The Mechanism Which Astragaloside ? Promotes Osteoblast And Its Effect On APN And MSH In Rabbits With Femoral Head Necrosis

Part 1 Mechanisms that Astragaloside ? promotes proliferation and differentiation of osteoblastsBackgroundNontraumatic osteonecrosis of the femoral head(N-ONFH)is a common and debilitating disorder which has a considerably high incidence from the young to aged adults.Osteoporosis is a generalized skeletal disorder characterized by the imbalance between bone resorption by osteoclasts and bone formation by osteoblasts.Astragaloside ?,one of the major active ingredients in Astragalus membranaceus,has been identified to be involved in regulating the bone metabolism both in vitro and in vivo.However,the beneficial effects of its osteogenic properties and the underlying mechanism remain largely unkown.ObjectiveThe present study is aimed to confirm whether Astragaloside ? can stimulates osteoblast proliferation and differentiation in vitro.Meanwhile,the underlying signaling pathway which mediated the osteogenic effects of Astragaloside ? on MC3T3-E1 cells will also be investigated.MethodsMC3T3-E1 cells were cultured in a-minimal essential medium(?-MEM)containing 10%FBS,1%penicillin and streptomycin at 37? in a humidified 5%CO2 incubator.Then cells were cultured in differentiation medium,followed by incubated with different concentrations of Astragaloside ? for indicated days.Then cell proliferation and ALP activity assay was performed.For mRNA expression analysis,MC3T3-E1 cells were treated as described above for 5 days,and total RNA was extracted by using the TRIZOL reagent according to the manufacturer's instructions and reversely transcribed to cDNA as required by the reverse transcription kit.Then,using SYBR dye method,real-time fluorescence quantitative PCR analysis was performed on ABI 9700 fluorescence quantitative PCR to investigate the effect of drugs on mRNA expression of bone metabolism-related genes.Finally,cells were treated as previously described for 5days and the protein was then extracted with RIPA buffer containing protease inhibitor cocktail(Sigma)and the protein expression levels of catenin,Runx2 and BGP were analyzed by westem blotting.ResultsFirstly,the effects of astragaloside ?(0,5,10or20 ?M)on the proliferation of MC3T3-E1 cells were analyzed by using the MTT assay.As compared with the control group,10 and 20?M of astragaloside ? significantly increased the proliferation of MC3T3-E1 cells at day 2.Similar results were observed when the cells were treated with the same concentrations of astragaloside ? for 3 and 7 days.Next,the effects of astragaloside ? on ALP activity in MC3T3-E1 cells were investigated.The results showed that the ALP activity in MC3T3-E1 cells was modestly increased at day 3 after treated with different concentrations of astragaloside ?.While,when the cells were incubated with astragaloside ? for 5 or 7 days,the ALP activity was markedly increased in a concentration-dependent manner.To further confirm whether the mineralization in MC3T3-E1 cells will be impaired by treating with astragaloside ?,the Alizarin red S staining was performed after the cells were treated with indicated concentrations of astragaloside ?.The results indicated that the mineralized nodule formation was significantly improved by astragaloside ? treatment for about 21 days,which was consistent with the ALP results.We also investigated the effect of astragaloside ? on osteoblastogenesis differentiation and found that treated with astragaloside ? only slightly increased the Runx2 and BGP mRNA levels at low concentration.However,20 ?M of astragaloside ? markedly induced the mRNA expression of Runx2 and BGP.Finally,the underlying mechanism of the effects of astragaloside ? on MC3T3-E1 cells was investigated.We found that Dkk-1,the specific Wnt/?-catenin signaling pathway inhibitor,significantly decreased the astragaloside ?-induced ALP activity in MC3T3-E1 cells.ConclusionThe results indicated that astragaloside ? increased the proliferation and differentiation of MC3T3-E1 cells.Moreover,astragaloside ? promoted ALP production,mineralization and mRNA expression of osteoblast-related genes.These effects may be mediated by activation of the Wnt/?-catenin signaling pathway.Part 2 Study on the correlation between adiponectin and N-ONFHBackgroundUntil now,the underlying mechanisms of pathogenesis are still unclear.Because of the multiple and complex pathological processes,the early diagnosis of this disease is very difficult.lt is necessary to look for a simple and effective biochemical index that is helpful for the early diagnosis of the disease.APN,as an independent predictor of bone mass,shows a negative effect on bone mass in humans.However,the correlation between APN and n-onfh has not been reported.ObjectiveThe purpose of this study was to explore the relationship between adiponectin(APN)level and the severity of the disease in N-ONFH patients,so as to provide an objective basis for early clinical diagnosis and assessment of its severity and prevention.Methods92 healthy normal people and N-ONFH patients with the same sample size were randomly selected to participate in this study.APN levels in serum of all samples were detected by enzyme-linked immunosorbent assay.The Ficat grading system was used to assess the radiographic progress on of the patients.The visual analog scale(VAS),Harris hip scores(HSSS)and Western Ontario and McMaster University Osteoarthritis Index(WOMAC)scores were developed to evaluate the clinical severity of the N-ONFH patients.ResultsThe independent sample t-test showed that the serum APN level in the N-ONFH group was significantly lower than that in the healthy group(t=10.896,P=0.000<0.05).There were significant differences among the three groups in FICAT staging by One-Way ANOVA(Welch=21.796,P=0.000<0.05).In addition,by Spearman correlation analysis,we also found that there was a significant negative correlation between serum APN level and FICAT staging(r=-0.472,P=0.000<0.05).Pearson correlation analysis showed that patients' serum APN level was significantly negatively correlated with VAS score(r=-0.556,P=0.000<0.05),and was also significantly negatively correlated with WOMAC score(r=-0.310,P=0.003<0.05),and significantly positively correlated with Harris(r=0.404,P 0.000<0.05).ConclusionThe decreased serum adiponectin levels were highly related to N-ONFH,and the serum adiponectin levels might be used a sindependent a potential indicator of the severity of the N-ONFH.Part 3 Study on the correlation between MSH and N-ONFHBackgroundIt has been demonstrated that the ?-melanocyte-stimulating hormone(MSH),an endogenous neuropeptide derived from pro-opiomelanocortin(POMC),can suppress inflammation and prevent osteoblast damage.However,the role of MSH in N-ONFH has not been investigated.ObjectiveThe present study is aimed to investigate the critical role of serum MSH in nontraumatic osteonecrosis of the femoral head patients.MethodsIn this study,90 patients diagnosed with N-ONFH and healthy individuals with the same sample size were randomly selected as subjects.The levels of MSH in 180 serum samples were determined by Elisa.Imaging progression in N-ONFH patients was assessed by FICAT staging system.VAS scoring,Harris hip scoring and WOMAC scoring were used to evaluate the severity of symptoms of N-ONFH.As potential biomarkers,serum APN and interleukin-33(IL-33)were also detected and analyzed.ResultsBy One-Way ANOVA,there were significant differences in MSH levels among the three groups divided by FICAT staging(Welch=11.820,P=0.000<0.05).In addition,by Spearman correlation analysis,there was a significant negative correlation between MSH and FICAT staging(r=-0.521,P=0.000<0.05).Pearson correlation analysis showed that MSH was negatively correlated with VAS score(r=00.497,P=0.000<0.05),positively correlated with Harris score(r=0.236,P=0.025<0.05),and not significantly correlated with WOMAC score(r=-0.170,P=0.108>0.05).By further comparing the correlation between MSH,IL-33 and APN,it was found that there was a negative correlation between MSH and IL-33(r=-0.312,P=0.003<0.05),while there was a positive correlation between MSH and APN(r=0.323,P=0.002<0.05).These correlations remained significant after adjustment for age and body mass index.Logistic regression analysis showed that APN and MSH were independent factors affecting the incidence of N-ONFH(OR=0.327[0.217,0.492],0.298[0.206,0.431];P=0.000;0.000<0.05).ConclusionAPN and MSH levels might be used as a potential protective biomarker for N-ONFH.It can also be used as an independent indicator to predict the incidence of N-ONFH.Part 4 Effects of astragaloside ? on APN and MSH in alcohol and steroid-induced femoral head necrosis in rabbitsBackgroundAlthough APN and MSH have a protective effect on non-traumatic femoral head necrosis,there is no evidence of longitudinal comparison before and after the disease.It was reported that astragaloside ? increased the proliferation and differentiation of MC3T3-E1 cells,promoted ALP production,mineralization in vitro,but there is no relevant animal experimental research support.ObjectiveThe main purpose of this study is to investigate the changes of APN and MSH before and after the onset of alcohol-induced and steroid-induced femoral head necrosis in Chinese white rabbits,and the effects of astragaloside ? on APN and MSH,so as to provide an objective basis for the clinical diagnosis of femoral head necrosis by APN and MSH,and to provide basic research for the clinical application of astragaloside ?.MethodsForty-eight rabbits to model alcoholic and steroid necrosis of femoral head respectively were selected and randomly divided into three groups:the normal group,the model group and the experimental group.The experimental group was treated with astragaloside ? lavage intervention on the basis of the model group.Serum APN and MSH levels were detected before experiment and 8 weeks after the experiment.At 8 weeks after the experiment,bone tissue of the femoral head of the experimental white rabbits was taken for pathological examination,and the ratio of empty bone lacuna of each group was recorded.The changes of serum APN and MSH in each group before and after the experiment and the differences of empty bone lacuna rate in each group construction were analysed.ResultsNecrosis of femoral head occurred in different degrees in each model group and experimental group.After the Paired t-test,APN and MSH in the model group and the experimental group were significantly lower than those before the intervention.After One-Way ANOVA,APN and MSH of each group were significantly different after intervention(P=<0.05).In addition,there was a significant difference in the ratio of empty bone lacuna in the alcohol model group and the steroid model group(P=<0.05).ConclusionAPN and MSH were closely related to the incidence of alcoholic and steroid femoral head necrosis in rabbits.Astragaloside ? could improve the level of MSH in alcohol and steroid-induced femoral head necrosis,but there was no effect on the level of adiponectin in alcoholic femoral head necrosis.