| OBJECTIVE: through yiqijianpihuoxue and regulation role of mitochondrial apoptosis research,explore yiqijianpihuoxue and the prevention and treatment of T2 DM targets and function law of skeletal muscle cell pathological changes,reveals the treatment mechanism,for replenishing yiqijianpihuoxue and provide a scientific basis for the clinical application,also offers a new research idea for TCM prevention and treatment of T2 DM skeletal muscle lesions.METHODS:SPF SD rats were randomly divided into two groups,20 in each group,namely the blank group and the traditional Chinese medicine group,and the traditional Chinese medicine group was given shenqi compound 3mL(2mg raw medicine /mL)traditional Chinese medicine extract by gavage.Normal group was given equal volume normal saline intragastric twice a day for 3 consecutive days.The blood was collected from the abdominal aorta after 1h of the last gavage,and then stood at room temperature for 2h.The blood was centrifuged at 4?.The serum was collected,inactivated by water bath at 56? for 30 min,and frozen at-80? for later use.The fourth generation of rat L6 muscle cells was digested with trypsin,and DMEM(containing 10% FBS)was added to the centrifuge tube,which was blown and mixed.When the cell density was close to 70%,the serum-free medium was replaced for 24 h to synchronize the cells.Each 6-well cell group was divided into groups and stimulated by the following methods: the 4th generation of rat L6 muscle cells were inoculated in T75 culture bottle.When the cell density was close to 70%,serums free medium was replaced for 24 h to synchronize the cells.(1)HBM hyperglycemia model group;Ii.HRM hyperglycemia control group;(3)HLS5% Chinese medicine serum group;(4)hms10% Chinese medicine serum group;(5)hhs15% Chinese medicine serum group;(6)HBL rosiglitazone group;(7)CTRL blank control group.After the preparation of the intervention fluid,0.22 micron filtration was performed.Cells with various stimuli were placed in incubators at 37? and 5%CO2 for culture,and the cells were collected at 24,48 and 72 hours for indicator detection.Mitochondrial swelling degree,changes of mitochondrial membrane potential,apoptosis rate detected by flow cytometry,and expression of caspase-3 and caspase-9 in the cytoplasm of gastrocnemial muscle were detected by rt-pcr and Western blot.Results: 1.The detection of apoptosis rate showed that the serum group(HMS)had the lowest apoptosis rate,followed by the rosiglitazone group(HBL),and the high glucose control group(HRM)and high glucose model group(HBM).2.L6 cells mitochondrial membrane potential delta ? m visible blank control group(CTRL)membrane potential is highest,traditional Chinese medicine medicated serum group(HMS)membrane potential times,rosiglitazone(HBL)third,high sugar model(HBM)and high sugar controls(HRM)membrane potential minimum.3.Mitochondrial swelling in the blank control group(CTRL),the mitochondrial swelling in the rosiglitazone containing serum group(HBL)was the lowest,the HRM in the high glucose control group and the high glucose model group(HBM),and the HMS in the traditional Chinese medicine containing serum group(HMS)was higher.4.Rt-qpcr showed that high glucose control group(HRM)caused caspase-3 mRNA,and caspase-9 mRNA expression level was significantly increased.5.Western blotting results showed that the protein expression levels of caspase-3 and caspase-9 in the high-glucose control group(HRM)and the high-glucose model group(HBM)were significantly increased.6.ELISA showed that the insulin content in the culture medium was basically the same and the results were negative.Conclusion: 1.In the high-glucose environment,Chinese medicine serum group and rosiglitazone serum group have certain inhibitory effects on mitochondrial cell apoptosis.2.The measurement of mitochondrial membrane potential(MMP)showed that the membrane potential of the serum group containing traditional Chinese medicine and the serum group containing rosiglitazone was higher than that of the high-glucose control group and the high-glucose model group,indicating that the serum group containing traditional Chinese medicine and the serum group containing rosiglitazone had certain inhibitory and delayed effect on the initiation of apoptosis.3.It can be seen from the measurement of mitochondrial swelling that all the test groups experienced mitochondrial swelling,and the rosiglitazone containing serum showed relatively low swelling,indicating that rosiglitazone containing serum had a prominent effect on controlling mitochondrial swelling.4.Western blotting assay: the protein levels of caspase-3 and caspase-9 in the hyperglycemia control group and the hyperglycemia model group were significantly higher than those in other groups,with enhanced activity,forming apoptotic bodies,leading to the occurrence of mitochondrial cell apoptosis,and even T2 DM skeletal muscle lesions.5.Rt-qpcr detection: the high-glucose control group and the high-glucose model group significantly increased caspase-3 mRNA and caspase-9 mRNA,and formed apoptotic bodies with other substances,leading to cell mitochondrial apoptosis and T2 DM skeletal muscle lesions 6.The insulin content in L6 skeletal muscle cell culture medium showed that the insulin content in each group was similar,which meant that there was little difference in the influence of all groups on the insulin content and no statistical significance In conclusion,shenqi compound formula based on the method of invigorating qi,strengthening spleen and activating blood can prevent T2 DM skeletal muscle diseases by regulating mitochondrial apoptosis... |
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