匹多莫德抑制地塞米松诱导的小鼠隐性弓形虫感染的活化

Pidotimod Inhibits Activation of Latent Toxoplasma gondii Infection Induced by Dexamethasone in Mice

目的观察匹多莫德(PT)对经地塞米松(Dem)诱导小鼠隐性弓形虫感染活化的抑制作用。方法96只BALB/c小鼠每鼠灌胃感染弓形虫TgCtwh6株包囊30个,感染后4周随机均分为3组:Dem组(A组)腹腔注射地塞米松6 mg/(kg·d),同时灌胃生理盐水200μl;Dem+PT组(B组)腹腔注射地塞米松6 mg/(kg·d),同时灌胃匹多莫德100 mg/(kg·d);对照组(C组)腹腔注射生理盐水200μl,同时灌胃生理盐水200μl。上述药物使用时均溶于200μl生理盐水中。3组小鼠均连续注射和灌胃5周。自治疗开始后每周各组随机处死3只小鼠,取小鼠眼窦血、脑组织和脾组织备用。荧光定量PCR检测小鼠虫血症出现时间,以及脑组织虫荷和弓形虫DNA拷贝数。流式细胞术(FCM)对小鼠脾单个核细胞Th1、Th2和调节性T细胞(Treg)进行检测。使用微量样本多指标流式蛋白定量术试剂盒(CBA)检测小鼠血清中Th1和Th2类细胞因子的表达情况。结果 A组小鼠在受治第2周时,虫血症再次出现,5周内有50%小鼠出现虫血症,死亡17只。而B组小鼠在受治第3周时,虫血症再次出现,5周内有25%小鼠出现虫血症,死亡7只。C组小鼠未见虫血症出现,生存状态良好。A组小鼠连续给予Dem 21 d后,脑组织中弓形虫DNA拷贝数为(209±12)×109,而B组接受PT治疗后弓形虫DNA拷贝数为(62±10)×109,前者显著高于后者(P<0.01);FCM检测显示,小鼠给予Dem 21 d后,小鼠脾脏Th1、Th2和Treg细胞占CD4+细胞百分比,A组的分别为(4.0±1.5)%、(0.6±0.1)%和(5.0±0.9)%,B组分别为(6.1±1.0)%、(0.5±0.2)%和(7.0±1.2)%,A组和B组小鼠Th1和Treg细胞百分比差异有统计学意义(P<0.01)。血清细胞因子检测结果显示,小鼠血清Th1型细胞因子γ干扰素(IFN-γ)和肿瘤坏死因子-α(TNF-α),A组的[(2.2±0.7)pg/ml、(20.1±5.0)pg/ml]均低于B组的[(3.6±0.6)pg/ml、(32.0±8.0)pg/ml](P<0.01)。Th2型相关细胞因子白细胞介素-4(IL-4)和IL-10,A组的[(2.6±0.4)pg/ml、(39.0±6.0)pg/ml]与B组的[(2.7±0.7)pg/ml、(40.0±8.0)pg/ml]相比差异无统计学意义(P>0.05)。结论匹多莫德能够显著激活地塞米松诱导的免疫抑制,抑制地塞米松诱导的小鼠弓形虫隐性感染活化,其免疫调节机制有Th1和Treg细胞的参与。

Objective To study the inhibition effect of pidotimod （PT） on dexamethasone （Dem）-induced reactivated toxoplasmosis in mice. Methods A total of 96 female BALB/C mice were infected orally with 30 cysts of Toxoplasma gondii TgCtwh6 strain（genotype Chinese 1）. 4 weeks later the mice were divided into three groups （A, B, and C）. Mice of group A （Dem±NS） were given Dem [6 mg/（kg.d）] intraperitoneally and 200 μl normal saline given orally. Mice of group B （Dem±PT） were orally given pidotimod [100 mg/（kg·d）] and intraperitoneally injected with Dem[6 mg/（kg.d）] simultaneously. Each mouse in group C received 200 μl normal saline intraperitoneally. The mice were injected and given by gavage for 5 weeks. After treatment, three mice in each group were scarified weekly, and the survival time of the mice was recorded in days. Brain parasite burden and T. gondii DNA copies in serum were detected by quantitative real-time PCR. T cell sub- sets, cytokine profiles in each group were analyzed by flow cytometry, and CBA kit, respectively. Results On the secondweek after Dem administration, parasitemia appeared in group A; in 5 weeks 50% mice had parasitemia again, and 17 mice died. Comparatively, in group B parasitemia appeared on tile third week after PT and Dem administration, in 5 weeks 25% mice had parasitemia again, and 7 mice died. Parasitemia did not appear in Group C. On the 21st day after Dem adminis- tration, T. gondii DNA copies in brain tissues of group A was（209±12）xl09, significantly higher than（62±10）×109 in group B treated with PT （n=3, P〈0.01）. Flow cytometry test showed that on the 21st day after Dem adminisration, the proportions of Thl, Th2 and Treg cells in groups A and B were （4.0±1.5）% and （6.1±1.0）%, （0.6±0.1）% and （0.5±0.2）%, and （5.0±0.9）% and （7.0±1.2）%, respectively. There was siginifieant difference in the percentages of Thl and Treg between group B and A （P〈0.01）. The levels of IFNγ TNF-α in group A were （2.2±0.7） pg/ml and （20.1±5.0） pg/ml, respectively, lower than that of group B [（3.6±0.6） pg/ml and （32.0-±8.0）pg/ml] （P〈0.01）. No statistical significance was found in the levels of IL-4 and IL-10 between group A[（2.6±0.4） pg/ml, （39.0-±6.0） pg/ml] and group B [（2.7±0.7） pg/ml, （40.0±8.0） pg/ml] （P〉0.05）. Conclusion Pidotimod can inhibit activation of latent Toxoplasma gondii infection induced by dexamethasone in mice. Thl and Treg cells may contribute to the pidotinmd/dexamethasone-induced immunoregulation.