Yang Qing-Li, Shen Ji-Qing, Jiang Zhi-Hua, Shi Yun-Liang, Wan Xiao-Ling, Yang Yi-Chao
Guangxi Key Laboratory for Viral Hepatitis Prevention and Control, Guangxi Zhuang Autonomous Region Center for Disease Prevention and Control, Nanning, 530028, Guangxi, People's Republic of China.
Department of Parasitology, Guangxi Medical University, Nanning, 530021, Guangxi, People's Republic of China.
Parasit Vectors. 2017 Aug 7;10(1):379. doi: 10.1186/s13071-017-2318-y.
Although the responses of inducible nitric oxide synthase (iNOS) and associated cytokine after Clonorchis sinensis infection have been studied recently, their mechanisms remain incompletely understood. In this study, we investigated the effects of toll-like receptor 2 (TLR2) signals on iNOS/nitric oxide (NO) responses after C. sinensis infection. We also evaluated the correlations between iNOS responses and worm development, which are possibly regulated by TLR2 signal.
TLR2 wild-type and mutant C57BL/6 J mice were infected with 60 C. sinensis metacercariae, and the samples were collected at 30, 60, 90 and 120 days post-infection (dpi). The total serum NO levels were detected using Griess reagent after nitrate was reduced to nitrite. Hepatic tissue samples from the infected mice were sliced and stained with hematoxylin and eosin (HE) to observe worm development in the intrahepatic bile ducts. The iNOS mRNA transcripts in the splenocytes were examined by real time reverse transcriptase polymerase chain reaction (qRT-PCR), and iNOS expression was detected by immunohistochemistry.
Developing C. sinensis juvenile worms were more abundant in the intrahepatic bile ducts of TLR2 mutant mice than those of TLR2 wild-type mice. However, no eggs were found in the faeces of both mice samples. The serum levels of total NO significantly increased in TLR2 mutant mice infected with C. sinensis at 30 (t = 2.595, P = 0.049), 60 (t = 7.838, P = 0.001) and 90 dpi (t = 3.032, P = 0.029). Meanwhile, no changes occurred in TLR2 wild-type mice compared with uninfected controls during the experiment. The iNOS expression in splenocytes showed unexpected higher background levels in TLR2 mutant mice than those in TLR2 wild-type mice. Furthermore, the iNOS mRNA transcripts in splenocytes were significantly increased in the TLR2 wild-type mice infected with C. sinensis at 30 (t = 5.139, P = 0.004), 60 (t = 6.138, P = 0.002) and 90 dpi (t = 6.332, P = 0.001). However, the rising of iNOS transcripts dropped under the uninfected control level in the TLR2 mutant mice at 120 dpi (t = -9.082, P < 0.0001). Both total NO and iNOS transcripts were significantly higher in the TLR2 mutant mice than those in the TLR2 wild-type mice at 30 (t = 3.091/2.933, P = 0.027/0.033) and 60 dpi (t = 2.667/6.331, P = 0.044/0.001), respectively. In addition, the remarkable increase of iNOS expressions was immunohistochemically detected in the splenic serial sections of TLR2 wild-type mice at 30 and 60 dpi. However, the expressions of iNOS were remarkably decreased in the splenocytes of both TLR2 wild-type and mutant mice at 120 dpi.
These results demonstrate that TLR2 signal plays an important role in the regulation of iNOS expression after C. sinensis infection. TLR2 signal is also beneficial to limiting worm growth and development and contributing to the susceptibility to C. sinensis in which the iNOS/NO reactions possibly participate.
尽管近年来对中华分支睾吸虫感染后诱导型一氧化氮合酶（iNOS）及相关细胞因子的反应进行了研究，但其机制仍未完全阐明。在本研究中，我们调查了Toll样受体2（TLR2）信号对中华分支睾吸虫感染后iNOS/一氧化氮（NO）反应的影响。我们还评估了iNOS反应与虫体发育之间的相关性，这可能受TLR2信号调节。
将60只中华分支睾吸虫尾蚴感染TLR2野生型和突变型C57BL/6 J小鼠，并在感染后30、60、90和120天（dpi）采集样本。用格里斯试剂在硝酸盐还原为亚硝酸盐后检测血清总NO水平。将感染小鼠的肝组织样本切片，用苏木精和伊红（HE）染色，以观察肝内胆管中的虫体发育情况。通过实时逆转录聚合酶链反应（qRT-PCR）检测脾细胞中iNOS mRNA转录本，并通过免疫组织化学检测iNOS表达。
在TLR2突变型小鼠的肝内胆管中，发育中的中华分支睾吸虫幼虫比TLR2野生型小鼠的更丰富。然而，两组小鼠粪便中均未发现虫卵。感染中华分支睾吸虫的TLR2突变型小鼠在感染后30天（t = 2.595，P = 0.049）、60天（t = 7.838，P = 0.001）和90天（t = 3.032，P = 0.029）时血清总NO水平显著升高。同时，在实验期间，TLR2野生型小鼠与未感染对照相比无变化。TLR2突变型小鼠脾细胞中的iNOS表达显示出比TLR2野生型小鼠更高的背景水平。此外，感染中华分支睾吸虫的TLR2野生型小鼠在感染后30天（t = 5.139，P = 0.004）、60天（t = 6.138，P = 0.002）和90天（t = 6.332，P = 0.001）时脾细胞中iNOS mRNA转录本显著增加。然而，在感染后120天，TLR2突变型小鼠中iNOS转录本的升高降至未感染对照水平以下（t = -9.082，P < 0.0001）。在感染后30天（t = 3.091/2.933，P = 0.027/0.033）和60天（t = 2.667/6.331，P = 0.044/0.001）时，TLR2突变型小鼠中的总NO和iNOS转录本均显著高于TLR2野生型小鼠。此外，免疫组织化学检测发现在感染后30天和60天，TLR2野生型小鼠脾连续切片中iNOS表达显著增加。然而，在感染后120天，TLR2野生型和突变型小鼠脾细胞中的iNOS表达均显著降低。
这些结果表明，TLR2信号在中华分支睾吸虫感染后iNOS表达的调节中起重要作用。TLR2信号也有利于限制虫体生长发育，并可能参与iNOS/NO反应，从而影响对中华分支睾吸虫的易感性。
