin score,upregulated expressions of lncRNA HAGLR,TXNIP,NLRP3,Caspase-1,GSDMD-N,IL-1β,and IL-18,and downregulated expression of miR-106b-5p,with all differences being statistically significant(P<0.05).In vitro experiments:compared with the Control group,the OA group exhibited upregulated expressions of lncRNA HAGLR,TXNIP,NLRP3,Caspase-1,GSDMD-N,IL-1β,and IL-18,significantly decreased cell proliferation activity,as well as downregulated expression of miR-106b-5p,with all differences being statistically significant(P<0.05).Compared with the si-NC group,the si-HAGLR group showed significantly downregulated expressions of the aforementioned pyroptosis and inflammation-related indicators,significantly increased cell proliferation activity,along with upregulated expression of miR-106b-5p,with all differences being statistically significant(P<0.05).Compared with the mimic-NC group,the miR-106b-5p-mimic group showed no significant change in lncRNA HAGLR expression(P>0.05),but an upregulation in miR-106b-5p expression(P<0.05),increased in cell proliferation activity(P<0.05),and downregulation in TXNIP,pyroptosis-related proteins and inflammation-related indicators(P<0.05).The si-HAGLR+miR-106b-5p-mimic group showed further downregulation of the above pyroptosis and inflammatory indicators and further increase of cell proliferation activity(P<0.05).Dual-luciferase reporter gene assay confirmed the existence of direct binding sites between lncRNA HAGLR and miR-106b-5p.Conclusion LncRNA HAGLR promotes TXNIP expression and activates the NLRP3/Caspase-1/GSDMD-N pathway by targeting miR-106b-5p,thereby inducing pyroptosis of OA chondrocytes and exacerbating chondrocyte inflammation and injury.