[Changes in expression of autophagy-related factors during acute contusion repair of skeletal muscle].

OBJECTIVES: 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. METHODS: 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, n=6). On the 3rd, 5th, 7th and 14th 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. RESULTS: The results of HE staining showed that compared with the control group, the inflammation reached its peak on the 5th 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 (P<0.01). Similarly, compared with the control group, the expression of P62 reached its peak on the 3rd day after injury (P<0. 01), and returned to normal level on the 14th 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 (P<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 (P<0.01, P<0.05, P<0.01). CONCLUSIONS: 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.

[The Effect of Electroacupuncture Combined with Treadmill Training on Skeletal Muscle Contusion in Rats].

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.

[Effects of Electroacupuncture on Apoptosis-related Protein Expression of Gastrocnemius Muscle Cells in Rats with Denervated Sciatic Nerve].

OBJECTIVE: To observe the anti-apoptosis effect of electroacupuncture (EA) on gastrocnemius muscle cells in rats with denervated sciatic nerve, so as to explore its possible mechanisms underlying delaying atrophy of skeletal muscle. METHODS: Sixty-three male Sprague-Dawley rats were randomly divided into sham operation group, model group, and EA group, and then further divided into 3 subgroups in each group(n=7/subgroup). Gastrocnemius muscle atrophy model was established by transecting the sciatic nerve of rat. EA (5 Hz, 1.5 mA) was applied to "Zusanli" (ST 36) and "Chengshan" (BL 57) acupoints at the affected side for 10 min, once a day for 1, 2, 3 weeks, respectively. The gastrocnemius muscles were sampled on the 7th, 14th, 21th d after modeling, separately. The apoptotic cells were detected by TUNEL staining. Bcl-2, Bax, Cyt-C and Caspase-3 protein expressions were determined by Western blot. RT-PCR was used to check Caspase-3 gene expression. RESULTS: Compared with the sham operation group, the cell apoptotic index in gastrocnemius was markedly higher, gastrocnemius Bcl-2 protein expression was markedly down-regulated, and gastrocnemius Bax, Cyt-C and Caspase-3 protein expressions were considerably up-regulated in the model group at all time-points (P<0.05). Compared with the model group, the cell apoptotic indexes in gastrocnemius were significantly lower in the three EA subgroups (P<0.05). Bcl-2 protein expressions were markedly up-regulated while Bax, Cyt-C and Caspase-3 protein expressions were significantly down-regulated in the EA group 14, 21 days after modeling compared with the corresponding model subgroups (P<0.05). The changes of Caspase-3 mRNA expression levels of gastrocnemius in all groups were similar to those of Caspase-3 protein expressions. CONCLUSIONS: Electroacupuncture intervention can effectively increase Bcl-2 expression and decrease Bax, Cyt-C and Caspase-3 expression in gastrocnemius muscle, and consequently reduce the apoptosis of muscle cell, which may contribute to its effect in delaying the skeletal muscle atrophy of denervated sciatic nerve.

[Effects of Electroacupuncture on Adaptive Hypertrophy of Gastrocnemius and Proliferation and Differentiation of Muscle Satellite Cells in Rats].

OBJECTIVE: To explore the effects and mechanism of different duration electroacupuncture (EA) on adaptive hypertrophy of gastrocnemius and the expressions of proliferation cell nuclear antigen (PCNA), paired box transcription factor 7 (Pax 7), myogenic differentiation antigen (MyoD 1), myogenin (MyoG), myosin heavy chain-Ⅰ(Myh 7), transforming growth factor-ß 1 (TGF-ß 1), Smad 3 in rats. METHODS: Twenty-four SD adult male rats were randomly divided into normal, two-week EA (A 2), four-week EA (A 4) and eight-week EA (A 8) groups, 6 rats in each group. Except the normal group, EA was applied at right "Zusanli" (ST 36) and "Huantiao" (GB 30) for 10 min in the other three groups, separately for 2 weeks, 4 weeks and 8 weeks, once a day, 6 times a week. The ratio of gastrocnemius wet weight was calculated after EA. The cross-sectional area and diameter of muscle fiber were measured after HE staining. The expression of PCNA protein was detected by immunofluorescence. The relative expressions of Pax 7, MyoD 1, MyoG, Myh 7, TGF-ß 1 and Smad 3 mRNA were tested with real-time PCR. RESULTS: The expressions of PCNA protein were obviously higher in the A 2 and A 4 groups than that in the normal group. The expression of genes in the EA intervention groups showed a trend of increasing first and then decreasing except Smad 3 mRNA. The relative expressions of Pax 7 and TGF-ß 1 mRNAs reached their peaks in the A 2 group compared with those in the normal group (P<0.01). The ratio of gastrocnemius wet weight, cross-sectional area and diameter of muscle fiber, and the relative expressions of MyoD 1, MyoG, Myh 7 mRNAs reached their peaks in the A 4 group (P<0.01), while the relative expression of Smad 3 mRNA showed a trend of decreasing first and then increasing and reached its bottom at the 4th week (P<0.01). CONCLUSIONS: EA at "Zusanli" (ST 36) and "Huantiao" (GB 30) may induce adaptive hypertrophy of gastrocnemius in rats by up-regulating the expressions of Pax 7, PCNA, TGF-ß 1, MyoD 1, MyoG, Myh 7 and down-regulating the expression of Smad 3, promoting the proliferation of skeletal satellite cells, myogenic differentiation, myotubes terminal differentiation, increasing the wet weight, fiber cross-sectional area and diameter of gastrocnemius.

[Effect of Electro-acupuncture on the Expression of Slit2 and HSPGs in Rats with Focal Cerebral Infarction].

OBJECTIVE: To determine the expression of Slit2 and heparan sulfate proteoglycans (HSPGs) in cortex of rats with focal cerebral infarction and the effect of acupuncture (EA) on the expression of Slit2 and HSPGs. METHODS: 40 male Sprague Dawley (SD) rats were equally randomized into four groups: Control group, model group, non-acupoint EA group, and EA group. Thread-tying method was used in the model group, non-acupoint EA group and EA group to clog arteries and then open up after 1.5 h. Morphology changes of tissues around the infarction area were observed 14 days later by Nissl staining. The expressions of Slit2 and HSPGs in the ischemic brain tissues were detected by indirect immunofluorescence staining (IF) and Western blot (WB). RESULTS: Modified neurologicalseverity scores (mNSS) showed zero in the control group, lower than the score of EA group. The EA group had lower mNSS score than the non-acupoint group. The highest mNSS score appeared in the model group. Paired comparisons showed statistical differences (P < 0.01). Nissl's staining showed that EA group had increased Nissl bodies in alignment; non-acupoint EA group had increased and disordered Nissl bodies; model group had decreased and disordered Nissl bodies with edema in the brain. IF and WB showed that non-acupoint EA group had higher levels of Slit2 and HSPGs than model group (P < 0.05); EA group had higher levels of Slit2 and HSPGs than non-acupoint EA group (P < 0.05); model group had higher levels of Slit2 and HSPGs than control group (P < 0.05). The results of Nissl' s staining, IF and WB were consistent. CONCLUSION: EA can enhance the expressions of Slit2 and HSPGs. This may be one of the mechanisms of EA promoting recovery of neural functions after cerebral infarction.

[Effect of Electroacupuncture on Expression of Cortical srGAP 1 and Cdc 42 in Rats with Focal Cerebral Infarction].

OBJECTIVE: To observe the effect of electroacupuncture (EA) intervention on the neurological function and the expression change of Slit-Robo GTPase-activating protein-1 (srGAP 1) and cell division-cycle 42 (Cdc 42) in the cortex of rats with cerebral ischemic injury (CIRI) , so as to explore the mechanism of EA in the management of cerebral infarction. METHODS: A total of 48 male Sprague Dawley (SD) rats were randomly and equally divided into control, model, non-acupoint EA and EA groups (n = 12/group). The CIRI model was established based on the modified Zea Longa method. EA intervention was applied for 30 min, once a day for 14 days. Modified neurologic severity scores (mNSS) were assessed on day 1,3,7 and 14 after mode- ling. Immunofluorescence assay was used to detect the immunoactivity and distribution of srGAP 1 and Cdc 42 in the cortical ischemic region. Western blot was employed to detect the expression of srGAP 1 and Cdc 42 in the affected cortex. RESULTS: The mNSS displayed that the neurological score in the EA group was significantly lower than that in the model group and non-acupoint EA group at the 7th d and 14th d (P<0. 01). Immunofluorescence results showed that cerebral srGAP 1 and Cdc 42 were ex- pressed mainly in the cytoplasm. The fluorescence intensity of srGAP 1 of the EA group was significantly lower than that of the model group and non-acupoint EA group(P<0. 01). Meanwhile the fluorescence intensity of Cdc 42 of the EA group was markedly higher than that in the model group and non-acupoint EA group(P<0. 01). Western blot assay indicated that the expression level of srGAP 1 in the model group was significantly higher than that of the control group( P<0. 01) ,and that of the EA group was much lower than those of the model group and non-acupoint EA group(P<0. 01). There was no significant difference of srGAP 1 expression levels between the non-acupoint EA group and the model group(P>0. 05). Additionally, the protein expression of Cdc 42 in the model group was slightly higher than that of the control group(P>0. 05), and that of the EA group was significantly higher than those of the model group and non-acupoint EA group(P<0. 01). There was no significant difference of Cdc 42 expression levels between the non-acupoint EA group and the model group(P>0. 05). CONCLUSION: Cerebral infarction induced increase of cerebral srGAP 1 and decrease of Cdc 42 can be reversed by acupoint EA intervention in CIRI rats, which may be responsible for its effect in improving impaired neurological function after cerebral infarction.

Adolescents'static behaviors and related factors in Shanghai / 上海预防医学

[Objective] To investigate and analyse the related factors of static behavior among ado-lescents , such as screen time , mild physical activity , moderate and vigorous physical activity , and nutri-tional status. [ Methods] In 2011, multistage sampling was done from 10 classes of three middle schools in three districts in Shanghai;the students filled the questionnaire on their own which recorded all of the activities of one school day and one weekend day .The data was analyzed by general describing analysis , correlation analysis, and Kruskal-Wallis test. [Results] The static time in weekend was 389.19 mi-nutes per day , the screen time was 122 .93 minutes per day , and both were over three times those on one school day .The static time was not related to moderate and vigorous physical activity , but was related to mild physical activity .The difference in mean time before the screen at weekend was statistically significant between obesity group , overweight group , normal group , low weight group and malnutrition group . [ Con-clusion] Adolescents lacked adequate physical activities in their spare time , and behavior before the screen might be related to their poor nutrition status .