[Tetrapeptide repeat domain 36 (TTC36) inhibits the inflammatory response of HK2 cells by enhancing the expression of IκBα to inhibit the activation of NF-κB signaling pathway].

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.