【作者】 赵丹；

【导师】 付晶；

【作者基本信息】 东北农业大学 ， 动物生产， 2019， 硕士

【摘要】 本试验在成功建立雏鸡肝脏损伤模型的基础上,从细胞自噬与凋亡的角度探讨白术多糖(Polysaccharide of Atractylodes Macrocephala Koidz,PAMK)对环磷酰胺(cyclophosphamide,CTX)诱导的雏鸡肝脏损伤的影响,应用酶联免疫吸附(ELISA)法检测雏鸡血清中免疫球蛋白含量、血清及肝脏中细胞因子含量,应用光镜和透射电镜对雏鸡肝脏进行形态学观察,应用实时荧光定量PCR法(Realtime PCR)和免疫印迹法(Western Blot)检测与细胞自噬及凋亡相关基因mRNA表达量和蛋白含量。研究结果如下:(1)血清中免疫球蛋白和细胞因子含量的研究结果显示:与对照组相比,环磷酰胺组雏鸡血清中免疫球蛋白(IgM和IgA)和细胞因子(IL-4和IL-6)含量显著降低(P<0.05)。与环磷酰胺组相比,白术多糖环磷酰胺联合组雏鸡血清中(IgM和IgA)和IL-6含量均显著升高(P<0.05)。该研究结果表明,环磷酰胺可导致雏鸡血清中免疫球蛋白和细胞因子含量下降,而白术多糖可缓解雏鸡血清中免疫球蛋白和细胞因子含量的下降。(2)肝脏中细胞因子含量的研究结果显示:与对照组相比,环磷酰胺组雏鸡肝脏中(IL-2、IL-4和IL-6)含量显著降低(P<0.05)。与环磷酰胺组相比,白术多糖环磷酰胺联合组雏鸡肝脏中(IL-2和IL-4)含量显著升高(P<0.05)。该研究结果表明,环磷酰胺可降低雏鸡肝脏中细胞因子含量,而白术多糖可促进肝脏细胞因子的分泌。(3)光镜结果显示:与对照组相比,环磷酰胺可破坏雏鸡肝脏细胞形态结构的完整性、使肝细胞索排列紊乱、并出现炎性细胞浸润。电镜结果显示:与对照组相比,环磷酰胺可导致雏鸡肝脏线粒体受损,并出现自噬小体和凋亡小体。该研究结果表明,自噬与凋亡参与肝脏损伤的病理过程。(4)对自噬相关通路的研究结果显示:与对照组相比,环磷酰胺组雏鸡肝脏中LC3-Ⅰ、LC3-Ⅱ、LC3-Ⅱ/LC3-Ⅰ、Beclin1、Dynein、ATG5和TOR的mRNA表达量显著增加(P<0.05),PI3K和AKT的mRNA表达量显著降低(P<0.05);同时Beclin1、Dynein和ATG5的蛋白含量也显著增加(P<0.05)。与环磷酰胺组相比,白术多糖环磷酰胺联合组雏鸡肝脏中LC3-Ⅱ、LC3-Ⅱ/LC3-Ⅰ和ATG5的mRNA表达量显著降低(P<0.05),PI3K和AKT的mRNA表达量显著增加(P<0.05)。该研究结果表明,环磷酰胺可诱导雏鸡肝脏发生自噬,而白术多糖能够缓解环磷酰胺诱导的雏鸡肝脏自噬的发生。(5)对凋亡中线粒体通路的研究结果显示:与对照组相比,环磷酰胺组雏鸡肝脏中促凋亡基因(Caspase-3、Caspase-9、Bax和p53)mRNA表达量显著增加(P<0.05),抑凋亡基因Bcl-2 mRNA表达量显著下降(P<0.05);同时Caspase-3、Caspase-9和Bax的蛋白含量显著升高(P<0.05),抑凋亡基因Bcl-2的蛋白含量显著降低(P<0.05)。与环磷酰胺组相比,白术多糖环磷酰胺联合组雏鸡肝脏中Caspase-3、Bax和抑凋亡基因Bcl-2的mRNA表达量均呈现出与环磷酰胺组相反的趋势(P<0.05)。此外,Caspase-3和Bcl-2蛋白表达水平的变化趋势和它们相应的mRNA表达水平变化趋势是一致的。该研究结果表明,线粒体通路参与了环磷酰胺诱导的雏鸡肝脏细胞凋亡,而白术多糖可通过调节线粒体通路关键基因及蛋白的表达缓解环磷酰胺诱导的雏鸡肝脏细胞凋亡。(6)对凋亡中内质网应激通路的研究结果显示:与对照组相比,环磷酰胺组雏鸡肝脏中ATF4、ATF6和GRP94 mRNA表达量显著增加(P<0.05);同时雏鸡肝脏中GRP94的蛋白含量也显著增加(P<0.05)。与环磷酰胺组相比,白术多糖环磷酰胺联合组雏鸡肝脏中ATF4和GRP94mRNA表达量和GRP94蛋白含量均显著降低(P<0.05)。该研究结果表明,内质网应激通路参与了环磷酰胺诱导的雏鸡肝脏细胞凋亡,而白术多糖可通过调节内质网通路关键基因及蛋白的表达缓解环磷酰胺诱导的雏鸡肝脏细胞凋亡。(7)对凋亡中死亡受体通路的研究结果显示:与对照组相比,环磷酰胺组雏鸡肝脏中FADD、Fas和Caspase-8的mRNA表达量显著增加(P<0.05);同时FADD和Caspase-8的蛋白含量也显著升高(P<0.05)。与环磷酰胺组相比,白术多糖环磷酰胺联合组雏鸡肝脏中Fas和Caspase-8的mRNA表达量显著降低(P<0.05);白术多糖环磷酰胺联合组雏鸡肝脏中FADD和Caspase-8蛋白含量显著降低(P<0.05)。该研究结果表明,死亡受体通路参与了环磷酰胺诱导的雏鸡肝脏细胞凋亡,而白术多糖可通过调节死亡受体通路关键基因及蛋白的表达缓解环磷酰胺诱导的雏鸡肝脏细胞凋亡。综上所述,环磷酰胺可导致雏鸡血清和肝脏组织中与免疫相关细胞因子含量下降、肝脏发生自噬和凋亡等形态学损伤,而白术多糖可以通过促进细胞因子的分泌、调节与自噬和凋亡相关基因和蛋白的表达来缓解该损伤。更多还原

【Abstract】 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-Ⅰ,LC3-Ⅱ,LC3-Ⅱ/LC3-Ⅰ,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-Ⅱ,LC3-Ⅱ/LC3-Ⅰ,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.更多还原