| Objective:To observe the effect of oblique needling Ashi point on exercise ability,cell morphology and specific marker proteins MyoD1 and Pax7 of MSC in mice with quadriceps femoris injury,so as to prove the therapeutic effect of oblique needling Ashi point on skeletal muscle injury and explore its possible mechanism.Methods:A total of 24 C57BL/6N male mice were randomly divided into control,model,shallow needling and deep needling groups,with 6 mice in each group.The quadriceps femoris injury model was produced by single intramuscular injection of 0.5%Bupivacaine.24h after modeling,the two treatment groups were intervened by oblique needling at Ashi point,and were divided into shallow needling and deep needling according to different needling levels.The oblique needling was lifted and inserted 3 times,and the treatment times were all 1.The overall situation,weight and quadriceps diameter of mice in each group were observed and recorded 24h before modeling,24h after modeling,and 24h after treatment;Behavioural tests and comparisons were performed on each group of mice 24h before and after treatment,including Pole Test,Weight Bearing,Suspension Test and Treadmill Running;After 24h of treatment,the quadriceps femoris tissues of each group of mice were dissected,and the wet weight ratio of the tissues was compared.Histopathological changes of quadriceps femoris was observed after H.E.Stain.The immunoactivity of MyoDl and Pax7 was detected by immunohistochemistry.The relative expression of MyoD1 and Pax7 protein in quadriceps femoris was detected by Western blot.Results:(1)Effect of oblique needling Ashi point on body weight and wet weight ratio of quadriceps femoris:The body weight of mice in the deep needling group was lower than that in the control group(P<0.05);The wet weight ratio of quadriceps femoris among the 4 groups were no statistically significant difference(P>0.05);(2)Effect of oblique needling Ashi point on the diameter of quadriceps femoris and behavioural tests:After modeling,compared with the control group,the diameter of quadriceps femoris in the model group,shallow needling group and deep needling group was increased(P<0.01),the score of Pole Test was decreased(P<0.01),the weight-bearing capacity of lower limbs was decreased(P<0.01),and the score of Suspension Test was decreased(P<0.01),the time of electric shocks in the Treadmill Test was increased significantly(P<0.01);After oblique needling Ashi point,compared with the model group,the diameter of quadriceps femoris of the mice in the shallow needling group and the deep needling group was decreased(P<0.01),the score of Pole Test was increased(P<0.01),the score of Suspension Test was increased(P<0.01),the time of electric shocks was significantly reduced(P<0.01),and there was no significant difference in the weight-bearing capacity of lower limbs of the mice in the shallow needling group(P>0.05),and that in the deep needling group was increased significantly(P<0.01);Compared with the shallow needling group,the diameter of quadriceps femoris in the deep needling group was decreased(P<0.01),the score of Pole Test was increased(P<0.05),the weight-bearing capacity of lower limbs was lifted(P<0.01),the score of Suspension Test was not statistically significant(P>0.05),and the time of electric shocks was decreased(P<0.01);Compared with before treatment,the diameter of quadriceps femoris was decreased(P<0.01),the score of Pole Test was increased(P<0.01),the weight-bearing capacity of lower limbs was increased(P<0.05,P<0.01),and the score of Suspension Test was increased(P<0.05,P<0.01)in the shallow needling group and the deep needling group after treatment,there was no significant difference in the time of electric shocks of the mice in the shallow needling group(P>0.05),and that in the deep needling group was decreased significantly(P<0.01);(3)Effect of oblique needling Ashi point on the morphology of muscle cells:HE staining showed that the muscle cells in the control group were closely arranged,surrounded by several nuclei,regular in shape and uniform in size;In the model group,there was a large area of inflammatory infiltration,muscle cells swelling,atrophy,rupture,degeneration and necrosis,enlarged cell compartments,nuclear aggregation,deep staining,pyknosis,and irregular overall arrangement with bleeding;In the shallow needling group,the muscle cells were swollen and ruptured,the cell septum was large,the nucleus was aggregated and stained deeply,accompanied with hemorrhage;In the deep needling group,the inflammation was alleviated,the size of muscle cells tended to be consistent,the defecation tended to be regular,the cell compartments became smaller,and the number of nuclei decreased;(4)Effect of oblique needling Ashi point on the immunoactivity of MyoD1 and Pax7:IHC staining results showed that the cumulative absorbance value of positive expression of MyoD1 and Pax7 were increased in model group,shallow needling group and deep needling group to compared with the control group(P<0.01),which were increased in shallow needling group and deep needling group to compared with the model group(P<0.01),and those in the deep needling group were increased to compared with the shallow needling group(P<0.01);(5)Effect of oblique needling Ashi point on the relative protein expression of MyoD1 and Pax7:WB results showed that compared with the control group,the relative expression of MyoD1 protein in model group,shallow needling group and deep needling group was increased(P<0.01),the relative expression of Pax 7 protein in model group was increased(P<0.05),and that in shallow needling group and deep needling group was increased(P<0.01);Compared with the model group,the relative expression of MyoD1 and Pax 7 in shallow needling group were increased(P<0.05),and those in deep needling group were increased(P<0.01);Compared with the shallow needling group,the relative expression of MyoD1 and Pax 7 in the deep needling group increased(P<0.01).Conclusion:(1)Oblique needling Ashi can improve the local swelling degree of quadriceps femoris injured mice,promote the repair of muscle cell morphology,contribute to the recovery of damaged muscle tissue structure and function,thus improving the exercise ability of quadriceps femoris injured mice;(2)Oblique needling Ashi point may accelerate the proliferation of MSC by up-regulating the expression of MyoD1 and Pax7 protein,so as to promote the repair of injured quadriceps femoris;(3)Oblique needling Ashi point,deep needling and shallow needling,both have therapeutic effect on quadriceps femoris injury.The effect of deep needling is better than that of shallow needling. |
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