【作者】 李有贵；

【导师】 时连根； 计东风；

【作者基本信息】 浙江大学 ， 特种经济动物科学， 2011， 博士

【摘要】 酗酒既是个社会问题也是个医学问题,因酗酒导致的急慢性酒精中毒、酒精性脂肪肝、慢性肝炎、肝硬化等疾病严重危害着人们的健康。大量饮酒过后,血液中乙醇浓度明显升高,出现各种醉酒症状。乙醇主要在肝脏中通过乙醇氧化系统进行代谢,同时产生大量自由基02-·、OH·,以及乙醇自身产生的自由基C2H50-和C2H5OH-,当产生自由基超出了机体的清除能力,便造成机体组织损伤。所以普遍认为乙醇导致肝损伤,其机制之一是通过激活O2产生自由基,导致肝细胞膜脂质过氧化及肝细胞损伤。竹节人参(Panax Japonics.C.A.Mey)系五加科植物,为《中华人民共和国药典》收载的名贵常用中药,通常被认为具有抗炎、镇痛、镇静、抗衰老、抗疲劳、抗病毒、抗肿瘤、保护中枢神经系统、保护心血管系统、保护内分泌系统、保护免疫系统等多方面的功能,其主要化学成份为竹节参皂苷。现代药理学研究发现,竹节人参总皂苷可显著提高多种模型(如：运动、衰老、脑缺血再灌注损伤、高脂血症、等)动物血清、肝脏中SOD、CAT、GSH-PX活性,同时降低MDA含量,从而抑制脂质过氧化。因此,本研究利用循环超声波提取了人工栽培的竹节人参总皂苷,高效液相色谱-蒸发光散射检测-质谱(HPLC-ELSD-MS)分析了竹节人参皂苷单体及含量组成,并以抗氧化性为着手点,研究了竹节人参总皂苷对模型小鼠、人胚肝细胞L-O2乙醇性损伤的保护作用及作用机理,同时分析了不同竹节人参皂苷单体对乙醇损伤肝细胞L-O2的保护作用及作用机理,主要研究结果如下：1、通过竹节人参总皂苷提取,HPLC-ELSD分离获得6个皂苷单体,通过标准品比对,结合HPLC-ESI-MS分析,可知分别为Rg1、Re、Rf、F3、Rg2和Rd,其在人参总皂苷中的含量分别为：12.23%、16.24%、16.95%、7.51%、8.53%和11.47%。2、给乙醇性肝损伤模型小鼠灌胃给予竹节人参总皂苷,气相色谱法(GC)测定小鼠血清、尿液中乙醇浓度,分光光度法测定小鼠血清谷丙转氨酶(ALT)、谷草转氨酶(AST)活性以及血清、肝组织中丙二醛(MDA)含量、还原型谷光甘肽(GSH)含量、超氧化物歧化酶(SOD)活性、过氧化氢酶(CAT)活性、谷胱甘肽过氧化物酶(GSH-PX)活性等生化指标,光镜、透射电镜观察肝组织超微病理变化,研究竹节人参总皂苷对小鼠乙醇性肝损伤的保护效果及作用机制；结果显示,竹节人参总皂苷不具有抑制乙醇在胃肠道吸收作用,但能显著降低乙醇肝损伤模型小鼠血清ALT、AST酶活性及血清、肝组织MDA含量,提高模型小鼠血清、肝脏中GSH含量和抗氧化酶GSH-PX、CAI、SOD的活性。在血清中,竹节人参总皂苷50 mg/kg剂量组小鼠血清GSH含量、GSH-PX、CAT和SOD活性与正常组小鼠无显著性差异(P>0.05)；在肝脏中,竹节人参总皂苷50 mg/kg剂量组小鼠肝脏GSH-PX、SOD活性可恢复至正常水平,与正常组小鼠无显著性差异(P>0.05)；GSH、CAT水平虽然显著提高,但仍显著低于正常组(P<0.01)；竹节人参总皂苷(50 mg/k)可降低肝组织病变,促进肝小叶结构恢复正常,肝索、肝窦无明显异常,肝细胞体积逐渐恢复正常,细胞核染色质分布较均匀,核膜整形光滑,线粒体结构基本恢复正常,内(嵴)外膜完整,基质均匀,但部分线粒体嵴仍有些紊乱。以上结果表明,竹节人参总皂苷具有提高机体抗氧化功能,特别是对肝细胞及细胞器线粒体的结构完整性具有明显的保护作用。3、通过台盼蓝染色计数观察竹节人参总皂苷对正常及乙醇损伤的肝细胞L-02活性的影响,分光光度法测定肝细胞内液MDA含量和SOD、GSH-PX、CAT活性变化,以及RT-PCR分析肝细胞内SOD1、SOD2、SOD3、GSH-PX1、GSH-PX2、CAT mRNA水平变化,研究竹节人参总皂苷对乙醇损伤人胚肝细胞L-02的保护作用及作用机制。结果发现,对正常肝细胞,竹节人参总皂苷(100μg/mL)表现出明显的促进L-02增殖作用,当竹节人参总皂苷浓度达400μg/mL时,则表现出明显的抑制细胞增殖作用；对乙醇损伤的肝细胞L-02,竹节人参总皂苷(100μg/mL)表现出明显的保护作用,其肝细胞内液MDA含量、SOD、GSH-PX活性可恢复至正常水平(P>0.05),CAT活性虽显著升高,但仍显著低于正常组(P<0.01)。RT-PCR分析发现,竹节人参总皂苷(100μg/mL)可促进SOD1、SOD2、SOD3、GSH-PX1、GSH-PX2、CAT mRNA表达,其中GSH-PX3显著高于正常组(P<0.01),SOD1、SOD3与正常组无显著差异(P>0.05),SOD2、CAT虽显著提高,但仍低于正常对照(P<0.01),该结果与体内实验结果一致,说明竹节人参总皂苷发挥抗氧化作用与促进抗氧化酶SOD、GSH-PX、CAT特别是SOD1、SOD3、GSH-PX3mRNA表达有关。4、通过清除羟自由基(OH·)和超氧阴离子自由基(02·)试验,进行竹节人参总皂苷体外抗氧化能力评价。结果竹节人参总皂苷呈现出明显的清除OH·效应,并且其效果优于Vc,当浓度为12.5-1600μg/mL时,竹节人参总皂苷和Vc对OH·清除率分别为3.37-59.47%和3.10-31.79%；竹节人参总皂苷对联苯三酚自氧化具有一定的抑制作用,能清除O2-·,但清除能力要低于Vc,当浓度为12.5-1600μg/mL时,竹节人参总皂苷和Vc清除率分别为1.01-6.87%和0.89-15.98%。研究结果表明,竹节人参总皂苷在乙醇性肝损伤保护作用中,不仅可提高抗氧化剂GSH含量以及抗氧化酶GSH-PX、CAT、SOD的活性,同时具有直接清除肝代谢过程产生的·OH、O2-·自由基,亦或者是两者的协同作用达到对肝细胞的保护。5、通过MTT法测定细胞存活率,分光光度法测定肝细胞内液MDA含量、SOD及GSH-PX活性,研究竹节人参皂苷单体对乙醇损伤肝细胞L-02的保护作用及作用机理。结果显示,Rg1 0.16 mg/mL、Re 1.28 mg/mL和Rf 0.64mg/mL可促进正常肝细胞L-O2增殖,增殖率分别为22.68%、34.80%和28.47%(P<0.01)；Rd 0.16mg/mL和F3 1.28 mg/mL对正常肝细胞L-O2生长表现出明显的抑制作用,抑制率分别为49.69%和43.33%(P<0.01)。对乙醇损伤的肝细胞L-O2,Rg1 0.16mg/mL、Re 1.28 mg/mL和Rf 0.64 mg/mL具有明显的保护作用,对细胞生长的抑制率由乙醇损伤模型组的50.37%分别降低为23.31%、26.90%和26.58%(P<0.01)；Rd 0.16mg/mL和F3 1.28 mg/mL则加剧乙醇对肝细胞的损伤,抑制率高达83.18%和64.79%(P<0.01)。乙醇损伤模型组肝细胞L-02内乙醇代谢产生MDA含量、抗氧化酶SOD和GSH-PX活性分别为1.35±0.05μmol/mL、26.45±2.78 U/mL和67.04±4.27U/mL, Rg1 0.16 mg/mL、Re 1.28 mg/mL和Rf 0.64mg/mL可降低乙醇损伤的肝细胞L-02内MDA含量,分别降低为1.17±0.04、1.21±0.03和1.21±0.05μmol/mL(P<0.01)；提高SOD活性,分别升高为38.32士4.85、35.58±4.43和38.96±3.27 U/mL(P<0.01)；增强GSH-PX活性,分别升高为86.30±5.72、78.40±3.54和84.25士4.35U/mL(P<0.01)。以上结果表明,人参皂苷单体Rg1、Re和Rf对乙醇损伤肝细胞L-02具有明显的保护作用,清除乙醇代谢过程中产生的MDA以及提高抗氧化酶SOD和GSH-PX的活性,可能是其发挥该保护作用的机制之一。更多还原

【Abstract】 Alcoholism is a social problem, and is also a medical issue. Acute alcoholism, chronic alcoholism, alcoholic fatty liver, chronic hepatitis and cirrhosis caused by prolong and heavy alcohol intake seriously harms people’s health. After heavy drinking, blood alcohol concentration was significantly increased, and emergence of various drunken symptoms. Ethanol is mainly metabolized in liver by ethanol oxidation system, while producing a large number of free radicals O2-·, OH·and ethanol production of free radicals C2H5O-, C2H5OH-. When free radicals beyond the body scavenging capacity, it will cause the body tissue injury. So generally considered that one of the mechanisms of the alcohol-induced liver damage is through activating the O2 and produce free radicals, then cause hepatic cell membrane lipid peroxidation and injury.Panax japonicus (Panax japonicus. C.A.Mey), as one of traditional Chinese medicines and recorded in "Chinese Pharmacopoeia", is usually considered to have many effects such as anti-inflammatory, analgesic, sedative, anti-aging, anti-fatigue, anti-virus, anti-tumor, and protection of central nervous system, cardiovascular system, endocrine system, immune system and so on, the main active components of Panax japonicus are the saponins. Pharmacological studies found that total saponins of Panax japonicus can significantly increased the activities of SOD, CAT, GSH-PX, while reducing the MDA content in serum and liver in various animal models (such as:sports, aging, cerebral ischemia reperfusion injury, hyperlipidemia, etc.), thus inhibiting lipid peroxidation. Therefore, the total saponins used in this study were extracted from artificial cultivated Panax japonicus by circular sonication, the composition and its content of different saponins were analyzed with high performance liquid chromatography- evaporative light scattering detector- mass spectroscope (HPLC-ELSD-MS). Antioxidant activities as a starting point, we investigated the protective effect of saponins from Panax japonicus on ethanol-induced mice liver and human hepatic cell L-O2 injury, and its potential mechanisms of hepatoprotection involved, the protective effect of different purity saponin from Panax japonicus on ethanol-induced hepatic cell L-O2 injury and its possible mechanism are also studied in this paper. The main results are as follows:1. Total saponins were extracted from Panax Japonics, tentative identification of the saponins was achieved by comparing their retention times with those of the authentic standards, and further identification of the structures of these saponins by LC-ELSD-MS. The results were that the saponins used in this paper were identified to be ginsenosides Rg1, Re, Rf, F3, Rg2 and Rd, and the contents of which were 12.23%,16.24%,16.95%, 7.51%,8.53% and 11.47%, respectively.2. Saponins from Panax Japonics (SPJ) were given to mice by gavage 1 hour prior the alcohol treatment, the alcohol concentrations in serum and urine were determined by GC; the levels of malondialdehyde (MDA), alanine transaminase (ALT), aspatate transaminase (AST), reduced glutathione (GSH), superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-PX) etc were measured by spectrophotometry; histopathological examination were observed through Lecia-DM2500 microscope and JEOL-1230 transmission electron microscopy; Results:SPJ may not inhibit ethanol absorption in the gastrointestinal tract. The AST, ALT activities and MDA content were decreased distinctly in serum in SPJ (50 mg/kg) treated mice, and the antioxidant as GSH, GSH-PX, CAT and SOD were evidently increased in serum and liver. Pretreated with SPJ, all the levels could restore to normal (50mg/kg b.w) in serum. In liver determination, the administration of SPJ significantly increased the activities of GSH-PX and SOD, and the final values were restored to normal at the dose of 50mg/kg b.w. (P>0.05). The levels of CAT and GSH were also significantly elevated compared to alcohol treatment mice, but still lower evidently than control (P<0.01). In histopathological studies, SPJ (50 mg/kg b.w.) pretreatment significantly prevented alcohol-induced abnormalities in dose-dependent manner, in which the hepatic lobule structure, liver cord and sinusoidal were remedied and showed normal histology. The cell structure were gradually restored nearly normal, karyon chromatin distributed uniform, nuclear membrane became smooth, mitochondrial structure returned to normal, the cristae and outer membrane became integrity, the persistent damage disappeared and most of mitochondria restored back to normal, whiles a few portion of mitochondria still showed with little distorted cristae. These results indicated that SPJ may exert a protective effect against lipid peroxidation by scavenging reactive oxygen species and elevating the activity of antioxidant enzymes, in consequence prevented the peroxidative deterioration of structural lipids in membranous organelles, especially mitochondria and karyon.3. To investigate the protective effect and its possible mechanism(s) of SPJ on alcohol-induced hepatic cell L-O2 damage, the normal and alcohol-induced injury cell were counted through trypan blue staining, the intracellular MDA, SOD, CAT and GSH-PX levels of L-02 cells were measured by spectrophotometry, the CAT. GSH-PX1, GSH-PX3, SOD1, SOD2 and SOD3 mRNA were detected by Quantitative real-time reverse transcription-PCR. Results:SPJ(100μg/mL) could promote normal cell (L-02) proliferation, but SPJ (400μg/mL) showed a evident toxicity to normal cell (L-02). SPJ (100μg/mL) also showed a significant protective effect on ethanol-induced hepatic cell L-02 injury, and presented evidently inhibitory ability on MDA in L-02 cells. Meanwhile, the SOD, GSH-PX and CAT activities were increased, in which the SOD and GSH-PX activities were restore to normal after treated with SPJ (100μg/ml), but the CAT was still lower than control (P<0.01). RT-PCR results showed that the expression of all the antioxidant enzymes were remarkably decreased with treated with ethanol (200 mmol/1) for 48h. Pretreated with SPJ (100μg/ml), these down-regulated tendencies of all the antioxidant enzymes were reversed, in which GSH-PX3 was evidently increased compared with normal control, SOD1 and SOD3 were restored to normal level, but the CAT and GSH-PX1 mRNA levels were still lower than evidently. These results of RT-PCR studies were in agreement with biochemical analyses in vivo, which indicated that the antioxidant effect of SPJ was associated with up-regulating the expression of SOD, GSH-PX and CAT, especially of GSH-PX 3, SOD1 and SOD3.4. Antioxidant activities of SPJ measured by hydroxyl radicals (·OH) and superoxide anion radical (O2·-) scavenge assays in vitro. Results:SPJ had a higher scavenging hydroxyl radical’s effect than Vc, and their scavenging effects increased with increasing concentration. Scavenging effects of SPJ was 3.37-59.47% at amount of 12.5-1600μg/ml, respectively, and that of Vitamin C was about 3.10-31.79%. Superoxide radicals were generated in biphenyl trihydroxybenzene autoxidation system; results showed that the SPJ has little scavenging activities on O2’-. At the amount of 12.5-1600μg/ml. the effect on scavenging superoxide of SPJ was just 1.01-6.87%, while the scavenging activity of Vitamin C for superoxide radical was 0.89-15.98%. These results indicated that the protective effect of SPJ on alcohol-induced damage not only just associated with increasing the activities of GSH-PX, CAT and SOD, but also related with directly scavenging free radical such as·OH、O2-·,or the synergy effect of the two.5. To study the protective effect of different purity saponins from Panax japonicus on ethanol-induced hepatic cell (L-O2) injury and its possible mechanism(s). Survival ratios of hepatic cell (L-02) were determined by MTT, the level of MDA, the activities of SOD and GSH-PX in cell L-02 were measured by spectrophotometer. Result:0.16 mg/mL Rg1、s 1.28 mg/mL Re and 0.64 mg/mL Rf could promote normal cell (L-02) proliferation, the proliferation ratio was 22.68%,34.80%,28.47%(P<0.01). respectively.0.16 mg/mL Rd and 1.28 mg/mL F3 showed a evident toxicity to normal cell (L-02), the inhibition ratio (%) was 49.69%,43.33%(P<0.01).0.16 g/L Rg1,1.28 mg/mL Re and 0.64 mg/mL Rf also showed a significant protective effect on ethanol-induced hepatic cell (L-02) injury, the inhibition ratio (%) was decreased to 23.31%,26.90%,26.58% compare to ethanol-induced control 50.37% (P<0.01), but 0.16 mg/mL Rd and 1.28 mg/mL F3 showed a more evident toxicity to ethanol-induced hepatic cell (L-02), the inhibition ratio was 83.18%,64.79%(P<0.01), respectively. Meanwhile,Rg1. Re and Rf also could decreased the level of MDA (1.17±0.04,1.21±0.03,1.21±0.05 compare to ethanol-induced control 1.35±0.05 mmol/; P<0.01) and increased the activities of SOD (38.32±4.85,35.58±4.43,38.96±3.27 compare to ethanol-induced control 26.45±2.78 kU/L; P<0.01) and GSH-PX (86.30±5.72,78.40±3.54,84.25±4.35 compare to ethanol-induced control 67.04±4.27 kU/L; P<0.01) in intracellular fluid of cell L-02. These results indicated that Rg1, Re and Rf have protective effect against ethanol-induced hepatic cells L-02 injury, the possible mechanism may relate to decline of MDA content, increase of antioxidant enzymes such as SOD and GSH-PX activities.更多还原