The Effect Of Celecoxib On The Proliferation And Differentiation Of Rat Skeletal Muscle Myoblasts

Objective:Stress Urinary Incontinence(SUI)is a common chronic disease among women around the world,which brings great troubles to the lives of patients.The pathogenesis of SUI is diverse.Among them,the damage or functional decline of pelvic floor muscles,urethral sphincter and other muscle tissues is one of the key factors that induce SUI.The repair of muscles mainly depends on the proliferation and differentiation of muscle satellite cells.At present,the non-surgical treatment of SUI has many limitations.Therefore,finding a safe and effective drug to treat SUI has always been a hot issue of research.Celecoxib is a kind of non-steroidal anti-inflammatory drug,which is widely used in anti-inflammatory and analgesic.At present,celecoxib has shown a beneficial effect in improving the symptoms of SUI in clinical studies and animal experiments,but its specific mechanism of action is still unclear,and its effect on muscle regeneration is also unclear.Therefore,exploring the pharmacological effects of celecoxib in muscle regeneration can lay a molecular basis for celecoxib in the treatment of SUI.This study aimed to investigate the effect of celecoxib on the proliferation and differentiation of rat skeletal muscle myoblasts(L6 cells)at the cellular level.Methods:L6 cells were cultured in vitro.In the experiment to explore the effect of celecoxib on the proliferation of L6 cells,treat L6 cells with serum-free medium containing different concentrations(0μmol/L,6.25μmol/L,12.5μmol/L,25μmol/L,50μmol/L)celecoxib,and the cell morphology and density were observed under a microscope.L6 cells were treated with growth medium containing celecoxib at different concentrations(0μmol/L,6.25μmol/L,12.5μmol/L,25μmol/L,50μmol/L)for 24h,48h,72h,and cell proliferation was detected by CCK-8 method.In an experiment to explore the effect of celecoxib on the differentiation of L6 cells,cells were treated with differentiation media containing different concentrations(0μmol/L,6.25μmol/L,12.5μmol/L;25μmol/L,50μmol/L)of celecoxib for 2 days,and then Western blot and qPCR were used to detect the expression of differentiation-related genes Myod,Desmin and α-SCA.After that,the cells were treated with differentiation medium with a concentration of 0μmol/L,25μmol/L celecoxib for 2 days,and the expression of Myod and Desmin was detected by immunofluorescence experiment.Results:In the experiment of the effect of celecoxib on the proliferation of L6 cells,the observation results of cell morphology and density showed that:The comparison of the drug treatment group(6.25μmol/L,12.5μmol/L,25μmol/L,50μmol/L celecoxib group)and the control group(0μmol/L celecoxib group)shows that the cell density of the 50μmol/L celecoxib group is significantly lower than that of the control group,and the cell state is the worst,the rest of the drug treatment group and the control group have little difference.The results of CCK-8 showed that compared with the control group(0μmol/L celecoxib group),cell proliferation was inhibited when the drug concentration reached 50μmol/L during the same culture period,and the difference was statistically significant(P<0.05),compared with the control group,the remaining groups had no statistically significant difference(P>0.05).In the experiment of the effect of celecoxib on the differentiation of L6 cells,the Western blot results showed that compared with the control group(0μmol/L celecoxib group),6.25μmol/L,12.5μmol/L,25μmol/L,50μmol/L celecoxib group Myod,Desmin Protein expression is up-regulated,and 25μmol/L celecoxib group Myod,Desmin protein expression up-regulated most significantly;Compared with the control group,the expression of α-SCA protein in the 12.5μmol/L,25μmol/L,and 50μmol/L celecoxib groups was up-regulated,and the expression of α-SCA protein in the 25μmol/L,50μmol/L celecoxib group was up-regulated significantly.The qPCR results showed that compared with the control group,the mRNA expression levels of Myod,Desmin,and α-SCA in the 6.25μmol/L,12.5μmol/L,25μmol/L,50μmol/L celecoxib groups were up-regulated,and the mRNA expression levels of Myod and Desmin in the 25μmol/L celecoxib group were up-regulated most significantly,and the mRNA expression levels of α-SCA in the 50μmol/L celecoxib group were up-regulated most significantly.Immunofluorescence results showed that the expression of Myod and Desmin in the 25μmol/L celecoxib group was higher than that of the control group(0μmol/L celecoxib group).Conclusions:1.The low concentration of celecoxib has no effect on the proliferation of L6 cells.When the drug concentration is 50μmol/L,it has an inhibitory effect on the proliferation.2.Celecoxib can promote the expression of Myod,Desmin and α-SCA,which are closely related to differentiation,which may promote the differentiation of L6 cells.3.The expression of Myod and Desmin was more significant when the concentration of celecoxib was 25μmol/L,while the expression of α-SCA was more significant when the drug concentration was 25μmol/L and 50μmol/L,and when the drug concentration is 50μmol/L,cell proliferation is inhibited,so the more appropriate drug concentration to promote the differentiation of L6 cells may be 25μmol/L.