Effect And Mechanism Of Salidroside On Skeletal Muscle Dysfunction In Rats With Chronic Obstructive Pulmonary Disease Induced By Cigarette Smoke

Objective:Chronic obstructive pulmonary disease（COPD）is a disease that is caused by persistent airflow limitation due to airway inflammation and airway remodeling.It is a common respiratory disease,which can be prevented and treated.It is estimated that the disease will be the third most common disease in the world by 2030.Except common complications such as respiratory failure,heart failure,and pneumothorax,COPD is far more harmful than that.It can also cause malnutrition,weight loss,skeletal muscle atrophy,and skeletal muscle dysfunction.There are exact reports that the incidence of COPD malnutrition can reach more than 50%.Malnutrition often causes significant depletion of skeletal muscle in patients with COPD,leading to skeletal muscle dysfunction,decreased patient endurance,and significantly reduced quality of life.The nutritional status of patients with COPD in the clinic is more and more concerned by patients and medical staff.Therefore,it is increasingly important to further study the underlying pathophysiology of COPD skeletal muscle dysfunction and explore new treatment methods.The main pathophysiological mechanism of COPD is the chronic airway inflammation caused by the human body to harmful gases such as smoking.In addition,long-term severe smoking and chronic obstructive pulmonary disease can also cause skeletal muscle atrophy and accelerate the pathogenesis of chronic obstructive pulmonary disease.Skeletal muscle dysfunction can also aggravate lung injury and worsen lung function,leading to morbidity and death in patients with COPD.However,the pathogenesis of skeletal muscle dystrophy associated with COPD is unclear.It has been reported that COPD-related hypoxia can cause inflammation,leading to an increase in the production of pre-inflammatory factors such as TNF-αand IL-6 in skeletal muscle tissue,thereby further damaging muscle cells.In addition,myogenesis is positively regulated by myogenic proteins such as myogen and negatively regulated by myostatin.Some literatures have confirmed that enhanced oxidative stress and inflammation can up-regulate the expression of myostatin,and down-regulate the expression of myostatin,and make the skeletal muscle atrophy process worse.Therefore,treatment strategies that regulate these targets may inhibit the process of COPD-related skeletal muscle atrophy.In addition,it is well known that muscle growth is a process of dynamic balance between protein synthesis and degradation.There are two important signal pathways involved in it.IGF1–PI3K–Akt–mTOR is a positive regulatory signal and myostatin–Smad2/3 is a negative regulatory signal.Rhodiola is one of the traditional Chinese herbal medicines.Rhodiola is a perennial herb or subshrub plant of the family Rhodiolaceae.The"Compendium of Materia Medica-the ministry of Grass"states that Rhodiola is a"herbal top grade",Nourishing qi and nourishing blood"and other effects.Salidroside is widely used as a traditional medicine in Eastern Europe and Asia.Previous studies have shown that salidroside has potential anti-oxidant,anti-inflammatory,anti-apoptotic,anti-fatigue and hypoxia,anti-COPD effects,and studies have confirmed that salidroside can reduce gastrocnemius loss in cachexia in lung cancer mice,increase expression of the gastrocnemius myosin heavy chain,and is achieved through the IGF1–PI3K–Akt–mTOR pathway.It has also been reported that gavage of Rhodiola and Salidroside can inhibit the expression of p-smad3 in gastrocnemius and soleus muscle for 4 weeks.However,whether salidroside treatment can improve skeletal muscle dysfunction associated with COPD and whether it can pass the two signaling pathways mentioned above has not been studied in depth.The purpose of this study is to simulate the process of smoking in vivo,establish a COPD rat model,observe the pathophysiology and functional changes of COPD-induced skeletal muscle dysfunction,and evaluate the protective effect of salidroside on COPD-related skeletal muscle dysfunction.To explore the mechanism of salidroside improving skeletal muscle dysfunction by evaluating the changes of inflammatory factors,oxidative stress and molecular biology methods from the expression levels of myostatin and myogenin genes and proteins.In addition,the role of mitochondrial ultrastructure,energy metabolism,and TGF-β-smad and PI3K-akt signaling pathways in COPD-related skeletal muscle dysfunction induced by cigarette smoke and the protective mechanism of salidroside were further investigated.Methods:1.Performance of skeletal muscle dysfunction caused by chronic smoke exposure in ratsSixteen male SPF Wistar rats were randomly divided into groups according to the random number table method,and were divided into a control group（control,n=8）and a model group（COPD,n=8）.The whole-body oral and nasal smoke toxicity experimental equipment was used.The whole body of the rat’s nose and nose was exposed to the poisoned inner cavity.The cigarette burner ignited 10 cigarettes at a time,and then successively lit 5-10 cigarettes to maintain the smoke concentration of 6%-8%,12 weeks,6 days a week,2 times a day,30 minutes each time.The COPD model of rats was copied,and the control group did not smoke.Throughout the process of COPD modeling,the rats’mental state,temperament changes,ability to respond to the outside world,grip and weight changes were closely observed.The grasping force of rats was measured with a YLS-13A grasping force tester for rats,and the muscle strength changes were measured by measuring the forced swimming time of rats with a self-made swimming facility.Rats were tracheally intubated and connected to the AniRes2005animal lung function analysis system.FEV_0.2,FVC and PEF were measured to evaluate the success of the model.HE staining was used to observe the structure and shape of lung tissue and gastrocnemius muscle tissue.Western blot was used to detect the expression of myostatin and myogenin.The changes of mitochondrial ultrastructure of skeletal muscle were observed under electron microscope.2.Salidroside improves the skeletal muscle dysfunction induced by long-term smoke exposure in rats by regulating myostatin and myogeninForty male SPF Wistar rats were randomly divided into five groups according to the random number table method,and each group was divided into 5 groups of 8（n=8）:control group（control）,COPD group（COPD）,COPD+Sal（50 mg/kg）group[COPD+Sal（50 mg/kg）],COPD+Sal（100 mg/kg）group[COPD+Sal（100 mg/kg）],and COPD+Sal（200 mg/kg）group[COPD+Sal（200 mg/kg）].The COPD rat model was prepared according to the first part of the method.The rats were smoked and treated continuously for 16 weeks.Each COPD group received different doses of salidroside（0,50,100,200 mg/kg,intraperitoneal injection）before daily smoking.At the end of the experiment,we observe the mental state,temperament changes,water intake,and weight changes of the rats,measure muscle strength and lung function.In the same way,HE staining was used to observe the structure and shape of lung tissue and gastrocnemius muscle tissue.Immunohistochemistry,Western blot,and real-time quantitative PCR were used to detect myostatin and myogenin protein and gene expression.3.Role of TGF-β-smad and PI3K-akt signaling pathway in skeletal muscle dysfunction induced by smoking in COPD rats and the protective mechanism of salidrosideCOPD models were grouped and prepared according to the second part of the method.Gastrocnemius muscle tissue was taken and embedded with resin.Ultrathin microtome sections were used to observe the ultrastructural changes of mitochondria in gastrocnemius muscle cells.Cytochrome C oxidase activity assay kit and ATP synthase activity assay kit were used to evaluate the degree of mitochondrial energy metabolism.Western blot was used to detect the differences in the expression of slow and fast proteins in gastrocnemius muscle cells,the expression of muscle atrophy-related proteins MaFbx and MuRF1,and the expression of key pathway proteins smad2/3,p-smad2/3,akt,and p-akt.Results:1.Performance of skeletal muscle dysfunction caused by chronic smoke exposure in ratsAn animal model of COPD in rats was prepared using the whole-body oral and nasal smoke toxicity experimental equipment.We observe that compared with the control group,the rats in the COPD group are thinner,poorer in general condition,with pale hair,weak spirits,dull eyes,significantly slower response to external stimuli,slow action,laziness.The above symptoms are gradually increasing with prolonged exposure to smoke.From the perspective of pulmonary function,compared with the control group,the COPD group rats had significantly lower PEF and FEV0.2/FVC,P<0.001.In general anatomy comparison,the control group was smooth and round in shape,bright in color,white to pale pink,and the COPD group was rough in shape,uneven in surface,dark in color,and gray to black.After HE staining,compared with light microscopy,the alveolar walls of the COPD group became thinner,the alveolar cavity expanded and ruptured,MAN was significantly reduced,and MLI was significantly increased,P<0.001.From the comparison of body weight,compared with the control group,the weight of rats in the COPD group was significantly reduced,and the difference reached statistical significance at the end of the first week of modeling,P<0.05,and the difference was more significant at the end of the second week,P<0.001,and there has gradually been a clear trend of slow weight gain week by week.The skeletal muscle weight（left gastrocnemius muscle weight）also reached a statistically significant difference at 12weeks of smoke exposure.Compared with the control group,the grip strength of the rats in the COPD group was significantly reduced,P<0.001,but the forced swimming time was not reduced,P>0.001.HE staining of the gastrocnemius muscle showed that CSA in the COPD group was significantly lower than that in the control group,suggesting that COPD was associated with skeletal muscle atrophy.Western blot results showed that myostatin protein expression in the COPD group was significantly higher than that in the control group,and myogenin protein expression was significantly reduced.The ultrastructure of mitochondria was observed under electron microscope.The mitochondria of gastrocnemius muscle cells in the control group were regular in shape,the membrane was smooth,the ridges were arranged longitudinally or concentrically,and the structure was clear.But the mitochondria of gastrocnemius muscle cells in the COPD group were Rupture,maggot damage or even disappear,the number of mitochondria decreases or disappears,forming vacuoles.2.Salidroside improves the skeletal muscle dysfunction induced by long-term smoke exposure in rats by regulating myostatin and myogeninThe salidroside intervention group can significantly improve the general state of COPD rats.The response to external stimuli is significantly enhanced compared with the simple COPD group.The high-dose（200mg/kg）salidroside group has the most obvious effect.Improved pulmonary function in COPD rats,with FEV0.2/FVC significantly improved,P<0.001 in the middle and high dose groups,PEF also improved compared to the COPD group,and the effect was highest in the high dose（200mg/kg）salidroside group P<0.001.Observed on HE staining of lung tissue,salidroside intervention group can significantly improve the histopathological morphology of COPD rats,manifested by improved alveolar cavity enlargement,MAN increased compared with COPD group,low dose group reached P<0.01,medium and high dose group reached P<0.001,MLI was lower than that in COPD group,P<0.05 in middle-dose group,and the effect was highest in high-dose（200mg/kg）salidroside group,P<0.001.In terms of body weight,after salidroside intervention,rat body weight increased compared with the simple COPD group,and reached statistical significance in the high-dose group（200mg/kg）,P<0.05,and muscle weight increased.P<0.01 in the high-dose group,suggesting that salidroside can alleviate weight gain in rats caused by COPD,and has a certain dose-effect relationship.Functionally,the salidroside intervention group can significantly improve the grip of COPD rats.The medium-dose group P<0.05 and the high-dose group P<0.001.The weight-bearing swimming time of the COPD group was longer than that of the control group,but did not reach a statistical difference.The forced swimming time of the salidroside intervention group was longer than that of the COPD group,and the middle-dose group reached statistical significance P<0.05,and the high-dose P<0.01.The structure of muscle tissue was observed by HE staining under the light microscope.The salidroside intervention group significantly increased the cross-sectional area of??muscle fibers in COPD rats,in medium-dose group P<0.01,and in high-dose group P<0.001.Changes in inflammatory factors were detected by ELISA.The salidroside intervention group significantly reduced the production of TNF-αin COPD rats.The serum effect was more pronounced in the high-dose salidroside group,reaching P<0.01,in the skeletal muscle tissue homogenate.In the medium-dose group,P<0.05,and in the high-dose group,P<0.001,the salidroside intervention group also significantly reduced the production of IL-6 in COPD rats.The serum and skeletal muscle tissue homogenate reached in the medium-dose group,P<0.01,in the high-dose salidroside group,P<0.001.The chemical reaction was used to detect the level of oxidative stress.The salidroside intervention group significantly reduced the MDA level in COPD rats.The serum reached P<0.001 in the medium and high dose groups;the tissue homogenate was in the medium dose group P<0.01 and the high dose group P<0.001.The salidroside intervention group significantly increased SOD activity,with serum levels of P<0.001 in the medium and high-dose groups;tissue homogenates in the medium-dose group P<0.01,and high-dose groups P<0.001.The salidroside intervention group significantly increased the level of GSH.The serum was in the medium-dose group P<0.05,the high-dose group P<0.01;the tissue homogenate was in the medium-dose group P<0.05,and the high-dose group P<0.001.Western blot was used to detect protein expression.Salidroside intervention group significantly reduced the protein expression of myostatin in COPD rats,P<0.01 in the high-dose group and P<0.001 in the high-dose group;increased protein expression of myogenin,and in the middle-dose group P<0.05,P<0.01 in the high-dose group.Real-time quantitative PCR was used to detect gene expression.The salidroside intervention group significantly reduced the gene expression of myostatin in COPD rats.The high-dose group reached statistical significance,P<0.01;the gene expression of myogenin was increased,and both in the high-dose group were P<0.01,consistent with protein expression.Immunohistochemical detection of protein expression revealed that salidroside intervention group significantly reduced the protein expression of myostatin in COPD rats,which was statistically significant in the high-dose group,P<0.001;increased protein expression of myogenin in the high-dose group was statistically significant,P<0.01.Consistent with the results obtained by Western blot and PCR methods.3.Role of TGF-β-smad and PI3K-akt signaling pathway in skeletal muscle dysfunction induced by smoking in COPD rats and the protective mechanism of salidrosideObserved from the ultrastructure,the mitochondria of the gastrocnemius muscle cells in the control group were regular in shape,the membrane was smooth,the ridges were arranged in a longitudinal or concentric circle,and the structure was clear.The mitochondria of the gastrocnemius muscle cells in the COPD model group were significantly swollen and degenerate,and the membrane was blurred.Rupture,maggot destruction or even disappearance,the number of mitochondria decreased or even disappeared,and vacuolation was formed;after the application of salidroside,the above-mentioned performance of mitochondria of gastrocnemius muscle cells of rats in the low to high dose group gradually reduced.From the perspective of energy metabolism,the activity of cytochrome C oxidase and ATP synthase in the mitochondria of rat gastrocnemius muscle tissue were（881.3±180.7）U/mgprot and（11.7±3.14）U/mgprot in the COPD group,respectively.In comparison,the activity of cytochrome C oxidase gradually increased after salidroside application,reaching P<0.01 in the high-dose group;the ATP synthase activity gradually increased,reaching（16.44±2.77）U/mgprot and（19.25±2.56）U/mgprot,the difference was statistically significant,P<0.05 in the middle dose group and P<0.001 in the high dose group.In terms of different types of protein expression,the content of slow and fast protein gradually increased in the three treatment groups compared with the model group.Slow was statistically significant in the high-dose group,P<0.01;fast was statistically significant in the high-dose group,P<0.05 and P<0.01,respectively.From the expression of dystrophy-related proteins,compared with the control group,the protein content of MaFbx and MuRF1 in the model group was significantly increased,and the differences were statistically significant,respectively P<0.01 and P<0.001.3 treatment groups compared with the model group The protein content of MaFbx and MuRF1 gradually decreased,and the difference of MaFbx in the high and high dose groups was statistically significant,respectively P<0.01 and P<0.001;the difference of MuRF1 in the high dose group was statistically significant,P<0.01.Western blot method was used to measure the expression of each pathway protein,and there was no significant change in smad2/3content in the five groups.Compared with the control group,the p-smad2/3 protein content in the model group was significantly increased,and the difference was statistically significant,P<0.001.The p-smad2/3 protein content in the three treatment groups was gradually decreased compared with the model group.The difference was statistically significant,P<0.01 and P<0.001,respectively.There was no significant change in akt content in the 5 groups.Compared with the control group,the p-akt protein content in the model group was significantly reduced,and the difference was statistically significant,P<0.001.The p-akt protein content in the three treatment groups was gradually increased compared with the model group,and the difference was statistically significant in the high-dose group.Significance,P<0.01.Conclusion:1.Long-term smoke exposure can cause emphysema,including reduction of MAN in lung tissue,increase of MLI,and decline in lung function.At the same time,it can cause skeletal muscle weight loss,muscle strength decline.But muscle endurance may not decline due to the weight gain.The cross-sectional area of the gastrocnemius muscle decreased,and the mitochondrial morphology changed.Myostatin and myogenin proteins are abnormally expressed.2.Salidroside played a protective role in COPD-induced skeletal muscle dysfunction,showing a certain dose-effect relationship.The protective mechanism is related to the decrease of inflammation-mediated myostatin expression and increase of myogenin expression.3.Salidroside can improve the mitochondrial ultrastructure of skeletal muscle,reduce mitochondrial vacuole degeneration,and improve energy metabolism.The protective effect of salidroside on skeletal muscle fiber cells is related to inhibiting the activation of the TGF-β-smad signaling pathway and enhancing the activation of the PI3K-akt signaling pathway.