Regulation And Mechanism Of Gamma-aminobutyric Acid On Muscle Growth And Development

Muscle growth and development are mainly divided into pre-birth myoblast differentiation stage and post-birth muscle hypertrophy stage.Post-birth muscle hypertrophy is mainly determined by activation of satellite cells and the level of muscle protein metabolism,both of which are regulated by Muscle regulatory factors(MRFs).growth hormone(GH)and insulin-like growth factor 1(IGF-1)are also involved in promoting muscle growth and development.In addition,PI3K-Akt-mTOR-p70S6 K and PI3K-Akt-GSK3β are involved in postnatal muscle growth and development by regulating myoglobin metabolism.Gamma-aminobutyric acid(GABA)is an inhibitory neurotransmitter widely found in the central nervous system and some peripheral tissues of the body,and has been proven to play an important regulatory role in the growth and development of the body.However,current studies on GABA and growth mainly focus on lipid metabolism,and there are relatively few studies on muscle growth.Therefore,in this study,GABA intervention model was constructed by intragastric administration of GABA and injection administration of GABAAR antagonist Bicuculine,aiming to explore whether exogenous GABA affects the muscle growth and development of mice,and explore the specific mechanism of its influence on muscle growth,so as to provide a theoretical basis for the application of GABA in culture production.In this study,male KM mice aged 4-5 weeks were selected and randomly divided into control group,GABA group and GABA+Bicuculine group according to their average body weight after three days of feeding.The control group was intragastrically injected with 0.9% normal saline,the GABA group was intragastrically injected with30mg/kg GABA and intrabitoneally injected with normal saline,and the GABA+Bicuculine group was intragastrically injected with 30mg/kg GABA and intrabitoneally injected with 4mg/kg Bicuculine suspension.Each mouse was given an intragastric and injection dose of 0.2 m L.Weight was weighed every other day,and the drug was reformulated according to the average weight of each group,and the concentration was strictly guaranteed.During the experiment,the mice had normal appetite,good spirit and smooth coat.Blood and muscle tissue samples were collected ten days later.The contents and results of this experiment were as follows:1.In order to explore the influence of GABA on the weight and skeletal muscle growth of mice,this experiment weighed the weight of mice and the wet weight of unilateral gastrocnemius muscle.Masson staining method was used to observe and statistically calculate the average cross-sectional area of the gastrocnemius muscle fibers of mice.The final body weight of the control group,GABA group and GABA+Bicuculine group was not significantly different(p > 0.5),but the wet weight of gastrocnemius muscle in GABA group was significantly lower than that in the control group,and the wet weight of gastrocnemius muscle in GABA+Bicuculine group was also significantly lower than that in the control group(p < 0.5).There was no significant difference in wet weight between GABA and GABA group(p > 0.5).The statistical results of muscle fiber CSA showed that the cross-sectional area of gastlarmius fibers in GABA and GABA+Bicuculine groups was significantly smaller than that in the control group(p < 0.5),and there was no significant difference in muscle fiber CSA between GABA and GABA+Bicuculine groups(p > 0.5).2.In order to explore the influence of GABA on the expression level of GABAA receptor in mouse skeletal muscle,immunohistochemistry and fluorescence quantitative PCR were used for detection.The results showed that GABAAR positive cells and mRNA expression levels in gastrocnemius of mice in GABA group were significantly higher than those in control group(p < 0.5).There were no significant differences in GABAAR positive cells and mRNA expression levels of gastrocnemius muscle in GABA+Bicuculine group compared with control group(p > 0.5).3.In order to explore the influence of GABA on the expression of GH in mouse serum and IGF-1 and IGF-1R in skeletal muscle,Elisa was used to detect the GH level in mouse serum.The results showed that the GH level in GABA group was significantly lower than that in the control group(p < 0.5).Serum GH level in GABA+Bicuculine group was significantly lower than that in control group(p < 0.5),but had no significant difference with that in GABA group(p > 0.5).In this study,the expression levels of IGF-1 and IGF-1R in the gastrocnemius of mice were also detected by immunohistochemical and fluorescent quantitative PCR methods.The results showed that the expression levels of IGF-1 positive cells and mRNA in GABA group were significantly lower than those in the control group(p < 0.5).The IGF-1positive cell expression and mRNA expression in GABA+Bicuculine group were significantly higher than those in control group(p < 0.5).The IGF-1R positive cell expression and mRNA expression in GABA group were significantly lower than those in the control group(p < 0.5),while the IGF-1R positive cell expression and mRNA expression in GABA+Bicuculine group were not significantly different from those in the control group(p > 0.5).4.In order to explore the effect of GABA on the expression of MyoD and MRF4 in the skeletal muscle of mice,immunohistochemical method was used to detect the expression of MyoD in the gastrocnemius of mice.The results showed that the expression of MyoD positive cells in the gastrocnemius of mice in the GABA group was significantly lower than that in the control group(p < 0.5).There was no significant difference in MyoD positive cell expression between GABA+Bicuculine group and control group(p > 0.5).In this study,fluorescence quantitative PCR was also used to detect MRF4 mRNA expression in the gastrocnemius of mice,and the results showed that there was no significant difference in the expression of MRF4 mRNA in the gastrocnemius of mice in the GABA group compared with the control group(p > 0.5).The mRNA expression level of MRF4 in GABA+Bicuculine group was significantly increased compared with control group(p < 0.5).5.In order to explore the effect of GABA on the expression of PI3 K,Akt,GSK3β,mTOR and p70S6 K in skeletal muscle of mice,immunohistochemical and fluorescent quantitative PCR methods were used to detect GABA.The results showed that the PI3 K,Akt,GSK3β,mTOR positive cell expression and mRNA expression of gastrocnemius in GABA group were significantly lower than those in control group(p< 0.5).The expression levels of PI3 K,Akt,GSK3β and mTOR positive cells in GABA+Bicuculine group were not significantly different from those in control group(p > 0.5),but mRNA expression levels were significantly higher than those in control group(p < 0.5).The mRNA expression level of p70S6 K in gastrocnemius of mice in GABA group was significantly lower than that in control group(p < 0.5),and that in GABA+Bicuculine group was significantly higher than that in control group(p < 0.5).In conclusion,30mg/kg GABA reduces the serum GH level,transmits signals through GABAAR,reduces the expression of IGF-1 and IGF-1R in skeletal muscle,and also reduces the expression of MyoD and MRF4 in skeletal muscle.Thus,the PI3K-Akt-mTOR-p70S6 K and PI3K-Akt-GSK3β pathways are affected,and ultimately the growth and development of skeletal muscle is inhibited.This is a new discovery for the physiological function of GABA,which is of great significance for further understanding the regulatory effect of GABA on body growth and development,and also provides a theoretical basis for the application of GABA in culture production.