清热解毒凉血化瘀法对内毒素肝损伤大鼠蛋白质表达的干预作用
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摘要
目的：探讨清热解毒凉血化瘀法对内毒素肝损伤大鼠蛋白质表达的干预作用。方法：(1)SD大鼠40只,随机分为4组：正常组、模型组、中药高剂量组、中药低剂量组。(2)模型组：模型组大鼠每天用蒸馏水2ml灌胃,每天1次,连续7天。于末次灌胃1小时后腹腔注射脂多糖(LPS) (10mg/Kg)。腹腔注射及取血前禁食12小时以上,自由饮水。注射脂多糖6小时后,用1%戊巴比妥钠(30mg/Kg)腹腔注射,麻醉动物后,酒精消毒皮肤,开胸,心脏取血。所有血液放入4℃冰箱保存。用表面增强激光解析电离飞行时间质谱技术(SELDI-TOF-MS技术)检测血清蛋白质谱的差异表达。用全自动生化仪检测肝功酶学指标。用显色基质鲎试剂盒检测内毒素浓度。取肝组织1.5cm×1cm×0.5cm,10%甲醛固定,常规病理切片,HE染色,光镜下观察肝组织病理变化。(3)中药组：成人中药用量为1.58g生药／kg／天,根据体表面积公式,大鼠常规用量为10.20g生药／kg／天。中药高剂量组用中药20.40g／kg灌胃,每天1次,连续7天,余实验方法同模型组；中药低剂量组用中药5.10g／kg灌胃,每天1次,连续7天,余实验方法同模型组。(4)正常组：正常组大鼠每天用蒸馏水2ml灌胃,每天1次,连续7天,于末次灌胃1小时后腹腔注射等量蒸馏水,余实验方法同模型组。结果：(1)各组内毒素水平：与模型组比较,中药高剂量组、中药低剂量组的血液内毒素水平降低,差异有统计学意义(P<0.05)。与中药低剂量组比较,中药高剂量组的血液内毒素水平降低,差异有统计学意义(P<0.05)。(2)肝脏生化检查结果：与模型组比较,中药高剂量组、中药低剂量组的血清谷丙转氨酶(ALT)、谷草转氨酶水平(AST)降低,差异有统计学意义(P<0.05)。与中药低剂量组比较,中药高剂量组的ALT、AST,差异有统计学意义(P<0.05)。(3)蛋白质芯片共捕获到115个差异表达蛋白峰,其中,11个差异表达蛋白峰有统计学意义(P<0.05)。相对分子质量为4200道尔顿的蛋白峰在中药干预组的血清中高表达；相对分子质量为8984道尔顿、9005道尔顿的蛋白峰在中药干预组低表达。(4)光学显微镜下观察：①正常组：肝板纹理清楚,肝细胞排列整齐,索状结构清晰。②模型组：可见炎细胞浸润,核固缩,较多脂肪滴,索状结构不清,肝窦狭窄,肝细胞排列紊乱。③中药高剂量组可见肝板纹理比较清楚,有少量炎细胞。肝细胞排列较整齐。索状结构清晰。④中药低剂量组可见肝板纹理比较清楚,肝细胞肿胀、炎细胞及中央静脉扩张淤血。结论：(1)清热解毒凉血化瘀中药能降低内毒素肝损伤大鼠的内毒素水平。(2)清热解毒凉血化瘀中药能降低内毒素肝损伤大鼠的谷丙转氨酶水平、谷草转氨酶水平。(3)清热解毒凉血化瘀中药能影响内毒素肝损伤大鼠的蛋白质表达,对内毒素肝损伤大鼠的蛋白质表达有一定的干预作用。蛋白峰的变化,可能是清热解毒凉血化瘀中药干预内毒素肝损伤大鼠的重要的分子基础。各组大鼠的血清内确实存在着差异表达蛋白,这可能有助于内毒素肝损伤的进一步研究。
Objective:To investigate the intervention of clearing away heat and removing toxic substances(CAHRTS), cooling blood to dissipate blood stasis (CBDBS) on expression of proteins in rats with endotoxemia and liver injury.Methods:(1) Forty SD rats were randomly divided into four groups:Normal group, Model group, High dose of traditional chinese medicine (TCM) group, Low dose of traditional chinese medicine (TCM) group. (2) Model group:Model group rats were administered with distilled water 2ml by gastric perfusion continue for 7 days,once daily. One hour after the last gastric perfusion, rats were injected with LPS (10mg/Kg) into its abdominal cavity. Except drinking water freely, rats must fast for more than 12 hours before intraperitoneal injection and blood sampling. Six hours after the intraperitoneal injection, rats were injected with 1% pentobarbital sodium (30mg/Kg) into its abdominal cavity. Anesthetic rats were sterilized with alcohol on the skin, opened chest and sampled blood from hearts. All of the blood was stored in 4℃refrigerator. The serum differential protein expression profiling was detected by SELDI-TOF-MS technology; Liver function enzyme index test was detected by automated hematology analyzer.Serum Endotoxin concentrations was measured by Quantitative Chromogenic Tachypleus Amebocyte Lysate.10% formalin fixed liver tissues (1.5cm×1cm×0.5cm), HE staining routine pathological section were used to observe the pathological changes of liver tissues under light microscope. (3) TCM group:Adult dosage of TCM is 1.58g/crude drug/Kg/per day. Therefore, the conventional dosage of rats are 10.20g/crude drug/Kg/per day which could be obtained by the equation of body surface area. High dose of TCM group were administered with 20.40g/Kg by gastric perfusion continue for 7 days,once daily. Low dose of TCM group were administered with 5.10g/Kg by gastric perfusion continue for 7 days,once daily. The rest of the experimental method was the same as the model group's.(4)Normal group:Normal group rats were administered with distilled water 2ml by gastric perfusion continue for 7 days,once daily. One hour after the last gastric perfusion, rats were injected with the same dose of distilled water into its abdominal cavity. The rest of the experimental method was the same as the model group's. Results:(1) Each group's serum Endotoxin level:compared with model group, the serum Endotoxin level was decreased in the high dose of TCM group and low dose of TCM group, the differences were statistical significance (P<0.05). Compared with low dose of TCM group, the serum Endotoxin level was decreased in the high dose of TCM group, the differences were statistical significance (P＜0.05). (2) Liver biochemistry test showed:compared with model group, the levels of serum ALT、AST were decreased in the high dose of TCM group and low dose of TCM group, the differences were statistical significance(P<0.05). Compared with low dose of TCM group, the levels of serum ALI、AST were decreased in the high dose of TCM group. (3) 115 differently expressed protein peaks were found by protein chip, among which 11 differently expressed protein peaks had statistical significance (P<0.05).Protein 4200Da was high expression in TCM group, protein 8984Da and 9005Da was low expression in TCM group. (4) Observed under optical microscope:①Normal group could be seen clean mark of hepatic plates.Liver.cell sarranged orderliness.Cord structure was clear.②Model group:Inflammatory cell、karyopyknosis and a lot of lipid droplets could be observed.Cord structure was not clear.Hepatic sinus was narrow. Liver cell sarranged disorderliness.③High dose of TCM group could be seen relatively clean mark of hepatic plates and a tiny amount of inflammatory cell.Liver cell sarranged relatively orderliness.Cord structure was clear.④Low dose of TCM group could be seen relatively clean mark of hepatic plates、oncotic liver cells、inflammatory celland congestion around the central veins. Conclusions: (1) The TCM of CAHRTS and CBDBS can reduce the serum Endotoxin level in rats with endotoxemia and liver injury. (2) The TCM of CAHRTS and CBDBS can reduce the levels of ALT、AST in rats with endotoxemia and liver injury. (3) The TCM of CAHRTS and CBDBS have certain intervention role to expression of proteins, which have an effect on expression of proteins in rats with endotoxemia and liver injury. The change of protein peaks may be a significant molecular basis which cause the TCM of CAHRTS and CBDBS intervene rats with endotoxemia and liver injury.The serum assuredly has differential expression protein in rats among groups, which may be of use for farther study.
Objective:To investigate the intervention of clearing away heat and removing toxic substances(CAHRTS), cooling blood to dissipate blood stasis (CBDBS) on expression of proteins in rats with endotoxemia and liver injury.Methods:(1) Forty SD rats were randomly divided into four groups:Normal group, Model group, High dose of traditional chinese medicine (TCM) group, Low dose of traditional chinese medicine (TCM) group. (2) Model group:Model group rats were administered with distilled water 2ml by gastric perfusion continue for 7 days,once daily. One hour after the last gastric perfusion, rats were injected with LPS (10mg/Kg) into its abdominal cavity. Except drinking water freely, rats must fast for more than 12 hours before intraperitoneal injection and blood sampling. Six hours after the intraperitoneal injection, rats were injected with 1% pentobarbital sodium (30mg/Kg) into its abdominal cavity. Anesthetic rats were sterilized with alcohol on the skin, opened chest and sampled blood from hearts. All of the blood was stored in 4℃refrigerator. The serum differential protein expression profiling was detected by SELDI-TOF-MS technology; Liver function enzyme index test was detected by automated hematology analyzer.Serum Endotoxin concentrations was measured by Quantitative Chromogenic Tachypleus Amebocyte Lysate.10% formalin fixed liver tissues (1.5cm×1cm×0.5cm), HE staining routine pathological section were used to observe the pathological changes of liver tissues under light microscope. (3) TCM group:Adult dosage of TCM is 1.58g/crude drug/Kg/per day. Therefore, the conventional dosage of rats are 10.20g/crude drug/Kg/per day which could be obtained by the equation of body surface area. High dose of TCM group were administered with 20.40g/Kg by gastric perfusion continue for 7 days,once daily. Low dose of TCM group were administered with 5.10g/Kg by gastric perfusion continue for 7 days,once daily. The rest of the experimental method was the same as the model group's.(4)Normal group:Normal group rats were administered with distilled water 2ml by gastric perfusion continue for 7 days,once daily. One hour after the last gastric perfusion, rats were injected with the same dose of distilled water into its abdominal cavity. The rest of the experimental method was the same as the model group's. Results:(1) Each group's serum Endotoxin level:compared with model group, the serum Endotoxin level was decreased in the high dose of TCM group and low dose of TCM group, the differences were statistical significance (P<0.05). Compared with low dose of TCM group, the serum Endotoxin level was decreased in the high dose of TCM group, the differences were statistical significance (P＜0.05). (2) Liver biochemistry test showed:compared with model group, the levels of serum ALT、AST were decreased in the high dose of TCM group and low dose of TCM group, the differences were statistical significance(P<0.05). Compared with low dose of TCM group, the levels of serum ALI、AST were decreased in the high dose of TCM group. (3) 115 differently expressed protein peaks were found by protein chip, among which 11 differently expressed protein peaks had statistical significance (P<0.05).Protein 4200Da was high expression in TCM group, protein 8984Da and 9005Da was low expression in TCM group. (4) Observed under optical microscope:①Normal group could be seen clean mark of hepatic plates.Liver.cell sarranged orderliness.Cord structure was clear.②Model group:Inflammatory cell、karyopyknosis and a lot of lipid droplets could be observed.Cord structure was not clear.Hepatic sinus was narrow. Liver cell sarranged disorderliness.③High dose of TCM group could be seen relatively clean mark of hepatic plates and a tiny amount of inflammatory cell.Liver cell sarranged relatively orderliness.Cord structure was clear.④Low dose of TCM group could be seen relatively clean mark of hepatic plates、oncotic liver cells、inflammatory celland congestion around the central veins. Conclusions: (1) The TCM of CAHRTS and CBDBS can reduce the serum Endotoxin level in rats with endotoxemia and liver injury. (2) The TCM of CAHRTS and CBDBS can reduce the levels of ALT、AST in rats with endotoxemia and liver injury. (3) The TCM of CAHRTS and CBDBS have certain intervention role to expression of proteins, which have an effect on expression of proteins in rats with endotoxemia and liver injury. The change of protein peaks may be a significant molecular basis which cause the TCM of CAHRTS and CBDBS intervene rats with endotoxemia and liver injury.The serum assuredly has differential expression protein in rats among groups, which may be of use for farther study.
引文
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5.汪承柏.重视慢性肝炎内毒素血症的防治[J].中西医结合肝病杂志,2001；11(4)：193-194
6.邓文龙.中药的抗内毒素作用研究进展[J].中药药理与临床, 1998；3(4)：58
7.王连江,冯乘安,陈佳宁,等.IL-18在内毒素诱导大鼠肝损中的变化及中药腑安的作用[J].中国中西医结合外科杂志,2001；7(5)：331-333
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10.丁荣华,王冬梅,钱俊芳.清热解毒凉血活血治疗慢性乙型肝炎重度黄疸30例[J].中国中西医结合杂志,2006；26(1)：84-85
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12.周鑫,韩德五,尹镭.从微循环角度探讨丹参素抗急性重型肝损伤的机制[J].山西医药杂志,2006；35(6)：507-508
13.欧阳钦,曹家麟.加减石灰散对亚急性重型肝炎内毒素血症的影响[J].江西中医药,2006；5(37)：24-25
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18.王莉,包旭,岑小波,等.巴巴多斯芦荟多糖对内毒素化大鼠和正常高龄大鼠血清及肝组织过氧化脂质的影响[J].华西药学杂志,2002；17(1)：21-22
19.毕铭华,张淑文,王宝恩,等.内毒素血症对大鼠肝细胞线粒体质子ATP酶的影响及中药912液的干预作用[J].中国危重病急救医学,2001；13(9)：533-535.
20.李琴,赵龙凤,韩德五.肠源性内毒素血症对急性肝损伤大鼠细胞免疫功能的影响[J].临床和实验医学杂志,2002；1(4)：220-221
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22.宋仕玲,黄团新,龚作炯,等.茵栀清肝汤对急性肝损伤大鼠细胞因子IL-6和IL-19的影响[J].中国中医基础医学杂志,2004；10(10)：12-15
23.吴建成,王泽荣,万千红,等.中药清热方及IL-18抗体抗急性免疫性肝坏死动物模型的实验性研究[J].苏州大学学报(医学版),2007；27(2)：203-206
24.胡岚,张淑文,阴赪宏.内毒素血症大鼠肝损伤及中药912液干预的研究[J].中国中西医结合杂志,2007；27(6)：523-525
25.高建蓉.50例重型肝炎的中药治疗疗效分析[J].中华肝脏病杂志,2004；12(10)：635