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

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.


Background
Sepsis is a systemic inflammatory response syndrome elicited by polymicrobial infections; it is the primary cause for mortality among hospital patients [1]. Active, excessive, and innate immune responses are considered the primary cause of septic damage, which can lead to endothelial dysfunction, imbalanced host homeostasis, and altered metabolism due to parenchymal cellular maladjustments [2].
Studies have increasingly found that circular RNAs (cir-cRNAs) have important functions in regulating tissue microenvironments. circRNAs belong to an endogenous RNA family, have variable full-length sequences, and are identified by their covalently closed-loop structure and lack of poly-adenylated tail. circRNAs are generated by a backsplicing event, and thus are different from linear RNAs such as mRNAs. circRNAs do not have 5′ cap or 3′ tail structures [14,15], which were discovered in the 1970s from viruses [14]. Initially, circRNAs were considered a low abundance RNA species [16]. Nevertheless, due to high-throughput sequencing and bioinformatics analyses, we now appreciate that circRNAs are substantial and common fraction of the transcriptome [16]. Previous studies have found that hsa_ circRNA_104670 and hsa_circRNA_104484 might function as therapeutic targets and candidate biomarkers for sepsis [17]. circRNA VMA21 ameliorates sepsis-associated organ injury through the miR-9-3p/SMG1/inflammation axis and by regulating oxidative stress [18]. Yet, the role of circRNA1 in sepsis-associated liver injury is still largely unknown.
Thus, we examined whether circRNAs could be utilized as novel diagnostic markers for sepsis [19]. To date, there has not been an adequate study of the altered expression of circRNAs or their role in sepsis. This study investigated the circRNA expression in liver tissues that underwent sepsisinduced damage to detect their roles in GSDMD regulation during sepsis and to determine if the altered circRNA expression could lead to therapeutic targets. 2.2. In Vivo Sepsis Model. We induced polymicrobial sepsis in mice via cecal ligation and puncture (CLP) following the published protocols [19]. We randomly divided the 6-to 8-week-old C57BL/6 mice into three groups (6 mice in each group). Mice were anesthetized with 1.5% pentobarbital sodium solution (30 mg/kg) and underwent CLP. A 2 cm incision was made into the abdominal wall, and the cecum was exposed and ligated 0.5 cm from the tip with a 4-0 silk suture. A 22-gauge needle was used to make one puncture through the distal cecum, extruding a small amount of fecal contents. The cecum was replaced in the abdominal cavity, and the exposed abdominal wall was closed in two layers with the running 4-0 silk suture. In sham-operated mice, only laparotomy was performed, but their cecum was not ligated and punctured. The mice were resuscitated with 1 mL of normal saline subcutaneously 12 h after CLP induction. The whole blood was collected through a cardiac puncture, and the mice were sacrificed. Then, the liver tissue was collected and fixed in a 4% paraformaldehyde solution. The blood was collected from the right ventricle after thoracotomy, placed for 2 h at room temperature, and centrifuged at 1300 rpm for 20 min at 4°C. The serum was collected, and the levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β), alanine aminotransferase (ALT), and aspartate transaminase (AST) were measured.

2.3.
High-Throughput RNA-Seq and Strand-Specific RNA-Seq Library. Total RNA from the sepsis model and shamoperated mouse liver tissues was extracted with TRIzol Reagent (Invitrogen, CA, USA). Further,~3 μg total RNA from every sample was subjected to a VAHTS Total RNA-Seq (H/M/R) Library Prep Kit (Vazyme Biotech Co., Ltd, Nanjing, China) to eliminate ribosomal RNA and retain other classes of RNAs such as noncoding RNAs and mRNAs. We treated purified RNA employing 40 U RNase R (Epicenter, New England Biolabs, MA, USA) at 37°C for 3 h, followed by TRIzol purification. Our lab used a KAPA Stranded RNA-Seq Library Prep Kit (Roche, Basel, Switzerland) to prepare an RNA-Seq library, which was subjected to deep sequencing with Illumina HiSeq 4000 (CA, USA) at Aksomics, Inc. (Shanghai, China).

Histological Analysis.
Liver tissues were fixed with 4% paraformaldehyde solution and embedded in paraffin. Then, the sections were obtained and stained with hematoxylin and eosin (H&E) to detect hepatocyte morphology and inflammatory cell infiltration. Liver tissue samples were assessed with the transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) staining using a fluorescence detection kit (Yeasen Biotech Co., Ltd., Shanghai, China). Five visual fields were observed in each group. The apoptotic rate in each visual field ð100%Þ = ðthe total number of apoptotic cells/total cellsÞ × 100%, and the average value were calculated.
2.5. Cell Culture and Transfection. We purchased RAW264.7 cells from Cell Bank in the Chinese Academy of Sciences, Shanghai, China, which were cultured in DMEM supplemented with 10% FBS in an incubator with 5% CO 2 at 37°C. We primed RAW264.7 cells with 1 μg/mL LPS for 6 h before stimulation with 5 mM ATP for 1 h. To silence circ-Katnal1, we employed control (si-NC) and circ-Katnal1-specific (si-circ-Katnal1) siRNAs, which were synthesized and designed by Shanghai Genepharma (Shanghai, China). We transfected RAW264.7 cells with si-NC and sicirc-Katnal1 utilizing Lipofectamine 3000 (Invitrogen,   2 Mediators of Inflammation Carlsbad, CA, USA). After 2 d, we analyzed knockdown efficiency by western blot and real-time quantitative PCR (qPCR). For the GSDMD overexpression, the cDNA of GSDMD was cloned into the cDNA3.1 vector, which was transfected into RAW264.7 cells using Lipofectamine 3000.

Serum ALT and AST Measurements.
Serum AST and ALT levels were detected utilizing assay kits (Nanjing Jiancheng Bioengineering Institute) following the manufacturer's protocol.
2.11. Enzyme-Linked Immunosorbent Assay (ELISA) for Inflammatory Factors. Blood was collected from the right ventricle after thoracotomy. After being placed for 2 h at room temperature, the blood was centrifuged at 1300 rpm for 20 min at 4°C, and the serum was collected. The levels of IL-1β, IL-6, or TNF-α in the serum were measured using inflammatory factor ELISA kits (eBioscience, San Diego, CA, USA) following the protocols. The results were determined spectrophotometrically using a microplate reader.

Statistical
Analysis. Data were shown as means ± standard deviation (SD). Statistical analyses were performed in GraphPad Prism 5.0 (GraphPad Software Inc., La Jolla, CA, USA) to determine significant differences among groups. P values ≤0.05 indicated statistically significant differences. Two-tailed Student t-tests were used to determine significant differences between the two groups, and one-way ANOVA with post hoc Bonferroni tests was used to determine significant differences among three or more groups.

CLP-Induced Sepsis Promoted Liver Injury through
Increased Inflammatory Pyroptosis. To examine liver injury resulting from sepsis, we investigated two biochemical serum markers of liver function, ALT and AST. Following CLP-induced sepsis, serum ALT and AST levels were elevated (Figures 1(a) and 1(b)). H&E (Figure 1(c)) staining showed that the liver cells from the sham group exhibited an intact cellular structure with round, large, and clearly visible nuclei. In contrast, CLP-induced sepsis showed obvious cell damage with irregularly shaped cells, concentrated cytoplasm, and nuclei. TUNEL (Figure 1(d)) staining showed that CLP-induced sepsis promoted liver injury and cell death. ELISA showed that CLP-induced sepsis promoted the expression of inflammatory factors TNF-α, IL-1β, and IL-6 (Figures 1(e) and 1(g)). The western blot showed that CLP-induced sepsis promoted GSDMD, caspase-1, and NLRP3 expression (Figures 1(h) and 1(i)). These data demonstrated that CLP-induced sepsis promoted liver injury by inducing liver pyroptosis.

Abnormal circ-Katnal1 Expression Was Important for CLP-Induced
Sepsis. An increasing number of studies found that ncRNAs had indispensable functions in regulating tissue microenvironments [20,21]. Studies have also suggested that circRNAs might function to regulate patient immune systems against various pathogens such as viruses and bacteria. Mounting evidence has revealed that circRNA dysregulation is an early event in various conditions such as sepsis [22]. We employed high-throughput RNA-Seq to identify abnormally expressed circRNAs between liver tissues of 3 Mediators of Inflammation septic and sham-operation mice. The results showed that CLP-induced sepsis resulted in abnormal circRNA expression in liver tissues, including increased expression of mmu_circ_0012734, mmu_circ_0001432, mmu_circ_ 0012767, mmu_circ_0012771, and mmu_circ_0012774 (Figure 2(a)). qPCR data confirmed that the mmu_circ_ 0001432 expression was significantly increased in liver tis-sues of CLP-induced septic mice compared with shamoperation mice (Figure 2(b)). This finding suggested that mmu_circ_0001432 played an important role in CLPinduced sepsis.
The detection of inflammatory factors showed that LPS pretreatment promoted IL-1β, TNF-α, and IL-6 secretion.
We discovered that mmu_circ_0001432 (circ-Katnal1) was abnormally expressed in a murine CLP-induced sepsis model. Downregulating circ-Katnal1 suppressed LPSinduced inflammatory pyroptosis in RAW264.7 cells. Macrophages circulating in blood or residing in tissues represent the first barrier against external infection through controlling both innate and acquired immunity. Because of their diversity and plasticity, macrophages undergo heterogeneous activation and polarization. M1 macrophages release proinflammatory molecules, which cause tissue damage [30]. Luciferase reporter assays showed that miR-31-5p was a downstream target of circ-Katnal1. Previous studies expression. Data were presented as mean ± SD. * * * P < 0:001, versus sham NC. ### P < 0:001, versus mimic. 9 Mediators of Inflammation showed a decrease in the miR-31-5p expression in mice with sepsis [31]. The upregulation of miR-31-5p inhibited inflammatory cytokine expression [32,33]. In this study, we also found that the miR-31-5p expression decreased in mice with CLP. The downregulation of circ-Katnal1 promoted the miR-31-5p expression. However, inhibiting miR-31-5p reversed the protective effects of silencing circ-Katnal1 on LPS treatment in RAW264.7 cells.
The luciferase reporter assays showed that GSDMD was the downstream target of miR-31-5p. The downregulation of circ-Katnal1 decreased the GSDMD expression. The upregulation of GSDMD reversed the protective effects of silencing circ-Katnal1 on LPS treatment in RAW264.7 cells. Previous studies found that the activation of proinflammatory caspases and their subsequent cleavage of gasdermin D (GSDMD) led to inflammatory apoptosis [34,35]. Upregulating GSDMD reversed the protective effects of miR-31-5p on LPS-induced inflammatory pyroptosis in RAW264.7 cells. These findings suggested that circ-Katnal1 enhanced inflammatory pyroptosis in sepsis-induced liver injury through the miR-31-5p/GSDMD axis.

Conclusions
In summary, downregulating circ-Katnal1 attenuated proinflammatory cytokine production and inflammatory pyroptosis during CLP-induced sepsis via upregulating miR-31-5p and downregulating GSDMD. This study indicated that circ-Katnal1 was a promising therapeutic biomarker for sepsis-induced liver injury.

Data Availability
The datasets used and/or analyzed during the current study are available from the corresponding authors on reasonable request.

Ethical Approval
The Ethics Committee at the First Affiliated Hospital of Harbin Medical University approved all animal experiments.