ABSTRACT
OBJECTIVE: To observe the therapeutic effect of electroacupuncture (EA) of "Zusanli" (ST36) and "Ashi"-point on the healthy side (opposing needling) on muscular injury and expression of myogenin (myoG) and fast myosin skeletal heavy chain (Fast MyHC) proteins in the gastrocnemius muscle (GM) tissues in skeletal muscle contusion rats，so as to explore its mechanism underlying improvement of skeletal muscle injury. METHODS: A total of 54 male SD rats were divided into normal control (n ＝ 6)，model (n＝24) and opposing needling (EA, n＝24) groups. The latter two groups were further randomized into 3, 5, 7 and 14 d subgroups (n＝6 per subgroup). The skeletal muscle contusion model of the hind-limb was established by using a self-made striking device. EA (1 Hz/3 Hz，1－2 mA) was applied to ST36 and "Ashi"-point on the uninjured side of the hind-limb for 15 min every time, once a day for 3, 5, 7 and 14 days, respectively. The injured GM was harvested on the 3rd, 5th, 7th and 14th day after muscular contusion. The morphological changes of the injured GM and the mean cross-sectional areas (CSAs) of the neonatal muscle cells were observed by microscope after H．E. staining. The immunoactivity of desmin protein (myogenic marker protein of myoblast cell) of GM was detected by immunofluorescence stain on the 7th day after injury, and the expression levels of myoG (on the 3rd and 5th day after injury) and fast MyHC protein of GM tissues (on the 7thand 14th day after injury) were detected by Western blot. RESULTS: H．E. staining of GS tissue showed fewer neuronal myocytes with disordered arrangement at different sizes, and appearance of some collagenous fibers among the mesenchyme on day 7 and 14 after muscular contusion, which was relatively milder in the EA group. In the EA group, the CSA values of the neonatal muscle cells were significantly larger than those in the model group on the day 7th (P0.05). On the 3rd and 5th day after muscular contusion, the expression level of myoG protein was significantly up-regulated in the model group compared with the normal control group (P<0.001), and significantly up-regulated in the EA group than that in the model group (P<0.001). On the 7th and14th day after contusion, the expression level of fast MyHC protein was significantly down-regulated in the model group relevant to the normal control group (P<0.001), and markedly up-regulated in the EA group relevant to the model group (P<0.01)．. CONCLUSION: EA of ST36 and "Ashi"-point on the contralateral limb can up-regulate the expression of myoG and fast MyHC proteins of GM in acute skeletal muscle contusion rats, which may contribute to its effect in promoting the repair of skeletal muscle injury.
ABSTRACT
<p><b>OBJECTIVES</b>To explore the possible biological mechanism of skeletal muscle contusion repair through researching the changes in expression of autophagy-related genes and proteins in SD rats with acute skeletal muscle contusion.</p><p><b>METHODS</b>Six rats were randomly selected as the control group from 30 male SD rats, acute skeletal muscle contusion model were established in the remaining 24 rats with self-made hitter, then the model rats were randomly divided into 4 groups (3 d, 5 d, 7 d, 14 d groups, =6). On the 3, 5, 7 and 14 day after injury, injured gastrocnemius of each group was harvested. The morphological and the ultra-microstructure changes of gastrocnemius after injury were observed by HE staining and transmission electron microscope (TEM) respectively. The relative protein levels of (LC3-Ⅱ) and P62 of each group were observed by Western blot. The relative mRNA levels of atg7, atg10, atg12, atg16L1 of each group were observed by RTPCR.</p><p><b>RESULTS</b>The results of HE staining showed that compared with the control group, the inflammation reached its peak on the 5 day after injury, new muscle fibers were clearly observed in 7 d group. The results of TEM showed that, compared with the control group, oncotic mitochondria could be clearly seen in the 3 d, 5 d, 7 d groups. Also, the Z line changed from disappearing to drift thickening, sarcoplasmic reticulum dilatation gradually improved, there was no evident difference between the 14 d group and the control group, suggesting that the damage has preliminarily healed. The results of Western blot showed that the expressions of LC3-Ⅱand P62 were increased at first and then decreased. The expression of LC3-Ⅱwas markedly up-regulated in the 3 d, 5 d, 7 d groups compared with the control group and the 14 d group (<0.01). Similarly, compared with the control group, the expression of P62 reached its peak on the 3 day after injury (<0. 01), and returned to normal level on the 14 day. The results of RT-PCR showed that the expression of atg10 mRNA in the natural recovery group of 3 d, 5 d, 7 d, 14 d was firstly decreased and then increased, the atg10 mRNA was markedly down-regulated in the 3 d, 5 d, 7 d groups compared with the control group and the 14 d group (<0. 01). The expression of atg7, atg12, atg16L1 mRNA was generally increased at first and then decreased, it was markedly up-regulated in the 3 d, 5 d, 7 d groups compared with the control group and the 14 d group (<0.01, <0.05, <0.01).</p><p><b>CONCLUSIONS</b>The above results indicate that the autophagy is involved in repair of skeletal muscle injury by its autophagyrelated factors,regularly changes after contusion, and the rate of damage repair may be related to the level of autophagy.</p>
Subject(s)
Animals , Male , Rats , Autophagy , Contusions , Muscle, Skeletal , Wounds and Injuries , RNA, Messenger , Rats, Sprague-DawleyABSTRACT
OBJECTIVE: To study the effect and mechanism of electroacupuncture(EA)combined with treadmill training in rats with skeletal muscle contusion. METHODS: A total of sixty Sprague Dawley rats were randomly divided into normal,model, EA, treadmill, and combination groups (n=12/group). The self-made striking device was used to establish skeletal muscle contusion model. EA was applied at "Zusanli" (ST 36) and Ashi point in the EA group. Treadmill was applied to train rats in the treadmill group. Rats in the combination group were received the above two methods. All the treatment was given once a day until the 3rd and 14th days after injury. The cross-sectional area and diameter of neonatal gastrocnemius muscle cells 14 days after injury were observed by HE staining. The expression levels of mammalian target of rapamycil (mTOR), myogenin (myoG) in gastrocnemius 3 days after injury and gastrocnemius mTOR, Fast myosin skeletal heavy chain (Fast MyHC) 14 days after injury were observed by Western blot. RESULTS: The cross-sectional area and diameter of neonatal muscle gastrocnemius cells in the model group were significantly lower than those in the normal group (P<0.01), and those in all the intervention groups were significantly higher than those of the model group (P<0.05), with better results in the combination group compared with those in the EA and treadmill groups (P<0.05). On the 3rd day after injury, the expressions of mTOR and myoG proteins in the model group were significantly higher compared with those in the normal group (P<0.01), and those in all the intervention groups were up-regulated in comparison with the model group (all P<0.01). The mTOR and myoG proteins in the treadmill group were lower than those in the EA group (P<0.01). The above two indexes in the combination group were higher than those in the EA and treadmill groups (P<0.05, P<0.01). On the 14th day after injury, the expression of mTOR was up-regulated in the model group compared with that in the normal group, but without significant difference (P<0.05), while the expression of Fast MyHC decreased (P<0.01). The expressions of mTOR and Fast MyHC in all the intervention groups increased compared with those in the model group (P<0.05, P<0.01). The expressions of mTOR and Fast MyHC proteins in the treadmill group were significantly down-regulated compared with those in the EA group (P<0.05, P<0.01). The two indexes in the combination group were higher than those in the other two intervention groups (P<0.01). CONCLUSIONS: EA combined with treadmill training can improve the myogenic differentiation and maturation, alleviate the injury of skeletal muscle, which may be related to its effect of increasing the expression of mTOR protein and positively regulating myoG and Fast MyHC proteins.