| Objective:Nowadays the incidence of osteoporosis has been increasing rapidly in the past few years,and it has become a most common disease in the elderly.The reason is due to the rapid development of social civilization and economy,the improvement of living standards,the change of lifestyles and habits,especially in developed cities and areas with serious aging population.It is well known that osteoporosis is seen as a typical aging-related and systemic bone disease,which is characterized by gradually reduced bone mass,bone fragility and susceptibility to fractures.Osteoblasts are functional cells of osteogenesis and responsible for bone formation.The hypoactivation of osteoblasts leads to aging-related osteoporosis.The increase in the number and function of osteoblasts can promote bone formation,which seems to be a primary strategy for osteoporosis treatment.Currently the drugs of prevention and treatment of osteoporosis are primarily focused on promoting the proliferation and differentiation of osteoblasts.Autophagy is the process by which eukaryotic cells degrade and recover damaged macromolecules or damaged cells.Under normal conditions,autophagy is at a basal level on most cells,but it can be activated to maintain cell survival in the case of stress.A large number of recent studies have shown that autophagy,as a cell survival pathway,plays a pivotal role in maintaining bone homeostasis,and the changes in this pathway are also related to the occurrence of osteoporosis to some extent.The silent information regulator of transcription1(SIRT1)plays a vital role in the regulation of various biological functions such as tumor suppression,maintenance of mitochondrial homeostasis,energy metabolism,cellular senescence and cell death.Recently,many studies have found that the activation of SIRT1 in mice is associated with delayed onset of various related diseases such as osteoporosis.Deletion or overexpression of SIRT1 reduces or increases bone mass,respectively.In recent years,resveratrol has attracted much attention because of its beneficial effects on antioxidants,anti-tumor and longevity.In addition,although resveratrol is not fully understood in its biological function,its activation of SIRT1 has been recognized as an important mechanism for improving cell function and health.However,the mechanism by which resveratrol activates SIRT1 on osteoblasts remains to be further elucidated. In this study,we detected the expression of SIRT1,PI3K/Akt/m TOR signaling pathway and autophagy related protein in resveratrol-treated osteoporosis rats,and observed the mechanism of SIRT1 on them in the osteoblasts in resveratrol-treated osteoporosis rats,which may provide a new direction for the prevention and treatment of osteoporosis.Methods: The first part: in vivo animal experiments 1.Osteoporotic animal model established Twenty male SD rats were randomly divided into three groups,the control group(n=5),the osteoporosis group(n=5),and the osteoporosis + resveratrol group(n=10).In the osteoporosis model group,rats were intramuscularly injected with dexamethasone 5 mg/kg twice a week for 6 weeks to induce osteoporosis,while the control group was given a normal saline control.2.Expressions in bone mineral density,proximal femur porosity,alkaline phosphatase,osteocalcin,SIRT1,autophagy-related proteins,and Akt and m TOR pathways in each group After successful modeling in the osteoporosis group,the osteoporosis + resveratrol groups were randomly divided into the low dose group(n=5)and the high dose group(n=5).Seven days after the successful modeling,resveratrol was administered at a dose of 5 mg/kg/d and 45 mg/kg/d(the low-dose group and the high-dose group).After satisfactory anesthesia,2 ml blood sample was collected from the right common carotid artery in a centrifuge tube,centrifuged,and then alkaline phosphatase(ALP)and osteocalcin(OC)were measured by ELISA.The femur was obtained and used for dual-energy X-ray absorptiometry and related software to detect bone mineral density and proximal femoral porosity in each group.The expressions of SIRT1,LC3,Beclin-1,p-m TOR,t-m TOR,p-Akt and t-Akt protein in bone samples of each group were detected by Western blot.The second part: in vitro cell experiments 1.Cell culture and treatment The well-grown mouse MC3T3-E1 osteoblasts were cultured in vitro,and then treated with different concentrations of resveratrol(10-8-10-6M)for 48 h and resveratrol(50 μM).After 5,30,60 and 120 minutes of the osteoblasts,resveratrol was added or not,and the expression of SIRT1 in osteoblasts was detected by RT-PCR and immunofluorescence.The well-grown osteoblasts were inoculated into 96-well plates and divided into normal control,dexamethasone group,resveratrol group,dexamethasone + resveratrol group,resveratrol + nicotinamide(or PI3 K specific inhibitor or m TOR inhibitor or p38 specific inhibitor or JNK specific inhibitor),for 48 h.2.Expressions of SIRT1,PI3K/Akt/m TOR signaling pathway and autophagy related protein in each group The proliferation and viability of each group were tested by Brd U and MTT assay.Western blot was used to detect the expressions of SIRT1,LC3,Beclin-1,Atg7,TOM20,Hsp60,p-m TOR,t-m TOR,p-Akt,t-Akt,p-p38 and p-JNK proteins.The expression of Beclin-1 m RNA was detected by RT-PCR.The morphological changes and the number of mitophagy in osteoblasts were observed by transmission electron microscopy.Results: 1.In vivo experimental osteoporosis rats,the bone mineral density was significantly lower than that of the control group,and that in the resveratrol-treated osteoporosis rats was significantly increased(P<0.05).In addition,the proximal femur porosity of the resveratrol-treated group decreased(P<0.05).Serum ALP and OC were significantly higher in osteoporotic rats than in the control group,and were significantly reduced after treatment with low or high doses of resveratrol(P<0.05).2.In vivo experimental,the results of Western blot showed that the expressions of SIRT1,LC3 and Beclin-1 in osteoporosis rats were significantly lower than those in the control group,and p-Akt and p-m TOR were significantly increased(P<0.05).The expression of SIRT1,LC3 and Beclin-1 were significantly increased in the high-dose resveratrol group,while p-Akt and p-m TOR were significantly decreased(P<0.05),but there were no change in t-Akt and t-m TOR expression between all the groups(P>0.05).3.In vitro study,the RT-PCR results showed that resveratrol promoted the expression of SIRT1 m RNA in osteoblasts in a dose-dependent way,and 10-6M resveratrol significantly enhanced the expression of SIRT1(P<0.05).Western blot results showed that resveratrol promoted the expression of SIRT1 m RNA in osteoblasts in a time-dependent manner,and reached the highest peak after 60 minutes(P<0.05),and then no obivious changes.Immunofluorescence showed that the expression of SIRT1 in osteoblasts treated with resveratrol was significantly higher than that in the blank group(P<0.05).4.Western blot analysis of osteoblasts in vitro showed that the dexamethasone group reduced the expression of SIRT1 in osteoblasts,and the resveratrol treatment group significantly increased the expression of SIRT1 in osteoblasts,while NAM abolish the effect of resveratrol on the expression of SIRT1 in osteoblasts.The Brd U and MTT result showed that compared with the control group,the proliferation and viability of dexamethasone-treated cells were significantly decreased(P<0.05),while the resveratrol-treated group significantly increased the proliferation and viability of osteoblasts(P<0.05).RT-PCR results showed that the expression of LC3 m RNA and Beclin-1 m RNA in the resveratrol-treated group was significantly increased(P<0.05),regardless of whether with dexamethasone or not,but there are not significant change between NAM-treated and control group(P>0.05).Western blot results showed that the expression of autophagy-related protein LC3,Beclin-1 and Atg7 protein were consistent with the above results.5.Transmission electron microscopy results showed that the dexamethasone + resveratrol treatment group showed more mitochondrial autophagic vesicles than the dexamethasone treatment group.Western blot analysis showed that the protein levels of TOM20 and Hsp60 in the resveratrol-treated group were significantly lower than those in the dexamethasone-treated group(P<0.05),but there was no significant change in the control group and NAM-treated group(P> 0.05).6.Western blot analysis showed that the expression of p-Akt and p-m TOR were down-regulated in the resveratrol-treated group compared with the dexamethasone-treated group,while rapamycin and LY294002 eliminated the effect of resveratrol on the phosphorylation of Akt and m TOR in the osteoblasts,but there was no significant difference in the expression of t-Akt and t-m TOR between the groups(P>0.05).The protein activity of p38 and JNK did not change significantly compared with the control group after treatment with or without resveratrol.Conclusions:1.In osteoporosis rats,resveratrol treatment can significantly improve bone mineral density and femur porosity in rats,and reduce the bone transformation and osteoporosis of the rats.2.In resveratrol-treated osteoporosis rats,SIRT1 and autophagy-related protein expression were increased,and the PI3K/Akt/m TOR signaling pathway was inhibited.It indicated that resveratrol may affect the occurrence of autophagy induced by PI3K/Akt/m TOR signaling pathway in osteoporosis rats by enhancing the expression of SIRT1.3.Resveratrol increased the activity of SIRT1 in osteoblasts.In the osteoblasts exposed to dexamethasone group,resveratrol increased the expression of SIRT1,resulting in inhibition of PI3K/Akt/m TOR signaling pathway,decreased the activity of TOM20 and Hsp60,and increased the mitophagy level.These results suggest that resveratrol can enhance the activity of SIRT1,inhibit PI3K/Akt/m TOR signaling pathway,induce mitophagy in osteoblasts,and improve the proliferation and viability of osteoblasts. |
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