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目的探讨大黄附子汤对重症急性胰腺炎(SAP)大鼠肠黏膜上皮细胞(IEC)结构的保护作用及IEC线粒体功能的影响。方法本研究为随机对照试验,将60只健康雄性SD大鼠(体重250~300 g),采用随机数字表法将大鼠分为对照组、重症急性胰腺炎组(SAP组)和大黄附子汤治疗组(大黄附子汤组),每组20只。每组再按建模后时间(6、12、24、48 h)分为4个亚组,每个亚组5只。SAP组和大黄附子汤组采用5%牛磺胆酸钠(0.1 m L/100g)胰胆管注射法诱导SAP模型,对照组开腹翻动胰腺数次后关腹。大黄附子汤组大鼠分别于术后0、15、30、45 h以大黄附子汤灌肠(10 m L/kg),对照组和SAP组在同时间点给予同体积0.9%氯化钠溶液灌肠。各组按各时间点处死动物,左侧股静脉取血测定血清淀粉酶和内毒素变化。采用分光光度计、荧光定量法检测IEC线粒体ATP合成酶、细胞色素C氧化酶(Cc O)含量变化,光镜观察小肠上皮组织的形态学变化。结果与SAP组相比,大黄附子汤可降低SAP后24 h、48 h的血清淀粉酶水平[24 h:(769.14±456.99)vs(1 399.85±623.40)U/L,P<0.01;48 h:(889.59±209.82)vs(1 562.00±316.19)U/L,P<0.001)。与SAP组相比,大黄附子汤组大鼠血清内毒素在建模后24 h、48 h均有不同程度下降[24 h:(108.78±22.16)vs(168.77±29.25)pg/m L,P<0.01;48 h:(242.90±45.12)vs(124.01±321.45)pg/m L,P<0.001]。与对照组相比,SAP后IEC线粒体ATP合成酶和Cc O活性均降低且随病程推移呈递减趋势;经大黄附子汤治疗后IEC线粒体ATP合成酶活性高于同时相点的SAP组[12 h:(19.60±1.67)vs(13.60±2.88)nmol·min(-1)·mg(-1)pro,P<0.05;24 h:(17.40±2.79)vs(11.2±2.78)nmol·min(-1)·mg(-1)pro,P<0.05;48 h:(18.60±2.07)vs(11.2±1.92)nmol·min(-1)·mg(-1)pro,P<0.01],且24 h、48 h线粒体Cc O活性高于同时相点SAP组[24 h:(1.56±0.11)vs(1.06±0.18)μmol·min(-1)·mg(-1);48 h:(1.04±0.31)vs(0.50±0.16)μmol·min(-1)·mg(-1),P均<0.01]。SAP后12 h小肠上皮损伤指数开始升高(2.2±0.84 vs 0.60±0.55,P<0.01),且24、48 h持续性升高;大黄附子汤组可显著降低SAP后48 h小肠上皮损伤指数(1.2±0.45 vs 4.0±1.0,P<0.001)。结论大黄附子汤能通过增强大鼠IEC线粒体ATP合成酶和Cc O活性,改善IEC线粒体能量代谢和呼吸功能,保护SAP后肠屏障的完整性和功能,降低血清淀粉酶和内毒素含量,缓解SAP病情。
Objective To investigate the protective effect of rhubarb and aconite decoction on the structure of IEC in severe acute pancreatitis (SAP) rats and the effect of IEC mitochondrial function. Methods This study was a randomized controlled trial in which 60 healthy male SD rats weighing 250-300 g were randomly divided into control group, severe acute pancreatitis group (SAP group) and Rhubarb Aconite Decoction Treatment group (rhubarb aconite soup group), each group of 20. Each group was divided into 4 sub-groups according to the time after modeling (6, 12, 24, 48 h), 5 in each sub-group. The SAP group and the rhubarb aconite decoction group were induced by 5% sodium taurocholate (0.1 m L / 100 g) into the pancreaticobiliary duct, and the control group was transabdominalized after turning the pancreas several times. Rhubarb aconite decoction group rats were given Rhubarb aconite decoction (10 m L / kg) at 0, 15, 30 and 45 h after operation, while 0.9% sodium chloride solution enema . Animals in each group were sacrificed at each time point, and the left femoral vein was taken for determination of serum amylase and endotoxin. The contents of ATP synthase and cytochrome C oxidase (CcO) in mitochondria of IEC were detected by spectrophotometer and fluorescence quantitative method. Morphological changes of intestinal epithelium were observed under light microscope. Results Compared with SAP group, Rhubarb aconite decoction could reduce serum amylase level 24 h and 48 h after SAP (24 h: (769.14 ± 456.99) vs (399.85 ± 623.40) U / L, P <0.01) : (889.59 ± 209.82) vs (5682.00 ± 316.19) U / L, P <0.001). Compared with the SAP group, the serum endotoxin of the rhubarb aconite decoction group decreased to different extents 24 h and 48 h [24 h: (108.78 ± 22.16) vs (168.77 ± 29.25) pg / m L, P <0.01; 48 h: (242.90 ± 45.12) vs (124.01 ± 321.45) pg / m L, P <0.001]. Compared with the control group, the ATP mitochondrial ATP synthase and CcO activity in IEC were decreased and decreased with the course of the disease; ATP mitochondrial ATP synthase activity in IEC was higher than that in the SAP group [12 h (19.60 ± 1.67) vs (13.60 ± 2.88) nmol · min -1 · mg -1 -1 pro, P <0.05; 24 h: (17.40 ± 2.79) vs (11.2 ± 2.78) nmol · min 1) · mg (-1) pro, P <0.05; 48 h: (18.60 ± 2.07) vs (11.2 ± 1.92) nmol · min -1 · mg -1 pro and P The activity of mitochondrial Cc O in h group was higher than that in SAP group at 24 h (24 h: (1.56 ± 0.11) vs (1.06 ± 0.18) μmol · min -1 · mg -1) 0.31) vs (0.50 ± 0.16) μmol · min -1 · mg -1, both P <0.01]. The injury index of small intestine began to increase 12 h after SAP (2.2 ± 0.84 vs 0.60 ± 0.55, P <0.01), and continued to increase at 24 and 48 h. Rhubarb aconite decoction significantly reduced the intestinal epithelial injury index (1.2 ± 0.45 vs 4.0 ± 1.0, P <0.001). Conclusion Rhubarb aconite decoction can improve IEC mitochondrial ATP synthase and Cc O activity, improve IEC mitochondrial energy metabolism and respiratory function, protect the intestine barrier integrity and function, reduce serum amylase and endotoxin levels, relieve SAP Condition.