| Objective Osteoporosis is a kind of chronic systemic disease with higher and higher incidence,with the progress of the disease,osteoporosis fractures are often associated with osteoporosis,which has become one of the main reasons for the decline in life quality or even death of the elderly.Resveratrol(RES)is a natural polyphenol compound with similar structure to diethylstilbestrol,which can promote the proliferation,differentiation and mineralization of osteoblasts,but the specific mechanism has not been studied clearly.Recent studies have confirmed that SIRT1/NF-?B signaling pathway plays a very important role in the occurrence and development of osteoporosis.therefore,this study intends to observe the effects of resveratrol on proliferation,differentiation and mineralization of osteoblast MC3T3-E1,as well as its protective effect on rat disused osteoporosis,and explore its mechanism of action through SIRT1/NF-?B signaling pathway regulation,so as to provide experimental basis for resveratrol widely used in clinical treatment of osteoporosis.Methods Osteoblasts MC3T3-E1 were divided into 4 groups,the control group was added with DMEM medium containing 1%fetal bovine serum,the low dose group was added a final concentration of 10-7 mol/L resveratrol in addition to DMEM medium containing 1%fetal bovine serum,the middle dose group was added a final concentration of 10-6 mol/L resveratrol in addition to DMEM medium containing 1%fetal bovine serum,the high dose group was added a final concentration of 10-5 mol/L resveratrol in addition to DMEM medium containing 1%fetal bovine serum.The proliferation rate of osteoblasts MC3T3-E1 at all groups were detected by MTT method,the activity of alkaline phosphatase of osteoblasts MC3T3-E1 at all groups were detected by BCA method,and the bone mineralization of osteoblasts MC3T3-E1 at all groups were detected by the calcium alizarin red staining.The expression of SIRT1/NF-?B signaling pathway related proteins(SIRT1,NF-?B,IkBa)of osteoblasts MC3T3-E1 at all groups were detected by Western Blotting method.60 male Wistar rats were randomly divided into six groups,the control group(n=10):saline gavage without osteoporosis model suscessfully established,the allen sodium phosphate group(n=10):allen sodium phosphate(10 mg/kg)gavage after osteoporosis model suscessfully established,osteoporosis group(n=10):saline gavage after osteoporosis model suscessfully established,low dose group(n=10):the resveratrol(5 mg/kg)gavage after osteoporosis model suscessfully established,middle dose group(n=10):the resveratrol(25 mg/kg)gavage after osteoporosis model suscessfully established,high dose group(n=10):the resveratrol(45 mg/kg)gavage after osteoporosis model suscessfully established.The treatment time of each group was 8 weeks.The bone mineral density and porosity of proximal femur,femoral shaft and distal epiphysis at all rats were measured by X-ray scan,the the histological changes of bone tissue in each group of rats were observe by HE staining,the expression of serum alkaline phosphatase and Osteocalcin(OC)of bone tissue in each group of rats were detected by ELISA,the peak load and the limit stiffness of bone tissue in each group of rats were evaluate by three point bending test,and the expression of SIRT1/NF-?B signaling pathway related proteins(SIRT1,NF-?B,IkBa)of bone tissue in each group of rats were detected by Western Blot.Results There was no significant difference about the proliferation rate,alkaline phosphatase activity of all groups 24 h after osteoblasts MC3T3-E1 cultured(P>0.05),the proliferation rate,alkaline phosphatase activity of the high,middle dose group were significantly higher than those in low dose group and the control group 48 h after cultured(P<0.05),and the proliferation rate,alkaline phosphatase activity of the high dose group were significantly higher than that of the middle dose group(P<0.05),but there was no significant difference between the low dose group and the control group(P>0.05).The proliferation rate,alkaline phosphatase activity of the high,middle,low dose group were significantly higher than that of the control group 72 h after cultured(P<0.05),and there were significant differences between each dose group(P<0.05).With the increased of the drug concentration,proliferation rate and alkaline phosphatase activity all significantly increased(P<0.05).The osteoblast MC3T3-E1 mineralized nodule count of high,middle,low dose group were significantly higher than that of the control group(P<0.05),and there were significant differences between each dose group(P<0.05).With the increased of the drug concentration,cell mineralized nodule count significantly increased(P<0.05).The SIRT1,NF-?B,IkBa protein levels of all groups compared has a significantly different(P<0.05),the SIRT1,IkBa protein levels of drug dose group were significantly higher than that of the control group(P<0.05),and the NF-?B protein level was significantly lower than that of the control group(P<0.05),and with the increased of the drug concentration,the SIRT1 and IkBa protein levels significantly increased(P<0.05),and NF-kB protein levels significantly decreased(P<0.05).The final weight of the normal control group was significantly higher than that of the other experimental group(P<0.05),and there was no significant difference between the other experimental groups(P>0.05).Compared with the normal control group,the bone mineralization density in the osteoporosis group decreased significantly,and the porosity increased significantly.No statistically significant difference between osteoporosis group and low dose of resveratrol group,and the bone mineral densitymiddle dose group,high dose group and sodium phosphate Allen rats significantly increased.The BMD of osteoporosis group was significantly lower than that of the normal control group(P<0.05),and there was no significant difference between the osteoporosis group and low dose group(P>0.05).The BMD of middle,high dose group increased more significantly than that of lower dose group and osteoporosis group(P<0.05),and there was no significant difference between the high dose group and Allen sodium phosphate group(P>0.05),but higher than that of the middle dose group(P<0.05).The serum ALP,OC levels of bone osteoporosis group were significantly lower than that of the normal control group(P<0.05),and there was no statistically significant between the osteoporosis group and low dose group(P>0.05).The serum ALP and OC levels of middle,high dose group increased significantly more than that of low dose group and osteoporosis group(P<0.05),there was no significant difference between the high dose group and Allen sodium phosphate group(P>0.05),but higher than that of the middle dose group(P<0.05).The porosity of osteoporosis group(proximal femur,middle femur and distal femur epiphysis)was higher than that of the normal control group(P<0.05),and there was not statistically significant between the osteoporosis group and low dose group(P>0.05).The porosity of middle dose group and high dose group(proximal femur,middle femur and distal femur epiphysis)decreased significantly than that of low dose group and osteoporosis group(P<0.05),there was no significant difference between the high dose group and Allen sodium phosphate group(P>0.05),but lower than that of middle dose group(P<0.05).The peak load and limit stiffness of osteoporosis group was significantly lower than that of normal control group(P<0.05),and there was no statistically significant between the osteoporosis group and low dose group(P>0.05).The peak load and limit stiffness of middle dose group and high dose group significantly increased more than that of the low dose group and osteoporosis group(P<0.05),there was no significant difference between the high dose group and Allen sodium phosphate group(P>0.05),but higher than that of middle dose group(P<0.05).There was no significant difference about SIRT1,NF-?B,IkBa protein levels between the normal control group,osteoporosis group and low dose group(P>0.05),the SIRT1,IkBa protein levels of the middle dose group and high dose group were significantly higher than that of normal control group,osteoporosis group and low dose group(P<0.05),and the NF-?B protein level was significantly higher than that of normal control group,osteoporosis osteoporosis group and low dose group(P<0.05),There was no statistically significant difference about SIRT1,NF-?B,IkBa protein levels between the high dose group and Allen sodium phosphate group(P>0.05),but the SIRT1 and IkBa protein levels were higher than that of middle dose group(P<0.05),and the NF-?B protein level was significantly lower than that of middle dose group(P<0.05).Conclutions Resveratrol can significantly promote the proliferation,differentiation and mineralization of osteoblasts MC3T3-E1,has an obvious protective effect on rat disuse osteoporosis model,and had dose dependent.It is presumed that this role may be played through the mediating of the SIRT1/NF-?B signaling pathway. |
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