桑黄酸性多糖对D-半乳糖诱导小鼠的免疫调节作用研究

Immunomodulatory effect of phellinus igniarius acid polysaccharide on D-galactose induced mice

目的:探讨桑黄酸性多糖(PIP-A)对D-半乳糖(D-gal)诱导小鼠的免疫调节作用。方法:采取水提醇沉法提取桑黄总多糖(PIP),并利用DEAE-纤维素柱(7.5 cm×30 cm,CI型)进行分级,获得PIP-A并测定其单糖组成;采用D-gal(200 mg/kg)构建小鼠衰老模型,将小鼠随机分成4组,分别为空白组(CON组,灌胃蒸馏水,背颈部皮下注射生理盐水)、模型组(MOD组,灌胃蒸馏水,背颈部皮下注射200 mg/kg D-gal)、阳性对照组(PC组,灌胃800 mg/kg吡拉西坦,背颈部皮下注射200 mg/kg D-gal)、PIP-A给药组(PIP-A,灌胃800 mg/kg PIP-A,背颈部皮下注射200 mg/kg D-gal),连续给药10周。造模结束后测定小鼠胸腺和脾脏脏器指数;制备小鼠脾淋巴细胞悬液,CCK-8法测定Con A诱导的小鼠脾淋巴细胞增殖转化能力;ELISA法测定血清中TNF-α、IL-6含量;通过HE染色观察小鼠脾脏病理学变化;采用RT-PCR法检测TLR4、MyD88、TRAF6、NF-κB、P-C-Jun因子的mRNA表达水平。结果:PIP-A主要由葡萄糖和甘露糖组成,其糖含量为64.64%,糖醛酸含量为30.30%;与MOD组相比,PIP-A组能增加小鼠的胸腺和脾脏指数,并且促进小鼠脾淋巴细胞增殖,提高小鼠血清中TNF-α、IL-6的含量;经PIP-A处理后,脾脏的显微照片显示红髓和白髓边界清晰,脾淋巴细胞增加;PIP-A显著提高TLR4、MyD88、TRAF6、NF-κB、P-C-Jun的mRNA表达水平。结论:PIP-A通过调节TLR4信号通路相关因子表达能够促进小鼠淋巴细胞增殖,对D-gal诱导小鼠具有免疫调节增强作用。

Objective:To investigate the immunoregulatory effect of Phellinus igniarius acid polysaccharide(PIP-A)on D-galactose(D-gal)induced mice.Methods:The crude polysaccharide of Phellinus(PIP)igniarius was extracted by the method of water extraction and alcohol precipitation.The acid polysaccharide of Phellinus igniarius(PIP-A)was obtained by DEAE cellulose column(7.5 cm×30 cm,CI type)and its monosaccharide composition was determined.The aging model of mice was constructed by D-gal(200 mg/kg).The mice were randomly divided into four groups:Blank group(CON group,gavage distilled water,subcutaneous injection of normal saline into the back and neck),model group(MOD group,gavage distilled water,subcutaneous injection of 200 mg/kg D-gal into the back and neck),positive control group(PC group,gavage 800 mg/kg piracetam,subcutaneous injection of 200 mg/kg D-gal into the back and neck),Phellodendron acid polysaccharide administration group(PIP-A,800 mg/kg PIP-A by gavage and200 mg/kg D-gal by subcutaneous injection in the back and neck)for 10 weeks.At the end of modeling,the indexes of thymus and spleen were measured;the proliferation and transformation ability of spleen lymphocytes induced by Con A was measured by CCK-8 method;the contents of TNF-αand IL-6 in serum were measured by ELISA method;the pathological changes of spleen were observed by HE staining;the mRNA expression levels of TLR4,MyD88,TRAF6,NF-κB and P-C-Jun were detected by RT-PCR method.Results:The PIP-A was mainly composed of glucose and mannose,the content of which was 64.64%and the content of glucuronic acid was 30.30%.Compared with the MOD group,PIP-A group could increase the thymus and spleen index of mice,promote the proliferation of spleen lymphocytes and increase the content of TNF-αand IL-6 in serum of mice.After PIP-A treatment,the micrograph of spleen was obtained.The results showed that the boundary of red pulp and white pulp was clear,the spleen lymphocyte was increased,and the expression levels of TLR4,MyD88,TRAF6,NF-κB,P-C-Jun mRNA were significantly increased.Conclusion:PIP-A of can promote the proliferation of lymphocytes in mice and enhance the immune regulation of mice induced by D-gal,which may be achieved by regulating the expression of TLR4 signal pathway related factors.