Effects of probiotics on loperamide-induced constipation in rats

Constipation, a common health problem, causes discomfort and affects quality of life. This study aimed to evaluate the potential effect of probiotics on loperamide (LP)-induced constipation in Sprague-Dawley (SD) rats, a well-established animal model of spastic constipation. In total, 100 male SD rats (age, 8 weeks; Japan SLC, Inc., Hamamatsu, Japan) were used in the experiments following a 12-day acclimatisation period. They were randomly divided into two treatment groups (groups 1 and 2) of 50 rats each. Spastic constipation was induced via oral administration of LP (3 mg/kg) for 6 days 1 hour before the administration of each test compound. Similarly, a probiotics solution (4 ml/kg body weight) was orally administered to the rats once a day for 6 days in group 2. In group 1, phosphate buffer solution was orally administered once a day for 6 days 1 hour after each LP administration. The changes in body weight, faecal parameters, short-chain fatty acid concentration in faeces, and faecal immunoglobulin (Ig)-A concentration were recorded. In the present study, the oral administration of probiotics improved faecal parameters, short-chain fatty acid concentration in faeces, and faecal IgA concentration. Our results indicate that probiotics increase the levels of intestinal short-chain fatty acids, especially butyric acid, thereby improving constipation and intestinal immunity.


Introduction 28
Constipation is a common problem, and probiotics have been reported to improve bowel motility 29 [1,2]. Symptoms of constipation include the following: a decrease in the frequency of bowel immunomodulatory effects by modulating the gut microbiota [17,18]. Thus, probiotic bacteria could . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in

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There have been many studies regarding the role of probiotics in the mitigation of constipation.

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However, to the best of our knowledge, only few studies have investigated the role of 56 safety-guaranteed probiotics in the mitigation of constipation and gut immunity. Therefore, this 57 study aimed to investigate the effects of safety-guaranteed probiotics on constipation relief and gut 58 immunity in a rat model.

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Ethical approval 62 This study was conducted at Inatomi Animal Clinic in Tokyo Prefecture, Japan. It was performed . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted November 23, 2020. ; https://doi.org/10.1101/2020.11.23.393843 doi: bioRxiv preprint Effects of probiotics on loperamide-induced constipation 6 81 82 Induction of constipation in the rats 83 Constipation was induced in all rats through the oral administration of 3 mg/kg of LP, once a day for

Short-chain fatty acid concentration in faeces 98
The short-chain fatty acid (SCFA) concentration in faeces of each rat was measured using gas . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted November 23, 2020. ; https://doi.org/10.1101/2020.11.23.393843 doi: bioRxiv preprint Effects of probiotics on loperamide-induced constipation 7 99 chromatography, as described previously [26]. Approximately 0.5 g of faeces from the dissections 100 described above was gently squeezed into a micro-centrifuge tube containing 1 mL of 10% 101 meta-phosphoric acid with 0.4 mL of 4-methyl valeric acid per mL added as an internal standard.

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The solution was thoroughly mixed using a vortex mixer and centrifuged at 5,700 ×g for 20 minutes

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The parameters of gas chromatography were as follows:  water contents of faeces were significantly higher in group 2 than in group 1 (p<0.01).

Short-chain fatty acid concentration in faeces 156
Short-chain fatty acid concentrations in faeces are shown in Table 4.

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159 . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted November 23, 2020. ; https://doi.org/10.1101/2020.11.23.393843 doi: bioRxiv preprint Table 4. Short-chain fatty acid concentration in faeces (mg/g).   Table 5.

Day
170 171 172 . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted November 23, 2020. ; https://doi.org/10.1101/2020.11.23.393843 doi: bioRxiv preprint

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The results of the faecal parameters of group 1 24 hours after the fourth administration suggested 187 that constipation was properly induced using LP, in accordance with previous studies [22][23][24][25]. The . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted November 23, 2020. ; https://doi.org/10.1101/2020.11.23.393843 doi: bioRxiv preprint body weight of rats was not markedly different between groups 1 and 2 in this study. These results

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are consistent with those of previous studies [22,23]. . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted November 23, 2020. ; https://doi.org/10.1101/2020.11.23.393843 doi: bioRxiv preprint Havenaar and Spanhaak demonstrated that probiotics stimulate the immunity of animals in two 207 ways: 1) flora from the probiotic migrate throughout the gut wall and multiply to a limited extent;

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and 2) antigens released by dead microorganisms are absorbed, thus stimulating the immune system In support of past studies, the current study findings . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted November 23, 2020. ; https://doi.org/10.1101/2020.11.23.393843 doi: bioRxiv preprint suggest that three bacteria contained in Bio-three H increased faecal IgA concentrations. However,

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the mechanism by which this probiotic contributes to the increase in the butyrate concentration in the 226 intestinal tract and stimulates IgA production, as well as the relationship between constipation and

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IgA production, needs to be studied in detail in the future. The authors have performed everything related to this study.

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. CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted November 23, 2020. ; https://doi.org/10.1101/2020.11.23.393843 doi: bioRxiv preprint S58-S61.

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Analysis of treatment effects on the microbial ecology of the human intestine. FEMS . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted November 23, 2020. ; https://doi.org/10.1101/2020.11.23.393843 doi: bioRxiv preprint