TLRs Promote Osteocyte Necroptosis Induced By TNF-α In Postmenopausal Osteoporosis

Part Ⅰ:The correlation between osteocyte necroptosis and TNF-α,TLRs in patients with PMOPObjective:To clarify that osteocyte necroptosis is involved in the occurrence and development of postmenopausal osteoporosis(PMOP),and to further explore the correlation between osteocyte necroptosis and TNF-α,TLRs in PMOP patients.Methods:1.The clinical data and postoperative femoral Ward’s triangle bone tissue specimens of 45 patients with PMOP who underwent hip replacement in the First Affiliated Hospital of Hainan Medical College from December 2021 to December2022 were collected.2.According to the dual-energy X-ray absorptiometry(DXA)identified by the International Society of Clinical Bone Mineral Density(BMD),the bone mineral density of PMOP patients was detected.According to the T value of bone mineral density detection,the patients were divided into osteopenia group,osteoporosis group and severe osteoporosis group,and 15 patients with normal bone tissue were collected as control group.3.The morphological characteristics of bone cells in PMOP patients were observed by transmission electron microscopy.4.HE staining was used to observe the pathological changes of bone tissue cells and the thickness,spacing,integrity and absence of bone cells in PMOP patients.5.The ultrastructure of trabecular bone was scanned by Micro-CT,and the changes of trabecular bone parameters(BV/TV,Tb.N,Tb.Th and Tb.Sp)in PMOP patients were compared.6.The expression of necroptosis-specific proteins(RIP1,RIP3,MLKL),inflammatory signal molecules(TNF-α)and pattern recognition receptors(TLR2,TLR4)in bone tissue of PMOP patients were detected by immunohistochemistry,and the differences were compared.7.The expression of TUNEL~+/RIP3~+,TUNEL~+/MLKL~+,TUNEL~+/TNF-α~+,TUNEL~+/TLR2~+and TUNEL~+/TLR4~+positive protein osteocytes in bone tissue of PMOP patients was detected by immunofluorescence technique,and the differences were compared.8.The co-localization expression of RIP3~+/TNF-α~+and RIP3~+/TLR2~+double positive protein osteocytes in bone tissue of PMOP patients was detected by double-labeled immunofluorescence staining,and the differences were compared.9.Pearson correlation analysis was used to analyze the correlation between the percentage of osteocyte necroptosis(RIP3)and the positive rates of TNF-α,TLR2and TLR4 expression in PMOP patients.Results:1.Under the observation of transmission electron microscope,compared with the normal bone mass(control)group,the osteocytes in the bone tissue of PMOP patients can find typical morphological characteristics of necroptosis.The volume of necroptosis osteocytes increases,the cells and organelles are obviously swollen,the cell membrane is ruptured,the cytoplasmic content overflows,accompanied by the formation of extensive vesicles in the cells.2.The results of HE staining showed that with the progression of osteoporosis in patients with PMOP,the trabecular space gradually increased,the trabecular bone became thinner,and even broken to varying degrees,and the loss of bone cells in bone lacunae increased.3.Micro-CT results showed that compared with the control group,the BV/TV(P<0.05),Tb.N(P<0.05)and Tb.Th(P<0.05)of trabecular bone in the osteopenia group,osteoporosis group and severe osteoporosis group of PMOP patients were significantly decreased,while the Tb.Sp value was significantly increased(P<0.05).4.Immunohistochemistry results showed that necroptosis-specific proteins RIP1,RIP3 and MLKL were mainly expressed in the cytoplasm of bone cells in PMOP patients.The expression of RIP1,RIP3 and MLKL in osteopenia group,osteoporosis group and severe osteoporosis group was significantly higher than that in control group(P<0.01).In addition,the expression levels of inflammatory signal molecule TNF-αand pattern recognition receptors TLR2 and TLR4 protein in osteopenia group,osteoporosis group and severe osteoporosis group were also significantly higher than those in control group(P<0.01).5.Immunofluorescence results showed that TUNEL~+/RIP3~+,TUNEL~+/MLKL~+,TUNEL~+/TNF-α~+,TUNEL~+/TLR2~+and TUNEL~+/TLR4~+proteins were mainly expressed in the cytoplasm of bone cells.Compared with the control group,the percentage of TUNEL~+/RIP3~+and TUNEL~+/MLKL~+positive osteocytes(programmed necroptosis osteocytes)in osteopenia group,osteoporosis group and severe osteoporosis group were significantly increased(P<0.01).In addition,the expression levels of TUNEL~+/TNF-α~+,TUNEL~+/TLR2~+and TUNEL~+/TLR4~+in osteopenia group,osteoporosis group and severe osteoporosis group were significantly higher than those in control group(P<0.01).RIP3~+/TNF-α~+and RIP3~+/TLR2~+proteins were co-localized in osteocytes with necroptosis,and significantly increased in osteopenia group,osteoporosis group and severe osteoporosis group(P<0.01).6.Pearson correlation analysis showed that the percentage of osteocyte necroptosis(RIP3)was positively correlated with the positive rate of TNF-α(r=0.950,P<0.01),TLR2(r=0.910,P<0.01)and TLR4(r=0.943,P<0.01)protein expression.Conclusions:.1.In the progression of PMOP,osteocytes in PMOP patients have undergone necroptosis,and the peak of necrosis may be in the stage of osteoporosis.2.The occurrence of osteocyte necroptosis in PMOP patients was positively correlated with the up-regulation of TNF-α,TLR2 and TLR4 in osteocyte cytoplasm.Part Ⅱ:TLRs enhance MLO-Y4 necroptosis induced by TNF-αin vitroObjective:To verify whether TNF-αpromotes osteocyte necroptosis by up-regulating TLRs at the cellular level.Methods:1.MLO-Y4 cells were divided into control group,TNF-α(vehicle)group,TNF-αgroup was divided into TNF-α+Nec-1s group,TNF-α+GSK,872 group.Nec-1s and GSK,872 were pretreated for 30 min and then treated with TNF-αfor 24h.The expression levels of TLR2 and TLR4 proteins in MLO-Y4 cells during necroptosis were detected by Western blot and compared.It was verified that TNF-αcould up-regulate the expression of TLR2 and TLR4 during osteocyte necroptosis.2.The MLO-Y4 cells were divided into control group and TNF-αgroup,and then the TNF-αgroup was divided into TNF-α+C29 group and TNF-α+TAK242group.After pretreatment with C29 and TAK242 for 30 min,the cells were treated with TNF-αfor 24h.The expression levels of necroptosis-specific proteins RIP3 and MLKL in MLO-Y4 cells were detected by Western blot and the differences were compared to verify that TNF-αtriggers osteocyte necroptosis by up-regulating TLRs.3.The morphological characteristics of MLO-Y4 cells in each group were observed by transmission electron microscopy.Results:1.Western blot results showed that compared with the control group,the expression levels of TLR2 and TLR4 protein in MLO-Y4 cells induced by TNF-αgroup were significantly increased(P<0.01),both Nec-1s and GSK,872 could effectively reduce the expression of TLR2 and TLR4 in MLO-Y4 cells during necroptosis(P<0.01).2.Western blot results showed that compared with the control group,the expression levels of necroptosis proteins RIP3 and MLKL in the TNF-αgroup were significantly increased(P<0.01),both C29 and TAK242 could effectively inhibit the expression of necroptosis proteins RIP3 and MLKL(P<0.01).3.The results of transmission electron microscopy showed that MLO-Y4 cells showed typical morphological characteristics of necroptosis in TNF-αgroup compared with control group.No typical necroptosis cells were observed in MLO-Y4cells treated with C29 or TAK24.Conclusions:1.TLRs are important signaling molecules in TNF-αinduced osteocyte necroptosis.2.The intervention of TLR2 and TLR4 may be one of the ways to effectively inhibit the occurrence of osteocyte necroptosis.