黄芪多糖对脓毒症小鼠肺组织炎症因子水平和细胞免疫的影响

Effect of Astragalus Polysaccharide on Inflammatory Cytokines and Cellular Immunity in Lung Tissue of Mice with Sepsis

目的观察黄芪多糖对脓毒症小鼠肺组织炎症因子水平和细胞免疫的影响。方法将90只C57BL/6J雄性小鼠随机分为假手术组、模型组、低剂量组、中剂量组和高剂量组,每组各18例。低剂量组、中剂量组和高剂量组连续6 d腹腔注射剂量为100 mg/kg、200 mg/kg和400 mg/kg的黄芪多糖(APS),第7天腹腔注射10 mg/kg的脂多糖(LPS);模型组连续6 d腹腔注射等体积的生理盐水,第7天腹腔注射10 mg/kg的LPS;假手术组连续7 d腹腔注射等体积的生理盐水。采用HE染色观察小鼠肺组织病理改变。采用酶联免疫试剂盒(Enzyme-Linked ImmunoSorbent Assay,ELISA)检测小鼠血清肿瘤坏死因子-α(Tumor necrosis factor,TNF-α)、白细胞介素-2(Interleukin-2,IL-2)、白细胞介素-4(Interleukin-4,IL-4)、白细胞介素-17(Interleukin-17,IL-17)、白细胞介素-10(Interleukin-10,IL-10)、转化生长因子-β(Transforming growth factor,TGF-β)水平。流式细胞术检测小鼠外周血Th1细胞、Th2细胞、Th17细胞、Treg细胞水平。采用western blot检测小鼠肺组织NF-κB磷酸化水平。结果病理检查结果显示,假手术组小鼠肺泡和细胞结构完整,模型组小鼠肺组织水肿严重,表面呈片状或点状出血,肺泡间隔断裂,并有大量炎性细胞浸润,低剂量组、中剂量组和高剂量组随着APS给药的增加,肺水肿和出血情况减轻,肺泡结构趋于正常,炎性细胞浸润程度下降。与假手术组比较,模型组小鼠IL-4、IL-17、TNF-α含量上升,IL-2、IL-10、TGF-β含量下降,差异有统计学意义(P<0.05);与模型组比较,低剂量组、中剂量组和高剂量组小鼠IL-4、IL-17、TNF-α含量降低,IL-2、IL-10、TGF-β含量回升,且呈明显的剂量依赖性,差异有统计学意义(P<0.05)。与假手术组比较,模型组小鼠Th2和Th17细胞水平上升,Th17/Treg比值上升;Th1和Treg细胞水平下降,Th1/Th2比值下降,差异有统计学意义(P<0.05);与模型组比较,低剂量组、中剂量组和高剂量组小鼠Th2细胞、Th17细胞、Th17/Treg比值剂量依赖性降低,Th1细胞、Treg细胞、Th1/Th2比值剂量依赖性上升,差异有统计学意义(P<0.05)。与假手术组比较,模型组小鼠肺组织IκB、NF-κB的磷酸化水平升高,差异有统计学意义(P<0.05);与模型组比较,低剂量组、中剂量组和高剂量组小鼠肺组织IκB、NF-κB的磷酸化水平剂量依赖性下降,差异有统计学意义(P<0.05)。结论黄芪多糖可通过抑制NF-κB的磷酸化下调脓毒症小鼠体内炎症反应,并改善细胞免疫的失衡。

Objective To observe the effect of Astragalus polysaccharide(APS)on inflammatory cytokines and cellular immunity in the lung tissue of mice with sepsis.Methods Ninety C57 BL/6 J male mice were randomly assigned into a sham group,a model group,and low-,medium-,and high-dose APS(L-APS,M-APS,and H-APS,respectively)groups,with 18 mice in each group.The mice in the L-APS,M-APS,and H-APS groups were injected intraperitoneally with APS at doses of 100 mg/kg,200 mg/kg,and 400 mg/kg,respectively,for 6 days and 10 mg/kg lipopolysaccharide(LPS)on day 7.The model group was injected with an equal volume of normal saline for 6 days,and 10 mg/kg LPS on day 7.The sham group was injected with equal volume of normal saline for 7 days.Hematoxylin-eosin(HE)staining was used for observation of the pathological changes of mouse lung tissue.The serum levels of interleukin-2(IL-2),tumor necrosis factor-alpha(TNF-α),IL-4,IL-10,IL-17,and transforming growth factor-beta(TGF-β)were measured by enzyme-linked immunosorbent assay(ELISA).The proportions of Th1,Th2,Th17,and Treg cells in peripheral blood were detected by flow cytometry.Western blotting was employed to determine the phosphorylation level of nuclear factor-κB(NF-κB)in the lung tissue.Results The results of pathological examination showed that the alveolar and cellular structures of mice in the sham group were intact.The model group presented severe edema in the lung tissue,patchy or scattered hemorrhage on the surface,broken alveolar septum,and massive infiltration of inflammatory cells.With the increase of APS administration in L-APS,M-APS,and H-APS groups,pulmonary edema and bleeding were alleviated,alveolar structure tended to be normal,and the degree of inflammatory cell infiltration was decreased.Compared with the sham group,the model group showed elevated the serum levels of IL-4,IL-17,and TNF-αand lowered those of IL-2,IL-10,and TGF-β(P<0.05).Compared with the model group,the serum levels of IL-4,IL-17 and TNF-αin the lL-APS,M-APS,and H-APS groups were decreased,and the serum levels of IL-2,IL-10 and TGF-βwere increased,which showed a significant dose-dependent effect(P<0.05).Compared with those in the sham group,the Th2 cells,Th17 cells,and Th17/Treg ratio increased,while the Th1 cells,Treg cells,and Th1/Th2 ratio decreased in the model group(P<0.05).Compared with the model group,the Th2 cells,Th17 cells and Th17/Treg ratio of mice in the lL-APS,M-APS,and H-APS groups were decreased in a dose-dependent manner,while Th1 cells,Treg cells and Th1/Th2 ratio were increased in a dose-dependent manner,(P<0.05).Compared with the sham group,the model group showed up-regulated phosphorylation of inhibitor of NF-κB(IκB)and NF-κB(P<0.05).Compared with the model group,the phosphorylation levels of IκB and NF-κB in lung tissue of mice in the lL-APS,M-APS,and H-APS groups decreased in a dose-dependent manner,(P<0.05).Conclusion APS can alleviate the inflammation and the imbalance of cellular immunity in mice with sepsis by inhibiting the phosphorylation of NF-κB.