大鼠胰腺损伤血清代谢组学特征分析

Serum metabolomics characteristics of pancreatic trauma in rats

目的 探究大鼠胰腺损伤(PT)后血清代谢特征的变化,为PT后的发展机制以及治疗靶点提供新的理论依据。方法 将12只SD大鼠随机分为Sham组和PT组,各6只。Sham组开腹后轻轻翻动胰腺后关腹,PT组开腹后利用多功能动物撞击仪以400 kPa压力冲击胰腺组织。禁食24 h后取血清及胰腺组织,HE染色观察胰腺组织损伤的情况,ELISA法检测淀粉酶、脂肪酶、炎症因子的表达水平。采用液相色谱串联质谱技术测定血清内源性代谢物的表达,通过多元统计分析筛选潜在差异代谢物,MetaboAnalyst 5.0进行代谢通路分析。结果 与Sham组比较,PT组胰腺腺泡细胞变性坏死,细胞间质水肿,间隙增大,坏死区域及间质伴有明显炎性细胞浸润。PT组病理学评分均高于Sham组[水肿(2.33±0.19)分vs.(0.24±0.07)分、出血(2.28±0.16)分vs.(0.16±0.06)分、坏死(2.52±0.09)分vs.(0.14±0.07)分、炎症浸润(2.31±0.13)分vs.(0.27±0.09)分,P<0.05],PT组淀粉酶、脂肪酶、IL-6和TNF-α表达水平较Sham组明显升高[(303.70±23.72)pg/mL vs.(204.70±17.67)pg/mL、(1207.00±89.52)pg/mL vs.(568.10±120.60)pg/mL、(63.00±4.63)pg/mL vs.(12.79±1.36)pg/mL、(446.50±27.96)pg/mL vs.(88.39±13.32)pg/mL,P<0.05],PT组IL-10、TGF-β表达水平较Sham组明显降低[(22.80±3.37)pg/mL vs.(63.69±0.80)pg/mL、(216.90±28.14)pg/mL vs.(1030.00±80.70)pg/mL,P<0.05];血清中鉴定出359个差异性代谢物,其中45个上调,314个下调;这些差异性代谢物与苯丙氨酸代谢、色氨酸代谢、组氨酸代谢、柠檬酸循环(TCA循环)、酪氨酸代谢以及苯丙氨酸、酪氨酸和色氨酸生物合成密切相关。结论 大鼠PT后血清代谢组发生明显变化,能量代谢、氨基酸代谢紊乱是PT最典型的代谢特征,可能与PT病情发展有关,靶向回调氨基酸代谢的相对丰度可能对PT有一定的治疗效果。

Objective To explore the changes in serum metabolic characteristics after pancreatic trauma(PT)in rats and to provide new theoretical basis for studies on the mechanism and therapeutic targets of PT.Methods A total of 12 SD rats were randomly divided into sham and PT groups(n=6 for each).After laparotomy,for the sham group,the pancreas of rats was gently flipped and then the abdomen was closed;while for the PT group,a modeling instrument was used to impact the pancreatic tissue at the pressure of 400 kPa.After fasting for 24 h,serum and pancreatic tissue sampling was conducted and HE staining was used to observe the damage to pancreatic tissues.The expression levels of amylase,lipase,and inflammatory factors were detected by ELISA.Liquid chromatography-tandem mass spectrometry was adopted to determine the expression of endogenous metabolites in serum,multivariate statistical analysis to screen potential differential metabolites,and MetaboAnalyst 5.0 to analyze metabolic pathways.Results In the PT group,pancreatic acinar cells underwent degeneration and necrosis,with interstitial edema and enlarged gaps.The necrotic area and interstitium were accompanied by significant inflammatory cell infiltration.Compared with the sham group,the PT group showed much higher pathological scores in terms of edema(2.33±0.19 vs.0.24±0.07),bleeding(2.28±0.16 vs.0.16±0.06),necrosis(2.52±0.09 vs.0.14±0.07),and inflammatory infiltration(2.31±0.13 vs.0.27±0.09,all P<0.05),indicating successful PT modeling.The PT group showed much higher expression levels(pg/mL)of amylase(303.70±23.72 vs.204.70±17.67)and lipase(1207.00±89.52 vs.568.10±120.6,both P<0.05),much higher levels(pg/mL)of IL-6(63.00±4.63 vs.12.79±1.36),TNF-α(446.50±27.96 vs.88.39±13.32)and much lower levels(pg/mL)of IL-10(22.80±3.37 vs.63.69±0.80)and TGF-β(216.90±28.14 vs.1030.00±80.70,all P<0.05).Altogether 359 differential metabolites were identified in serum,of which 45 were upregulated and 314 were downregulated.These differential metabolites were closely related to phenylalanine metabolism,tryptophan metabolism,histidine metabolism,tricarboxylic acid cycle,and tyrosine metabolism,as well as phenylalanine,tyrosine,and tryptophan biosynthesis.Conclusion After PT in rats,there are significant changes in serum metabolomics.Energy metabolism and amino acid metabolism disorders are the most typical metabolic characteristics of PT,which may be related to the development of PT.Targeted callback of the relative abundance of amino acid metabolism may have certain therapeutic effects on PT.