论文部分内容阅读
目的通过Western blot法和气相色谱-质谱(GC-MS)联用技术观察大黄灵仙胶囊对小鼠胆结石肝细胞转运蛋白表达及胆汁代谢谱的影响,探讨其可能机制。方法将50只雄性C57BL/6小鼠随机分为正常对照组(N组)、模型组(M组)、熊去氧胆酸(UDCA)对照组(U组)、药物对照组(Y组)及大黄灵仙胶囊治疗组(D组),每组10只。N组和Y组小鼠普通饲料喂养,M、U、D组给予高脂、高热量、高胆固醇饲料诱导胆囊结石的形成。同时U组每天给予130mg/kg UDCA溶液灌胃;Y组和D组每天给予大黄灵仙胶囊汤药13g/kg灌胃,N组和M组给予相同体积的生理盐水灌胃。干预8周后,观察小鼠成石率,利用Western blot法观察肝胆管侧膜细胞转运蛋白三磷酸腺苷结合盒转运子B亚族成员11(ATP binding cassette subfamily B member 11,Abcb11)和三磷酸腺苷结合盒转运体c2(ATP binding cassette subfamily C member 2,Abcc2)变化,GC-MS技术检测小鼠胆汁代谢组学特征。结果造模期间因灌胃操作不慎,U组1只小鼠挣扎时灌胃针扎破小鼠食管导致死亡。M组成石率为100.00%,高于N组和Y组(均为0.00%,χ2=20.00,P<0.01);与M组比较,U组及D组成石率降低(44.44%,χ2=7.54,P=0.011;30.00%,χ2=10.77,P=0.003)。经红外光谱分析检测后在2939、1 446、1 382、1 056cm-1处可见胆固醇特有吸收峰。5组小鼠Abcb11和Abcc2转运蛋白总体比较差异有统计学意义(F=41.89;P<0.01;F=90.01,P<0.01)。与N组比较,M组Abcb11和Abcc2表达降低(P<0.01);与M组比较,U、Y、D组转运蛋白Abcb11和Abcc2表达均升高(P<0.01)。与其他组比较,模型组小鼠胆汁内源性代谢物丙氨酸、柠檬酸、赖氨酸、蛋氨酸、苯丙氨酸、酪氨酸、胆固醇、LDL、甘油、苹果酸及丙酮浓度升高,乳酸、谷氨酰、胺甘氨酸、胆碱及牛磺酸浓度降低,差异有统计学意义(P<0.05,P<0.01)。结论大黄灵仙胶囊具有稳定肝胆管侧膜细胞转运蛋白表达、改变或改善病理性胆汁的分泌及其代谢物的作用,从而达到防治胆结石的目的。
OBJECTIVE: To observe the effect of rhubarb and Lingxian capsule on the expression of hepatic cell transporter protein and bile metabolism in gallstone by Western blot and GC-MS, and to explore its possible mechanism. Methods Fifty male C57BL / 6 mice were randomly divided into normal control group (N group), model group (M group), ursodeoxycholic acid (UDCA) control group (U group) and drug control group (Y group) And rhubarb Lingxian capsule treatment group (group D), 10 rats in each group. N and Y mice were fed the normal diet, M, U, D groups were given high fat, high calorie, high cholesterol feed to induce gallstones formation. At the same time, U group was given intragastric administration of 130mg / kg UDCA solution every day. Y group and D group were given Rhubarb Lingxian Decoction 13g / kg intragastrically every day. N and M groups were given the same volume of normal saline. After intervention for 8 weeks, the rate of stone formation in mice was observed. Western blotting was used to observe the transport of ATP binding cassette subfamily B member 11 (Abcb11) and adenosine triphosphate binding cassette The change of bcc in mice was analyzed by GC-MS. Results During the modeling period, due to inadvertent operation, one mouse in U group was killed by gavage of the mouse esophagus while struggling. The stone ratio of M group was 100.00%, which was higher than that of N group and Y group (all 0.00%, χ2 = 20.00, P <0.01). Compared with M group, the stone formation rate of U group and D group decreased 44.44%, χ2 = 7.54 , P = 0.011; 30.00%, χ2 = 10.77, P = 0.003). The specific absorption peak of cholesterol was observed at 2939, 1 446, 1 382 and 1 056 cm-1 after IR analysis. The Abcb11 and Abcc2 transporters in the 5 groups showed statistically significant differences (F = 41.89; P <0.01; F = 90.01, P <0.01). Compared with N group, the expression of Abcb11 and Abcc2 in M group decreased (P <0.01). Compared with M group, the expression of Abcb11 and Abcc2 in U, Y and D groups increased (P <0.01). Compared with other groups, the concentrations of alanine, citrate, lysine, methionine, phenylalanine, tyrosine, cholesterol, LDL, glycerol, malic acid and acetone in bile of model mice increased (P <0.05, P <0.01). The concentrations of lactate, glutamyl, glycine, choline and taurine decreased. Conclusion Rhubarb Lingxian Capsule has stable hepatocyte bile duct cell membrane transporter protein expression, change or improve the secretion of pathological bile and its metabolites, so as to achieve the purpose of prevention and treatment of gallstones.