节点文献
电针对大鼠非酒精性脂肪性肝炎的治疗作用及机制研究
【作者】 程井军；
【导师】 孙国杰；
【作者基本信息】 湖北中医学院 ， 针灸推拿学， 2007， 博士
【摘要】 目的随着社会的发展及人民生活水平的不断提高，非酒精性脂肪性肝炎(nonalcoholic steatohepatitis，NASH)发病逐渐增多。其病理机制较为复杂，至今尚未完全阐明。其中，游离脂肪酸过多、氧化应激、细胞因子相关性损害等是其重要的病理环节。已有的临床和实验研究表明，脂肪性肝炎与NF-κB、PPAR r的相关性研究文献较少，有必要深入研究，进一步阐明核因子-κB(nuclear factorKappa B NF-κB)、过氧化物酶体增殖物激活受体r(peroxisome prolifera-tor-activated receptor gamma，PPAR r)与NASH之间的关系。NASH尚无理想的治疗药物。中医药在治疗NASH方面具有一定的优势。导师孙国杰教授根据长期临床实践，认为NASH多因长期过食肥甘、体胖少动、七情内伤，致脾失健运，肝失疏泄，痰湿内聚，郁久化热，阴精暗耗，终至形成本虚标实的病理特点。治宜补益肝肾、健脾利湿、化痰逐瘀，穴选肝俞、足三里、丰隆、太冲。我们应用电针刺激患者肝俞、足三里、丰隆、太冲治疗非酒精性脂肪性肝炎，发现电针具有明显的改善非酒精性脂肪性肝炎患者的临床症状、改善肝功能、降低血脂及抗肝纤维化的作用，对非酒精性脂肪性肝炎治疗有较好的临床疗效。本课题采用国内外公认的经典NASH动物模型，结合已有的关于NASH的研究成果，用病理学、生物化学、分子生物学等技术，探讨NASH状态下肝组织NF-κB和PPARrmRNA的表达情况，阐明电针治疗NASH的作用机制，为临床应用电针治疗NASH提供科学依据。方法将48只SD大鼠称重标记后按随机数字表随机分为2组：正常对照组12只；造模组36只。正常对照组采用普通饲料喂养；造模组采用高脂饲料喂养。实验依据范建高经典造模方法(标准饲料的基础上加2％胆固醇、13％猪油)复制NASH动物模型。12周后，从正常对照组随机抽取2只，造模组随机抽取6只，肝脏病理切片证实造模成功后，取剩余30只造模组大鼠称重，再随机分为模型组10只，电针组10只，药物组10只。正常组不进行电针和药物治疗，同前普通饲料喂养4周；模型对照组，不进行电针和药物治疗，同前高脂饲料喂养4周。针刺组取穴参照新世纪全国高等中医药院校规划教材《实验针灸学》大鼠针灸穴位定位方法及拟人比照法定位，选取双侧肝俞、足三里、丰隆、太冲，自第12周开始每天治疗1次，连续6天后休息1天，共治疗4周；药物组，自第12周开始每天以西药熊去氧胆酸(UDCA)250mg／kg．d的水溶液灌胃，继前高脂饲料喂养4周。各组大鼠在实验第16周隔夜空腹以2％戊巴比妥钠按2ml／kg量麻醉，经下腔静脉采血，迅速按常规分离血清；摘取肝脏。观察动物体重、食欲、行为、状态、毛发及死亡情况，计算肝指数(肝脏湿重／体重×100％)；采用Olympus Au560全自动生化分析仪测定血清ALT、AST、TG、TC、FFA、空腹血糖(fasting blood glucose，FBG)；放射免疫分析法测定TNF-α、空腹血清胰岛素(fasting Insulin，FNS；计算胰岛素抵抗指数(HOMA-IR=FBGx FINS)／22.5)；肝组织匀浆生化测定MDA、SOD；电泳迁移率改变分析(EMSA)检测NF-KB活性；采用逆转录聚合酶链反应法(RT-PCR)检测PPARrmRNA；将肝组织用4℃生理盐水冲洗，在肝脏最大叶距边缘5mm处取小块肝组织，中性福尔马林固定，石蜡包埋，切片，HE染色，光镜下观察肝组织普通病理变化。另取1mm~3肝组织，2.5％戊二醛及1％锇酸双固定，梯度酒精脱水，环氧树脂包埋，超薄切片，HITACHI H7500透射电镜下观察肝细胞超微结构变化。结果1成功复制大鼠NASH模型。与空白模型组相比，电针组大鼠肝指数明显降低(P＜0.05)，炎症活动程度皆有明显改善(P＜0.05)；血清ALT、AST明显降低(P＜0.05)。除正常对照组外各组大鼠肝组织均表现有不同程度的脂肪变性、炎症、坏死。电针组肝组织脂肪变性及炎症程度明显减轻，肝组织形态结构改善。2模型组血清TG、TC、FFA水平比正常组显著增高(P＜0.05)；模型组比电针组显著增高(P＜0.05)；电针组血清TG、TC水平较之药物组有下降趋势，但二者比较无明显差异(P＞0.05)；药物组血清FFA水平较之电针组有下降趋势，但二者比较无明显差异(P＞0.05)。3模型组肝组织MDA含量显著高于正常组(P＜0.05)，SOD活性显著低于正常组(P＜0.05)；电针组与模型组比较，MDA含量显著降低(P＜0.05)，而SOD活性明显升高(P＜0.05)；电针组与药物组比较，MDA含量有下降趋势，有统4计学差异(P＜0.05)；而SOD活性有下降趋势，但无统计学差异(P＞0.05)。4模型组血清TNF-α水平比正常组显著增高(P＜0.05)；电针组血清TNF-α水平比模型组显著降低(P＜0.05)；电针组血清TNF-α水平与药物组比较无明显差异(P＞0.05)。5模型组胰岛素抵抗指数水平比正常组显著增高(P＜0.05)；模型组比电针组增高(P＜0.05)；电针组与药物组比较无明显差异(P＞0.05)。6模型组和药物组的NF-KB活性明显高于正常对照组(P值均＜0.01)和电针组(P值均＜0.05)。模型组、电针组和药物组大鼠肝组织PPAR rmRNA的表达均较正常对照组有不同程度的减弱(P值均＜0.01)，模型组大鼠肝组织PPAR rmRNA的表达明显低于电针组(P＜0.01)。结论氧化应激进而引起脂质过氧化和肝损伤，最终引起前炎症细胞因子产生，启动肝脏炎症反应；在NASH状态下NF-κB活性增强、PPAR rmRNA表达减弱，显示了三者之间的相关性。电针具有多靶位治疗NASH的作用：①电针能明显降低NASH大鼠肝指数，改善肝组织脂肪变性、炎症活动程度及肝功能。②电针能降低血清脂质水平、减轻FFA的肝脏毒性、改善肝脏对脂质代谢的调节、减轻肝脏炎症反应。③电针能够提高肝组织抗氧化能力，减轻脂质过氧化。④电针能降低TNF-α的表达，减少细胞因子相关性损害。⑤电针能改善NASH大鼠IR状态。⑥电针能够抑制NF-KB的活性、促进PPARrmRNA表达。电针治疗NASH的作用可能是通过腧穴-经络-脏腑相关性及神经-内分泌-免疫网络整体调节实现的。
【Abstract】 Objective With the dramatic development of people’s living standards, more and more people are suffering from non-alcoholic steatohepatitis or NASH as time goes by. Its mechanism is complex and still unknown. Several factors such as extra free fatty acids or FFA, oxidative stress, injuries caused by cytokines or CKs are considered to be pathologically relative.Some existed clinical and experimental researches have shown that steatohepatitis is in correlation with NF-κB as well as PPAR r, but pertinent literature is still not enough. It is necessary to do further research to clarity the relations between NASH and NF-κB as well as PPAR r. At present, it seems to be hard to choose some effective medications to treat NASH. TCM which has some predominances can be turned to concerning its treatment. According to his long term of clinical experiences, my tutor, professor Sun Guo-jie claimed that NASH might be caused by intaking excess fat and sweet foods for a long time, which lead to phlegm and blood stasis generating from within, constrained liver qi, heat caused by permanent stagnation, Yin deficiency due to internal heat. All in all, all these pathological factors lead to such a pathological characteristic as being insufficient in nature but the signs and symptoms are seemed to be excess. Its therapeutical principle should be supplementing the kidney and liver, strengthening the spleen and drain the excess water and phlegm, expelling the blood stasis. Pro. Sun Guo-jie chose Gan-shu acupoint, Zu Sanli acupoint, Feng Long acupoint and Tai Chong acupoint to treat NASH. We needled the acupoints mentioned above to treat patients who suffered NASH and gained significant effects concerning relieving the symptoms, improving the liver function, decreasing the blood fat and resisting fibrosis. In this research, we used classical animal models which are accepted worldwide, armed with the technologies in the fields of pathology, biochemistry, molecular biology, aiming to probe the activity of NF-κB and the expression of PPARrmRNA, make a clear explanation on the therapeutical mechanism of electric acupuncture on NASH in order to offer strong proofs for the clinical use of electric acupuncture in treating NASH.Methods 48 SD rats were divided them into 2 groups, normal group with 12 rats and modeling group with 36 rats according to random digits table. The rats of the normal group were fed with common food;while the rats of the model group were fed with fat-rich diet, i.e,normal diet plus 13% lard oil and 2% cholesterol to rebuild NASH animal models according to the classical method offered by Fan Jian-gao. All the rats took food and drank water in a free way and the dairy staffs were changed on a daily basis. They were fed for 12 weeks in total. After 12 weeks, 2 rats of the normal group and 6 rats of the modeling group were picked out randomly. Pathological sections were made to show that animal model had been built successfully. Then the rest 30 rats of the modeling group were randomly divided into3 groups: model group, electric acupuncture group and the western Medicine group.The rats of both the normal group and the model group were treated as before continuously for another 4 weeks;the rats of the acupuncture group were punctured with electric acupuncture at such acupoints as Ganshu （BL 18）, Zusanli （ST 36）, Fenglong （ST 40） and Taichong （LR 3） bilaterally and also fed with a fat-rich diet continuously for another 4 weeks; the rats of the western medicine group were lavaged with UDCA 250mg/kg.d and also fed with a fat-rich diet continuously for another 4 weeks.All the rats were anesthetized with 2% Pentobarbitalum Natricum through peritoneal injection with a regular standard dose after a night at the end of the 16th week. Blood was collected from the inferior vena and Serum was separated with regular methods. The weight, appetite, behavior, state,hair and death rate of the rats were closely observed.Liver index numbers were calculated by humid weight/body weight x 100%.Serum alanine aminotransferase or ALT, aspartate aminotransferase or AST, triglyceride or TG, total cholesterol or TC, Free fatty acid or FFA, fasting blood glucose or FBG were tested through Olympus Au560 Automatic biochemistry analyzer. Tumor necrosis factor-αor TNF-αand fasting Insulin or FINS were tested by radio immunoassay.As a result, Insulin resistance index or IR index could be calculated through the formula of FBGx FINS/22.5. Fresh liver tissue was made into tissue homogenate. Malondialdehyde or MDA and superoxide dismutase or SOD were tested by way of tissue homogenate biochemistry. NF-kappa B Binding Activity detected by electrophoretic mobiLity shift assay （EMSA）: According to the procedure in the manual of the kits, took 1.75μmol/L unlabelled double-stranded oligonucleotide probe, T4polynucleotide kinase 1μ1,γ<sup>32p ATP （0.37 GBq/ml） 1μ1 to llabel. Took 20μg ribonucleoprotein and put into 10μl gel retardation binding buffer 10mINS at 37℃, added 1μ132p-labelled oligonucleotide probe 30miNS at 37℃, then carried out 6% native polyacrylamide gel electrophoresis and autoradiography 72 hs after gel vaccum dehydration, Observed the results and gray scale scanning was carried out with BIO-PROFIL image analysis system, assayed coloration positive area in comparison with the background negative area and calculated the mean gary scale values. PPAR rmRNA was detected by RT-PCR. Part of the liver tissues were flushed with 4℃normal saline and small scraps of liver tissues were taken at a farmost distance of 5mm to the rim of the liver lobes.Histological specimens were stained with HE after being fixated with 10% netural Formalin and made into slices buried in Paraffin and then studied. 1 mm3 Hepatic slices were fixed with 2.5% Glutaric Dialdehyde and 1% osmic acid and stained with plumbum and uranium and the ultrastructures of the liver tissues was observed through HITACHI H7500 electron microscope.Results1 NASH animal models were replicated successfully. In comparison to the rats of model group, the body weight number of the rates of the normal group were significantly decreased （P<0.05）; so were serum ALT and AST.Steatosis, inflammation,necrosis and fibrosis were present in the livers of the rats of all the modelling groups, and were most severe in the liver tissues of the rats of the model group while the conditions mentioned above were significantly improved in the livers of the rates of the electric acupuncture group. The morphosis of the hepatic tissues of the rats of the electric acupuncture group were markedably improved.2 The serum TG, TC and FFA contents were markedly higher in the model group than those of the normal group （P<0.05）; There was also a significant difference between the model group and the electric acupuncture group （P<0.05）; Concerning the serum TG and TC contents, there was a downward trend in the electric acupuncture group compared with the western medicine group, but there was no difference between them （P>0.05）; Concerning the serum FFA contents, there was a downward trend in the western medicine group compared with the electric acupuncture group, but there was no difference between them （P>0.05）.3 The MDA contents were markedly higher in the model group than those of the normal group （P<0.05）; The MDA contents were markedly lower in the model group than those of the normal group （P<0.05）; The MDA and SOD contents were markedly lower and higher respectively in the livers of the electrnic acupuncture group in comparison to the livers of the model group （P<0.05）; There was a significant difference between the electric acupuncture group and the western medicine group concerning the MDA contents （P<0.05）;Regarding the SOD contents,there was a downward trend in the livers of the electric acupuncture group, but there was no significant difference between the electric acupuncture group and the western medicine group （P>0.05）.4 The serum TNF-αcontents were markedly higher in the model group than those of the normal group （P<0.05）; There was also a significant difference between the model group and the electric acupuncture group （P<0.05）; Concerning the serum TNF-αcontents, there was a downward trend in the western medicine group, but there was no difference between the electric acupuncture group and the western medicine group （P>0.05）.5 Concerning the IR levels, IR levels were markedly higher in the model group than those of the normal group （P<0.05）; There was also a significant difference between the model group and the electric acupuncture group （P<0.05）; there was a downward trend in the western medicine group, but there was no difference between the electric acupuncture group and the western medicine group （P>0.05）.6 NF-kappa B binding activity was obviously higher in the model group and the western medicine group than those in the normal group （P<0.01） and the electric acupuncture group（P<0.05）.The expression of P P A RrmRNA of the model group, electric group and western medicine group was weaker than that of the normal group （P<0.01） and the level of P P A RrmRNA was lower in the livers of the model group than that of electric acupuncture group（P<0.01）.Conclusion Oxidative stress can leads to lipid peroxidation and hepatic injuries as well. In the end, it can cause the appearance of the pre-inflammatory cell factors which might initiate inflammatory reactions in the liver.In the state of NASH, NF-kappa B binding activity will be significantly enhanced while the levels of the expression of PPAR rmRNA will be lowered, which shows the correlatin among NASH, NF-kappa B binding activity and PPAR rmRNA. Electric acupuncture can be used to treat NASH at multiple target sites:It can decrease the liver index of the rats, improve adipoe degeneration, inflammation degree and liver function; it can lower serum lipid, decrese the hepatotoxity caused by FFA, improve the regulation of liver on the metabolim of lipid, relieve the inflammatory reaction; it can strengthen the hepatic tissues against oxidation,relieve lipid peroxidation; it can decrease the expression of TNF-αand as a result it can decrease the injuries caused by cytokines; it can inhibit the binding activity of NF-KB and promote the expression of PPARrmRNA. It is a possibility that electric acupuncture can be used as a therapeutical method in treating NASH through such two ways as the correlaton among acupoints, channels and collaterals and visceras as well as the interaction between nerve, endocrine and immunological network.