Effect Of Necroptosis Mediated RIP1/3 On The Progression Of Postmenopause Osteoporosis And Its Possible Mechanism

PART ⅠNecroptosis mediated RIP1/3 contributes to osteocyte excessivedeath after ovariectomy in ratsObjective:To clarify necroptosis participates in the osteoporosis pathogenesis and it possible mechanisms, we detect the expression of RIP1、RIP3(necroptotic key signaling molecules) in bone of OVX rats, and assess the number of necroptotic osteocyte at different stage of OVX rats and compares their differences.Methods:1. Twelve-week-old healthy female Sprague-Dawley rats were used in this study. Ninety-six rats were randomly divided the ovariectomy(OVX) group(n=48) and the sham group(n=48). SD rats were subjected to ovariectomized surgery in the OVX group and sham surgery in the sham group. Then, rats in the OVX group and the sham group were divided respectively into 4 sub-groups by the time points after surgery: 0 week、4 weeks、8 weeks、12 weeks(n=12).2. Bone mineral density(BMD) was measured by DXA. Ward’s triangle was scanned with a micro-CT system to analyze the 3D microarchitecture of the bone.3. Western blotting, quantitative real-time PCR and immunohistochemical assessments determinated the levels of TNF-α、RIP1、RIP3.The coexpression of RIP1 and RIP3 was observed by laser scanning confocal microscopy. Osteocyte apoptosis and necroptosis were evaluated via TUNEL and immunofluorescence staining for cleaved caspase-3 and transmission electron microscopy.Results:1. BMD of the proximal femur in the OVX rats was significantly reduced by 8 weeks after surgery compared with those of 0 week(P<0.05) and attained the further reduction by 12 weeks. In the OVX group, a 3D reconstruction of Ward’s triangle showed that BV/TV and Tb.N were significantly lower at 8 weeks than at 0 weeks(P<0.05) and attained the further elevation at 12 weeks. Tb.Th of the OVX group achieved a significantly greater at 12 weeks than at 0 weeks(P<0.01). Tb.Sp in Ward’s trigonum of bone tissue of the OVX group rats achieved a significantly greater by 8 weeks after surgery compared with those of 0 week(P<0.05) and attained the further elevation by 12 weeks.(P<0.01).2. Following OVX for 8 weeks, we recorded an interesting phenomenon that most of the osteocytes displayed a necrotic morphology. Furthermore, we observed that RIP1 colocalized with RIP3 in some osteocytes from OVX rats at 8weeks after surgery, especially in the osteocytes cytoplasm, however, obvious necrotic changes and colocalization of RIP1 and RIP3 were not found at 0, 4, 12 weeks. The percentage of TUNEL-positive cells and cleaved caspase-3-positive osteocytes were significantly higher in the OVX rats by 4 weeks than those of the rats from the sham groups(P<0.05), and attained the peak elevation by 8 weeks. After that, a sharp decrease of the percentage of TUNEL-positive cells and cleaved caspase-3-positive osteocytes was found by 12 weeks.3. TNF-α 、 RIP1 and RIP3 protein expression in the osteocytes cytoplasm was assessed by semiquantitative estimation of immunohistochemistry. RIP1 and RIP3 protein expression was no higher in OVX rats at 4weeks than at 0 week, but the expression of TNF-α was significantly higher(P<0.01). TNF-α、RIP1 and RIP3 protein expression were significantly higher in OVX rats at 8 weeks than at 0 week(P<0.01) and reduced at 12 weeks.4. TNF-α、RIP1 and RIP3 protein expression was no higher in OVX rats at 4weeks than at 0 week according to Western blotting analysis, but the expression of them was significantly higher in OVX rats at 8weeks than at 0 week(P<0.05) and reduced at 12 weeks. RIP3 mRNA expression was no higher in OVX rats at 4weeks than at 0 week, but the expression of TNF-α、RIP1 was significantly higher(P<0.01). TNF-α、RIP1 and RIP3 mRNA expression were significantly higher in OVX rats at 8 weeks than at 0 week(P<0.01) and reduced at 12 weeks.Conclusion:1. Necroptotic osteocyte is involved in the process of estrogen deficiency-induced bone loss in this OVX rat mode, and the highest peak of osteocyte necroptosis in the bone of OVX rat was at 8 weeks after ovariectomy.2. TNF-α may be a trigger to the osteocyte necroptosis mediated RIP1 and RIP3.Part ⅡInhibition of necroptosis mediadted RIP1 and RIP3by necrostatin-1 can alleviate progress of osteoprosis in OVX ratsObjective:Nec-1 was administered to rats underwent OVX surgery for exploring the effect of osteocyte necroptosis on contributing to the progression of osteoporosis.Methods:1. Sixty female Sprague-Dawley rats were randomly divided into the OVX group(n=48) and the control group(n=12). All animals were subjected respectively to ovariectomized surgery in the OVX group and sham surgery in the control group. Then, rats in the OVX group were divided into 4 sub-groups: the OVX +vehicle group,the OVX +Nec-1 group,the OVX +Z-VAD group,the OVX+E2 group(n=12). Rats in the SNX+Nec-1 group, the SNX+Z-VAD group and the OVX+E2 group were intraperitoneally injected respectively with Nec-1, Z-VAD, E2. They were injected for 4 weeks once a day.2. Osteocyte apoptosis and necroptosis in the bone tissue of OVX rats submitted to different intervention were evaluated via TUNEL and immunofluorescence staining for cleaved caspase-3. The morphological features of osteocyte death were observed by transmission electron microscopy. Ward’s triangle was scanned with a micro-CT system to analyze the 3D microarchitecture of the bone. The changes of serum PINP and CTX were analyzed by ELISA. The gene and protein expression of caspase-3, RIP1, RIP3 and MLKL were investigated by Western blotting and quantitative real-time PCR, and the localisation of RIP1 and RIP3 in osteocyte was observed by immunofluorescence analyses.Results:1. Nec-1 could inhibit significantly osteocyte necroptotic death, thus reducing the percentage of TUNEL-positive cells(P<0.01).2. Nec-1 inhibited significantly the elevated mRNA and protein expression of RIP1 in the bone tissue of OVX rats(P<0.01). Furthermore,Nec-1 inhibited significantly the protein expression of MLKL. Z-VAD inhibited significantly the elevated mRNA and protein expression of caspase-3 in the bone tissue of OVX rats(P<0.01). however, Z-VAD did not reduce elevated RIP1 and RIP3 levels(P>0.05). The percentage of the localisation of RIP1 and RIP3 was dramatically reduced with Nec-1 treatment(P<0.05).3. Treatment with Nec-1 or Z-VAD obviously decreased the bone resorption and increased the bone formation, thus obviously inhibiting degeneration of the bone microarchitecture(P<0.01).Conclusions:1. Nec-1 could inhibit significantly osteocyte necroptotic death, thus delaying degeneration of the bone microarchitecture in the progression of osteoporosis.2. In addition to apoptosis, necroptosis also participated in the excessive loss of osteocyte in the progression of postmenopause osteoporosis in the OVX rats.PART ⅢNecroptosis mediated RIP1/3 might be induced byTNF-α in murine long bone osteocyte Y4 and possible mechanismObjective:To clarify TNF- a may be a main cause of osteocyte necroptotic death from the cellular level, we investigate whether TNF-α could induce necroptosis in murine long bone osteocyte Y4(MLO-Y4) and its possible mechanism.Methods:At 12、24、36 and 48 hours after cells are pretreated by TNF-α, specimens were respectively harvested. We found out the peak time of cell death by flow cytometry to determine the death rate of MLO-Y4. Nec-1、Z-VAD and RIP3-siRNA were used to pretreat MLO-Y4 cells, then the cells were induced by TNF-α. The flow cytometry was used to detect death rate of the pretreated MLO-Y4 cells and morphological features of the cells was observed by transmission electron microscopy. The protein expression of RIP1、RIP3 、cleaved caspase-3、MLKL and Drp1 were detected by western blot. Finally, RIP1 colocalized with RIP3 was observed using a fluorescence microscopy. The ROS level of cells was detected by DCFH-DA analysis.Results:The percentage of apoptotic or necroptotic MLO-Y4 cells induced by TNF-α increased to the peak at 24 hours. The rate of apoptotic or necroptotic MLO-Y4 cells was dramatically reduced with Nec-1 or Z-VAD or RIP3-siRNA treatment(P<0.01). In the TNF-α group and the Z-VAD group, we could observe a lots of MLO-Y4 cells with typical necroptotic morphological features by TEM. However, obvious necroptotic cells were not found in the Nec-1 and RIP3-siRNA treatment croup. Western blot results revealed that the protein level of RIP1 、MLKL、Drp1 treated by Nec-1 was sharply lower than the TNF-α group(P<0.01).However, Z-VAD did not reduce elevated the level of them. RIP3-siRN effectively down-regulated the protein levels of RIP3、MLKL、Drp1 compared with the TNF-α group(P<0.01). Nec-1 effectively down-regulated the protein levels of RIP1 colocalized with RIP3 compared with the TNF-α group(P<0.01); however, Z-VAD did not reduce levels of RIP1 colocalized with RIP3(P>0.05). Nec-1、Z-VAD and RIP3 siRNA significantly decreased the ROS levels(P< 0.01)Conclusion:1. TNF-α could induce the necroptosis of murine long bone osteocyte Y4 cells.2. RIP1 and RIP3 play vital roles in the necroptotic cell pathway.3. Necroptotic cell death in MLO-Y4 cells might be driven by TNF-α activating the downstream signaling molecules--MLKL、Drp1and ROS.