【作者】 宋雨鸿；

【导师】 吕志平； 刘强；

【作者基本信息】 南方医科大学 ， 中西医结合临床， 2010， 博士

【摘要】 研究背景：我国肝病的主要发病原因是感染肝炎病毒。流行病学资料表明,我国每年有50万～100万新发病例,而且在全球约3.5亿以上慢性乙型肝炎病毒携带者中,中国人占80%。在HBV感染者中,有10%～15%的患者可发展为慢性HBV感染,从慢性肝炎发展为纤维化一般只需2～6年时,其中25%～40%的病例最终发展为肝硬化乃至肝癌。研究肝炎的发生机理及寻找有效的治疗措施是国内外学者共同研究的热门课题,大量研究表明,免疫功能的异常改变和氧自由基的大量产生是引起肝组织损伤的重要原因。西医用于肝炎的治疗多见于抗病毒药、免疫调节药及护肝降酶类药物,目前临床上常用的品种有干扰素、阿糖腺苷、干扰素诱导剂聚肌胞、无环鸟苷、左旋咪唑、转移因子、辅酶Q、胸腺素、右旋儿茶素、甘草甜素、齐墩果酸、葫芦素B、葫芦素E、联苯双酯、维生素类等。临床治疗结果表明,这些药物多具有良好的抑制病毒复制,恢复正常肝功能,保护肝细胞等作用,并且疗效迅速,不足之处为远期疗效不稳定,易反复,HBsAg转阴率低,有些药品价格昂贵和表现一定的副作用。中医对肝炎的认识常见于胁痛、黄疸、积聚及虚劳等病症中。其病因为湿热疫毒或先天胎毒,病机特点为湿热羁留、肝胆不疏,脾胃受损。蔡春江等提出慢性乙肝属伏邪致病,伏邪为“浊”、“毒”之邪,病位在肝,邪伏部位为血分,浊、毒之邪与慢性乙肝关系密切,既是病理产物,又是致病之因。刘兴家等认为乙型肝炎病毒的侵入是致病的主因,而湿热与肝郁是一种诱因。陈良金等认为慢性乙肝的病理基础可概括为郁、湿、热、毒、痰、瘀、虚；病位主要在肝,涉及到脾、肾二脏。裴建宏从中医病因理论着眼,认为慢乙肝的病因当属疫气范畴,其病理属性为湿热疫气侵袭,胶固难解,损及肝脾,伤及气血阴阳,并在病情发展过程中出现正邪盛衰演变的复杂病理格局,病势迁延难愈。陈华东等认为病毒性肝炎急性期以湿热疫毒为主,尤其热重于湿；慢性期则因失治、误治、久治不愈而致毒邪滞留,以湿为主。以具有解毒活血祛湿、滋补肝肾、健脾益气之效的药物予以治疗。中医中药具有不良反应较少,可长期服药的特点。近年来,中药治疗HBV感染取得了良好的疗效。多聚糖(polysaccharide),简称多糖,常由一百个以上甚至几千个单糖基通过糖苷键连接而成的,其性质已大不同于单糖,如甜味已经基本消失,广泛存在于动物细胞膜和植物、微生物的细胞壁中,是构成生命的四大基本物质之一,与生命功能的维持密切相关。到目前为止,已有几百种多糖从自然界被分离和提纯,多糖不仅是生命有机体的重要组成成分,还具有广泛的生物活性,如免疫调节、抗炎、抗病毒、抗肿瘤、抗凝血、抗氧化、抗辐射、抗衰老、降血糖、降血脂、保肝、抗寄生虫等。多糖作为非细胞毒性物质,对正常细胞几乎无毒副作用。目前已对数十种多糖进行了含量测定,乌头多糖、东当归多糖、石斛多糖、桔梗多糖、人参多糖、细叶蓟多糖、柴胡多糖、猪苓多糖、红松果多糖、菟丝子多糖、胎盘脂多糖、黔产党参多糖、文蛤多糖、石仙桃多糖、肉苁蓉多糖、胖大海多糖、黄精多糖、耳叶牛皮消多糖、石菖蒲多糖、海藻多糖、黄花倒水莲多糖、商陆多糖、金线莲多糖、灵芝多糖、白花蛇舌草多糖、米糖多糖、芦荟多糖、山药多糖、大枣多糖等进行了较系统的含量研究。应用于临床取得较好疗效的有黄芪多糖、猪苓多糖、茯苓多糖、香菇多糖等。文献已有报道许多多糖对肝脏具有保护作用,并且它们的保肝作用多与免疫因素联系密切例如：虫草多糖对小鼠免疫性肝损伤的保护作用,天麻多糖GEP-2对BCG+LPS致小鼠免疫性肝损伤的保护作用,大蒜多糖C对免疫性肝损伤小鼠外周血淋巴细胞亚群的影响,紫菜多糖对肝损伤小鼠的保护作用,琐琐葡萄多糖对大鼠体外免疫性肝损伤保护作用的研究。丹参为唇形科植物丹参Salvia miltiorrhiza Bge.的干燥根及根茎,性味苦,微寒,归心、肝经。始载于《神农本草经》,列为草部上品,名为郄蝉草。为多年生直立草本,高40-80cm,全株密被黄白色柔毛及腺毛；根肉质,圆柱形,外皮朱红色。丹参具有活血祛瘀、通经止痛、清热安神的功效。其“善治血分,去滞生新,调经顺脉”之功效尤佳。故有“一味丹参,功同四物”之誉,广泛应用于临床。经现代研究证明,丹参有效化学成分为脂溶性菲醌色素丹参酮类(如：丹参酮ⅡA等)和一些水溶性的酚性化合物(如丹酚酸类、原儿茶醛等),水溶性和脂溶性成分均有药理活性。由于丹参及其复方制剂对许多疾病如冠心病、中风、肝炎、肿瘤、糖尿病等有良好的疗效,国内外对丹参化学成分、药理作用、临床应用、制剂、质量控制等方面进行了系统的研究,取得了显著的进展。本课题组在进行药物成分分离的实验中提取到了丹参中的多糖成分,经文献检索发现丹参中作为重要有效部位的多糖成分研究较少。现有文献报道的丹参多糖提取方法为水提醇沉法,其提取方法的缺点是操作复杂、多糖提取率较低,而且关于丹参多糖药效和药理方面的研究更少。本课题组进一步将丹参多糖的提取工艺进行优化,找到一种多糖提取率高、成本低廉、操作简单的提取方法。鉴于大多数多糖的疗效及丹参的临床应用,我们将对丹参多糖对免疫性肝损伤的影响及免疫调节机制进行初步研究,从而为其进一步研究开发奠定理论和实验基础。研究目的：通过对丹参多糖提取工艺、丹参多糖抗免疫性肝损伤及其免疫调节机制的实验研究,提供一种从丹参中提取多糖的方法并阐明其抗免疫性肝损伤及免疫调的作用机制,为丹参多糖的进一步开发利用奠定实验基础。研究方法：1.丹参多糖的提取1.1粉碎：将丹参粉碎至粒径为70～90μm的微粉。1.2脱脂：将丹参微粉用10倍体积的浓度大于或等于85%乙醇回流2小时,过滤,取滤渣挥干溶剂后备用。1.3酶处理：将脱脂处理后的丹参微粉加到水中,再加入酶,然后用碱将pH值调节至5.5,在70℃下保温1小时；所述的酶可以是纤维素酶、木瓜蛋白酶、果胶酶中的一种,用量为丹参重量1.5%,也可以纤维素酶、木瓜蛋白酶、果胶酶中的两种以上,每种酶的用量为丹参重量1%,最好是纤维素酶和木瓜蛋白酶,每种酶的用量均为丹参重量的1.5%。1.4超声提取：在频率为59kHz的超声波下处理,过滤,收集滤液,滤液浓缩至1：2(每2ml药液相当于1克药材)。1.5醇沉：加入70ml的乙醇使其在滤液中的浓度至85%,滤去乙醇即得丹参多糖粗品,再经洗涤得丹参多糖,75℃真空干燥。2.丹参多糖抗卡介苗(BCG)加脂多糖(LPS)诱导的小鼠免疫性肝损伤的实验研究2.1实验分组昆明小鼠,SPF级,体质量18～22g,60只(南方医科大学实验动物中心提供)。采用随机分组的方法分为正常组、模型组、丹参多糖高(360mg/kg)、中(180mg/kg)、低(90mg/kg)剂量组、联苯双酯组(200mg/kg),每组10只,雌雄各半。除正常组小鼠外,其余小鼠在造模的同时灌胃给药,灌胃剂量为0.5ml/20g,正常组小鼠给同等剂量生理盐水。2.2造模方法采用BCG加LPS诱导小鼠免疫性肝损伤。小鼠尾静脉注射BCG浆液0.2 ml/只(含菌量5×107单位),致敏后12 d,尾静脉注射LPS 0.2 ml/只(7.5μg)以诱导肝损伤,正常组小鼠尾静脉注射同等剂量生理盐水。2.3指标检测2.3.1摘眼球采血后,离心获取血清,按试剂盒说明操作检测血清ALT、AST、NO活性。2.3.2小鼠处死后,立即取出所需肝脏,称取0.25g肝组织用冷生理盐水洗去浮血,配成10%肝组织匀浆,匀浆液经500转离心20min去胞核碎片后,再经3000转离心15min,取上清液,ELISA方法检测TNF-a、IL-1β含量。2.3.3肝组织固定、切片和肝组织病变程度分级方法处死动物,取出肝脏,用冷生理盐水洗去浮血,拭干。于肝门处及右肝叶取材4块,每块大小约为1.5cm×1.5cm×0.4cm,立即用10%中性福尔马林固定。70%、80%、90%、95%、100%乙醇脱水,二甲苯透明,石蜡包埋,切片机作4μm切片,常规脱蜡,HE染色,中性树胶封片。用显微镜观察,以及PM-20型显微摄像系统照相。光镜下检查,按肝脏坏死程度分级:“-”,未见明显病理改变；“+”,部分肝组织肿胀伴有散在的点状坏死,汇管区有少许炎性细胞浸润；“++”,肝细胞肿胀有点状坏死及小灶性坏死,可见散在的肉芽肿形成,汇管区有大量炎性细胞浸润；“+++”,肝细胞肿胀,有大片状坏死,有肉芽肿形成,汇管区及周围有大量炎性细胞浸润。3.丹参多糖对免疫功能调节的机制研究3.1 BALB/c小鼠,SPF级,中山大学实验动物中心提供。分离BALB/c鼠脾细胞,不同浓度(1、10、50、100、200mg/l)丹参多糖对淋巴细胞(浓度1×106cell/ml)作用48h,MTT法检测细胞增殖。3.2分离B、T淋巴细胞,不同浓度(1、10、50、100、200mg/l)丹参多糖分别对B、T淋巴细胞(浓度1×106cell/ml)作用48h并与分裂素ConA(T细胞)、LPS(B细胞)(最终浓度5ug/ml)作比较,MTT法检测细胞增殖。3.3不同浓度(1、10、50、100、200mg/l)丹参多糖作用于T淋巴细胞72h,流式技术分析CD4+、CD8+的变化。3.4 200mg/l浓度丹参多糖作用于T细胞不同时间(12、24、48、72h),ELISA分析IL-2、IL-4浓度。3.5 200mg/l浓度丹参多糖作用于T细胞不同时间(24、48、72h),荧光定量PCR分析IL-2、IL-4水平变化。4.统计处理实验结果用均数(x)±标准差(s)表示,组间比较用one-way ANOVA。方差齐性时,采用LSD方法进行组间多重比较；方差不齐时,采用Welch法,组间多重比较采用DunnettT3方法进行。两组等级资料的比较采用两个独立样本的非参数检验Mann-WhitneyU法,多组等级资料的比较采用多个独立样本的非参数检验KrusKal-WallisH法,用统计软件SPSS13.0进行分析,以P<0.05定为差异有统计学意义。结果：1.采用粉碎、脱脂、酶处理、超声提取、醇沉的方法对丹参中的多糖成分进行提取,采用正交设计法进一步优化超声法的工艺条件,最终经统计学分析确定最优工艺条件为：以加水为12倍药材重量,粒度为过20目筛,超声提取时间20min为最佳提取条件。2.在丹参多糖抗免疫性肝损伤的实验中,血清ALT、AST水平丹参多糖高、中、低剂量组与模型组相比有显著差异(P=0.000,P=0.003,P=0.044)。阳性药对照组与模型组相比有显著差异(P=0.000)；血清NO和肝组织匀浆IL-1β、TNF-α的水平丹参多糖高剂量组与模型组相比有显著差异(P=0.000),丹参多糖中剂量组与模型组相比TNF-α、IL-1β有显著差异(P=0.048),但是NO差异不显著(P=0.057)。丹参多糖低剂量组与模型组相比TNF-α(P=0.187)、NO(P=0.097)、IL-1β(P=0.145)差异不显著。联苯双酯滴丸组与模型组相比NO有显著差异(P=0.037),但是TNF-a(P=0.064)、IL-1β(P=0.882)差异不显著；脏器系数丹参多糖高剂量组与模型组相比有显著差异(P=0.000,P=0.005,P=0.018)。丹参多糖中剂量组与模型组相比肝脏系数(P=0.043)、脾脏系数(P=0.016)有显著差异。丹参多糖低剂量组与模型组相比无显著差异(P=0.178,P=0.175,P=0.329)。阳性药物对照组与模型组相比除脾脏系数差异不显著外(P=0.059),其两项均有显著差异(P=0.000,P=0.042)；与正常组相比较,模型组肝脏病理变化显著(P=0.000)。丹参多糖高剂量组(P=0.032)、阳性药物对照组(P=0.016)与模型组相比肝脏病理变化显著。丹参多糖中剂量组(P=0.240)、丹参多糖低剂量组(P=0.547)与模型组相比肝脏病理变化不显著。3.在丹参多糖免疫调节机制的研究中,在10～200mg/l剂量范围内,丹参多糖不同剂量组均能显著促进脾细胞的增殖(P=0.000),与对照组比有显著性差异(P=0.020,P=0.000),丹参多糖200mg/l组显示出更高的活性；实验结果显示在100～200mg/l剂量范围内,丹参多糖组能显著促进T淋巴细胞的增殖,与对照组比较有显著性差异(P=0.042,P=0.000),同时丝裂原ConA与对照组比较有显著性差异(P=0.000)。丹参多糖浓度在50～200mg/l范围内,丹参多糖组能显著促进B淋巴细胞的增殖,与对照组比较有显著性差异(P=0.033,P=0.000),同时丝裂原LPS与对照组比较有显著性差异(P=0.000)；用200mg/l浓度的丹参多糖对小鼠淋巴细胞干预12h、24h、48h、72h后,IL-2水平升高,各时间段之间比较具有显著差别(P=0.000)。IL-4水平也有显著增强,各时间段之间比较具有显著差别(P=0.005),但是升高趋势较IL-2水平平缓；荧光定量PCR检测结果显示,经浓度为200mg/l的丹参多糖干预24h、48h、72h后IL-2 mRNA和IL-4 mRNA表达存在差异(P=0.016,P=0.169)；与空白对照相比,干预72h后IL-2 mRNA和IL-4mRNA表达均有所有提高,差异有统计学意义(P=0.022,P=0.036)；干预24h、48h后的IL-2mRNA和IL-4 mRNA表达差异不显著(P=0.753,P=0.400,P=0.177,P=0.208)。说明丹参多糖在干预72h后可有效增强IL-2 mRNA和IL-4 mRNA的表达；丹参多糖在1～200 mg/l剂量范围内,随着剂量的升高对CD4+、CD8+比例的影响逐渐增强,显示出明显的剂量依赖性,同时与对照组比较较高浓度组差异有统计学意义(P=0.028,P=0.007,P=0.008,P=0.027,P=0.000)。结论：1.确定最优丹参多糖提取工艺条件为：以加水为12倍药材重量,粒度为过20目筛,超声提取时间20min为最佳提取条件,本提取方法具有低耗能、提取率高、操作简单等优点。2.结果显示丹参多糖有抗免疫性肝损伤的作用,这种作用的实现不仅表现在直接的保肝降酶方面,还对肝损伤后小鼠的免疫功能起到调节作用,通过免疫功能的调控进一步发挥保肝降酶作用。3.体外实验说明丹参多糖在一定浓度范围内对淋巴细胞有较强的增殖作用,并且还有促进相关细胞因子表达及分泌的作用,与体内试验的结果相一致,进一步说明丹参多糖在免疫调节方面有很好的疗效。更多还原

【Abstract】 Background:The main causes of liver disease in China are infected with hepatitis virus. Epidemiological data show that our country have about 50 million to 1 million new cases every year, also in the world for more than about 350 million chronic hepatitis B virus carriers, accounts for 80% in China. In HBV infection,10% to 15% of patients may develop into chronic HBV infection, from chronic hepatitis develop into liver fibrosis only need 2 to 6 years in general.25%～40% of the patients eventually develop into cirrhosis of the liver and even hepatocellular carcinoma. The of study occurrence of hepatitis mechanism and to find effective treatment measures are the most popular in domestic and foreign scholars, a large number of studies have shown that abnormal changes in immune function and oxygen free radicals production are the cause of a large number of important reasons for the liver tissue damage.Doctors prevalently use anti-viral drugs, immunomodulatory drugs and liver protection drugs to treat hepatitis in western medicine. These drugs commonly contain varieties interferon, vidarabine, interferon-inducing agent polyinosinic, acyclovir, levamisole, transfer factor, coenzyme Q, thymosin, cianidanol, glycyrrhizin, oleanolic acid, cucurbitacin B, cucurbitacin E, bifendate, vitamins and so on. Clinical results show that many of these drugs have good control of virus replication and restore normal liver function, protect liver cells and so on with efficacy of rapid. Disadvantage is unstable long-term efficacy, easily repeated, low rate of HBsAg darkening, expensive and the certain side effects.Knowledge of traditional Chinese medicine for hepatitis common is Xie Tong, jaundice, accumulation and consumption diseases. The cause is hot and humid disease or congenital erupted fetal disease, pathogenesis characteristic is hot and humid detention, liver and gallbladder blockage, the spleen and stomach damage. Caichun jiang et al proposed an Fuxie pathogenesis of chronic hepatitis B, Fuxie is "muddy" and "poisonous",sickness locate in the liver, disease sub-site in the blood, "muddy" and "poisonous" are closely related to chronic hepatitis B both the pathological product and the result of illness. Liu xing jia presume that the intrusion of hepatitis B virus is the main cause of disease, while the hot and humid with liver depression is a kind of inducement. Chen Jin presume that the pathological basis of chronic hepatitis B can be summarized as depression, wet, heat, poison, phlegm, stasis, weak; Disease localization is mainly in the liver, involving the spleen and kidney. Pei Jianhong presumes the cause of chronic hepatitis B epidemic course ought to YiQi from the theory of traditional Chinese medicine. The pathological properties is hot and humid YiQi attack, damage liver and spleen, cause injuries to yin and yang, qi and blood, and the progression of the disease relapse during the evolution of the intricately illness.Chen Huadong et al presume that the acute phase of viral hepatitis is base on wet and heat; chronic phase due to incurable disease, mistreatment and long term therapy. They usually use HuoXueQuSi drug, ZhiBuGanShen drug, JianPiYiQi drug to treat this disease.Chinese medicine has fewer side effects and long-term medication characteristics. In recent years, treatment of Chinese medicine in HBV infection achieved good effect.Polysaccharide referred to as polysaccharides, often composed by more than one hundred and even thousands of simple sugars through glycosidic, its nature has been very different from simple sugars, such as sweet has basically disappeared, widely found in animal and plant cell membrane, microorganisms of the cell wall, one of the four basic material of constitute life, and is closely related to the maintenance of life functions. So far, hundreds of kinds of polysaccharides have been isolated and purified from nature, polysaccharide is not only an important component of living organisms, but also has a wide range of biological activity, such as immune regulation, anti-inflammatory, anti-virus, anti-tumor, anti-coagulation, anti-oxidation, anti-radiation, anti-aging, lowering blood sugar and blood fat, liver protection, anti-parasites. Polysaccharide as non-cell-toxic substances has almost innocuity effects on normal cells.Many polysaccharide has been carried out assaying, aconitane polysaccharide, east angelica polysaccharide, Dendrobium polysaccharide, Campanulaceae polysaccharide, ginseng polysaccharides, thistle polysaccharide, Bupleurum polysaccharide, grifola polysaccharide, red cone polysaccharide, Dodder polysaccharide, placenta polysaccharide, Codonopsis polysaccharide, hard-shelled clam polysaccharide, Chinese Pholidota Pseudobulb or Herb polysaccharide, Desertliving Cistanche Herb polysaccharide, Sterculia lychnophora polysaccharide, Siberian Solomonseal Rhizome polysaccharide, Cynanchum auriculatum Royle ex Wight polysaccharide, Tatarinow Sweetflag Rhizome polysaccharides, seaweed polysaccharides, False-yellowflower Milkwort Root polysaccharide, Indian Pokwees Root Polysaccharide, Roxburgh Anoectochilus Herb polysaccharide, Ganoderma lucidum polysaccharides, Spreading Hedyotis Herb polysaccharide, Mi Tang polysaccharides,aloe polysaccharide,Common Yam Rhizome polysaccharide,jujube polysaccharide and so on. Astragalin polysaccharide, grifola polysaccharide, pachyman polysaccharide, Lentinan polysaccharide has been applied to achieve better clinical efficacy. Documents has been reported a number of polysaccharides have a protective effect on the liver, and their role of protecting liver is more closely with immune factors, for example:Cordyceps polysaccharide on immunological liver injury in rats, Tall Gastrodia Rhizome polysaccharide GEP-2 against BCG+ LPS-induced immune liver injury in rats, garlic polysaccharide C on immunological liver injury in mice peripheral blood lymphocyte subsets, Sweet Porphyra polysaccharides on hepatic injury of mice, Haloxylon grape polysaccharides’ protective effect on immunological liver injury in rats in vitro.Salvia miltiorrhiza belongs to the plant family oregano’s dried root and rhizome, sexual bitter, slightly cold, ascription heart and liver. It was first indexed in the Shen Nong’s Materia Medica as a promoting blood flow-removing blood stasis and tonic herb of the nontoxic superior class. It is a erect perennial herb, high 40-80cm, whole-plant density is yellow-white hair and soft glandular hairs; roots fleshy, cylindrical, red skin. Salvia miltiorrhiza has effects of promoting blood flow-removing blood stasis, inducing menstruation to stop pain, clearing heat tranquilization. It has better efficacy of curing postamenorrhea edema, freshing new, activating blood circulation to dissipate blood stasis. And therefore, Salvia miltiorrhiza is considered equivalently with the SiWu tang, and is widely used in clinical.Modern studies have shown that the effective chemical composition of fat-soluble pigment phenanthraquinone tanshinone classes (such as tanshinone II A, etc.), and some water-soluble phenolic compounds (such as Salvia miltiorrhizanolic acid, protocatechualdehyde, etc.), both water-soluble and fat-soluble constituents have pharmacologically active. Because Salvia miltiorrhiza and its compound preparation have a good effect on many diseases such as coronary heart disease, stroke, hepatitis, cancer, diabetes. At domestic and abroad, Salvia miltiorrhiza’s chemical composition, pharmacological effects, clinical application, formulation, quality control and other aspects have made remarkable progress.Our research group extracts the polysaccharide components from Salvia miltiorrhiza when carrying out drug ingredients separate experiments. We found that polysaccharide as important and effective parts of Salvia miltiorrhiza have been studied less. Current reported method of Salvia miltiorrhiza polysaccharides extraction is water extraction and alcohol precipitation, the shortcomings of its extraction method is complicated to operate, low rates of polysaccharide extraction, and the efficacy and pharmacological research of Salvia miltiorrhiza polysaccharides have been studied less. Our research group’s purpose is to find a method to extract Salvia miltiorrhiza polysaccharide with a high rate, low cost,easy-to-extraction. Because of most the efficacy of polysaccharides and Salvia miltiorrhiza’s clinical application, we will study Salvia miltiorrhiza polysaccharides on immunological liver injury in rats and the immune regulatory mechanisms as a preliminary research in order to establish its further research and development of theoretical and experimental basis.Objectives:Through the experimental research of extraction technology, immunological liver injury and its immune regulation mechanism to provide a polysaccharide extracted method from Salvia miltiorrhiza, and to clarify its anti-immune liver injury and the role of the immune adjustment mechanism of Salvia miltiorrhiza polysaccharides to establish the experimental basis for further development and utilization.Methods:1.Extraction of Salvia miltiorrhiza polysaccharides1.1 Crush:The Salvia miltiorrhiza is crushed to 70～90μm particle size of the powder.1.2 Degreasing:The Salvia miltiorrhiza powder with 10 times the volume of the concentration that is greater than or equal to 85% ethanol, reflux 2 hours, filter, take residues to dry and to reserve.1.3 Enzyme treatment:The treatments of Salvia miltiorrhiza degreasing powder added to the water, then add enzymes, and then adjust pH to 5.5,at 70℃for 1 hour; The enzymes could be one of cellulase, papain, pectinase, and the dosage is the weight of 1.5% Salvia miltiorrhiza.The enzymes could be two or more of cellulase, papain, pectinase, and each enzyme dosage is the weight of 1% Salvia miltiorrhiza. The best method is cellulase and papain, and each enzyme dosage is the weight of 1.5% Salvia miltiorrhiza.1.4 Ultrasonic extraction:Under the 59 kHz of ultrasound, filtration, collecting the filtrate, the filtrate concentrated to 1:2 (equivalent to 1 gram per 2ml physic liquor).1.5 Alcohol precipitation:Add 70ml ethanol to the filtrate for the density to 85%, filter ethanol for gaining crude Salvia miltiorrhiza polysaccharide, and then wash and gain Salvia miltiorrhiza polysaccharides,75℃vacuum drying.2. This study was designed to evaluate the hepatoprotective effects of Salvia miltiorrhiza polysaccharides in immunological liver injury induced by Bacille-Calmette-Guerin (BCG) and lipopolysaccharide (LPS) in mice.2.1 Experiment groupingKunming mice, SPF grade,18～22g,60 (purchased from the Laboratory Animal Center of Southern Medical University). A randomized method was divided into normal group, model group, Salvia miltiorrhiza polysaccharide high-dose (360mg/kg), Salvia miltiorrhiza polysaccharide middle-dose(180mg/kg), Salvia miltiorrhiza polysaccharide low-dose (90mg/kg) dose group, Bifendate group (200mg/kg) (n=10 in each group), evenly divided male and female. In addition to normal mice, the remaining mice were gavage and the dose was 0.5ml/20g. Normal mice were given the same dose of saline.2.2 Method0.2mL BCG culture (approximately 5×107 viable units per mouse) was injected via the tail vein into mice, with the exception of the control group, who received saline alone. The control group was given an equal volume of saline. Twelve days later, the mice were given 7.5μg of LPS in 0.2ml saline (control received saline alone) via lateral tail vein injection.2.3 Index Detection2.3.1 The mice were weighed and the blood was collected from mice orbit, the blood was centrifugated to obtain serum, according to kit instructions to study serum ALT, AST,NO activity.2.3.2 After the mice were killed and immediately took out the necessary liver,0.25g liver tissue was obtained and the blood were washed away with cold saline, dubbed into 10% liver tissue homogenate, homogenate centrifugated for 20min at 500 rotation speed, then at 3000 rotation speed for 15min, getted the supernatant, ELISA to detected TNF-a, IL-1βlevels.2.3.3 Liver tissue fixation, sectioning and liver tissue pathological classificationAnimals were killed, taken out the liver, washed floating blood with cold saline for drying. The right hepatic leaves were drawn into four. Each block size was about 1.5cm×1.5cm×0.4cm, and immediately fixed with 10% neutral formalin.70%, 80%,90%,95%,100% ethanol dehydration, xylene transparent, embedded in paraffin, slicing machine for 4μm slices, conventional dewaxing, HE staining, neutral gum mounting. As well as taked pictures with PM-20 type microscope camera system, observed with a microscope.Using light microscope to examine, the degree of liver damage was categorized according to the degree of liver necrosis rating:"-",no significant pathological changes in liver tissue;"+",part of the scattered point-like necrosis, a little periportal infiltration of inflammatory cells;"++",liver cell necrosis and swelling and scattered granuloma formation can be seen, periportal inflammatory cell infiltration;"+++",a large number of liver cell swelling,necrosis, and granuloma formation of periportal areas is surrounded by a large number of inflammatory cell infiltration.3.Salvia miltiorrhiza polysaccharides on immunomodulatory mechanism3.1 BALB/c mice, SPF grade, purchased from Experimental Animal Center of Sun Yat-sen. Separated BALB/c mouse spleen cells, at different concentrations (1,10,50,100,200 mg/1) of Salvia miltiorrhiza polysaccharides on lymphocytes (concentration of 1×106 cell/ml) for 48h, MTT assayed cell proliferation.3.2 Separated B, T lymphocytes, at different concentrations(1,10,50,100,200 mg/1) of Salvia miltiorrhiza polysaccharides respectively on B, T lymphocytes (concentration of 1×106cell/ml) for 48h and with the mitogen ConA (T cell),LPS (B cells) (final concentration 5mg/l) for comparison, MTT assayed cell proliferation.3.3 Used different concentrations(1，10，50，100，200, mg/l) of Salvia-miltiorrhiza polysaccharides on T lymphocytes for 72h, streaming technology assayed CD4+ and CD8+.3.4 200mg/l concentration of Salvia miltiorrhiza polysaccharides acted on T cells at different time(12,24,48,72 h), ELISA analyzed IL-2 and IL-4 concentration.3.5 200mg/l concentration of Salvia miltiorrhiza polysaccharides acted on T cells at different time (24,48,72 h), fluorescence quantitative PCR analyzed IL-2, IL-4.4. Statistical analysisAll experiments quantitative data were expressed as mean±S.D and assessed by the one-way analysis of variance (ANOVA).If the variances between groups were homogenous (Levene’s test), groups were subjected to the multiple comparison Least Significant Differences (LSD) test. In case of no homogeneity variances, differences were evaluated by Welch and the groups were subjected to the multiple comparisons Dunnett’s T3 test. The Mann-Whitney rank-sum test was used for the degree of histopathological liver injury. The statistical significance between groups was indicated using superscript signs in the figures and tables. Significance was defined as P<0.05.Results:1.With the method of grinding, degreasing, enzyme treatment, ultrasonic extraction, alcohol precipitation, the polysaccharide was extracted from Salvia miltiorrhiza. Using orthogonal design method to optimize the ultrasound process conditions and ultimately by the statistical analysis to determine the optimal process conditions:The water is 12 times of the herbs weight, particle size is 20 mesh sieve, ultrasonic extraction time is 20min for the best extraction conditions.2.In the experiment of Salvia miltiorrhiza polysaccharide against immunological liver injury in mice. The serum levels of ALT, AST in Salvia miltiorrhiza polysaccharides high-dose, middle-dose, low-dose group compared with the model group were significantly different(P=0.000,P=0.003,P=0.044). Control group and model group were significantly different(P=0.000);The levels of serum NO and liver tissue homogenate IL-1β, TNF-a in Salvia miltiorrhiza polysaccharide high-dose group and model group were significantly different(P=0.000), and Salvia miltiorrhiza polysaccharides middle dose group compared with model group, TNF-a, IL-1βwere significantly different(P=0.048), but NO was not significantly different(P=0.057). Salvia miltiorrhiza polysaccharide low dose group compared with model group TNF-a(P=0.187), NO(P=0.097)and IL-1β(P=0.145)were not significantly different. Bifendate Pills group compared with model group NO was significantly different (P=0.037), but TNF-a(P=0.064)and IL-1β(P=0.882)were not significantly different; organ coefficient of Salvia miltiorrhiza polysaccharide high dose group and model group were significant difference(P=0.000,P=0.005,P=0.018). Salvia miltiorrhiza polysaccharide middle dose group compared with model group, liver index(P=0.043), spleen index(P=0.016)were significantly different. Salvia miltiorrhiza polysaccharide low dose group and model group showed no significant difference(P=0.178,P=0.175, P=0.329). Except spleen index(P=0.059), control group compared with model group were significantly different(P=0.000,P=0.042); Compared with the normal group, model group pathological changes in liver was significantly. Compared with model group, Salvia miltiorrhiza polysaccharide high dose group(P=0.032) and control group(P=0.016) pathological changes in liver were significantly. Compared with model group, Salvia miltiorrhiza polysaccharide middle dose group(P=0.240) and Salvia miltiorrhiza polysaccharide low dose group(P=0.547) showed no significant pathological changes in liver.3.In the experiment of Salvia miltiorrhiza polysaccharide on immunomodulatory mechanism, in the 10～200mg/l range of different doses of Salvia miltiorrhiza polysaccharides can significantly promote the proliferation of spleen cells, compared with the control group were significant difference(P=0.020,P=0.000), Salvia miltiorrhiza polysaccharides 200mg/l group showed higher activity; Experimental results showed that 100～200mg/l dose range Salvia miltiorrhiza polysaccharide group could significantly promote the proliferation of T lymphocytes, compared with the control group were significant difference (P=0.042,P=0.000), while mitogen ConA compared with the control group was significant difference(P=0.000). In the concentration of 50-200mg/l, Salvia miltiorrhiza polysaccharide group could significantly promote the proliferation of B lymphocytes,compared with-the-control group there was significant difference(P=0.033,P=0.000), while mitogen LPS and the control group were significant difference (P=0.000); 200mg/l concentrations of Salvia miltiorrhiza polysaccharides on mouse lymphocytes for 12h,24h,48h,72h, IL-2 levels increased,each time showed significant difference(P=0.000). IL-4 levels significantly increased, making comparisons between different time periods with a significant difference(P=0.005), but mildly compared with the levels of IL-2; Fluorescence quantitative PCR results showed that, after 200mg/l concentration of Salvia miltiorrhiza polysaccharides interfereing for 24h,48h,72h,IL-2 mRNA and IL-4 mRNA expression were differences; Compared with the control, IL-2 mRNA and IL-4mRNA expression increased after 72h, and the difference was statistical significance(P=0.022,P=0.036); IL-2 mRNA and IL-4 mRNA expression were no significant difference after 24h,48h(P=0.753,P=0.400,P=0.177,P=0.208). It was accounted that Salvia miltiorrhiza polysaccharides effectively increased IL-2 mRNA and IL-4 mRNA expression after interfereing for 72h; With the higher dose in 1～200mg/l range of Salvia miltiorrhiza polysaccharides, the ratio of CD4+, CD8+ gradual enhanced, showing a clear dose-dependent, while higher concentrations group compared with control group showed statistically significant differences(P=0.028,P=0.007,P=0.008,P=0.027,P=0.000).Conclusion:1.To determine the optimal extraction conditions of Salvia miltiorrhiza polysaccharides:The water was 12 times of the herbs weight, particle size was 20 mesh sieves, ultrasonic extraction time was 20 min for the best extraction conditions, and the extraction method had low energy consumption, high extraction rate, advantages of simple operation.2.The results showed that Salvia miltiorrhiza polysaccharides had the role of anti-immune liver injury. This role displayed not only in direct hepatoprotection, but also in regulatory role of immune function in liver injury, through the immune function further played the role of hepatoprotection.3.Salvia miltiorrhiza polysaccharides in vitro showed a stronger proliferation of lymphocytes in certain concentration,and also promoted expression and secretion of cytokines. Consistent with the results in vivo experiments to further clarify the Salvia miltiorrhiza polysaccharides had immune regulation function.更多还原