Chen Chuan-Bao, Liu Long-Shan, Zhou Jian, Wang Xiao-Ping, Han Ming, Jiao Xing-Yuan, He Xiao-Shun, Yuan Xiao-Peng
Third Division of Organ Transplant Center, Eastern Campus of First Affiliated Hospital, Sun Yat-Sen University, Guangzhou, China.
Second Division of Organ Transplant Center, First Affiliated Hospital, Sun Yat-Sen University, Guangzhou, China.
Cell Physiol Biochem. 2017;41(6):2447-2460. doi: 10.1159/000475914. Epub 2017 May 3.
BACKGROUND/AIMS: The aim of this study was to elucidate how high-mobility group box 1 (HMGB1) exacerbates renal ischemic-reperfusion injury (IRI) by inflammatory and immune responses through the toll-like receptor 4 (TLR4) signaling pathway.
A total of 30 wild-type (WT) mice and 30 TLR4 knockout (TLR4-/-) mice were selected and then randomly assigned to the Sham, I/R or HMGB1 groups. The serum and kidney tissues of all mice were collected 24 h after the perfusion. The fully automatic biochemical detector and ELISA were applied to determine the blood urea nitrogen (BUN) and serum creatinine (Scr) levels, and TNF-α, IL-1β, IL-6, IFN-γ and IL-10 levels, respectively. HE staining was used to evaluate kidney tissue damage, immunofluorescence and immunohistochemical staining were performed to observe CD68 and MPO cell infiltration, and flow cytometry was applied to detect immune cells. qRT-PCR and Western blotting were used to detect the expressions of TLR signaling pathway-related genes and proteins, respectively.
Compared with the Sham group, the levels of BUN, Scr, TNF-α, IL-1β, IL-6, IFN-γ and IL-10, kidney tissue damage score, CD68 and MPO cell infiltration, the numbers of immune cells, and the expressions of TLR signaling pathway-related genes and proteins in the I/R and HMGB1 groups were significantly up-regulated. In the I/R and HMGB1 groups, the levels of BUN and Scr, TNF-α, IL-1β, IL-6 and IFN-γ, kidney tissue damage score, CD68 and MPO cell infiltration, immune cell numbers, and TLR signaling pathway-related gene and protein expressions in the WT mice were all higher than those in the TLR4-/- mice, but IL-10 level was significantly lower. Similarly, all aforementioned indexes but IL-10 level in the WT and TLR4-/- mice were higher in the HMGB1 group than in the I/R group.
Our study indicated that the up-regulation of HMGB1 could exacerbate renal IRI by stimulating inflammatory and immune responses through the TLR4 signaling pathway.c.
背景/目的：本研究旨在阐明高迁移率族蛋白B1（HMGB1）如何通过Toll样受体4（TLR4）信号通路引发炎症和免疫反应，从而加剧肾脏缺血再灌注损伤（IRI）。
选取30只野生型（WT）小鼠和30只TLR4基因敲除（TLR4-/-）小鼠，然后随机分为假手术组、缺血再灌注组或HMGB1组。灌注24小时后收集所有小鼠的血清和肾脏组织。使用全自动生化检测仪和酶联免疫吸附测定法分别测定血尿素氮（BUN）和血清肌酐（Scr）水平，以及肿瘤坏死因子-α（TNF-α）、白细胞介素-1β（IL-1β）、白细胞介素-6（IL-6）、干扰素-γ（IFN-γ）和白细胞介素-10（IL-10）水平。采用苏木精-伊红（HE）染色评估肾脏组织损伤，进行免疫荧光和免疫组化染色观察CD68和髓过氧化物酶（MPO）细胞浸润情况，并应用流式细胞术检测免疫细胞。分别采用实时定量聚合酶链反应（qRT-PCR）和蛋白质免疫印迹法检测TLR信号通路相关基因和蛋白的表达。
与假手术组相比，缺血再灌注组和HMGB1组的BUN、Scr、TNF-α、IL-1β、IL-6、IFN-γ和IL-10水平、肾脏组织损伤评分、CD68和MPO细胞浸润、免疫细胞数量以及TLR信号通路相关基因和蛋白的表达均显著上调。在缺血再灌注组和HMGB1组中，野生型小鼠的BUN和Scr水平、TNF-α、IL-1β、IL-6和IFN-γ、肾脏组织损伤评分、CD68和MPO细胞浸润、免疫细胞数量以及TLR信号通路相关基因和蛋白表达均高于TLR4-/-小鼠，但IL-10水平显著降低。同样，野生型和TLR4-/-小鼠中，除IL-10水平外，HMGB1组上述所有指标均高于缺血再灌注组。
我们的研究表明，HMGB1的上调可通过TLR4信号通路刺激炎症和免疫反应，从而加剧肾脏IRI。
