基于非靶向代谢组学技术探讨Yes相关蛋白1改善马兜铃酸I诱导小鼠肝损伤的机制

Mechanism of Yes-Associated Protein 1 Ameliorating Aristolochic Acid 1-Induced Liver Injury in Mice Based on Untargeted Metabolomics Techniques

目的基于非靶向代谢组学技术探讨Yes相关蛋白1(YAP1)改善马兜铃酸I(Aristolochic acid I,AAI)诱导小鼠肝损伤的机制。方法随机选取8只3周龄雄性肝细胞特异性Yap1基因敲除(基因型为Yap1^(Flox/Flox),Albumin-Cre,简称Yap1^(LKO))小鼠作为Yap1^(LKO)+AAI组,8只Yap1Flox对照小鼠作为Yap1^(Flox)+AAI组。2组小鼠均按照2.5 mg·kg^(-1)·d^(-1)剂量腹腔注射AAI,连续14 d。采用鼠尾PCR法鉴定基因型;微板法测定血清丙氨酸氨基转移酶(ALT)、天门冬氨酸氨基转移酶(AST)活性;HE染色法观察肝脏组织病理变化;免疫组织化学法测定肝组织中YAP1蛋白表达情况。采用非靶向代谢组学方法分析肝脏组织差异代谢物,利用超高效液相色谱-四极杆-静电场轨道阱高分辨质谱联用技术分析样品,通过主成分分析(PCA)、偏最小二乘法判别分析(PLS-DA)和正交偏最小二乘法判别分析(OPLS-DA)筛选差异代谢物,利用HMDB数据库、METLIN数据库对代谢物进行鉴定,通过KEGG数据库对差异代谢物进行通路富集分析。结果(1)AAI诱导14 d,Yap1^(LKO)小鼠的体质量增长低于Yap1^(Flox)小鼠,但差异无统计学意义(P>0.05)。在第14天,与Yap1^(Flox)+AAI组比较,Yap1^(LKO)+AAI组小鼠血清中的ALT、AST酶活性明显升高(P<0.05),肝脏组织病理损伤明显加重。Yap1^(Flox)小鼠肝脏有YAP1蛋白阳性表达,而Yap1^(LKO)小鼠无YAP1蛋白阳性表达。(2)通过代谢组学分析共筛选出139种显著变化(VIP>1且P<0.05)的差异代谢物,与Yap1^(LKO)+AAI组小鼠相比,Yap1Flox+AAI组小鼠的62种肝脏代谢物上调,包括胆碱、牛磺酸、亚牛磺酸、α-亚麻酸、桐油酸、鹅去氧胆酸等;77种代谢物下调,包括甘油磷酸胆碱、L-磷脂酰胆碱、L-谷氨酰胺、L-丝氨酸、L-谷胱甘肽、5-甲硫腺、苯丙氨酸、6-磷酸葡萄糖、乳酸、尿酸苷等。KEGG富集通路主要有胆碱代谢、甘油磷脂代谢、胰岛素抵抗、谷胱甘肽代谢等。结论肝细胞特异性Yap1基因敲除加重了AAI诱导的小鼠肝脏损伤,YAP1通过上调胆碱、牛磺酸等不饱和脂肪酸,参与调控胆碱代谢、甘油磷脂代谢等,从而改善AAI诱导的小鼠肝损伤。

Objective To investigate the mechanism of Yes-associated protein 1(YAP1)ameliorating aristolochic acid 1(AAI)-induced liver injury in mice based on untargeted metabolomics techniques.Methods There were 83-week-old male hepatocyte-specific Yap1 gene knockout mice(genotyped as Yap1^(Flox)/Flox,Albumin-Cre,aka.Yap1LKO)were randomly selected as the Yap1LKO+AAI group,and 8 Yap1^(Flox)control mice as the Yap1^(Flox)+AAI group.Both groups were injected intraperitoneally with AAI at a dose of 2.5 mg·kg^(-1)·d^(-1)for 14 consecutive days.Genotypes were identified by tail PCR;serum alanine transaminase(ALT)and aspartate transaminase(AST)activities were determined by microplate assay;histopathological changes of liver tissue were observed by HE staining;and the protein expression of YAP1 in liver tissue was determined by immunohistochemistry.The untargeted metabolomics approach was used to analyze the liver tissue differential metabolites,and the samples were analyzed by ultra performance liquid chromatography-quadrupole-electrostatic field orbit trap high-resolution mass spectrometry,and the differential metabolites were screened by principal component analysis(PCA),Partial least square-discriminant analysis(PLS-DA),and orthogonal partial least squares-discriminant analysis(OPLS-DA);using HMDB database and METLIN database to identify metabolites,and the pathway enrichment of differential metabolites was analyzed by KEGG database.Results(1)After 14 days of AAI induction,the increase of body mass in Yap1^(LKO)mice was lower than that in Yap1^(Flox)mice,but there was no statistical significance(P>0.05).On day 14,compared with the Yap1^(Flox)+AAI group,the serum ALT and AST enzyme activities in the Yap1^(LKO)+AAI group of mice were significantly increased(P<0.05),and the histopathological damage of the liver was significantly aggravated.The livers of the Yap1^(Flox)mice had a positive protein expression of YAP1,whereas the Yap1^(LKO) mice did not have a positive protein expression of YAP1.(2)A total of 139 differential metabolites with significant changes(VIP>1 and P<0.05)were screened by metabonomic analysis;compared with Yap1^(LKO)+AAI group,62 liver metabolites in Yap1^(Flox)+AAI group were up-regulated,including choline,taurine,hypotaurine,α-linolenic acid,eleostearic acid,chenodeoxycholic acid and so on.Seventy-seven metabolites were down-regulated including glycerophosphocholine,L-phosphatidylcholine,L-glutamine,L-serine,L-glutathione,5-methionine,phenylalanine,glucose 6-phosphate,lactic acid,uric acid glycosides,etc..KEGG-enriched pathways were mainly choline metabolism,glycerophospholipid metabolism,insulin resistance,glutathione metabolism,etc..Conclusion Hepatocyte-specific Yap1 gene knockout exacerbated AAI-induced liver injury in mice,and YAP1 was involved in the regulation of choline metabolism and glycerophospholipid metabolism through the up-regulation of unsaturated fatty acids,such as choline and taurine,which ameliorated AAI-induced liver injury in mice.