| 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.
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