Epidemiological Investigation Of Chicken Selenium Deficiency Disease In Harbin And Preliminary Study On Biological Function Of Selenoprotein K

Selenium(Se),as an essential micronutrient,plays an important role in many physiological processes both in human and animals.In chicken,Se deficiency induces damage of tissues and organs including brains,hearts,livers,pancreas and muscles and even death.So Se deficiency disease has become one of the major nutritional metabolic diseases in the poultry industry.Because the primary Se deficiency disease has obvious geographical characteristics,the epidemiological survey for regional chicken Se deficiency disease will provide some reasonable references for the prevention and treatment of this disease.The major biological form of Se was selenoprotein and the selenoprotein K(Selk),as a single spanning integral protein,which locates in the endoplasmic reticulum(ER)was revealed in recent years.Studies have shown that Selk deficiency inhibited the proliferation and migration of T cell,and disordered the function of macrophage and the secretion of cytokines,and damaged the body immune function and anti-virus ability in mammalian.In poultry,the preliminary studies have shown that Selk had the function of antioxidation and regulation of calcium homeostasis,which involved in skeletal muscle injury induced by Se deficiency,however,the specific biological function of Selk has not been revealed in chickens.Harbin is located in the lack of Se,which is a high incidence area of Se deficiency disease.In this study,to clarify the incidence profile of this disease in Harbin and clarify the biological function of Selk,epidemiological investigation was conducted on this disease in Harbin and a series of experiments were explored to investigate the biological function of Selk.The Se deficiency chicken model was reproduced and the indexes involving in inflammation,endoplasmic reticulum stress and apoptosis in skeletal muscles were detected.The Selk deficiency chicken myoblasts model was built and the indexes involving in oxidative stress,selenoproteins,inflammatory factor,heat shock protein,calcium homeostasis,glucose metabolism,and muscle development were detected.Micro RNA(mi RNA)targeting Selk and long non coding RNA(lnc RNA)targeting stearoyl-Co A desaturase(SCD)were predicted by bioinformatics and thetargeting relationship between mi RNA and Selk was detected by double luciferase reporter gene technique.The lnc RNA silencing chicken embryo myoblasts model was established and the targeting relationship between lncRNA and SCD,and the related indexes involving in inflammation were detected.The gga-let-7f-3p overexpression and knockdown chicken myoblasts model and the treatment model with N-acetylcysteine(NAC)and hydrogen peroxide(H2O2)were established and the indicators involving in oxidative stress,endoplasmic reticulum stress and apoptosis were detected.The gga-let-7a-3p overexpression and knockdown chicken myoblasts model was established,and treated with avian influenza virus(H5N1),then the virus replication ability and autophagy index were detected.The results showed that:(1)The results of epidemiological survey showed that starting from September,the percentage of Se deficiency disease in the case of admissions were increased sharply(0.77% in 2014,0.71%in 2015.0.52% in 2016),and the rate reached a peak in December(3.39% in 2014,3.24% in 2015.2.47% in 2016)from 2014 to 2016 in Harbin.The rate in January was reduced to 1.01% in 2014,1.68% in 2015,and 2.04% in 2016,respectively.These results suggested that the incidence of the disease was increased significantly after the winter and that this disease had obvious seasonality,and specific climatic factors(especially low temperature)had an important role in the occurrence and development of this disease.In addition,the incidence numbers of broiler chickens were significantly higher than that in laying hens.Further study has found that 4-week-old broiler and egg laying have the highest incidence of this disease.These results suggested that it is important to monitor the contents of vitamin E and Se in diet after September in Harbin.At the same time,it is also important to add the vitamin and Se to diet precisely when the deficiency was found.(2)Selk siliencing significantly affected the expressions of 25 selenoproteins in chicken myoblasts.After 24 h of si RNA treatment,Gpx1,Gpx2,Gpx4,Dio3,Selw,Selh and Sepp1 mRNA levels were significantly lower,while Txnrd1,Txnrd3,Seln,Sels,Selt,Selm,Sep15,Seli,Selo and Sepx1 mRNA levels in the si RNA group were significantly higher compare to the values of each corresponding control group.After 48 h of si RNA treatment,the Gpx1,Gpx2,Gpx4,Txnrd1,Txnrd2,Txnrd3,Dio3,Seln,Sels,Selw,Selt,Selh,Selm,Sep15,Seli,Selu,Sepp1,Selo and Sepx1 mRNA levels were significantly lower,while Gpx2 and SPS2 m RNA levels in the si RNA group were significantly higher compared to the values of each corresponding control group.After 72 h of siRNA treatment,the Gpx3,Dio2,Dio3 and Selh m RNA levels were significantly lower,while the Txnrd1,Txnrd2,Txnrd3,Sepn1,Selt,Selm,Seli,Selo,Sepx1 and SPS2 m RNA levels in the si RNA group were significantly higher compared to the values of each corresponding control group.These results suggested that Selk can regulate the expression of other selenoproteins.(3)Se deficiency caused the typical pathological changes including inflammation,hemorrhage and necrosis in skeletal muscles,and significantly down-regulated the expression ofSelk,ALDBGALG0000005049,SCD,PPAR?,PPAR? and PPAR?,and up-regulated IL-1?,IL-2,IL-6,IL-7,IL-8,IL-17 and CCL4.Selk silencing significantly affected the expressions of iNOS,NF-?B,HO-1,COX-2,PTGEs,IL-1?,IL-6,IL-7,IL-8,IL-17 and IFN-?,and down-regulated the expressions of ALDBGALG0000005049 and SCD.ALDBGALG0000005049 regulated SCD expression.Knocking down ALDBGALG0000005049 significantly up-regulated the expressions of IL-1?,IL-6,IL-8 and CCL4,and down-regulated the expression of SCD,PPAR?,PPAR? and PPAR? in chicken myoblasts.These results indicated that Selk was involved in skeletal muscle inflammation induced Se deficiency by regulating the ALDBGALG000000504-SCD pathway.(4)Se deficiency significantly up-regulated the expressions of ATF4,ATF6,XBP1,CHOP and Bax and down-regulated the expressions of Bcl2.Gga-let-7f-3p regulated Selk expression.Overexpression of gga-let-7f-3p induced apoptosis by increasing the activity of SOD and CAT and the levels of ROS and MDA,up-regulating the expressions of ATF4,ATF6,XBP1,CHOP and Bax and down-regulating the expression of Bcl2.However,this effect could be reversed by the NAC.Similarly,knocking down gga-let-7f-3p blocked apoptosis induced by H2O2.The results indicated that gga-let-7f-3p-Selk-ERS pathway was involved in skeletal muscle apoptosis induced by Se deficiency.(5)H5N1 could infect chicken myoblasts,and induced autophagy in chicken myoblasts.Gga-let-7a-3p regulated Selk expression.Knocking down gga-let-7a-3p up-regulated the expression of BECN1,and down-regulated the expression of HA,M1,M2,mTOR,LC3-1 and P62 in chicken myoblasts.The results suggested that knocking down gga-let-7a-3p could inhibit the expression of M2 by targeting Selk,and removed the obstruction of M2 to formation of autosomal lysosome to inhibit the replication of H5N1 in chicken myoblasts.(6)After 24 h and 72 h of si RNA treatment,mRNA levels of ITPR1,ITPR2,ITPR3,Ry R1,RyR3,NCX,PMCA1,CACNA1 S,TRPC1,TRPC3,STIM1,ORAl1,CaLM,Ca SR,MSTN,and P21 in the si RNA group were significantly higher compared to the values of each corresponding control group.After 24 h of si RNA treatment,SERCA1 s and CRT m RNA levels were significantly higher,while after 72 h of si RNA treatment,that were significantly lower compared to the values of each corresponding control group.After 24 h of si RNA treatment,the Myo D and Myf-5 m RNA levels were significantly lower,while after 72 h of si RNA treatment,that were significantly higher compared to the values of each corresponding control group.After 72 h of si RNA treatment,the IRS1,IRS2,IGF1,GLUT1,GLUT3,GLUT8,MRF4 and Myogenin mRNA levels were significantly lower compared to the values of each corresponding control group.Correlation analysis and principal component analysis(PCA)showed that IRS1,IRS2,IGF1,GLUT1,GLUT3 and GLUT8 significantly affected MRF4 and Myogenin.ITPR3,RyR1,PMCA1,CACNA1 S,STIM1,Ca LM and CRT significantly affected Myogenin.Therefore,Selk siliencing significantly affected the carbohydrate metabolism and calcium homeostasis in chicken myoblasts,whichaffected the proliferation and differentiation of chicken myoblasts,especially the differentiation stage.These results suggested that Selk exerted the function of maintaining the development of chicken myoblasts normally.