【作者】 魏翠萍；

【导师】 张一昕；

【作者基本信息】 河北医科大学 ， 中西医结合临床， 2008， 硕士

【摘要】 目的:消瘀化痰饮(由丹参、郁金、泽泻、制半夏、柴胡、决明子、生黄芪、炒白术等药物组成)具有消瘀化痰、疏肝健脾功效,为导师经临床观察筛选的效方,对非酒精性脂肪肝(NAFLD)具有较好的治疗效果,为了进一步探讨其作用机理,参照相关文献运用高脂饮食复制NAFLD大鼠模型,同时施加药物干预,观察了其对NAFLD大鼠脂质代谢、肝功能的影响,探讨了其促进脂质代谢、保护肝功能的作用机制;运用免疫组化和逆转录聚合酶链反应(RT-PCR)方法观察了其对实验大鼠肝组织解偶联蛋白2(UCP2)表达的影响,探讨了其抑制UCP2过度表达的作用机制;并运用光镜和电镜观察了本方对肝组织形态学的影响。方法第一部分消瘀化痰饮对NAFLD大鼠脂质代谢和肝组织形态学的影响选用SD大鼠50只,体重160～180g,雄性。大鼠自由饮水进食,饲养于18℃～22℃明暗各24小时的清洁级动物实验室内。正常喂养1周后,随机分为5组:正常对照组(简称正常组,normal control group,N)、模型对照组(简称模型组,model group,M)、消瘀化痰饮高剂量组(简称高剂量组,high-dose Xiao-Yu-Hua-Tan-Yin group,H)、消瘀化痰饮低剂量组(简称低剂量组,low-dose Xiao-Yu-Hua-Tan-Yingroup,L)、阳性药(东宝甘泰)对照组(简称对照组,Dong-Bao-Gan-Tai control group,D)。除正常组喂饲普通饲料外,其余各组均喂饲高脂饲料。同时各用药组灌服治疗药或对照药,正常组与模型组灌服等量生理盐水,每天上午灌胃1次,每次用药体积均按1ml/100g计算,持续灌胃8周。9周末,于最后1次给药后禁食12h麻醉状态下股动脉取血,将血样低温离心,分离血清,密封,-20℃冷贮备用。于肝脏最大叶距边缘0.5cm处取0.1×0.1×0.1cm3肝组织浸泡于4%戊二醛中固定,以备电镜观察。另取少许肝组织液氮冷冻以备检测肝匀浆TG、TC ,然后取相同部位肝组织0.5×0.5×0.5cm3置于4%多聚甲醛液中固定,HE染色,以备光镜观察。第二部分消瘀化痰饮对NAFLD大鼠肝组织UCP2表达的影响实验动物分组、用药情况同前。连续喂养9周,确定脂肪肝形成后,于最后1次给药禁食12h后麻醉动物,剖取肝脏,在肝脏最大叶距边缘0.5cm处取适量肝组织置于液氮中冷冻,以备运用RT-PCR法观察肝组织UCP2mRNA的表达变化;再取相同部位0.5cm×0.5cm×0.5cm3大小的肝组织于4%多聚甲醛中固定,以备运用免疫组化法观察肝组织UCP2的表达变化;另取相同部位适量肝组织,制作肝组织匀浆测定ATP的含量。结果第一部分消瘀化痰饮对NAFLD大鼠脂质代谢和肝组织形态学的影响1消瘀化痰饮对NAFLD大鼠血清TG、TC、FFA含量的影响模型组大鼠血清TG、TC、FFA的含量(1.17±0.096、3.27±0.18、480.475±18.94)明显高于正常组(0.56±0.084、1.45±0.20、225.71±15.98)(P<0.01)。经用药干预,各用药组均能显著降低模型大鼠血清TC、TG、FFA含量,与模型组比较均具有显著性差异(P<0.01)。其中高剂量组大鼠血清TG、TC、FFA含量(0.52±0.065、1.44±0.11、221.43±15.32)和低剂量组(0.57±0.32、1.56±0.63、241.42±16.96)较对照组(0.76±0.07、2.06±0.28、331.90±16.32)均明显降低(P<0.01),而高、低剂量组之间经统计学处理无明显差异(P>0.05)。2消瘀化痰饮对NAFLD大鼠AST、ALT活性的影响模型组大鼠血清AST、ALT的活性(32.37±2.45、15.84±1.01)明显高于正常组(26.59±2.33、10.89±0.86)(P<0.01)。经用药干预,各用药组大鼠血清AST、ALT的活性均有明显的下降,与模型组比较,差异有显著性(P<0.01)。其中低剂量组AST的活性(19.45±1.90)较对照组(23.19±1.78)低(P<0.05),高剂量组ALT活性(4.16±0.89)高于对照组(7.32±0.45)(P<0.05);而高、低剂量组之间AST、ALT的活性经统计学处理无明显差异(P>0.05)。3消瘀化痰饮对NAFLD大鼠肝脏TG、TC的影响模型组大鼠肝脏TG、TC的含量(0.82±0.08、0.44±0.07)明显高于正常组(0.45±0.05、0.27±0.04)(P<0.01)。经用药干预,各用药组均能显著降低模型大鼠肝组织TG、TC的含量(P<0.01)。其中高剂量组TC的含量(0.27±0.04)低于对照组(0.34±0.05)(P<0.05),而高、低剂量组TC的含量经统计学处理无明显差异(P>0.05);各用药组TG的含量经统计学处理无明显差异(P>0.05)。4肝组织形态学改变光镜可见:正常组大鼠肝组织结构完整、清晰,肝小叶结构正常,中央静脉大而壁薄,肝细胞排列成肝索,在中央静脉周围呈放射状分布,细胞呈多边形。模型组大鼠肝细胞中度细胞水肿,少量的肝细胞坏死,多数肝细胞内可见大小不等、数量不一的脂滴空泡(脂肪变性),重度变性者,脂滴空泡融合,呈现中至重度脂肪变性,但未见明显的纤维化改变。各用药组动物肝小叶和肝血窦结构清晰,高、低剂量组肝细胞内脂滴空泡基本消失,对照组中少量的肝细胞内仍有脂滴空泡,并有轻度的细胞水肿。透射电镜可见:正常组大鼠肝脏细胞质中线粒体、粗面内质网等基本正常;模型组细胞质内充满了大、小不同的脂滴,线粒体全部脊融合、消失;粗面内质网有轻度脱颗粒现象。对照组细胞胞浆内可见大、小不等脂滴,线粒体肿胀、内质网有部分断裂现象。高、低剂量组上述情况明显改善,肝细胞胞浆有少量脂滴,线粒体形态基本正常,脊数目明显增多,仅部分可见轻度肿胀变形,内质网基本正常。第二部分消瘀化痰饮对NAFLD大鼠肝组织UCP2表达的影响1各组大鼠肝组织ATP含量的变化模型组大鼠肝组织ATP的含量(2.78±0.16)明显低于正常组(4.66±0.12)(P<0.01)。经用药干预后,各用药组大鼠ATP的含量均高于模型组(P<0.01)。其中,高、低剂量组大鼠肝组织ATP的含量(4.57±0.13、4.39±0.19)明显高于对照组(4.24±0.16)(P<0.01);高剂量组大鼠肝组织ATP的含量高于低剂量组(P<0.01)。2各组大鼠肝组织UCP2表达的变化半定量分析显示:模型组大鼠肝组织UCP2表达的平均灰度(0.0883±0.0079)明显低于正常组(0.2170±0.0106)(P<0.01)。经用药干预后,各用药组大鼠肝组织的平均灰度均高于模型组(P<0.01)。其中,高、低剂量组平均灰度(0.1627±0.0130、0.1310±0.0046)高于对照组(0.1103±0.0064)(P<0.01);高剂量组平均灰度高于低剂量组(P<0.01)。3各组大鼠肝组织UCP2mRNA表达的变化RT-PCR后,琼脂糖凝胶电泳,在479bp、587bp处分别显示出UCP2mRNA和β-actin电泳带,与预期结果一致。模型组大鼠肝组织UCP2mRNA的表达(0.4065±0.0281)显著高于正常组(0.0944±0.0102)(P<0.01)。给药后,各用药组UCP2mRNA的表达显著低于模型组(P<0.01),其中,高、低剂量组UCP2mRNA的表达(0.1846±0.01301、0.1790±0.0134)低于对照组的表达(0.2669±0.0208)(P<0.01),而高、低剂量组之间无显著性差异(P>0.05)。结论1消瘀化痰饮能显著降低NAFLD大鼠血清和肝组织内异常升高的TC、TG、FFA的含量,抑制血清内ALT和AST活性的异常升高,呈现出良好的调脂护肝作用。2消瘀化痰饮可明显改善NAFLD大鼠肝组织的病变程度,表现为用药后大鼠肝小叶和肝血窦结构清晰,肝细胞内脂滴空泡基本消失;线粒体形态基本正常,脊数目明显增多。3消瘀化痰饮能抑制NAFLD大鼠肝组织UCP2的过度表达,增加ATP的合成和线粒体的能量贮备,促进脂肪酸代谢,以达到治疗NAFLD的目的。更多还原

【Abstract】 Objective: Xiao-Yu-Hua-Tan-Yin (It was composed of danshen, yujin, zexie, zhi-banxia, chaihu, juemingzi, sheng- huangqi, chao-baizhu medicine etc.) has the function of regulating liver and removing stasis and phlegm, strengthening spleen. And it has been proved significantly effective in treating nonalcoholic fatty liver disease (NAFLD) through years of clinical observation. In order to futher probe its action mechanism, we duplicated the NAFLD animal model with high- fat forage. At the same time, the animal was interfered with medicines. And to explore the action mechanism of this formula in regulating lipid metabolism, protecting hepatic function to observe its effect on lipid metabolism. The expression of uncoupling protein2 (UCP2) in the hepatocytes was examined by immunohistochemistry and reverse transcript-polymerase chain reaction (RT-PCR). To observe Xiao-Yu-Hua-Tan-Yin restraining the overexpression of UCP2 in the hepatocytes. Futhermore, we made observation with light microscope and transmission electronic microscope (TEM) on effect of this formula on pathomorphology of hepatic tissue. MethodsPart I Effect of Xiao-Yu-Hua-Tan-Yin on lipid metabo- lism and pathomorphology of NAFLD RatSelect male SD rats 50, weight from 160g to 180g. Rats drunk water and took food freely, and were raised in sanitary experiment lab that temperature was 18℃～22℃, bright and dim alternate every 12 hours. After 1 week, they were divided into 5 groups at random: Normal control group, Model control group, High-dose of Xiao-Yu-Hua-Tan-Yin group, Low-dose of Xiao-Yu-Hua-Tan-Yin group, Dong-Bao-Gan-Tai control group. Rats except for normal control group are treated as well as set up models. Except for the ordinary forage in normal group, the rest of groups were all fed with high-fat forage. Meanwhile treatment drug or control drug was given in each treatment group and Saline was given to normal and model group, which lasted for 8 weeks of stomach-perfusion. Rats in each group were given stomach-perfusion once a day and every time the drug volume was calculated by 1ml/100g. Confirmed the formation of fatty liver, then 12 hours after fasting of the last time, the rats were anaesthetized and their blood were drawn out, the blood sample was made centrifugation with low temperature and the blood serum was isolated. Then unfold abdominal cavity quickly to get liver, finally the contents of TC, TG and FFA were measured with enzyme method; the contents of AST and ALT were measured with Lai’s method. In addition, to obtain 0.1×0.1×0.1cm3 hepatic tissue at the 0.5cm borderline of the max leaf of the liver, soak in 4% Glutaric Dialdehyde, affix to provide for the observation of the light microscope. To obtain 0.5×0.5×0.5cm3 hepatic tissue at the same genetic locus, soak in 4% paraform, affix, paraffin imbedding, microtome section, HE dyeing to provide for the observation of TEM. In the end, the rest of the max leaf of the liver was made into 10% liver tissue homogenate and then the supernatant fluid was drawn out for measurement of contents of TC, TG with color compared method.Part II Effect of Xiao-Yu-Hua-Tan-Yin on hepatic tissue UCP2 of NAFLD RatThe group distribution and drug-given were the same as above. At the 9th week since the experiment formally started, when confirmed the formation of fatty liver, the rats were killed 12 hours after fasting of the last time. The rats were anaesthe- tized, then unfold abdominal cavity quickly to get liver to obtain a little hepatic tissue at the 0.5cm borderline of the max leaf of the liver, liquid nitrogen frozen, to detect the expression of UCP2mRNA in liver through ways of RT-PCR. To obtain 0.5×0.5×0.5cm3 hepatic tissue at the same genetic locus, soak in 4% paraform, to detect the expression of UCP2 in liver through ways of immunohistochemistry. Another from the same location suitable hepatic tissue, liver tissue production of ATP content.ResultsPart I Effect of Xiao-Yu-Hua-Tan-Yin on lipid metabo- lism and pathomorphology of NAFLD Rat 1 Effect of Xiao-Yu-Hua-Tan-Yin on contents of TC, TG, FFA of Nonalchoholic-fatty liver disease ratThe contents of TG, TC, FFA (1.17±0.096, 3.27±0.18, 480.475±18.94) in serum of model rats were significantly increased than normal group (0.56±0.084, 1.45±0.20, 225.71±15.98) (P < 0.01), which showed that NAFLD rats had significant blood-fat metabolic disorder. After the intervention of medicine, the contents of TG, TC, FFA in serum of each treatment group were significantly reduced. There was signifi- cant difference between treatment group and model group (P<0.01). And the contents of TG, TC, FFA in high-dose group (0.52±0.065, 1.44±0.11, 221.43±15.32) and low-dose group (0.57±0.059, 1.56±0.13, 241.42±16.96) were obviously reduced than the control group (0.76±0.070, 2.06±0.28, 331.90±16.32) (P<0.01). There was no marked difference between the high- dose and low-dose group (P>0.05).2 Effect of Xiao-Yu-Hua-Tan-Yin on contents of ALT and AST in serum of NAFLD ratThe contents of AST and ALT in the rats’serum of model group (32.37±2.45, 15.84±1.01) was markedly increased than the normal group (26.59±2.33, 10.89±0.86) (P<0.01). After the intervention of medicine, the contents of AST and ALT in the rats’serum of each treatment group were all obviously reduced, and had significant difference with model group (P<0.01). The AST contents in the rats’serum of low-dose group (19.45±1.90) was obviously reduced than the control group (23.19±1.78) (P< 0.05); The ALT contents in the rats’serum of High-dose group (4.16±0.89) was obviously reduced than the control group (7.32±0.45) (P<0.05); There was no marked difference between the high-dose and low-dose group (P>0.05).3 Effect of Xiao-Yu-Hua-Tan-Yin on contents of TC, TG in liver tissue of NAFLD ratThe contents of TG and TC in liver tissue of model rats (0.82±0.08, 0.44±0.07) were obviously increased than normal group (0.45±0.05, 0.27±0.04) (P<0.01), which showed that NAFLD rats had significant blood-fat metabolic disorder in liver tissue. After the intervention of medicine, low-dose group, high-dose group and positive drug control group could all significantly reduced the contents of TC, TG in liver tissue and all had significant difference with model group (P<0.01). And the contents of TC in liver tissue of high-dose group (0.27±0.04) were obviously reduced than control group (0.34±0.05) (P<0.05); There was no marked difference between the high-dose and low-dose group at the contents of TC, TG (P>0.05).4 Effect of Xiao-Yu-Hua-Tan-Yin on hepatic tissue morpho- logy of NAFLD ratIt was clear that by light microscope: in the normal group, constitution of hepatic tissue was clear and integrity, consti- tution of hepatic lobules was normal, central veins was big and had thin paries. Hepatic cells were polygon which were arranged hepatic cord, which were distributed as radial around central veins. As for the rats in model group, they have unclear limit of hepatic lobules and disordered arrangement of hepatic cord; major part of the hepatic sinus disappeared; majority of the hepatic cells appeared severe fatty degeneration, especially in the area around the lobules; hepatic cells became extremely swollen and formed round shape; lipid droplet vacuoles (fatty degeneration) in different sizes and numbers was visible inside the cytoplasm, and sometimes the lipid droplets merged together and squeezed the nucleus to the cell side, which made it look very similar with fatty cells; the rest of the hepatic cells had focal necrosis and hepatic interstitials had no fibrosis changes; While as for the rats in high-dose and low-dose group, they had very clear structure of hepatic lobules and sinuses; the cells arranged orderly; lipid droplet vacuoles basically disappeared; the morphology and structure of nucleus became normal and regenerated hepatic cells were visible; and as for the rats in the control group, they had clear structure of hepatic lobules; hepatic sinus became mildly expanded; fatty degeneration of hepatic cells became mild; scanty lipid droplet vacuoles were still visible; the structure of nucleus was comparatively clear. Compared with the model group, the degree of fatty dege- neration in each drug group got significant improvement, but the high and low-dose group were superior to the control group.It was clear that by TEM: as for the rats in normal group, they had basically normal chondrosom and rough endoplasmic reticulum. In the model group, lipid droplet vacuoles (fatty degeneration) in different sizes and numbers was visible inside the cytoplasm, all lophos of chondrosome confluence or disappeared. Rough endoplasmic reticulum had degranulation. Intracytoplasm were full of big or small lipoids droplet, chondrosome tumescenced, part of rough endoplasmic reticulum were disrupted. As is mentioned above between the high-dose and the low-dose group obviously improved, endochylema had small amounts lipids droplet, chondrosome was normal, the number of lophos were mult, only portion of which lightly tumescenced or deformed, rough endoplasmic reticulum were basically normal.Part II Effect of Xiao-Yu-Hua-Tan-Yin on expressing UCP2 of NAFLD Rat1 Effect of every group on hepatic tissue ATP of NAFLD rat The contents of ATP in hepatic tissue of model rats (2.78±0.16) were obviously increased than normal group (4.66±0.12) (P<0.01); After the intervention of medicine, high-dose group, low-dose group and positive drug control group, could all significantly reduced the contents of ATP in hepatic tissue and all had significant difference with model group (P<0.01). And the contents of ATP in hepatic tissue of high-dose group and low-dose group (4.57±0.13, 4.39±0.19) were obviously reduced than control group (4.24±0.16) (P<0.01); there was marked difference between the high-dose and low-dose group at the contents of ATP (P<0.01).2 Effect of every group on hepatic tissue gray scale of UCP2 of NAFLD rat by immunohistochemistry The contents of gray scale of UCP2 on hepatic tissue of model rats (0.0883±0.0079) were obviously reduced than normal group (0.2170±0.0106) (P < 0.01); after the intervention of medicine, high-dose group, low-dose group and positive drug control group (0.1627±0.0130, 0.1310±0.0046, 0.1103±0.0064) could all significantly increased the contents of gray scale of UCP2 in hepatic tissue and all had significant difference with model group (P<0.01). And the contents of gray scale of UCP2 on hepatic tissue of high-dose and low-dose group were obviously increased than control group (P<0.01); there was marked difference between the high-dose and low- dose group at the contents of gray scale of UCP2 (P<0.01).3 Effect of every group on hepatic tissue UCP2mRNA of NAFLD ratWith RT-PCR, electrophoresis band of UCP2mRNA andβ-actin were showed respectively at 479bp and 587bp, which were coincident with the respected result. The contents of UCP2mRNA in hepatic tissue of model rats (0.4065±0.0281) were obviously increased than normal group (0.0944±0.0102) (P<0.01); After the intervention of medicine, high-dose group, low-dose group and positive drug control group could all significantly reduced the contents of UCP2mRNA in hepatic tissue and all had significant difference with model group (P<0.01). And the contents of UCP2mRNA in hepatic tissue of high-dose and low-dose group (0.1846±0.0130, 0.1790±0.0134) were obviously reduced than control group (0.2669±0.0208) (P <0.01); There was no marked difference between the high-dose and low-dose group at the contents of UCP2mRNA (P>0.05).Conclusions1 Xiao-Yu-Hua-Tan-Yin could significangtly reduce the content- s of TC, TG, FFA in model group, restrain the activity of ALT, AST in serum. It had marked function in regulating blood fat and protecting liver.2 Xiao-Yu-Hua-Tan-Yin could obviously improve the hepatic tissue lesion degree of NAFLD, it manifested like this: after the drug was given, the hepatic lobule and sinus got clear structure; the cells arranged orderly, no obvious lipid droplet vacuoles inside the hepatic cells; only scanty hepatic cells appeared mild swollen, chondrosom were basically normal, and the amounts of lophos were increased.3 Xiao-Yu-Hua-Tan-Yin which could promote conveying of lipid and oxygenolysis of fat, could refrain the overexpression of UCP2 in hepatic tissue. At the same time it could increase ATP synthesis and energy storage of mitochondria, promote metabolism of fatty acid. In a word, it has the function of treating NAFLD.更多还原