| Objective: With the development of The Times,obesity as a global public health problem has attracted more and more people's attention.Metabolic surgery,as an effective way of weight loss,can not only improve the weight of obese patients but also reduce the occurrence and development of obesity-related diseases.However,the profound abnormal bone metabolism caused by postoperative surgery can not be ignored.There is growing evidence that two of the most common metabolic procedures,gastric bypass surgery(RYGB)and sleeve gastric surgery(SG),can cause abnormalities in bone metabolism.According to the current studies,the possible changes in bone metabolism after metabolism include changes in intestinal structure that affect the absorption of nutrients,decreased mechanical load caused by weight loss,and changes in hormones and adipokines in and out of the gastrointestinal tract.Intestinal flora has been closely related to human beings after a long process of co-evolution with human beings.In recent years,intestinal flora has been proved to be closely related to the occurrence and development of many diseases.Intestinal flora regulates the bone metabolism of the host through a variety of ways,which are inextricably linked with the host.The colonization of intestinal flora itself and the metabolites it produces can act on intestinal endocrine cells and nervous system,and then change intestinal permeability and immune system.At the same time,changes in intestinal flora diversity and phyla can be caused by changes in intestinal anatomical structure and physiology after metabolism.Many studies have confirmed that the changes of intestinal flora after metabolism affect the early and long-term weight loss,the improvement of lipid metabolism and the changes of pancreatic islet function.However,there is still no direct evidence about its relationship with postoperative bone metabolism.Autophagy,as one of programmed cell death,plays an important role in many physiological and pathological processes.Recent studies have confirmed that autophagy is closely related to bone metabolism,and many factors can act on osteoblasts or osteoclasts to play different roles in bone metabolism by activating or inhibiting autophagy pathway.At present,the results of studies on autophagy on bone metabolism are inconsistent.It may be that overactivation or inhibition of autophagy will cause damage to cells,and appropriate autophagy level will promote the functional activity of osteoblasts.Therefore,the selection of a reasonable autophagy regulator and the adjustment of autophagy in an appropriate range is the key issue of autophagy pathway in the prevention and treatment of osteoporosis.As an important pathway of bone metabolism regulation,Wnt signaling pathway can promote the mature differentiation and mineralization of osteoblasts.However,its relationship with autophagy has not been confirmed.Intestinal flora itself and its metabolites affect host bone metabolism through various mechanisms,as a member of the short chain fatty acid butyrate,formed in various diseases as well as the change has played a key role,its role as intestinal flora in metabolites produced by dietary fiber,has been shown to have the effect that the prevention of osteoporosis in vivo and in vitro.Its protective value is closely related to osteoblast differentiation and mineralization.However,detailed mechanism still needs comprehensive analysis.On the basis of the above studies,this study intends to further investigate the changes of intestinal flora and metabolites caused by metabolic surgery,understand the relationship between the above changes and bone metabolism,and study the role of autophagy and its related pathways in it.Further improve the cause and mechanism of abnormal bone metabolism after metabolism.It has important theoretical and practical significance for the prevention and treatment of abnormal bone metabolism after metabolic surgery.Methods:1.Male SD rats induced by high fat were randomly divided into 4 groups,which were divided into group A: sleeve gastric sham operation;Group B: sleeve gastric surgery;Group C: Gastric bypass sham operation;Group D: gastric bypass;2.Serum OCN and CTX1 levels of rats were determined by enzyme-linked ELISA.3.Dual energy X-ray was used to determine the bone mineral density and bone mineral salt content in each part of the rats after metabolism.4.The maximum bending load,maximum bending stress and elastic modulus of tibia of rats in each group were determined by bone biomechanics.5.Micro CT tomography showed bone volume/tissue volume(BV/TV),trabecular thickness(Tb.Th),trabecular number(Tb.N)and trabecular separation(Tb.Sp(mm).6.He staining was performed to observe the changes of bone structure,ALP staining was performed to observe the activity of osteoblasts,TRAP staining was performed to observe the activity of osteoclasts,and the expression of RANKL and OPG was detected by immunohistochemistry.7.The protein expressions of LC3 I/II,P62 and Beclin-1 in each group were determined by Western blot.8.Beclin-1 expression level and location were detected by immunohistochemistry.9.16 s RNA sequencing technology was used to detect the changes in intestinal flora caused by metabolic surgery.10.Liquid chromatography-mass spectrometry was used to detect the changes of intestinal microbiota metabolites in RYGB operation group and sham operation group.11.Alizarin staining and ALP staining were used to observe the bone differentiation and mineralization of osteoblasts in each group.12.The protein expressions of autophagy related proteins and the phosphorylation changes of m TOR were determined by Western blot.Results:1.Both SG and RYGB metabolism surgery can cause a decrease in serum OCN level of bone metabolism marker and an increase in CTX1 level.2.RYGB surgery can cause the decrease of bone mineral density in the whole body and lower limbs of rats.Both SG and RYGB metabolic surgery can cause the decrease of bone mineral salt content in the spine and the whole body.RYGB surgery not only affected the strength,i.e.,the maximum bending load(N),but also affected the stiffness of the femoral tibia,i.e.,the maximum bending stress(MPa),while SG surgery only affected the strength.Both metabolic procedures altered bone microstructures in various aspects in rats.3.Both metabolic procedures can induce bone pathological staining,decrease the expression of ALP,and significantly increase TRAP staining.RANKL expression was significantly up-regulated and OPG expression was significantly down-regulated.4.Both metabolic procedures induced the expression of autophagy related proteins,LC3II/I and Beclin 1 expression were significantly decreased,and p62 protein level was increased.5.Metabolism of gut bacteria detection results suggest that RYGB surgery group and its control of the species diversity decreased,mainly for the low abundance of reduced Numbers of species At the gate level,according to the analysis of thick wall after RYGB fungus door is reduced,and deformation of bacteroidetes bacteria door and warts micro bacteria increased Analysis shows that in the level difference of statistical analysis,visible RYGB surgery decreased significantly higher abundance and is consistent with the negative bone metabolism index changes of several kinds of bacteria are both belong to the thick wall door clostridium outline,cut through the literature is closely connected with change of bone metabolism Therefore,the reduction of related bacteria genera in Firmicutes may be related to the changes in bone metabolism after metabolic surgery,such as the intervention of related bacteria or the treatment target of postoperative bone metabolism abnormalities.6.RYGB can cause metabolic changes in intestinal flora,and these metabolites with differences are mainly classified into lipids,lipid molecules,organic acids and their derivatives.Obviously down-regulated spermidine,isoocholic acid and ursodeoxycholic acid are expected to be metabolic markers of abnormal bone metabolism after metabolism.7.At the same time,the above metabolites were further analyzed through KEGG database,and the metabolic pathways affected were mainly focused on the metabolism of the three nutrients.It was also found that the metabolic pathway of butyric acid was significantly affected,which may be related to abnormal bone metabolism after metabolic surgery.8.Butyric acid significantly increased the expression of key autophagy proteins,such as Beclin 1 and LC3,while significantly decreased the expression of P62 and other proteins.In addition,butyric acid also inhibited the phosphorylation of m TOR protein to some extent.The autophagy level of osteoblasts was significantly changed,and m TOR signaling pathway was involved.9.Both butyric acid and rapamycin induced enhanced staining of ALP,a marker of osteoblast differentiation,and alizarin,a marker of mineralization.These changes can be attenuated by adding 3-MA.Conclusion:1.The changes of bone metabolism after metabolism may be related to the changes of autophagy level of osteoblasts.2.The change of the intestinal flora may be one of the reasons for the abnormal metabolism of the bone after metabolism.3.After metabolism,intestinal flora affects the metabolism of butyric acid through its metabolites,which regulates autophagy through the m TOR signaling pathway and affects bone differentiation and mineralization of osteoblasts. |
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