摘要
目的:观察中药柴胡、丹参复方治疗非酒精性脂肪肝大鼠血脂、脂质过氧化系统、肝功能、过氧化物酶体增殖物激活受体γ蛋白和mRNA表达、肝组织病理变化及炎症反应的影响,分析影响非酒精性脂肪肝大鼠过氧化物酶体增殖物激活受体γmRNA表达相关因素,了解柴胡、丹参复方治疗非酒精性脂肪肝的作用途径。方法:实验于2004-09/2005-03在大连医科大学附属第二医院中心实验室完成。①选用雄性SD大鼠30只,体质量(130±10)g。正常喂养1周后,按随机抽签法将大鼠分为3组:正常组、模型组、柴胡、丹参复方组,每组10只。正常组:普通饲料喂养;模型组和柴胡、丹参复方组:喂高脂饲料(普通饲料加质量分数0.1猪油、质量分数0.02胆固醇)造成非酒精性脂肪肝模型。②造模开始后第13周,正常组和模型组:灌胃生理盐水10mL/kg,1次/d;柴胡、丹参复方组:灌胃柴胡、丹参复方溶液10mL/kg(由柴胡15g,丹参15g,泽泻15g,半夏15g,生山楂15g组成,药材购自大连药材集团公司,水煎剂制成浓浸膏,加蒸馏水稀释成含生药0.17g/mL灌胃溶液),1次/d。③实验第16周末处死大鼠。用全自动生化分析仪检测血清丙氨酸氨基转移酶、天冬氨酸氨基转移酶活力和总胆固醇、三酰甘油水平。采用放免法测定血清肿瘤坏死因子α水平。按照试剂盒说明测定肝组织匀浆总胆固醇、三酰甘油、丙二醛含量和超氧化物歧化酶活力。④苏木精-伊红染色,光镜下评估脂肪变性程度(判断标准:以肝小叶内未见脂滴肝细胞为阴性,脂滴肝细胞占肝细胞总数<1/3为+,1/3～2/3为,>2/3为,几乎均呈脂滴肝细胞为)和计算炎症活动度计分[计分标准为汇管区炎症(P)、小叶内炎症(L)、碎屑坏死(PN)及桥接坏死(BN)4项指标,每项依据病变程度分别计为1,2,3,4分,计分公式为:P+L+2(PN+BN)]。⑤采用免疫组织化学染色检测肝组织过氧化物酶体增殖物激活受体γ的表达。计算每张切片阳性细胞所占百分率进行分级:0为-,<10%为+,10%～50%为,>50%为。⑥采用蛋白质免疫印迹法检测肝组织过氧化物酶体增殖物激活受体γ蛋白表达。⑦采用反转录聚合酶链反应检测肝组织过氧化物酶体增殖物激活受体γmRNA表达。⑧两组间实验数据的比较采用成组设计的两样本均数t检验;等级资料采用秩和检验;两变量间相关性分析采用直线相关分析法。结果:大鼠30只均进入结果分析。①柴胡、丹参复方组肝脂肪变性程度明显轻于模型组(P<0.01)。正常组基本无炎症细胞浸润;模型组肝小叶内出现汇管区炎症,炎症活动度计分明显高于正常组(P<0.01);柴胡、丹参复方组炎症活动明显轻于模型组(P<0.01)。②模型组血清丙氨酸氨基转移酶、天冬氨酸氨基转移酶活力和总胆固醇、肿瘤坏死因子α水平明显高于其他2组(P<0.01)。③模型组大鼠肝匀浆总胆固醇、三酰甘油、丙二醛含量明显高于其他2组(P<0.01),超氧化物歧化酶活力明显低于其他2组(P<0.01)。④正常组见棕黄色颗粒主要分布在汇管区周围肝细胞胞核内,中央静脉周围肝细胞阳性表达少;模型组过氧化物酶体增殖物激活受体γ阳性表达细胞明显少于其他2组(P<0.05～0.01)。⑤模型组大鼠肝组织过氧化物酶体增殖物激活受体γ的表达明显低于正常组和柴胡、丹参复方组(P<0.01)。⑥模型组肝组织过氧化物酶体增殖物激活受体γmRNA表达与血清肿瘤坏死因子α和肝匀浆三酰甘油、丙二醛含量呈显著负相关(-0.917,-0.727,-0.763,P<0.05～0.01),与肝匀浆超氧化物歧化酶活力呈显著正相关(r=0.859,P<0.05)。结论:①柴胡、丹参复方可使肝组织过氧化物酶体增殖物激活受体γ的表达增强,能显著降低肝脏内三酰甘油储存、减轻脂质过氧化反应,使肝细胞脂肪变性及炎症程度显著减轻。②柴胡、丹参复方可通过增强核转录因子过氧化物酶体增殖物激活受体γ的表达,抑制非酒精性脂肪肝大鼠肝组织脂质过氧化反应和炎症介质的释放,有效逆转高脂饮食造成的肝损伤。
AIM： To observe the effects and mechanism of Chinese tborowax root and compound dan-shen ropt in treating rats with nonalcoholic fatty liver （NAFL） disease in aspects of blood lipid, lipid peroxidation system, hepatic function, expressions of poroxiseme proliferator-activated receptor gamma （PPARγ） protein and mRNA, pathological change of liver tissues and inflammatory reaction, and to analyze the influencing factors of mRNA expression.
METHODS： The experiment was performed between September 2004 and March 2005 in the Central Laboratory of the Second Affiliated Hospital of Dalian Medical University,①Thirty male SD rats of （130±10） g weight were divided into three groups randomly with 10 in each group： Control group were fed with normal diet; Model group and treatment group were fed with high-rich diet （normal diet addition of 0,1 lard oil and 0.02 cholesterol in mass fraction） to establish NAFL models. ②From the 13^th week of model establishment, the rats in normal group and model group were treated with intragastric infusion of 10 mL/kg saline, once a day; While 10 mL/kg solution （consisted of Chinese thorowax root, dan-shen root, oriental waterplantain rhizome, pinellia tuber and huwtbom fruit of 15 g respectively, which were bought from Dalian Medicinal Materials Group Company, then decoction was applied to prepare strong extract and dilute into solution containing 0.17 g/mL crude drug） given in treatment group, once a day. ③Sixteen weeks later, all the rats were sacrificed to detect levels of serum alanine amlnotransferase （ALT）, aspartate aminotransferase （AST）, total cholesterol （TC） and triacylglycerol fiG） by using automatic biochemistry analyzer. Serum tumor necrosis factor alpha （TNFα） was tested with radioimmunoassay. Kit was used to detect TC, TG, malondialdehyde （MDA） content and superoxide dismutase （SOD） activity in liver homogenate. ④The level of steatosis was evaluated under light microscope after haemutoxylin-eosin （HE） staining （Judging standard： negative if no lipid droplet hepatocyte was present in hepatic lobule. ＋ as lipid droplet hepatoeyte was less than 1/3 of total hepatocyte; ＋＋ as 1/3- 2/3＋＋ as 〉2/3;＋＋＋ as almost all）, and inflammation activity was also scored [4 score codes： portal area inflammation （P）, lobule inflammation （L）, piecemeal necrosis （PN） and bridging necrosis （BN）; scored asl,2,3 and 4 according to lesion degree, with the formula of P＋L＋2 （PN＋BN）].⑤The expressions of PPARγ in liver tissues were detected by immunohistochemical staining. The percentages of positiye cells in each section were recorded and graded： - as no positive cells, ＋ as 〈 10%, ＋＋ as 10%-50%, ＋＋＋ as 〉 50%.⑥Furthermore, Western blotting was applied to sense PPAR＇y protein expression in liver tissues. ⑦PPARγ mRNA expression were assayed with reverse transcription-polymerase chain reaction （RT-PCR）. ⑧The experimental data between both groups was compared by two-sample average t test, while ranked data was adopted with rank sum test and two-variance correlation was analyzed with linear correlation analysis.
RESULTS： Totally 30＇rats were involved into the result analysis. ①The level of steatosis was notably slighter in treatment group than in model group （P 〈 0.01）. No inflammation cell infiltrated in normal group, while portal inflammation occurred in hepatic lobule of model group, with the higher score of inflammation activity than normal group （P 〈 0,01）; In addition, the inflammation activity was remarkably slighter in treatment group than in model group （P 〈 0.01）.②Compared with normal group and treatment group, the rats in model group showed a higher ratio in the serum levels of TC, ALT, AST, TNFα, MDA and TG content in the liver homogenate; The activity of SOD was decreased significantly （P 〈 0.01）.③ In normal group, buffy particles mainly distributed in periportal hepatocyte nucleus and there was few positive expression in hepatocyte around central vein; The PPARγ positive cells were significantly fewer in model group than in other two groups （P 〈 0,05-0.01）. ④Compared with normal group and treatment group, the expression of PPARγ in liver tissue was obviously decreased in model group（P 〈 0.01）.⑤In model group, the expressions of PPARγ protein and mRNA were negatively associated with serum TNFα, contents of TG and MDA in the liver homogenate （-0.917,-0.727,-0.763,P 〈 0.05-0.01）, but positively with the activity of SOD in the liver homogenate（r=0.859,P 〈 0.05）.
CONCLUSION： ①The Chinese thorowax root and Compound dan-shen root can enhance the expression of PPARγ in liver tissues, strikingly decrease TG deposition and lipid peroxidation as well as relieving steatosis and inflammation level in hepatocyte. ②By increasing the expression of transcription factor PPARγ, the Chinese thorowax root and compound dan-shen root can inhibit the lipid peroxidation and release of inflammation medium in liver tissues of NAFL rats so as to effectively reverse the hepatic injury caused by fat-rich diet,
出处
《中国临床康复》
CSCD
北大核心
2006年第15期114-117,F0003,共5页
Chinese Journal of Clinical Rehabilitation
