Melatonin Reduces Inflammation in Intestinal Cells, Organoids and Explants


 Inflammatory bowel diseases (IBDs) are chronic and recurrent diseases that often occur in young people and place a heavy burden on public health in both developed and developing countries. Melatonin has been confirmed to be useful in various diseases, including Alzheimer’s disease, liver injuries and diseases, and cancers, while its role in IBDs remains unclear. To uncover the function of melatonin in IBDs, three intestinal models, including Caco-2 cells, 3D intestinal organoids and intestinal explants, were used. It was found that different concentrations of melatonin could significantly inhibit the expression levels of NFκB in Caco-2 cells, 3D intestinal organoids and intestinal explants. It was also found that melatonin could significantly inhibit the expression levels of NFκB downstream cytokines, including IL6 and IL8, in Caco-2 cells, 3D intestinal organoids and intestinal explants. Melatonin abolished the activation of LPS on the expression levels of NFκB, IL6, and IL8 in three intestinal models. Importantly, it was found that the function of melatonin in the regulation of inflammation was dependent on its receptor. Herein, the findings in this study might provide useful information for understanding the pathogenesis of IBDs and developing novel drugs to treat the diseases.


Introduction
In ammatory bowel diseases (IBDs) mainly include Crohn's disease (CD) and ulcerative colitis (UC), which are chronic and recurrent diseases and often occur in young people [1]. IBDs have brought a heavy burden to public health in both developed countries, including Canada, the United States, and Western Europe, and developing countries, such as India and China [2]. Especially in developed countries, the incidences of IBDs reach as high as 20 and 24 cases per 100,000 person-years for CD and UC, respectively [2]. Although advances in IBD studies have been achieved, the etiology and pathogenesis are not completely identi ed, as the diseases are considered to be caused by complicated factors, including abnormal intestinal immunity and altered gut microbiota caused by environmental factors such as diet and infection in genetically susceptible individuals [3]. New evidence indicates that IBDs might be closely linked to enteric virus infection [4]. It was found that more than genes were involved in the regulation of CD and UC [3]. Thus, more efforts should be made to understand the pathogenesis of these broad diseases.
The intestine is the main target organ in IBD patients, although some other gastrointestinal tract-related organs are reported to be involved in the IBD process [5]. When the intestinal mucosa barrier is attacked to increase permeability, it may result in a greater antibody antigen reaction and generate an exacerbated in ammatory response, thus increasing the risk of IBDs [1]. Dysregulated host/enteric microbial interactions are closely related to the development of IBDs [6]. The gut microbiome can directly regulate the metabolism of the host, intestinal barrier integrity, and innate immunity to in uence the development of IBDs [7]. The NFκB signaling pathway plays an important role in innate immunity in IBD patients and is linked to the rapid, acute production of diverse proin ammatory mediators, including COX-2, interleukin-1β (IL-1β), and IL-6 [8]. Herein, the regulation of innate immunity-related targets might be useful for the development of novel drugs treating IBDs.
Melatonin (N-acetyl-5-methoxytryptamine) is secreted by the pineal gland and other organs in the body to exert regulatory effects on circadian rhythms and is often used to treat insomnia, jet lag, migraine and headaches [9]. Melatonin has been con rmed to be useful in various diseases, including Alzheimer's disease [10], liver injuries and diseases [11], and cancers [12]. Emerging evidence indicates that melatonin plays a crucial role in regulating immunity [13]. Thus, in the present study, we investigated the effects of melatonin on in ammation in intestinal cell lines, organoids and explant models. The ndings in the study should provide important information for enhancing the understanding of the mode of action of the regulatory effects of melatonin on in ammation and developing novel drugs against IBDs.

Ethical statement
The animal experiment was performed in strict compliance with the animal experiment guidelines of Tangshan Workers' Hospital (2020001). The study was approved by the ethics committee of Tangshan Workers' Hospital (TWH-2020001).

Drugs
Melatonin was purchased from Beyotime Technology (Catalog No. ST1497-5g). Melatonin powder was dissolved in ethanol to 50 mg/mL as a stock solution and was stored in a -20 °C freezer for subsequent experiments. Lipopolysaccharide (LPS) was purchased from Beyotime Technology (Catalog No. ST1470-50mg).

Cell line culture
Caco-2 cells have been used in the present study since they are an excellent human enterocyte-like model to study intestinal epithelial physiology [14]. Caco-2 cells were purchased from ATCC. Caco-2 cells were maintained according to a previous protocol [14].
Intestinal organoid culture Human intestinal organoids were cultured according to previous studies [15,16]. Brie y, human intestinal tissues were obtained during surgery from Tangshan Workers' Hospital. Informed consent was obtained from the patients, and this study was approved by the ethical committee of Tangshan Workers' Hospital. Tissues were minced and incubated in PBS containing 10 mM EDTA for 30-60 min at 4 °C to loosen crypts from tissues, followed by ltering through a 70-μM cell strainer (Catalog: 08-771-1, Thermo Fisher). Crypts were embedded in Matrigel and cultured in a cell incubator.
Intestine explant isolation and culture Gut explants have been reported to be a proper model for studying gut in ammation [17]. The isolation and culture of intestinal explants were followed and modi ed based on a previous study [18]. Brie y, intestinal tissue was rinsed with PBS containing 10% Pen/Strep, and then the mucosa layer was separated from the submucosa using sterile scissors and forceps in a Petri dish containing HBSS buffer. The large piece tissue was mechanically split (e.g., cutting or chopping) into small pieces of no more than a few millimeters in length, followed by gently washing the clean mucosa in HBSS and extending it on a Petri dish with the apical side facing upwards. Intestinal explant culture medium (denoted as IECM) was prepared using DMEM supplemented with 2 mM glutamine, 1 mM NaPyr, 15% FBS-Na (fetal bovine serum -North America), 1% ITS-X and 200 ng/ml EGF. Wells of 24-well plates precoated with 50% Matrigel were seeded with 3-5 pieces of intestinal tissues on Matrigel, followed by incubation of the tissue in a cell culture incubator for 10 min to solidify the Matrigel. Then, 1 mL of IECM was added to the wells of a 24-well plate, and 3-5 pieces of intestinal tissue were transferred to the wells. Tissues were incubated in a conventional cell culture incubator (37 °C in an atmosphere of 5% CO 2 ).

MTNR1A knockdown using siRNA
The siRNA against the melatonin receptor MTNR1A was designed using GenScript siRNA Target Finder online tool (https://www.genscript.com/tools/sirna-target-nder). The sequence of siRNA against MTNR1A was AACTGAAACCACAGGACTTCA. siRNA was synthesized by Sangon Biotechnology (Shanghai, China). One microgram and two micrograms of siRNA were transfected into Caco-2 cells and 3D intestinal organoids using Lipofectamine TM 2000 (catalog: 11668019, Thermo Fisher). Knockdown effects were examined using qRT-PCR and western blot assays.
Quantitative real-time polymerase chain reaction (qRT-PCR) Total RNA was isolated from Caco-2 cells, 3D intestinal organoids, and intestinal explants using an RNase mini kit (Qiagen, Valencia, CA, United States) according to the manufacturer's protocol. A total of 500 ng of RNA was reverse transcribed using a BeyoRT™ cDNA kit (Beyotime Technology, Beijing, China).

Real-time PCR was performed with a One
Step TB Green® PrimeScript™ RT-PCR Kit (Perfect Real Time, TAKARA, Beijing, China). All reactions were performed in triplicate. mRNA expression was normalized to endogenous glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression. Gene expression was quanti ed using the ΔΔCt method. The primers used in the study were listed in Table 1. Anti-sense GACATCAGCACCAACGGGTA

Statistical analysis
All results are expressed as the means ± the standard deviation (SD). Statistical analysis was carried out using the computer-assisted GraphPad Prism program (Prism version 5.0, GraphPad Software, San Diego, CA). P < 0.05 was considered statistically signi cant.

Results
Melatonin signi cantly inhibited the expression of NFκB in Caco-2 cells, 3D intestinal organoids and intestinal explants The NFκB signaling pathway plays a central role in in ammation [19]. To evaluate the effects of melatonin on in ammation in the intestine, the expression of NFκB in intestinal cell lines, 3D intestinal organoids, and intestinal explants was tested under melatonin treatment. It was found that different doses of melatonin (0.1, 1, 5, and 10 μM) could signi cantly inhibit NFκB expression in Caco-2 cells in a dose-dependent manner ( Figure 1A and 1B). Intestinal organoids have been broadly used in various types of studies, including agriculture [20], drug discovery [21], nutrition [22], infectious diseases [23], and cancer studies [24]. Thus, the effects of melatonin on NFκB expression were further measured in 3D intestinal organoids, which indicated that 1, 5, and 10 μM could signi cantly inhibit NFκB expression in 3D intestinal organoids in a dose-dependent manner ( Figure 1C and 1D). To further con rm the effects of melatonin on in ammation in the intestine, intestinal explants were treated with different concentrations of melatonin, which indicated that 0.01, 0.1, 1, 5, and 10 μM melatonin could signi cantly suppress NFκB expression in intestinal explants in a dose-dependent manner ( Figure 1E and 1F). Taken together, melatonin was able to suppress the expression of NFκB in intestinal cell lines, 3D intestinal organoids, and intestinal explants.

Melatonin signi cantly inhibited the expression of NFκB downstream cytokines in Caco-2 cells, 3D intestinal organoids and intestinal explants
To further demonstrate the effects of melatonin on in ammation in the intestine, the effects of melatonin on two important NFκB downstream cytokines, IL6 and IL 8, were examined. It was found that 0.1, 1, 5, and 10 μM melatonin could signi cantly inhibit IL6 mRNA expression levels in Caco-2 cells (Figure 2A

Melatonin abolished LPS-induced activation of in ammation in Caco-2 cells, 3D intestinal organoids, and intestinal explants
LPS is a strong activator that induces in ammation in various organs or tissues. Consistent with previous studies, we found that LPS could strongly induce in ammation in Caco-2 cells, 3D intestinal organoids, and intestinal explants (Figure 3). To further explore the regulation of in ammation by melatonin in the intestine, cotreatment with melatonin and LPS was performed in three intestinal models. The results indicated that 10 μM melatonin signi cantly compromised the effect of stimulation with 10 and 100 ng/mL LPS on NFκB expression in Caco-2 cells ( Figure 3A). Consistently, 10 μM melatonin signi cantly compromised the stimulation of Caco-2 cells with 10 and 100 ng/mL LPS on the expression of NFκB downstream cytokines, including IL6 ( Figure 3B) and IL8 ( Figure 3C). Similarly, 10 μM melatonin signi cantly compromised the stimulation of NFκB expression by 10 and 100 ng/mL LPS in 3D intestinal organoids ( Figure 3D). In parallel, it was found that 10 μM melatonin signi cantly compromised the stimulation of 10 and 100 ng/mL LPS on the expression of NFκB downstream cytokines, including IL6 ( Figure 3E) and IL8 ( Figure 3F), in 3D intestinal organoids. Taken together, melatonin abolished LPSinduced activation of in ammation in Caco-2 cells, 3D intestinal organoids, and intestinal explants.

Silencing the melatonin receptor (MTNR1A) abolished the reduction in in ammation induced by melatonin in Caco-2 cells and 3D intestinal organoids
The function of melatonin depends on its receptor. To further demonstrate the effects of melatonin on in ammation in the intestine, a knockdown assay based on siRNA technology was performed. It was indicated that an siRNA designed against MTNR1A could signi cantly inhibit MTNR1A expression in Caco-2 cells (Figure 4A), and the knockdown effect was further con rmed using western blot assay ( Figure 4B). Notably, knockdown of MTNR1A compromised the reduction in NFκB expression induced by melatonin in Caco-2 cells ( Figure 4C). To further demonstrate the effects of MTNR1A on the regulatory effects of melatonin on in ammation in the intestine, 3D intestinal organoids were transfected with siRNA against MTNR1A, which indicated successful knockdown, as tested using qRT-PCR ( Figure 4D) and western blot ( Figure 4E). Consistent with observations in Caco-2 cells, knockdown of MTNR1A compromised the reduction in NFκB expression induced by melatonin in 3D intestinal organoids ( Figure  4F).

Discussion
Chronic in ammatory disorders of the gastrointestinal tract are the main reason for the pathogenesis of IBDs [25]. Thus, targeting in ammation regulation signaling should be an important approach for developing therapies for IBDs. In the present study, we investigated the effects of a multiple-effect chemical, melatonin, on in ammatory activity in three different intestinal models, including Caco-2 cells, 3D intestinal organoids, and intestinal explants. Melatonin inhibited the expression of NFκB and its downstream cytokines. Melatonin was shown to compromise the activation of LPS during in ammation in three intestine models. The reduction in in ammation by melatonin was compromised by silencing the melatonin receptor. Thus, the results obtained in the present study shed light on the development of novel anti-in ammatory drugs for treating IBDs.
As a hormone secreted by the pineal body, melatonin has been con rmed to exert a variety of physiological functions, including immunoregulatory effects [13]. Melatonin could closely regulate immune signaling pathways since the pineal gland is also an immune target [13]. Emerging evidence indicates that melatonin exerts anti-in ammatory activities [26]. In the pancreas of animals, it was shown that melatonin could signi cantly reduce the plasma morphometric indicators of pancreatic in ammation severity and blood levels of proin ammatory cytokines, including TNFα, IL-1β, and IL-6 [27]. Song et al found that melatonin suppressed the production of NO, protected neural stem cells from LPSinduced in ammatory stress, promoted the expression of SOX2 and activated PI3K/Akt/Nrf2 signaling under LPS-induced in ammatory conditions [28]. In another study, melatonin was found to relieve in ammation-induced apoptosis in human umbilical vein endothelial cells by inhibiting the Ca2+-XO-ROS-Drp1-mitochondrial ssion axis by stimulating the AMPK/SERCA2a pathway [29]. In the present study, we found that melatonin could reduce the expression level of the important in ammation regulator NFκB in different intestine models (Figure 1), which was consistent with ndings from other groups. Similarly, melatonin inhibited the expression levels of NFκB downstream cytokines (i.e., IL6 and IL8) in intestinal models ( Figure 2).
Melatonin was found to be closely linked to NFκB; for example, melatonin was found to reduce NF-κB activation in pinealocytes and immune competent cells [30]. Liu et al demonstrated that melatonin potently inhibited androgen receptor splice variant-7 (AR-V7)-induced NFκB activation in prostate cancer cells [31]. Shrestha et al found that melatonin inhibited IKKβ/NF-κB/COX-2 signaling to exert anticancer effects against bladder cancer [32]. Interestingly, Mannino et al demonstrated that melatonin robustly suppressed the in ammatory response induced by interleukin-1β in human intestinal epithelial cells [33].
In the present study, it was demonstrated that melatonin signi cantly compromised the activation of NFκB, IL6 and IL8 induced by LPS ( Figure 3). Melatonin receptor signaling plays an important role in the regulation of the physiological activities of melatonin [34]. It was found that melatonin exerted inhibitory effects on oxidative stress-induced mitochondrial dysfunction depending on its receptor 1 (MT1) [35]. In the present study, we also found that knockdown of the melatonin receptor potently abolished the reduction in in ammation induced by melatonin in intestinal models ( Figure 4).
In conclusion, melatonin plays a crucial role in regulating in ammatory activities in the intestine ( Figure  5). It can signi cantly inhibit the expression levels of NFκB and its downstream cytokines, including IL6 and IL8. Melatonin can also abolish the activation of LPS during in ammation. Importantly, it was found that the function of melatonin in the regulation of in ammation was dependent on its receptor. Herein, the ndings in the study might provide useful information for understanding the pathogenesis of IBDs and developing novel drugs to treat these diseases.

Declarations
Author Contributions X.Z. and X.M. conceived the study. X.Z., X.Y., and X.Z. designed experiments, and X.Z. and X.Z. performed most of the experiments, data collection and data analysis. X.Z. and X.Z. wrote the manuscript, which was reviewed by all authors.

Disclosures
The authors have no competing nancial interests.