【作者】 梁国栋；

【导师】 冯翠萍；

【作者基本信息】 山西农业大学 ， 食品科学与工程， 2024， 硕士

【摘要】 [目的]在铅致小鼠肝损伤模型下,探究广叶绣球菌多糖对其调控作用的具体机制,为探究广叶绣球菌多糖在生物活性的作用提供理论依据。[方法]将75只小鼠随机分为5组,每组15只,分别为空白对照组(NC组),模型组(Pb组),低剂量组(L-SLPs组),中剂量组(M-SLPs组)和高剂量组(H-SLPs组)。除空白对照组外,其他各组小鼠给与1.84 g/L醋酸铅水溶液,低、中、高剂量组分别每天灌胃剂量为100、200、400 mg/kg?BW的广叶绣球菌多糖(SLPs)溶液,空白对照组和染铅模型组灌胃等量生理盐水,连续喂养8周,处死采集肝脏样品测定相关指标:(1)ICP/OES测定肝脏的重金属含量,HE染色观察肝脏组织形态结构;(2)试剂盒测定GSH和MDA的含量以及SOD、CAT和GSH-Px的活性;(3)超高效液相色谱串联质谱法检测肝脏代谢物含量;(4)实时荧光定量聚合酶链式反应(q RT-PCR)检测肝脏Cyclin E1、NF-κB、stat3、SHP-1、TGF-β1、SPHK2和MCL-1的mRNA表达量;蛋白免疫印迹检测肝脏SPHK2和p-stat3的蛋白表达量;ELISA法测定神经酰胺(Cer)和1-磷酸鞘氨醇(S1P)。[结果](1)与NC组相比,Pb组的铅含量显著升高(P<0.05),Pb组的Zn、Mg、Fe和Cu的含量显著降低(P<0.05);与Pb组相比,广叶绣球菌多糖剂量组的铅含量显著降低(P<0.05),广叶绣球菌多糖剂量组的Zn、Mg、Fe和Cu的含量显著升高(P<0.05)。HE染色结果:NC组肝小叶结构正常、轮廓清晰,未见炎性细胞浸润;Pb组肝小叶轮廓模糊、结构紊乱,肝细胞结构异常,伴有大量炎性细胞浸润;广叶绣球菌多糖均能不同程度地改善肝脏组织病理严重程度。(2)与NC组相比,Pb组小鼠肝脏SOD、CAT、GSH-Px活性和GSH含量均显著下降(P<0.05);MDA含量显著升高(P<0.05)。与Pb组相比,广叶绣球菌多糖剂量组SOD、CAT、GSH-Px的活性和GSH含量显著上升(P<0.05),MDA含量显著下降(P<0.05)。(3)通过主成分分析(PCA)和正交偏最小二乘方判别分析(OPLS-DA)发现与NC组相比,Pb组有一部分内源性代谢物浓度发生了显著变化,通过对筛选鉴定出的25种差异代谢物进行通路富集分析,结果表明染铅小鼠肝损伤主要涉及鞘脂代谢、甘油磷脂代谢和亚油酸代谢途径;与Pb组相比,广叶绣球菌多糖干预后显著影响了鞘脂代谢来发挥保护肝脏的作用。(4)与NC组相比,Pb组小鼠Cyclin E1、NF-κB、stat3、TGF-β1、SPHK2和MCL-1 mRNA表达量及SPHK2、p-stat3和S1P蛋白表达量升高,差异显著(P<0.05);SHP-1mRNA表达量和Cer的蛋白含量均显著下降(P<0.05)。与Pb组相比,广叶绣球菌多糖剂量组Cyclin E1、NF-κB、stat3、TGF-β1、SPHK2和MCL-1 mRNA表达量及SPHK2、p-stat3和S1P蛋白表达量呈下降趋势,其中高剂量组差异均显著(P<0.05);SHP-1mRNA表达量和Cer的蛋白含量均呈上升趋势,高剂量组差异显著(P<0.05)。[结论]广叶绣球菌多糖能够降低肝脏中铅的含量,提高其他2价金属离子的含量,并改善肝脏组织病理学变化;可提高SOD、CAT和GSH-Px活性和GSH含量,降低MDA含量,通过提高抗氧化酶的活性,来提高染铅小鼠肝脏的抗氧化能力;通过调节鞘脂代谢途径中S1P和Cer等代谢物的含量来减轻肝脏代谢紊乱,抑制炎症相关基因Cyclin E1、NF-κB、TGF-β1和MCL-1的表达水平,实现肝脏保护作用。更多还原

【Abstract】 [Objective] To explore the specific mechanism of the regulatory effect of Sparassis latifolia polysaccharides on lead-induced liver injury in mice,and to provide a theoretical basis for exploring the role of Sparassis latifolia polysaccharides in biological activity.[Methods] Seventy-five mice were randomly divided into 5 groups: blank control group(NC),model group(Pb),low-dose group(L-SLPs group),medium-dose group(M-SLPs group),high-dose group(H-SLPs group).Except for the blank control group,the mice in other groups were given 1.84 g / L lead acetate aqueous solution,and The rats in the low-dose group,medium-dose group and high-dose group were gavaged with a daily dose of 100mg/kg·BW,200mg/kg·BW and 400 mg/kg·BW,respectively.The blank control group and The lead-exposed model group received intragastric administration of an equivalent volume of normal saline.After 8 weeks of continuous feeding,the liver samples were collected to determine the relevant indicators :(1)ICP / OES was used to determine the content of heavy metals,and The morphological structure of liver tissue was examined using HE staining.(2)The contents of GSH and MDA and the activities of SOD,CAT and GSH-Px were determined by kits.(3)The content of liver metabolites was detected using ultra-high performance liquid chromatography tandem mass spectrometry;(4)The mRNA expression was detected using real-time fluorescence quantitative polymerase chain reaction of Cyclin E1,NF-κB,stat3,SHP-1,TGF-β1,SPHK2 and MCL-1 in liver;western blot was used to detect the protein expression of SPHK2 and p-stat3 in liver.Ceramide and sphingosine-1-phosphate were determined by ELISA.[Results](1)Compared with the NC group,the lead content in the Pb group was significantly increased(P < 0.05),and the contents of Zn,Mg,Fe and Cu in the Pb group were significantly decreased(P < 0.05).Compared with the Pb group,the lead content in the Sparassis latifolia polysaccharide dose group was significantly decreased(P < 0.05),the contents of Zn,Mg,Fe and Cu in the Sparassis latifolia polysaccharide dose group were significantly increased(P < 0.05).HE staining results : the structure of hepatic lobules in NC group was normal,the outline was clear,and no inflammatory cell infiltration was observed.In Pb group,the outline of hepatic lobules was blurred,the structure was disordered,the structure of hepatocytes was abnormal,accompanied by a large number of inflammatory cell infiltration;Sparassis latifolia polysaccharide can improve the pathological severity of liver tissue to varying degrees.(2)Compared with NC group,the activities of SOD,CAT,GSH-Px and the content of GSH in liver of Pb group were significantly decreased(P<0.05).MDA content was significantly increased(P < 0.05).Compared with the Pb group,the activity of SOD,CAT,GSH Px and GSH content in the polysaccharide dose group of Sparassis latifolia significantly increased(P<0.05),while the MDA content significantly decreased(P<0.05).(3)Through PCA and OPLS-DA,it was found that compared with NC group,the concentration of many endogenous metabolites in Pb group changed significantly.Through pathway enrichment analysis of25 differential metabolites screened and identified,the results showed that liver injury in lead-exposed mice mainly involved sphingolipid metabolism,glycerophospholipid metabolism and linoleic acid metabolism.The intervention of Sparassis latifolia polysaccharide significantly affected sphingolipid metabolism to protect the liver.(4)Compared with NC group,the mRNA expression levels of Cyclin E1,NF-κB,stat3,TGF-β1,SPHK2 and MCL-1 and the protein expression levels of SPHK2,p-stat3 and S1P(sphingosine-1-phosphate)in Pb group were significantly increased(P < 0.05).The mRNA expression of SHP-1 and the protein content of Cer were significantly decreased(P < 0.05).Compared with the Pb group,the mRNA expression levels of Cyclin E1,NF-κB,stat3,TGF-β1,SPHK2 and MCL-1 and the protein expression levels of SPHK2,p-stat3 and S1P(sphingosine-1-phosphate)in each polysaccharide dose group showed a downward trend,and the differences in the high-dose group were significant(P < 0.05).The expression of SHP-1 mRNA and the protein content of Cer(ceramide)showed an upward trend,and the difference was significant in the high-dose group(P < 0.05).[Conclusion] Sparassis latifolia polysaccharide can reduce the content of lead in liver,increase the content of other metal 2 valence ions,and improve the pathological changes of liver tissue.The activities of SOD,CAT and GSH-Px and the content of GSH were increased,and the content of MDA was decreased.The liver antioxidant capacity of mice exposed to lead was improved by increasing the activities of antioxidant enzymes.By regulating the content of metabolites such as S1 P and Cer in the sphingolipid metabolic pathway,it can alleviate liver metabolic disorders,inhibit the expression levels of inflammationrelated genes Cyclin E1,NF-κB,TGF-β1 and MCL-1,and achieve liver protection.更多还原