miR-128 participates in the pathogenesis of slow transit constipation by regulating the p38α/M-CSF inflammatory signaling pathway

Successful construction of cathartic colon STC model

The barium enema experiment confirmed the presence of significantly slower colonic transit (p < 0.05; Figure 1A, 1B) in the rhubarb and morphine+rhubarb groups. Compared with the saline group, mice that only received subcutaneous injections of morphine also exhibited a delay in colonic transit. This represented a case of morphine simulation of gastrointestinal disorders similar to that observed in case of chronic laxative use by STC patients.

Figure 1. Construction of cathartic colon mice models. (A) anatomical appearance of four groups. I saline group, II: morphine group, III rhubarb group, IV morphine+rhubarb group; Red A vermiform appendix, Red B rectum, Red C anus. (B) measurement of stool-transit distance after barium enema examination. I saline group (a) vs morphine group (b), II saline group (a) vs rhubarb group (b), III morphine group (a) vs morphine+rhubarb group (b), IV measurement of stool-transit distance; the forceps show the positions of transit barium, *p<0.05, versus saline group; #p<0.05, versus morphine group. (C) collection of stool samples. I appearance of stools in morphine group(L) and morphine+rhubarb group (R), II dynamic changes of the moisture content of stools in 65° C oven.

Additionally, comparisons of the gross appearance and water content of stool samples collected from the mouse models revealed that the morphine+rhubarb group had significantly drier and smaller stools with a significantly lower water content compared to the morphine group (Figure 1C). Taken together, we have successfully established a cathartic STC mouse model based on morphine and rhubarb.

Hematoxylin and eosin (HE) staining of colonic tissues (Figure 2A) revealed that the morphine group exhibited clear structures in the various colonic tissue layers, and regular and organized arrangement of muscle fibers, which did not differ significantly from the saline group. However, both the rhubarb group and morphine+rhubarb group exhibited thinner circular muscle-layer structures and poor structural damage in the colonic mucosal tissue relative to the saline group (Figure 2A). As the characteristics of the morphine+rhubarb group were more similar to the characteristics of disease progression observed in patients with STC, we defined the morphine+rhubarb group as the STC group and the morphine group as the control group for subsequent analyses.

Figure 2. Differential expression of SCF, NSE in colonic samples. (A) HE staining performed on colonic tissues [original magnification ×40]. saline group (A), morphine group (B), rhubarb group (C), morphine+rhubarb group (D). (B) IHC staining (anti-SCF) performed on colonic tissues [original magnification ×40]. I morphine group, II: morphine+rhubarb group (STC). (C) the mRNA expression of SCF, NSE expression in cathartic colon mice models. (D) the protein expression of SCF, NSE in cathartic colon mice models. *P<0.05 versus saline group, #P<0.05 versus morphine group.

Enteric nerve injury in the STC model

Stem cell factor (SCF) was used as the marker for slow wave-generating ICCs in the gastrointestinal tract and neuron-specific enolase (NSE) was a key marker of the enteric nervous system (ENS). In our cathartic colon model, both protein and gene-expression levels of NSE and SCF were significantly decreased in the STC group as compared with those in the morphine group (p < 0.05; Figure 2B–2D). Additionally, the expression of ICC marker TMEM16A decreased in the submucosal border (SMB) of the circular muscle layer, and the internal circular muscle (CM) layers in STC group (Supplementary Figure 2). Taken together, decreased ICC count and developmental and functional disorders of the ENS were observed in the cathartic colon model.

Decreased miR-128 expression and prediction of downstream target genes in the STC model

Changes in miRNA expression in the morphine and STC groups were measured using the miRNA array. The expression of 12 miRNAs was downregulated (including miR-193a-5p) and that of 45 miRNAs was upregulated (including miR-134-5p) (Figure 3D). Subsequent qPCR revealed that miR-128-2-5p expression in the STC group was significantly decreased compared to that in the morphine group (p<0.05; Figure 3C). These results confirmed the downregulation of miR-128-2-5p in the STC mouse model, a result that is consistent with our previous findings in STC patients.

Figure 3. Confirmation miR-128 expression and prediction of downstream target genes in STC model. (A) The venn diagram showing the overlapping genes among databases. (B) KEGG pathway enrichment of miR-128 interactive genes. (C) the mRNA expression miR-128-2-5p in cathartic colon mice models. (D) heat map show the up-regulated and down-regulated miRNAs in cathartic colon mice models. (E) the mRNA expression p38α, M-CSF in cathartic colon mice models. (F) the protein expression of p38α, M-CSF in cathartic colon mice models. *P<0.05.

To predict the downstream target genes of miR-128, we analyzed five miRNA databases (i.e., TargetScan, PicTar, miRanda, microT, and miRmap); 264, 676, 1439, 1138, and 1319 target genes were identified using TargetScan, PicTar, miRanda, microT, and miRmap, respectively. A Venn diagram revealed that 56 target genes [including mitogen-activated protein kinase (Mapk)14] were common across all five databases (Figure 3A). Enrichment analysis of these predicted miR-128-target genes using starBase revealed a greater distribution of genes within the MAPK signaling pathway, suggesting that the MAPK pathway was a key downstream pathway affected by miR-128 (Figure 3B).

miR-128 regulates the p38α/M-CSF signaling pathway

The p38 MAPK plays an essential role in responding to environment stress and contributes to the pathology of inflammation. Measurement of p38α and M-CSF expression in the colonic tissues by western blotting indicated significantly higher p38α and M-CSF levels in STC group relative to those in the morphine group (p < 0.05; Figure 3E). Additionally, qPCR indicated significantly upregulated M-CSF expression in the STC group (p < 0.05; Figure 3F), whereas p38α expression did not differ significantly different between the two groups.

To elucidate the effects of miR-128 on p38α/MAPK and the downstream genes encoding AKT and M-CSF, we separately trans-infected CT26.WT cells with the micrON mmu-miR-128-2-5p mimic, micrON-mimic NC, micrOFF mmu-miR-128-2-5p inhibitor, and micrOFF-inhibitor NC. Successful and effective trans-infection was confirmed by measuring post-transfection miR-128-2-5p expression (Figure 4A, 4C). In cells trans-infected with the mmu-miR-128-2-5p mimic, qPCR analysis revealed that expression of p38α, AKT and M-CSF decreased significantly (p < 0.05; Figure 4B). Subsequent confirmation by western blotting revealed significant decrease in p38α and M-CSF protein levels along with an increase in miR-128-2-5p expression (p < 0.05). The trans-infection of the mmu-miR-128-2-5p inhibitor significantly increased the expression of p38α, AKT and M-CSF (p < 0.05; Figure 4D). Subsequent confirmation by western blotting revealed a significant increase in p38α and M-CSF levels (p < 0.05; Figure 5E).

Figure 4. miR-128 interference in CT26.WT cells affect expression of miR-128 toward p38α/MAPK and the downstream genes AKT and M-CSF. (A, B) miR-128-2-5p, M-CSF, p38α and AKT mRNA expression levels in cells trans-infected with micrON mmu-miR-128-2-5p mimic/micrON mimic NC; (C, D) miR-128-2-5p, M-CSF, p38α and AKT mRNA expression levels in cells trans-infected with mmu-miR-128-2-5p inhibitor/ micrOFF inhibitor NC. (E) protein expression levels of M-CSF, p38α in CT26.WT cells trans-infected with micrON mmu-miR-128-2-5p mimic/micrON mimic NC/ micrOFF mmu-miR-128-2-5p inhibitor/ micrOFF inhibitor NC. *P<0.05, **P<0.01.

These results showed that the expression of miR-128 decreases in the STC model, and the treatment with miR-128 mimics and inhibitors confirmed that miR-128-2-5p negatively regulates the p38α/AKT/M-CSF pathway.

miR-128-induced effects of M-CSF in colonic epithelial cells on colonic macrophages

Next, we measured the levels of the pro-inflammatory cytokines IL-6, IL-8, and TNF-α and anti-inflammatory cytokines IL-10 and transforming growth factor (TGF)-β in the colonic tissues of the morphine and STC groups to determine inflammation levels in the cathartic colon model Supplementary Figure 1. qPCR revealed significantly elevated Il6 and Il8 expression, significantly decreased TGF-β expression, and no significant changes in TNF-α or IL-10 expression in STC group compared with those in morphine group (p < 0.05; Figure 5A). However, at the protein level, TNF-α level increased significantly (p < 0.05), whereas IL-6 expression did not change significantly relative to the morphine group (Figure 5B). Measurement of apoptosis-related markers in the tissue samples revealed a significant downregulation of B-cell lymphoma-2 (Bcl-2) levels in STC mice but no significant changes in Bax or Caspase-3 levels, suggesting that STC pathogenesis is unrelated to apoptosis (Figure 5C). These results indicated that the chronic effects of rhubarb might lead to the release of substantial quantities of inflammatory mediators in intestinal tissue, which is indicative of the inflammatory status of the colonic tissue.

Figure 5. Measurement of the inflammatory cytokines, anti-inflammatory cytokines and apoptosis-related markers. (A) mRNA expression of IL-6, IL-8, TNF-α, IL-10 and TGF-β in STC model. (B) The protein expression levels of IL-6 and TNF-α. (C) mRNA expression of Bax, Bcl-2 and Caspase-3 in STC model. *P<0.05, **P<0.01.

To determine the miR-128-induced effects of M-CSF in colonic epithelial cells on colonic macrophages, we co-cultured CT26.WT together with RAW246.7 cells. Compared with the untreated NC group, CT26.WT cells trans-infected with the miR-128 inhibitor showed a significant increase in M-CSF levels in the supernatant (p < 0.01), whereas cells transfected with the miR-128 mimic showed a significantly lower M-CSF levels in the supernatant relative to cells transfected with mimic NC (p < 0.05). (p < 0.05; Figure 6B). Moreover, measurement of TNF-α, IL-6 in the RAW264.7 cells indicated that compared with the mimic NC group, the miR-128-mimic group showed lower levels of TNF-α and IL-6, (p < 0.05), whereas RAW264.7 cells of the miR-128-inhibitor group showed significantly higher levels of TNF-α and IL-6 relative to the inhibitor NC group (p < 0.05; Figure 6A). Western blotting showed results similar to those observed for TNF-α and IL-6 expression in RAW264.7 cells (Figure 6C).

Figure 6. The results of co-culturing RAW264.7 cell CT26.WT cell. (A) The mRNA expression levels of IL-6 and TNF-α in RAW264.7 cells after co-culturing. (B) M-CSF level in the supernatant after trans-sfected with the miR-128 mimic/inhibitor. (C) The protein expression levels of IL-6 and TNF-α in RAW264.7 after co-culturing. I-NC, micrOFF inhibitor NC; I-128, micrOFF mmu-miR-128-2-5p inhibitor; m-NC, micrON mimic NC; m-128, micrON mmu-miR-128-2-5p mimic. *P<0.05, **P<0.01.

These results demonstrated that changes in M-CSF expression induced by changes in miR-128 levels in the colonic epithelium might promote altered expression of inflammatory mediators in colonic macrophages, which could ultimately lead to the onset of inflammation due to the increased expression of proinflammatory cytokines, such as IL-6 and TNF-α Supplementary Figure 3.

Cells and culture

The mouse colorectal carcinoma cell line CT26.WT (RRID: CVCL-7256) and mouse monocyte/macrophage cell line RAW264.7 were purchased from ATCC (Manassas, VA, USA). RPMI 1640 medium, 10% fetal bovine serum, and 1% penicillin-streptomycin were used for cell culture.

miRNA interference

The following miRNA interferents were purchased from Guangzhou RiboBio Co., Ltd. (Guangzhou, China): micrOFF mmu-miR-128-1-5p inhibitor, micrOFF mmu-miR-128-2-5p inhibitor, micrOFF-mimic NC, micrON miR-128-1-5p mimic, micrON miR-128-2-5p mimic, and micrON-mimic NC. CT26.WT cells were separately transfected with the aforementioned interferents and miR-NC using Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA USA). At 72-h post-transfection, cells were collected for use in subsequent experiments.

Animal models

128 SPF-grade ICR female mice were purchased from Hubei Provincial Center for Disease Control and Prevention (Wuhan, China) and randomly allocated to the morphine group and untreated group. Model construction was performed over a 1-week period by daily subcutaneous injections of 10 mg/kg morphine for the morphine group and equivalent volumes of saline for the untreated group. Subsequently, mice were randomly selected from the two groups and subjected to barium enema examination for the measurement of stool-transit distance. The model construction took more than 4 months. Following establishment of the morphine-stimulated acute STC model, mice in the untreated group were randomly sub-divided into the control group and rhubarb group, and mice in the morphine group were randomly sub-divided into the morphine group and morphine+rhubarb group. The rhubarb group and morphine+rhubarb group were administered rhubarb decoction in drinking water starting from a dose of 300 mg/kg•day (~5 mL/day based on the average body weight of the mice). Rhubarb was extracted from natural rhubarb, the rhubarb was concentrated to 1 g/mL using a 40 degree water bath. The dose of rhubarb decoction was doubled daily until ~50% of the mice exhibited loose stools at a dose of 2000 mg/kg•day. Subsequently, the dose level was maintained until >80% of the mice no longer exhibited loose stools, which occurred on day 107 of rhubarb administration. After the rhubarb decoction had been administered for 120 days, mice were randomly selected and subjected to barium enema examination to measure stool-transit distance. 2mL 60% (W/V) Barium Sulfate for Suspension (Type I) (Shengli Co., Ltd, Shandong, China) was injected to stomach through oral gavage for stool-transit measurement. The transmission of barium was measured 2 hours after injection and ileocecum was defined as the starting position of colonic transmission. The stools of the mice were collected, dried in a 65° C oven, and weighed every 20 min in a cryopreservation tube. When the mass of the stool sample no longer changed, the dry weight of stools was calculated as total weight − cryopreservation tube weight, and the moisture content of the stools was calculated as (wet weight of stools − dry weight of stools) / wet weight of stools × 100%.

Experiments were performed under a project license (No.2018039) granted by the ethics committee of Zhongnan Hospital of Wuhan University, Wuhan, China, in compliance with Animal Research Center of Wuhan University guidelines for the care and use of animals.

Immunohistochemistry (IHC)

The tissues were fixed in 10% neutral buffered formalin and then subjected the 5μm thick slices were subjected to heat-induced epitope retrieval by immersion in a heat-resistant container filled with citrate buffer solution (pH 6.0), placed in a pressure cooker, and microwaved for 20 min. Endogenous peroxidase activity was blocked by employing 3% H₂O₂ incubation for 5 min. M-CSF staining (rabbit monoclonal antibody against M-CSF, ab233387, dilution 1:500; Abcam, Inc., Cambridge, MA, USA) was used to evaluate the number of stromal cells. The number of mast cell was evaluated using anti-Mast Cell Tryptase (rabbit monoclonal antibody against mast cell tryptase, ab151757, dilution 1:100; Abcam, Inc., Cambridge, MA, USA). ICCs were detected using the ICC marker SCF (rabbit polyclonal antibody against SCF, 26582-1-AP, dilution 1:100, Proteintech Group, Inc, IL, USA) and TMEM16A (rabbit polyclonal antibody against TMEM16A, 12652-1-AP, dilution 1:50, Proteintech Group, Inc, IL, USA). The number of positive cells was calculated and expressed as the mean number of the results from all slides. HMIAS-2000 (Wuhan, China) imaging system was used for recording.

Real-time fluorescence-based quantitative PCR (qPCR)

Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and reverse transcribed into cDNA. qPCR was performed using U6 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as reference genes to measure relative changes in the expression of miRNA and target genes. miRNA expression was measured using a Taqman assay under the following PCR amplification conditions: initial denaturation at 50° C for 2min and 95° C for 10 min, followed by 45 cycles of denaturation at 95° C for 15s, and annealing at 60° C for 60s. Gene expression was measured using SYBR Green under the following PCR amplification conditions: initial denaturation at 95° C for 30s, followed by 40 cycles of denaturation at 95° C for 30s, annealing at 60° C for 30s, and extension at 72° C for 30s.

Primer sequences for qPCR were obtained from the GenBank and miRbase databases, and the primers were designed using Primer Premier 5 (Premier Biosoft, San Francisco, CA, USA). The sequences of the primers are shown in Supplementary Table 1.

Western blotting

Total protein was extracted from cells and tissues using a lysis buffer. After volume adjustment, the total protein was resolved via sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to a methanol-activated polyvinylidene difluoride membrane (Millipore Sigma, Billerica, MA, USA). The membrane was incubated with antibodies, and the protein was visualized based on enhanced chemiluminescence, with GAPDH used as a loading control. The primary antibody (Supplementary Table 2 for detailed antibody information) and secondary antibody (Abcam Cat# ab6728, RRID: AB-955440) were purchased from Abcam (Cambridge, MA, USA). Experimental results were processed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Enzyme-linked immunosorbent assay (ELISA)

Mouse TNF-α and IL-6 ELISA kits were purchased from Proteintech (Proteintech Group, Rosemont, IL, USA). Equivalent amounts of mouse colonic tissue were weighed, washed with phosphate-buffered saline, and sufficiently homogenized. The supernatant was then obtained for the measurement of TNF-α (Cat No. KE10002) and IL-6 (Cat No. KE10007) levels according to the manufacturer’s instructions.

Cell co-culturing

Cells were co-cultured using 0.4-μm pore size Transwell inserts, with CT26.WT cells in the top layer and RAW246.7 cells in the bottom layer. Samples were collected for testing after the co-culturing of CT26.WT cells that had been subjected to miRNA interference with RAW264.7 cells for 48 h.

Statistical analysis

Data analysis was performed using GraphPad Prism software (v.8.0; GraphPad Software, La Jolla, CA, USA). All data were expressed as the mean±standard deviation. Differences in miRNA array data were calculated using the limma algorithm, with an adjusted p < 0.05 and −1 ≤ fold change ≤ 1 used as thresholds, and differences in miRNA between groups were compared using Student's t test. p < 0.05 was considered statistically significant.