The Study Of Effect Of Jianpiyifei-Ⅱ On COPD Rat Skeletal Muscle Atrophy

Objective:1.To observe the effects of cigarette smoke on lung and skeletal muscle in rats at different time.2.To investigate the effect of Chinese herbal medicine "Jianpi Yifei-II"on cigarette smoking induced skeletal muscle atrophy in COPD rats.And the mechanism of "Jianpi Yifei-Ⅱ,on treatment skeletal muscle atrophy in COPD rats by interfering with AMPK/ULK-1 signaling pathway.3.To investigate the molecular mechanism of "Jianpi Yifei-Ⅱ" on treatment skeletal muscle atrophy in vitro experiments.Method:1.96 SD rats were randomly divided into 6 groups according to weight by randomized controlled trial,16 rats in each group.They were 1 months normal group(Con-1m),1 months model group(CS-1m),3 months normal group(Con-3m),3 months model group(CS-3m),6 months normal group(Con-6m),6 months model group(CS-6m).COPD rat models were established by cigarette smoking stimulation at different months time.By observing the general condition,body weight,water intake,pulmonary function,pathological changes of lung tissue and bronchoalveolar lavage fluid protein and superoxide dismutase content,changes of blood and skeletal muscle tissue pathology and ultrastructure of rats to evaluate the COPD model.The effects of cigarette smoking on skeletal muscle in rats were investigated by detecting autophagy related proteins and gene expression in skeletal muscle.2.45 SD rats were divided into 3 groups according to the random number table,15 rats in each group.They were normal group(Con group),model group(CS group),and Chinese medicine Jianpi Yifei-Ⅱ group(JY-Ⅱ).Except for Con group,CS group and JY-Ⅱ group were treated with cigarette smoking to establish COPD rat model.After 6 months later,we observed the effect of JY-II on of the general condition of about body weight,water intake,lung function,lung tissue pathological changes,BALF protein and oxidative stress index,changes of blood and skeletal muscle autophagy related proteins and genes of cigarette smoking stimulated rats.3.In vitro experiments,using rat myoblast cells(L6 cell line),using cigarette smoke extract(CSE)and/or JY-II to treatment L6 cells.Observing the expression of autophagy related protein Lc3B,Beclin-1,AMPK a and pAMPK a and ULK-1 in L6 cells induced by the CSE and the influence of JY-II on the protein changes after intervention.AMPK agonists(AICAR)and inhibitors(BML275)were applied to the cells to observe the changes of these proteins at the same time.Results:1.With the stimulation of cigarette smoking,the CS group rats mental state became worsed,and the fur became yellow,withered and rough gradually,and diet gradually decreased.When cigarette smoking,CS group rats curled up and moved less.The weight gain in CS group was slower than that in Con group(P<0.05),and the difference in weight remained with the duration of smoking(P<0.05).After cigarette smoking stimulation,CS group rats water intake increased,compared with Con group,the difference was statistically significant(P<0.05).The total lung volume of CS group rats was higher than that of Con group(P<0.05).And with the increase in the number of days of cigarette smoking stimulation,the higher the total lung volume.At first months and third months after cigarette smoking,the pulmonary airway resistance(RI)in CS group rats was higher than that in Con group(P<0.05).After third months of cigarette smoking stimulation,FEV0.1/FVC in CS group was decreased,which compared with Con group rats,the difference was statistically significant(P<0.05).Under the microscopy,the lung structure of CS rats gradually changed,and the alveolar walls ruptured and formed lung bullae.Alveolar septum and thickening of alveolar wall.After stimulated with smoking in three months,the alveolar space of CS group was more than that of Con group(P<0.05).After 3 months of cigarette smoking stimulation,the gastrocnemius muscle tissue structure of Con group rats was normal,and the muscle fibers in CS group were scattered,and the muscle filber area was decreased compared with the normal group(P<0.05).With the increase of smoking stimulation time,the muscle fiber area of CS group decreased gradually(P<0.05).After 3 months of cigarette smoking stimulation,the muscle fibers of CS group were arranged neatly and the ridges were less wrinkled under the electron microscope.A few vacuolar lesions were observed at high magnification.With the increase of cigarette smoking stimulation time,vacuole shape gradually increased compared with the Con group.At 3 months,the white blood cells(WBC)of rats in CS group,the total number of mononuclear cells(MONO),the total number of red blood cells(RBC)and total hemoglobin(HGB)were higher than that of Con group,the difference was statistically significant(P<0.05);The number of red blood cells(RBC)and blood red protein(HGB)of CS rats at 6 months the number of is higher than that of Con group,the difference was statistically significant(P<0.05).After the stimulation of cigarette smoking,the protein of bronchoalveolar lavage fluid(BALF)of CS rats was higher than that in Con group(P<0.05).The SOD in the CS group had statistically difference significant with Con group only in the first month(P<0.05),while the LDH in the CS group had statistically difference significant with Con group only in the sixth month(P<0.05).After cigarette smoking stimulation,the autophagy related proteins Beclin-1 and LC3B(especially LC3B-II),p-AMPK and ULK-1 in skeletal muscles of CS rats were increased compared with the Con group(P<0.05).After cigarette smoking stimulation,the relative expression of Lc3B and mRNA in gastrocnemius muscle of CS group was higher than that in Con group(P<0.05).2.After 6 months of smoking,the weight of JY-II rats was higher than that of CS group(P<0.05),the difference was statistically significant.The alveolar space in group JY-II was lower than that in CS group(P<0.05).The area of gastrocnemius muscle fiber in JY-II group decreased slowly compared with CS group,and the number of scattered muscles also decreased(P<0.05).Ultrastructural observation showed that the muscle fibers of JY-II group were arranged neatly,the line of Z was clear,and the cristae of wrinkles disappeared.The vacuolar lesions were less than that in CS group at high power microscope.The levels of RBC and HGB and serum LDH in group JY-II were lower than those in CS group(P<0.05).The relative expression of autophagy protein Lc3B and Lc3B mRNA in gastrocnemius muscle of JY-II rats was lower than that in CS group(P<0.05).3.Rat myoblasts(L6 cell lines)showed different trends after intervention with different concentrations of cigarette smoke extract(CSE),24h,48h and 72h.Among them,100%CSE inhibited L6 cells vitality in 24h(P<0.05).And it also showed different trends after intervention with different concentrations of JY-II,respectively in 24h,48h and 72h.The 6-10mg/ml of JY-II Chinese medicine were inhibited L6 cells vitality,and with time increasing,inhibition rate also increased,which present a time and dose dependence.1-4mg/ml has a proliferation effect on L6 cells.Different concentrations of CSE can increase the expression of Lc3B protein in L6 cells,especially the expression of Lc3B-II(P<0.05).After induction of L6 cells by CSE,the relative content of autophagy proteins Beclin-1,p-AMPK and its downstream protein(ULK-1)was higher than that of Con group,and the difference was statistically significant(P<0.05).The p-AMPK and ULK-1 protein in group JY-II were significantly lower than those in CS group.Conclusion:1.Cigarette smoking can cause pathological changes of COPD lung and skeletal muscle in SD rats,2.COPD rats had skeletal muscle atrophy,which due to the the upregulation of autophagy protein by cigarette smoking stimulation.3.CSE inhibited the proliferation of L6 cells in a concentration dependence,and the higher concentration,the stronger inhibition.4.CSE can activate and up regulate the expression of autophagy protein in L6 cells.5.JY-II can retard skeletal muscle atrophy in COPD rats,possibly by down regulation of AMPK/ULK-1 signaling pathway to inhibit Lc3B in certain extent.