miR-138-5p ameliorates intestinal barrier disruption caused by acute superior mesenteric vein thrombosis injury by inhibiting the NLRP3/HMGB1 axis

Animal model and treatment

Six- to eight-week-old male Sprague Dawley rats (weight, 300–350 g) were supplied by Hunan SJA Laboratory Animal Co., Ltd. and housed individually in a temperature-controlled barrier facility with light-dark (12:12) cycles at 22 °C with 50% humidity and received ad libitum food and water. The animal experiments were approved by the Animal Ethics Committee of Kunming University of Science and Technology.

The ASMVT rat model was established on the foundation of a previous study (Badripour et al., 2022). The rats were fasted for 12 h before the operation and were given free access to water. In the lower abdomen of the rat, the rat was anesthetized using intraperitoneal injection of ketamine (90 mg/kg) in combination with xylazine (12 mg/kg), and a midline incision of approximately two cm in length was made in the upper abdomen. The position of the cecum was determined after entering the abdominal cavity. The small intestine was pulled five cm away from the ileocecal region, and the mesentery was exposed. The main branches of the mesenteric vein and the ends of the arch vein were ligated with 7-0 nylon thread. During mesenteric vein clamping, approximately 15–20 ml/kg saline was injected intraperitoneally intermittently to prevent transient hypovolemia after loosening of the arterial clamp. Mesenteric vein dilatation and intestinal congestion were dark red, indicating successful modeling. After closing the abdomen, 25–30 m/kg saline (containing gentamicin 320,000 U/L) was added into the abdominal cavity to prevent abdominal surgical infection. After surgery, the rats were placed back into the feeding cage for observation, one rat per cage, and were allowed to eat and drink freely.

Thirty rats were randomly divided into the following groups: (1) Sham group: after surgical exposure of the superior mesenteric vein; (2) ASMVT group; (3) ASMVT+miR-138-5p mock group: the rats received 100 nM miR-138-5p mimic treatment (intraperitoneal injection, 0.5 mL). Rats were sacrificed by the spinal dislocation method under deep anesthesia. The affected intestine was cut down from the middle, and the intestinal contents were washed with normal saline. Small intestinal tissues were stored at −80 °C until use.

Animal care, feeding, housing, and enrichment

The rat feeding room was disinfected twice a week with 0.1% peroxyacetic acid spray, and the rat box and drinking water bottles were soaked in 0.2% peroxyacetic acid for 3 min or autoclaved every month. Rat feed was added according to the principle of small amount and multiple times, soft food was changed daily, and drinking water was autoclaved water and melon seeds were fed to ensure the welfare of experimental animals during the experiment. One week before the surgery, we started one cage for one rat and observed the rats’ food and water intake, activity level, whether the eyes were alert, tail color, etc., and recorded the temperature, humidity and ventilation of the feeding room. After the surgery, the rats were kept in one cage and allowed to eat and drink freely, and the condition of the rats was observed every day.

In this study, the experiment was divided into three groups, and considering the failure of the experiment in the modeling process and the death of the rats, 10 rats were needed for each group, and the total number of experimental rats needed was 30. The number of samples was calculated based on (the method of comparison between two groups was chosen): the formula N =2[(a+b)² × σ²]/(µ1-µ2)² was chosen to calculate the number of rats.

Details of euthanasia method(s) used

When loading the rat into the container, the animal is induced with a low concentration of carbon dioxide and then switched to a high concentration to cause rapid unconsciousness. When the rat appears to be dead, the gas infusion is continued for 2 min, and the rat must be confirmed dead before it is removed from the euthanasia container. After the rat is removed, the rat is confirmed dead by methods such as decannulation.

Criteria established for euthanizing animals prior to the planned end of the experiment and whether this was needed

The rats were unable to eat, were depressed and trembling after surgery, showed anxiety, decreased physical condition, ulceration of body surfaces, and extreme behavior during the experiment and were euthanized using carbon dioxide to reduce nonessential pain.

What happened to any surviving animals at the conclusion of the experiment

The experimental endpoint of the animals was to remove the small intestine of the rats to observe the pathological condition and to make sections for subsequent experiments. Therefore, our rats were sacrificed before the small intestine was removed, and no rats survived at the end of the experiment.

Hematoxylin and eosin (H&E) staining

Tissues were harvested and subjected to 4% paraformaldehyde (PFA) fixation for 24 h. The tissue blocks were sectioned at 5-µm thickness for subsequent staining. These sections were stained with hematoxylin-eosin (H&E) for 10 min at room temperature. H&E was observed by a Nikon Eclipse 80i microscope (Nikon Corporation).

Immunofluorescence

These sections and cells were fixed with 4% paraformaldehyde for 15 min and blocked with 3% BSA in phosphate buffer solution (PBS) for 30 min at room temperature. Then, the cells were incubated with rabbit antibodies against NLRP3 (dilution 1:500; cat. no., ab263899; Abcam, Cambridge, UK), HMGB1 (dilution 1:500; cat. no., ab18256; Abcam, Cambridge, UK), ZO-1 (5 µg/ml; cat. no., 40-2300; Thermo Fisher Scientific, Waltham, MA, USA), occludin (dilution 1:100; cat. no., ab216327; Abcam, Cambridge, UK) and Claudin-18 (dilution 1:500; cat. no., ab203563; Abcam, Cambridge, UK) overnight at 4 °C and Alexa Fluor® 488 goat anti-rabbit IgG secondary antibody (dilution 1:400; cat. no., dilution 1:500; cat. no., ab203563; Abcam, Cambridge, UK) for 1 h at room temperature. The cell nucleus was stained using 0.1% DAPI for 5 min at room temperature. Staining was observed by a Nikon Eclipse 80i microscope (Nikon Corporation, Tokyo, Japan).

Western blot

The tissues and cells were isolated and denatured via RIPA and BCA Protein Determination (Pierce Biotechnology, Waltham, MA, USA) for total protein extraction and quantification, respectively. Protein samples were separated by 10% SDS–PAGE and transferred to polyvinylidene difluoride membranes. After blocking in 5% skim milk. Subsequently, the membrane was incubated with primary antibodies: NLRP3 (1:1000; cat. no. ab26389; Abcam, Cambridge, UK), Caspase-1 (1:1000; cat. no. ab286125; Abcam, Cambridge, UK), ASC (1:500; cat. no. ab180799; Abcam), GSDMD (1:1000; cat. no. ab219800; Abcam, Cambridge, UK), IL-18 (1:1000; cat. no. ab191860; Abcam, Cambridge, UK), IL-1β (1:1000; cat. no. ab254360; Abcam, Cambridge, UK), HMGB1 (dilution 1:1000; cat. no., ab18256; Abcam), ZO-1 (1 µg/ml; cat. no., 40-2300; ThermoFisher Scientific, Waltham, MA, USA), occludin (dilution 1:1000; cat. no., ab216327; Abcam, Cambridge, UK) and Claudin-18 (dilution 1:1000; cat. no., ab203563; Abcam, Cambridge, UK) and GAPDH (1:5000; cat. no. ab8245; Abcam, Cambridge, UK) antibodies were applied to membranes, followed by HRP-conjugated secondary antibody (1:5000; cat. no. ab20272; Abcam, Cambridge, UK). Antibody binding was detected by enhanced chemiluminescence reagent (Thermo Fisher Scientific, Waltham, MA, USA). ImageJ (V version 1.47; National Institutes of Health) was used to analyze the gray value of each band on the membrane.

miRNA sequencing

Total RNA was extracted from three tissues from each group. The RNA amount and purity of each sample were quantified using a NanoDrop ND-1000 (NanoDrop, Wilmington, DE, USA). The RNA integrity was assessed by Agilent 2100 with RIN >7.0. The small RNA sequencing library was prepared by TruSeq Small RNA Sample Prep Kits (Illumina, San Diego, CA, USA). Next, deep sequencing was performed with an Illumina HiSeq2500 following the vendor’s recommended protocol. The differentially expressed miRNAs were selected with statistical significance (p value <0.05) by using Student’s t test. Two computational target prediction algorithms (TargetScan, v5.0, and Miranda, v3.3a) were used to determine which genes are targeted by the most abundant miRNAs. We then calculated overlaps between the predicted data from both algorithms. Additionally, these most abundant miRNAs and miRNA targets were annotated with GO terms and KEGG pathways. An RNA-Seq analysis was conducted by Hangzhou Lianchuan Biotechnology Co., Ltd. (Hangzhou, China).

RNA extraction and reverse transcription-quantitative PCR (RT–qPCR)

TRIzol® reagent (Invitrogen; Thermo Fisher Scientific) was used to extract total RNA from tissues and cells. In this study, the cDNA of the total RNA was generated using the PrimeScript™ RT reagent kit (Takara Biotechnology Co., Ltd., San Jose, CA, USA). After this, RT-qPCR was performed using the SYBR-Green qPCR kit (Thermo Fisher Scientific) according to the instructions provided by the manufacturer. RT-qPCR was conducted using the following primer sequences: miR-138-5p (forward 5′-CCAGCGTGAGCTGGTGTTGTGAATC-3′ and reverse 5′-AGCAGGGTCCGAGGTATTC-3′). U6 was used as a reference gene. The RT-qPCR experiments were performed on an Applied Biosystems 7900HT Fast Real-time PCR system (Applied Biosystems; Thermo Fisher Scientific). The relative expression levels were calculated using the 2^−ΔΔCq method (Livak & Schmittgen, 2001) and normalized to those of the internal reference gene U6.

Cell culture and transfection

IEC-6 cells were obtained from Beijing Beina Chuanglian Biotechnology Research Institute (Beijing, China) and were cultured at 37 °C with 5% CO₂. Cells were cultured in Dulbecco’s modified Eagle’s medium with 4 mM L-glutamine, adjusted to contain 1.5 g/L sodium bicarbonate and 4.5 g/L glucose, supplemented with 10% fetal bovine serum (Gibco, USA). To construct the HMGB1 overexpression vector (oe-HMGB1), the HMGB1 full-length sequence was inserted into pcDNA3.1 (Invitrogen, Waltham, MA, USA). NLRP3 siRNA (si-NLRP3), miR-138-5p mimic, inhibitor and corresponding negative controls were synthesized from RiboBio (Guangzhou, China). IEC-6 cells (1 × 10⁵) were transfected with si-NLRP3, oe-HMGB1, miR-138-5p mimic, inhibitor or corresponding negative controls using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific) following the manufacturer’s instructions. After transfection for 48 h, the efficiency of transfection was detected by RT–qPCR and western blot analysis.

Cell viability

IEC-6 cell viability was assessed using the Cell Counting Kit-8 (CCK-8, Beyotime, Shanghai, China). Briefly, cells were seeded in 96-well plates and treated with experimental requirements.

CCK-8 solution was added to each well. Absorbance was detected using an enzyme marker (BioTeke, Beijing, China).

Detection of cell apoptosis

Assays for cell apoptosis were carried out using an Annexin V fluorescein isothiocyanate/propidine iodide (Solarbio, Beijing, China). Briefly, a cold PBS buffer was used to collect IEC-6 cells, followed by 15 min of culture with Annexin VFITC/PI (1:1) at room temperature. Subsequently, flow cytometry analysis was performed with a FACS Verse flow cytometer (Becton Dickinson Biosciences, NJ, USA) and FlowJo software (version 10; Treestar, OR, USA).

Luciferase reporter analysis

PmirGLO-NLRP3-wild (NLRP3-WT)/-mutant (NLRP3-MUT) type reporter plasmids were provided by Shanghai GenePharma Co., Ltd. HEK293 cells (2 × 10⁵/well) were co-transfected with NLRP3-WT/-MUT plasmid and miR-NC mimic or miR-138-5p mimic using Lipofectamine 2000 reagent (Invitrogen) at 37 °C. At 48 h post-transfection, luciferase activity was determined using the dual-luciferase reporter assay system (Promega Corporation, Madison, WI, USA). Firefly luciferase activities were normalized to Renilla luciferase activities.

Statistical analysis

All experiments were performed in triplicate. Statistical analysis was performed using GraphPad Prism 7 (GraphPad Software Inc., San Diego, CA, USA). The results are expressed as the mean ± standard deviation (SD). Two groups were compared with Student’s t test, and three or more treatments or groups were compared with one-way ANOVA followed by Tukey–Kramer post hoc analysis. A P value <0.05 was defined as statistically significant.

Experimental plan

The ASMVT rat model was established by artificial surgery. IEC-6 was chosen as the experimental cell line. Dual-luciferase reporter assays prove that miR-138-5p targets NLRP3. Transcriptome sequencing, RT–qPCR, western blot, immunofluorescence and tissue staining were used to confirm that miR-138-5p inhibited pyroptosis of intestinal cells, promoted intestinal epithelial tight junctions and alleviated ASMVT injury-induced intestinal barrier disruption via the NLRP3/HMGB1 axis.

ASMVT injury induced intestinal barrier disruption

To determine the impacts of ASMVT injury on intestinal barrier disruption, intestinal tissues were tested by histopathological examination. Figure 1A shows a pictorial of the ASMVT injury induction method. H&E staining showed notable damage to the intestinal structure, and denuded villi with exposed lamina propria and dilated capillaries were observed in the intestinal histopathology of the ASMVT group (Fig. 1B). Western blot results showed that pyroptosis-related proteins (NLRP3, ASC, cleaved caspase 1, GSDMD-N, IL-18 and IL-1β) were elevated after ASMVT injury (Fig. 1C). The tight junctions between intestinal epithelial cells are a key part of intestinal permeability, so we examined the expression of tight junction-related proteins. Western blot and immunofluorescence results showed that the levels of ZO-1, occludin and Claudin-18 were reduced after ASMVT injury (Figs. 1D and 1E). Accumulating evidence shows that HMGB1 modulates the epithelial barrier (Kodera et al., 2020; Miyakawa et al., 2020; Peng et al., 2020), which is increased in intestinal tissues after ASMVT injury (Fig. 1F). To identify miRNAs involved in intestinal barrier disruption, we conducted RNA-Seq analysis of intestinal tissues from ASMVT mice. Sixty miRNAs were aberrantly expressed in normal intestinal tissues compared with ASMVT-induced intestinal tissues (Figs. S1A and S1B). The enrichment-based clustering analyses (Gene Ontology and KEGG pathway) of differentially expressed miRNA-related target genes are presented in Figs. S1C and S1D. The genes were associated with the cytoplasm, cytosol, protein binding, nucleoplasm and nucleus (Fig. S1C). The pathways of pathways in cancer, MAPK signaling pathway, mTOR signaling pathway, proteoglycans in cancer and neurotrophin signaling pathway were enriched in the differentially expressed miRNA-related target genes (Fig. S1D). Among these differentially expressed miRNAs, we focused on miR-138-5p. miR-138-5p is the only differentially expressed target of NLRP3. Therefore, we focused on the miR-138-5p/NLRP3 axis for detailed research into its role in intestinal barrier disruption in ASMVT injury.

miR-138-5p directly targets NLRP3

The binding sites between miR-138-5p and NLRP3-WT/MUT are shown in Fig. 2A. The expression of miR-138-5p was reduced in the intestinal tissues of ASMVT injury (Fig. 2B). To confirm whether miR-138-5p could bind to the 3′UTR of NLRP3. First, we overexpressed miR-138-5p through transfection of a miR-138-5p mimic (Fig. 2C). Subsequently, we cotransfected NLRP3-WT/MUT and miR-138-5p mimic into HEK293 cells and found that miR-138-5p mimic decreased the luciferase activity of NLRP3-WT in 293T cells but had no effects on NLRP3-MUT compared to the NC mimic group (Fig. 2D). Overexpression of miR-138-5p significantly reduced NLRP3 protein expression (Fig. 2E). Altogether, the aforementioned results indicated that miR-138-5p directly targeted NLRP3 genes.

Suppression of miR-138-5p promotes NLRP3-related pyroptosis and destroys tight junctions between IEC-6 cells

We next downregulated miR-138-5p in IEC-6 cells and established miR-138-5p stably downregulating cell lines (Fig. 3A). Suppression of miR-138-5p dramatically decreased the viability (Fig. 3B) and increased the apoptosis (Fig. 3C) of IEC-6 cells. Notably, miR-138-5p knockdown dramatically increased pyroptosis-related protein levels (Fig. 3D). Western blot results showed that the level of HMGB1 was increased after miR-138-5p knockdown and that the levels of ZO-1, occludin and Claudin-18 were reduced (Fig. 3E). Immunofluorescence results showed that suppression of miR-138-5p increased NLRP3 levels and decreased tight junction-related protein levels in IEC-6 cells (Fig. 3F). Taken together, these data indicate that miR-138-5p plays a critical role in pyroptosis and tight junctions between IEC-6 cells.

miR-138-5p targets NLRP3 expression to regulate NLRP3-related pyroptosis and tight junctions in IEC-6 cells

To explore the effect of the miR-138-5p/NLRP3 axis on NLRP3-related pyroptosis and tight junctions between IEC-6 cells. IEC-6 cells were transfected with si-NLRP3 to downregulate NLRP3 expression. si-NLRP3-2 achieved more effective knockdown efficiency (Fig. 4A). The results indicated that suppression of miR-138-5p inhibited cell viability but was reversed with knockdown of NLRP3 (Fig. 4B). In contrast, cell apoptosis was elevated after miR-138-5p downregulation, which was reversed with NLRP3 knockdown (Fig. 4C). The levels of pyroptosis-related proteins were significantly increased by downregulating miR-138-5p but decreased with knockdown of NLRP3 (Fig. 4D). HMGB1 protein levels were increased by downregulating miR-138-5p, which was decreased with knockdown of NLRP3 (Fig. 4E). In contrast, the levels of ZO-1, occludin and Claudin-18 were reduced by downregulating miR-138-5p but elevated after knockdown of NLRP3. Immunofluorescence results showed that suppression of miR-138-5p increased NLRP3 levels and decreased tight junction-related protein levels in IEC-6 cells, and these effects were reversed by NLRP3 knockdown (Fig. 4F). Therefore, these results indicated that miR-138-5p regulates NLRP3-related pyroptosis and tight junctions between IEC-6 cells by targeting NLRP3 expression.

miR-138-5p regulates tight junctions between IEC-6 cells via the NLRP3/HMGB1 axis

To investigate the effect of HMGB1 on the miR-138-5p/NLRP3 axis, tight junctions between IEC-6 cells were regulated. We next overexpressed HMGB1 in IEC-6 cells and established HMGB1 stably overexpressing cell lines (Fig. 5A). Western blot results showed that the levels of ZO-1, occludin and Claudin-18 were reduced after miR-138-5p knockdown, which was reversed with knockdown of NLRP3, but they were finally repressed by upregulating HMGB1 (Fig. 5B). Similar results were also observed in IEC-6 cells by immunofluorescence staining (Fig. 5C). Therefore, these results indicated that miR-138-5p regulates tight junctions between IEC-6 cells via the NLRP3/HMGB1 axis.

miR-138-5p ameliorates ASMVT injury-induced intestinal barrier disruption

To further explore the role of miR-138-5p in intestinal barrier disruption after ASMVT injury, we overexpressed miR-138-5p in ASMVT mice (Fig. 6A). H&E staining showed that overexpression of miR-138-5p ameliorated ASMVT injury-induced intestinal structure damage (Fig. 6B). Western blot results showed that the levels of pyroptosis-related proteins were significantly increased but were inhibited with overexpression of miR-138-5p (Fig. 6C). The level of HMGB1 was increased after ASMVT injury but was reversed with overexpression of miR-138-5p (Fig. 6D). In contrast, the levels of ZO-1, occludin and Claudin-18 were reduced after ASMVT injury and were increased with miR-138-5p overexpression. Immunofluorescence results showed that the expression of NLRP3 and HMGB1 was increased after ASMVT injury but was reversed with overexpression of miR-138-5p (Fig. 6E). In contrast, the expression of ZO-1, occludin and Claudin-18 was reduced after ASMVT injury and was increased with miR-138-5p overexpression. Therefore, these results indicated that miR-138-5p ameliorates ASMVT injury-induced intestinal barrier disruption by inhibiting NLRP3-related pyroptosis and promoting tight junctions between intestinal epithelial cells.

ASMVT: Acute superior mesenteric venous thrombosis; H&E: hematoxylin and eosin; miRNAs: microRNAs; miR-138: miR-138-5p; mim: mimic; inh: inhibitor.