Effect Of Polysaccharide Of Atractylodes Macrocephala Koidz On Autophagy And Apoptosis In Chicken Liver Induced By Cyclophosphamide

Based on the successful establishment of liver damage model in chicken,effects of Polysaccharide of Atractylodes macrocephala Koidz(PAMK)on liver damage which induced by CTX in chicken were investigated from the perspective of autophagy and apoptosis.ELISA was used to detect the content of serum immunoglobulin and cytokines in chicken serum and the content of cytokines in chicken liver,light microscopy and electron microscopy were used to observe the morphological changes,Realtime PCR and Western Blot were used to detect the expression of autophagy and apoptosis-related genes and protein content.The results are as following.(1)The results of serum immunoglobulin and cytokine content showed that,compared with the control group,treatment with CTX significantly decreased the content of immunoglobulin(Ig A and Ig M)and cytokines(IL-4 and IL-6)in chicken serum(P<0.05).Compared with the CTX group,treatment with PAMK+CTX significantly increased the content of immunoglobulin(Ig A and Ig M)and cytokines(IL-6)in chicken serum(P<0.05).It indicated that CTX decreased the content of immunoglobulin and cytokine in chicken serum,while PAMK resisted the decreased of content of immunoglobulin and cytokine in chicken serum.(2)The results of cytokine content in liver showed that,compared with the control group,treatment with CTX significantly decreased the content of cytokines(IL-2,IL-4 and IL-6)in chicken liver(P<0.05).Compared with the CTX group,treatment with PAMK+CTX significantly increased the content of cytokines(IL-2 and IL-4)in chicken liver(P<0.05).It indicated that CTX reduced the cytokine content in chicken liver,while PAMK promoted the secretion of cytokines in chicken liver.(3)The results of light microscope showed that,compared with the control group,CTX induced irregularities in liver cell morphology of chicken and the hepatocyte cords were disorderly arranged and inflammatory cell infiltration.The results of electron microscope showed that,compared with the control group,CTX caused damage to chicken liver mitochondria and autophagy and apoptotic bodies were observed.It was proved that autophagy and apoptosis participate in the pathological process of liverdamage.(4)The results of autophagy-related pathways showed that,compared with the control group,treatment with CTX significantly increased(P<0.05)the m RNA expression levels of LC3-?,LC3-?,LC3-?/LC3-?,Beclin1,Dynein,ATG5,TOR and significantly decreased(P<0.05)the m RNA expression levels of PI3 K and AKT in liver.Meanwhile,the protein content of Beclin1,Dynein and ATG5 were also significantly increased(P<0.05).Compared with the CTX group,treatment with PAMK+CTX significantly decreased(P<0.05)the m RNA expression of LC3-?,LC3-?/LC3-?,ATG5 and significantly increased(P<0.05)the m RNA expression of PI3 K and AKT in liver.It indicated that CTX induced autophagy in liver of chicken,while PAMK resisted the autophagy in liver of chicken induced by CTX.(5)The results of mitochondrial pathway in apoptosis showed that,compared with the control group,treatment with CTX significantly increased(P<0.05)the m RNA expression levels of pro-apoptotic genes(Caspase-3,Caspase-9,Bax and p53)and significantly decreased(P<0.05)the m RNA expression levels of Bcl-2 in liver.Meanwhile,the protein content of Caspase-3,Caspase-9 and Bax were significantly increased(P<0.05)and the protein content of anti-apoptotic gene Bcl-2 was significantly decreased(P<0.05).Compared with the CTX group,the m RNA expression levels of Caspase-3,Bax and Bcl-2 in the liver of PAMK+CTX exhibited the opposite results.In addition,the results of protein levels of Caspase-3 and Bcl-2 were consistent with their corresponding m RNA expression levels.It indicated that mitochondrial pathway mediates CTX-induced liver apoptosis in chicken,while PAMK resisted CTX-induced liver apoptosis in chicken by regulating the expression of key genes and proteins in mitochondrial pathway.(6)The results of endoplasmic reticulum stress pathway in apoptosis showed that,compared with the control group,treatment with CTX significantly increased(P<0.05)the m RNA expression levels of ATF4,ATF6 and GRP94 in liver,Meanwhile,the protein content of GRP94 was also significantly increased(P<0.05).Compared with the CTX group,the m RNA expression levels of ATF4 and GRP94 and the protein of GRP94 were significantly decreased(P<0.05).It indicated that endoplasmic reticulum stress pathway mediated CTX-induced liver apoptosis in chicken,while PAMK resisted CTX-induced liver apoptosis in chicken by regulating the expression of key genes and proteins in the endoplasmic reticulum pathway.(7)The results of death receptor pathway in apoptosis showed that,treatment with CTX significantly increased the m RNA expression levels of FADD,Fas and Caspase-8 in liver(P<0.05).Meanwhile,the protein content of FADD and Caspase-8 were also significantly increased(P<0.05).Compared with the CTX group,the m RNA expression levels of Fas and Caspase-8 and the protein contents of FADD and Caspase-8 were significantly decreased(P<0.05).It indicated that the death receptor pathway mediated CTX-induced liver apoptosis in chicken,while PAMK resisted CTX-induced liver apoptosis in chicken by regulating the expression of key genes and proteins in the death receptor pathway.In summary,CTX caused a decrease in the levels of immune-related cytokines in serum and liver of chicken,induced autophagy and apoptosis,while PAMK resisted the autophagy and apoptosis of chicken liver induced by CTX through promoting cytokine secretion and regulating the expression of autophagy and apoptosis-related genes and protein expression.