Heat-inactivated Lactobacillus plantarum nF1 promotes intestinal health in Loperamide-induced constipation rats

Abstract

Constipation is a common condition that affects individuals of all ages, and prolonged constipation needs to be prevented to avoid potential complications and reduce the additional stress on individuals with pre-medical conditions. This study aimed to evaluate the effects of heat-inactivated Lactobacillus plantarum (HLp-nF1) on loperamide-induced constipation in rats. Constipation-induced male rats were treated orally with low to high doses of HLp-nF1 and an anti-constipation medication Dulcolax for five weeks. Study has 8 groups, control group; loperamide-treated group; Dulcolax-treated group; treatment with 3.2 × 1010, 8 × 1010 and 1.6 × 1011, cells/mL HLp-nF1; Loperamide + Dulcolax treated group. HLp-nF1 treated rats showed improvements in fecal pellet number, weight, water content, intestinal transit length, and contractility compared to the constipation-induced rats. Also, an increase in the intestine mucosal layer thickness and the number of mucin-producing crypt epithelial cells were observed in HLp-nF1-treated groups. Further, the levels of inflammatory cytokines levels were significantly downregulated by treatment with HLp-nF1 and Dulcolax. Notably, the metagenomics sequencing analysis demonstrated a similar genus pattern to the pre-preparation group and control with HLp-nF1 treatment. In conclusion, the administration of >3.2 × 1010 cells/mL HLp-nF1 has a positive impact on the constipated rats overall health.

Fig 1. Effect of HLp-nF1 on constipation parameters.

Eight-week-old male rats were treated with loperamide along with 3.2 × 10¹⁰, 8 × 10¹⁰, and 1.6 × 10¹¹ cells/mL HLp-nF1, and Dulcolax individually. A single treatment with 1.6 × 10¹¹ cells/mLHLp-nF1 or Dulcolax was used as the control group. (A) The fecal pellets were counted at the indicated time points. (B) The fecal pellets collected prior to sacrifice, (C) the number of remaining fecal pellets in the colon, and (D) fecal water content is shown. Each value represents the mean ± SD. A t-test was used to determine the means of two groups (n = 11). *P< 0.05 vs. control group, ^#P< 0.05 vs. loperamide-treated group. Con, control group; Lop, loperamide-treated group; Dul, Dulcolax-treated group (0.75 mg/kg); HHL, treatment with 1.6 × 10¹¹ cells/mL HLp-nF1; Lop+LHL, treatment with loperamide and 3.2 × 10¹⁰ cells/mL HLp-nF1; Lop+MHL, treatment with loperamide and 8 × 10¹⁰ cells/mL HLp-nF1; Lop+HHL, treatment with loperamide and 1.6 × 10¹¹ cells/mL HLp-nF1; Lop+Dul treated group.

Fig 2. Effects of HLp-nF1 on a small intestinal transit parameter in a loperamide-induced constipation rat model.

Eight-week-old male rats were treated with loperamide along with 3.2 × 10¹⁰, 8 × 10¹⁰, and 1.6 × 10¹¹ cells/mLHLp-nF1, and Dulcolax, individually. A single treatment with 1.6 × 10¹¹ cells/mLHLp-nF1 or Dulcolax was used as the control group. The intestinal transit length (A) and intestinal transit percentage (B) were measured and quantified as described in the Materials and Methods. The quantification analysis is shown. Data represent the mean ± SD. A t-test was used to determine the means of two groups (n = 7). *P< 0.05 vs. control group, ^#P< 0.05 vs. loperamide-treated group. Con, control group; Lop, loperamide-treated group; Dul, Dulcolax-treated group (0.75 mg/kg); HHL, treatment with 1.6 × 10¹¹ cells/mL HLp-nF1; Lop+LHL, treatment with loperamide and 3.2 × 10¹⁰ cells/mL HLp-nF1; Lop+MHL, treatment with loperamide and 8 × 10¹⁰ cells/mL HLp-nF1; Lop+HHL, treatment with loperamide and 1.6 × 10¹¹ cells/mL HLp-nF1; Lop+Dul treated group.

Fig 3. Effects of HLp-nF1 on inflammation state in loperamide-induced constipation.

Eight-week-old rats were treated with loperamide along with 3.2 × 10¹⁰, 8 × 10¹⁰, and 1.6 × 10¹¹ cells/mL HLp-nF1, and Dulcolax, individually. A single treatment with 1.6 × 10¹¹ cells/mL HLp-nF1 or Dulcolax was used as the control. (A) Immunoblotting for anti-IL-6, IL-1β, TNF-α, PGE2, COX-2, and β-actin antibody was performed using the ileum samples. (B) The expression of these proteins were quantified. A t-test was used to determine the means of two groups (n = 4). *P< 0.05 vs. control group, ^#P< 0.05 vs. loperamide-treated group. Con, control group; Lop, loperamide-treated group; Dul, Dulcolax-treated group (0.75 mg/kg); HHL, treatment with 1.6 × 10¹¹ cells/mL HLp-nF1; Lop+LHL, treatment with loperamide and 3.2 × 10¹⁰ cells/mL HLp-nF1; Lop+MHL, treatment with loperamide and 8 × 10¹⁰ cells/mL HLp-nF1; Lop+HHL, treatment with loperamide and 1.6 × 10¹¹ cells/mL HLp-nF1; Lop+Dul treated group.

Fig 4. Effects of HLp-nF1 on the histological parameters in rats with loperamide-induced constipation.

Eight-week-old rats were treated with loperamide along with 3.2 × 10¹⁰, 8 × 10¹⁰, and 1.6 × 10¹¹ cells/mL HLp-nF1, and Dulcolax individually. A single treatment with 1.6 × 10¹¹ cells/mL HLp-nF1 or Dulcolax was used as the control. (A) H&E-staining of the ilium and (B) the thickness of the muscularis externa layer was analyzed. (C) Alcian blue staining was performed as described in Materials and Methods, and (D) blue positive-mucin expression was quantified. A t-test was used to determine the means of two groups (n = 3). *P< 0.05 vs. control group, ^#P< 0.05 vs. loperamide-treated group. Scale bar, 10 μm. Con, control group; Lop, loperamide-treated group; Dul, Dulcolax-treated group (0.75 mg/kg); HHL, treatment with 1.6 × 10¹¹ cells/mL HLp-nF1; Lop+LHL, treatment with loperamide and 3.2 × 10¹⁰ cells/mL HLp-nF1; Lop+MHL, treatment with loperamide and 8 × 10¹⁰ cells/mL HLp-nF1; Lop+HHL, treatment with loperamide and 1.6 × 10¹¹ cells/mL HLp-nF1; Lop+Dul treated group.

Fig 5. Effect of HLp-nF1 on intestinal contractility.

Eight-week-old male rats were treated with loperamide with or without 8 × 10¹⁰ cells/mL HLp-nF1 and Dulcolax individually. (A) After isolation from the treated rats, the ileum was treated with 1 μM acetylcholine, and then with 1 μM phenylephrine. (B) Quantification of acetylcholine-induced intestine contraction. (C) Quantification of phenylephrine-induced intestine relaxation and recovery. A t-test was used to determine the means of two groups (n = 4). *P< 0.05 vs. control group, ^#P< 0.05 vs. loperamide-treated group). Con, control group; Lop, loperamide-treated group; Lop+MHL, treatment with loperamide and 8 × 10¹⁰ cells/mL HLp-nF1; Lop+Dul treated group.

Fig 6. Changes of the genus pattern and microbiome composition in Extracellular Vesicles (EVs) and bacteria in rat feces.

Eight-week-old rats were treated with loperamide along with 3.2 × 10¹⁰, 8 × 10¹⁰, and 1.6 × 10¹¹ cells/mL HLp-nF1, and Dulcolax, individually. A single treatment with 1.6 × 10¹¹ cells/mL HLp-nF1 or Dulcolax was used as the control. (A) The genus pattern in each group is graphically shown. The average relative density of the dominant bacterial phyla. (B) The X-axis shows the groups, and the Y-axis displays the average percentage of sequence reads. The cutoff point for selecting the dominant phyla was set at ≥ 1%. “Others” indicates minor phyla, and “Unassigned” indicates non-identified phyla. PRE, pre-preparation group; Con, control group; Lop, loperamide-treated group; Dul, Dulcolax-treated group (0.75 mg/kg); HHL, treatment with 1.6 × 10¹¹ cells/mL HLp-nF1; Lop+LHL, treatment with loperamide and 3.2 × 10¹⁰ cells/mL HLp-nF1; Lop+MHL, treatment with loperamide and 8 × 10¹⁰ cells/mL HLp-nF1; Lop+HHL, treatment with loperamide and 1.6 × 10¹¹ cells/mL HLp-nF1; Lop+Dul treated group.