【作者】 李艳；

【导师】 王金明；

【作者基本信息】 山西农业大学 ， 兽医学， 2024， 硕士

【摘要】 【目的】本试验旨在探讨氟暴露致小鼠肝脏线粒体自噬紊乱的确切机制以及几丁聚糖-纳米硒（CS-Se Nps）对氟致小鼠肝脏损伤的缓解效果。【方法】拟开展以下两方面的研究:（1）以C57BL/6野生型（WT）雄性小鼠为受试对象,随机分为C组（饮用蒸馏水）、F组（饮用100 mg/L Na F）、Se组（灌胃1μg/g bw/d的CS-Se Nps）、FSe组（饮用100 mg/L Na F并灌胃1μg/g bw/d的CS-Se Nps）,试验连续处理三个月后,观察小鼠肝脏组织的病理形态,检测肝功能和氧化应激状态、线粒体自噬相关因子和NLRP3炎症小体的表达;（2）以Parkin^-/-的C57BL/6雄性小鼠为受试对象,将其分为Cq组（饮用蒸馏水）、Fq组（饮用100 mg/L Na F）、Seq组（灌胃1μg/g bw/d的CS-Se Nps）、FSeq组（饮用100 mg/L Na F并灌胃1μg/g bw/d的CS-Se Nps）,试验连续处理三个月后,检测小鼠肝脏损伤情况、氧化应激状态、线粒体自噬相关因子表达。【结果】试验结果如下:（1）野生型小鼠试验结果1)与C组相比,F组小鼠精神沉郁,活动迟缓,肝脏显微及超微结构损伤显著,肝脏中AST和ALT活性显著增高（*P<0.05）;与F组相比,FSe组小鼠毛色光亮,且活泼敏捷,肝脏显微及超微结构损伤有所缓解。2)与C组相比,F组小鼠肝脏MDA（**P<0.01）、ROS（*P<0.05）含量显著增高,T-SOD活性显著降低（*P<0.05）;与F组相比,FSe组小鼠肝脏MDA、ROS含量显著降低（^#P<0.05）,GSH-Px活性极显著升高(^###P<0.001)。3)与C组相比,F组小鼠肝脏PINK1、Atg5、LC3、TAX1BP1、NLRP3、IL-1βmRNA表达水平显著升高（*P<0.05）,Parkin、Atg3、OPTN、P62、IL-18 m RNA表达水平极显著升高（**P<0.01）;与F组相比,FSe组小鼠肝脏LC3、Atg3、Atg5、NLRP3、IL-1β、IL-18 m RNA表达水平显著降低（^#P<0.05）,Parkin m RNA表达水平极显著降低(^##P<0.01)。4)F组小鼠肝脏PINK1、Parkin、P62、LC3-Ⅰ、LC3-Ⅱ、ASC、Pro-IL-1β、IL-18蛋白表达水平较C组显著升高（*P<0.05）;与F组相比,FSe组小鼠肝脏Parkin、P62、LC3-Ⅰ、NLRP3、ASC蛋白表达水平显著降低（^#P<0.05）,PINK1、LC3-Ⅱ、Pro-IL-1β蛋白表达水平极显著降低(^##P<0.01)(^###P<0.001)。（2）Parkin^-/-型小鼠试验结果1)与Cq组相比,Fq组小鼠状态萎靡不振,反应迟钝,肝索排列紊乱,中央静脉周围部分肝细胞局部坏死和肿胀,肝脏中AST和ALT活性无显著变化（P>0.05）;FSeq组小鼠毛色光亮,且活泼敏捷,肝脏显微中病理现象有所缓解,AST和ALT活性较Fq组也无显著变化（P>0.05）。2)Fq组小鼠肝脏中T-SOD水平较Cq组显著降低（*P<0.05）;与Fq组相比,FSeq组小鼠肝脏中T-SOD(^##P<0.01)、GSH-Px（^#P<0.05）活性显著升高,MDA极显著降低(^##P<0.01)。3)与Cq组相比,各处理组间线粒体自噬相关基因均无显著变化（P>0.05）;FSeq组小鼠肝脏Atg3 m RNA表达水平较Fq组显著降低（^#P<0.05）,其余各处理组无显著变化（P>0.05）。4)Fq组小鼠肝脏P62蛋白表达水平较Cq组极显著升高（**P<0.01）;与Fq组相比,FSeq组小鼠肝脏PINK1、P62、LC3-Ⅰ蛋白表达水平显著降低（^#P<0.05）,LC3-Ⅱ蛋白表达水平极显著降低(^##P<0.01)。【结论】（1）氟暴露可诱导小鼠肝细胞线粒体自噬紊乱、组织与超微结构破坏,激活NLRP3炎症小体,导致小鼠肝脏受损,而1μg/g bw/d的CS-Se Nps补充可缓解氟诱导的肝脏损伤。（2）氟化钠通过促进PINK1/Parkin自噬通路中Parkin的表达导致小鼠肝脏线粒体自噬紊乱,CS-Se Nps通过降低自噬相关因子表达缓解氟致肝损伤。更多还原

【Abstract】 [Objective] This study was conducted to elucidate the precise mechanism of liver mitochondrial autophagy disorder induced by fluoride exposure and to assess the impact of CSSe Nps on liver injury induced by fluoride in mice.[Methods] This study will investigate the following two aspects of research:（1）C57BL/6 WT male mice were randomly divided into four groups: control group（drinking distilled water）,fluoride group（drinking 100 mg/L Na F）,CS-Se Nps group（gavage 1 μg/g bw/d of CS-Se Nps）,fluoride + CS-Se Nps group（drinking 100 mg/L Na F and gavage 1 μg/g bw/d of CSSe Nps）.After three months of feeding,observe the pathological morphology of mouse liver tissue,detect liver function and oxidative stress status,mitochondrial autophagy-related factors,and NLRP3 inflammasome expression.（2）Parkin^-/-C57BL/6 male mice were divided into four groups: control group（Cq,drinking distilled water）,fluoride group（Fq,drinking 100 mg/L Na F）,and CSSe Nps group（Seq,gavage 1 μg/g bw/d of CS-Se Nps）,fluoride + CS-Se Nps group（FSeq,drinking 100 mg/L Na F and gavage 1 μg/g bw/d of CS-Se Nps）.After three months of feeding,liver damage,oxidative stress status,and the expression of mitochondrial autophagy-related factors were detected in mice.[Results] The test results are as follows:（1）WT mouse test results1)Compared with group C,mice in group F showed mental depression,delayed activity,and significant damage to liver microstructure and ultrastructure.AST and ALT activities were significantly increased in group F（*P < 0.05）compared with group C.Compared with the F group,mice in the FSe group had brighter coats and were more agile,and their liver microscopic and ultrastructural damage was alleviated.2)Compared with group C,MDA（**P < 0.01）and ROS（*P < 0.05）levels were significantly increased in the liver of group F mice,while T-SOD activity was significantly decreased（*P < 0.05）.Compared with the F group,MDA and ROS levels in the liver of FSe group mice were significantly reduced（#P < 0.05）,and GSH-Px activity was significantly increased（###P < 0.001）.3)Compared with group C,the m RNA expression level of liver PINK1,Atg5,LC3,TAX1BP1,NLRP3,and IL-1β in group F mice was significantly increased（*P < 0.05）,while the m RNA expression levels of Parkin,Atg3,OPTN,P62,and IL-18 were extremely significantly increased（**P < 0.01）.Compared with the F group,the m RNA expression levels of LC3,Atg3,Atg5,IL-1β,IL-18,and NLRP3 in the liver of mice in the FSe group were significantly reduced（#P < 0.05）,while the m RNA expression level of Parkin was extremely significantly reduced（##P < 0.01）.4)The expression levels of PINK1,Parkin,P62,LC3-Ⅰ,LC3-Ⅱ,ASC,Pro-IL-1β,and IL-18 proteins in the liver of the mice in the F group were significantly increased compared with those in the C group（*P < 0.05）.Compared with group F,the expression levels of Parkin,P62,LC3-Ⅰ,NLRP3,and ASC proteins in the liver of FSe group mice were significantly reduced（#P < 0.05）,while the expression levels of PINK1,LC3-Ⅱ,and Pro-IL-1β proteins were significantly reduced（##P < 0.01）（###P < 0.001）.（2）Parkin^-/-mouse test results1)Compared to the Cq group,the Fq group was depressed,slow in response,had disordered arrangements of liver cords,local necrosis,and swelling of some hepatocytes around the central vein.There were no significant changes in AST and ALT activity in the liver of the Fq group compared to the Cq group（P > 0.05）.The mice in the FSeq group had bright fur,were lively and agile,and showed alleviation of pathological phenomena in liver microscopy.There were no significant changes in the activities of AST and ALT in the liver of the FSeq group mice compared to the Fq group（P > 0.05）.2)The level of T-SOD in the livers of mice in the Fq group was significantly lower than that of the Cq group（*P < 0.05）.Compared to the Fq group,the liver of FSeq group mice showed a significant increase in the activities of T-SOD（##P < 0.01）and GSH Px（#P < 0.05）,and a significant reduction in MDA（##P < 0.01）.3)There were no significant changes in mitochondrial autophagy-related genes among the other treatment groups compared to the Cq group（P > 0.05）.Compared to the Fq group,the expression level of Atg3 m RNA in the liver of FSeq group mice was significantly reduced（#P < 0.05）,while there was no significant change in the other treatment groups（P > 0.05）.4)The expression level of P62 protein in the liver of Fq group mice was significantly higher than that of Cq group（**P < 0.01）.Compared to the Fq group,the liver of FSeq group mice showed significantly reduced expression levels of PINK1,P62,and LC3-I proteins（#P < 0.05）,as well as an extremely significant reduction in the expression levels of LC3-II proteins（##P < 0.01）.[Conclusion]（1）Exposure to fluoride has been found to cause mitochondrial autophagy disorders,tissue and ultrastructural damage in mouse liver cells,and activation of NLRP3 inflammasomes,leading to liver damage in mice.However,supplementing CS-Se Nps with 1 μg/g bw/d has been shown to alleviate fluoride-induced liver damage.（2）Sodium fluoride induces liver mitochondrial autophagy disorders by promoting the expression of Parkin within the PINK1/Parkin autophagy pathway.CS-Se Nps alleviate liver injury caused by fluoride by reducing the expression of autophagy-related factors.更多还原