摘要
目的:研究不同相对分子质量黄芪多糖(APS)对哮喘小鼠肺组织炎症的抑制作用,并阐述其作用机制。方法:选取30只BALB/c雌性小鼠,随机分为正常对照组,哮喘模型组(模型组),低、中和高相对分子质量APS组,每组6只。模型组和APS组小鼠采用卵白蛋白(OVA)制备哮喘小鼠模型。低、中和高相对分子质量APS治疗组小鼠OVA雾化激发前30min给予腹腔注射0.1mL相对分子质量为4 500、15 000和30 000的APS,正常对照组小鼠采用等量生理盐水代替雾化致敏液和腹腔注射。雾化期间观察各组小鼠行为学变化,光学显微镜观察支气管肺泡灌洗液(BALF)中白细胞(WBC)总数和炎症细胞分类计数,HE染色观察各组小鼠肺组织病理形态表现,流式细胞小球微阵列术(CBA)检测小鼠血清和BALF中白细胞介素4 (IL-4)和γ-干扰素(IFN-γ)水平;提取分选脾脏CD4+T细胞,培养后流式细胞术检测Th1、Th2、Th17和Treg细胞比例,CBA检测细胞培养上清液中IL-4、IFN-γ、白细胞介素17 (IL-17)和白细胞介素10 (IL-10)水平。结果:与正常对照组比较,模型组小鼠出现明显打喷嚏、抓口鼻和气促等哮喘样症状,肺组织炎性浸润明显,气道黏膜水肿,平滑肌增厚,血清和BALF中IL-4水平明显升高(P<0.05),IFN-γ水平明显降低(P<0.05),细胞培养上清液中IL-4和IL-17水平明显升高(P<0.05),IFN-γ和IL-10水平明显降低(P<0.05),Th2和Th17细胞比例明显升高(P<0.05),Th1和Treg细胞比例明显降低(P<0.05)。与模型组比较,APS组小鼠抓口鼻、气促和烦躁等哮喘样症状有不同程度缓解,肺组织炎性细胞浸润和管壁增厚等明显改善,血清和BALF中IL-4水平明显降低(P<0.05),IFN-γ水平明显升高(P<0.05),细胞培养上清液中IL-4和IL-17水平明显降低(P<0.05),IFN-γ和IL-10水平明显升高(P<0.05),Th2和Th17细胞比例明显降低(P<0.05),Th1和Treg细胞比例明显升高(P<0.05)。不同相对分子质量APS组间比较,低相对分子质量APS组小鼠哮喘症状和肺组织炎症浸润改善最明显,血清和BALF中IL-4水平及Th2和Th17细胞比例降低最明显(P<0.05),血清和BALF中IFN-γ水平及Th1和Treg细胞比例升高最明显(P<0.05)。结论:APS通过调节Th1/Th2及Th17/Treg细胞平衡、降低IL-4和IL-17水平、增加IFN-γ和IL-10水平发挥抗哮喘作用,且低相对分子质量APS作用最明显。
Objective:To investigate the inhibitory effects of astragalus polysaccharides(APS)with different molecular weights on the pulmonary inflammation in the asthmatic mice,and to elucidate their mechanisms.Methods:A total of 30 female BALB/c mice were selected.They were randomly divided into normal control group,asthma model group(model group),low molecular weight APS(APS-low)group,midde molecular wieght APS(APS-middle)and high molecular weight APS(APS-high)group,with 6 mice in each group.The mouse models of asthma in model group and APS groups were established by injecting and inhaling ovalbumin(OVA).The mice in APS-low,APS-middle and APS-high groups were separately given 0.1 mL of 4 500,15 000,30 000 molecular weight APS for intraperitoneal injection before inhaled OVA.The mice in normal control group were treated with the same amount of normal saline instead of OVA injected and inhalled.The behavioral changes of the mice were observed during the atomization with OVA,and the total number of WBC and the counts of inflammatory cells in the bronchoalveolar lavage fluid(BALF)were observed with light microscope;the pathological changes of lung tissue was observed by HE staining.Cytometric Bead Array(CBA)method was used to detect the levels of IL-4 and IFN-γin the serum as well as BALF.The spleen CD4+T cells were extracted and cultured,and the ratios of Th1,Th2,Th17 and Treg cells were detected by flow cytometry.CBA method was used to detect the levels of IL-4,IFN-γ,IL-17,and IL-10 in the culture supernatant.Results:Compared with normal control group,the mice in model group showed asthma-like symptoms such as sneezing,snouting nose and shortness of breath,inflammatory infiltration of lung tissue,airway mucosal edema,smooth muscle thickening;the IL-4 levels in serum and BALF were increased(P<0.05),and the IFN-γlevels in serum and BALF were significantly decreased(P<0.05);IL-4 and IL-17 levels in cell culture supernatant were significantly increased(P<0.05),and the IFN-γand IL-10 levels were significantly decreased(P<0.05);the ratios of Th2 and Th17 cells were significantly increased(P<0.05),and the ratios of Th1 and Treg cells were significantly decreased(P<0.05).Compared with model group,the asthmatic symptoms of the mice in APS groups,for example,scratching nose and mouth,shortness of breath,and irritability,were relieved to varying degrees;the inflammatory cell infiltration and wall thickening were significantly improved;the levels of IL-4 in serum and BALF were significantly reduced(P<0.05),and the IFN-γlevels were increased(P<0.05);the levels of IL-4 and IL-17 in the culture supernatant were significantly reduced(P<0.05)and the levels of IFN-γand IL-10 were increased(P<0.05);the ratios of Th2 and Th17 cells were significantly decreased(P<0.05),and the ratios of Th1 and Treg cells were increased(P<0.05).Compared between three APS groups,the asthmatic symptoms and lung tissue inflammatory infiltration were lightened obviously in APS-low group,the IL-4 levels and the ratios of Th2 and Th17 cells in serum and BALF were significantly decreased(P<0.05),and the IFN-γlevels in serum and BALF and the ratios of Th2 and Th17 cells were significantly increased(P<0.05).Conclusion:APS can significantly improve the situation of airway inflammation infiltration and the symptoms of the asthmatic mice.APS plays a therapeutic role for asthma by regulating the Th1/Th2 and Th17/Treg cell balance,decreasing the levels of IL-4 and IL-17 and increasing the levels of IFN-γand IL-10 level;the low molecular weight APS shows an obvious effect.
引文
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