Peng Rui, Yan Xin, Li Qianyin
The Ministry of Education Key Laboratory of Clinical Diagnostics, College of Laboratory Medicine, Chongqing Medical University, Chongqing 400016, China.
The Ministry of Education Key Laboratory of Clinical Diagnostics, College of Laboratory Medicine, Chongqing Medical University, Chongqing 400016, China, *Corresponding author, E-mail:
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2024 Aug;40(8):673-680.
Objective To investigate the effects and underlying mechanisms of tetratricopeptide repeat domain 36 (TTC36) on injury of HK2 renal tubular epithelial cell. Methods HK2 stable cell lines expressing either TTC36 and an empty vector control-CMV-Flag were generated with lentivirus . The mRNA expression level of tumor necrosis factor α (TNF-α), inducible nitric oxide synthase(iNOS), interleukin 6(IL-6), C-C motif chemokine ligand 2(CCL2), IL-1β, inhibitor of nuclear factor κB α(IκBα) and nuclear factor κB p65(NF-κB p65) were analyzed by real time quantitative PCR (qRT-PCR). Flow cytometry was used to quantify cell apoptosis. Cell proliferation was evaluated by using cell counting kit-8(CCK-8) assay. The protein expression levels of iNOS, TNF-α, caspase-3, cleaved-caspase-3(c-caspase-3), Bcl2 associated X protein(BAX), proliferating cell nuclear antigen (PCNA), zonula occludens 1(ZO-1), IκBα, NF-κB p65, and phosphorylated NF-κB p65(p-NF-κB p65) were determined by Western blot analysis. IκBα protein expression level was further analyzed by Western blot after being treated with cycloheximide (CHX) and MG132. Results Compared with the control group, the expression of inflammatory molecules were reduced after the overexpression of TTC36 in HK2 cells. TTC36 inhibited the apoptosis of HK2 cells, and the expression of apoptosis-related proteins c-caspase-3 and BAX were significantly decreased in the TTC36 overexpression group. Upregulation of TTC36 promoted cell proliferation and strengthened the expressions of PCNA and ZO-1. Meanwhile, the expression of IκBα was significantly increased, while that of NF-κB p65 and p-NF-κB p65 was markedly downregulated. Furthermore, TTC36 overexpression substantially prolonged the half-life of IκBα in HK2 cells after being treated with CHX. MG132 could restore the changes of IκBα caused by overexpression of TTC36. Conclusion Overexpression of TTC36 inhibits the inflammatory response of HK2 cells, reduces cell apoptosis, promotes proliferation, and strengthens tight junctions. The mechanism may be to inhibit the activation of NF-κB signaling pathway by enhancing the expression of IκBα, thereby reducing the cell damage caused by inflammatory response.
目的 探讨四肽重复结构域36（TTC36）对HK2肾小管上皮细胞损伤的影响及其潜在机制。方法 利用慢病毒构建表达TTC36的HK2稳定细胞系以及空载体对照-CMV-Flag。采用实时定量PCR（qRT-PCR）分析肿瘤坏死因子α（TNF-α）、诱导型一氧化氮合酶（iNOS）、白细胞介素6（IL-6）、C-C基序趋化因子配体2（CCL2）、IL-1β、核因子κBα抑制因子（IκBα）和核因子κB p65（NF-κB p65）的mRNA表达水平。运用流式细胞术定量细胞凋亡。使用细胞计数试剂盒-8（CCK-8）法评估细胞增殖。通过蛋白质免疫印迹分析检测iNOS、TNF-α、半胱天冬酶-3（caspase-3）、裂解的半胱天冬酶-3（c-caspase-3）、Bcl2相关X蛋白（BAX）、增殖细胞核抗原（PCNA）、紧密连接蛋白1（ZO-1）、IκBα、NF-κB p65和磷酸化的NF-κB p65（p-NF-κB p65）的蛋白表达水平。用放线菌酮（CHX）和MG132处理后，通过蛋白质免疫印迹进一步分析IκBα蛋白表达水平。结果 与对照组相比，HK2细胞中过表达TTC36后炎症分子表达降低。TTC36抑制HK2细胞凋亡，TTC36过表达组中凋亡相关蛋白c-caspase-3和BAX的表达显著降低。TTC36上调促进细胞增殖并增强PCNA和ZO-1的表达。同时，IκBα的表达显著增加，而NF-κB p65和p-NF-κB p65的表达明显下调。此外，CHX处理后，TTC36过表达显著延长了HK2细胞中IκBα的半衰期。MG132可恢复TTC36过表达引起的IκBα变化。结论 TTC36过表达抑制HK2细胞的炎症反应，减少细胞凋亡，促进增殖，并增强紧密连接。其机制可能是通过增强IκBα的表达抑制NF-κB信号通路的激活，从而减轻炎症反应引起的细胞损伤。
