| Chronic alcoholic myopathy (CAM) is a kind of myopathy because of long time alcohol misuse. It has been reported that there are about 40%-60 % alcoholists whose muscle was affected. Their frequently clinical appearance was having difficulties in gait, various muscle symptoms such as cramps, local pain and reduced muscle mass. So, chronic alcoholic myopathy affects the quality of patients'life severely. Lots of researchers have observed that long time alcohol misuse can induce the imbalance of oxidation and anti-oxidation system, and lead the excessive apoptosis of skeletal muscle cells. EGb761 is a standardized extract of Ginkgo biloba leaves containing 24% flavone glycosides and 6% terpene lactones. Studies have found that EGb761 has antioxidative and antiapoptotic effects. EGb761 is mostly used to treat cardiac and cerebral vascular diseases as a free-radical scavenger and anti lipid peroxidation presently in China and Western countries. However, the application of EGb761 on CAM hasn't been reported domestically and abroad. Thus, with the increasing daily of the CAM patients, it has an important social significance to research the pathogenesis and also the prevention and treatment of chornic alcohol myopathy. In our research, we studied the role of alcohol-induced oxidation impairment in skeletal muscle apoptosis of rats and its mechanism concerned with Bcl-2,Bax and oxidative free radicals and studied the effect in this mechanism of EGb761 on alcohol-induced skeletal muscles apoptosis. We hope our research can provide theoretic and experimental evidence for its exertion. Objectives: The project is designed from vivo and vitro two parts. The vivo experiment is on the basis of chronic alcoholic myopathy model of rat, using EGb761 for the prevention and treatment at the same time, choose the plantaris (typeⅡfibre predominated) as the research object to investigate whether EGb761 can protect and treat against the oxidative and apoptotic injury in rat skeletal muscles of CAM and the possible mechanism responsible for it through observing the change of oxidatant-antioxidant status, apoptosis rate, Bax and Bcl-2 and pathology. The vitro experiment is on the basis of skeletal muscle primary cell cultures, treated with ethanol accompanied with or without EGb761, to determine the concentration-dependent and time-dependent effects of ethanol on skeletal muscle cells apoptosis, meanwhile, to observe the protective effect of the extract of Ginkgo biloba (EGb761) against ethanol induced apoptotic injury in skeletal muscle cells.Methods: The vivo experiment. (1) 70 health, male, Sprague-Dawley rats (weight 180~200g), were separated randomly into four groups (control, alcohol, treatment, prevention+treatment), treated with EGb761 for prevention (96μg/g bw per day) and treatment (192μg/g bw per day) on the basis of setting up the CAM rat myopathy model, at the same time, they are feeding the same improved food with high fats and sufficient nutrients, to observe the rat body weight, the change of praxiology and the pathologic change of muscle tissue. (2) At the end of 12 weeks, rats were killed, taken the muscle of posterior limb, after separating plantaris, to prepare the frozen section of the plantaris to be confirmed to form the myopathy model by the histochemical stain. (3) Annexin V-Biotin Apoptotic Detection Kit was used to detect the apoptosis of different groups by flow cytometry;immunohistochemistry of SABC to measure different groups of plantaris Bcl-2 and Bax expression; RT-PCR was used to detect the expression of Bax and Bcl-2 of each group; Spectrophotometer was used to measure the activities of superoxide dismutase (SOD), Glutathione peroxidase (GSH-Px), inducible nitric-oxide synthase (iNOS) and the content of malondialdehyde (MDA) in the muscle specimens of each group respectively. The vitro experiment (1) Choose the health, male, Sprague-Dawley rats (weight 150~180g) to do the skeletal muscle primary cell cultures, then use the desmin antibody to identify the skeletal muscle satellite cells (SMSCs)by immunohistochemistry stain. (2) Trypan Blue staining was used to detect the cytotoxicity of ethanol with different concentration and time;Annexin V/PI double staining by flow cytometry and nuclear fluorophore Hoechst 33258 staining were used to detect the apoptotic cells induced by ethanol. (3) On the basis of cells treated with 75mM ethanol, added EGb761, for 48h, then use the 3-(4,5-dimethylthiazo-2-yl)-2, 5diphenyl-tetrazolium bromide (MTT) to assay the cell viability, flow cytometry to detect the change of apoptotic rate.Results: The vivo experiment (1) The body weight (BW) of rats shows: Alcohol induced a gradual decrease of the BW, at the end of 12 weeks, it was significantly lower than the control group. However, EGb761-treated group were significantly upper than the alcohol group.(2) The CAM rat shows typical pathological change in the skeletal muscle, such as myofibrosis,necrosis, the shape of the muscle fiber atrophy becomes triangle,strip or irregular, and the connective tissue among the muscle tissue become hyperplasia, and so on. EGb761 can attenuate the degree of pathological change.(3) Annexin V/PI double staining by flow cytometry shows: It is clear that alcohol was able to induce increase apoptosis compared with control. However, the apoptotic rate in EGb761 group was significantly lower than alcohol group.(4) Immunohistochemistry of SABC stain shows: Comparing between alcohol group and control group, Bax and Bcl-2 expression significant increase, the ratio of Bcl-2/Bax decrease; the EGb761 treated group shows Bax expression down-regulated and Bcl-2 up-regulated compared with alcohol group, the ratio of Bcl-2/Bax increase.(5) RT-PCR method shows: With the alcohol taking, the expression of Bax and Bcl-2 increase in plantaris. However, EGb761-treated group shows Bax expression down-regulated and Bcl-2 up-regulated compared with alcohol group.(6) Spectrophotometer method shows: MDA and iNOS in alcohol group were significantly higher than control group, activity of SOD and GSH-Px were lower than control group. However, EGb761-treated groups show a increase in the level of SOD and GSH-Px and a reduction in the level of MDA and iNOS compared with CAM alcohol group in the plantaris.The vitro experiment (1) Immunohistochemistry stain shows that about 90% were skeletal muscle satellite cells(cytoplasmic positive reactivity).(2) Trypan blue stain shows that ethanol can induce a dose-dependent reduction in skeletal muscle cells survival and a time-course cell death.(3) Annexin V/PI double staining by flow cytometry and nuclear fluorophore Hoechst 33258 staining show that it is clear that ethanol was able to induce progressive apoptosis in a time-dependent manner, at the concentration of 75mM.(4) MTT assay shows that 40μg/ml EGb761 played a protective role in the damage induced by 75mM ethanol for 48 h, it can increase the viability of cells treated with 75mM for 48h. Annexin V/PI double staining shows that 40μg/ml EGb761 can decrease the apoptosis of cells treated with 75mM for 48h.Conclusions:(1) Long time alcohol misuse can induce the skeletal muscle apoptosis, its mechanism relates to the imbalance of oxidation and anti-oxidation system, increasing of iNOS and change of Bax and Bcl-2 expression.(2) EGb761 can induce the decrease of apoptosis in CAM, its mechanism may relate to inhibit the generation of free radical, decrease the activity of iNOS, up-regulated anti-apoptosis gene Bcl-2 and down-regulated pro-apoptosis gene Bax.(3) EGb761 can not only treat the CAM but also prevent the CAM. |
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