Tissue-Specific Expression Of Thermosensitive TRP Channel Genes In Chickens And A Preliminary Investigation Of Their Functions

In mammals,transient receptor potential(TRP)ion channel proteins could respond to changes in ambient temperature,mediating the formation of temperature sensation.Due to the evolution of vertebrates,the type and structure of TRP protein are significantly different from that of mammals.To better understand the physiological functions of temperature sensitive TRP protein in the central and peripheral tissues within chicken and offer new ideas and targets for studying body heat and immunoregulation of chicken,the tissue distribution of temperature sensitive TRP protein and its differences between species were determined,those results explained the regulation of TRP protein in intestinal epithelial cell inflammation and heat stress of chicken.Trial 1 was conducted to investigate the distribution of several kinds of temperature sensitive ion channel proteins in chickens and to analyze the differences among varieties.Hypothalamus,hippocampus,liver,heart,lung,kidney,intestine,oviduct and ovary tissue samples of Nongda 3 layers and Hyline brown layers with differences in body size were collected.qPCR were carried out to detect the expressions of temperature sensitive ion channel proteins on mRNA level respectively in various tissue.By the same time,immunohistochemistry technology was applied to locate the six kinds of temperature sensitive ion channel proteins in each tissue.The results showed that there are 5 temperature-sensitive ion channel proteins mainly expressed in laying hens,they are TRPV1,TRPV2,TRPV3,TRPA1 and TRPM8.The expression of TRPV4 is extremely low,close to zero.In general,the expression of TRP in hypothalamus and hippocampus was high and mainly distributed in the nerve cell,the expression of TRPV3 was relatively high among the six channels proteins.In some tissue,the expression of TRPV1,TRPV2,TRPA1 and TRPM8 have significant differences in different varieties.Trial 2 was conducted to investigate the changes of TRP ion channel proteins in the process of broilers growth and its correlation with body temperature.Day-old chicken hatchlings were selected,conventional breeding,along with the age growth,choose the chickens that have big differences in body temperature per week,they were divided into two groups according to the temperature differences,there are eight chickens in every group.All these chickens were slaughtered and collected the hypothalamus.Then,the expression of TRPV2 and TRPV3 on protein levels in the hypothalamus were detected.Experimental results show that the chicken body temperature varies with growth increased,and the individual temperature difference is narrowing.The expression of TRPV3 ion channels in the different age exist significant differences.The protein expression of TRPV3 channel and TRPV2 channel shows consistency with high and low temperature in the early In the early and late development of broilers,namely the high expression of high temperature,and vice versa.Trial 3 was conducted to evaluate the role of TRP protein in the inflammatory response mediated by heat stress and immune stress in intestinal epithelial cells.The test is divided into two parts: small intestinal epithelial cells in chicken embryos were cultured,randomly divided into three groups,six repeat in every group,respectively incubation for 1 hour at 34oC,40oC and 43oC,the total protein was extracted.In addition,small intestinal epithelial were cultured and divided into three groups,six repeat in every group: The control group cultivated with 37oC;The heat temperature group cultured with 43oC;The heat temperature with TRP inhibitor group was added to the inhibitor and cultured at 43oC,the inhibitor was added an hour earlier,cells in each temperature environment for an hour.Further,detect the expression of proinflammatory factor TNF-α,IL-1 and inflammatory factor IL-10.The results showed that the expression of proinflammatory factor in heat temperature group quantity higher than the other two groups,the expression of inflammatory factor in heat temperature group quantity lower than the other two groups.And the expression of TNF-α and IL-1 in heat temperature group are significantly higher than heat temperature with TRP inhibitor group(P<0.05).The cultured intestinal epithelial cells were randomly divided into four groups: control group(treated with physiological saline),lipopolysaccharide group(LPS,0.5 μg/m L,2 h),TRP ion channel protein inhibitor group(ruthenium red,10 μmol/L,3 h),LPS with TRP inhibitor group(incubated with ruthenium red for an hour,then incubated for two hours),there are six repeat in every group.The m RNA expression of inflammatory factors of TNF-α and IL-1,inflammatory factor IL-10,TRPV1 and TRPV3 were detected.The results showed that after LPS processing,the expression of TRPV1 and TRPV3 significantly lower than the control group,the expression of IL-1 and IL-10 is significantly higher than the control group.IL-1 in LPS with TRP inhibitor group is significantly lower than the LPS group.In conclusion,the distribution of temperature sensitive chicken TRP ion channel protein has specificity in tissue and species.The expression of TRPV3 and TRPV2 in hypothalamus is involved in the central control of poultry and the expression of TRPV1 and TRPV3 in intestine is related to the inflammation mediated by heat stress and immunological stress.