circ-Katnal1 Enhances Inflammatory Pyroptosis in Sepsis-Induced Liver Injury through the miR-31-5p/GSDMD Axis

Abstract

Background: Sepsis is a systemic inflammatory response that can elicit organ dysfunction as well as circulatory diseases in serious cases. When inflammatory responses are especially dysregulated, severe complications can arise, including sepsis-induced liver injury. Various microRNAs along with circular (circ) RNAs are involved in inflammatory responses; nevertheless, their functions in regulating sepsis-induced liver injury remain unknown. The cecal ligation and puncture (CLP) procedure can induce liver injury as well as polymicrobial sepsis.

Methods: In this study, CLP was used to induce liver injury as well as polymicrobial sepsis. Then, liver function, inflammatory cytokine expression, and hepatic histopathology were evaluated. High-throughput sequencing was employed to investigate the abnormal hepatic circRNA expression after CLP. Raw264.7 cells were utilized to simulation an in vitro sepsis inflammation model with LPS induce. The relative mRNA as well as protein levels of TNF-α, IL-1β, and IL-6 was explored by quantitative polymerase chain reaction (PCR) and enzyme-linked immunosorbent assays. We explored functional connections among circRNAs, miR-31-5p, and gasdermin D (GSDMD) using dual-luciferase reporter assays. Western blot was employed to test GSDMD, caspase-1, and NLRP3 expression in mice and cell models.

Results: Our results showed that CLP-induced sepsis promoted liver injury via increasing inflammatory pyroptosis. The abnormal expression of circ-Katnal1 played an important role in CLP-induced sepsis. Downregulating circ-Katnal1 suppressed LPS-induced inflammatory pyroptosis in Raw264.7 cells. Bioinformatics and luciferase reporter results confirmed that miR-31-5p and GSDMD were downstream targets of circ-Katnal1. Inhibiting miR-31-5p or upregulating GSDMD reversed the protective effects of silencing circ-Katnal1.

Conclusion: Taken together, circ-Katnal1 enhanced inflammatory pyroptosis in sepsis-induced liver injury through the miR-31-5p/GSDMD axis.

Figure 1

CLP-induced sepsis promoted liver injury by increasing inflammatory pyroptosis. Mice were sacrificed 12 h after CLP, and liver tissues and serum were collected for further analysis. (a, b) ALT and AST expression levels were measured using ELISA. Data were presented as mean ± SD. ^∗∗∗P < 0.001, versus the sham group. (c, d) HE and TUNEL staining showed the liver injury (magnification, ×200). (e)–(g) ELISA detection showed the expression of inflammatory factors TNF-α, IL-1β, and IL-6. Data were presented as mean ± SD. ^∗∗∗P < 0.001, versus the sham group. (h, i) Western blot detection showed GSDMD, caspase-1, and NLRP3 expression. Data were presented as mean ± SD. ^∗∗∗P < 0.001, versus the sham group.

Figure 2

Abnormal expression of circ-Katnal1 played an important role in CLP-induced sepsis. (a) Heat map showing the expression of circRNA following 12-h induction after CLP in liver tissues. (b) RT-qPCR detection showed the expression of mmu_circ_0012734, mmu_circ_0001432, mmu_circ_0012767, mmu_circ_0012771, and mmu_circ_0012774 in normal and CLP-induced mouse liver tissues. Data were presented as mean ± SD. ^∗∗∗P < 0.001, versus the sham group. (c) Genomic loci of the Katnal1 gene and mmu_circ_0001432 (circ-Katnal1). (d) The luciferase activity of circ-Katnal1 in HEK293 T cells transfected with different miRNA mimics, which were putative binding sites for the circ-Katnal1 sequence. Luciferase activity was normalized by Renilla luciferase activity. Data were presented as mean ± SD. ^∗∗∗P < 0.001. (e) RT-qPCR detection showed the expression of miR-31-5p in normal and CLP-induced mouse liver tissues. Data were presented as mean ± SD. ^∗∗P < 0.01 versus the sham group. (f, g) Prediction of the binding sites of miR-31-5p in circ-Katnal1. The MUT version of circ-Katnal1 is presented. The relative luciferase activity determined 48 h after the transfection of HEK293 T cells with miR-31-5p mimic/NC or circ-Katnal1 WT/Mut. Data were presented as means ± SD. ^∗∗P < 0.01. (h, i) Prediction of the binding sites of miR-31-5p in 3′UTR-GSDMD. The MUT version of 3′UTR-GSDMD is presented. Relative luciferase activity determined 48 h after the transfection of HEK293 T cells with miR-31-5p mimic/NC or 3′UTR-GSDMD WT/Mut. Data were presented as means ± SD. ^∗∗∗P < 0.001.

Figure 3

Downregulation circ-Katnal1 suppressed LPS-induced inflammatory pyroptosis in RAW264.7 cells. However inhibiting miR-31-5p or upregulating GSDMD reversed the protective effect after circ-Katnal1 silencing. (a)–(c) RT-qPCR detection showed the expression of circ-Katnal1, miR-31-5p, and GSDMD. (d)–(f) ELISA detection showed the expression of inflammatory factors TNF-α, IL-1β, and IL-6 under LPS condition (1 μg/mL). Data were presented as mean ± SD. ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001, versus sham NC. ^##P < 0.01, ^###P < 0.001, versus si-circ-Katnal1 (si-mmu_circ_0001432). (g)–(j) Western blot detection showed GSDMD, caspase-1, and NLRP3 expression. Data were presented as mean ± SD. ^∗∗∗P < 0.001, versus the sham group. Data were presented as mean ± SD. ^∗P < 0.05, ^∗∗∗P < 0.001, versus sham NC; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001, versus si-circ-Katnal1 (si-mmu_circ_0001432).

Figure 4

Upregulation of GSDMD reversed the protective effect of miR-31-5p on LPS-induced inflammatory pyroptosis in RAW264.7 cells. (a, b) RT-qPCR detection showed the expression of miR-31-5p and GSDMD. (c)–(e) ELISA detection showed the expression of inflammatory factors TNF-α, IL-1β, and IL-6 under LPS pretreatment (1 μg/mL). Data were presented as mean ± SD. ^∗∗∗P < 0.001, versus sham NC. ^###P < 0.001, versus mimic. (f)–(i) Western blot detection showed the expression of GSDMD, caspase-1, and NLRP3 expression. Data were presented as mean ± SD. ^∗∗∗P < 0.001, versus sham NC. ^###P < 0.001, versus mimic.