Polyphenol Extract of Moringa Oleifera Leaves Alleviates Colonic Inflammation in Dextran Sulfate Sodium-Treated Mice

Moringa oleifera Lam. is an essential herb used for the treatment of inflammation, diabetes, high blood pressure, and other diseases. In this study, phenolic extracts of M. oleifera leaves were obtained and analyzed. The results showed that the main identifiable phenols were astragalin, chlorogenic acid, isoquercitrin, kaempferitrin, luteolin, quercetin, and rutin. The effects of M. oleifera polyphenol extract (MOPE) on experimental colitis induced by 3% dextran sulfate sodium (DSS) were investigated. The results showed that oral administration of MOPE significantly alleviated the symptoms of DSS-induced colitis. MOPE significantly reduced weight loss, the disease activity index, colon shortening, and mucosal damage. In addition, MOPE attenuated the infiltration of CD3+ T cells, CD177+ neutrophils, and F4/80+ macrophages and significantly inhibited the secretion of IL-6 and TNF-α. After the MOPE administration, the expression of proteins associated with the NF-κB signaling pathway changed. Specifically, compared with that of the DSS group, the protein expression of NF-κB p65 and p-IκBα was downregulated, and the expression of IκBα was upregulated. This study revealed the anti-inflammatory effects and mechanisms of MOPE in the colon, indicating its potential use in preventing inflammation-driven diseases.


Introduction
Moringa oleifera Lam. has been historically used as nutritious food and traditional medicine in China [1]. In 2012, M. oleifera leaves were approved by the Chinese Ministry of Health as a new resource food. M. oleifera is also an essential traditional herb used to treat inflammation, diabetes, hypertension, anemia, hypoimmunity, and other diseases [2][3][4][5]. ere are some flavonoid pigments, such as alkaloids, kaempferol, rhamnose, and isoquercitrin, and various antioxidant compounds, such as ascorbic acid, flavonoids, phenols, and carotenoids, which occur naturally in M. oleifera [6] and may contribute to the anti-inflammatory activity of M. oleifera. Additionally, it has been previously reported that some active components in M. oleifera mitigate some chronic inflammation [7]. However, there have been few reports on the improvement and alleviation of inflammation by M. oleifera polyphenols.
Inflammatory bowel disease (IBD), including ulcerative colitis (UC) and Crohn's disease (CD) [8], is a chronic recurrent disease of the intestine [9]. IBD is exceptionally harmful and is associated with an increased risk for colon cancer [10]. IBD is affected by diets such as high-fat diets, and the incidence is high in some developed countries, such as Europe [8]. Recently, the incidence of IBD in Asia has increased rapidly due to the improvements in living conditions and the adoption of a Western lifestyle. e pathogenesis of IBD is not yet fully clear; it is generally considered to be related to the environment, infection, genetics, psychology, and immunity. In addition, the intestinal mucosal immune system plays an essential role in the pathogenesis of IBD [11]. Several studies have shown that there is a high level of activated transcription factorkappa B in the intestinal mucosa of IBD patients, and nuclear transcription factor kappa B (NF-κB) is considered to be one of the most critical signaling pathways in IBD. High expression of NF-κB can enhance the release of proinflammatory cytokines, such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), which leads to colonic tissue damage [12]. It has been suggested that the activation of NF-κB plays a crucial role in the regulation of immune and inflammatory responses, and thus inhibiting the NF-κB signaling pathway is a novel therapeutic strategy in IBD treatment research.
To date, there are no ideal drugs for the treatment of IBD. Existing treatments mainly include resection and relief medication, such as 5-aminosalicylic (5-ASA), corticosteroids, immunomodulatory drugs, and biological agents. However, long-term administration of these drugs has various side effects, including drug resistance and drug tolerance [13]. erefore, it is necessary to find natural treatments with fewer side effects. Although M. oleifera has been reported to exhibit various bioactivities, knowledge about the effects of M. oleifera polyphenol extract (MOPE) on gut health is limited. e present study aimed to evaluate the effects of MOPE in an established model of acute ulcerative colitis induced with dextran sulfate sodium (DSS) and further explore its underlying mechanisms.

Preparation of MOPE.
Dried M. oleifera leaves (Yunnan Shenbaofu Technology Development Co., Ltd., Dehong, China) were crushed and then extracted by the ultrasoundassisted method as described by Fei et al. [14]. e extraction process was performed three times under the same conditions with 70% ethanol as the solvent, a solid/solvent ratio of 1/30, and 250 W of ultrasonic power for 20 min. ese three extraction supernatants were combined and passed through a D101 macroporous resin, followed by elution with an 80% ethanol solution to obtain an ethanol eluate. e eluate was vacuum freeze-dried to obtain the MOPE.

Animal Treatments.
e animal experiments were performed according to international guidelines. e protocol was approved by the Animal Care and Use Committee of Yunnan Agricultural University (No. YNAU-2017-011). Six-week-old male C57BL/6 mice (18-20 g, Liaoning Changsheng Biotechnology Co. Ltd., Liaoning, China) were maintained on a 12/12 h light/dark cycle at 25 ± 1°C and 55% humidity. All mice were housed 5/cage and had free access to standard mouse feed and tap water. e mice were allowed to acclimate for one week before the study began.

Induction of Colitis and MOPE Treatment.
Colitis was induced with dextran sulfate sodium (DSS, 36000-50000, MP Biomedicals, USA, 2160110). e mice were randomly divided into five groups (n � 10 per group): the control group was given ultrapure (UP) water for 14 days; the DSS group was given UP water for the first seven days and then 3% DSS for the following seven days; the 5-aminosalicylic acid (5-ASA, positive control) group and the MOPE groups (50 mg/kg and 200 mg/kg, respectively) were given 5-ASA and MOPE individually for 14 days and were simultaneously given the water containing 3% DSS starting on the eighth day ( Figure 1(a)).

Evaluation of the Disease Activity Index (DAI).
Mice were monitored daily for the development of colitis based on body weight changes, gross rectal bleeding, and stool consistency. e disease activity index (DAI) scores were measured for each animal according to a previously described method [15]. e details of the DAI grading standards are listed in the Supplementary Materials (Table S1).

Histopathology.
e colon tissues 1 cm from the distal colon were rinsed with ice-cold PBS, fixed in 10% (v/v) neutral formalin, embedded in paraffin, cut into 5 µm slices (Leica, RM2126RT, Germany), and stained with hematoxylin and eosin (H&E). Histological analysis was performed according to previously described methods [16]. A detailed description of the score standards is listed in the Supplementary Materials (Table S2).

Immunohistochemistry.
e tissue slices were deparaffinized by xylene for 0.5-1 h and rehydrated sequentially with 100%, 100%, 95%, and 80% ethanol and water for 5 min each. en, the slices were immersed in methanol containing 3% hydrogen peroxide, followed by high-pressure antigen repair. e slides were incubated with primary antibodies against CD3 + , CD177 + , and F4/80 + (Abcam, Cambridge, MA) overnight and then incubated with the appropriate secondary antibodies, followed by diaminobenzidine (DAB) color development, hematoxylin staining, and xylene clearing. Immunostained tissue slices were visualized under a microscope (Olympus, Japan).

Determination of Serum Inflammatory Factors.
At the end of the experiment, serum samples were prepared and subjected to enzyme-linked immunosorbent assay (ELISA) to determine serum levels of IL-6 and TNF-α using ELISA kits from BD Pharmingen. e procedure was performed according to the instructions.
2.10. Statistical Analysis. GraphPad Prism software version 5.0 was used to graph the results. e data are presented as the mean ± SEM. Statistical analysis was performed by SPSS 19.0. e differences between multiple groups were analyzed by one-way analysis of variance (ANOVA), followed by  Duncan's test. e results were considered statistically significant when p < 0.05.

MOPE Ameliorated Colitis Symptoms in Mice.
e mice were treated with a low (50 mg/kg) or a high dose (200 mg/kg) of MOPE for 2 weeks (Figure 1(a)). Bodyweights and DAI scores of the mice were monitored every day throughout the experiment.
e DSS-only treatment group exhibited severe bodyweight loss, while treatment with 5-ASA (positive control) and 50 mg/kg and 200 mg/kg MOPE significantly alleviated weight loss (Figure 1(b)). Additionally, mice in the DSS group exhibited high DAI scores, while 5-ASA and MOPE treatment significantly decreased the DAI scores compared to those of the DSS group (Figure 1(c)). ese results indicated that 5-ASA and MOPE treatments alleviated colonic inflammatory symptoms in mice. e length of the colon is an important indicator for indirectly assessing the severity of colitis. In this study, fecal residues in the colons of control mice were normal and granular.
e DSS group was induced to develop colitis, exhibiting hyperemic colons and very severe colon shortening. Furthermore, fecal residue in the colon in the 5-ASA group and MOPE groups was loose and less hyperemic (Figure 1(d)). 5-ASA and MOPE treatment significantly prevented the colonic shortening compared to that of the DSS-treated group (Figure 1(e)).

MOPE Ameliorated Colonic Injury in Mice with DSS-Induced Colitis.
e histological examination of colonic tissue from healthy mice revealed that the epithelial cells and crypts were structurally intact, the glands were arranged neatly, and there was no inflammatory cell infiltration (Figure 3(a)). In contrast, in the DSS-treated group, the lamina propria was damaged, the glands were destroyed and arranged irregularly, the epithelial cells and crypt structures were destroyed, and a large number of inflammatory cells infiltrated the muscle floor (Figure 3(b)). is damage was significantly alleviated after treatment with 5-ASA and MOPE (Figures 3(c)-3(f )).

MOPE Attenuated the Infiltration of Inflammatory Cells in Mice with DSS-Induced Colitis.
e expression of CD3 + , CD177 + , and F4/80 + in the distal colon was measured by immunohistochemistry to detect T cell, neutrophil, and macrophage infiltration, respectively. e results indicated that T cells, neutrophils, and macrophages were increased in DSS-treated mice compared to normal mice, indicating that these inflammatory cells infiltrated the colonic injury area. 5-ASA and MOPE treatment reduced the infiltration of T cells, neutrophils, and macrophages ( Figure 4).

MOPE Decreased the Cytokine Levels in Mice with DSS-Induced
Colitis. IL-6 and TNF-α are two typical inflammatory cytokines. e serum levels of IL-6 and TNF-α were measured in the present study. Compared with those of the control group, substantial increases in IL-6 ( Figure 5(a)) and TNF-α ( Figure 5(b)) levels were observed in the DSS group (p < 0.001). However, MOPE treatment significantly reduced the serum levels of IL-6 (p < 0.01) and TNF-α (p < 0.05), and the effect was the same as that of 5-ASA (positive control). Moreover, MOPE treatment significantly inhibited the protein expression of TNF-α in colon tissue after DSS-induced colitis (Figures 5(c) and 5(d)).
e NF-κB signaling pathway strongly influences the pathogenesis of colitis. In the present study, the protein expression of NF-κB p65 in the nucleus and p-IκBα in the colon tissue of DSS model mice was higher than that of control mice, and the protein expression of IκBα was decreased ( Figures 5(d)-5(g)). is result indicated that the NF-κB signaling pathway was activated in the colonic mucosal. After treatment with MOPE, the protein expression of NF-κB p65 and p-IκBα was significantly downregulated, and the expression of IκBα was upregulated. ese results suggested that the anti-inflammatory effects of MOPE were related to inhibiting NF-κB pathway activation.

Discussion
M. oleifera has been reported to have beneficial effects on various diseases, including diabetes, hypertension, hyperlipidemia, and other chronic inflammation. ere are some individual bioactive components in M. oleifera extracts that have potential preventive and therapeutic effects on inflammation [17]. Studies have shown that extracts of M. oleifera inhibit the production of NO and proinflammatory cytokines in LPS-induced macrophages [7,18]. e pods of M. oleifera can inhibit the LPS-induced expression of IL-6 and TNF-α and inhibit LPS-induced IκB activation [19]. e ethyl acetate extract of M. oleifera can significantly inhibit LPS-induced TNF-α, IL-6, and IL-8 production in human monocyte-derived macrophages (MDMs). M. oleifera extracts effectively inhibit the expression of inflammatory mediators that may be related to the inhibition of the NF-κB signaling pathway [3,20]. In addition, it has been reported that the hydroalcoholic extract of M. oleifera seeds can reduce acetic acid-induced colitis in rats [21]. Moreover, the isothiocyanate extracted from M. oleifera seeds can effectively alleviate DSS-induced colitis [22]. However, knowledge about the effects of MOPE on gut health is limited. erefore, the present study aimed to  Here, we showed that MOPE could significantly ameliorate the symptoms of DSS-induced colitis, including mitigating body weight loss, colonic tissue damage, the secretion of inflammatory cytokines, and the infiltration of inflammatory cells. IL-6 and TNF-α are proinflammatory cytokines that mediate inflammatory responses in the UC model. Excessive secretion of these inflammatory cytokines can cause colitis [15]. Many studies have reported that the levels of IL-6 and TNF-α in IBD mice are increased [23][24][25]. IL-6 is an interleukin produced by a variety of cells and is closely related to inflammation and the immune response. IL-6 stimulates neutrophil chemotaxis and causes tissue destruction in the colon. Some studies have found that IL-6 is elevated in many IBDs and may be associated with the NF-κB signaling pathway [26,27]. TNF-α, a cytokine with multiple effects that is produced by activated T cells, plays an essential role in the pathogenesis of colitis by triggering the accumulation and activation of leukocytes. TNF-α overexpression is vital for the pathogenesis of the intestinal mucosa [28,29]. In the present study, DSS induced an increase in TNF-α and IL-6 in mice, whereas MOPE treatment significantly reduced the increases in both inflammatory factors.
NF-κB is a classic signaling pathway associated with inflammation. Several studies have shown that some natural compounds, such as caffeic acid, blueberry polyphenols, and Aster glehni extract, can improve UC by inhibiting the NF-κB pathway [25,[30][31][32]. It was shown that the ethyl acetate extract of M. oleifera inhibited NF-κB p65 in RAW 264.7 cells, and it exerted anti-inflammatory effects by upregulating the expression of IκB inhibitor (IκBα) and blocking the nuclear translocation of NF-κB [3]. NF-κB family members are retained in the cytoplasm bound to a class of inhibitory proteins termed IκBs [33]. When cells are activated, IκB is phosphorylated by IκB kinase (IKK) and degraded, after which the released NF-κB translocates to the nucleus and activates transcription. In other words, phosphorylation of IκB (p-IκB) is a crucial step in NF-κB activation, which leads to activation of the NF-κB signaling pathway [34]. e increase in TNF-α-related indicators is an essential manifestation of NF-κB pathway activation. Many treatments target TNF-α, and some related drugs, including TNF-α blockers, have been successfully used in the treatment of patients with IBD [31,35]. For these reasons, the proteins related to the NF-κB signaling pathways were investigated in the present study. Our results showed that the protein expression of NF-κB p65 in the nucleus and p-IκBα and TNF-α in colon tissue of DSS model mice was higher than that of control mice, and the expression of IκBα was decreased. is result indicated that the NF-κB pathway was activated in the colonic mucosa. After MOPE administration, the protein expression of NF-κB p65 and p-IκBα was downregulated, and the expression of IκBα was upregulated. Furthermore, MOPE treatment inhibited the high expression of TNF-α in colon tissue. ese results indicate that the anti-inflammatory effects of MOPE may be related to the inhibition of the NF-κB signaling pathway.
In the present study, MOPE is a mixed extract. e main phenolic substances in MOPE are kaempferol, quercetin, chlorogenic acid, isoquercitrin, rutin, and luteolin, as determined by UPLC-QTOF-MS/MS analysis, which is consistent with the results of the study by Zhu et al. [36]. In the future, we will focus on the separation and purification of M. oleifera polyphenols to determine which phenolic substance has antiinflammatory or multicomponent synergistic effects.  In conclusion, our research shows that MOPE can alleviate DSS-induced colitis, including mitigating body weight loss, colon shortening, the secretion of inflammatory cytokines, colon tissue damage, and inflammatory cell infiltration. Further study examination shows that MOPE may alleviate colitis by inhibiting NF-κB signaling pathway activation. ese results indicate that M. oleifera can be developed as a potential health food for preventing colitis.
Data Availability e data to support the findings of this study are included within the article. Other data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare that there are no conflicts of interest regarding the publication of this paper.

Authors' Contributions
Yunjuan Zhang and Lei Peng contributed equally to this article.  (Table S1). A detailed description of  the Histological Scores of Colon Damage is listed in the Supplementary Materials (Table S2). (Supplementary Materials)