Effect Of Chronic Stress On Rat Gastrocnemius And Soleus Muscle And Its Molecular Mechanism

In the process of modern scale breeding,livestock and poultry suffer from a variety of adverse environmental stresses for a long time.This chronic stress can lead to reduced animal feed intake,increased feed-to-meat ratio,reduced immunity,and reduced resistance to disease.Skeletal muscle,as an important metabolic organ of the body,is also most directly affected by chronic stress in animals.According to the type of muscle fiber metabolism,skeletal muscle can be divided into oxidative,intermediate and fermentative muscles.Previous studies have been limited to muscle tissue,however,it is not clear whether skeletal muscle of different metabolic types exhibits different behaviors under chronic stress and whether the molecular regulatory mechanisms are the same.As two typical muscle tissues,gastrocnemius muscle and soleus muscle have different metabolic types and muscle fiber types,so they have become the main research objects in this experiment.We compared the effects of chronic stress on gastrocnemius and soleus muscle histomorphology and related gene expression patterns by constructing a rat model of chronic stress,and explored the potential mechanisms of its downstream regulatory genes in response to chronic stress in different metabolic types of muscles around glucocorticoid receptors.1 Effects of chronic stress on tissue morphology and gene expression patterns of gastrocnemius and soleus muscles in ratsIn this experiment,12 adult male healthy SD rats were selected and randomly divided into control group and experimental group.Rats in the experimental group were subjected to 2 hours of random stress daily,including restraint stress,cold stress,rotational stress,greenhouse swimming,cold water swimming and water-cut stress.Continuous chronic stress treatment was carried out for 3 weeks,fasting for 8 hours before sampling.After anesthesia with intraperitoneal injection of sodium pentobarbital(40 mg/kg),eyeball blood was collected and frozen tissue and molecular samples of soleus and gastrocnemius muscles were collected.The results showed that chronic stress did not affect the feed intake of rats,but significantly reduced the body weight(p<0.05)and gastrocnemius muscle weight(p<0.05),but had no significant effect on soleus muscle weight.In addition,oil red staining showed that chronic stress did not cause fat deposition in gastrocnemius and soleus muscles.The results of hematoxylin-eosin staining showed that chronic stress caused a significant reduction in the cross-sectional area of gastrocnemius muscle fibers compared with the control group(p<0.01);the cross-sectional area of soleus muscle fibers was significantly increased(p<0.01).In addition,we compared the expression of genes and proteins related to protein synthesis and decomposition in gastrocnemius and soleus muscles under chronic stress.The results showed that the mRNA levels of FADD in Gastrocnemius after chronic stress were significantly higher than those in the control group(p<0.01),the mRNA levels of Cyes,p62,LC3a and ATG3 were significantly higher(p<0.05),the protein expression of MuRF was significantly lower(p<0.05),and ATG7(p=0.06)protein expression showed a decreasing trend;the mRNA levels of Sqstm1 and BCLx1 in soleus muscle were significantly decreased compared with the control group(p<0.05)after chronic stress,the protein expression of LC3b and MuRF was significantly increased(p<0.05),and the protein expression of mTOR was significantly decreased(p<0.05).2 Effects of chronic stress on muscle fiber types and gene expression of gastrocnemius and soleus muscles in ratsTo analyze the differential responses of gastrocnemius and soleus muscles under chronic stress,we used immunofluorescence staining of tissue sections,real-time quantitative fluorescence PCR and Western blot to detect the differences.Immunofluorescence results showed that chronic stress caused a very significant increase in the proportion of type Ⅰ muscle fibers in gastrocnemius(p<0.01),which had little effect on the proportion of type 2b muscle fibers,while the proportion of type 2b muscle fibers in soleus decreased significantly(p<0.05),which had little effect on the proportion of type Ⅰ muscle fibers.After chronic stress,the protein expression of MyHC-Ⅰin gastrocnemius muscle was significantly higher than that in control group(p<0.01),and the protein expression of MyHC-2b was significantly lower than that in control group(p<0.05);while the protein level of MyHC-I in soleus was also significantly higher than that in control group(p<0.01),and there was no difference in the protein level of MyHC-2b.In addition,the mRNA levels of NFATc4 and Myoz2 related to myofiber type transformation in gastrocnemius muscle of rats were significantly increased after chronic stress treatment(p<0.05),and the mRNA levels of NFAT5 tended to be increased;the mRNA levels of NFATc1,Rcan1 and Myoz1 in soleus muscle were significantly decreased(p<0.05).3 Effect and mechanism of glucocorticoid receptor in gastrocnemius muscle after chronic stressIn response to chronic stress,the sensitivity to negative feedback regulation of the hypothalamic-pituitary-adrenal axis decreases,but greatly enhances the ability of tissues and organs other than the adrenal gland to synthesize glucocorticoids.The results showed that there was no significant difference in CYP21 among the key enzymes related to corticosterone synthesis in gastrocnemius muscle and soleus muscle caused by chronic stress,but the mRNA contents of StAR and 11β-HSD1 in gastrocnemius muscle were significantly increased(p<0.05);the mRNA contents of StAR and 11β-HSD2 in soleus muscle were significantly decreased by chronic stress(p<0.05).The mRNA level of GR in gastrocnemius muscle after chronic stress was not different from that in control group,but the protein expression of GR was significantly decreased(p<0.05);the mRNA level of GR in soleus muscle after chronic stress was significantly lower than that in control group(p<0.05),but the expression of GR protein was not different.To test whether GR targeting is involved in the regulation of skeletal muscle fiber type transition by chronic stress,we performed chromatin immunoprecipitation,and the results showed that the enrichment of GR on the promoters of NFATC1,NFATC4,KLF15,LC3a and MyoD genes in gastrocnemius muscle after chronic stress was significantly higher than that in the control group(p<0.05),and the enrichment on the promoter of P62 gene was significantly higher than that in the control group(p<0.01).In conclusion,chronic stress significantly reduced gastrocnemius muscle weight with less effect on soleus muscle;chronic stress did not promote fat deposition in the two muscle tissues;although the muscle fiber composition of gastrocnemius and soleus muscle differed greatly,chronic stress promoted the expression of type Ⅰ muscle fibers in the two muscles;through chromatin immunoprecipitation we found that chronic stress may be promoted by GR The expression of NFATc1,its downstream target,leads to the expression of type Ⅰ muscle fibers.