ABCG2-mediated AMPK Pathway Regulates Muscle Growth And Development In Broilers Under Heat Stress

Heat stress has a negative impact on the growth and development of broilers,which leads to the decline of growth performance and slow development.At the same time,it also affects the slaughter performance of broilers,reduces meat quality and causes the damage of antioxidant capacity.It has become one of the important environmental factors causing economic losses in the breeding industry.ATP-binding cassette transporters G2 (ABCG2) can utilize the energy produced by hydrolysis of ATP to excrete cells from its bound substrates,which can transport and excrete harmful substances.ABCG2 plays an important role in promoting and maintaining the stability of cells and tissues under various stress states.Therefore,it is important to study the effect of ABCG2 on heat stress and its regulation mechanism in clinical application.The research contents of this paper include three parts:Firstly,in order to study the effects of didderent heat stress time on growth performance,meat quality and antioxidant capacity of broilers,the experiment was divided into control group (25 ± 2℃) and heat stress group (37±2℃).Each group was fed water freely.The heat stress test period was 14 days,and was analyzed by slaughter sampling in 1 week and 2 weeks of heat stress respectively.The results showed that heat stress significantly reduced feed intake and daily gain,increased feed-to-weight ratio and reduced feed conversion in broilers.Muscle pH and water holding capacity decreased significantly,shear stress increased significantly, while meat color almost had no difference.Meanwhile,MDA increased significantly,SOD,GSH-Px and T-AOC contents decreased significantly,and antioxidant capacity decreased.It indicated that heat stress could reduce the growth performance of broilers.Secondly,in order to study the changes of expression and location of ABCG2 in broiler muscle at different heat stress time,RNA was extracted from the muscle tissue of control group and heat stress group,retranscribed into cDNA as template,ABCG2 gene primers were synthesized,and ABCG2 gene was expressed and localized by fluorescent quantitative PCR and immunohistochemical methods.Immunohistochemical results showed that ABCG2 location and cell membrane were related to ABCG2 expression.RT-PCR and fluorescence quantitative PCR results showed that ABCG2 gene was expressed in both control and heat stress groups,and the expression level was significantly different.After one week of heat stress,the expression level of ABCG2 gene was not significant.The difference was significant,but after 2 weeks of heat stress,the chesthi muscle (P <0.01) and leg muscle (P <0.05) were significantly higher than those of the control group.Thirdly,in order to explore how heat stress regulates the growth and development of skeletal muscle under the influence of heat stress through the change of ABCG2 expression, myoblasts were isolated and cultured in vitro.The cells were then divided into control group (37±1℃),heat stress group (42±℃) and inhibitor group (42±1℃,20 μMK0143).The results showed that the expression of genes selected from ABCG2 and AMPK signaling pathway was significantly decreased after being affected by heat stress (P<0.01);after adding inhibitors,the expression of ABCG2 gene was significantly lower than that of heat stress group (P <0.05).There was no significant difference between TAK1,AMPK and mTOR genes after adding inhibitors and heat stress group (P> 0.05),while the eEF2 gene downstream of AMPK was not significantly different from that of heat stress group (P> 0.05).Compared with heat stress group,the expression level was significantly lower (P <0.01).In conclusion,this experiment found that heat stress could lead to the decline of growth performance,meat quality and antioxidant capacity of yellow-feathered broilers.Immunohistochemistry and fluorescence quantitative PCR showed that ABCG2 gene was located in the cell membrane and was affected by heat stress,and the expression of ABCG2 was up-regulated.After adding KO143,the downstream genes eEF2 of ABCG2 and AMPK were further down-regulated.It is demonstrated that the ABCG2 gene locus is between AMPK and eEF2 and regulates protein synthesis through this pathway to regulate the growth and development of broilers.