2.1 Strain and chemicals

L. plantarum X7022 is a novel strain isolated from Chinese traditional fermented food, stinky tofu. The strain was deposited in China Center for Type Culture Collection with the code CCTCC NO: M 2016505, and the complete genome was sequenced and uploaded to GenBank as No. CP048921.1 (https://www.ncbi.nlm.nih.gov/nuccore/ CP048921.1). The strain was stored at −80°C in the sterilized mixture of man rogosa sharpe (MRS) broth (Aobox Biotechnology) and glycerol (75:25, v/v). L. plantarum X7022 was inoculated in MRS broth at 2% (v/v) and activated at 37°C for 18 h. Bacterial culture was centrifuged at 5000 rpm, 4°C for 10 min, then the cells were washed twice with normal saline and resuspended in normal saline to the cell density of 1 × 10⁹ and 1 × 10⁶ CFU/ml ready for the animal experiments.

Phenolphthalein was purchased from Linfen Qilin Pharmaceutical Co., Ltd. Loperamide hydrochloride was purchased from Xian Janssen Pharmaceutical Co., Ltd. Polyethylene glycol 4000 (PEG4000) was purchased from Hunan HuaNa Pharmaceutical Factory Co., Ltd. All the medicines were dissolved in saline when they were used for treatment. All other reagents were of analytical grade.

2.2 Animal experiment

Five-week-old male Institute of Cancer Research mice at average body weight of 20–25 g were purchased from Shanghai Slack Laboratory Animal Co., Ltd. The mice were acclimatized for 1 week with controlled temperature (20–22°C), humidity (45%–55%) and a light/dark cycle of approximately 12 h each day, and were allowed freely access to standard chow and water. The animal experiments in this study were performed according to the Guidelines for Care and Use of Laboratory Animals of the East China University of Science and Technology and were approved by the Animal Ethics Committee of East China University of Science and Technology.

2.2.1 Effect of L. plantarum X7022 on acute constipation mice

Sixty mice were used. Constipation was induced as described by Kim et al. (2021) and Gao et al. (2021) with minor modifications. After 7 days of adaptation, 48 mice among them were administered with 5 mg/kg BW loperamide hydrochloride by gavage daily to induce constipation for 1 week, and the rest 12 mice were orally administrated with equal volume of normal saline and used as normal group. Constipation symptoms were confirmed by measuring the amount of feces. After modeling, the 48 mice were randomly divided into four groups: model group, positive group treated with 20 mg/kg BW phenolphthalein, X7022L group treated with low-dose of L. plantarum X7022 (2 × 10⁷ CFU/kg BW), and X7022H groups treated with high-dose of L. plantarum X7022 (2 × 10¹⁰ CFU/kg BW). The gavages continued for 14 successive days (Figure 1). In each group, six mice were used to determine the fundamental indices for the laxative experiment and the rest were used for the small intestine propulsion test. The overall experimental procedure is shown in Figure 1.

2.2.2 Effect of L. plantarum X7022 on subacute constipation mice

A total of 60 mice were used. Constipation was induced as described by C. L. Chen et al. (2020) and Kim et al. (2021) with minor modifications. After 7 days of adaptation, 48 mice among them were administered with 5 mg/kg BW loperamide hydrochloride by gavage daily to induce constipation for 1 week, and the rest 12 mice were orally administrated with equal volume of normal saline and used as normal group. The 48 mice were randomly divided into four groups: model group, positive group, X7022L group, and X7022H group. During the 14 days of treatment, except for the normal group, which was administered with 5 ml/kg BW normal saline by gavage, the other groups were administered with the same dose of loperamide hydrochloride by gavage. Therefore, it was replaced by polyethylene glycol 4000 dispersion as the positive drug used to treat constipation due to that Phenolphthalein tablets have been stopped production and sales in China because of significant adverse effects. The normal group and model group were given normal saline at a dose of 20 ml/kg. The positive group was treated with polyethylene glycol 4000 at a dose of 3000 mg/kg. The X7022L and X7022H group were treated with low-dose of L. plantarum X7022 (2 × 10⁷ CFU/kg BW) and high-dose of L. plantarum X7022 (2 × 10¹⁰ CFU/kg BW), respectively. Six mice in each group were used to determine the fundamental indices for the laxative experiment, and the other six mice were used for the small intestine propulsion test. The detailed experimental arrangement is shown in Figure 2.

2.3 Determination of constipation indices

The method described by H. Wang et al. (2020) was modified to measure the relative indices of constipation (i.e., fecal weight, the gastrointestinal transit ratio, and the first black stool defecation time). Six mice from each group were selected for this determination. On Day 22 of the experiment, all mice were fasted for 16 h, and then administered with 500 μl of ink suspension with 5% (w/v) activated carbon powder and 10% (w/v) Arabic gum powder. Then each mouse was immediately moved into an individual clean and empty cage and allowed food and water, ad libitum. The time for the first black stool defecation, defecation number, and defecation weights after 6 h of the ink suspension administration were recorded.

2.4 Analysis of gastrointestinal transit ratio

2.5 Gut microbiota profiling

Total genomic DNA samples were extracted by the OMEGA Soil DNA Kit (Omega Bio-Tek) according to the instruction of the manufacturer and stored at −20°C for further analysis. An agarose gel electrophoresis and NanoDrop NC2000 spectrophotometer (Thermo Fisher Scientific) were used to measure the quality and quantity of extracted DNA. Polymerase chain reaction (PCR) amplification of the bacterial 16S rRNA genes V3–V4 region was performed using the forward primer 338F (5'-ACTCCTACGGGAGGCAGCA-3') and the reverse primer 806R (5'-GGACTACHVGGGTWTCTAAT-3'). Purification and quantitation of PCR amplicons were conducted using Vazyme VAHTSTM DNA Clean Beads (Vazyme) and Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen), respectively.

Raw reads were further filtered according to the following rules using FASTP (version 0.18.0). The clean tags with ≥97% similarity were assigned to the same OTUs. The abundance of bacterial DNA was analyzed at different levels for an independent sample. The α-diversity and β-diversity of different groups were analyzed to show the difference of intestinal microbial composition.

2.6 Statistical analysis

The results were expressed as mean ± standard deviation (SD). All statistical analyses were performed using the SPSS software package. Significant differences between mean values were determined by analysis of variance (ANOVA). Values of p < 0.05 were regarded as statistically significant.

3.1 Treatment of L. plantarum X7022 on different constipation mice

In the acute constipation model mice, Figure 3a showed that the first black stool defecation time was considerably shorter in the positive group (69 min) and the L. plantarum X7022H group (78 min) compared with that in the model group (132 min; p < 0.001). The shorter the time to defecate the first black stool, the more effective the tested drug was in treating “acute” constipation. The first black stool defecation time in the X7022H group was quite similar to the normal group (74 min, p > 0.05). As indicated in Figure 3b, the 6 h defecation number of the positive group, X7022L and X7022H group were significantly different from that of the model group (p < 0.001), with an increase of 273%, 266%, and 328%, respectively. In addition, the 6 h defecation number in the X7022L group was similar to that of normal mice, and high dose of X7022 gavage promoted defecation remarkably, which was 1.21 times of the defecation number in normal mice. Furthermore, compared with the model group, the 6 h defection weight was increased by 225% and 331% in the X7022L and X7022H groups, respectively. Mice administrated with X7022 exhibited higher 6 h defection weight compared with normal mice (Figure 3c). In addition, the gastrointestinal transit ratio showed a significant increase (p < 0.05) to 57%, 53%, and 56% in the positive, X7022L, and X7022H group, respectively, compared with that of 42% in the model group (Figure 3d). Meanwhile, the gastrointestinal transit ratio of mice in both the positive and X7022H groups was restored to the level of normal mice (55%). Therefore, L. plantarum X7022 was efficient in increasing the number and weight of defecation in the acute constipation model, which improved the defecation condition of constipated mice. Moreover, a high dose of L. plantarum X7022 administration was almost as effective as the positive drug in treating model mice with acute constipation.

On the other hand, Figure 3a'–d' showed the treatment effects of L. plantarum X7022 on “subacute” constipation. Compared with the model group (180 min), the first black stool defecation time in the positive, X7022L and X7022H groups was significantly decreased (p < 0.001), which were 118, 96, and 88 min, respectively (Figure 3a'). And after gavage with a high dose of X7022, the first black stool defecation time could be recovered to the normal condition (89 min). Furthermore, the positive and X7022H groups showed a significant difference in the 6 h defecation number compared with the model group, with an increase of 129% and 128%, respectively (Figure 3b').

As shown in Figure 3c', the fecal weight of mice in the positive group (0.47 g) and X7022H group (0.41 g) was significantly higher than that in the model group (0.18 g), and increased by 170% and 136%, respectively. In addition, no significant difference in the defecation weight between the X7022H group and the normal group (p > 0.05), which indicated that gavage of L. plantarum X7022 at high dose had a positive effect on defecation weight in mice, effectively increasing the defecation weight in constipated mice. Figure 3d' showed that mice's gastrointestinal transit ratio in the positive, X7022L and X7022H group were 62.2%, 59.2%, and 63.2%, respectively, which were higher than that of the model group (45.4%) and showed no significant difference with the normal group (59.3%). Therefore, the low and high-dose of L. plantarum X7022 effectively improved the small intestinal propulsion ratio to normal levels throughout the 2-week treatment period. It could be concluded that L. plantarum X7022 can effectively treat “subacute” constipation by improving the defecation problems and bring the defecation condition to normal healthy mice.

Comparing the treatment effect of L. plantarum X7022 on “acute” and “subacute” model mice, the level of the three indicators (first black stool defecation time, 6 h defecation number and weight) were different between acute and subacute model mice. Under the situation of same administration dosage and duration, the improvement played by L. plantarum X7022 showed more effective on acute model than subacute model, which implied that L. plantarum X7022 might be administrated for longer time for the persistent constipation mice. While the gastrointestinal transit ratio showed a similar level in acute and subacute model mice (Figure 3d,d'), which suggested that L. plantarum X7022 could improve gastrointestinal motility in both constipation models. Overall, L. plantarum X7022 could be used to treat acute and subacute constipation by improving the defecation condition.

3.2 Intestinal microbial diversity in different constipation mice

Figure 4 showed the diversity of intestinal flora of two types of constipation mice. For the treatment trials on the acute constipation model, the Chao1 index of the model group was significantly reduced compared to the normal group (p < 0.001), and that of the three treatment groups did not show significant changes compared to the model group (Figure 4a). The Shannon index was not significantly different among all the groups (Figure 4c). For the treatment trials on the subacute constipation model, the Chao1 index showed a significant decrease in the positive and X7022H groups compared with the model group (Figure 4b, p < 0.05), which suggested that a high dose of X7022 treatment might change the intestinal microbial abundance in constipated mice. Furthermore, the Shannon index was significantly lower in the positive group (p < 0.001) compared with the model group, while there was no significant difference between the X7022 treatment groups and the model group (Figure 4d). In general, gut microbial α diversity was a little lower in both acute and subacute constipation mice compared with the normal group.

Figure 4e,f showed the β diversities of gut microbiota in acute and subacute constipation model mice based on unweighted UniFrac distance matrices, respectively, which revealed differences and similarities among the groups. In acute model, the microbial communities of mice treated with a high dose of X7022 showed a separation with that of the model mice, and was close to that of the normal group (Figure 4e), which indicated that treatment with X7022, especially at high dose, could regulate the bacterial community composition of constipated mice to the normal level. In the subacute constipation model, the microbial communities of mice treated with low and high doses of X7022 and the mice in normal group overlapped to some extent (Figure 4f), which implied a high degree of similarity in their microbial communities. However, in both constipation models, the microbiota community of mice treated with positive drug distinguished from those of the normal group, showing that the application of drugs played more impact on the diversity of the gut microbiota in the mice.

3.3 The gut microbial composition of mice in different constipation models

Figure 5a,b showed the composition of gut microbiota at the phylum level in the acute and subacute constipation models. The intestinal microflora was dominated by Bacteroidetes, Firmicutes, and Proteobacteria, accounting for more than 90%. For the treatment trials on the acute constipation model, Bacteroidetes significantly decreased, and Firmicutes and Proteobacteria significantly increased in the model group compared with that of the normal group. There was no significant difference in the abundance of Bacteroidetes and Firmicutes between the model and the X7022 treatment groups (Figure 5c,d, p > 0.05), and the ratio of Firmicutes to Bacteroidetes (F/B) also showed the same trend. Furthermore, the abundance of Bacteroidetes and Firmicutes, and the ratio of F/B in the X7022L group was close to that of the normal group. High dose of X7022 treatment significantly decreased the abundance of Bacteroidetes and increased both the abundance of Firmicutes and the ratio of F/B compared with that of the model group, which suggested that high dose of X7022 treatment could significantly influence the structure of the gut microbiota in the mice.

For the treatment trials on the subacute constipation model, compared to the model group, there was a significant increase in the abundance of Bacteroidetes and decrease in the abundance of Firmicutes (p < 0.05, Figure 5c,d). As a result, the ratio of F/B was also decreased (Figure 5e). Moreover, there was no significant difference in the abundance of Bacteroidetes and Firmicutes, and the ratio of F/B between the X7022L, X7022H groups and the normal group, which verified that X7022 treatment might regulate the gut microbiota in the constipated mice to normal level.

Previous work has reported that probiotics can decrease inflammation and regulate intestinal microecological balance by increasing the abundance of Bacteroidetes (Stojanov et al., 2020). Yoghurt containing Lactococcus lactis, Lactobacillus plantarum, and Lactobacillus casei promoted higher relative abundance of Bacteroidetes phylum and lower abundance of Firmicutes phylum in mice intestine (Liu et al., 2017). Kim et al. (2021) found that intake of multistrain probiotics increased the abundance of Bacteroidetes in the intestinal microbiota of Sprague–Dawley rats induced by loperamide. In addition, decreasing the ratio of F/B in the intestine, regulating intestinal pH, and improving the environment for beneficial bacteria to flourish can correct intestinal microecological disorders in constipated patients (Li et al., 2019).

Additionally, the effects of X7022 treatment on modulating the structure of gut microbiota in constipated mice were more pronounced in the subacute constipation model than the acute model. The different modeling time in acute and subacute constipated mice might account for the results. Although there were differences in the composition of the intestinal microbiota between acute and subacute constipation mice, L. plantarum X7022 has led to an improvement in their intestinal microbiota.

Figures 6 and 7 showed several significantly changed genera resulted by the X7022 treatment in both the acute and subacute models, respectively. In the acute constipation model, the relative abundance of Bacteroides increased and Adlercreutzia decreased in the X7022L group comparing with that in the model group (Figure 6a,b), while the X7022H group showed significantly reduction in the relative abundance of Adlercreutzia (Figure 6b, p < 0.001) and Odoribacter (Figure 6c, p < 0.001), and increase in the abundance of Rumminococcus (Figure 6d, p < 0.01). Furthermore, the proportions of Bacteroides, Adlercreutzia, and Odoribacter at the genus level were much closer to that of the normal mice after the X7022 treatment. L. Wang et al. (2019) found that Bifidobacterium could alleviate constipation by increasing the abundance of Bacteroides and Rumminococcus and decreasing the abundance of Odoribacter, which is consistent with our results. In addition, the X7022H group behaved significantly increase in the abundance of Rumminococcus, which was claimed to associate with fecal short-chain fatty acids and endocrine factors (luteinizing hormone, serum testosterone levels) affecting constipation by a previous study (Lin et al., 2021). Based on the above results, it suggested that gavage of L. plantarum X7022 is a potential method to effectively relieve acute constipation.

The genera which varied significantly in acute constipation mice, did not change significantly in subacute constipation mice. Figure 7 showed a significant difference in the relative abundance of Alloprevotella, Lachnospiraceae_NK4A136, and Desulfovibrio in the model group compared with the normal group. Moreover, compared with the model group, X7022 treatment significantly increased the abundance of Alloprevotella (p < 0.05) and Rikenellaceae_RC9 (p < 0.05), and decreased the abundance of Lachnospiraceae_NK4A136 (p < 0.001) and Desulfovibrio (p < 0.001). An et al. (2021) showed that Alloprovotella and Rikenellaceae_RC9 were two beneficial flora in the gut, the gavage of X7022 might improve intestinal dysbiosis concerning with constipation by increasing such beneficial microflora. Xie et al. (2022) demonstrated that Alloprevotella exhibited relative enrichment in the mice intestine at the colitis treatment, which reflected the positive role of Alloprevotella in treating intestinal diseases. Lachnospiraceae_NK4A136 is an indicator of dysbiosis of the intestinal flora, and a previous study reported that the decreasing abundance of this genus was possible to alleviate constipation symptoms (R. Wang et al., 2021). Desulfovibrio could convert sulfate into a toxic gas (H₂S), and release large amounts of lipopolysaccharide (LPS) at high concentrations, disrupt the intestinal barrier, and increase intestinal permeability. Ingestion of L. plantarum X7022 decreased the abundance of Lachnospiraceae_NK4A136 and Desulfovibrio at the genus level, and subsequently, enhance the intestinal barrier function and improve intestinal integrity, consequently suppress the inflammatory response (Cui et al., 2019).