Melatonin Alleviates Chronic Intermittent Hypoxia-induced Microbiota Dysbiosis and Attenuates Intestinal Barrier Dysfunction via STAT3/Th17 signalling pathway

doi:10.21203/rs.3.rs-2715995/v1

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Melatonin Alleviates Chronic Intermittent Hypoxia-induced Microbiota Dysbiosis and Attenuates Intestinal Barrier Dysfunction via STAT3/Th17 signalling pathway

https://doi.org/10.21203/rs.3.rs-2715995/v1

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Background: Chronic intermittent hypoxia (CIH) triggers subclinical intestinal barrier disruption prior to systemic low-grade inflammation. Increasing evidence suggests therapeutic effects of melatonin on systemic inflammation and gut microbiota remodelling. However, whether and how melatonin alleviates CIH-induced intestinal barrier dysfunction remains unclear.

Methods: C57BL/6J mice and Caco-2 cell line were treated. We evaluated gut barrier function spectrophotometrically using fluorescein isothiocyanate (FITC)-labelled dextran. Immunohistochemical and immunofluorescent staining were used to detect morphological changes in the mechanical barrier. Western blotting (WB) and quantitative real-time polymerase chain reaction (qRT-PCR) revealed the expression of tight junctions, signal transducer and activator of transcription 3 (STAT3) levels. 16S rRNA analysis of the colonic contents microflora. Flow cytometry was used to detect cytokines and Th17 cells with and without melatonin supplementation.

Results: We found that CIH could induce colonic mucosal injury, including reduction in the number of goblet cells and over expression of intestinal tight junction proteins

CIH could decrease the abundance of the beneficial genera Clostridium, Akkermansia, and Bacteroides, while increasing the abundance of the pathogenic genera Desulfovibrioand Bifidobacterium. Finally, CIH facilitated Th17 differentiation via the phosphorylation of signal transducer and activator of transcription 3 (STAT3) in vitro and elevated the circulating pro-inflammatory cytokine including interleukin (IL)-1β, IL-6, tumor necrosis factor-α, tumor growth factor-β, IL-17A, IL-17F, IL-21, IL-22, IL-23, and C-C motif chemokine ligand 20 in vivo. Melatonin supplementation ameliorated CIH-induced intestinal mucosal injury, gut microbiota dysbiosis, enteric Th17 polarization, and systemic low-grade inflammation reactions mentioned-above.

Conclusions: Melatonin attenuated CIH-induced intestinal barrier dysfunction by regulating gut flora dysbiosis, mucosal epithelium integrity, and Th17 polarization via STAT3 signalling.

Melatonin

chronic intermittent hypoxia

intestinal barrier dysfunction

Th17 differentiation

STAT3 signalling

Chronic intermittent hypoxia (CIH) is a hallmark of obstructive sleep apnoea (OSA) which is one of the most common sleep-related diseases and affect approximately 936 million adults in the general population.^{1, 2} CIH and/or OSA-induced neuropathologies, cognitive deficits are often attributed to CIH/OSA-induced CNS inflammation^{3, 4}. In turn, inflammation plays a crucial role in the occurrence and development of OSA.⁵ However, Mechanisms of CIH-induced inflammation have scarcely been studied.

The intestine is crucial for immunity; its intestinal flora are involved in maintaining intestinal barrier homeostasis.^{6, 7} CIH may damage the integrity of the intestinal barrier and alter the gut microbiota composition.^{7, 8} In particular, the diversity and abundance of the gut microflora, particularly Firmicutes and Bacteroidetes, were altered in humans and rodents with CIH.⁹ Gut microbial dysbiosis is also associated with CIH and/or OSA-related comorbidities, including insulin resistance¹⁰, hypertension^{11, 12}, obesity¹³, and atherosclerosis.¹⁴

Melatonin, which is a potent peroxide scavenger, is synthesized from tryptophan and has antioxidant and anti-inflammatory effects.¹⁵ In Zucker diabetic fatty rats, melatonin ameliorated low-grade inflammation and oxidative stress.¹⁶ Melatonin is also important with respect to the permeability^17–19, energy utilization²⁰, and tight junctions of the intestinal epithelium.¹⁸ Under CIH, melatonin ameliorates endothelial dysfunction, vascular inflammation, systemic hypertension²¹, hypoxic pulmonary hypertension²² hypoxia-induced lipid peroxidation, and local inflammation²³, and exerts cardioprotective effects against myocardial injuries.²⁴

Melatonin supplementation significantly influences the metabolism of the intestinal microbiota, as well as sleep deprivation-induced flora dysbiosis, by providing probiotics^{25, 26} and improving intestinal barrier dysfunction.^{27, 28} Our combined analysis of microbiomes and metabolomics illustrated that the metabolic levels of indole derivatives, represented by melatonin and serotonin, were significantly reduced by CIH-induced intestinal flora disturbance.²⁹ Furthermore, our previous randomized controlled trial showed that oral melatonin supplementation improved the apnoea-hypopnea index (AHI) in insomniacs.³⁰

We hypothesized that CIH exposure may affect intestinal barrier integrity, microbial colonies, and the immune micro-environment, and that exogenous melatonin supplementation could reverse these effects. No previous clinical or experimental studies have evaluated the effects of melatonin on gut microbial dysbiosis and CIH-related intestinal injuries. In the present study, we found that CIH-induced intestinal barrier dysfunction and low-grade inflammation might be therapeutic targets through which melatonin can improve OSA and related complications.

Animals and treatments

Twenty-eight male C57BL/6J mice (6 weeks old) were purchased from Shanghai Laboratory Animal Centre and housed in six cages (4–5 mice/cage) in a controlled environment (temperature: 21 ± 1°C; relative humidity: 50 ± 10%) with regular 12-h light-dark cycles. After a 2-week adaptation period with ad libitum access to a normal mouse diet and water, the mice (8 weeks old) were randomly divided into control (CON; n = 8), CIH (n = 10) and CIH + melatonin (CIH + MT; n = 10) groups (Fig. 1A).

The CIH intervention was performed in identical chambers (Oxycycler model A84; BioSpherix, Parish, NY, USA) for 8 h/day (8:00 a.m. to 4:00 p.m.) over 10 consecutive weeks. The O2 concentration inside the chamber was continuously measured using an O2 analyser. Hypoxia was induced with 8–10% O2 for 240 s, followed by 21% O2 for 120 s, as described previously.³¹ In the CON group, 8–10% O₂ (hypoxia) was replaced with 21% O₂ (air), whereas the other conditions followed those for the CIH groups. Melatonin (M5250; Sigma-Aldrich, St. Louis, MO, USA) was dissolved in absolute ethanol and diluted in sterile water to the final concentration in light-resistant bottles³² according to the weight and average water consumption of the mice (Fig. 2B, F). The CIH group received 0 mg/kg (vehicle) of melatonin, while the CIH + MT group received 30 mg/kg of melatonin. After the experiment ended and following an overnight fast, all mice were euthanized with an overdose of 1% sodium pentobarbital (100 mg/kg, intraperitoneally). This animal study protocol was approved by the Institutional Animal Care and Use Committee of Shanghai Jiao Tong University Affiliated Sixth People's Hospital. All animal experiments were conducted following the Guide for the Care and Use of Laboratory Animals, published by the Animal Welfare Committee of the Agricultural Research Organization.

Cell culture and treatments

The Caco-2 human colon cancer cell line was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in high-glucose Dulbecco’s modified Eagle’s medium supplemented with 10% foetal bovine serum (FBS), 1% antibiotics (100 U/mL of penicillin and 100 mg/mL of streptomycin) and 1% nonessential amino acids at 37°C in 5% CO₂. The culture medium was replaced every 2 days. The Caco-2 cells were randomly divided into five groups: CON, CIH, CIH + MT, CIH + Janus-activated kinase-2 (JAK2)/signal transducer and activator of transcription3 (STAT3) inhibitor, and CIH + MT + STAT3 activator. CIH was induced in identical cell culture chambers (Oxycycler model C42; BioSpherix) for 48 h. As described previously ³³, hypoxia was induced with 0.5–1% O₂ for 30 min followed by 21% O₂ for 30 min. To further confirm the roles of melatonin and JAK2/STAT3 signalling in Caco-2 cells exposed to CIH, melatonin (20 ng/mL), the JAK2/STAT3 inhibitor FLLL32 ³⁴ (5 µM; 4434-05-3; Selleckchem, Houston, TX, USA) and the STAT3 signal activator Colivelin ³⁵ (1 µM; 867021-83-8; Santa Cruz Biotechnology, Dallas, TX, USA) were added to the culture medium before the CIH intervention. Melatonin, FLLL32, and Colivelin were dissolved in absolute ethanol. The CON group consisted of < 2.5% ethanol.

Intestinal paracellular permeability assay

A dose of 750 mg/kg of fluorescein isothiocyanate (FITC)-labelled dextran (60842-46-8; Sigma-Aldrich) suspended in phosphate-buffered saline (PBS) was gavaged in four mice per group at the end of modelling ³⁶. After 4 h, the serum FITC-dextran fluorometry density was measured spectrophotometrically (excitation: 485 nm, emission: 528 nm; Varioskan Flash; Thermo Electron Corp., Waltham, MA, USA).

The colon (from the ileocecum to the terminal bowel) and spleen were collected and photographed under sterile conditions. Then, spleen specimens were homogenized in sterile saline (1 mL/0.1 g), and a 100-µL suspension was cultured on agar plates for 24 h and 72 h at 37°C under anaerobic conditions. The growth of nonspecific bacterial clusters was evidence of bacterial translocation ³⁷.

Enzyme-linked immunosorbent assay (ELISA)

All mouse serum samples were assayed for melatonin concentration. Cell supernatant samples from polarized CD4⁺ T cells (n = 4 per group) were analysed for interleukin (IL)-17A levels. These analyses were conducted using a competitive ELISA (USCN Life Science, Wuhan, China) following the manufacturer’s instructions. The intra-assay coefficient of variation was 7.9%. Each sample was tested in duplicate.

Antibody array assay

A mouse Th1/Th2/Th17 cytokine antibody array (QAM-TH17-1-1; Ray Biotech, Norcross, GA, USA) that simultaneously detects 18 cytokines (C-C motif chemokine ligand 20, interferon-γ, IL-28A, IL-10, IL-12, IL-13, IL-17A, IL-17F, IL-1β, IL-2, IL-21, IL-22, IL-23, IL-4, IL-5, IL-6, transforming growth factor-β, and tumour necrosis factor-α) was used. Briefly, serum samples were incubated with primary antibodies in the arrays overnight at 4°C. Then, a biotin-conjugated detection antibody mixture was added to the array pools and incubated for 2 h at RT. Cy3-conjugated streptavidin was used to bind the biotin-conjugated detection antibodies for 2 h. The slides were scanned using an InnoScan 300 Microarray Scanner (Innopsys, Carbonne, France). The signal values were measured using Mapix software (Ray Biotech) and normalized using the internal positive controls in the array. Annotation enrichment bubble diagrams of the expressed genes were based on the Kyoto Encyclopaedia of Genes and Genomes (KEGG) and Gene Ontology_biological process (GO_BP) databases.

Microbial sequencing

The colonic contents were immediately frozen in liquid nitrogen for 16S rRNA analysis of the microflora. Bacterial genomic DNA was obtained using the QIAamp DNA Stool Mini Kit (51504; Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. Bacterial operational taxonomic units (OTUs) were created by clustering the reads at 97% identity in the Quantitative Insights into Microbial Ecology (QIIME) database ³⁸. Subsequently, the OTUs were reduced to alpha diversity indices, including Shannon and Chao1. Principal coordinate analysis and UPGMA clustering were used for the beta diversity analysis. Linear discriminant analysis effect size analysis was performed to quantify the biomarkers in each group ³⁹ using the nonparametric Kruskal–Wallis and rank tests. Then, the unpaired Wilcoxon rank-sum test was used to identify the most abundant taxa. Additionally, characteristic sequences of the OTUs were compared to the Greengenes database to predict the phenotypes of three BugBase phenotypes: Gram-negative, potential pathogenicity, and stress tolerance ⁴⁰.

Haematoxylin-eosin (HE) staining and Alcian blue-periodic acid-Schiff (AB-PAS) staining

Intestinal segments were fixed in 4% paraformaldehyde overnight, embedded in paraffin for sectioning (5 µm cross-sections) and stained with HE or AB-PAS. At least 25 random fields in each colonic section were analysed from AB-PAS staining at 400× magnification to calculate the number of goblet cells/µm². Caco-2 cells were fixed in acetone and methanol (1:1) for 15 min at RT and stained with HE solution. We calculated the numbers of injured Caco-2 cells/µm² in at least 25 random fields of every slice at 400× magnification.

Immunohistochemical staining

Paraffin slices of the intestines were incubated overnight at 4°C with the monoclonal rabbit anti-mouse primary antibodies IL-17A (1:500; ab79056; Abcam, Cambridge, UK) and IL-23 (1:250; PA5-20239; Thermo Fisher Scientific, Waltham, MA, UK). Next, the sections were rinsed with PBS and incubated with biotinylated goat anti-rabbit immunoglobulin (Ig)-G H&L (1:250; ab205718; Abcam) for 2 h at RT. After washing, the tissues were incubated with streptavidin-horseradish peroxidase (1:250; Sigma-Aldrich) for 2 h at RT. Immunoreactivity was visualized by incubating the tissue sections in PBS containing 0.05% 3’,3-diaminobenzidine tetrahydrochloride (Sigma-Aldrich) and 0.003% hydrogen peroxide for 10 min in the dark. Control slides without primary antibodies were examined in all cases. The immunoreactive portions stained yellow-brown from the haematoxylin.

Immunofluorescent staining

The mouse colons were embedded in an optimal cutting temperature compound (Thermo Fisher Scientific) and stored at − 80°C until use. Serial sections (6 µm) were cut using a cryostat, fixed in 2% paraformaldehyde for 15 min, permeabilized in 0.2% Triton X-100 for 10 min and then blocked with 5% bovine serum albumin for 60 min. The Caco-2 cells were fixed in acetone and methanol (1:1) for 15 min at RT. After three washes in PBS for 10 min each, the cells were blocked with 10% donkey serum for 1 h at RT. Then, the colonic slices and cells were incubated overnight at 4°C with primary rabbit anti-mouse ZO-1 (1:100; ab221547; Abcam), rabbit anti-mouse occludin (1:100; ab216327; Abcam) or rabbit anti-mouse claudin-1 (1:1,000; ab211737; Abcam) in a humidified chamber. After three washes in PBS on the following day, the slides and cells were incubated with goat anti-rabbit IgG (H&L) secondary antibody (Alexa Fluor647, 1:1,000; ab150079; Abcam) for 1 h at RT in the dark. Nuclei were stained with 4’,6-diamidino-2-phenylindole, and the slices were analysed using confocal microscopy (LSM 780; Carl Zeiss, Zena, Germany).

Scanning electron microscopy of the processed samples

One-centimetre colonic sections were cut longitudinally to expose the intraluminal biofilm and fixed in 2.5% w/v glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2) overnight. Next, the samples were placed in fresh 0.1 M cacodylate buffer before dehydration through an ethanol series (25, 50, 75, 95, 95 and 100% w/v, 1-h incubations of each sample). The sections were air-dried using carbon cement (Agar Scientific, Stansted, UK) and sputter-coated with platinum under a high vacuum using the Q150T ES system (Quorum Technologies, Chico, CA, USA) before being viewed in a chamber pressure (3.5 × 10^− 3 Pa; 0% humidity) using an accelerating voltage of 5 kV at extra high tension with a secondary electron detector.

Western blotting (WB)

Total protein of the colonic segments was extracted using a lysis buffer (62.5 mmol/L Tris-HCl, 2% SDS, 10% glycerol, pH 6.8), and centrifuged at 12,000 g for 10 min at 4°C to collect the supernatant. The protein concentration was determined using a bicinchoninic acid protein assay kit (ab102536; Abcam). Then, 20 µg of boiled proteins in a loading buffer was electrophoresed by 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis. After electro-transferring the samples onto a polyvinylidene difluoride membrane (Millipore, Milford, MA, USA), they were blocked with 5% skim milk in 1× Tris-buffered saline with Tween (TBST) for 2 h at RT. The membranes were incubated with the following monoclonal rabbit anti-mouse primary antibodies (β-actin, 1:2,000, ab8226; ZO-1, 1:1,000, ab221547; occludin, 1:2,000, ab216327; claudin-1, 1:500, ab211737; STAT3, 1:1,000, ab68153; phosphorylated (p)-STAT3, 1:2,000, ab76315; Abcam) overnight at 4°C. After washing with TBST, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:5,000, CW0103; CoWin Biosciences, Cambridge, MA, USA) for 2 h at 37°C. Immunoblotting was performed using an enhanced chemiluminescence WB kit (CW0049; CoWin Biosciences). The bands were scanned, and the integral optical density was measured using ImageJ software (version 1.8.0; NIH, Bethesda, MD, USA). The results were obtained from two experiments.

Preparation of lamina propria mononuclear cells (LPMCs) for flow cytometry

The mouse colonic tissues were cleaned of mesentery, opened longitudinally and fragmented with scissors. Next, they were incubated in Hank’s balanced salt solution (HBSS) without Ca²⁺ and Mg²⁺ containing 10 mM HEPES, 5 mM ethylenediaminetetraacetic acid, 5% FBS, and 1 mM dithiothreitol for 20 min with continuous shaking at 37°C. The supernatants containing the intraepithelial lymphocytes were removed. The residual lamina propria were cut into smaller pieces and digested in HBSS with Ca²⁺ and Mg²⁺ containing 10 mM HEPES, 10% FBS, 1 mg/mL collagenase D, 0.1 mg/mL deoxyribonuclease I and 0.1 U/mL dispase at 37°C for 30 min with persistent shaking. The LPMCs were isolated from ileal specimens using the murine Lamina Propria Dissociation Kit (130-097-410; Miltenyi Biotec, Charlestown, MA, USA), washed with PBS (with 0.5% bovine serum albumin), and re-suspended in PBS for further analysis.

In vitro CD4⁺ T cell polarization assay

After preparing the single-cell suspension of the spleen from 6-week-old mice, naïve CD4⁺ T cells (CD4⁺ CD45RA⁺ and CD45RO⁻) were negatively selected using the mouse naïve CD4⁺ T Cell Isolation Kit (130-104-453; Miltenyi Biotec) and cultured in TexMACS medium (130-097-196; Miltenyi Biotec) with 50 µM 2-mercaptoethanol and penicillin/streptomycin (100 IU/mL). Naïve CD4⁺ T cells were stimulated with anti-CD3/CD28-coated microbeads at a 1:1 bead-to-cell ratio (11132D; Thermo Fisher Scientific) in the presence of IL-1β (10 ng/mL; 200-01B; PeproTech, Rocky Hill, NJ, USA), and IL-6 (20 ng/mL; 200-06; PeproTech) to polarize the Th17 cells for 3 days, along with exposure to CIH (3% O₂ for 10 min followed by 21% O₂ for 5 min, for 12 cycles per day) ⁴¹. The polarized CD4⁺ T cells were randomly divided into five groups: CON, CIH, CIH + MT, CIH + JAK2/STAT3 inhibitor and CIH + MT + STAT3 activator. Melatonin, FLLL32 and Colivelin were added to the culture medium of the naïve CD4⁺ T cells at the same concentrations as Caco-2 cells before differentiation.

Flow cytometry to detect Th17 cells

LPMCs and polarized CD4⁺ T cells (1 × 10⁶/mL) for intracellular cytokine staining were stimulated at 37°C for 5 h using the Leukocyte Activation Cocktail (550583; BD Biosciences, San Diego, CA, USA) in the presence of the GolgiStop™ Protein Transport Inhibitor (Brefeldin A, 554724; BD Biosciences) and then pre-treated with Fc block CD16/CD32 antibodies (553142, BD Biosciences; clone: 2.4G2) to block nonspecific binding. After staining for surface markers (CD45, APC-Cy7, 557659; CD3, APC, 565643; CD4, FITC, 553046; and CD8, Percp-cy5.5, 551162; BD Biosciences), the cells were fixed and permeabilized using Cytofix/Cytoperm (555028; BD Biosciences) and then stained with fluorochrome-coupled antibodies against IL-17A (PE, 559502; BD Biosciences) and FVS510 living/dead cell dye (BV510, 564406; BD Biosciences). Fluorescence data were collected using the FACS Canto II instrument (BD Biosciences) and analysed using FlowJo software (Ashland, OR, USA).

Quantitative real-time polymerase chain reaction (qRT-PCR)

Total RNA from the colon and polarized CD₄⁺ T cells was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). RNA purity and concentration (260 nm/280 nm) were determined using the NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific). Then, total RNA (1 µg) was reverse-transcribed into complementary DNA for mRNA detection using the PrimeScript RT Master Mix kit (Takara Bio, Shiga, Japan) at 37°C for 30 min and 85°C for 15 s, and stored at 4°C. qRT-PCR was performed using the ABI PRISM 7500 real-time PCR Detection System (Life Technologies, Carlsbad, CA, USA) and SYBR Green Master Mix (Takara Bio). The PCR cycle was pre-treatment at 95°C for 10 min followed by 40 cycles at 95°C for 30 s, 60°C for 30 s, and 68°C for 30 s, with storage at 4°C. Validated qRT-PCR (mouse/human specific) primers for ROR-γt, JAK1, JAK2, JAK3, Toll-like receptor (TLR) 2, TLR4, nuclear factor-κB, mitogen-activated protein kinase phosphatase, phosphatidylinositol kinase/Akt, adenosine monophosphate-activated protein kinase, sirtuin 1, STAT3 and β-actin were obtained from SA Biosciences (Qiagen). mRNA expression was calculated using the 2^−ΔΔCT method relative to the β-actin reference gene using CFX Manager software (version 1.6).

Effects of melatonin on CIH-altered body weight, body length, and intake amount, cytokine levels in mice

All mice survived CIH exposure with or without melatonin treatment for 10 consecutive weeks (Fig. 1A). Body weight was significantly decreased after 2 weeks of CIH, whereas it was considerably increased after oral supplementation with 30 mg/kg of melatonin for 5 weeks (Fig. 1B). The changes in solid and liquid intake were almost proportional to the body weight during the early and middle stages of modelling (Fig. 1C, D). CIH exposure significantly decreased body length in the CIH group (Fig. 1E). The body length of CIH + MT mice was similar to that of the controls, despite the former appearing thinner (Fig. 1F).

We observed a significant reduction in serum melatonin levels (by almost 24.2%; p < 0.001) in the CIH group, which returned to the normal level after oral melatonin supplementation (Fig. 2A). Compared to the CON group, the CIH group showed 1.2–2.6-fold increased expression of pro-inflammatory cytokines (Fig. 2B–C, E–L), including IL-1β, IL-6, TNF-α, TGF-β, IL-17A, IL-17F, IL-21, IL-22, IL-23, and CCL20 by (p < 0.05), and a 19% reduction in the expression of the anti-inflammatory cytokine IL-10 (p < 0.001) (Fig. 2D). In the CIH + MT group, all pro-inflammatory cytokines except TGF-β (Fig. 2F) were significantly decreased (p < 0.05), whereas the level of the anti-inflammatory cytokine IL-10 (Fig. 2D) was increased (p = 0.003). These results indicate that exogenous melatonin significantly ameliorated CIH-induced inflammation.

Mediating role of melatonin in the relationships of CIH with the morphology and permeability of the intestinal mucosa in mice

We evaluated gut barrier function by gavage of FITC-dextran in mice 4 h prior to sacrifice. Figure 3A shows that the optical density of CIH and CIH + MT mice was increased 2.7-fold and significantly decreased (p < 0.001), respectively, compared to the normoxia-conditioned mice. In the CIH group, the colon length was significantly shortened (by 19.3%; p = 0.008) (Fig. 3B–C). HE staining (Fig. 3D) showed that mucosal epithelial and lamina propria were damaged; additionally, a large number of inflammatory cells infiltrated regions adjacent to the submucosa in the CIH group. AB-PAS staining (Fig. 3E–F) showed that the number of goblet cells that stored and secreted acidic mucopolysaccharides was dramatically reduced in the CIH group. Scanning electron microscopy was used to evaluate intestinal permeability and bacterial translocation (Supplementary Fig. 1). WB assay demonstrated that the expression levels of ZO-1, occludin, and claudin-1 (Fig. 4A–B) were decreased by 64.7% (p < 0.001), 89.2% (p < 0.001), and 75.3% (p < 0.001), respectively, in the colonic tissues of CIH mice. Melatonin supplementation significantly increased the amounts of these tight junction proteins, particularly ZO-1 and claudin-1, by 1.2–3.1 fold (p < 0.01) compared to the CIH group. The immunofluorescence analysis (Fig. 4C) showed similar results to WB analysis with respect to changes in the amounts of the aforementioned tight junction proteins under CIH exposure, with or without melatonin administration.

Effect of melatonin on CIH-altered gut microbiota composition in mice

16S rRNA high-throughput pyrosequencing demonstrated the composition of the colonic microbiota, which was clustered into characteristic operational taxonomic units. Supplementary Fig. 2 shows the richness and diversity of the intestinal flora in three groups. Principal coordinate analysis (PCoA) plots of β-diversity and intergroup similarities revealed by hierarchical clustering are shown in Fig. 5A–B. CIH significantly reduced the abundance of Bacteroidetes, whereas it significantly upregulated the populations of Firmicutes, Proteobacteria, and Actinobacteria, as well as the Firmicutes/Bacteroidetes ratio. Exogenous melatonin significantly attenuated the increases in abundance of these phyla (Fig. 5C–H). Moreover, genus analysis showed that melatonin supplementation dramatically reversed the changes in abundance of Desulfovibrio and Duboslella due to CIH (Fig. 6A). Based on the linear discriminant analysis effect size (LEfSe) results of representative bacterial taxa among the three communities (Fig. 6B), CIH exposure significantly increased the levels of the pathogens Desulfovibrio and Bifidobacterium (Fig. 6C, F) and decreased the levels of Clostridium, Akkermansia, and Bacteroides (Fig. 6D–E, G). Additionally, we estimated the influence of melatonin supplementation on CIH-related microbiota dysbiosis, and found that melatonin significantly reduced the abundance of gram-negative bacteria and potentially pathogenic bacteria, but promoted the expression of stress-tolerant bacteria (Fig. 6H–J).

Melatonin prevented Th17 polarization to ameliorate CIH-induced immune barrier dysfunction in rodent intestines

We investigated the beneficial effects of melatonin on CIH-related immune dysfunction. Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology biological process (GO-BP) analysis of differentially expressed genes in the antibody array for the CIH and CIH + MT groups (Fig. 7A–B) revealed significant enrichment in inflammatory bowel disease, Th17 cell differentiation, STAT cascade, and JAK-STAT signaling pathway. Flow cytometry analysis of LPMCs demonstrated a 2.49-fold increase in Th17 subsets in the colons (p < 0.001) of CIH mice compared to those of controls (Fig. 8A–B). Melatonin treatment significantly reduced the proportion of Th17 cells from 8.42–4.05% (p < 0.001). A similar pattern was observed for the expression of RORγt mRNA, which is a crucial transcription factor of Th17 cells (Fig. 8I). Furthermore, the expression of p-STAT3, which is essential for Th17 cell polarization, was decreased by 77.8% in melatonin-replenished mice with CIH (p < 0.001) (Fig. 8C–D). We also evaluated the relative concentrations of the major Th17-related pathogenic cytokines IL-17A and IL-23 using immunohistochemical staining (Fig. 8E–H). In contrast to the high expression in the CIH group, melatonin administration significantly decreased the relative expression levels of IL-17A and IL-23 by 70.9% and 88.4%, respectively (p < 0.001), in rodent colons. Ten-week CIH exposure promoted intestinal Th17 polarization by increasing p-STAT3 expression, resulting in increased secretion of Th17-correlated pro-inflammatory molecules such as IL-17A and IL-23. The aforementioned changes in mouse colons were completely reversed by melatonin treatment.

Melatonin suppressed Th17 differentiation under CIH by inhibiting STAT3 signaling

To understand the Th17 depression-dependent effects of melatonin, we examined the effect of exogenous melatonin on naïve CD4⁺ T cells in vitro under Th17-polarizing conditions in the presence of CIH. To clarify the role of JAK2/STAT3 in CD4 + T cell differentiation in the presence of CIH, we administered a JAK2/STAT3 inhibitor, STAT3 signal activator, and melatonin under Th17-polarizing conditions.

The level of IL-17A, which is a characteristic marker of mature Th17 cells, was assessed to confirm the efficiency of the differentiation process. The IL-17A level in the culture supernatants was significantly higher in the CIH than CON group (Fig. 9A), reaching a peak of 357 pg/mL on day 3. Similar results were observed for STAT3 mRNA expression (Fig. 9B). After 3-day CIH exposure, the proportion of Th17 cells was markedly upregulated (Fig. 9C–D). JAK2/STAT3 suppression in CD4⁺ T cells significantly downregulated Th17 polarization, STAT3 mRNA expression, and IL-17A secretion in the CIH + JAK2/STAT3 inhibitor group, similar to the changes in the levels of these indicators seen in the CON group. In comparison with melatonin administration, JAK2/STAT3 suppression was more effective for reversing the effects of CIH-induced Th17 differentiation. Furthermore, the addition of a STAT3 signal activator to CD4⁺ T cells significantly reduced the ameliorative effect of melatonin on CIH-induced Th17 polarization in the CIH + MT + STAT3 activator group, as shown by ELISA, qRT-PCR, and flow cytometry (Fig. 9). Additionally, we evaluated the efficacy of JAK2/STAT3 inhibitor, STAT3 activator, and melatonin treatment in Caco-2 cells, and found that the protective effects of melatonin against CIH-induced impairment of intestinal mucosal epithelium were mediated by STAT3 inhibition (Supplementary Fig. 3). These results provide insight into the role of JAK2/STAT3 in CD4 + T cell differentiation under CIH with or without melatonin administration. CIH accelerated Th17 differentiation by activating the JAK2/STAT3 signalling pathway, whereas melatonin restrained CIH-elevated Th17 differentiation by blocking that pathway.

The intestinal barrier is essential for maintaining intestinal and systemic homoeostasis, and is particularly sensitive to hypoxia.^{42, 43} CIH might be involved in the pathogenesis and exacerbation of intestinal barrier dysfunction. In the present study, we found that CIH could trigger dysfunction of the intestinal mucosal, microbial, and immune barriers, subsequently leading to systemic inflammation in vivo and in vitro. Additional melatonin supplementation could reverse CIH-induced intestinal barrier damage by regulating gut dysbacteriosis, mucous epithelium integrity, and Th17 polarization by suppressing STAT3 signalling.

CIH affected the thickness and appearance of the colonic mucosa, tight junction membrane proteins and the circulating level of FITC-dextran was increased after CIH induction. These results indicate that CIH disrupts the intestinal mechanical barrier and increases colonic permeability. Intestinal barrier destruction also reduced the capacity for digestion and absorption⁴⁴, consistent with the weight loss and stunted growth in CIH mice. Previous studies found that tight junctions are sensitive to hypoxia, where a loss of hypoxia-inducible factor signalling results in a loss of tight junction function and disruption of the epithelial barrier.^{45, 46}

In the present study, microbial composition analysis revealed a substantial decrease in the diversity and richness of the faecal microbiome in CIH mice. CIH significantly reduced the abundance of Clostridium, which is involved in the absorption and conversion of raw tryptophan into indole metabolites, including melatonin.⁴⁷ To a certain extent, a reduction in the abundance of Clostridium genus reduced melatonin synthesis. Additionally, the populations of Akkermansia and Bacteroides were significantly downregulated under CIH, which was related to the decrease in enteric goblet cells and intercellular tight junctions.^{48, 49} Furthermore, prominent taxa in the CIH group belonged to the phyla Proteobacteria and Actinobacteria, corresponding to the genera Desulfovibrio and Bifidobacterium. The genus Bifidobacterium produces endogenous ethanol via fermentation.⁵⁰ Bacterial ethanol directly promotes intestinal permeability⁵⁰, and serves as a ligand for Toll-like receptors (TLRs) to accelerate chronic inflammation.⁵¹ Activated TLRs (pattern recognition receptors) significantly increase the expression levels of pro-inflammatory factors, such as IL-1β, IL-6, and TNF-α, which activate the NF-κB and JAK-STAT pathways; in turn, this excites pathogenic CD4⁺ T cells during inflammation.^{51, 52}

We observed higher circulating cytokine levels, and intestinal Th17 aggregation and proliferation, in the colons of CIH mice. Under physiological conditions with stimulation of IL-6 and TGF-β production, mucosal Th17 cells modulate intestinal integrity through the chemotaxis of neutrophils and macrophages, to in turn stimulate epithelial cells to produce antimicrobial peptides.⁵³ These peptides play an important role in maintaining the intestinal mucosa. However, under pathological conditions, such as CIH-induced leaky gut and an underlying pro-inflammatory microenvironment, Th17 cells secrete inflammatory mediators to promote the progression of inflammation, which worsens the prognosis.^{53, 54} Th17 cells play a central role in inflammatory bowel disease and depend on the production of downstream effector cytokines.⁵⁵ The IL-23 receptor further activates downstream effectors (STAT3 or MAPK) via downstream signaling of JAK2 to induce IL-17production.⁵⁶ Consistent with this, we found that the expression of IL-17A, IL-23, p-STAT3, and JAK2 was significantly upregulated in the colonic tissues of CIH mice, suggesting that CIH promoted pathological Th17 cell-mediated intestinal immune barrier dysfunction via the JAK2/STAT3 signaling pathway. Moreover, higher circulating levels of Th17-related cytokines and CCL20/MIP-3α served as pro-inflammatory reactions due to CIH-induced bacterial translocation and Th17 activation and immigration. Previous studies have found that Th17 subsets migrate into joints to accelerate inflammation⁵⁷. Thus, CIH-overactivated Th17 cells trigger the expression of pro-inflammatory cytokines and chemokines, leading to intestinal and peripheral inflammation.^{55, 58}

Interestingly, along with CIH-induced injury to the intestinal barrier, we found that extracorporeal melatonin supplementation significantly inhibited Th17 polarization by inhibiting the JAK2/STAT3 signaling, maintained the Th17 balance, enhanced anti-inflammatory effects and enterocyte renewal, and improved CIH-induced mechanical barrier dysfunction in the intestine. Melatonin has antioxidant and anti-inflammatory effects.^{15, 59} Previous studies reported an antagonistic relationship of Th17 and regulatory T (Treg) cells with the progression and outcome of inflammatory diseases⁶⁰, and found that melatonin could affect the Th17/Treg cell ratio by activating the AMPK/SIRT1 signaling pathway to improve the gut barrier.^{61, 62} Melatonin protects against CIH-induced hypertension and endothelial dysfunction via its effects on NADPH oxidase, pro-inflammatory mediators, nitric oxide (NO), and endothelial NO synthase.²¹ Melatonin also ameliorates impaired Ca2 + homeostasis, myocardial injury, and vulnerability to ischemia-reperfusion injury to prevent CIH-induced myocardial dysfunction.²⁴ Melatonin might prevent hypoxic pulmonary hypertension by suppressing hypoxia-induced expression of proliferating cell nuclear antigens, hypoxia-inducible factor-1a, and nuclear factor-jB, as well as by attenuating elevated ventricular systolic pressure.²² Additionally, melatonin can ameliorate FD/CIH-induced hepatocellular damage by activating the SIRT1-mediated autophagy signaling pathway.⁶³

This study focused on CIH-related intestinal barrier dysfunction and promoted JAK2/STAT3 signaling to improve our understanding of the mechanisms underlying the beneficial effects of melatonin. Although we evaluated several phenotypes and the mechanisms underlying the effects of melatonin, overactivation of the JAK2/STAT3 signaling pathway and JAK2/STAT3 conditional knockout mice models were not used to verify the effects of melatonin. Also, other molecules involved in the signaling pathway need further exploration. Melatonin is safe and effective in CIH models; therefore, it should be evaluated as an adjuvant treatment in large-scale clinical trials of OSA.

In conclusion, CIH exposure affects the intestinal barrier integrity, gut microbial colonies, enteric immune microenvironment, and systemic pro-inflammatory responses by inhibiting melatonin secretion. Melatonin may be useful as an anti-inflammatory agent to reverse CIH-induced intestinal barrier dysfunction by regulating gut dysbacteriosis, mucous epithelium integrity, and Th17 polarization via the suppression of STAT3 signaling.

OSA, obstructive sleep apnoea; CIH, chronic intermitten t hypoxia; AHI, apnoea-hypopnea index; PSG, polysomnography; ZO-1, zonula occludens-1; LPS, lipopolysaccharide; CRP, C-reactive protein; WB, Western blotting; qRT-PCR, quantitative real-time polymerase chain reaction; JAK2, Janus-activated kinase-2; STAT3, signal transducer and activator of transcription 3.

Ethics approval and consent to participate

Not applicable

Consent for publication

All authors consent to the publication of this study

Competing interests

The authors declare no competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Funding

This study was supported by grants-in-aid from STI2030-Major Projects 2021ZD0201900, the National Natural Science Foundation of China (82071030, 82071029, 82000967 and 81970869), and Shanghai Municipal Commission of Science and Technology (18DZ2260200).

Authors' contributions

Huajun Xu, Xinyi Li, Xiaolin Wu and Shankai Yin designed the study. Zhenfei Gao, Weijun Huang, Xiaoman Zhang, Xinyi Li, Feng Liu, Xiaolin Wu and Hongliang Yi assisted with animal experiments; Fan Wang, Xinyi Li and Huajun Xu analyzed date. Fan Wang, Xinyi Li and Xiaolin Wu wrote the paper. Huajun Xu, Jian Guan and Shankai Yin provided financial support and reviewed the paper. All authors read and approved the final manuscript.

Acknowledgements

We thank all the research subjects for their participation and acknowledge the skillful work of the entire medical staff.

Availability of data and material

The data that support the findings of this study are available from the corresponding author upon reasonable request. The raw metagenomics sequences are deposited at NCBI’s Bioproject database under accession PRJNA934121.

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SupplementaryMaterial.pdf
Graphicalfigure.pdf
Graphical figure. Schematic of the mechanistic model. Schematic of the therapeutic effects of melatonin on CIH-induced intestinal barrier dysfunction, which are achieved through the regulation of gut dysbacteriosis, mucous epithelium integrity, and Th17 polarization via the STAT3 signalling pathway.

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Melatonin Alleviates Chronic Intermittent Hypoxia-induced Microbiota Dysbiosis and Attenuates Intestinal Barrier Dysfunction via STAT3/Th17 signalling pathway

Status:

Version 1

Abstract

Figures

Introduction

Materials And Methods

Animals and treatments

Cell culture and treatments

Intestinal paracellular permeability assay

Enzyme-linked immunosorbent assay (ELISA)

Antibody array assay

Microbial sequencing

Haematoxylin-eosin (HE) staining and Alcian blue-periodic acid-Schiff (AB-PAS) staining

Immunohistochemical staining

Immunofluorescent staining

Scanning electron microscopy of the processed samples

Western blotting (WB)

Preparation of lamina propria mononuclear cells (LPMCs) for flow cytometry

In vitro CD4⁺ T cell polarization assay

Flow cytometry to detect Th17 cells

Quantitative real-time polymerase chain reaction (qRT-PCR)

Results

Effect of melatonin on CIH-altered gut microbiota composition in mice

Melatonin prevented Th17 polarization to ameliorate CIH-induced immune barrier dysfunction in rodent intestines

Melatonin suppressed Th17 differentiation under CIH by inhibiting STAT3 signaling

Discussion

Conclusions

Abbreviations

Declarations

References

Supplementary Files

Status:

Version 1

Melatonin Alleviates Chronic Intermittent Hypoxia-induced Microbiota Dysbiosis and Attenuates Intestinal Barrier Dysfunction via STAT3/Th17 signalling pathway

Status:

Version 1

Abstract

Figures

Introduction

Materials And Methods

Animals and treatments

Cell culture and treatments

Intestinal paracellular permeability assay

Enzyme-linked immunosorbent assay (ELISA)

Antibody array assay

Microbial sequencing

Haematoxylin-eosin (HE) staining and Alcian blue-periodic acid-Schiff (AB-PAS) staining

Immunohistochemical staining

Immunofluorescent staining

Scanning electron microscopy of the processed samples

Western blotting (WB)

Preparation of lamina propria mononuclear cells (LPMCs) for flow cytometry

In vitro CD4+ T cell polarization assay

Flow cytometry to detect Th17 cells

Quantitative real-time polymerase chain reaction (qRT-PCR)

Results

Effect of melatonin on CIH-altered gut microbiota composition in mice

Melatonin prevented Th17 polarization to ameliorate CIH-induced immune barrier dysfunction in rodent intestines

Melatonin suppressed Th17 differentiation under CIH by inhibiting STAT3 signaling

Discussion

Conclusions

Abbreviations

Declarations

References

Supplementary Files

Status:

Version 1

In vitro CD4⁺ T cell polarization assay