TRIM31 promotes apoptosis via TAK1-mediated activation of NF-κB signaling in sepsis-induced myocardial dysfunction

Abstract

Sepsis is a major condition caused by an overwhelming inflammatory response to an infection. Sepsis-induced myocardial dysfunction (SIMD) is a common complication in septic patients and a major predictor of morbidity and mortality. Here, we investigated the role of tripartite motif 31 (TRIM31) protein in sepsis progression in vitro and in vivo. Quantitative real-time PCR and western blot were used to detect the expression levels of relative genes and proteins. Cell proliferation and apoptosis were evaluated to determine cell viability. H&E and IHC staining were performed to examine morphological and pathological changes in mice. ELISA assay was used to detect inflammatory factors. TRIM31 was upregulated in septic patients compared with normal people. TRIM31 depletion reduced LPS-induced apoptosis whereas TRIM31 overexpression-elevated LPS-induced apoptosis. Furthermore, TRIM31 interacted with and ubiquitinated transforming growth factor-β-activated kinase-1 (TAK1), resulting in TAK1 activation and subsequent induction of NF-κB signaling. Of note, Trim31 depletion or blockade by PDTC treatment inhibited LPS-induced apoptosis in vivo. In conclusion, TRIM31 played an important role in SIMD by activating TAK1-mediated NF-κB signaling pathway.

Keywords: NF-κB; TAK1; TRIM31; sepsis.

Figure 1.

LPS-induced TRIM31 upregulation and cell apoptosis. (a) Real-time PCR was performed to measure the relative mRNA expression of TRIM31 in peripheral blood samples from 35 septic patients and 25 normal patients. *, P < 0.05, ***, P < 0.001 vs normal persons. (b) Cell proliferation was analyzed after stimulation with LPS (three repetitions). (c) Cell apoptosis was analyzed after LPS treatment. (d, e): The mRNA and protein levels of TRIM31 were measured by qRT-PCR (d) and Western blot (e). ***, P < 0.001 vs 0 μg/ml LPS

Figure 2.

TRIM31 knockdown inhibited cell apoptosis induced by LPS. (a, b): The mRNA and protein levels of TRIM31 were measured by qRT-PCR (a) and Western blot (b). (c): Cell apoptosis was measured by flow cytometry. (d): ELISA was used to measure the concentrations of IL-1β and TNF-α. (e): The protein levels of NF-κB signaling related protein were measured by Western blot

Figure 3.

TRIM31 elevated cell apoptosis induced by LPS. (a, b): AC16 cells were transduced with TRIM31 overexpression or vector lentivirus. Overexpression efficiency was analyzed by qRT-PCR (a) and Western blot (b). (c): Annexin V/PI assay was used to assess cell apoptosis. (d): ELISA was used to measure the concentrations of IL-1β and TNF-α. (e): The protein levels of NF-κB signaling related protein were measured by Western blot

Figure 4.

TAK1-mediated activation of NF-κB signaling pathway induced by TRIM31. (a): Western blot was used to determine the protein levels of TAK1 and p-TAK1. (b): Cell apoptosis was measured by flow cytometry. (c): The protein levels of cleaved-caspase-3, p-IKKβ and p-IκBα were measured by Western blot. (d): ELISA was used to measure the concentrations of IL-1β and TNF-α. (e): Co-immunoprecipitation assay was used to examine the interaction between TRIM31 and TAK1. (f): The ubiquitin level of TAK1 was measured

Figure 5.

TRIM31 deletion suppressed apoptosis in mice model of sepsis induced by LPS. (a) H&E staining was performed on heart tissue samples of animal models. (b) IHC was used to detect the protein level of NF-κB (c) TUNEL assay was performed to measure apoptosis in heart tissue samples of animal models. a, control; b, LPS + vector; c, LPS + shTRIM31; d, LPS + PDTC

Figure 6.

IHC was used to detect the protein levels of p-IKKβ (a) and p-IκBα (b). a, control; b, LPS + vector; c, LPS + shTRIM31; d, LPS + PDTC

Figure 7.

TRIM31 deletion blocked LPS-induced activation of NF-κB signaling pathway. (a): ELISA was used to measure the concentrations of IL-1β and TNF-α. (b) The protein levels of NF-κB signaling related protein were measured by Western blot