Effects And Mechanism Investigation Of Cigarette Smoke Exposure Of Various Intensity Ranks On Muscle Atrophy And Fiber Type Shift In Rat Diaphragm And Skeletal Muscles

In addition to pulmonary symptoms such as dyspnea,chronic obstructive pulmonary disease(COPD)patients is oftenly accompanied with diaphragm and skeletal muscle dysfunction,which is associated with the prognosis of COPD patients.However,the morphological and functional changes during the development and progression of COPD are rather complex and implicated mechanisms remain unknown.Smoking is the most important and common risk factor of COPD.The research was aimed the effects of cigarette smoke(CS)exposure on morphology and fiber type distribution of diaphragm and skeletal muscles and underlying mechanisms.In the present study,rats were randomly divided into 4 groups and received normal air or one of three durations of cigarette smoke exposure(4 weeks,8 weeks and 12 weeks respectively).Hematoxylin and eosin(H&E)staining of lung tissue and eletron microscopy of diaphragm and skeletal muscle were performed to assess alveolar destruction and endoplasmic reticulum(ER)damage and autophagosome contents respectively.Sections were stained for myosin ATPase with alkaline preincubation in order to fulfill fiber typing and determination of fiber type-specific diameters.Western blot and quantitative real-time polymerase chain reaction(q RT-PCR)were adopted to assay the expression of the hallmarkers of ERS,caspase-12,and the possible upstream and downstream molecules of Gadd45 a in rat muscles.The terminal deoxynucleotidyl transferase-mediated DNA nick-end labeling(TUNEL)staining was used to identify the apoptotic level.H&E staining revealed CS exposure caused inflammatory infiltration and pulmonary emphysema.Swollen and distended ER and more autophagosomes were observed using electron microscopy during muscles following CS exposure.ATPase staining analysis demonstrated CS exposure induced muscle atrophy in diaphragm and peripheral skeletal muscle,where type Ⅰ muscle fiber percentages were increased and reduced respectively.With the prolongation of CS exposure,the expression of glucose-regulating protein78(GRP78)was revealed an increase and a decrease,whereas the expression of C/EBP homologous protein(CHOP)and caspase12 and apoptotic cell percentage were permanently enhanced in a time-dependent fashion and peaked during the 12-week periods in rat diaphragm and extensor digitorum longus(EDL).The possible upstream regulators of Gadd45 a may be Akt and Foxo3 a,whose relative protein ratio of phosphorylated to total forms were markedly reduced in rat EDL.Also,the expression of speculated downstream targets including LC3 B,Bnip3and caspase3 were consistently upregulated at the transcript levels.The relative protein ratio of LC3Ⅱ to LC3Ⅰ together with the expression of Bnip3,caspase3 and p21 at the protein levels was significantly upregulated.Besides,the protein ratio of phosphorylated to total form for m TOR coupled with the expression of peroxisome proliferator-activated receptor γ coactivator 1(PGC-1α)at both protein and m RNA levels was obviously decreased.In conclusion,ERS induced apoptosis may be implicated in the atrophic progression of diaphragm and skeletal muscle induced by CS exposure.Meanwhile,Gadd45 a might be involved in CS exposure-induced fiber type shift and skeletal muscle atrophy and thus providing promising intervention targets of clinic therapy for diaphragm and skeletal muscle dysfunction in COPD patients.