Lactiplantibacillus plantarum 124 Modulates Sleep Deprivation-Associated Markers of Intestinal Barrier Dysfunction in Mice in Conjunction with the Regulation of Gut Microbiota

L. plantarum 124 was isolated from feces sampled from longevous people in the World’s Longevity Township and stored in Guangdong Microbial Species Preservation Center; its preservation number was GDMCC61123 [15]. The frozen strains were inoculated into 10 mL of Man Rogosa Sharpe (MRS, Guangdong Huankai Biotechnology Co., Ltd., Guangzhou, China) and incubated anaerobically at 37 °C for 24 h. Activated L. plantarum 124 was inoculated into 50 mL of MRS meat broth and incubated anaerobically at 37 °C for 24 h. Then, the samples were centrifuged at 5500× g for 10 min at 4 °C, and then the cells were retained, resuspended with normal saline, and prepared into a bacterial suspension of 10¹⁰ CFU/mL for subsequent studies.

All experiments were approved by the Management and Ethics Committee of Experimental Animals of the Institute of Microbiology, Guangdong Academy of Sciences. Two batches of specific pathogen-free (SPF) male C57/BL6J mice (25 ± 2.0 g, aged 6 weeks) were purchased from Guangdong Medical Experimental Animal Center (Guangzhou, Guangdong, China). The mice could eat and drink freely and were kept under conventional conditions for 1 week of acclimatization.

After the 7 days of acclimatization, the first batch of 48 mice was divided into four groups: the CON group consisted of mice placed in a sleep deprivation cage (Shanghai Xinruan Information Technology Co., Shanghai, China) and that had normal sleep conditions; the SD-3 group consisted of mice that usually slept for 6 weeks and then slept for 4 h (10:00–14:00) every day for 3 weeks in a sleep-deprived cage, while in the SD-6 group and the SD-9 group the mice had the same sleep deprivation as did those in the SD-3 group, but the duration was 6 weeks and 9 weeks, respectively (Figure 1A). After sleep deprivation, the mice were fasted, anesthetized with Shutai 50 (Virbac Group. Carros, France) the next day, and dissected. Serum, colon tissue, and intestinal contents were collected and divided into three parts for the subsequent measurement of indicators.

The second batch of 48 mice was divided into 6 groups and placed in sleep deprivation cages. Mice in the CON group were fed and slept freely every day; the SD group mouse slept for 4 h (10:00–14:00) a day for 6 weeks; the SD + LP group mice slept for 4 h a day, and then orally consumed L. plantarum 124 at a dosage of 2 × 10 ⁹ CFU/day for 5 weeks; the SD + LP + Ati group slept for 4 h per day and was pretreated with antibiotics for one week before the oral administration of L. plantarum 124 at a dosage of 2 × 10 ⁹ CFU/day for 5 weeks. In the SD + FMT group, the sleep-deprived mice were transplanted with fecal microbiota from normal sleep mice every other day for five weeks. The CON-FMT group mice were normal sleepers and transplanted fecal microbiota from the sleep-deprived mice every other day for five weeks. Two days after the last fecal microbiota transplantation, feces were collected from mice (Figure 1B). The mice were anesthetized with Shutai 50 (Virbac Group. Carros, France) and dissected. Serum, colon tissue, and intestinal contents were collected and divided into three parts for the subsequent determination of indicators.

Modified according to the previous literature [16,17], the feces of sleep-deprived mice were collected once every other day, all feces were pooled together, and then homogenized at 100 mg/mL and supplemented with saline (Solarbio Science & Technology Co., Ltd., Beijing, China). The homogenized solution was centrifuged at 500 rpm for 10 min, and the supernatant was the bacterial suspension. An amount of 100 μL/10 g of the bacterial suspension was transplanted into normal sleeping mice via oral gavage three times a week for 6 weeks. Before FMT, recipient mice were treated with 200 μL of an antibiotic mixture (ampicillin (Shanghai Macklin Biochemical Co., Ltd., Shanghai, China), 1 g/L; metronidazole (Shanghai Macklin Biochemical Co., Ltd., Shanghai, China), 1 g/L; neomycin (Shanghai Macklin Biochemical Co., Ltd., Shanghai, China), 1 g/L; vancomycin (Shanghai Macklin Biochemical Co., Ltd., Shanghai, China), 0.5 g/L) three times, one day apart. FMT was stopped two days before dissection. Fecal samples from the donor and recipient were collected in a sample stabilizer and cryopreserved for subsequent gut microbiota analysis.

Colonic morphology was observed using Hematoxylin–Eosin (H&E, Solarbio Science & Technology Co., Ltd., Beijing, China) and periodic acid–Schiff (PAS, Solarbio Science & Technology Co., Ltd., Beijing, China) staining [18]. After anesthesia, mice were dissected, and colon tissue was fixed with a 10% formaldehyde solution (Guangzhou yongjin Biotechnology Co., Ltd., Guangzhou, China). After 48 h, the colon tissue was dehydrated in an ethanol gradient, embedded in paraffin, cut into 5 μm thin sections, and finally stained with H&E or PAS. Intestinal morphology was observed and photographed using an inverted optical microscope.

Mouse colonic tissue was collected, diluted with PBS (Solarbio Science & Technology Co., Ltd., Beijing, China) 10 times, and then placed in a low-temperature grinder for grinding. Under the conditions of 4 °C at 5000 rpm centrifugation for 10 min, the supernatant was transplanted on a new EP tube. After dilution, the levels of the protein content, superoxide dismutase (SOD), glutathione (GSH), catalase (CAT), malondialdehyde (MDA), and total antioxidant capacity (T-AOC) were determined using a BAC, SOD, GSH, CAT, MDA, and T-AOC determination kit (Nanjing Jiancheng Bio Co., Nanjing, China).

In accordance with the manufacturer’s instructions (Beijing winter song Boye Biotechnology Co., Ltd., Beijing, China), the content of inflammatory cytokines interleukin (IL)-1β, IL-6, IL-10, tumor necrosis factor-α (TNF-α), Occludin-1, and Claudin-1 were measured in intestinal samples. Colon samples were weighed at 50 mg, then 9 times the volume of physiological saline was added, grinded (at −30 °C, and at 60 Hz for 30 s, three times), then centrifuged at 13,000 rpm for 15 min at 4 °C, and the supernatant was transferred to a new EP tube for subsequent experiments. An amount of 50 μL of the supernatant was sucked into the sample well, and 100 μL of a horseradish-peroxidase-labeled detection antibody was added to each well. The samples were incubated at 37 °C for 60 min, and then the liquid was discarded and washed 5 times with washing solution. Briefly, 50 μL of substrate A (containing hydrogen peroxide) and B (containing TMB) was added and incubated at 37 °C in the dark for 15 min. Then, 50 μL of a termination solution was added and optical density was determined at a wavelength of 450 nm. In addition, serum levels of D-lactic acid (D-LA) and diamine oxidase (DAO) were measured using an ELISA kit (Beijing winter song Boye Biotechnology Co., Ltd., Beijing, China) in accordance with the above procedures.

The mucin MUC2 in colon tissue was stained via immunohistochemistry. Briefly, paraffin sections of colon tissues were dewaxed with xylene and rehydrated through different concentrations of ethanol, then antigen repair was performed. After blocking with BSA, they were mixed with clonal rabbit anti-mouse primary antibody (MUC2, 1:1000; Beyotime) and incubated overnight at 4 °C. The cells were washed three times with PBS and incubated with goat anti-rabbit IgG H&L (Alexa Fluor^®488) for 2 h at room temperature. After final washing as described above, tissue sections were sequentially stained with DAB and hematoxylin, dehydrated, and sealed with neutral resin. Intestinal morphology was observed and photographed using an inverted optical microscope.

Bacterial DNA was extracted from the fecal samples (0.2–0.3 g) using the E.Z.N.A. soil DNA kit (Omega Bio-tek, Norcross, GA, USA), and the V3–V4 variable regions of bacterial 16S rRNA genes were amplified using an ABI GeneAmp 9700 PCR thermocycler (ABI, Waltham, MA, USA). PCR products were purified using the AxyPrep DNA gel Extraction Kit (AxyPrep Biosciences, Union City, CA, USA). The purified products were quantified with a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and a quantum fluorometer (Promega, Madison, WI, USA). Libraries were constructed, then sequenced using the Illumina MiSeq PE300 platform (Illumina, San Diego, CA, USA).

All sequences were merged, quality-filtered, denoised, and flattened, and the ASV table was obtained in accordance with the Silva 16S rRNA gene database (v138) [19]. Based on the ASV table, the samples were analyzed through the online platform Majorbio Cloud Platform (www.majorbio.com↗, accessed on 28 September 2022). The analysis content included Alpha diversity analysis and Beta diversity analysis. Multivariate principal component analysis (PCA) and non-metric multidimensional scaling (NMDS) analysis were used to investigate the similarities or differences in community composition and to identify the potential principal components that affected the differences in community composition. A Circos diagram was used to visualize the distribution of dominant gut microbiota species at the genus level. A community heat map was used to visually visualize the differences in gut microbiota abundance at thew genus level. Finally, accordingly, the Bar map was used to explore the differences in microbial community abundance between each sample group at the phylum level and genus level.

Based on Majorbio Cloud Platform (www.majorbio.com↗), the non-parametric Kruskal–Wallis test and Mann–Whitney test or Dunn test were used to analyze the data on gut microbiota composition. The data statistics involved using GraphPad Prism (GraphPad Software v8.0.2). All data were expressed as means ± SD. Significant differences among groups were determined via a one-way analysis of variance (ANOVA) with Duncan’s range tests. The statistical significance was set to p < 0.05.

Studies have reported that intestinal inflammation and sleep deprivation have a high degree of clinical comorbidity [20]. Alexandra Vaccaro et al. revealed that sleep deprivation causes early death through the accumulation of oxidative stress in the gut [6]. To investigate the effects of SD on intestinal oxidative stress and the inflammatory response, we first constructed a sleep-deprived mouse model through a sleep deprivation cage and examined the changes in antioxidant enzymes and inflammatory factors in colon tissues. Compared with the CON group, the levels of antioxidant enzymes (SOD, CAT, and T-AOC) in the SD group were significantly decreased, while the level of MDA was significantly increased (p < 0.05, Figure 1C–F). The changes in colonic antioxidant enzymes correlated with the sleep deprivation duration. In addition, measurements of intestinal inflammatory factors showed that proinflammatory factors (IL-1β, IL-6, TNF-α) were significantly increased, and anti-inflammatory factors (IL-10) were significantly decreased in the sleep-deprived group compared with those in the CON group (p < 0.05, Figure 1G–J). These results indicated that insufficient sleep is associated with intestinal oxidative stress and inflammation.

Next, we assessed whether or not SD affected intestinal barrier permeability. DAO and D-LA serum levels are biomarkers of intestinal barrier permeability and intestinal wall integrity [21,22]. The results showed that compared with those in the CON group, the serum levels of DAO and D-LA in the SD group were significantly increased, indicating that intestinal permeability was increased in the sleep-deprived mice (p < 0.05, Figure 2A,B). In addition, the results of the intestinal tight junction protein measurement showed that the contents of Claudin-1 and Occludin-1 in the SD group decreased compared to those in the CON group, especially in the SD-6 and SD-9 group which showed a significant decrease (p < 0.05, Figure 2C,D). These results indicate that the integrity of the intestinal barrier decreases with the prolongation of sleep deprivation. In summary, we tentatively judged that SD was associated with increased intestinal barrier permeability.

To investigate whether or not supplementation with L. plantarum 124 and intestinal microbiota transplantation affect SD-associated colonic injury, we examined changes in intestinal inflammation and oxidative stress levels in sleep-deprived mice after L. plantarum 124 supplementation or transplanting gut normal microbiota from normal-sleeping mice, respectively. Compared with the SD group, L. plantarum 124-supplemented mice had a significant decrease in colonic proinflammatory factor IL-1β and a significant increase in anti-inflammatory factor IL-6 (p < 0.05, Figure 3A,B). In terms of intestinal oxidative stress, L. plantarum 124 supplementation reversed the SD-associated decrease in SOD and GSH in the colon of mice (p < 0.05, Figure 3C,D). These data suggested that L. plantarum 124 can enhance the antioxidant effect of colon tissue, maintain antioxidant balance in SD mice, and partially reversed the intestinal oxidative stress and inflammation response associated with SD. In addition, intestinal oxidative stress and inflammation were significantly alleviated in the SD + FMT group (p < 0.05, Figure 3A–D). The above results suggest that the gut microbiota has a specific function in alleviating intestinal oxidative stress and inflammation associated with SD.

The results showed that sleep deprivation is associated with increased intestinal barrier permeability. Therefore, we further analyzed the effect of L. plantarum 124 intervention on the intestinal barrier. Compared with the SD group, H&E staining showed that the mucosal thickness (red line), crypt depth (blue line), and muscle layer thickness (white arrow) in the SD + LP group were significantly improved (p < 0.05, Figure 4A). Intestinal permeability was measured by evaluating the leakage of DAO and D-LA. Results revealed a significant increase in DAO and D-LA levels in the serum of mice in the SD group. However, with the administration of L. plantarum 124, these levels significantly decreased (p < 0.05, Figure 4D,E). Interestingly, normal sleeping mouse fecal recipient SD mice also exhibited a similar phenomenon. In addition, we evaluated the effects of L. plantarum 124 supplementation or transplanted normal-sleeping mouse intestinal microbiota on the intestinal integrity of SD mice by measuring the content of tight junction proteins (Occludin-1, and Claudin-1) in colon tissue. Compared with that in the CON group, tight junction protein content in the SD group was significantly decreased, whereas that in the SD + LP group and SD + FMT group were significantly up-regulated (p < 0.05, Figure 4F,G). Moreover, the effect of the transplantation of normal microflora on SD-associated injury was superior to that of L. plantarum 124 supplementation. Of note, goblet cells and MUC2 protein are essential for maintaining the integrity of the intestinal barrier. Goblet cells in the colon were visualized with PAS staining (Figure 4A,B). The results showed that the number of goblet cells in mice fed L. plantarum 124 significantly increased than that in the SD group. In addition, the immunohistochemical staining of the colon tissues of mice in each group showed that the expression of MUC2 protein was decreased in sleep-deprived mice. At the same time, it was increased with the L. plantarum 124 supplement (p < 0.05, Figure 4A,C). Taken together, these results suggested that L. plantarum 124 intervention or the transplantation of gut microbiota from normal sleeping mice was effective in repairing the intestinal barrier disruption associated with sleep deprivation. However, whether or not L. plantarum 124 was directly related to gut microbiota was still unknown.

Previous studies have confirmed the association between sleep deprivation and the gut microbiome. This study found that transplanting the gut microbiota of normal-sleeping mice alleviated SD-associated changes in colonic phenotypes. This provides evidence that gut microbes may have a function in the treatment of sleep deprivation-related injuries. Therefore, we identified the effect of L. plantarum 124 intervention on microbiota composition via 16S rRNA gene amplicon pyrosequencing. The chimeric sequences were identified and removed, resulting in a total of 10,966 ASVs. An average of 3527, 2463, 2071 and 2905 ASVs were obtained in the CON, SD, SD + LP and SD + FMT groups, respectively. A total of 614 genera were identified, and a Venn diagram showed that on average 167, 150, 144, and 153 genera were identified in the CON, SD, SD + LP, and SD + FMT groups, respectively (Figure 5A). Moreover, principal component analysis (PCA, Figure 5B) and non-metric multidimensional scaling (NMDS, Figure 5C) showed the obvious clustering of gut microbiota composition in the four groups. The changes in the intervention group tended toward the CON group, indicating that the supplementation of L. plantarum 124 and FMT changed the structure of SD-associated gut microbiota in mice. The Circos chart showed that the most dominant genera in all experimental groups were Dubosiella, Faecalibaculum, Turicibacter, norank f Muribaculaceae, Bacillus, and unclassified f Lachnospiraceae (Figure 5D). At the genus level, a cluster analysis of the top 50 species showed that the SD + LP group and SD + FMT group were clustered into the same group and then with the CON group (Figure 5E).

The phylum- and genus-level analysis showed that sleep deprivation significantly increased the relative abundance of Bacteroidetes and decreased the relative abundance of Firmicutes, and that the supplementation of L. plantarum 124 or FMT showed improvement (Figure 5F). At the genus level, compared to the levels of those in the CON group, a significant decrease in Dubosiella, Faecalibaculum, Bacillus, Bifidobacterium, Akkermansia, Intestinimonas, Lactobacillus, Actinobacteriota, Colidextribacter, and Oscillospiraceae levels was observed in the SD group. In the SD + LP and SD + FMT groups, compared with those in the SD group, the horizontal Dubosiella, Faecalibaculum, Bifidobacterium, Colidextribacter, Akkermansia, and Lactobacillus levels were significantly enriched (Figure 5G). In addition, compared to that in the CON group, the SD treatment resulted in a higher abundance of Erysipelotrichaceae, Bacteroides, Odoribacter, Romboutsia, Streptococcus, Clostridium_sensu_stricto_1 Erysipelatoclostridium, Prevotellaceae, and Enterorhabdus. In contrast, L. plantarum 124 administration or FMT reduced the abundance of Erysipelotrichaceae, Odoribacter, Romboutsia, Streptococcus, and Erysipelatoclostridium. These results suggested that L. plantarum 124 supplementation mitigated SD-associated colonic microbiota disorder in mice.

In the previous study, L. plantarum 124 reversed the colonic oxidative stress, inflammation (Figure 3A–D), intestinal barrier damage (Figure 4), and gut microbiota disorder caused by SD to a certain extent. We used antibiotic treatment and fecal microbiota transplantation pretreatment on sleep-deprived mice to elucidate further the causal relationship between L. plantarum 124, gut microbiota, and intestinal injury. As shown, after transplanting the gut microbiota of sleep-deprived mice, intestinal oxidative stress levels, inflammatory factors, and intestinal permeability indexes of the CON + FMT group tended to reflect those of sleep-deprived mice (Figure 6). The result suggested that gut microbiota mediated the gut damage caused by sleep deprivation. Compared with the SD group, L. plantarum 124 supplementation alleviated intestinal damage. Interestingly, pretreatment with a quadruple antibiotic eliminated the repair effects of L. plantarum 124 on intestinal barrier damage in sleep-deprived mice. Therefore, L. plantarum 124 ameliorated intestinal injury in sleep-deprived mice, possibly by regulating gut microbiota.

16S amplicon data from animal feces of C57/BL6J mice https://dataview.ncbi.nlm.nih.gov/object/PRJNA997797?reviewer↗ (accessed on 24 July 2023). Submission ID: SUB13702060, BioProject ID: PRJNA997797.