【作者】 杨鑫；

【导师】 欧德渊； 洪猛；

【作者基本信息】 贵州大学 ， 兽医， 2023， 硕士

【摘要】 目的:H6N6亚型禽流感病毒常引起家禽呼吸道感染,导致家禽生产性能下降,给养殖业带来严重危害,甚至对人类的健康构成威胁。自噬是一种内在的宿主防御机制,在宿主抗感染免疫应答中发挥重要作用。中药黄芩苷具有良好抗病毒的作用,但是尚未见到通过调节自噬来减少H6N6亚型禽流感病毒感染机体损伤方面的研究。本论文通过体内外试验结合研究黄芩苷对H6N6亚型禽流感病毒诱导小鼠巨噬细胞及肺自噬的影响,以揭示黄芩苷抗禽流感病毒感染与自噬之间的关系,为黄芩苷抗禽流感病毒感染的临床应用提供试验依据。方法:体外试验建立H6N6亚型禽流感病毒感染RAW264.7细胞模型;细胞活性实验筛选出黄芩苷在RAW264.7细胞上的最大药物安全浓度;设置对照组、模型组、高剂量黄芩苷组（50μg/m L）、中剂量黄芩苷组（25μg/m L）和低剂量黄芩苷组（12.5μg/m L）。CCK8法检测细胞活性的变化;HE染色与透射电镜观察细胞形态学变化;ELISA检测细胞培养上清液IL-1β、IL-2、IL-6及TNF-α的含量;Western blot和免疫荧光检测细胞LC3蛋白表达水平以及q RT-PCR检测自噬相关基因PI3K、m TOR、LC3、Beclin-1、ATG5及ATG12 m RNA转录水平。体内试验选用40只6周龄昆明小鼠随机分为对照组、模型组、黄芩苷治疗组（1.86 mg/kg·BW）和3-MA抑制剂组（15 mg/kg·BW）,连续治疗6d眼球采血脊椎处死。HE染色观察肺组织病理变化;ELISA法检测血清中IL-1β、IL-2、IL-6及TNF-α的含量;透射电镜观察肺组织中自噬体的水平;Western blot、免疫荧光和免疫组化法检测肺组织LC3、Beclin-1、ATG5及ATG7蛋白表达水平;q RT-PCR检测肺组织自噬相关基因PI3K、m TOR、LC3、Beclin-1、ATG3、ATG5、ATG7及ATG12 m RNA转录水平。结果:1.体外试验结果显示:（1）经测定H6N6亚型禽流感病毒的TCID50为10^-3.73/0.1 m L,PCR特异性扩增出N6基因,细胞线粒体肿胀明显,细胞炎性因子IL-1β、IL-2、IL-4、IL-6和TNF-αm RNA转录水平显著升高（P<0.01）,成功构建H6N6亚型禽流感病毒体外感染RAW264.7细胞模型;（2）黄芩苷作用于感染H6N6亚型禽流感病毒RAW264.7细胞12 h、24 h和48 h时,能够显著提高细胞的活力（P<0.01）,细胞培养上清液IL-1β、IL-2、IL-6及TNF-α的含量显著降低（P<0.01）;（3）H6N6亚型禽流感病毒感染RAW264.7细胞24 h时,细胞中的自噬体数量增多,经黄芩苷作用后自噬体数量减少,细胞损伤程度减轻;（4）黄芩苷作用于感染H6N6亚型禽流感病毒RAW264.7细胞24 h时,细胞中LC3蛋白水平以及PI3K、m TOR、LC3、Beclin-1、ATG5及ATG12 m RNA转录水平显著降低（P<0.01）,三个不同剂量组间差异显著（P<0.01或P<0.05）。2.体内试验结果显示:（1）经黄芩苷治疗后小鼠肺组织炎性细胞浸润减少以及肺泡壁充血减少,缓解患鼠肺组织的损伤;（2）血清中IL-1β、IL-2、IL-6及TNF-α含量显著下降（P<0.01）;（3）肺组织中自噬体数量减少;（4）肺组织中LC3、Beclin-1、ATG5及ATG7蛋白表达水平显著下降（P<0.01）;（5）肺组织中PI3K、m TOR、LC3、Beclin-1、ATG3、ATG5、ATG7及ATG12 m RNA转录水平显著下降（P<0.01）。结论:黄芩苷能缓解H6N6亚型禽流感病毒对RAW264.7细胞的炎性损伤和肺脏损伤,减少IL-1β、IL-2、IL-6及TNF-α炎性因子的含量,下调RAW264.7细胞和肺脏自噬相关基因表达及自噬体的水平,对H6N6亚型禽流感病毒诱导小鼠RAW264.7细胞和肺脏自噬具有抑制作用,提示黄芩苷抗小鼠肺损伤机制可能与调控巨噬细胞自噬有关。更多还原

【Abstract】 Objective:The H6N6 subtype avian influenza virus often causes respiratory tract infection in poultry,which leads to the degradation of poultry performance,brings serious harm to the breeding industry,and even poses a threat to human health.Autophagy is an intrinsic host defense mechanism that plays an important role in the host’s anti-infection immune response.The traditional Chinese medicine baicalin have good antiviral effects,but there is no research on reducing the damage of H6N6subtype avian influenza virus by regulating autophagy.In this thesis,the effects of baicalin on H6N6 subtype avian influenza virus-induced autophagy in mouse macrophages and lungs were investigated through a combination of in vivo and ex vivo experiments to reveal the relationship between baicalin anti-avian influenza virus infection and autophagy,and to provide an experimental basis for the clinical application of baicalin anti-avian influenza virus infection.Methods:In vitro assay to establish a model of H6N6 subtype avian influenza virus infection of RAW264.7 cells.Cellular activity assay screening for maximum drug safe concentration of baicalin on RAW264.7 cells.A control group,a model group,a high-dose baicalin group（50μg/m L）,a medium-dose baicalin group（25μg/m L）and a low-dose baicalin group（12.5μg/m L）were set up.CCK8 assay to detect changes in cellular activity.The changes of cell activity were detected by CCK8 method.Cell morphology was observed by HE staining and transmission electron microscopy.The contents of IL-1β,IL-2,IL-6 and TNF-αin the supernatant of cell culture were determined by ELISA.The LC3 protein expression levels were detected by Western blot and immunofluorescence,and the m RNA transcription levels of autophagy related genes PI3K,m TOR,LC3,Beclin-1,ATG5 and ATG12 were detected by q RT-PCR.In vivo,40 6-week-old Kunming mice were randomly divided into control group,model group,baicalin treatment group（1.86 mg/kg·BW）and 3-MA inhibitor group（15 mg/kg·BW）.HE staining was used to observe the pathological changes of lung tissue.The contents of IL-1β,IL-2,IL-6 and TNF-αin serum were determined by ELISA.The level of autophagosomes in lung tissue was observed by transmission electron microscopy.The protein expression levels of LC3,Beclin-1,ATG5 and ATG7 in lung tissues were detected by Western blot,immunofluorescence and immunohistochemistry.m RNA transcription levels of autophagy related genes PI3K,m TOR,LC3,Beclin-1,ATG3,ATG5,ATG7 and ATG12in lung tissue were determined by q RT-PCR.Results:1.In vitro experiment results show that:（1）The TCID50 of H6N6subtype avian influenza virus was determined to be 10^-3.73/0.1 m L.The N6 gene was specifically amplified by PCR.The mitochondrial swelling of cells was obvious,and the m RNA transcription levels of cytokines IL-1β,IL-2,IL-4,IL-6 and TNF-αwere significantly increased（P<0.01）.RAW264.7 cells infected by H6N6 subtype influenza virus in vitro were successfully constructed.（2）Baicalin treated RAW264.7cells infected with H6N6 subtype avian influenza virus for 12 h,24 h and 48 h,significantly increased cell viability（P<0.01）,and significantly decreased the contents of IL-1β,IL-2,IL-6 and TNF-αin the supernatant of cell culture（P<0.01）.（3）When H6N6 subtype avian influenza virus infected RAW264.7 cells for 24 h,the number of autophagosomes in the cells increased,and the number of autophagosomes decreased after baicalin treatment,and the degree of cell damage was reduced.（4）After baicalin treated RAW264.7 cells infected with H6N6 subtype avian influenza virus for 24 h,LC3 protein level and the m RNA transcription levels of PI3K,m TOR,LC3,Beclin-1,ATG5 and ATG12 were significantly decreased（P<0.01）.There were significant differences among the three different dose groups（P<0.01 or P<0.05）.2.In vivo experiment results show that:（1）After baicalin treatment,the inflammatory cell infiltration and alveolar wall congestion of mice lung tissue were reduced,which alleviated the lung tissue injury of mice.（2）Serum levels of IL-1β,IL-2,IL-6 and TNF-αwere significantly decreased（P<0.01）.（3）The number of autophagosomes in lung tissue decreased.（4）The expression levels of LC3,Beclin-1,ATG5 and ATG7 proteins in lung tissue were significantly decreased（P<0.01）.（5）The m RNA transcription levels of PI3K,m TOR,LC3,Beclin-1,ATG3,ATG5,ATG7 and ATG12in lung tissue were significantly decreased（P<0.01）.Conclusion:Baicalin could alleviate the inflammatory damage and lung damage of RAW264.7 cells induced by H6N6 subtype avian influenza virus,reduce the contents of IL-1β,IL-2,IL-6 and TNF-αinflammatory factors,and down-regulate the expression of autophagy related genes and autophagosome levels in RAW264.7 cells and lung.H6N6 subtype avian influenza virus has an inhibitory effect on RAW264.7cells and lung autophagy in mice,suggesting that baicalin’s anti-lung injury mechanism may be related to the regulation of macrophage autophagy.更多还原