Correlation between slow transit constipation and spleen Qi deficiency, and gut microbiota: a pilot study

doi:10.19852/j.cnki.jtcm.20220408.002

Abstract

Abstract:

OBJECTIVE: To investigate the effect of slow transit constipation (STC) and spleen Qi deficiency on gut microbiota, and the mechanism underlying the action that the positive drug Maren Runchang (MR) alleviates STC.

METHODS: STC was induced, using the cathartic method of Senna and the hunger-fullness disorder method, in ICR mice; one group of model mice was treated with MR (6.24 g/kg). The changes in the general condition, fecal parameters, D-xylose content in the serum, intestinal propulsion rate, and histopathology of the colon were assessed after STC induction in the control, model, and MR groups. Fecal microbiota transplantation (FMT) was performed from STC mice into pseudo germ-free mice. Changes in the contents of substance P (SP), vasoactive intestinal peptide (VIP), and gut microbiota in STC mice and pseudo germ-free mice were assessed after FMT.

RESULTS: Compared with the control group, the model mice showed the following results: the time of the first black stool was significantly longer (P < 0.01), the number and weight of black stools were significantly reduced within 6 h (P < 0.05), the D-xylose content in the serum was significantly reduced (P < 0.05), the intestinal propulsion rate decreased (P < 0.01), the content of VIP in colon tissue significantly increased (P < 0.05), and SP content in the colon tissue significantly decreased (P < 0.01); moreover, the colon showed significant inflame-mation and injury. Furthermore, the abundance of Firmicutes was increased, the abundance of Bacteroides decreased, and the abundance of Alistipes, unclassified_ o__Bacteroidales, Alloprevotella, Bilophila, and Anaero-truncus decreased, while the abundance of the conditional pathogenic bacteria Escherichia and Klebsiella increased. However, after treatment with MR, the time of the first black stool decreased (P < 0.01), the number of black stools within 6 h increased, and the intestinal propulsion rate increased (P < 0.05). Moreover, the content of D-xylose in the serum and the content of VIP in colon tissue significantly decreased (P < 0.05), the content of SP in colon tissue significantly increased (P < 0.01), and colon inflammation significantly improved. Additionally, the abundance of Firmicutes decreased, and the abundance of Bacteroides increased. The abundance of unclassified_o__Bacteroidales and Bilophila increased, and the abundance of Klebsiella decreased. In the model + FMT group, compared with control + FMT group, the content of VIP in colon tissue decreased (P < 0.05), the content of SP in colon tissue significantly increased (P < 0.01), and the abundance of probiotics, such as Lactobacillus and Akkermansia, decreased. In the MR + FMT group, compared with the model + FMT group, the content of VIP in colon tissue increased, the content of SP in colon tissue significantly decreased (P < 0.01), and the abundance of probiotics increased.

CONCLUSIONS: STC mice with spleen Qi deficiency show a decreased abundance of beneficial bacteria, such as Alistipes, unclassified_o__Bacteroidales, Allopr-evotella, Bilophila, and Anaerotruncus, and an increased abundance of the conditional pathogenic bacteria Escherichia coli and Klebsiella. Furthermore, the mechanism of action of MR in treating STC may involve the regulation of intestinal movement, reduction of intestinal inflammation, elevation of intestinal absorption, and regulation of gut microbiota.

Key words: spleen Qi deficiency, gastrointestinal microbiome, probiotics, fecal microbiota transplantation, substance P, vasoactive intestinal peptide, slow transit constipation, Maren Runchang pills

YU Zeyue, HAO Liyu, LI Zongyuan, SUN Jianhui, CHEN Hongying, HUO Hairu, LI Xiaoqin, SHAN Zhongchao, LI Hongmei. Correlation between slow transit constipation and spleen Qi deficiency, and gut microbiota: a pilot study[J]. Journal of Traditional Chinese Medicine, 2022, 42(3): 353-363.

Figures/Tables 11

Table 1 Body weight of female mice which had STC with deficiency of spleen Qi (g, $\bar{x}\pm s$)

Group	Dosage	n	Body weight
Group	Dosage	n	Female	Male
Control	-	5	24.5±2.2	31.6±2.2
Model	-	5	20.0±1.2^a	19.7±0.9^a
MR	6.24 g/kg	5	19.9±1.8	20.4±1.0
Control+ FMT	200 μL per mouse	5	25.5±2.4	33.9±2.9
Model+ FMT	200 μL per mouse	5	19.7±1.0^b	22.9±1.7^b
MR+ FMT	200 μL per mouse	5	18.7±1.8	24.1±1.4

Table 2 Fecal parameters measurement of mice which had STC with deficiency of spleen Qi ($\bar{x}\pm s$)

Group	Dosage （g/kg）	n	Time for the first black bowel movement (min)	Number of black stools	Black stools weight (g/6 h)
Control	-	10	75.50±29.30	34.10±6.80	0.42±0.10
Model	-	10	139.80±26.80^a	23.60±4.60^c	0.28±0.11^e
MR	6.24	10	74.80±56.00^b	31.90±8.40^d	0.36±0.08

Figure 1 HE staining to observe the pathological changes and characteristics of colon tissue and inflammatory cell infiltration in mice A-C: hematoxylin-eosin (HE) staining of colon tissue (× 200). A-C: pathological changes of control group, model group, MR group. A1, A2: control group: normal mice without treatment (n = 10); B1, B2: model group: Slow transit constipation with deficiency of spleen Qi mice without treatment (n = 10); C1, C2: MR group: Slow transit constipation with deficiency of spleen Qi mice with Maren Runchang pills intragastrically at a daily dose of 6.24 g/kg (n = 10).

Figure 2 Evaluation index of pseudo germfree mice A-F: direct fecal smear and gram stain method (× 400). A-C: fecal of normal mice (n = 10), there were lots of gram-negative bacteria (blue) and gram-positive bacteria (red); D-F: fecal of pseudo germfree mice which prepared by gavaging combined antibiotics for 7 days (n = 30), there were hardly any gram-negative bacteria (blue) and gram-positive bacteria (red).

Table 3 The SP, VIP content of mice which had STC with deficiency of spleen Qi in 5 % colon homogenate (pg/mg, $\bar{x}\pm s$)

Group	Dosage	n	SP content	VIP content
Control	-	10	0.45±0.08	0.60±0.13
Model	-	10	0.32±0.05^a	0.80±0.27^e
Mr	6.24 g/kg	10	0.44±0.11^b	0.57±0.19^f
Control+ FMT	200 μL per mouse	10	0.42±0.09	0.50±0.11
Model+ FMT	200 μL per mouse	10	0.35±0.04^c	0.65±0.25
Mr+ Fmt	200 μL per mouse	10	0.42±0.10^d	0.47±0.04^g

Figure 3 Significant difference of relative abundance of gut microbiota in slow transit constipation mice====A-C: described relative abundance of gut microbiota at the phylum and genus level respectively, which could present 2 aspects of information intuitively: which dominant species were contained in each sample at a given taxonomic level, and the relative abundance (proportion of the sample) of each dom89*9inant species.

Figure 4 Relative abundance of gut microbiota in pseudo germfree mice A-C: described relative abundance of gut microbiota at the phylum and genus level respectively, which could present 2 aspects of information intuitively: which dominant species were contained in each sample at a given taxonomic level, and the relative abundance (proportion of the sample) of each dominant species.

Figure 5 Principal co-ordinates analysis at operational taxonomic unit (OTU) level of slow transit constipation with deficiency of spleen Qi mice P = 0.001 and R = 0.4139 for control, model, MR groups. The points with different shapes and colors in the figure represented samples in different groups. The distance between samples in the same group represented the repeatability of this group of samples; The distance of samples between different groups represented the difference between different groups.

Table 4 PerMANOVA analysis results between different groups at OTU level of slow transit constipation with deficiency of spleen Qi mice

Group	R value	P value
Control & Model	0.2018	0.001
Model & MR	0.1024	0.044
Control & Model& MR	0.2321	0.001

Figure 6 Principal co-ordinates analysis at operational taxonomic unit (OTU) level of pseudo germfree mice

Table 5 PerMANOVA analysis results between different groups at OTU level of pseudo germfree mice

Group	R value	P value
Control + FMT & model + FMT	0.5012	0.006
Model + FMT& MR + FMT	0.5498	0.007
control + FMT& model + FMT& MR + FMT	0.3904	0.002

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