Role Of ArfGAP3 In Acute Mechanical Injury Repair Of Levator Ani Muscle In Mice

Part 1: Establishment and evaluation of acute mechanical injury model of levator ani muscle and C2C12 myoblast in mice Objective: To establish the animal and cell models of acute mechanical injury of levator ani muscle and C2C12 myoblasts,and determine the effective modeling parameters to study the injury repair.Methods: Vaginal balloon dilatation and distal traction(VDT)were used to simulate birth injury.Female C57 BL / 6 mice were randomly divided into 6 groups: CON(control group,without any operation),Sham(sham operation group,only inserting catheter in vagina,without dilation and traction),VD(vaginal balloon dilation group),VDT-1,VDT-2 and VDT-3 were vaginal balloon dilation with 10 g,30g and 60 g distal traction,respectively.The levator ani muscle was sectioned on the third day after modeling.The injury state of levator ani muscle were observed by HE staining and transmission electron microscope.The expression of 8-OHd G in levator ani muscle was detected by immunohistochemistry,and the protein levels of XO and NOX2 were detected by Western blot.The acute mechanical injury model of C2C12 myoblasts was established by four-point bending device.C2C12 myoblasts were randomly divided into four groups: CON(cells were cultured on strain plate without mechanical strain)、CMS-1(cyclic mechanical strain loading with parameters of 1,333 μ(1 mm),4h,1 Hz)、CMS-2(cyclic mechanical strain loading with parameters of 1,333 μ(1 mm),8 h,1 Hz)、CMS-3(cyclic mechanical strain loading with parameters of 5,333 μ(4mm),4 h,1 Hz).The mitochondrial membrane potential,ROS level and apoptosis rate of C2C12 myoblasts were detected immediately after loading.Results: The results of HE staining showed that the levator ani muscle injury was not obvious in VD group and VDT-1 group.The levator ani muscle injury was obvious in VDT-2 group,with muscle bundle disorder,increased muscle space with a large number of inflammatory cells infiltration.The levator ani muscle injury in VDT-3group was serious and irreversible,which was not conducive to the repair of levator ani muscle.The results of transmission electron microscopy showed that the muscle fibers of levator ani muscle in VDT-2 group were arranged irregularly with multiple focal necrosis of sarcomere,and the mitochondria were vesicular dilation.It was further confirmed that VDT-2 group could produce effective levator ani muscle injury.Immunohistochemistry and Western blot results showed that the expression levels of8-OHd G,XO and NOX2 in levator ani muscle of VDT-2 group were significantly increased(all P<0.01).The results of cell experiment suggest that compared with the CON group,there was no significant difference in mitochondrial membrane potential,ROS level and apoptosis rate between CMS-1 and CMS-2 groups(all P>0.05),but the mitochondrial membrane potential of CMS-3 group was significantly decreased(P<0.01),and ROS level and apoptosis rate were significantly increased(all P<0.05).Conclusion: Vaginal balloon dilatation with 30 g weight distal traction for 1h is the effective parameter to construct the acute mechanical injury model of levator ani muscle in mice,and can induce oxidative stress in levator ani muscle;5,333μ × 4h,1Hz is the effective mechanical parameter to construct C2C12 myoblast acute mechanical injury model by four-point bending device.Part 2: Bioinformatics analysis and validation of Arf GAP3 gene related to muscle injury repair Objective: Obtaining the differentially expressed gene related to muscle injury repair by Bioinformatics and verify it.Methods: The muscle injury repair associated data sets GSE469 、 GSE5413 and GSE45577 were obtained from the GEO database to extract and analyze the co-differentially expressed genes.PCR was used to detect the ten most significant genes.According to the relationship between the expression level and C2C12 myoblast differentiation time line,Arf GAP3 were selected as the research object.C57 BL / 6 mice were randomly divided into CON(control group),Sham(only inserting catheter in vagina,without dilation and traction)and VDT(vaginal balloon dilation with 30 g distal traction).On the third day after successful modeling,the levator ani muscle was taken to detect the expression of Arf GAP3 by Western blot.Results: Bioinformatics analysis showed that Arf GAP3 was related to the repair of muscle injury.Western blot results showed that the expression of Arf GAP3 in levator ani muscle of VDT mice was significantly higher than that in CON and Sham groups(P<0.05).Conclusion: Arf GAP3 is a gene related to the repair of muscle injury.Acute mechanical injury can up-regulate the expression of Arf GAP3 in levator ani muscle.Arf GAP3 involves in the repair process of acute mechanical injury of levator ani muscle.Part 3: Mechanism of Arf GAP3 in the acute mechanical injury repair of C2C12 myoblasts Objective: To explore the effect of acute mechanical injury on oxidative stress,differentiation and Arf GAP3 expression of C2C12 myoblasts,and the mechanism of Arf GAP3 in the acute mechanical injury repair of C2C12 myoblasts Methods: In order to detect the expression of Arf GAP3 and My HC in C2C12 myoblasts during differentiation,C2C12 myoblasts were induced to differentiate and were randomly divided into four groups: CON(undifferentiated),D1(on the first day of differentiation),D3(on the third day of differentiation)and D5(on the fifth day of differentiation).PCR and Western blot were used to detect the expression of Arf GAP3 and My HC.To detect the effect of acute mechanical injury on Arf GAP3 expression,differentiation and cytoskeleton of C2C12 myoblasts,C2C12 myoblasts were randomly divided into three groups: CON DO(no mechanical strain,undifferentiated),CON D3(no mechanical strain,on the third day of differentiation)and CMS-3 D3(on the third day of differentiation after mechanical strain treatment).The expression levels of Arf GAP3 and My HC were detected by PCR and Western blot.The expression and distribution of Arf GAP3,My HC and F-actin were detected by immunofluorescence.In order to detect the oxidative stress of C2C12 myoblasts induced by acute mechanical injury,C2C12 myoblasts were randomly divided into two groups: CON(control group,without mechanical strain)and CMS-3(with cyclic mechanical strain treatment).The expression levels of XO and NOX2 were detected by Western blot.To investigate the effect of Arf GAP3 on oxidative stress in C2C12 myoblasts after acute mechanical injury,the Arf GAP3 knockdown cell line(sh-Arf GAP3)and Arf GAP3 overexpression cell line(Arf GAP3)were constructed by lentivirus vector transfection.C2C12 myoblasts were randomly divided into four groups: CON(control group),CMS-3(cyclic mechanical strain treatment group),sh-Arf GAP3 CMS-3(Arf GAP3 knockdown with cyclic mechanical strain treatment)and Arf GAP3 CMS-3(Arf GAP3 over-expression with cyclic mechanical strain treatment).ROS,MDA level and SOD enzyme activity were detected respectively.Results: The m RNA and protein expression levels of Arf GAP3 and My HC were significantly increased on the 1st,3rd and 5th day of differentiation culture,and the expression level of Arf GAP3 reached the peak on the 3rd day.Compared with CON DO group,the m RNA and protein expression levels of Arf GAP3 and My HC were significantly increased in CON D3 and CMS-3 D3 group(all P<0.01),and the expression levels of Arf GAP3 and My HC in CMS-3 D3 group were significantly higher than that in CON D3 group(all P<0.01).Immunofluorescence results showed that compared with CON D3 group,the expression level of Arf GAP3 in CMS-3 D3 group was significantly increased(P<0.01),and the fusion index also increased apparently(P<0.05),but there was no significant difference in the expression of F-actin(P>0.05).Compared with CON group,XO and NOX2 protein expression levels in CMS-3 group were significantly increased(P<0.05).Compared with CON group,ROS and MDA levels in CMS-3,sh-Arf GAP3 CMS-3 and Arf GAP3 CMS-3groups were significantly increased(all P < 0.05),while the SOD activity was significantly decreased(all P<0.05).However,compared with CMS-3 group,the degree of oxidative stress in sh-Arf GAP3 CMS-3 group was significantly higher(all P<0.01),while that in Arf GAP3 CMS-3 group was significantly lower(all P<0.05).Conclusion: The expression levels of Arf GAP3 and My HC increased during the first three days of C2C12 myoblast differentiation.Acute mechanical injury can induce oxidative stress in C2C12 myoblasts,promote their differentiation and the expression of Arf GAP3.Arf GAP3 knock-down can aggravate oxidative stress,while Arf GAP3over-expression can inhibit oxidative stress to a certain extent,reduce the decrease of SOD antioxidant enzyme activity after C2C12 myoblast injury,and reduce the levels of ROS and MDA,so as to reduce the oxidative damage of ROS to My HC,promote C2C12 myoblast differentiation and myotube formation,which is conducive to the repair of muscle injury.