Zhang Liukai, Shao Dingwei, You Junyao, Liu Penggang, Deng Fang, Zhang Jianping
College of Life Sciences and Technology, Tarim University, Alar, Xinjiang, China.
Xinjiang Production & Construction Corps Key Laboratory of Protection and Utilization of Biological Resources, Tarim University, Alar, Xinjiang, China.
Biochem Genet. 2024 Sep 30. doi: 10.1007/s10528-024-10925-z.
Lepus yarkandensis live year-round in harsh desert environments and are less susceptible to enteritis. The living conditions of Oryctolagus cuniculus in captivity were suitable, but they were highly susceptible to death by Gram-negative bacteria infected with inflammatory bowel disease complex.TLR4 is closely related to the occurrence of enteritis, and the neighbor-joining topology based on the 12S rDNA sequences showed that the relationship between O. cuniculus and L. yarkandensis is as high as 98%.Therefore, we chose O. cuniculus and L. yarkandensis for comparative study.The purpose of this study was to investigate the role of Toll-like receptor 4 (TLR4) in the regulation of immunity and inflammation in the intestinal tract of L. yarkandensis. In this study, the TLR4 gene was cloned for the first time in the colon of L. yarkandensis. The expression of TLR4 in the intestinal tissues of L. yarkandensis and O. cuniculus was detected by histological observation, real-time fluorescence quantification PCR(qRT-PCR), and protein blotting (Western blot).An LPS-induced cell inflammation model was constructed in vitro, and ELISA was used to examine the effect of pEGFP-N1-TLR4 and siRNA knockout on the anti-inflammatory ability of the TLR4 gene. The results showed that the open reading frame of the L. yarkandensis TLR4 gene was 2520 bp in length. Compared with the sequence of O. cuniculus, there were 15 differences in the TLR4 amino acid sequence of L. yarkandensis, 12 of which occurred in the LRR domain and 2 in the TIR domain, and the sequence changed from G to D at position 298. Immunohistochemistry showed that TLR4 was mainly expressed in the epithelial cells of the colon L. yarkandensis, and the expression level of TLR4 in the cecum and colon was significantly lower compared with that of O. cuniculus. qRT-PCR and Western blot results showed that the expression level of TLR4 in the colon of L. yarkandensis was significantly lower than that of O. cuniculus. At the cellular level, ELISA showed that overexpression of the TLR4 protein in L. yarkandensis could reduce the LPS-induced inflammatory response. Therefore, according to the above results, the protein structure and function of L. yarkandensis TLR4 may be different due to the change of nucleotide, which affects its binding with LPS and the activation of downstream molecules, so that L. yarkandensis is not prone to enteritis and can adapt to the harsh desert environment for a long time. This study also laid the foundation for improving the disease resistance of O. cuniculus and promoting the development and utilization of genes in L. yarkandensis.
塔里木兔常年生活在恶劣的沙漠环境中，对肠炎的易感性较低。圈养穴兔的生活条件适宜，但它们极易死于感染炎症性肠病复合体的革兰氏阴性菌。Toll样受体4（TLR4）与肠炎的发生密切相关，基于12S rDNA序列的邻接法拓扑结构显示，穴兔与塔里木兔之间的关系高达98%。因此，我们选择穴兔和塔里木兔进行比较研究。本研究的目的是探讨Toll样受体4（TLR4）在塔里木兔肠道免疫和炎症调节中的作用。在本研究中，首次在塔里木兔的结肠中克隆了TLR4基因。通过组织学观察、实时荧光定量PCR（qRT-PCR）和蛋白质印迹法（Western blot）检测了塔里木兔和穴兔肠道组织中TLR4的表达。体外构建了脂多糖（LPS）诱导的细胞炎症模型，并用酶联免疫吸附测定（ELISA）检测了pEGFP-N1-TLR4和小干扰RNA（siRNA）敲除对TLR4基因抗炎能力的影响。结果表明，塔里木兔TLR4基因的开放阅读框长度为2520 bp。与穴兔的序列相比，塔里木兔TLR4氨基酸序列有15处差异，其中12处发生在富含亮氨酸重复序列（LRR）结构域，2处发生在TIR结构域，第298位的序列由G变为D。免疫组织化学显示，TLR4主要在塔里木兔结肠的上皮细胞中表达，与穴兔相比，盲肠和结肠中TLR4的表达水平显著降低。qRT-PCR和Western blot结果显示，塔里木兔结肠中TLR4的表达水平显著低于穴兔。在细胞水平上，ELISA显示塔里木兔中TLR4蛋白的过表达可以降低LPS诱导的炎症反应。因此，根据上述结果，塔里木兔TLR4的蛋白质结构和功能可能由于核苷酸的变化而不同，这影响了其与LPS的结合以及下游分子的激活，从而使塔里木兔不易患肠炎，并能长期适应恶劣的沙漠环境。本研究也为提高穴兔的抗病能力和促进塔里木兔基因的开发利用奠定了基础。
