Scorpion Venom Heat-Resistant Synthetic Peptide Alleviates DSS-Induced Colitis via α7nAChR-Mediated Modulation of the JAK2/STAT3 Pathway

Abstract

Background: Inflammatory bowel disease (IBD) is a chronic relapsing inflammatory disorder with limited treatment options. Emerging evidence reveals bidirectional crosstalk between gut and brain through inflammatory signaling, leading us to hypothesize that anti-neuroinflammatory agents may concurrently ameliorate intestinal inflammation. The scorpion venom-derived heat-resistant synthetic peptide (SVHRSP), a bioactive peptide initially identified in scorpion venom and subsequently synthesized by our laboratory, possesses neuroprotective, anti-inflammatory, and antioxidative activities. Its properties make SVHRSP a promising candidate for investigating the therapeutic potential of anti-neuroinflammatory strategies in mitigating intestinal inflammation.

Methods: Using a chronic dextran sodium sulfate (DSS)-induced colitis model in wild-type and α7 nicotinic acetylcholine receptor (α7nAChR) knockout mice, along with lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages, we assessed SVHRSP's effects on inflammation, histopathology, gut permeability, oxidative stress markers, and α7nAChR-Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) signaling.

Results: SVHRSP treatment significantly ameliorated colitis symptoms in wild-type mice by reducing inflammation, repairing histological damage, restoring gut barrier function, and attenuating oxidative stress, with these effects abolished in α7nAChR knockout mice. Mechanistically, SVHRSP activated JAK2/STAT3 signaling through α7nAChR engagement, suppressing proinflammatory cytokine production in macrophages.

Conclusion: These results demonstrated that SVHRSP alleviated intestinal inflammation via α7nAChR-dependent JAK2/STAT3 activation. Combined with its known neuroprotective properties, our findings support the repurposing of this neuroactive peptide, SVHRSP, for treating intestinal inflammatory disorders.

Keywords: CAIP; IBD; JAK2/STAT3; gut–brain axis; scorpion venom; α7nAChR.

Figure 1

Therapeutic effects of synthetic peptide-scorpion venom heat-resistant synthetic peptide (SVHRSP) in a mouse model of dextran sodium sulfate (DSS)-induced chronic colitis. (A) Animal experiment arrangement. (B) Changes in body weight (n = 6–7). (C) Disease Activity Index (DAI) scores (n = 6–7). (D) Colon pictures and (E) length (n = 6–7). (F) Colon tissue H&E staining and (G) scored (n = 6). Results were presented as the mean ± standard error of the mean (SEM). * p < 0.05, ** p < 0.01, and *** p < 0.001. 5-ASA, 5-aminosalicylic acid.

Figure 2

Synthetic peptide-scorpion venom heat-resistant synthetic peptide (SVHRSP) protected the integrity of the intestinal barrier in mice with colitis. (A) Fluorescein isothiocyanate (FITC)-dextran (4 kDa) detection of intestinal permeability (n = 6). (B) Alcian blue staining to observe mucin (blue) secreted by goblet cells in the colon tissues, with the pink color serving as the nuclear counterstain. Immunohistochemistry staining to detect the expression of mucin 2 (MUC2) (brown) in colon tissues (n = 3). (C) The number of Alcian blue-positive cells was counted by randomly selecting nine crypts in each section, three in each group. (D) MUC2-positive cells per crypt (n = 9). (E) Reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis of Occludin and zonula occludens-1 (ZO-1) (n = 5). (F) Western blot analysis of Occludin and ZO-1 and (G) quantitative analysis in colon tissues (n = 3). (H) Immunohistochemistry staining analysis of Occludin and ZO-1 (brown) in colon tissues and (I,J) quantitative analysis (n = 3). Results were expressed as mean ± standard error of the mean (SEM). * p < 0.05, ** p < 0.01.

Figure 3

Synthetic peptide-scorpion venom heat-resistant synthetic peptide (SVHRSP) reduced macrophage accumulation and the secretion of inflammatory mediators in colitis mice. (A,B) Immunohistochemistry analyses of F4/80⁺ cells (brown) in the colonic tissues and quantitative analysis (n = 4). (C,D) Flow cytometry analyses of F4/80⁺CD11b⁺ labeled macrophages in colonic tissues (n = 3). (E) Reverse transcription quantitative polymerase chain reaction (RT-qPCR) was used to assess the mRNA levels of interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), monocyte chemoattractant protein-1 (MCP-1), and inducible nitric oxide synthase (iNOS) in colon tissues (n = 6). (F) Enzyme-linked immunosorbent assay (ELISA) was employed to measure the serum levels of IL-6, TNF-α, and IL-1β in the mice (n = 6). Results were expressed as mean ± standard error of the mean (SEM). * p < 0.05, ** p < 0.01 and *** p < 0.001.

Figure 4

Synthetic peptide-scorpion venom heat-resistant synthetic peptide (SVHRSP) modulated oxidative stress and reactive oxygen species (ROS) production in dextran sodium sulfate (DSS)-induced colitis. (A,B) The colonic concentrations of catalase (CAT), superoxide dismutase (SOD), malondialdehyde (MDA), and myeloperoxidase (MPO) (n = 6). (C) Western blot analysis of nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2) and (D) quantitative analysis in colon tissues (n = 3). (E) Immunohistochemistry staining analysis of 8-hydroxy-2′-deoxyguanosine (8-OHdG) (brown) in colon tissues and (F) quantitative analysis (n = 5). (G) SVHRSP pre-treatment of wild-type (WT) peritoneal macrophages to analyze ROS production and (H) quantitative analysis of the dihydroethidium (DHE)-positive area (n = 5). Results were expressed as mean ± standard error of the mean (SEM). * p < 0.05, ** p < 0.01 and *** p < 0.001.

Figure 5

Synthetic peptide-scorpion venom heat-resistant synthetic peptide (SVHRSP) inhibited dextran sodium sulfate (DSS)-induced chronic colitis in mice via a cholinergic anti-inflammation pathway. (A) Expression of acetylcholine (Ach) in colonic tissues by enzyme-linked immunosorbent assay (ELISA) (n = 6). (B) Immunohistochemistry analysis of α7 nicotinic acetylcholine receptor (α7nAChR) (brown) in colon tissues and (C) quantitative analysis (n = 5). (D) Western blot analysis of α7nAChR and (E) quantitative analysis in colon tissues (n = 5). The original blots are shown in Supplementary Figure S3. (F) Western-blot analysis of α7nAChR in RAW264.7 cells and (G) quantitative analysis (n = 3). The original blots are shown in Supplementary Figure S4. (H) Reverse transcription quantitative polymerase chain reaction (RT-qPCR) was used to measure mRNA levels of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in RAW264.7 cells (n = 6). (I) Overall docking model of SVHRSP with α7nAChR. (J) Enlarged view showing hydrogen-bond interactions with residues THR483, THR479, and ALA246. α7nAChR is depicted as a slate cartoon, SVHRSP as cyan sticks, binding site residues as magenta sticks, and hydrogen bonds as yellow dashed lines. Predicted binding energy is –5.4 kcal/mol. Results were expressed as mean ± standard error of the mean (SEM). * p < 0.05, ** p < 0.01 and *** p < 0.001.

Figure 6

Knockout of α7 nicotinic acetylcholine receptor (α7nAChR) attenuated the protective effect of synthetic peptide-scorpion venom heat-resistant synthetic peptide (SVHRSP). (A) The Body weight changes, (B) disease activity index (DAI) scores, (C,D) colon length (n = 7). (E,F) Colon tissue H&E staining and scored (n = 4). (G) Reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis of the mRNA expression levels of interleukin-1 beta (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) in colon tissues (n = 6). (H,I) Flow cytometry analysis of F4/80⁺CD11b⁺ labeled macrophages in colon tissues (n = 3). (J) Level of Acetylcholine (Ach) in colon tissues by enzyme-linked immunosorbent assay (ELISA) (n = 7). The data were expressed as the mean ± standard error of the mean (SEM). * p < 0.05, ** p < 0.01.

Figure 7

Synthetic peptide-scorpion venom heat-resistant synthetic peptide (SVHRSP) suppressed inflammation by activating the Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) signaling pathway. Western-blot analysis of JAK2/STAT3 and quantified in wild-type (WT) mice (A–C) and in α7 nicotinic acetylcholine receptor (α7nAChR) KO mice (D–F) (n = 4). Results were expressed as mean ± standard error of the mean (SEM). * p < 0.05, ** p < 0.01.