摘要
目的研究乳源六肽脯氨酸-甘氨酸-脯氨酸-异亮氨酸-脯氨酸-天冬酰胺(PGPIPN)和其截短五肽脯氨酸-甘氨酸-脯氨酸-异亮氨酸-脯氨酸(PGPIP)缓解小鼠慢性酒精性肝损伤及其相关的分子机制。方法60只昆明小鼠,随机均分为对照组、模型组、谷胱甘肽(GSH)组、PGPIPN组、截短五肽PGPIP组。采用梯度酒精灌胃的方法建立小鼠慢性酒精性肝损伤模型,造模的同时给予药物干预,共12周。肝脏HE染色分析各处理组对小鼠酒精性肝损伤的病理学影响。体外分离培养小鼠原代肝细胞和人正常肝细胞系L-02,水溶性四氮唑-1(WST-1)细胞增殖及细胞毒性检测确定各种细胞合适的PGPIPN诱导浓度。持续诱导L-02细胞不同时间,Western blot检测人叉头框蛋白O3(FoxO3a)和磷酸化FoxO3a蛋白质的表达,确定合适的诱导时间。免疫荧光染色检测FoxO3a在L-02细胞中的亚细胞定位。实时荧光定量PCR(qRT-PCR)检测不同处理组小鼠原代肝细胞和L-02细胞FoxO3a和锰超氧化物歧化酶(MnSOD)基因mRNA的变化。结果PGPIPN组和PGPIP组病理学检查类似GSH组,小鼠肝脏损伤明显减轻。分别选择中浓度和高浓度PGPIPN诱导小鼠原代肝细胞和L-02细胞。在16 h,L-02细胞FoxO3a蛋白表达显著增加,并且FoxO3a蛋白主要表达于细胞核内。此外,在相应剂量PGPIPN诱导后,两种类型细胞中mRNA水平的显著增加。结论PGPIPN和截短五肽PGPIP能减少小鼠慢性酒精性肝损伤,其机制可能是通过FoxO3a-MnSOD信号通路,减少酒精诱导的氧化应激从而发挥作用。
Objective To investing ate the milk-derived hexapeptide PGPIPN and its truncated pentapeptide PGPIP to alleviate chronic alcoholic liver injury in mice and its associated molecular mechanisms.Methods Sixty Kunming mice were randomly divided into control group,model group,GSH group,PGPIPN group,and truncated pentapeptide PGPIP group.The model of chronic alcoholic liver injury in mice was established by gavage with gradient alcohol.Drug intervention was given at the same time for 12 weeks.Liver HE staining was used to analyze the pathological effects of each treatment group on alcoholic liver injury in mice.Primary mouse hepatocytes and human normal hepatocyte line L-02 were isolated and cultured in vitro.The appropriate PGPIPN induction concentrations of both cells were determined by WST-1 method.L-02 cells were induced at different times.The expression of FoxO3a and phosphorylated FoxO3a protein were detected by Western blot to determine the appropriate induction time.The subcellular localization of FoxO3a in L-02 cells was detected by cellular immunofluorescence.The mRNA changes of FoxO3a and MnSOD genes in primary hepatocytes and L-02 cells of mice in different treatment groups were detected by qRT-PCR.Results The pathological examination of PGPIPN group and PGPIP group was similar to that of GSH group,and the liver injury of mice was significantly reduced.Medium and high concentrations of PGPIPN were respectively selected to induce mouse primary hepatocytes and L-02 cells.At 16 hours,the expression of FoxO3a protein in L-02 cells increased significantly.FoxO3a protein was mainly expressed in the nucleus.In addition,mRNA levels in both types of cells increased significantly after induction with the corresponding dose of PGPIPN.Conclusion PGPIPN and truncated pentapeptide PGPIP can reduce chronic alcoholic liver injury in mice.The mechanism may be to reduce alcohol-induced oxidative stress through FoxO3a-MnSOD signaling pathway.
作者
李安琪
祝晓梅
黄九九
秦宜德
戚楠
Li Anqi;Zhu Xiaomei;Huang Jiujiu;Qin Yide;Qi Nan(Dept of Biochemistry and Molecular Biology,School of Basic Medical Science,Anhui Medical University,Hefei 230032;Dept of Histology and Embryology,School of Basic Medical Science,Anhui Medical University,Hefei 230032)
出处
《安徽医科大学学报》
CAS
北大核心
2022年第12期1864-1869,共6页
Acta Universitatis Medicinalis Anhui
基金
国家自然科学基金(编号:81472448)
安徽省自然科学基金(编号:1908085MH280)。
