| Part 1:D-galactose induced sarcopenia animal model and mechanism exploreObjectiveSarcopenia is a chronic degenerative disease that seriously affects the life quality of the elderly by increasing the fall rate and the all-cause mortality in the elderly.So,more attention needs to be paid to the study of muscle wasting caused by aging.This study first designed to establish the model of sarcopenia based on the D-galactose aging model,and explore the drug dosage,treatment cycle and appropriate evaluation method for the diagnostic criteria of sarcopenia.By studying the changes in muscle mass,muscle strength and physical performance induced by D-galactose in C57 mice,a fast and convenient modeling method of sarcopenia was obtained and the changes in its biological indexes were explored.In addition,Wnt signaling pathway is an important signal pathway in the aging process of skeletal muscle and affects the myoblast differentiation process.So,this study was designed to explore the role of this pathway in the process of muscle wasting in the sarcopenia model.Methods18 male C57 aged 8 weeks were divided into 3 groups:control group:0.9%intraperitoneal injection of saline solution,low dose group(D-GAL-L:200 mg/kg-galactose saline solution),high dose group(D-GAL-H:400 mg/kg/D-galactose saline solution),continuous intraperitoneal injection for 8 weeks.Food intake,body weight and grip strength were measured once a week.24 h after the last administration,the rota drop times of the mice were measured with the rota meter,and the body composition of the fraction was measured by DXA after anesthesia the next day,so as to comprehensively evaluate the muscle mass and physical performance of the mice.After the muscle mess measured,abdominal aorta blood was collected.The content of myostatin(GDF-8)in serum was detected by ELISA kit.After blood collection.tibial anterior muscle,gastrocnemius,quadriceps femoris.and biceps femoris were obtained.The changes of muscle fiber were observed by HE stains of tibial anterior muscle.Then,western blot and Real-T ime PCR were used to detect the differences in Wnt signal-related protein expression levels in skeletal muscles of the three groups.ResultsThe different concentrations of D-galactose model group mice have all been apparent to aging condition.The handgrip strength of mice in the treatment group was lower than that in the control group,but there was no statistical significance(P=0.07).Meanwhile,the rod-drop time of mice in the treatment group was significantly lower than that in the control group(P<0.05).The weight of the mice kept increasing during the treatment,and there was no significant difference between the groups after 8 weeks.The ratio of lean body weight and lean body weight to total body weight in the D-galactose model group was significantly lower than that in the control group(P<0.05).HE stains of the model group tibial anterior muscle showed that the intermuscular space was enlarged.In the model group,serum SOD decreased significantly(P<0.01)and serum MDA increased significantly(P<0.05).In addition,the serum levels of myostatin(GDF-8)in mice were significantly increased,while the relative mRNA expression of GDF-8 was significantly up-regulated.However,the IGF-1 receptor and IGF-1 of quadriceps femoris had no significant change.In addition,the expression level of GSK-3 in the D-galactose treated model group was increased,and β-catenin was also increased.Meanwhile,the expression of c-myc was also slightly up-regulated.ConclusionContinuoued D-galactose intraperitoneal injection for eight weeks with a dose of 400 mg/kg,can result in C57BL/6 mice aging with muscle atrophy,accompanied by a drop-in muscle strength,muscle mass decreases and motion ability decreases.D-galactose treatment may inhibit myoblast differentiation and reduce myoblast proliferation by activating the Wnt classical pathway.Meanwhile,increased expression of genes associated with muscle atrophy led to sarcopenia in mice.Part 2:D-galactose induced sarcopenia in vitro model and mechanism researchObjectiveC2C12 is a muscle to generate skeletal muscle cells in the process of precursor cells,and muscle growth repair process requires muscle satellite cells are activated to become into muscle cells further differentiated into muscle fiber cells.In animal models,we have found that D-galactose treated mice show symptoms of reduced muscle mass,reduced muscle strength,and decreased motor ability,but the mechanism of D-galactose leading to sarcopenia is not clear.Therefore,we hope to use C2C12 myoblasts to discuss the effect of D-galactose on muscle in vitro environment.At the same time,explore the mechanism of D-galactose causing sarcopenia in mice.MethodsC2C12 myoblast in different concentrations in D-galactose,with CCK8 method to detect the change of the cell proliferation activity,with the change of flow cytometry instrument to detect the cell cycle,with galactose glucoside enzyme detection into whether the muscle cell senescence.The changes of Pax7 and MyoD were detected by western blot and immunosilver light.ResultsCompared with the control group.the proliferation activity of D-galactose treatment group decreased in concentration and time dependence.The proportion of G1 and G2 cells in the D-galactose treated group increased.The smaller S phase ratio indicates the weaker proliferation activity of C2C12.In addition,cells in the D-galactose treated group showed significantly more blue staining in D-galactose group,indicating that D-galactose accelerated the aging of myoblasts C2C12.D-galactose can reduce the expression of MyoD and Pax7 in myoblasts C2C12 cells in a concentration-dependent manner,and 20g/LD-galactose can maximize the inhibitory effect.ConclusionStable cell models were established with D-galactose and C2C12,which can be used for high-throughput screening of anti-myosenia nutritional factors.Part 3:Effect of Ursolic acid on sarcopenia in vivo and in vitroObjectiveIn our population based survey,we found that the proper hawthorn intake is associated with the lowest skeletal muscle to reduce disease prevalence,this suggests that hawthorn intake in the diet,may help prevent rheological skeletal sarcopenia,so we hope to further explore whether the effectiveness of hawthorn ingredients can reduce the attenuation of the skeletal muscle in the process of aging.There are also foreign studies that show that the green apple peel has the potential to protect skeletal muscle,and its possible effective ingredient is ursolic acid.However,as far as we know,no researchers have fully evaluated the effect of ursolic acid on muscle strength,muscle mass and motor ability of animals,so as to evaluate the effect of ursolic acid on sarcopenia.Therefore,we evaluated the effect of ursolic acid on muscular dysprasia in vivo and in vitro.Methods10 C57 mice in each group,a total of four groups,continuous subcutaneous injection plus lavage 8 weeks.The dosage was 400mg/kg D-galactose and 100mg/kg cellulose sodium ursolic sodium suspension.The treatment group was divided into three groups:galactose-ursolic acid treatment group,D-galactose-ursolic acid treatment group,galactose-ursolic acid treatment group,and normal saline control group.At the end of the experiment,the mice were measured for weight,grip strength,and the time of the dropper.Body composition was measured with DXA the next day after anesthesia.Ursolic acid was used to interfere with D-galactose treated myoblast C2C12.the proliferation activity of cells was determined by CCK8fanfa method,and the effect of ursolic acid on the differentiation trend of myoblasts was demonstrated by eMyHC immunofluorescence staining.ResultsAfter joining ursolic acid in mice can obviously alleviate the D-galactose and exercise capacity(rod drop time)damage(P<0.05),but in lean body mass and grip strength relief effect is no statistical differences.Ursolic acid significantly increased the cell proliferation activity and cell differentiation level of D-galactose intervention.ConclusionUrsolic acid to a certain extent,ease the D-galactose animal models in mice caused by muscle symptoms of attenuation.Ursolic acid may alleviate the progression of sarcopenia by promoting the proliferation and differentiation of myoblasts.Part 4:Population based study of relationship between the frequency of hawthorn intake and sarcopeniaObjectivePathogenesis is not yet reached a consensus.Studies have shown that nutrition is an important factor affecting muscle atrophy in the elderly.A population-based survey of dietary intake and lifestyle could better express the correlation between nutritional factors and certain diseases.Therefore,it may be more efficient and accurate to screen out the relevant potential nutritional factors from the population survey and then evaluate the efficacy in vivo and in vitro experiments.Therefore,we designed the cross-sectional study to investigate the relationship between dietary intake and the prevalence of sarcopenia in elderly Chinese population.Taking hawthorn as an example,the relationship between different intake frequency and sarcopenia prevalence in the elderly was discussed.MethodsFor the older population hawthorn intake data were obtained by the food frequency questionnaire,the criteria for sarcopenia in the elderly were based on the diagnostic criteria recommended by Asian Working Group for Sarcopenia.The potential nutrients found in the population survey were given to animal models and cell models,and the anti-sarcopenia effect of the nutrients was judged according to the changes of animal body composition and the ability of cell proliferation and differentiation.ResultHawthorn intake 4-6 times per week was associated with lower prevalence of sarcopenia in elderly population,and the correlation still existed after adjustment of population characteristic information(P<0.05).ConclusionAppropriate intake of hawthorn is associated with lower prevalence of sarcopenia in elderly population.Part 5:Vitamin D is related to hand grip strength in adult men aged 50 years and overObjectiveHand grip strength(HGS)begins an accelerating decline around 50y.Many of the studies performed in old adults have demonstrated a significant relation between vitamin D and HGS,but the studies performed in participants with a broad age range drew conflicting results.The purpose of the study was to investigate the relationship between vitamin D and HGS using age 50 as a specific cutoff.MeasurementsSerum concentration of 25-hydroxyvitamin D(25(OH)D)was measured using an enzyme immunoassay.We divided participants into quartiles according to 25(OH)D,HGS was measured with a hydraulic hand-held dynamometer.Analysis of covariance was employed to explore the relationship.ResultsAmong males aged above 50y,the means(95%confidence interval)for HGS per body weight across the categories of serum 25(OH)D concentration were 0.523(0.430-0.638),0.545(0.447-0.664),0.543(0.446-0.661),0.546(0.449-0.664)(Ptrend<0.01)after adjustment for potential confounding factors.However,no relationships were observed between serum 25(OH)D concentration and HGS in males aged below 50y and females in the whole age range.ConclusionsSerum 25(OH)D concentration was significantly related to HGS in males aged above 50y,independent of confounding factors.Future studies are needed to clarify the age and sex relationship between serum 25(OH)D concentration and HGS. |
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