20250327_TmLp_constipation_Figure_v1.pptx

Figure 1. Experimental design and the body weight changes of loperamide-induced constipated Institute of Cancer Research (ICR) mice. (A) Schematic diagram of the experimental protocol. After a 7-day acclimation period, constipation was induced in all groups except the normal control (CONT) group by oral administration of loperamide (LPA; 3 mg/kg) once daily for 10 days. Test samples (T. molitor hydrolysate [TmLP] and non-fermented hydrolysate [Tm]) were administered orally once daily for 28 days, starting before and continuing after constipation induction. (B) Body weight changes were measured weekly over the 28-day experimental period. Data are presented as the mean ± standard deviation (SD) (n = 5 per group). Asterisks indicate statistically significant differences compared to the control group (*p < 0.05).Figure 2. SDS-PAGE and peptide profile of T. molitor protein hydrolysates. (A) SDS-PAGE analysis was conducted using a 15% gel to compare the protein profiles of HeTm (lane 1) and TmLP (lane 2). M represents the molecular weight protein marker. The boxed region indicates the gel section (< 15 kDa) excised for LC-MS/MS analysis. (B) Bar graph showing the top peptides identified in the TmLP group based on LC-MS/MS analysis (Table 1). The protein score reflects the confidence and abundance of peptide identification, with keratin, mucin-3A-like, and chemosensory proteins being the most prominent.Figure 3. Evaluation of the growth-promoting efficacy of fermented T. molitor hydrolysate (TmLP) and non-fermented hydrolysate (Tm) on beneficial intestinal bacteria, Lactobacillus plantarum (KCTC 21024) and Bifidobacterium bifidum (KCTC 3202). Bacterial cultures (5 × 10⁵ CFU/mL) were incubated with varying concentrations (0.002, 0.02, 0.25, 0.5, and 1 mg/mL) of Tm or TmLP for 24 h. PBS was used as a control. Bacterial growth was measured at 600 nm. Data are presented as the mean ± SD (n = 3). Asterisks indicate statistically significant differences between the experimental groups and the control (*p < 0.05; **p < 0.01; ***p < 0.001).Figure 4. Anti-inflammatory effects of T. molitor hydrolysate (Tm) and fermented T. molitor hydrolysate (TmLP) on LPS-stimulated RAW 264.7 cells. RAW 264.7 cells were stimulated with LPS (1 µg/mL) and treated with Tm or TmLP (1,000 μg/mL) for 24 h. Cytotoxicity (A) and nitric oxide (NO) production (B) in the culture supernatant. The expression levels of the pro-inflammatory cytokines TNF-α (C) and IL-6 (D) were analyzed using qPCR. Data are presented as the mean ± SD (n = 3). Hashtags indicate statistically significant differences between the experimental groups and the control or positive control (#p < 0.05, ##p < 0.01). Asterisks indicate statistically significant differences between the experimental groups and the LPS-treated negative control (*p < 0.05).Figure 5. Evaluation of dietary intervention effects on LPA-induced constipation. (A) Fecal pellet count per cage (A), fecal pellets per unit of feed intake (B), and fecal water content (C) in experimental mice administered the interventions for 10 consecutive days. LIC-0, 5, and 10 = days after loperamide-induced constipation. Data are presented as the mean ± SD (n = 5 per group). Asterisks indicate statistically significant differences compared to the control group (*p < 0.05; **p < 0.01).Figure 6. Intestinal transit time assessment and charcoal meal experiment. (A) Small intestine transit rate after oral administration of a 3% charcoal meal on ten consecutive days. (B) Number of charcoal-stained fecal pellets collected from the colon following the sacrifice of experimental mice. Data are presented as the mean ± SD (n = 5 per group). Hashtags indicate statistically significant differences between the experimental groups and the control and negative control groups (#p < 0.05). Asterisks indicate statistically significant differences between the experimental groups and the control and experimental groups (*p < 0.05; **p < 0.01).