| Osteoporosis is listed by the World Health Organization as one of the "three biggest killers" of the elderly.Although there are a variety of causes of osteoporosis,the imbalance between bone formation and bone resorption mediated by osteoblasts and osteoclasts during bone remodeling is the most common pathological mechanism.Most current osteoporosis treatments focus on anti-bone resorption drugs.Although these drugs may prevent further bone loss,they do not solve the problem of impaired bone formation in patients with osteoporosis.Recent studies suggest that inducing mesenchymal stem cells to differentiate into osteoblasts and promote bone formation may be a new method of treatment.At present,there are few reports on effective tools to promote osteogenic differentiation of mesenchymal stem cells,and the mechanism of osteogenic differentiation remains to be further studied.Therefore,it is of great significance to find the molecular mechanism of regulating osteogenic differentiation of mesenchymal stem cells from the important factors that promote osteogenic differentiation for the treatment of osteoporosis.In recent years,more and more attention has been paid to the improvement of osteoporosis by the intestinal flora,which maintains the number of osteoblasts through derived metabolites.However,the reason why intestinal flora maintains the number of osteoblasts and whether it is involved in osteogenic differentiation of mesenchymal stem cells has not been reported.The purpose of this study was to explore the role of intestinal flora in osteogenic differentiation and related mechanisms,and to lay a foundation for the adjuvant treatment of OP with intestinal flora.This research adopts the scheme of mice celiac injection of LPS,successfully established osteoporosis model.HE staining was used to detect bone trabeculae,and it was clear that fecal microbiota transplantation had a good effect on reducing LPS induced osteoporosis.In order to determine whether fecal microbiota transplantation protects osteoporosis by regulating the balance of intestinal flora,the composition and abundance of intestinal flora were evaluated by 16S rRNA sequencing.The a and βdiversity analysis showed that the fecal microbiota transplantation treatment group had higher microflora richness than the LPS group,and there were significant differences between the fecal microbiota transplantation treatment group and the LPS group.The relative abundance analysis of main groups showed that fecal transplantation increased the abundance of Bacteroides and Lactobacillus.These results suggest that fecal microbiota transplantation protects LPS-induced osteoporosis by regulating the balance of intestinal flora,especially bacteroides and Lactobacillus.Damage to osteoblasts is one of the causes of osteoporosis.In order to determine whether intestinal flora plays a protective role by targeting osteoblasts,the expression level of RUNX2,a specific marker for osteoblasts,was detected by immunofluorescence staining in mouse femur sections,and it was found that the expression level of RUNX2 increased after fecal microbiota transplantation.This suggests that fecal microbiota transplantation plays a protective role by maintaining osteoblast numbers.Studies have shown that Bacteroides and Lactobacillus can regulate the taurine level,and the change of taurine content directly affects the expression level of TUG1.Therefore,in order to explore whether the intestinal flora maintains the number of osteoblasts through TUG1,we respectively detected the level of TUG1 in the intestine and blood,and found that the expression level of TUG1 increased after fecal transplantation.These results suggest that fecal microbiota transplantation may have a protective effect by increasing TUG1 levels to maintain osteoblast numbers.Osteoblasts are derived mainly from mesenchymal stem cells.Therefore,the number of osteoblasts is directly affected by the osteogenic differentiation of mesenchymal stem cells.To investigate whether TUGI maintains osteoblast numbers by participating in osteogenic differentiation,we used human umbilical cord mesenchymal stem cells(hUC-MSCs)in vitro.TUG1 was found to promote osteogenic differentiation using overexpressed plasmid and siRNA techniques.ENCORI software found that TUG1 had strong binding ability with miR-138.and double luciferase reporter gene experiment was used to confirm the direct binding between TUG1 and miR-138.and a negative correlation between miR-138 and TUG1 was determined by siRNA and overexpression.To determine the role of miR-138 in promoting osteogenic differentiation of TUG1,mimic and inhibitor transfected cells were used to regulate the function of miR-138.and miR-138 was found to inhibit osteogenic differentiation.The downstream target of miR-138 was further explored by TargetScan prediction,and SIRT1 was found to be the downstream of miR-138.and the dual luciferase reporter gene experiment proved that miR-138 and SIRT1 could bind to each other.Finally,in order to confirm that TUG1 promotes osteogenic differentiation by targeting miR-138 and SIRT1,we co-transfected TUG1 overexpression vector and miR-138 mimic and found that miR-138 mimic reduced the upregulation of SIRT1 and osteogenic marker genes induced by TUG1.These results suggest that TUG1 may competitively bind miR-138 and target the expression of SIRT1,thus promoting the osteogenic differentiation of hUC-MSCs.In this study,we demonstrated that intestinal flora protects LPS-induced osteoporosis in mice by regulating intestinal flora balance and increasing TUG1 level.In vitro experiments,TUG1 was found to promote osteogenic differentiation by targeting miR-138 and SIRT1.This study provides a reference for the development of intestinal flora transplantation as a treatment for osteoporosis,and provides a potential target for the study of osteogenic differentiation of mesenchymal stem cells. |
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