| Objective:To observe the efficacy of acupuncture on swelling degree, behavior, histomorphlolgy, HGF growth factor protein and mRNA expression in the reparative process of rats soft tissue after acute contusion.To explore the mechanism of the reparative process of acute blunt muscle being effectively promoted with acupuncture.Methods:80 SD rats were randomly divided into normal control group (n=8),model group (n=24), acupuncture treatment group(n=24),acupuncture control group(n=24),and the latter 3 groups were further divided into Oh,24h and 48h subgroups(n=8) according to the different sampling time.The method of free falling weight hitting was used to make the model of acute soft tissue contusion for model and acupuncture control group.24 hours after modeling, obliquely puncturing Ashi point was used in acupuncture treatment group.For acupuncture control group.acupuncture was performed in the same Ashi-point in normal rats,but without modeing. Measure the weight of rats,gastrocnemius muscle swelling, locomotor activity and gait respectively at the time before modeling,24h,48h and 72h after modeling.For acupuncture treatment group and acupuncture control group,the injured part of gastrocnemius muscle were withdrawn at 0h,24h and 48h after acupuncture;for model group,the injured part of gastrocnemius muscle were withdrawn at the same time;for normal control group the,the sampling time was the same to the 48h goup.Light microscopy and transmission electron microscope morphology were used to observe the changes of gastrocnemius muscle in rats. The expression amount of HGF protein and mRNA were detected with methods of Western blot and Real-time PCR.Results:1.The swelling degree and behavioral resultsThere were no statistical difference of weight among groups at the same time(p>0.05);there were also no statistical difference of weight among each time in the same group(p>0.05).At the time before modeling, there were no significantly difference of indicators among groups(p>0.05) At the time 24-72 hours after modeling, comparing with the normal control group,model groups and acupuncture treatment groups’gastrocnemius muscle swelling and gait score increased significantly(p<0.01),number of activities significantly reduced (p<0.01),acupuncture control groups showed no significantly difference (p>0.05).Compared with model group, no statistical difference of indicators was observed among acupuncture treatment groups(p>0.05); acupuncture control groups’gastrocnemius muscle swelling and gait score decreased significantly(p<0.01), number of activities significantly increased(p<0.01).Compared with the time before modeling,model groups and acupuncture treatment groups’gastrocnemius muscle swelling and gait score increased significantly (p<0.01),number of activities significantly reduced(p<0.01);compared with the time point 24 hours after modeling, model groups’behavior index slightly improved, but no statistical difference was observed(p> 0.05),rat’s activities increased significantly at the time 48 hours after modeling in 24h and 48h acupuncture treatment group (p<0.05),in 48h acupuncture treatment group.rats’gastrocnemius muscle swelling, gait score significantly reduced (p<0.05, p<0.01),number of activities increased singificantly(p<0.01) at the time 72 hours after modeling.compared with the time point 48 hours after modeling, model group and acupuncture treatment group’s behavior index slightly improved without statistical difference (p> 0.05).Acupuncture control group showed no significant change of swelling degree and behavioral indicators in the time window (p>0.05).2.The morphology Results:Light microscopy results:In the normal control group,the transverse section of muscle fibers were polygonal and at equality size. The longitudinal section of muscle fibers was strip tape and arranged parallel to each other. Model groups and acupuncture treatment groups showed different degree of edema,fracture melting and inflammatory cells infiltration. Within the time window, damage degree aggravating gradually,compared with the normal control group, tissue injury score increased significantly(p<0.01);Compared with model group,at the time right after acupuncture and 24 hours later,acupuncture treatment groups’ tissue injury score were reduced without statistical difference(p>0.05),damage level of muscle fiber were equal to model group;acupuncture treatment groups at 48 hours after acupuncture showed that inflammatory cells infiltration decreased significantly and distribution in muscle fiber breakup,the degree of muscle fiber fracture and dissolved reduced,the tissue injury score decreased significantly(p<0.01). At the time point right after acupuncture and 24 hours later, acupuncture control groups’fibers appeared mild edema and a small amount of inflammatory cells infiltration,compared with the normal control group,tissue injury score was significant difference (p<0.01),then muscle fiber morphology returned to normal level at the time 48 hours after acupuncture,compared with the normal control group,there was no statistical difference of tissue injury score(p>0.05).Electron microscopy results:in the normal control group,sarcomeres and myofilaments arranged neatly, sarcomeres were completed, Z-lines, and M-lines were clear and continuous.Model group and acupuncture treatment group appeared different degree of fracture of muscle fiber,swelling,and unable to distinguish between A and I take, M line and Z line fuzzy, discontinuous,sarcomere sizes,etc..within the time window,damage degree aggravating gradually; Oh and 24h acupuncture treatment groups’ damage level were equal to model group,48h acupuncture treatment group showed a small amount of muscle fiber fracture and swelling,a little fuzzy and displacement in M line and Z line, compared with model group, the damage was lighter.At the time right after acupuncture and 24h later, acupuncture control group appeared a small amount of fiber breakage,muscle wire overall order,a slight displacement in Z line,muscle fiber morphology returned to normal level at the time 48 h after acupuncture. 3.HGF mRNA and protein expressionWestern Blot results:compared with the blank group, model groups’HGF protein expression were significantly higher at the time 48h and 72h after hitting(p<0.05, p<0.01),the rest of the groups’ HGF protein expression increased but without statistical difference (p>0.05).Compared with model group,the acupuncture treatment group and acupuncture control groups’HGF protein expression decreased at the time right after acupuncture (p> 0.05),24h and 48h after acupuncture, the two groups’HGF protein expression were significantly decreased(p<0.05), compared with the time 24h after hitting,model groups’HGF protein expression significantly increased at the time 72h after modeling(p<0.05); Within the time window, Acupuncture treatment group and acupuncture group’s HGF protein expression slow increased,but showed no statistical difference (p>0.05).RT-PCR results:compared with the blank group,except acupuncture treatment immediate group,other groups’HGF mRNA expression level were significantly higher (p<0.01);compared with model group, acupuncture treatment groups and acupuncture control groups’HGF mRNA expression were significantly lower at the time 24h and 48h after acupuncture(p< 0.01).Compared with the time 24 hours after modeling, groups’HGF mRNA expression significantly increased at the time 48h and 72h after hitting (p< 0.01);Within the time window,acupuncture treatment group and acupuncture control groups’HGF mRNA expression level increased without statistical differences(p>0.05).Conclusion:1. Acupuncture intervention can effectively relieve local swelling of skeletal muscle after blunt trauma, improve the motor function, the effect is obvious at the time 48 h after acupuncture intervention. Acupuncture stimulation shows no significantly effect on the local swelling and behavior of normal rats.2.By inhibiting the inflammatory cell infiltration,acupuncture intervention can promote the recovery of injuried skeletal muscle’s structure,the effect is obvious at the time 48 h after acupuncture intervention.Acupuncture stimulation has no obvious effect on the local skeletal muscle morphology of normal rats.3. Acupuncture intervention can effectively promote the repair of acute blunt injury of skeletal muscle and its mechanism may be related to that acupuncture lowers damage excessive HGF protein and gene expression in skeletal muscle (48 h after acupuncture significantly), providing appropriate HGF protein and gene concentration for satellite cell activation and proliferation. Acupuncture stimulation can increase local HGF protein and gene expression of the normal rat,its internal mechanism needs further research. |
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