| Objective: To investigate the role of sodium butyrate(Sodium butyrate,Na B)in the mechanism of osseointegration of titanium nails after osteoporotic fracture surgery.Methods: In this experiment,we used a rat model of osseointegration of titanium nails after debulking osteoporotic fractures and a carbonyl cyanide m-chlorophenylhydrazone(CCCP)-induced oxidative stress model of MC3T3-E1 cells to elaborate the protective effects and molecular mechanisms of sodium butyrate on osseointegration of titanium nails after osteoporotic fractures.1.Cellular experiments: CCCP-induced C3 cells were used to establish Oxidative stress cell model,cells were treated with sodium butyrate and PMA/TPA,and the cells were divided into 4 groups: normal group with titanium plates(CON),CCCP,CCCP+Na B,CCCP+Na B+TPA group and normal group without titanium plates(CON),CCCP,CCCP+Na B,CCCP+Na B+TPA group.Alkaline phosphatase staining(ALP)was applied to each group of cells in the titanium plate-free state using the BCIP/NBT alkaline phospholipase chromogenic kit,and the activity of ALP in each group of cells was detected using the ALP assay kit;each group of cells in the titanium plate-free state was stained using the osteoblast mineralized nodule staining kit,and the calcium nodules were lysed with 10% cetylpyridinium chloride,and then fully lysed in The absorbance(570 nm)of each group of cells was measured under the enzyme standardization instrument.Using the ROS assay kit,each group of cells was treated in advance,and the probe and each group of cells in the state without titanium plates were fully contacted and washed,and directly observed by laser confocal microscope;each group of cells in the state with titanium plates was treated in advance,and each group of cells was digested with trypsin and collected,and the probe and the digested cells were fully contacted and washed,and then detected in flow cytometry.Each group of cells in the titanium plate condition was fixed with electron microscope fixative,stored at 4°,dehydrated with different concentration gradients of alcohol,treated with gold spray,and then observed under scanning electron microscope.Each group of cells in the titanium plate condition was intervened in advance and the distribution of SOD and MDA contents in each group was detected using SOD and MDA assay kits.Immunoblotting technique(Western blot)was used to detect the expression of osteogenic protein BMP2/OCN/COLL-1,apoptosis-related protein BAX,BCL2,CASPASE3,oxidative stress channel protein,PKC,NOX4,NF-κB,etc.2.Animal experiment: 3-week-old female SD rats were taken,and all rats were randomly divided into two groups,sham-operated All rats were randomly divided into two groups: 20 rats in the sham-operated group(Sham group)and 40 rats in the model group(OVX group).The model group was subjected to bilateral ovarian removal(OVX),while the sham-operated group was treated with an equal amount of fat around the ovaries,and ceftiofur sodium was injected intramuscularly for three days after surgery.After three months,the femoral trunks of five female rats in the sham-operated group and the model group were randomly removed,and Mirco-CT bone trabeculae examination and HE staining were performed to establish the postmenopausal osteoporosis model.The rats in the sham-operated and model groups were implanted with titanium nails into the distal femur,and the animals were subsequently divided into three groups,the CON-Ti group(n=15),the OVX-Ti group(n=15)and the OVX-Ti+Na B group(n=15).the CON-Ti group was gavaged with an equal amount of saline,the rats in the OVX-Ti+Na B group were gavaged with Na B(280 mg/kg)The rats in the OVX-Ti group were gavaged with equal volume of saline for 30 days.30 days later,the femoral trunks of the rats were removed and scanned for bone quality around the titanium nail by mirco-CT,and the scanned femoral trunks were decalcified for HE staining and Masson staining,and the remaining femoral trunks were extracted for bone histone protein,and the PKC,PKCα,and NF-κB proteins in bone tissue were detected by WB technique.content.Results: 1.Expression of alkaline phosphatase(ALP)and mineralized nodules in MC3T3-E1 cells in each group:Comparison of the groups of cells in the state without titanium plates:In the CCCP group compared with the CON group,ALP staining showed a lighter blue-purple color,ALP enzyme activity decreased,and its ability and number of expressed mineralized nodules decreased.In the CCCP+Na B group compared with the CCCP group,the shade of blue-purple color of ALP staining,ALP enzyme activity,and ability and number of expressed mineralized nodules were higher than those in the CCCP group.When comparing the CCCP+Na B group with the CCCP+Na B+TPA group,the shade of ALP staining blue-purple,ALP enzyme activity,and the ability and number of expressed mineralized nodules were higher than those of the CCCP+Na B+TPA group.When comparing the CON group with the CCCP+Na B group,the shade of ALP staining blue-purple,ALP enzyme activity,and the ability and number of expressed mineralized nodules were higher than those of the CCCP+Na B+TPA group.The shades of blue-violet color,ALP enzyme activity,and the ability and number of expressed mineralized nodules were higher in the CON group compared with the CCCP+Na B+TPA group than in the CCCP+Na B+TPA group.The shades of blue-violet color,ALP enzyme activity,and the ability and number of expressed mineralized nodules were higher in the CCCP and CCCP+Na B+TPA groups.The shade of blue-purple color,ALPase activity,ability and number of expressed mineralized nodules were lower than those of CCCP+Na B+TPA group.2.the performance of the level of oxidative stress in each group of MC3T3-E1 cells.ROS measurement in the state without titanium plate: four groups of cells compared with each other for ROS fluorescence intensity,where the fluorescence intensity was the highest in the CCCP group,followed by the CCCP+Na B+TPA group,then the CCCP+Na B group,and finally the CON group.The level of oxidative stress in the presence of titanium plates: the peak of ROS detected by flow cytometry was high,SOD content was low and MDA content was high in the CCCP group compared with the CON group.the peak of ROS detected by flow cytometry was low,SOD content was high and MDA content was low in the CCCP+Na B group compared with the CCCP group;the peak of ROS detected by flow cytometry was low,SOD content was high and MDA content was low in the CCCP+Na B group compared with the CCCP+Na B+TPA group.The peak of ROS detected by flow cytometry was low,SOD content was high and MDA content was low;comparing the CON group and CCCP+Na B group,the peak of ROS detected by flow cytometry was low,SOD content was high and MDA content was low;comparing the CON group and CCCP+Na B+TPA group,the peak of ROS detected by flow cytometry was low,SOD content was high and MDA content was low;comparing the CCCP and CCCP+Na B+TPA group,the peak of ROS detected by flow cytometry was high,SOD content was low,and MDA content was high.3.the performance of apoptosis rate of MC3T3-E1 cells in each group: comparison of cells in each group in the titanium plate state: the flow cytometry assay presented a high apoptosis rate in the CCCP group compared with the CON group,both in early apoptosis and late apoptosis.the flow cytometry assay presented a lower apoptosis rate in the CCCP+Na B group compared with the CCCP group,both in early Comparing the CCCP+Na B group with the CCCP+Na B+TPA group,the flow cytometry assay presented a lower rate of apoptosis,both in early and late apoptosis.comparing the CON group with the CCCP+Na B group,the flow cytometry assay presented a lower rate of apoptosis,both in early and late apoptosis.comparing the CON group with the CCCP+Na B group,the flow cytometry assay presented a lower rate of apoptosis,both in early and late apoptosis.comparing the CON group with the CCCP+ Na B+TPA groups compared,flow cytometry assays presented a lower rate of apoptosis,both in early and late apoptosis.CCCP and CCCP+Na B+TPA groups compared,flow cytometry assays presented a high rate of apoptosis,both in early and late apoptosis.4.Effect of protein expression in MC3T3-E1 cells in each group: comparison of protein in each group in the titanium plate state: compared to the CCCP group and the CON group,the expression levels of the osteogenic proteins BMP2、OCN and COLL-1were decreased,the apoptotic protein BAX was increased,Caspase3 was increased and BCL2 was decreased,which subsequently brought about an increase in PKC、NOX4、NF-κB and a decrease in Nrf2 and HO-1 in the CCCP+Na B group,the expression levels of osteogenic proteins BMP2、OCN and COLL-1 were increased,apoptotic protein BAX was decreased,Caspase3 was decreased,and BCL2 was increased,followed by decreased PKC,NOX4,NF-κB and increased Nrf2 and HO-1 in the CCCP+Na B group compared with the CCCP group.In comparison with the CCCP+Na B group,the expression levels of osteogenic proteins BMP2、OCN and COLL-1 were increased,apoptotic protein BAX was decreased,Caspase3 was decreased,BCL2 was increased,and oxidative stress pathway proteins PKC、NOX4、NF-κB were decreased and Nrf2 and HO-1 were increased.In comparison with the CCCP+Na B group,the expression levels of osteogenic proteins BMP2、OCN、COLL-1 expression level was increased,apoptotic protein BAX was decreased,Caspase3 was decreased,BCL2 was increased,oxidative stress pathway proteins PKC、 NOX4、 NF-κB were decreased and Nrf2,HO-1 were increased.comparing CON group with CCCP+Na B+TPA group,expression level of osteogenic protein BMP2、OCN,、COLL-1 was increased,apoptotic protein BAX was decreased,In the CCCP and CCCP+Na B+TPA groups,the expression levels of osteogenic proteins BMP2,OCN and COLL-1 were decreased,apoptotic protein BAX was increased,Caspase3 was increased,BCL2 was decreased,and oxidative stress channel proteins PKC,NOX4,NF-κB were increased and Nrf2,HO-1 were decreased.5.Establishment of osteoporosis model: Mirco-ct assay:Three months after bilateral ovarian removal in rats,Micro-CT examination in OVX group compared with Sham group showed that bone mineral density(BMD),bone volume fraction(BV/TV),bone trabecular thickness(Tb.Th),bone trabecular number(Tb.N)were decreased,while bone trabecular separation(Tb.Sp)HE staining: Tb.N was significantly reduced in the OVX group compared to the Sham group.6.SD rats were established as a titanium nail osseointegration model,and after 1month of sodium butyrate(280 mg/kg)gavage,Micro-CT results showed that BMD,BV/TV,Tb.Th,Tb.N were lower in OVX-Ti group rats and Tb.Sp were higher in Con-Ti group rats,while after administration of Na B,BMD,BV/TV,Tb.Th,Tb.N were higher in Na B-Ti group rats and Tb.Sp were lower than in Con-Ti group rats.The expression of PKC,PKCα and NF-κB in the OVX-Ti group was higher than that in the Con-Ti group,while the expression of PKC,PKCα and NF-κB in the Na B-Ti group was lower than that in the OVX-Ti group,but the expression of PKC,PKCα and NF-κB in the NAB-Ti group was higher than that in the Con-Ti group.were lower than those of the OVX-Ti group but higher than those of the CON-Ti group.7.HE staining showed that the number of bone trabeculae around the titanium nail in the OVX-Ti group was much lower than that in the CON-Ti group,while the number of bone trabeculae around the titanium nail in the Na B-Ti group was higher than that in the OVX-Ti group but lower than that in the CON-Ti group.Masson staining showed that the number of new bone around the titanium nail in the OVX-Ti group was much lower than that in the CON-Ti group,while the number of new bone around the titanium nail in the Na B-Ti group was The number of new bone in the Na B-Ti group was higher than that in the OVX-Ti group but lower than that in the CON-Ti group.Conclusion: Sodium butyrate enhances titanium nail osseointegration in devitalized rats by inhibiting PKC/NOX4 and NF-κB oxidative stress channels. |
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