【作者】 隋晓丹；

【导师】 刘铁军；

【作者基本信息】 长春中医药大学 ， 中医内科学， 2013， 博士

【摘要】 目的：通过高脂饮食诱导非酒精性脂肪肝大鼠模型，观察中药肝脂溶颗粒对实验性大鼠血清HOMA-IR、Leptin及肝脏组织TNF-α、PPARα表达的影响，探讨肝脂溶颗粒对非酒精性脂肪肝干预的作用机制。方法：将清洁级Wistar大鼠60只，适应环境7天后随机分为2组，正常组9只，高脂组51只。正常组给予普通饲料喂养，高脂组给予高脂饲料喂养。12周后，在正常组中随机选出1只大鼠，在高脂组中随机选出6只大鼠，处死后取肝脏组织进行HE染色。正常组剩余大鼠继续给予普通饲料喂养。高脂组剩余大鼠随机分为5组：模型组、对照组、肝脂溶颗粒高、中、低剂量组，每组9只大鼠。继续高脂饲料喂养同时每日给予不同药物进行灌胃，正常组和模型组给予生理盐水灌胃（lml/100g）；对照组给予多烯磷脂酰胆碱胶囊水溶液灌胃（0.14g/kg）；中药组分别给予不同剂量肝脂溶颗粒水溶液灌胃（3g/kg、2.1g/kg、0.9g/kg）。连续灌胃4周后取材，取肝脏组织行HE染色，观察肝脏病理形态学改变；运用放射免疫分析法检测血清HOMA-IR、酶联免疫吸附测定血清Leptin表达、免疫组织化学染色检测肝脏组织TNF-α表达及PCR技术检测肝脏组织PPARα表达。结果：1肝脂溶颗粒对NAFLD大鼠肝指数的影响高剂量组肝指数显著低于对照组，有显著统计学意义（P<0.05）；中剂量组肝指数与对照组相比，无统计学意义（P>0.05）；低剂量组肝指数高于高、中剂量及对照组，有显著的统计学意义（P<0.05）。2肝脂溶颗粒对NAFLD大鼠肝脏病理形态学的影响模型组大鼠肝脏组织脂肪变程度最高，与正常组相比有显著性差异(p<0.01)；高、中、低剂量组及对照组大鼠肝脏组织的病理改变均有不同程度的改善，肝细胞脂肪变数目和胞浆内脂滴均有不同程度的减少，与模型组相比，差异具有统计学意义(p<0.05)；高剂量组大鼠肝脏组织病理学改善明显优于对照组(p<0.05)；中剂量组大鼠肝脏组织脂肪变性程度与对照组相比，无明显统计学意义(p>0.05)；低剂量组与高、中剂量组及对照组相比，有统计学意义(p<0.05)。3肝脂溶颗粒对NAFLD大鼠血清生化指标及HOMA-IR表达影响的研究3.1血清生化指标模型组和药物干预各组大鼠血清TG、CHOL均高于正常组（P<0.01）；高、中、低剂量组和对照组均低于模型组（P<0.01），有显著性统计学意义；高剂量组明显低于对照组（P<0.05），有显著性统计学意义；中剂量组与对照组相比（P>0.05），无显著统计学意义；低剂量组高于高、中剂量组及对照组（P<0.05），有统计学意义。各组NAFLD大鼠血清ALT、AST较正常组均有显著的升高（P<0.01）；模型组大鼠血清ALT、AST水平最高，与其它药物干预组相比（P<0.01），有显著统计学意义；高剂量组大鼠血清ALT、AST水平低于对照组（P<0.05），有显著统计学意义；中剂量组大鼠血清ALT、AST水平与对照组相比（P>0.05），无统计学意义；低剂量组大鼠血清ALT、AST水平明显高于高、中剂量组及对照组（P<0.01），有统计学意义。说明在改善NAFLD大鼠肝功能（ALT、AST）方面，肝脂溶颗粒效果优于多烯磷脂酰胆碱胶囊。血清游离脂肪酸FFA以正常组最低，与其它各组相比（P<0.01），有显著统计学意义；以模型组最高，与其它药物干预各组相比（P<0.01），有显著统计学意义；高剂量组血清FFA含量明显低于对照组（P<0.05），有显著统计学意义；中剂量组血清FFA含量与对照组相比（P>0.05），无显著统计学意义；低剂量组血清FFA含量均高于高、中剂量组及对照（P<0.01），有统计学意义。3.2血清HOMA-IR表达各组NAFLD大鼠空腹血糖、胰岛素和IR均明显高于正常组（P<0.01），有显著统计学意义；而模型组大鼠空腹血糖、胰岛素和IR最高，明显高于各药物干预组（P<0.01），有显著统计学意义；高剂量大鼠空腹血糖、胰岛素和IR明显低于对照组（P<0.01），有显著统计学意义；中剂量组大鼠空腹血糖、胰岛素和IR低于对照组（P<0.05），有统计学意义；低剂量组大鼠空腹血糖、胰岛素和IR与对照相比较（P>0.05），无统计学意义。4肝脂溶颗粒对NAFLD大鼠血清Leptin表达影响的研究各组NAFLD大鼠血清Leptin的含量均明显高于正常组（P<0.01），有显著统计学意义；模型组大鼠血清Leptin的含量最高，与其它药物干预组相比有显著差异（P<0.01），有统计学意义；高剂量组大鼠血清Leptin的含量明显低于对照组（P<0.05），有统计学意义；中剂量组大鼠血清Leptin的含量与对照组相比较（P＞0.05），无显著统计学意义；低剂量组大鼠血清Leptin的含量与高、中剂量组及对照组相比（P<0.05），有统计学意义。5肝脂溶颗粒对NAFLD大鼠肝脏组织TNF-α表达影响的研究各NAFLD模型组大鼠肝脏组织内TNF-α的表达明显高于正常组（P<0.01），具有显著性统计学意义；模型组大鼠肝脏组织内TNF-α的表达明显高于各药物干预组（P<0.01），具有显著性统计学意义；高剂量组大鼠肝脏组织内TNF-α的表达低于对照组（P<0.05），具有显著性统计学意义；中剂量组大鼠肝脏组织内TNF-α的表达与对照组相比较（P>0.05），无显著性统计学意义；低剂量组大鼠肝脏组织内TNF-α的表达与高、中剂量组及对照组相比较（P<0.05），有统计学意义。6肝脂溶颗粒对NAFLD大鼠肝脏组织PPARα表达影响的研究正常组明显高于其它各组(P<0.01)，有显著统计学意义；模型组明显低于其它各组(P<0.01)，有显著统计学意义；高剂量组最接近正常组，显著高于其它药物干预组(P<0.05)，有显著统计学意义；中剂量组与对照组相比(P>0.05)，无显著统计学意义；低剂量组低于高、中剂量组及对照组(P<0.05)，有显著统计学意义。结论：1肝脂溶颗粒能改善实验性NAFLD大鼠的一般状态，减轻肝内脂质沉积，改善肝脂肪变性程度。2肝脂溶颗粒有一定保护肝功能、降低转氨酶、调节血脂异常、改善胰岛素抵抗，恢复正常糖脂代谢的作用。3肝脂溶颗粒明显降低血清Leptin表达、降低肝脏组织TNF-α表达、增强肝脏组织PPARαmRNA表达。4肝脂溶颗粒通过减轻肝内脂质沉积，改善肝脂肪变性程度；保肝、降酶、降脂，而体现对非酒精性脂肪肝有良好的防治作用。5肝脂溶颗粒通过改善IR、抑制血清Leptin表达、抑制肝脏组织TNF-α表达、增强肝脏组织PPARαmRNA表达，增加胰岛素敏感性，调节脂质代谢，促进脂肪酸氧化分解，减轻肝脏脂肪变性，从而发挥对非酒精性脂肪肝的治疗作用，这可能是肝脂溶颗粒防治非酒精性脂肪肝的机制之一。更多还原

【Abstract】 Purposes：Through the high fat diet-induced non-alcoholic fatty liver rat model,observation on liver lipid soluble granule on experimental rat serum HOMA-IR, Leptin andliver tissue TNF-α, PPAR α expression effects on hepatic lipid soluble, particles ofnon-alcoholic fatty liver intervention mechanism.Methods: Will clean in60Wistar rats, to adapt to the environment after7days wererandomly divided into2groups, the normal group9, high fat group51.Normal group weregiven general feeding, high lipid group were fed high fat diet.12weeks later, in the normalgroup selected randomly from1rats, in hyperlipidemia group selected randomly from6rats,were killed after liver tissues were stained with HE.The normal group the remaining ratscontinued to give ordinary feed.High lipid group remaining rats were randomly divided into5groups: control group, model group, hepatic lipid soluble granule high, low dose group,9ratsin each group.Continue the feeding high-fat diet and daily administration of different drugs togastric lavage, normal group and model group were given saline (lml/100g); the control groupwas given the polyene phosphatidylcholine capsule solution intragastrically (0.14g/kg);traditional Chinese medicine group were treated with different doses of liver fat solubleparticles in aqueous solution (3g/kg,2.1g/kg, gastric perfusion0.9g/kg).Consecutive gavagefor4weeks after operation, the liver tissue stained with HE, were observed in the liverpathological change; by radioimmunoassay in detection of serum HOMA-IR, enzyme linkedimmunosorbent assay of Leptin expression, immunohistochemical staining for detection ofliver tissue TNF-α expression and PCR technique for the detection of liver tissue PPAR αexpression.Result：1Liver lipid soluble particle effects on NAFLD rats liver indexThe high dose group of liver index was significantly lower than the control group, therewas significant（P<0.05）; the middle dose group of liver index compared with the controlgroup, no statistical significance（P>0.05）; low dose group of liver index higher than high,middle dose and control group, there was a statistically significant difference（P<0.05）.2Liver fat soluble granule on NAFLD rat liver pathological and morphological effectsThe model of liver tissue steatosis degree is the highest in rats of group, there wassignificant difference compared with the normal group (p<0.01); high, medium, low dosegroup and the control group of rats liver tissue pathological changes were improved, liversteatosis and reduce the number of cytoplasmic lipid droplets in varying degrees, comparedwith with the model group, the difference was statistically significant (p<0.05); high dosegroup rats liver tissue were significantly better than the control group (p<0.05); the middledose group rats liver tissue steatosis compared with the control group, no statisticalsignificance (p>0.05); low dose group, middle dose group compared with control group, therewas statistical significance (p<0.05).3Liver fat soluble granule on NAFLD rat serum biochemical index and HOMA-IRexpression of 3.1Serum biochemical indexesThe model group and drug intervention group rats serum TG, CHOL, FFA were higherthan those in the normal group (P<0.01); high, low dose group and control group were lowerthan those in the model group (P<0.01), there is statistical significance; the high dose groupwas significantly lower than that of the control group (P<0.05), a significant; compared withmiddle dose group and the control group (P>0.05), no significant difference; low dose groupwas higher than that of high, middle dose group and the control group (P<0.05), there wasstatistical significance.Detection results show that the groups of NAFLD rats serum ALT and AST weresignificantly increased in the normal group (P<0.01); Model group rats serum ALT, AST levelthe highest, compared with other drug intervention group (P<0.01), there is significantstatistical significance; High dose group of rats serum ALT, AST level is lower than thecontrol group (P<0.05), there is significant statistical significance; Dose group of rats serumALT, AST level compared with the control group (P>0.05), no statistical significance; Lowdose group of rats serum ALT, AST level significantly higher than the high, middle dosegroup and control group (P<0.01), with statistical significance. Instructions to improveNAFLD rats liver function (ALT, AST), liver lipid soluble particle effect is better than that ofpolyene phosphatidylcholine capsules.Serum free fatty acid FFA in normal group were the lowest, compared with the othergroups (P<0.01), there was significant; the model group was the highest, compared with otherdrug intervention group (P<0.01), there was significant; high dose group, serum FFA wassignificantly lower than that of the control group (P<0.05), there was significant statisticalsignificance the serum content of FFA; the middle dose group compared with the controlgroup (P>0.05), no significant difference of serum FFA content; low dose group were higherthan those of high, middle dose group and control (P<0.01), there was statistical significance.3.2Expression of serum HOMA-IRGroups of NAFLD rats fasting blood glucose, insulin and IR were significantly higherthan that in normal group (P<0.01), there was significant; while the fasting blood glucose,insulin and IR in the rats of model group was significantly higher than that of the highest,drug intervention group (P<0.01), there was significant; high dose rats fasting blood glucose,insulin and IR significantly lower than the control group (P<0.01), there was significant; themiddle dose group rats, fasting blood glucose, insulin and IR lower than that of the controlgroup (P<0.05), there was significant; low dose group rats, fasting blood glucose, insulin andIR compared with the control (P>0.05), no statistical significance.4Liver fat soluble granules on NAFLD rat serum Leptin express impact studiesThe Leptin content in serum of rats in each group of NAFLD were significantly higherthan normal group (P<0.01), there was significant; the serum Leptin in the rats of modelgroup was the highest, compared with other drug intervention group were significantlydifferent (P<0.01), there is statistical significance; the Leptin content in serum of rats in highdose group was lower than that of the control group (P<0.05), there is statistical significance;serum Leptin rats dose content compared with the control group (P>0.05), no statistical significance; the content of serum Leptin in rats of low dose compared with high, middle dosegroup and the control group (P<0.05), there was statistical significance.5Liver fat soluble granules on NAFLD rat liver tissue TNF alpha express impactstudiesThe expression of NAFLD in liver tissue of rats in model group of TNF-α was higherthan that in normal group (P<0.01), with significant statistical significance; expression ofliver tissue in rats of model group was significantly higher than that of TNF-α in the drugintervention group (P<0.01), with significant statistical significance; expression in liver tissueof rats in high dose group of TNF-alpha is lower than that of the control group (P<0.05), withsignificant statistical significance; liver tissue dose group rats TNF-expression compared withthe control group (P>0.05), no significant difference; the expression of liver tissue in rats oflow dose group, TNF-α with high, middle dose group and the control group. Comparison(P<0.05), there was statistical significance.6liver fat soluble granules on NAFLD rat liver tissue PPAR α express impact studiesThe normal group was significantly higher than that in the other groups (P<0.01), therewas significant; the model group was significantly lower than that of other groups (P<0.01),there was significant; high dose group was the most close to the normal group, theintervention group was significantly higher than that of other drugs (P<0.05), there wassignificant; compared with middle dose group and the control group (P>0.05), no statisticalsignificance; low dose group were lower than those in high, middle dose group and thecontrol group (P<0.05), there was significant.Conclusion:1. Liver fat soluble granules can improve experimental NAFLD rat general condition,reduce the intrahepatic lipid deposition, improve the degree of hepatic steatosis.2Liver fat soluble granules have certain protection liver function, reduce thetransaminase, regulating dyslipidemia, improving insulin resistance, restore normal glucolipidmetabolism role.3Liver fat soluble granules obviously decrease serum Leptin expression, reduce the livertissue TNF alpha expression, strengthen liver tissue PPAR α mRNA expression.4Liver fat soluble granules through can reduce intrahepatic lipid deposition, improvehepatic steatosis degree; Protect liver, reduce enzyme, fall fat, and reflect on nonalcoholicfatty liver disease have good control effect.5Liver fat soluble granules by improving IR, inhibition of serum Leptin expression,inhibiting liver tissue TNF alpha expression, strengthen liver tissue PPAR α mRNAexpression, increase insulin sensitivity, regulate lipid metabolism, promote the fatty acidoxidation decomposition, reduce the liver steatosis, thus play on nonalcoholic fatty liverdisease treatment effect, this may be liver fat soluble particle control nonalcoholic fatty liverdisease is one of the mechanisms.更多还原