Effects Of ABCG2-mediated ERK1/2 Pathway On Growth And Development Of IEC In Broilers Under Heat Stress

Heat stress seriously affects the production performance of poultry,leading to growth and metabolic disorders,immunosuppression and meat quality decline.ATP-binding cassette transporters G2(ABCG2),located in the cell membrane,is widely distributed in normal tissues,mainly concentrated in small intestine,liver,placenta and other structures with secretion and excretion.As a tissue barrier,ABCG2 participates in the regulation of various stresses.Extracellular regulated protein kinase(ERK1/2)is the key to signal transduction from surface receptors to nuclei.Heat stress can activate ERK1/2 signaling pathways in many cells and play a protective role.Therefore,it is of theoretical significance to study how heat stress regulates the growth and development of intestinal cells of yellow feather broilers by regulating ABCG2 through ERK1/2 signaling pathway.This experiment carried out the following three aspects of research:1.The effects of heat stress on small intestinal development,digestive enzymes and antioxidant capacity of yellow feather broilers were divided into control group and heat stress group.The heat stress test lasted 14 days.Broilers were slaughtered and sampled after 1 and 2 weeks of heat stress respectively.The results showed that heat stress damaged the intestinal mucosal morphology of broilers to varying degrees,and the villi were broken,shortened and swollen;the activities of lipase,amylase and trypsin were decreased;the content of malondialdehyde(MDA)was increased,and the activity of glutathione peroxidase(GSH-PX)was decreased.The above phenomena were alleviated after 2 weeks of heat stress compared with 1 week of heat stress.This indicated that heat stress affected the structure and function of intestinal epithelial cells in broilers,and the effects of different heat stress time were different.2.Changes of expression and localization of ABCG2 in small intestine under heat stress.The expression of ABCG2 was studied by fluorescence quantitative PCR and the location of ABCG2 was studied by immunohistochemistry.Fluorescence quantitative results showed that there was no significant difference in the expression of ABCG2 between the control group and the heat stress group(P>0.05),but the expression of ABCG2 gene in the heat stress group was significantly higher than that in the control group(P<0.05).Immunohistochemical analysis showed that ABCG2 was mostly located in small intestinal cell membrane after 1 and 2 weeks of heat stress,and the brown area of ABCG2 in heat stress group was higher than that in control group.3.Heat stress activated ABCG2 regulated the growth and development of IECs through ERK1/2 signaling pathway,which was divided into control group(culture temperature 37?),heat stress group(culture temperature 42?)and ABCG2 inhibitor group(culture temperature 42?+Ko143).Intestinal epithelial cells were cultured in the same humidity and 5%C02 incubator for 3 hours.RT-PCR and qRT-PCR analysis showed that RAF1,ERK1 and C-fos were significantly lower under heat stress than those in the control group(P<0.01).There was no significant difference in ATF2 expression between the two groups,which may be due to the activation of ERK1/2 signaling pathway by heat stress.After adding ABCG2 inhibitor,there was no significant difference in the expression level of RAF1 upstream gene between heat stress group and inhibitor group,but there was significant difference in the expression level of ERK1 between heat stress group and inhibitor group.It was speculated that ABCG2 gene was located between the upstream gene RAF1 and ERK1,and participated in the regulation of downstream gene expression of ERK1/2 signaling pathway in IECs.In conclusion,the results of this experiment showed that heat stress could lead to morphological damage of small intestinal mucosa,decrease the activities of digestive enzymes in small intestine,and decrease the antioxidant capacity of yellow feather broilers.ABCG2 was localized on the small intestinal cell membrane,and the expression of ABCG2 was increased in the small intestine of yellow-feathered broilers under long-term heat stress.IECs ABCG2 gene located between RAF1 and ERK1 in ERK1/2 signaling pathway and participate in the regulation of downstream gene expression during heat stress.