Immunomodulatory effect of pachymaran on cyclosporine A(CsA)-induced lung injury in mice

茯苓多糖对环孢菌素A所致的肺损伤小鼠的免疫调节作用

Objective To investigate the immunomodulatory effect of pachymaran on cyclosporine A(CsA)-induced lung injury in mice.Methods(i) Fifty male BALB/c mice were randomly divided into five groups(10 mice in each group): normal control(NC) group, 30, 45, and 60 mg/kg CsA groups, and lipopolysaccharide(LPS) group. Except for the NC group, other groups underwent CsA modeling. The NC group was treated with phosphate-buffered saline(PBS), the LPS group with 10 mg/kg LPS eight hours before mice euthanized, and the 30, 45, and 60 mg/kg CsA groups with corresponding doses of CsA for seven consecutive days. After treatment, the body and organ mass of each group were weighed, and the lung, thymus, and spleen indexes were calculated. Hematoxylin-Eosin(HE) staining was performed to observe histopathological changes in the lungs of the mice. The protein expression levels of interleukin(IL)-2 and IL-1β in the blood were detected using enzyme-linked immunosorbent assay(ELISA), and those of surfactant protein D(SP-D), IL-2, and IL-6 in lung tissues were detected by immunohistochemistry(IHC). The mRNA expression levels of SP-D, IL-1β, IL-6, and myeloperoxidase(MPO) in the lung tissues were detected by quantitative reverse transcriptase-polymerase chain reaction(q RT-PCR).(ii) Another 60 BALB/c mice were divided into six groups(10 mice in each group) : NC group,model control(MC) group, 50, 100, and 200 mg/kg pachymaran groups, and polyinosinicpolycytidylic acid [poly(I:C)] group. Except for the NC group, other groups underwent45 mg/kg CsA modeling. The NC and MC groups were treated with distilled water, the pachymaran groups with corresponding doses pachymaran, and the poly(I:C) group with 0.1 mg/kg poly(I:C) for seven days.The mice were euthanized to obtain tissues and serum for detection.Detection methods were identical to those described in(i) above.Results(i) CsA(30 mg/kg) increased the lung index of mice(P < 0.001), and decreased the spleen index(P < 0.01), thymus index(P < 0.05), and the serum level of IL-2(P < 0.05). CsA(45 mg/kg) decreased the spleen, thymus indexes, and the serum level of IL-2(P < 0.01) in mice, and increased the serum level of IL-1β(P < 0.05) and the protein level of lung SP-D(P <0.001). CsA(60 mg/kg) increased the lung index of mice(P < 0.01), the serum level of IL-1β(P < 0.05), the protein level of lung SP-D(P < 0.01), and the mRNA levels of lung MPO and SP-D( P < 0.05), and decreased the thymus index of mice(P < 0.01). HE staining showed that 30, 45, and60 mg/kg CsA, and LPS caused pathological changes in the lung tissue of mice.(ii) After pachymaran intervention in MC mice, the spleen and thymus indexes(P < 0.05) were increased in the 100 and 200 mg/kg pachymaran groups, and the lung index was decreased(P < 0.05).Moreover, 50 mg/kg pachymaran increased the thymus index(P < 0.05) and decreased the lung index(P < 0.01) in MC group. Pachymaran(50, 100, and 200 mg/kg) improved lung tissue injury, reduced the serum level of IL-1β(P < 0.001), and the mRNA levels of MPO and SPD in lung tissues(P < 0.05) of mice. Pachymaran(100 mg/kg) increased the protein level of lung IL-2(P < 0.01), decreased the protein level of lung SP-D(P < 0.01), and the mRNA level of IL-1β(P < 0.001) in the lung tissues of mice. Pachymaran(200 mg/kg) increased the serum level of IL-2(P < 0.01) and lung IL-6 of mice(P < 0.05). Pachymaran(50 and 200 mg/kg) increased the mRNA level of IL-6 in the lung tissues of mice(P < 0.05).Conclusion While the immune function of mice was suppressed by CsA, the lung tissue was also damaged. Pachymaran can improve the immunosuppression induced by CsA and improve the lung tissue injury in immunosuppressed mice.

目的 探讨茯苓多糖对环孢菌素A(Cs A)所致的肺损伤小鼠的免疫调节作用。方法 (i)将50只BALB/c小鼠随机分为5组,每组10只:正常对照(NC)组、30、45、和60 mg/kg Cs A组和脂多糖(LPS)组。除NC组,其他各组进行Cs A造模。NC组使用磷酸盐缓冲盐水(PBS);LPS组在安乐死前8小时注射10 mg/kg LPS;30、45和60 mg/kg Cs A组连续7天注射相应剂量的Cs A。上述处理后,称量小鼠体质量和脏器质量,计算肺指数、胸腺指数和脾指数。采用苏木精-伊红(HE)染色观察小鼠肺组织病理学变化;酶联免疫吸附法(ELISA)检测血清中白细胞介素(IL)-2和IL-1β蛋白表达水平;免疫组织化学染色(IHC)检测表面活性蛋白D(SP-D)、IL-2和IL-6的表达水平;定量逆转录-聚合酶链反应(q RT-PCR)检测肺组织中SP-D、IL-1β、IL-6和髓过氧化物酶(MPO)的表达水平。(ii)另取60只BALB/c小鼠,随机分为6组,每组10只:NC组、模型对照(MC)组、50、100和200 mg/kg茯苓多糖组和聚肌苷酸-聚胞苷酸[poly(I:C)]组,除NC组外,其他组均进行Cs A造模。NC和MC用蒸馏水处理,茯苓多糖各组用相应剂量的茯苓多糖处理,poly(I:C)组用0.1 mg/kg poly(I:C)处理,14 d后小鼠实施安乐死。检测方法与上述(i)中描述的方法相同。结果 30 mg/kg Cs A能增加小鼠肺指数(P <0.001),降低脾指数(P <0.01)、胸腺指数(P <0.05)和血清IL-2水平(P <0.05)。45 mg/kg Cs A能降低小鼠脾指数、胸腺指数和血清IL-2水平(P <0.01),升高血清IL-1β水平(P <0.001)和肺组织SP-D蛋白水平(P <0.05)。60 mg/kg Cs A可提高小鼠肺指数(P <0.01)、血清IL-1β水平(P <0.05)、肺组织SP-D蛋白水平(P <0.01)及肺组织MPO和SP-D mRNA水平(P <0.05),降低小鼠胸腺指数(P <0.01)。HE染色显示,30、45、60 mg/kg Cs A和LPS可引起小鼠肺组织的病理改变。茯苓多糖干预后,100和200 mg/kg剂量组小鼠的脾指数(P <0.05)和胸腺指数(P <0.05)增加,肺指数降低(P <0.05)。此外,50 mg/kg茯苓多糖还能增加小鼠的胸腺指数(P <0.05),降低肺指数(P <0.01)。50、100和200 mg/kg茯苓多糖可改善小鼠肺组织损伤,降低血清IL-1β水平(P <0.001),降低肺组织MPO和SP-D mRNA水平(P <0.05)。100 mg/kg茯苓多糖可增加小鼠肺组织IL-2蛋白水平(P <0.01),降低肺组织SP-D蛋白水平(P <0.01)和IL-1βmRNA水平(P <0.001)。200 mg/kg茯苓多糖可提高小鼠血清IL-2(P <0.01)和肺组织IL-6蛋白水平(P <0.05)。50和200 mg/kg茯苓多糖可增加小鼠肺组织IL-6 mRNA水平(P <0.05)。结论 Cs A所致小鼠免疫功能抑制的同时,肺组织也受到损伤。茯苓多糖可以纠正Cs A所致的免疫功能抑制,同时改善免疫抑制小鼠的肺组织损伤情况。