Study on Immune Organs injuries Mechanism of Selenium Deficiency in Chicken

摘要

Nutritional trace element selenium(Se)is a valuable component for the normal immune function in various life forms.Selenoproteins and Se play important roles in the immune system.Se is necessary for the immune system in chicken and mediates its physiological functions through selenoproteins.Cytokines are soluble,low molecular weight polypeptides and glycopeptides produced by a broad range of cell types of haematopoietic and nonhaematopoietic origin that have suppressive or enhansive effects on cellular proliferation,differentiation,activation,and motility.Heat shock proteins(Hsps)are present nearly in all species with their conserved nature,and Hsps are used as biomarkers of various stresses condition in animals as well as birds.Autophagy is a process which exerts effect of targeted component and its action in particular cells and system.Selenoprotein,cytokine,Hsps,autophagy related genes and ultra structures in the immune system of mammals are sensitive to dietary Se levels;however,little is known about the expression of selenoproteins,Hsps mRNAs,Hsp proteins level,mRNAs expression of autophagy-related genes,protein levels of autophagy-related genes,effects on cytokines,and on internal structures of in the chicken spleen,bursa of Fabriciuos and thymus with Se deficiency.We assessed selenoproteins mRNAs expression of(Txnrd1,Txnrd2,Txnrd3,Dio1,Dio2,Dio3,GPx1,GPx2,GPx3,Gpx4,Sepp1,Selo,Sep15,Sepx1,Sels,Seli,Selu,Selh,and SPS2),Hsps mRNAs expression of(Hsp27,Hsp40,Hsp60,Hsp70,and Hsp90),Hsp proteins level of(Hsp40,Hsp60,Hsp70,and Hsp90),autophagy-related genes mRNAs expression of(LC3-Ⅰ,LC3-Ⅱ,Beclin1,Dynein,ATG5,TOR1),protein levels of autophagy related genes(LC3-Ⅱ,Beclin1,Dynein),cytokine content(IL-2,IL-6,IL-8,IL-10,IL-17,IL-1β,IFN-α,IFN-β,IFN-γand TNF-α)and change in structural integrity in the tissues in the chicken spleen,bursa of Fabricius and thymus in this study.The animals were randomly assigned into two groups as follows:the Se-deficient group(L group)was fed a diet containing0.033mg Se/Kg,and the control group was fed the same basal diet supplemented with Se at0.15mg/kg(sodium selenite).Real-time qPCR was used to investigate the expression level of selenoproteins,Hsps and autophagy-related genes on days15,25,35,45,and55,protein level of heat shock protein and autophagy genes were check through western blot analysis on days35,45and55,ELISA was used to evaluate the cytokine content on days15,35,and55,an electron microscope was used to observed the structural change in chicken immune organs.
　　The result revealed that selenoprotein messenger RNA(mRNA)levels of Txnrd1,Txnrd2,Txnrd3,Dio1,Dio2,Dio3,GPx1,GPx2,GPx3,Gpx4,Sepp1,Selo,Sep15,Sepx1,Sels,Seli,Selu,Selh,and SPS2were all significantly decreased in the Se deficiency group compared to the control group in spleen,bursa of Fabricius and thymus of chicken.
　　Cytokines study showed that in spleen IL-2,IL-6,IL-17,IL-1β,IFN-αwere significantly decreased and there was also significant increased in IL-8,IL-10,IFN-β,IFN-γand TNF-αin Se deficiency group.In bursa of Fabricius the IL-2,IL-6,IL-8,IL-10,IL-17,IL-1β,IFN-α,IFN-β,IFN-γwere significantly decreased,and TNF-αwas significantly increased in the Se deficiency.In thymus a significant decrease in IL-2,IL-10,IL-17,IL-1β,IFN-α,IFN-βwas observed in the Se deficiency group,and there also a significant increase in IL-6,IL-8,IFN-γ，and TNF-αin the L group in thymus of chicken.
　　Result of Hsps messenger RNA(mRNA)levels showed that(Hsp27,Hsp40,Hsp60,Hsp70,and Hsp90)were significantly increased in spleen,bursa of Fahricius and thymus of the Se deficient group.The proteins levels of Hsps(Hsp60,Hsp70,and Hsp90)were significantly increased in spleen,bursa of Fabrieiuos and thymus of the Se deficient group.
　　Autophagy-related gene expression results showed that in spleen mRNA levels of LC3-Ⅰ,LC3-Ⅱ,Beclin1,Dynein,ATG5,TOR1were high and protein levels LC3-Ⅱ,Beclin1and Dynein was also observed high in Se deficiency group as compared to control group in chicken spleen.In bursa of Fabricius as compared to the control group mRNA level of LC3-Ⅰ,LC3-Ⅱ,Beclin1,Dynein,ATG5,TOR1was increased and protein level of Beclin1and Dynein were increased and LC3-2was low in Se deficiency group.In thymus as compared to the control group mRNA levels of LC3-Ⅰ,Beclin1and AGT5were high and LC3-Ⅱ,Dynein,TOR1were observed low and protein level ofBeclin1was high and LC3-Ⅱand Dynein were recorded low in Se deficiency group.Further cellular morphological changes such as autophagy vacuole,autolysosome and lysosomal degradation were observed in the spleen,bursa of Fabricius and thymus of Se deficiency group.
　　In summary,Se deficiency results in significant decreased in the expression of selenoproteins,change in the cytokine,and change in autophagy-related genes.Further ultra structure in chicken immune organs showed histopathology,immuno-supersion,and increased in the Hsps participated in protective mechanism in tissues of spleen,bursa of Fabricius and thymus of Se deficiency group.Our results provide a comprehensive tool of structural injuries in the chicken immune organs with Se deficiency.

目录

CONTENTS

声明

Abstract

1 Introduction

1．1 Effect of Se on immune organs

1．2 Selenoprotein function and expression

1．3 Cytokines function and expression

1．4 Hsps function and expression

1．5 Autophagy function and expression

1．6 Objectives of Research

2 Materials and Methods

2．2 Determination of mRNAs levels by Quantitative Real-Time PCR

2．3 Western blot analysis

2．4 Determination of Immune Function

2．5 Determination of Ultra structure

2．6 Statistical Analysis

3 Results

3．1 Result of selenoproteins mRNA level in chicken spleen

3．2 Result of selenoproteins mRNA level in chicken bursa of Faricius

3．3 Result of selenoproteins mRNA level in chicken thymus

3．4 Result of cytokines in chicken spleen

3．5 Result of cytokines in chicken bursa of Fabricius

3．6 Result of cytokines in chicken thymus

3．7 Result of Hsp mRNA levels in chicken spleen

3．8 Result of Hsps mRNA levels in the chicken bursa of Fabricius

3．9 Result of Hsp mRNA levels in chicken thymus

3．10 Result of Hsps protein levels in chicken spleen

3．11 Result of Hsps protein levels in the chicken bursa of Fabricius

3．12 Result of Hsps protein levels in chicken thymus

3．13 Result of autophagy genes mRNA levels in chicken spleen

3．14 Result of autophagy genes mRNA levels in chicken bursa of Fabricius

3．15 Effect of Se deficiency on autophagy genes mRNA levels in chicken thymus

3．16 Result of autophagy genes protein level in chicken spleen

3．17 Result of autophagy genes protein level in chicken bursa of Fabricius

3．18 Result of autophagy genes protein level in chicken thymus

3．19 Result of ultrastrcuture expression in spleen，bursa of Fabricious and thymus of chicken

4 Discussion

4．2 Effect of dietary Se on cytokines expression level in chicken immune organs

4．3 Effect of dietary Se on Hsps expression level in chicken immune organs

4．4 Effect of dietary Se on autophagy related genes level in chicken immune organs

4．5 Effect of dietary Se on ultrastructure of immune organs of chicken

5 Conclusions

Acknowledgements

References

List of Abbreviation

Peer Reviewed Publications