The Effect And Mechanism Of TXNIP/NLRP3 Inflammasome Pathway In Osteoporosis

Objective:This study aimed to investigate the role of the thioredoxin interacting protein（TXNIP）/nucleotide-binding oligomeric domain-like receptor family containing pyrin domain 3（NLRP3）inflammasome pathway in ovariectomized osteoporotic rats,and explore the effect and mechanism of this pathway in mouse osteoblastic precursor cells（MC3T3-E1）under oxidative damage induced by hydrogen peroxide（H₂O₂）,to provide new insights into the prevention and treatment of osteoporosis（OP）.Method:1.An osteoporosis rat model established by ovariectomy operating in a total of 32healthy 12-week-old female SPF SD rats,divided into the sham-operated group（Sham group）,ovarian surgery group（OVX group）,estradiol group（E2 group）with a dosage of 50μg/kg/d,and naringin group（NAR group）with a dosage of 300mg/kg/d.Hematoxylin and Eosin（H&E）staining was used to observe the pathological changes of the left femoral trabecular bone.The left femur underwent micro-computed tomography（micro-CT）scanning and 2D/3D bone reconstruction to assess trabecular bone mineral density（BMD）and bone parameters including bone volume fraction（BV/TV）,trabecular number（Tb.N）,trabecular thickness（Tb.Th）,and trabecular separation（Tb.Sp）.Immunohistochemical（IHC）staining was used to detect the protein expression of TXNIP,NLRP3,apoptosis-associated speck-like protein containing a CARD（ASC）,cysteine-requiring aspartate protease-1（Caspase-1）,and gasdermin D（GSDMD）in femoral tissue.2.To explore the molecular mechanism of TXNIP/NLRP3 inflammasome pathway in osteoblast oxidative damage,MC3T3-E1 cells were treated with different concentrations of H₂O₂（0μM,100μM,200μM,400μM）.The cell viability and the NLRP3 inflammasome pathway-related factors protein expressions were detected.Finally,200μM concentration was selected and used for the subsequent experiment.（1）MC3T3-E1 cells were pretreated with NLRP3 inhibitor MCC950,and then intervened by 200μM H₂O₂ screened above,and divided into Con group（Control group）,H group（200μM H₂O₂group）,and MCC950+H group（NLRP3 inhibitor MCC950+200μM H₂O₂ group）.MC3T3-E1 cell viability,NLRP3 inflammasome pathway proteins expression,inflammatory factor IL-1βlevel,LDH activity,osteogenic differentiation markers,and mineralization indicators were all detected.（2）MC3T3-E1 cells were pretreated with NAC,and then intervened by 200μM H₂O₂,divided into NAC group（N-acetylcysteine group）,H group（200μM H₂O₂ group）,and NAC+H（NAC+200μM H₂O₂ group）.The ROS level,cell viability,NLRP3inflammasome pathway proteins expression,osteogenic differentiation markers,and mineralization indicators in MC3T3-E1 cells were all detected.（3）TXNIP gene in MC3T3-E1 cells was knocked down and overexpressed by lentivirus（LV）and plasmid transfection technology respectively and then intervened by 200μM H₂O₂,divided into PEX-3 group（empty plasmid group）,PEX-TXNIP group(plasmid TXNIP passed expression group,Con group（Control group）,H group（200μM H₂O₂ group）,LV-NC+H group（lentivirus negative control+200μM H₂O₂group）,and LV-TXNIP+H group（lentivirus TXNIP knockdown+200μM H₂O₂ group）.The protein expression of TXNIP,NLRP3 inflammasome pathway,GSDMD,and cell microstructure in MC3T3-E1 cells were all detected.Cell counting kit-8（CCK-8）was used to detect cell viability.Western Blot was used to detect TXNIP,NLRP3 inflammasome pathway-related factors such as NLRP3,ASC,Caspase-1 precursor（pro-Caspase-1）and its activator Caspase-1,interleukin 1βprecursor（pro-IL-1β）and its activator（cleaved-IL-1β）,GSDMD and the N-terminal of GSDMD（GSDMD-N）,and the protein expression of osteogenic differentiation markers such as RUNX2（runt-associated transcription factor 2）and COL1（type I collagen）.Immunofluorescence（IF）combined with the high-content cell imaging system was applied to observe the fluorescence expression of TXNIP,NLRP3,and Caspase-1 in cells.Flow cytometry and 2’,7’-Dichlorofluorescein diacetate（DCFH-DA）probe methods were used to detect the level of intracellular reactive oxygen species（ROS）.Enzyme-linked immunosorbent assay（ELISA）was used to determine the level of IL-1βin the cell supernatant.Lactate dehydrogenase（LDH）kit was to determine the activity of LDH in the cell supernatant.Alkaline phosphatase（ALP）staining and alizarin staining were used to detect the ALP activity and mineralization degree,respectively.At the same time,real-time fluorescent quantitative reversed transcription polymerase chain reaction（RT-PCR）and Western Blot were used to verify the overexpression and knockdown efficiency.Transmission electron microscopy（TEM）was used to observe the changes in the microstructure of cell mitochondria.Results:1.Compared with the Sham group,the femoral trabecular bone in the OVX group decreased,the density decreased and the arrangement was disordered,and more vacuolar cells were seen.The BMD of the OVX group was significantly lower than that of the Sham group.The parameters of the trabecular bone in the OVX group such as BV/TV,Tb.N,and Tb.Th were significantly lower than those in the Sham group,while Tb.Sp was significantly higher than that in the Sham group.Compared with the Sham group,the protein expression TXNIP and NLRP3 inflammasome pathway-related factors such as NLRP3,ASC,Caspase-1,and GSDMD in the femur of the OVX group were up-regulated.The above indexes in the OVX group were significantly different from those in the Sham group（all P<0.05）.After the intervention of estrogen and naringin,compared with the OVX group,the number of bone trabeculae in the E2 group and the NAR group increased significantly,the density increased and the arrangement was regular and orderly,and the vacuolar cells decreased.The BMD of the E2 group and the NAR group was higher than that of the OVX group.Trabecular bone parameters such as BV/TV,Tb.N,and Tb.Th were significantly higher than those of the OVX group,while Tb.Sp was significantly lower than that of the OVX group.The protein expressions of TXNIP and NLRP3 inflammasome pathway-related factors such as NLRP3,ASC,Caspase-1,and GSDMD were down-regulated in the femur of the E2group and NAR group compared with those in the OVX group.The above indexes in the E2 group and NAR group were significantly different from those in the OVX group（all P<0.05）.There was no statistically significant difference in the above indicators between the E2 group and the Sham group.2.（1）Compared with the Con group,the viability of MC3T3-E1 cells intervened by H₂O₂ was significantly reduced,but the NLRP3 inflammasome pathway-related factors in the cells,such as NLRP3,ASC,pro-Caspase-1,Caspase-1,pro-IL-1β,and cleaved-IL-1βprotein expressions were significantly increased,and the above factors increased most significantly when 200μM H₂O₂ was intervened.At the same time,the fluorescence intensity of NLRP3 and Caspase-1 proteins in the cells of the H group was significantly enhanced.In addition,the inflammatory factor IL-1βlevel and LDH activity in the cell supernatant of the H group were significantly increased,while the protein expression levels of the bone differentiation markers COL1 and RUNX2 were significantly reduced,and the ALP activity and mineralization degree were weakened in the H group.There were statistically significant differences in the above indicators between the H group and the Con group（all P<0.05）.After MCC950 pretreatment,the above effects of H₂O₂ intervention on MC3T3-E1 cells were partially alleviated.Compared with the H group,the cell viability in the MCC950+H group was significantly increased,while the protein expression of the NLRP3,ASC,pro-Caspase-1,and Caspase-1 was significantly reduced.At the same time,the fluorescence intensity of the Caspase-1 protein in the cells of the MCC950+H group was significantly weakened.In addition,the level of inflammatory factor IL-1βand LDH activity in the supernatant of cells of the MCC950+H group were significantly reduced.While the expression of bone differentiation marker RUNX2 protein,ALP activity,and mineralization degree was all significantly increased in the MCC950+H group.The above indicators of the MCC950+H group were significantly different from those of the H group（all P<0.05）.（2）After MC3T3-E1 cells were pretreated with antioxidant NAC,the effects of H₂O₂ intervention on MC3T3-E1 cells were partially alleviated.Compared with the Con group,the ROS level of MC3T3-E1 cells intervened by H₂O₂ was significantly increased.Compared with the H group,the cell viability in the NAC+H group increased significantly,while the protein expressions of NLRP3,ASC,pro-Caspase-1,and Caspase-1 in the NAC+H group decreased significantly.At the same time,the Caspase-1 protein fluorescence in the cells of the NAC+H group was significantly weakened.While the protein expression of bone differentiation markers COL1 and RUNX2 in the NAC+H group was significantly increased,and the ALP activity and mineralization degree were all increased（all P<0.05）.There was no statistically significant difference in the above indicators between the NAC+H group and the NAC group.（3）The protein expression of TXNIP was significantly increased in the H group and the fluorescence intensity of TXNIP in the cytoplasm was significantly enhanced.Overexpression of TXNIP up-regulated the protein expression of NLRP3 and ASC in MC3T3-E1 cells,and the expression of NLRP3 and ASC in the PEX-TXNIP group were all significantly higher than that in the PEX-3 group,and the differences were statistically significant（all P<0.05）.Knockdown of TXNIP inhibited the activation of the NLRP3 inflammasome in MC3T3-E1 cells.Compared with the LV-NC+H group,the protein expression of TXNIP,NLRP3,ASC,pro-Caspase-1,Caspase-1,and IL-1βproteins were significantly down-regulated in the LV-TXNIP+H group.Meanwhile,the fluorescence intensity of Caspase-1 protein in cells of the LV-TXNIP+H group was significantly weaker than that of the LV-NC+H group.The protein expressions of GSDMD and GSDMD-N in the LV-TXNIP+H group were also significantly down-regulated.The above indicators in the LV-TXNIP+H group were significantly different from those in the LV-NC+H group（P<0.05）.In addition,MC3T3-E1 cells intervened by H₂O₂ had obvious damage and mitochondrial swelling,while the cell damage and mitochondrial swelling in the LV-TXNIP+H group were significantly less than those in the LV-NC+H group.Conclusion:1.The TXNIP/NLRP3 inflammasome pathway is activated in the femur of ovariectomized osteoporosis rats,which is involved in the occurrence of osteoporosis.2.TXNIP positively regulates the activation of NLRP3 inflammasome pathway and pyroptosis in the process of H₂O₂-induced osteoblast oxidative damage,which suggests that targeted inhibition of TXNIP/NLRP3 inflammasome pathway can reduce H₂O₂-induced osteoblast oxidative damage and pyroptosis,thereby improving osteoblast differentiation.