Effect And Mechanism Study Of Gut Probiotic Akk On GC-induced Osteoporosis

BackgroundGlucocorticoid-induced osteoporosis(GIOP)is one of the most common and serious adverse reactions to glucocorticoid therapy.Patients with GIOP have a high incidence of fractures,ranging from 30-50%,which significantly increases the risk of death for patients receiving glucocorticoid therapy and also brings heavy medical burden to families and society.Therefore,it is particularly important to clarify the pathogenesis of GIOP and explore effective prevention and treatment measures.Gut microbiota(GM)are the microorganisms(including bacteria,fungi,viruses,etc.)that colonize our digestive tract.The homeostasis of GM is closely related to host health;while GM dysbiosis may lead to various diseases.In recent years,accumulating evidence has shown that GC can cause GM dysbiosis through multiple pathways.However,whether GC-induced GM dysbiosis is related to the pathogenesis of GIOP remains elusive.Akkermansia muciniphila(Akk)is a probiotic that accounts for 5% of the total gut microbiota in humans.A decrease or lack of Akk is closely related to various diseases.Our previous research has shown that Akk can regulate osteoclast differentiation and exert bone-protective effects by transmitting its secreted extracellular vesicles(EVs)to bone tissue.However,there have been no reports on the changes in Akk after glucocorticoid treatment or whether it is involved in the GIOP process.Many nursing intervention strategies based on regulating gut probiotics(such as directly supplementing a specific probiotic or regulating multiple probiotics through measures such as fecal microbiota transplantation and intermittent fasting)are believed to have health-promoting effects.However,whether these strategies can exert bone-protective effects in GIOP and the mechanism of the gut microbiota in this process remains to be explored.Objectives1.To clarify the changes in the types and abundance of gut microbiota,especially Akk,during the course of GIOP.2.To clarify the effect of various gut microbiota improvement measures(including direct probiotic supplementation,fecal microbiota transplantation and fasting therapy)in increasing Akk abundance and alleviating GIOP.3.To clarify the role of Akk-EVs in entering bone tissue and alleviating the osteoporotic phenotype caused by glucocorticoids.Methods1.Co-housing experiments:(1)Intraperitoneal injection of GC-like drug methylprednisolone induced the GIOP phenotype in mice(GC group),and the GC group mice were caged with normal mice(Veh group)to achieve GM sharing.After the model was constructed,the changes of bone volume,bone strength and bone microstructure were compared among the groups.(2)The changes of GM components in GC and Veh group mice were compared using 16 s RNA sequencing and q PCR.2.Intervention experiments of multiple GM modulation measures:(1)Culturing Akk to intervene in GC group mice,and compare the changes of bone volume,bone strength and bone microarchitecture in different groups of mice.(2)The feces of GC and Veh mice were collected and made into GM suspension,and the above suspension was used to gavage the GC mice to construct the FMT model,and the changes of bone volume,bone strength and bone microstructure of the mice in the different groups were compared.(3)The mice were fasted every other day while the GC intervention was performed to construct the IF intervention model,and the changes of bone volume,bone strength and bone microstructure were compared among different groups of mice.3.Mechanistic experiments for Akk-EVs:(1)After isolation of Akk-EVs from Akk bacteria cultures,(1)their morphological and physicochemical properties were identified.(ii)Fluorescently labeled Akk-EVs were administered by gavage for 3 h and then subjected to NIR tracer analysis.(iii)BMSCs and RAW264.7 were co-cultured with Di O-labeled Akk-EVs for 3h and observed under fluorescence microscopy.(2)In vitro and in vivo functional assays(1)GC or solvent control was given during osteogenesis/osteolysis-induced differentiation of BMSCs and RAW264.7,respectively,while Akk-EVs were used for intervention on this basis.(2)Akk-EVs were used to intervene in GC group and normal mice to observe the changes of bone volume,bone strength and bone microarchitecture in mice after Akk-EVs intervention.Results1.GM dysbiosis and Akk reduction in GIOP:(1)After GC intervention,mice showed obvious OP symptoms,while caging with mice in the Veh group improved the above OP symptoms.(2)Compared with the Veh group,the GM of mice in the GC group showed significant changes,and the persistent reduction of probiotic Akk was especially obvious.In contrast,the number of Akk was significantly increased in the aforementioned GC intervention plus co-housing mice.2.Multiple GM regulation measures can effectively increase the abundance of intestinal Akk bacteria and alleviate GIOP: After GC treatment,Akk in mice were significantly reduced and showed obvious OP phenotype.The reduction of Akk and the phenotype of OP induced by GC could be alleviate by intragastric administration of Akk,supplementation of normal GM and IF intervention.3.Extracellular vesicles(EVs)are the key mechanism by which Akk exert osteoprotective effects in GIOP.(1)Akk can release Akk-EVs into bone tissue and be absorbed by osteogenic/osteoblastic precursor cells:(i)Akk-EVs show a spherical appearance under transmission electron microscopy,with an average diameter between 100-200 nm.(ii)Akk-EVs were able to enter several organs such as bone,brain and spleen and were highly enriched in bone,and a large amount of fluorescent signals were seen on the surface of trabecular bone under fluorescence microscopy.(3)Fluorescence microscopy showed that there was a large amount of fluorescent signal distributed around the nucleus in BMSCs and RAW264.7 cells.(2)Akk-EVs can regulate osteoblast/osteoclast formation and thus produce osteoprotective effects.(1)Akk-EVs can promote osteogenic differentiation and inhibit osteoclastic differentiation,and it is more obvious in the environment of GC presence.(ii)Akk-EVs increased bone formation and decreased bone resorption in mice,and were more pronounced in GIOP.Conclusions1.Glucocorticoids can induce a decrease in the abundance of gut probiotic Akk,which in turn causes imbalance in bone metabolism and leading to GIOP;2.Direct supplementation of probiotic Akk,or improving gut microbiota through bacterial transplantation and intermittent fasting can effectively increase the abundance of gut Akk and alleviate GIOP;3.Akk can release Akk-EVs,which are able to penetrate the intestinal barrier and enter bone tissue to exert bone-sparing effects in GIOP through enhancing bone formation and inhibiting bone resorbing.