| Purpose:Taking autophagy and chronic obstructive pulmonary disease(COPD)skeletal muscle atrophy as the entry points to explore the intervention effects and mechanisms of Buyuan Decoction regulating autophagy to improve skeletal muscle atrophy in COPD through in vivo and in vitro experiments,so as to provide an experimental basis for Buyuan Decoction to prevent and treat skeletal muscle atrophy in COPD and enrich the scientific content of tonifying Zongqi therapy in the treatment of COPD skeletal muscle atrophy.Chapter 1.Theoretical discussionBy combing the academic development of ancient and modern physicians’ understanding of the pathogenesis of COPD and skeletal muscle atrophy,this paper summarized the TCM pathogenesis of COPD skeletal muscle atrophy as lung heat scorching the lobes,lung and spleen Qi deficiency,liver and kidney deficiency,and phlegm and blood stasis blocking collaterals.In combination with Professor Hong Guangxiang’s academic thought of "supplementing Zongqi to treat COPD",we proposed that the key pathogenesis of COPD skeletal muscle atrophy was "deficiency of Zongqi as Ben,phlegm and blood stasis blocking collaterals as Biao",and the method of invigorating Zongqi was an important therapeutic principle,which provided a theoretical basis for the clinical application of Buyuan Decoction in the treatment of skeletal muscle atrophy in COPD.Autophagy is the key mechanism of skeletal muscle atrophy in COPD.By deeply connecting the theory of " Zongqi deficiency" in TCM with the regulation mechanism of autophagy,we proposed that supplementing Zongqi can improve the body’s defense ability,promote the body’s gasification and enhance the body’s regulation ability,so as to regulate autophagy,inhibit inflammatory response,mitochondrial autophagy and skeletal muscle protein degradation to improve skeletal muscle atrophy in COPD,which layed a theoretical foundation for experimental research.Therefore,a scientific hypothesis was put forward: Buyuan Decoction may improve skeletal muscle atrophy in COPD by regulating autophagy.Chapter 2.Experimental researchMethod: 1.Effect and mechanism of Buyuan Decoction on cigarette smoke extract induced L6 myoblasts injuryThe concentration of scientific intervention was determined by flow cytometry to detect the effect of different concentrations of cigarette smoke extract(CSE)(5%,10%,20%,40% and 60%)and Buyuan Decoction(50mg/m L,5mg/m L,1mg/m L,0.5mg/m L,0.25mg/m L and 0.1mg/m L)of test medium treatment for 48 h on the apoptosis rate of L6 myoblasts.Subsequently,the experiments were divided into the following groups: control,CSE and Buyuan Decoction groups,the cell viability of each group was detected by CCK-8 assay,the apoptotic rate and cell cycle of each group were detected by flow cytometry,the ultrastructural changes of each group were observed by transmission electron microscopy,and the microtubule associated protein 1 light chain 3(LC3B),autophagy related gene 7(Atg7)and autophagy adaptor protein p62(sequestosome 1,p62)protein expression were detected by Western blot(WB).2.Effects of cigarette smoke exposure combined with lipopolysaccharide induction on atrophy in COPD skeletal muscle wasting ratsA rat model of COPD was constructed by cigarette smoke exposure combined with an intra airway injection of lipopolysaccharide(LPS)via a cervical incision,and randomly divided into4-week control(4W-CG),8-week control(8W-CG),12-week control(12W-CG),4-week model(4W-MG),8-week model(8W-MG)and 12-week model(12W-MG)groups respectively.Body and gastrocnemius muscle mass changes were observed,lung function indexes were detected by whole body plethysmography(WBP)system: ratio of the time of inspiration to expiration,Ti/Te,50% expiratory flow rate(EF50)and airway narrowing index(Penh),Serum levels of the inflammatory cytokine tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6)were measured by enzyme-linked immunosorbent assay(ELISA)levels,and hematoxylin eosin(he)staining was performed to observe the histopathological changes of lung tissue and gastrocnemius muscle morphologically.3.Intervention effects and mechanisms of Buyuan Decoction on skeletal muscle atrophy induced by cigarette smoke exposure combined with LPS in COPD ratsThe COPD rat model was replicated by cigarette smoke exposure combined with intraairway LPS injection via a cervical incision,and the experiment was divided into control,model,lowdose Buyuan decoction,medium dose Buyuan Decoction,and high-dose Buyuan Decoction groups,and the intervention was performed by intragastric administration of Buyuan Decoction in the 10 th week,which lasted for 4 weeks,until the end of the intervention in the 14 th week to detect the indicators.Body and gastrocnemius muscle mass were observed,lung function was assessed by WBP: Ti/Te,EF50 and Penh,TNF-α and IL-6 levels were detected by ELISA,lung tissue and gastrocnemius muscle histopathological morphological changes were observed by HE staining,gastrocnemius muscle autophagy related protein LC3 B,Atg7 and p62 expression and atrophy related protein myostatin and muscle ring finger protein 1(Mu RF1)expression were detected by WB,and gastrocnemius muscle ultrastructural changes were detected by transmission electron microscopy.Result:1.Effects and mechanisms of Buyuan Decoction on CSE induced L6 myoblast injury(1)L6 myoblasts treated with different concentrations of CSE(5%,10%,20%,40% and60%)for 48 hours showed no significant difference(P>0.05)in the apoptotic rate compared with the control group,while the apoptotic rate was significantly higher in the 20%,40%,60% group(P<0.0001).Among them,40% and 60% groups had apoptosis rates higher than 80%,which was not conducive to subsequent intervention experiments,so 20% CSE was chosen to induce L6 myoblast injury.L6 myoblasts were treated with different concentrations of Buyuan Decoction(50mg/m L,5mg/m L,1mg/m L,0.5mg/ml,0.25mg/m L and 0.1mg/m L)for 48 h,compared with the 20% CSE group,the apoptosis rate in the 50mg/m L group was significantly increased(P<0.0001),that in the 5mg/m L,1mg/m L group was significantly decreased(P<0.0001)and that in the 0.5mg/m L,0.25mg/m L and 0.1mg/m L group were not significantly different(P>0.05).The apoptosis rate of the 1mg/m L group was lower than that of the 5mg/m L group,so 1mg/m L Buyuan decoction was selected for subsequent experimental intervention.(2)The results of cell viability assays using CCK8 showed that cell viability was significantly decreased in the 20% CSE group compared with the control group(P<0.0001);Compared with the 20% CSE group,cell viability was significantly higher in the 1mg/m L Buyuan Decoction group(P<0.0001).The results of flow cytometry analysis showed that the apoptosis rate of cells in the 20% CSE group was significantly higher compared with the control group(P<0.0001);Compared with the 20% CSE group,the apoptosis rate in the 1mg/m L Buyuan Decoction group decreased significantly(P<0.0001).The cell cycle results showed that compared with the control group,the 20% CSE group showed no significant difference in the number of cells in G1/G0 phase(P>0.05),a significant increase in the number of cells in S phase(P<0.001),and a significant decrease in the number of cells entering G2/M phase(P<0.0001);Compared with the 20% CSE group,cells in the G1/G0 phase were slightly increased(P<0.05),cells in the S phase were significantly decreased(P<0.0001),and cells in the G2/M phase were significantly increased(P<0.0001).Compared with the control cells,the protein expression of LC3 B and Atg7 in cells treated with 20% CSE increased significantly(P<0.0001),while the protein expression of p62 decreased significantly(P<0.0001);Compared with the 20% CSE group,the protein expressions of LC3 B and Atg7 of cells in the 1mg/m L Buyuan Decoction group were significantly decreased(P<0.0001),and the protein expression of p62 was significantly increased(P<0.0001).Electron microscopy showed a large number of vacuole like changes in the cytoplasm and autophagosomes in the 20% CSE group,while no autophagosomes were observed in the 1mg/m L Buyuan Decoction group.2.Effects of cigarette smoke exposure combined with LPS induction on atrophy in COPD skeletal muscle wasting ratsCompared with the control group in the same period,in the model group,the spirit and activity status gradually become poor,the body size is gradually thin,the resistance is gradually weakened,and the hair gradually turns yellow;The lung function indicators of Ti/TE and EF50 decreased significantly(P<0.05),while Penh increased significantly(P<0.05);The bronchial lumen was distorted,epithelial cells were sloughed off,bronchial smooth muscle was thickened,lung interstitium was slightly congested,alveolar wall thickening was aggravated,and a large number of inflammatory cells were infiltrated around bronchial mucosa and among lung tissues,and the pathological score was significantly higher(P<0.0001);Serum TNF-α and IL-6 levels were significantly higher(P<0.05);body mass decreased significantly(P<0.0001);There was no significant difference in gastrocnemius muscle mass in 4W-MG(P>0.05),but 8W-MG and 12 WMG decreased significantly(P<0.05,P<0.01);There was no significant difference in CSA between 4W-MG and 8W-MG gastrocnemius muscle fibers(P>0.05),but 12W-MG decreased significantly(P<0.0001).3.Intervention effects and mechanisms of Buyuan Decoction on skeletal muscle atrophy induced by cigarette smoke exposure combined with LPS in COPD rats(1)Compared with the control group,the model group had poor mental and activity status,thin body,weak resistance,yellow hair,shortness of breath and slow recovery after smoking;Ti/Te and EF50 decreased significantly(P<0.0001),Penh increased significantly(P<0.0001);There were exfoliation of bronchial epithelial cells,thickening of bronchial smooth muscle,mild congestion of lung stroma,thickening and aggravation of alveolar wall,infiltration of a large number of inflammatory cells around bronchial mucosa and between lung tissues,and the pathological score increased significantly(P<0.001);The level of serum TNF-α and IL-6 were significantly higher(P<0.0001);Body mass and gastrocnemius muscle mass decreased significantly(P<0.0001);CSA of gastrocnemius muscle fibers decreased significantly(P<0.0001);Under electron microscope,the gastrocnemius muscle showed structural disorder,expansion of sarcoplasmic reticulum,a large number of vacuolar changes and autophagosome,swelling or cavitation of mitochondria,fracture or disappearance of cristae;The expression of LCB and Atg7 protein in gastrocnemius muscle increased significantly(P<0.0001),the expression of p62 protein decreased significantly(P<0.0001),and the relative expression of myostatin and Mu RF1 protein increased significantly(P<0.0001).(2)Compared with the model group,the general condition of the low dose group did not improve significantly,while the mental and activity status of the medium and high dose group improved,the body shape increased,the resistance increased,and the shortness of breath decreased;There was no significant difference in the levels of Ti/Te,EF50 and Penh in the low dose group(P>0.05).The levels of Ti/Te and EF50 in the medium and high dose group increased significantly(P<0.05),and Penh decreased significantly(P<0.0001);There was no significant difference in the pathological score between the low and middle dose groups(P>0.05).In the high dose group,the infiltration of inflammatory cells,the thickening of bronchial smooth muscle and alveolar wall,and the congestion of pulmonary interstitium decreased significantly(P<0.01);TNF-α and IL-6 in low,medium and high dose groups decreased significantly on average(P<0.05),especially in the high dose group;There was no significant difference in body mass and gastrocnemius muscle mass in the low dose group(P>0.05),but the body mass and gastrocnemius muscle mass in the medium and high dose group increased significantly(P<0.5),especially in the high dose group;CSA of gastrocnemius muscle fiber in low and middle dose group had no significant difference(P>0.05),but CSA in high dose group increased significantly(P<0.05);Vacuolar degeneration and autophagosome were observed in gastrocnemius muscle tissue of low dose group by transmission electron microscope.Autophagosome were not found in medium and high dose group,and the morphology of mitochondria was regular;There was no significant difference in the expression of LC3 B,Atg7 and p62 proteins in gastrocnemius muscle in the low dose group(P>0.05),there was no significant difference in LC3 B in the medium dose group(P>0.05),the expression of Atg7 protein decreased significantly(P<0.0001),p62 increased significantly(P<0.0001),the expression of LC3 B and Atg7 protein decreased significantly in the high dose group(P<0.01,P<0.0001),the expression of p62 protein increased significantly(P<0.0001).There was no significant difference in Mu RF1 and myostatin protein(P>0.05).The expression of myostatin protein decreased in the medium dose group(P<0.05),but there was no difference in Mu RF1 protein expression(P>0.05).The expression of myostatin and Mu RF1 protein decreased significantly in the high dose group(P<0.0001,P<0.001).Conclusion: 1.Buyuan Decoction has a protective effect on L6 myoblast cell injury induced by CSE,which can increase cell viability,reduce apoptosis,reduce cell cycle arrest in S phase and promote cells to enter G2/M phase for differentiation and proliferation,and the protective mechanism may be related to the inhibition of cell autophagy by down regulating LC3 B,Atg7and up regulating p62 protein expression levels.2.Buyuan Decoction can effectively attenuate skeletal muscle atrophy induced by cigarette smoke combined with LPS in COPD rats,and the protective mechanism may be related to the reduction of serum inflammatory factor TNF-α and IL-6 levels,down-regulation of LC3 B,Atg7and up regulation of p62 protein expression inhibit autophagy activation and autophagy lysosome system(ALS),while down-regulation of myostatin and Mu RF1 protein inhibits ubiquitin proteasome system(UPS),which in turn alleviated protein degradation in skeletal muscle. |
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