| Objective1.This study explains and enriches the theory of "equal emphasis on bones,tendons and muscles",and explores the clinical efficacy of Bushen Qiangjin capsule(BSQJ)in treating disuse muscular atrophy(DMA)after Total knee arthroplasty(TKA)in elderly with end-stage knee osteoarthritis,and provides an evidence-based basis for the clinical application of BSQJ for the solution of DMA.2.The DMA mouse model was established to observe the effect of BSQJ on the muscle mass of DMA mice.In addition,it seeks to explore the possible mechanism of BSQJ in alleviating DMA.Methods1.Clinical research sectionFifty-eight patients with knee osteoarthritis(end-stage)who need TKA from June 2020 to February 2021 were included,and randomly allocated to two groups:Bushen Qiangjin capsule group(BSQJ group,mean age(mean±SD)=70.00±7.85 years)and Control group((mean±SD)=69.31±5.87 years),29 patients each.Each patient in BSQJ group was provided Bushen Qiangjin capsule(6 pills daily,3 times per day)from one day before surgery to four weeks after surgery.Both groups received the same postoperative guided functional exercise.Pain score,knee joint function,and muscle thickness of rectus femoris,gastrocnemius and soleus were recorded before surgery,1,2,and 4 weeks after surgery.The pain was assessed by visual analogue scale(VAS),joint function was assessed by the score of Hospital for Special Surgery(HSS),and muscle thickness of rectus femoris,gastrocnemius and soleus were measured by ultrasound.2.Animal Experiment Section(1)Establishment of animal model of DMAForty male C57BL/6(11-13 weeks)were randomly divided into 4 groups:Control group,1 week hindlimb suspension(1W HS),2 weeks hindlimb suspension(2W HS),4 weeks hindlimb suspension(4W HS)groups,10 mice each.After hindlimb suspending,quadriceps,gastrocnemius,and soleus were collected and weighed.The right side of soleus muscle was subjected to histology analysis.Soleus muscle fiber cross-sectional area was recorded.(2)Mechanism of prevention and treatment of DMA with BSQJFifty male C57BL/6(11-13 weeks)were randomly divided into 5 groups①Control group:no suspension;②Hindlimb suspension(HS)group:hindlimb suspension,③ Bushen Qiangjin capsule group(BSQJ group):hindlimb suspension+BSQJ capsule;④Wortmannin group(wort group):Hindlimb suspension+intraperitoneal injection of Wortmannin;⑤Wort+BSQJ group:hindlimb suspended+BSQJ capsule+Wortmannin,10 mice each.The dose of BSQJ was 0.351g/Kg by gavage.Wortmannin is a PI3K specific inhibitor,and the intraperitoneal dose of Wortmannin was 0.5mg/kg.Once daily for 4 weeks.The quadriceps femoris,gastrocnemius,and soleus of each group were collected and weighed.Meanwhile,the right soleus was stained with HE and the cross-sectional area(CSA)of muscle fibers of soleus was calculated.Using western blotting to detect soleus protein anabolic PI3K/Akt/mTOR/P70S6K1/EIF4EBP1 and their phosphorylated products.Using western blotting to detect protein catabolism FOXO1/FOXO3A and their phosphorylated products.The expression levels of Atrogin-1 and MuRF-1 mRNA were detected by RT-qPCR.Results1.Clinical research section(1)VAS Score:Compared with VAS before surgery,VAS score decreased significantly in both groups at 1,2,and 4 weeks after surgery.Compared with control group,VAS of BSQ J group was lower at 2 and 4 weeks after surgery(P<0.05).(2)HSS score:Compared with HSS before surgery,the HSS score was increased in both groups at 2 and 4 weeks after surgery(P<0.05).The HSS in BSQJ group were higher than those in Control group at 2 and 4 weeks after surgery(P<0.05).(3)Rectus femoris thickness:On the operative side,the thickness of rectus femoris at 4 weeks after surgery was decreased by 12.7%in the BSQJ group,while the thickness of rectus femoris at 4 weeks after surgery was decreased by 17.7%(P<0.05)in the Control group.On the non-operative side,the thickness of rectus femoris at 4 weeks after surgery was decreased by 10.1%in the BSQJ group,while the thickness of rectus femoris on 4 weeks after surgery was decreased by 15.2%in the Control group,but there were no difference(P>0.05).Compared with rectus femoris thickness before surgery,there were significant differences in the thickness of operated side rectus femoris at 2 weeks and 4 weeks after surgery(P<0.05)in both groups,and there were significant differences in the thickness of non-operated side rectus femoris at 1 week,2 weeks and 4 weeks after surgery(P<0.05)in both groups.(4)Gastrocnemius muscle thickness:On the operative side,the thickness of gastrocnemius muscle in BSQJ group at 1 week after surgery was decreased by 3.7%,while that in the Control group was decreased by 7.95%(P<0.05);the thickness of gastrocnemius muscle in BSQJ group at 2 week after surgery was decreased by 8.3%,while that in the Control group was decreased by 11.9%(P<0.01);the thickness of gastrocnemius muscle in BSQJ group at 4 week after surgery was decreased by 10.8%,while that in the Control group was decreased by 14.6%(P<0.01).On the non-operated side,the thickness of gastrocnemius muscle in BSQJ group at 2 week after surgery was decreased by 7.6%,while that in the Control group was decreased by 10.8%(P<0.05).Compared with thickness of Gastrocnemius muscle before surgery,there were significant differences in the thickness of operated side Gastrocnemius muscle at 1 week,2 weeks and 4 weeks after surgery(P<0.05)in both groups,and there were significant differences in the thickness of non-operated side Gastrocnemius muscle at 2 weeks and 4 weeks after surgery(P<0.01)in both group.(5)Soleus thickness:On the operative side,Soleus thickness in BSQJ group at 1 week after surgery was decreased by 4.2%,while that in the Control group was decreased by 8.65%(P<0.05);Soleus thickness in BSQJ group at 2 weeks after surgery was decreased by 8.6%,while that in the Control group was decreased by 14.0%(P<0.05);Soleus thickness in BSQJ group at 4 weeks after surgery was decreased by 10.7%,while that in the Control group was decreased by 15.3%(P<0.05).On the non-operative side,Soleus thickness in BSQJ group at 2 weeks after surgery was decreased by 7.2%,while that in the Control group was decreased by 10.3%(P<0.05).Compared with Soleus thickness before surgery,there were significant differences in the thickness of operated side Soleus at 2 weeks and 4 weeks after surgery(P<0.01)in both groups,and there were significant differences in the thickness of the non-operated side Soleus at 2 weeks and 4 weeks after surgery(P<0.01)in both groups.2 Animal Experiment Section(1)Establishment of an animal model of disuse muscular atrophy①Quadriceps femoris-to-body weight ratio(g/mg):Compared with the Control group,quadriceps femoris-to-body weight ratio in 2W HS and 4W HS decreased by 7.65%(P<0.05)and 16.6%(P<0.01),respectively.The quadriceps femoris-to-body weight ratio in the 4W HS was significantly decreased compared with that in the 1W HS(P<0.05)and 2W HS(P<0.05).There was no significant difference between 1W HS and 2W HS(P>0.05).②Gastrocnemius muscle-to-body weight ratio(g/mg):Compared with Control group,the Gastrocnemius muscle-to-body weight ratio in 1W HS,2W HS and 4W HS decreased by 5.38%(P<0.05),16.5%(P<0.01)and 19.0%(P<0.01),respectively.There was a significant difference between the 4W HS and 1W HS(P<0.05).③Soleus muscle-to-body weight ratio(g/mg):Compared with Control group,Soleus muscle-to-body weight ratio in 1W HS,2W HS and 4W HS decreased by 13.6%(P<0.05),26.5%(P<0.01)and 41.6%(P<0.01),respectively.There was a significant difference between 4W HS and 1W HS group(P<0.01)as well as 2W HS(P<0.05).④Cross-sectional area of soleus muscle fiber(μm2):Compared with the Control group,the cross-sectional area of soleus muscle fiber in 1W HS,2W HS,and 4W HS decreased by 12.6%(P<0.01),25.2%(P<0.01)and 26.4%(P<0.01),respectively.There was a significant difference between the 4W HS and 1W HS(P<0.01).(2)The mechanism of prevention and treatment of DMA with BSQJThe time of hindlimb suspension was selected at 4 weeks.①Effect of BSQJ on Muscle MassQuadriceps femoris-to-body weight ratio(g/mg):Compared with the Control group,quadriceps femoris-to-body weight ratio in the HS group was decreased by 18.1%(P<0.01),and the BSQJ group was decreased by 9.17%(P<0.01),the Wort group was decreased by 23.8%(P<0.01),and the Wort+BSQJ group was decreased by 18.0%(P<0.01).Compared with HS group,there were significant differences in BSQJ group(P<0.01).Compared with BSQJ group,there were significant differences in Wort group(P<0.01)and Wort+BSQJ group(P<0.01).Gastrocnemius muscle-to-body weight ratio(g/mg):Compared with Control group,gastrocnemius muscle-to-body weight ratio in HS group was reduced by 22.2%(P<0.01),BSQJ group was decreased by 9.78%(P<0.05),Wort group was decreased by 23%(P<0.01),and Wort+BSQJ group was decreased by 27.5%(P<0.01).Compared with HS group,there was significant difference in BSQJ group(P<0.05).Compared with gastrocnemius muscle-to-body weight ratio of BSQJ group,there were significant differences in Wort group(P<0.01)and Wort+BSQJ group(P<0.01).Soleus muscle-to-body weight ratio(g/mg):Compared with the Control group,the Soleus muscle-to-body weight ratio in HS group was decreased by 40.2%(P<0.01),BSQJ group was decreased by 23.5%(P<0.01),Wort group was decreased by 40.4%(P<0.01),and Wort+BSQJ group was decreased by 37.9%(P<0.01).Compared with HS group,there was a significant difference in BSQJ group(P<0.05).Compared with Soleus muscle-to-body weight ratio of BSQJ group,there were significant differences between in Wort group(P<0.05)and in Wort+BSQJ group(P<0.05).Cross-sectional area of soleus muscle fiber(μm2):Compared with Control group,the cross-sectional area of soleus muscle fiber in HS group was reduced by 28.2%(P<0.01),BSQJ group was decreased by 12.8%(P<0.01),Wort group was decreased by 28.1%(P<0.01),and that in Wort+BSQJ group was decreased by 31.3%(P<0.01).Compared with HS group,there were significant differences in BSQJ group(P<0.05).Compared with Cross-sectional area of soleus muscle fiber of BSQJ group,there were significant differences in Wort group(P<0.01)and Wort+BSQJ group(P<0.01).②Effects of BSQJ on protein synthesis catabolic pathway-related proteins and muscle atrophy related genesa.Effect of BSQJ on Protein Anabolism PathwayCompared with Control group,the protein expression levels of p-PI3K/PI3K,p-AKT/AKT,p-mTOR/mTOR,p-S6K1/S6K1,p-ELF4EBP1/ELF4EBP1 in HS group were significantly decreased(P<0.01);Compared with the HS group,the expression of p-PI3K/PI3K protein in BSQJ group was significantly increased(P<0.05),and the expression of p-AKT/AKT,p-mTOR/mTOR,p-S6K1/S6K1,p-ELF4EBP 1/ELF4EBP1 protein synthesis related proteins were also increased(P<0.05).Compared with Wort+BSQJ group,p-PI3K/PI3K in BSQJ group was significantly increased(P<0.05).Meanwhile,the expression levels of p-Akt/Akt,p-mTOR/mTOR,p-S6K1/S6K1,p-ELF4EBP1/ELF4EBP1 in BSQJ group were also increased(P<0.05).b.Effects of BSQJ on protein catabolism p-FoxO1/FoxO1,p-FoxO3a/FoxO3aCompared with the Control group,the protein levels of p-FoxO1/FoxO1 and p-FoxO3a/FoxO3a in HS group were significantly decreased(P<0.05);Compared with HS group,the protein levels of p-FoxO1/FoxO1 and p-FoxO3a/FoxO3a in BSQJ group were significantly increased(P<0.05);Meanwhile,the protein level of p-FoxO3a/FoxO3a of BSQJ group was lower than that of model group(P<0.05).c.The expression of Atrogin-1 and MuRF-1 mRNA in muscular atrophy related gene by BSQJCompared with the Control group,the mRNA expressions of Atrogin-1 and MuRF-1 in all groups were significantly different(P<0.05).Compared with HS group,the expression of muscular atrophy related genes in BSQJ group was significantly decreased(P<0.01);The mRNA expression levels of Atrogin-1 and MuRF-1 in BSQJ group were significantly lower than those in Wort group(P<0.05)and Wort+BSQJ group(P<0.05).Conclusion1.Clinical TrialBSQJ may reduce early postoperative pain,improve the activity function and decrease the loss of muscle mass(Rectus femoris,gastrocnemius,and soleus)in elderly patients with TKA.2.Animal ExperimentHS method is a reliable method for making DMA,which can cause atrophy of quadriceps femoris,gastrocnemius,and soleus.DMA is associated with decreased protein anabolism and increased protein catabolism.BSQJ may reduce the loss of muscle mass by increasing the phosphorylation expression of protein anabolism-related proteins PI3K,Akt,mTOR,S6K1,and EIF4EBP1,reducing dephosphorylation of FoxO1 and FoxO3a,and decreasing the expression of Atrogin-1 and MURF-1 mRNA which are related to muscle atrophy. |
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