Vinegar Treatment Prevents the Development of Murine Experimental

Article pubs.acs.org/JAFC

Vinegar Treatment Prevents the Development of Murine Experimental Colitis via Inhibition of Inflammation and Apoptosis Fengge Shen,† Jiaxuan Feng,§ Xinhui Wang,† Zhimin Qi,† Xiaochen Shi,† Yanan An,† Qiaoli Zhang,† Chao Wang,† Mingyuan Liu,†,# Bo Liu,*,† and Lu Yu*,† †

Key Laboratory for Zoonosis Research, Ministry of Education, Institute of Zoonosis, The First Hospital of Jilin University, College of Veterinary Medicine and College of Animal Science, Jilin University, Changchun 130062, China § College of Medicine, Yanbian University, Yanji 133000, China # Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou 225009, China ABSTRACT: This study investigated the preventive effects of vinegar and acetic acid (the active component of vinegar) on ulcerative colitis (UC) in mice. Vinegar (5% v/v) or acetic acid (0.3% w/v) treatment significantly reduced the disease activity index and histopathological scores, attenuated body weight loss, and shortened the colon length in a murine experimental colitis model induced by dextran sulfate sodium (DSS). Further mechanistic analysis showed that vinegar inhibited inflammation through suppressing Th1 and Th17 responses, the NLRP3 inflammasome, and MAPK signaling activation. Vinegar also inhibited endoplasmic reticulum (ER) stress-mediated apoptosis in the colitis mouse model. Surprisingly, pretreatment with vinegar for 28 days before DSS induction increased levels of the commensal lactic acid-producing or acetic acid-producing bacteria, including Lactobacillus, Bifidobacteria, and Enterococcus faecalis, whereas decreased Escherichia coli levels were found in the feces of mice. These results suggest that vinegar supplementation might provide a new dietary strategy for the prevention of UC. KEYWORDS: vinegar, colitis, inflammation, apoptosis, intestinal microbiota

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INTRODUCTION Ulcerative colitis (UC) is a chronic and remittent−relapsing inflammation of the gastrointestinal tract that affects millions of people worldwide. UC is thought to result from activation of the mucosal immune system and disruption of the epithelial barrier by the enteric microbiota and is likely influenced by genetic factors.1 To investigate this disease, a mouse model of acute colonic inflammation has been developed by orally administering dextran sulfate sodium (DSS). The histopathology of this model showed some resemblance to human UC and enabled research into the pathogenesis of this disease. This model is characterized by a general inflammatory process that is associated with diarrhea, bloody stools, growth failure, and weight loss.2 UC has long been regarded as a problem of developed countries, with a Western lifestyle contributing to the pathogenesis. For UC therapy, biological agents such as infliximab, certolizumab pegol, and adalimumab cost several thousand to several tens of thousands of dollars per UC patient per year. With the rapid modernization and Westernization of the population, UC has now emerged in developing nations; thus, there is an urgent need for appropriate treatments that are highly effective and have low costs. The use of food additives of natural origin to prevent and treat diseases has increased significantly in recent years; however, there has been a lack of analyses of the medical benefits and related molecular mechanisms. From a healthcare standpoint, vinegar is much more conventional than pharmaceutical interventions due to its widespread availability and low cost. Vinegar that is derived from grain fermentation (sorghum, soybean, or rice is generally employed as a raw material) is used as an important condiment in © XXXX American Chemical Society

daily life and can be used for treating diseases in traditional Chinese medicine (TCM).3 The main component of vinegar is acetic acid, which gives vinegar its sour taste and pungent smell. The most common vinegars tested contained from 4 to 6% acetic acid. Shizhen Li stated in his book that vinegar can disperse blood stasis, cure jaundice, attenuate yellow perspiration, and treat inflammatory diseases.4 Vinegar has been used extensively since the era of Hippocrates as an antifungal and antibacterial agent for the treatment of numerous infections and ailments, including persistent coughs, head lice, insect bites, warts, ear infections, and wounds.5 Recently, studies have shown that acetic acid exhibits important physiological activities, including metabolism promotion, detoxification, liver function improvement,6 and especially antihypertensive and antihyperglycemic effects. Moreover, kurozu (a Japanese black vinegar) has been shown to have an anticolitis activity.7 Although this study was very interesting, it was also cursory; thus, we intend to do in-depth research on the molecular mechanisms of the protective effects of vinegar against UC. Chronic mucosal inflammation produces high pro-inflammatory cytokine levels, including tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β), and interferon-gamma (IFN-γ), by innate leukocytes and T cell subsets, thereby resulting in colonic tissue damage. A study by Bauer et al. demonstrated a role of the NLRP3 inflammasome in experimental colitis, suggesting that Received: November 15, 2015 Revised: January 19, 2016 Accepted: January 21, 2016

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DOI: 10.1021/acs.jafc.5b05415 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

component, acetic acid, were counted with differential media on the 28th day. Intestinal bacteria of mice were cultivated with differential media, including MRS medium, Bifidobacterium BS medium, MacConkey agar, and Enterococcus faecalis agar, which were purchased from Hopebio Co., Ltd. (Qingdao, China). The plates were incubated anaerobically for 2−3 days at 37 °C with the exception of the MacConkey and E. faecalis plates, which were incubated aerobically overnight at 37 °C. Cytokine Analysis by ELISA. Colons from mice in each group were cultivated in RPMI 1640 medium with 100 U/mL penicillin and 100 mg/mL streptomycin under a humidified 5% (v/v) CO2 atmosphere at 37 °C for 24 h. The culture medium was centrifuged at 12000g at 4 °C for 10 min. The IFN-γ, IL-1β, IL-18, IL-12/IL-23p40, IL-17A, and TNFα autocrine levels in the colon were quantified with ELISA kits (BioLegend, Inc., San Diego, CA, USA) according to the manufacturer’s instructions. Preparation of Cecal Bacterial Lysates. Cecal bacterial lysates (CBL) were prepared as described by Dieleman et al.14 Briefly, the cecal contents from the mice were solubilized by vortexing the contents in RPMI, incubating them with 10 μg/mL DNase and 0.01 M MgCl2, and then homogenizing them for 3 min using 0.1 mm glass beads in a MiniBead Beater (Biospec Products, Bartlesville, OK, USA). After centrifuging at 10000g for 10 min, the supernatant was filtered through a 0.45 mM syringe filter. The amount of total extracted protein was determined with a BCA protein assay kit (Dingguo, Beijing, China). Mesenteric Lymph Node Cell Cultures. Mesenteric lymph nodes (MLNs) were removed from mice, and single-cell suspensions were prepared by gentle teasing. MLN cells (4 × 105) and 10 μg/mL CBL were cultured in 96-well flat-bottom microplates, in 0.2 mL of complete medium (RPMI 1640 plus 5% heat-inactivated fetal calf serum and 50 mg/mL gentamicin) for 72 h. The culture medium was then collected and centrifuged at 2000g at 4 °C for 10 min. The culture supernatants were collected for cytokine detection and stored at −20 °C. Western Blotting. Both adherent and floating cells were collected, and Western blotting was performed as previously described. Briefly, THP-1 cells were solubilized in radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime, Wuhan, China). Protein concentrations were determined with the Bio-Rad DC protein assay (Bio-Rad Laboratories, Hercules, CA, USA). The protein lysates were separated on 12% SDSPAGE gels and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, USA). The membranes were soaked in blocking buffer (5% skim milk) for 2 h and incubated with primary antibodies at 4 °C overnight, followed by horseradish peroxidase-conjugated secondary antibodies for 2 h. Protein bands were detected using the enhanced chemiluminescence (ECL) system (Beyotime), and the images were obtained using a CanoScan LiDE 100 scanner (Canon, Tokyo, Japan). Mouse anti-mouse β-actin (Zhongshan Jinqiao, Beijing, China) was used as an internal reference to ensure equal loading. Protein blots were measured using ImageJ software to obtain accurate results. Statistical Analysis. The results were expressed as the mean ± SEM of three independent experiments, and each experiment included triplicate measurements. The data were statistically evaluated with oneway ANOVA followed by Dunnett’s test between the control and multiple-dose groups. The significance level was set at a p value of 0.05.

the NLRP3 inflammasome complex may serve as a potential target for the development of novel therapeutics for patients with UC.8 Previous papers suggest that the endoplasmic reticulum (ER) stress response is also associated with the development of UC.9 UC accumulation leads to ER stress and triggers the unfolded protein response (UPR), which initiates the development of apoptosis.10 The frequency of apoptosis and its contribution to the loss of epithelial cells are also considerably increased in UC.11 On the basis of the above-mentioned studies, we decided to examine the protective effects of vinegar or acetic acid in a rodent model of DSS-induced colitis, focusing on possible anti-inflammation and anti-apoptosis effects.

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MATERIALS AND METHODS

Ethics Statement. Male C57BL/6 mice (6−8 weeks old, 20−22 g) were obtained from the Experimental Animal Centre of Jilin University. The mice were housed in microisolator cages and received food and water. The laboratory temperature was 24 ± 1 °C, and relative humidity was 40−80%. All of the animal studies were conducted according to the experimental practices and standards that were approved by the Animal Welfare and Research Ethics Committee at Jilin University (no. IZ2009-008). The protocols were reviewed and approved by the committee. All of the animal studies were performed under isoflurane anesthesia, and every effort was made to minimize suffering. Cell Culture. Human THP-1 cells were purchased from the Shanghai Institute of Cell Biology (Shanghai, China) and maintained in RPMI 1640 medium supplemented with 100 U/mL penicillin, 100 mg/mL streptomycin (Dingguo Biosciences, Beijing, China), and 10% fetal calf serum (Gibco, Grand Island, NY, USA) under a humidified 5% (v/v) CO2 atmosphere at 37 °C. In this study, vinegar is derived from sorghum fermentation containing 6% acetic acid (Beikang Brewing Food Co., Ltd., Changchun, China). The THP-1 cells (1 × 106) were treated with 500 ng/mL LPS for 6 h in the presence or absence of vinegar (0.5 or 0.25% v/v) or acetic acid (0.03% w/v) at 37 °C. The LPS-primed THP-1 cells were treated with vinegar (0.5 or 0.25% v/v) or acetic acid (0.03%) for 5 h, followed by 5 mM ATP treatment for 30 min. According to our preliminary experiments, we found vinegar (0.5 or 0.25% v/v) had good anti-inflammatory effects. In vitro, acetic acid in 0.5% vinegar would be the equivalent of 0.03% acetic acid. Colitis Induction and Evaluations. Acute colitis was induced by feeding mice 3% (w/v) DSS (molecular weight 40 kDa; TdB Consultancy, Uppsala, Sweden), which was dissolved in drinking water, continuously for 5 days. In vivo, acetic acid in 5% vinegar would be the equivalent of 0.3% acetic acid. Normal C57BL/6 mice received the same drinking water without DSS (n = 8 mice in each group). The two groups were treated with vinegar (5% v/v) or acetic acid (0.3% w/v) that was dissolved in drinking water continuously from days 0 to 28. The other two groups were treated with drinking water continuously for 28 days. After 28 days, the three groups (including the vinegar and acetic acid groups) were induced with 3% (w/v) DSS from day 29 to 33. To avoid chemical interaction between vinegar (acetic acid) and DSS during the DSS treatment period, vinegar (acetic acid) was administered by intragastric infusion according to the mean daily dose of vinegar (acetic acid). The weights of animals were collected at days 0, 7, 14, 21, 28, 29, 31, and 33. The disease activity index (DAI) was calculated by assigning well-established and validated scores, as previously described.12 After 33 days, the animals were sacrificed and rapidly dissected. The entire colon was quickly removed, and macroscopic scores were blindly determined. Colon segments, which were taken for histopathological assays, were fixed in 10% normal buffered formalin, embedded in paraffin, sectioned at 4 μm thickness with a paraffin microtome, and mounted onto microscope slides. The sections were stained with hematoxylin and eosin (H&E), and histological scores were calculated by evaluating the damage to the epithelial mucosa and inflammatory infiltration. Macroscopic and histological grading was conducted according to a previously described method.13 Intestinal Bacteria Cultivation and Quantitation. Representative bacteria in the feces of the mice treated with vinegar or its main

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RESULTS Vinegar and Acetic Acid Attenuated DSS-Induced Murine Experimental Colitis. It is well-known that DSS induces a severe illness in mice that is characterized by dramatic body weight loss, diarrhea, and bloody stools. Compared with the vehicle-treated group, vinegar at 5% v/v or acetic acid at 0.3% w/ v significantly attenuated the body weight loss during experimental colitis disease progression in mice (Figure 1A). The colitis model had significant diarrhea, loose feces, and visible fecal blood, whereas the vinegar or acetic acid treatment resulted in a significant DAI elevation (Figure 1B−D). DSS typically causes colonic shortening; however, such changes were also B


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cytokine expression in the acute DSS colitis model, IL-12/IL23p40, INF-γ, IL-17A, and TNF-α levels in the MLN cells and colons were measured in parallel following the induction of colitis. As shown in Figure 3, the IL-12/IL-23p40, INF-γ, IL-17A, and TNF-α levels were remarkably increased after CBL or DSS challenge. The spontaneous IL-12/IL-23p40, INF-γ, IL-17A, and TNF-α levels in the colon from each group were analyzed. The inflammatory cytokine autocrine levels were significantly suppressed by the vinegar or acetic acid treatments (Figure 3A). Dieleman et al. reported that the MLNs drained the inflamed cecum and proximal colon.14 We then analyzed the in vitro cytokine responses of the MLN cells to CBL. Both vinegar and acetic acid significantly inhibited the IL-12/IL-23p40, INF-γ, IL17A, and TNF-α release from the MLN cells after CBL challenge in vitro (Figure 3B). Thus, our ex vivo and in vitro investigations showed that vinegar and acetic acid suppressed Th1 and Th17 responses in mice with DSS-induced colitis (Figure 3). Vinegar and Acetic Acid Reduced NLRP3 Inflammasome Activation in Vitro and in Vivo. A study by Bauer et al. demonstrated that the NLRP3 inflammasome complex may serve as a potential target for the development of novel therapeutics for patients with inflammatory bowel diseases using a murine experimental colitis model.8 IL-1β and IL-18 are processed as inactive cytoplasmic precursors (pro-IL-1β and proIL-18), which must be cleaved by caspase-1 to produce the mature active form. As shown in Figure 4A, the vinegar (acetic acid) treatments significantly inhibited spontaneous IL-1β/IL-18 release into the colon in murine experimental colitis. Macrophage activation in the colon mucosa was thought to play an essential role in UC pathogenesis. To investigate the inhibition effect of vinegar on activated macrophages, THP-1 cells were used for in vitro experiments. In vitro, we investigated the activation (phosphorylation) of the NLRP3 inflammasome (NLRP3, ASC, caspase-1, IL-1β, and IL-18; Santa Cruz Biotechnology, Santa Cruz, CA, USA) in macrophages after treatment with ATP. Due to differences in cells in vitro and in vivo, 0.5% vinegar or 0.03% acetic acid was used in vitro. Our results showed that vinegar (0.5 or 0.25% v/v) or acetic acid (0.03% w/v) inhibited ATP-induced NLRP3 inflammasome activation in vitro (Figure 4B). Moreover, 5% vinegar or 0.3% acetic acid treatment markedly inhibited ASC, caspase-1, IL-1β, and IL-18 activation in the colon and spleen of colitis mice in comparison with vehicle-treated mice (Figure 4C,D). This result showed that vinegar or acetic acid had an anti-inflammatory effect by inhibition of the NLRP3 inflammasome in macrophages or DSS-induced colitis mice. Vinegar and Acetic Acid Reduced MAPK Signaling Activation in DSS-Induced Colitis Mice. Activation of the mitogen-activated protein kinase (MAPK) pathways plays essential roles in transcriptional induction of various genes that are involved in inflammation, such as TNF-α, IL-1β, IFN-γ, and IL-17A.18 Next, we assessed the in vitro anti-inflammatory activities of vinegar or acetic acid in THP-1 cells that were activated by LPS. As shown in Figure 5A, the p-JNK, p-ERK, and p-P38 (Cell Signaling Technology, Beverly, MA, USA) levels in THP-1 cells were increased by LPS, whereas vinegar (0.5 or 0.25% v/v) or acetic acid (0.03% w/v) inhibited the levels of the three MAPK proteins in THP-1 cells that were induced by LPS (Figure 5A). As shown in Figure 5B,C, DSS treatment markedly induced the p38, ERK, and JNK phosphorylation levels in the spleens and injured colons from the mice. On the contrary, both vinegar and acetic acid treatment markedly reduced the ERK, JNK, and p38 phosphorylation levels (Figure 5B,C). These

Figure 1. Vinegar and acetic acid treatment ameliorated DSS-induced experimental colitis in mice: (A) loss of basal body weight of each group (n = 8 per group) during the disease process; (B, C) diarrhea and hematochezia evaluations of mouse feces (n = 8 per group); (D) calculated disease activity index (DAI) (n = 8 per group) [□, control; ■, DSS treated; ▲, DSS + vinegar (5% v/v); ○, DSS + acetic acid (0.3% w/ v)]; (E) Macroscopic appearances and (F) measured length of colons from each group of mice. Data are presented as the mean ± SEM; (∗) p < 0.05 and (∗∗) p < 0.01 versus DSS-treated group on the same day.

improved by the vinegar or acetic acid treatments (Figure 1E,F). Histopathological analysis of the proximal, mid, and distal colon segments showed that there were distorted crypts, goblet cell loss, mononuclear cell infiltration, and severe mucosal damage in the colitis mouse colon specimens (Figure 2A−C). These pathological changes were significantly improved after the vinegar or acetic acid treatments (Figure 2D−F). These results showed that the vinegar or acetic acid treatments attenuated DSS-induced murine experimental colitis. Vinegar and Acetic Acid Suppressed Th1 and Th17 Responses in Mice with DSS-Induced Colitis ex Vivo and in Vitro. Until recently, classical T cells have been considered the major players involved in UC development. However, there is an increasing body of evidence showing the importance of the T helper 17 (Th17) pathway in UC.15 Th17 cells are characterized by IL-17 production.16 Inflammasome-derived IL-1β and IL-18, which are likely to be essential in the early phase of the inflammatory cascade leading to inflammation in the colon,8 are required for the differentiation of Th17 and T helper 1 (Th1) cells.17 To examine the influence of vinegar or acetic acid on C


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Figure 2. Vinegar and acetic acid prevented DSS-induced colon damage in mice: (A−C) representative histological sections of colon from untreated, DSS, vinegar, and acetic acid dietary groups, respectively, examined microscopically after H&E staining in proximal (A), mid (B), and distal (C) colon with original magnification ×200; (D−F) histopathological scores of proximal (D), mid (E), and distal (F) colon determined using a colitis score as previously described by Dieleman et al.13 Data are reported as the mean ± SEM (n = 5); (∗) p < 0.05 and (∗∗) p < 0.01 versus DSS-treated control group.

proximal effectors of the UPR exist in cells that sense the accumulation of misfolded proteins. These include eukaryotic inositol, which requires transmembrane kinase-endoribonuclease-1 (IRE1α/β), pancreatic ER kinase (PERK), and activated transcription factor 6 (ATF6).19 To identify the UPR pathways that are inhibited by vinegar or acetic acid, we examined markers of the ATF6 branch of the UPR signaling pathway. As shown in Figure 6A, the phosphorylation levels of ATF6, GRP78, BiP, and CHOP (Cell Signaling Technology) were decreased by vinegar

results showed that vinegar or acetic acid reduced the MAPK signaling activation in the macrophages or DSS-induced colitis mice. Vinegar and Acetic Acid Reduced ER Stress in Mice with DSS-Induced Colitis. ER stress represents a newly recognized pathway that can occur in the intestinal epithelium, and several studies suggest that the UC is associated with ER stress induction.9 The unfolded protein response initiates the mechanisms that are required to resolve ER stress. Three D


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Figure 3. Vinegar and acetic acid suppressed pro-inflammatory cytokine production in colon tissues from DSS-colitis mice: (A) spontaneous IL-17A, TNF-α, INF-γ, and IL-12/IL-23p40 levels in colonic cultures determined by ELISA; (B) MLN cells from each group were stimulated with 10 μg/mL CBL for 72 h, and IL-17A, TNF-α, INF-γ, and IL-12/IL-23p40 secretions were measured. Data are presented as the mean ± SEM (n = 5); (∗) p < 0.05 and (∗∗) p < 0.01 versus DSS-treated group.

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DISCUSSION The organic acids in dietary vinegar include acetic acid, lactic acid, formic acid, citric acid, malic acid, tartaric acid, and acetic acid, and these acids account for 90% of all of the organic acids in vinegar.21 In the past decade, continuous intake (on a daily basis) of a drink containing 15 mL vinegar (750 mg of acetic acid) was reported to improve lifestyle-related diseases, such as hypertension,22 hyperlipidemia,23 and obesity.24 Furthermore, animal studies demonstrated that acetic acid was the active ingredient responsible for the effects.25−27 Acetic acid is absorbed immediately in the upper digestive tract after vinegar intake, especially the stomach and jejunum, and then circulates as acetate to the whole body.28 Acetate is adequately absorbed by the body, and serum acetate levels rise above background levels.28 The interplay between dietary nutrients, gut microbiota, and mammalian host tissues of the gastrointestinal tract has been recognized as highly important for host health. To evaluate the effect of a moderate intake of vinegar on host health, we investigated gut microbiota before DSS treatment and after 28 days. The result showed that commensal bacteria, including Lactobacillus, Bif idobacteria, and Enterococcus faecalis, were increased by the vinegar or acetic acid treatments, and commensal Escherichia coli levels were decreased at day 28 (Table 1). Other studies showed that pigs that were fed a wood vinegar diet had a higher Lactobacillus population in their ileal contents compared with pigs fed antibiotic and control diets.29 These results implied that vinegar might provide an acidic environment in the intestines, leading to changes of intestinal bacteria. Lactic acid produced by Lactobacillus, Bifidobacteria, and Enterococcus faecalis and acetic acid produced by Bif idobacteria were very important energy sources for the host, which might benefit the host by regulating colonic pH, ion transport, cellular proliferation, and gene expression.30 Additionally, vinegar contains short-chain fatty acids (SCFA; e.g., acetic acid). Lactic acid and acetic acid produced by these bacteria or vinegar decreased intraluminal colonic pH, causing inhibition of Escherichia coli proliferation. In the past few decades, it has become apparent that SCFAs might play a key role in the prevention and treatment of metabolic syndrome and bowel disorders.31,32 In clinical studies, SCFA administration positively influenced the treatment of UC, Crohn’s disease, and antibioticassociated diarrhea.33−35 This study showed that vinegar or

or acetic acid in LPS-stimulated THP-1 cells. In our study, the induction of colitis by DSS resulted in significantly increased ATF6, GRP78, BiP, and CHOP levels at day 5, whereas the values were significantly lower in mice that received DSS plus vinegar or acetic acid (Figure 6B,C). These results showed that vinegar or acetic acid attenuated ER stress through modulation of the ATF6-CHOP branch of the UPR signaling pathway in DSSinduced colitis. Vinegar and Acetic Acid Reduced DSS-Induced Cell Apoptosis in Vivo. In UC, the frequency of apoptosis is considerably increased, and epithelial cell loss appears to occur mainly by apoptosis.20 To identify the apoptotic pathways that are inhibited by vinegar or acetic acid, we examined different markers of apoptosis. The phosphorylation levels of Bax, caspase3, caspase-8, and caspase-9 in the colon and spleen tissues were measured by Western blot analyses. As shown in Figure 7A,B, the phosphorylation levels of cleaved Bax, caspase-3, caspase-8, and caspase-9 (Cell Signaling Technology) in the DSS mouse group were significantly higher than those in the control group (p < 0.05). Treatment with vinegar or acetic acid markedly decreased cleaved Bax, caspase-3, caspase-8, and caspase-9 in the colitis mice. The results showed that vinegar or acetic acid reduced DSS-induced cell apoptosis in vivo. Vinegar and Acetic Acid Regulated the Intestinal Bacteria of Mice. The intestinal microbiota plays a pivotal role in maintaining a healthy status. These bacteria may modulate epithelial cell function and innate immune cell activation, which contribute to maintaining intestinal immune homeostasis. To analyze whether vinegar (acetic acid) had potentially protective effects on commensal microbiota, representative bacteria in the feces of vinegar/acetic acid-treated mice were counted with differential media. From the acidic environment provided by vinegar, we detected well-grown commensal bacteria (e.g., Lactobacillus, Bifidobacteria, and Enterococcus faecalis) and poorly grown commensal bacteria (e.g., Escherichia coli). As shown in Table 1, the commensal Lactobacillus, Bif idobacteria, and Enterococcus faecalis were increased with the 5% v/v vinegar or 0.3% w/v acetic acid treatments, and the commensal Escherichia coli levels were decreased after 28 days. The results showed that vinegar (acetic acid) could regulate the intestinal bacteria of mice. E


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Figure 4. Vinegar and acetic acid reduced ATP- and DSS-induced activation of the NLRP3 inflammasome in vitro and in vivo: (A, B) spontaneous IL-1β and IL-18 levels in colonic cultures determined by ELISA; (C) LPS-primed THP-1 cells treated with vinegar (5%v/v) and acetic acid (0.3% w/v) for 5 h, followed by 5 h of incubation with 5 mM ATP for 30 min [protein levels of NLRP3, caspase-1, ASC, IL-1β, and IL-18 determined by Western blot]; (D, E) protein levels of caspase-1, ASC, IL-1β, and IL-18 determined from colon (D) and spleen (E) of DSS-induced mice by Western blot. Phosphorylation levels of the NLRP3 inflammasome were normalized to β-actin. Data are presented as the mean ± SEM (n = 3); (∗) p < 0.05 and (∗∗) p < 0.01 versus DSS-treated group.

acetic acid consumption may support gut health through their ability to exert probiotic actions. When DSS was administered to mice pretreated with vinegar or acetic acid, we observed significant benefits from the vinegar or acetic acid treatments regarding diarrhea, loose feces, visible fecal blood, and colon shortening, and our DAI analysis results

suggested that vinegar or acetic acid might have contributed to reducing the symptoms of murine experimental colitis. Preventive effects of vinegar or acetic acid might be connected with changes in intestinal bacteria in DSS-induced colitis mice. Moreover, previous studies have also shown that Lactobacillus, Bif idobacteria, and Enterococcus faecalis have beneficial effects on F


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Figure 5. Vinegar and acetic acid decreased activations of MAPK signaling pathways in THP-1 cells induced by LPS and colon tissues from DSS-colitis mice: (A) PMA-primed THP-1 cells treated with vinegar (5% v/v) and acetic acid (0.3% w/v) in the presence of 500 ng/mL LPS for 6 h [phosphorylation levels of MAPK proteins determined by Western blot]; (B, C) colonic homogenate and homogenate of spleen from each group of mice subjected to Western blot. Phosphorylation levels of MAPK proteins were normalized to β-actin. Data are presented as the mean ± SEM (n = 3); (∗) p < 0.05 and (∗∗) p < 0.01 versus DSS-treated group.

experimental colitis in mice. 36 Some of the proposed mechanisms by which commensal bacteria may exert beneficial effects are (1) the acetic acid and lactate produced by Lactobacillus and Bifidobacteria, which inhibit the growth of potentially pathogenic organisms (e.g., Escherichia coli); (2) the increased transit time by the net flow of water from the blood to the intestinal lumen, which influences the adherence of bacteria to the intestinal wall; and (3) the reduced production of noxious substances.37,38 In previous studies, some researchers used 3−4% (v/v) acetic acid to induce colitis by intracolonic instillation. This did not conflict with our research results that high concentrations acetic acid (3−4% v/v) induced colitis by intracolonic instillation and low concentrations of acetic acid (0.3% w/v) or vinegar (5% v/v; acetic acid in 5% vinegar would be the equivalent of 0.3% acetic acid) in drinking water prevented experimental colitis in our study. Oral vinegar is mainly absorbed in the stomach and

intestine, but locally instilled high concentrations acetic acid are largely smoldered in blood flow, leading to hemorrhage and tissue necrosis. Macrophage infiltration and activation in the colon are central features of UC, and inflammatory macrophages in the mucosa are thought to play an essential role in UC pathogenesis.39,40 In UC and experimental colitis, blood monocytes are recruited to the mucosa and differentiate into activated macrophages that produce pro-inflammatory cytokines, such as TNF-γ, IL-1, and IL-6.41 Recently, the role of the Th17 cytokines, IL-17, IL-22, and often IFN-γ, and the cells capable of producing them, including T cells and innate lymphoid cells, have become a focus in UC and colitis mouse models.42 Our ex vitro and in vivo investigations showed that vinegar or acetic acid suppressed Th1 and Th17 responses in the MLN and colonic tissue of mice with DSSinduced colitis (Figure 3). This result demonstrated vinegar or G


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Figure 6. Vinegar and acetic acid reduced the ER stress in THP-1 cells induced by LPS and mice with DSS-induced colitis: (A) PMA-primed THP-1 cells treated with vinegar (5% v/v) and acetic acid (0.3% w/v) in the presence of 500 ng/mL LPS for 6 h [representative Western blot photographs for BiP, GRP78, CHOP, ATF6, and β-actin]; (B, C) protein levels of BiP, GRP78, CHOP, ATF6, and β-actin determined from colon (B) and spleen (C) of DSS-induced mice by Western blot. Phosphorylation levels of ER proteins were normalized to β-actin. Data are presented as the mean ± SEM (n = 3); (∗) p < 0.05 and (∗∗) p < 0.01 versus DSS-treated group.

strated significant efficacy in experimental colitis models, making them potential targets of anti-inflammatory therapeutics.44 Our study suggested that the phosphorylation of ERK, p38, and JNK MAPKs was decreased by vinegar- or acetic acid-supplemented water in colitis mice. Recent findings strongly support the involvement of ER stress in a plethora of human diseases, including UC, either as a causative agent or as a complication.45 The transcription factor CHOP is a good ER stress marker because it is specifically expressed under ER dysfunction conditions.10 It has been shown that CHOP is up-regulated following DSS or TNBS administration, and CHOP-null mice are resistant to experimental colitis development in these models.46 The chaperone BiP has been recently been shown to play a central role in modulating the sensitivity and duration of the UPR.47 At the final step of the mammalian ER stress response, the apoptotic response is initiated to terminate cells. CHOP is involved in ER stress-

acetic acid could suppress inflammatory response by inhibiting Th1 and Th17 responses in DSS-induced colitis mice. IL-1β is a pro-inflammatory cytokine that is mainly produced by activated macrophages and monocytes, and it is likely essential in the early phase of the inflammatory cascade that leads to an inflamed colon. Additionally, IL-18 is also important for intestinal inflammation.43 Pro-IL-1β/pro-IL-18 cleavage requires caspase-1 activation.8 Our further in vitro and in vivo experiments showed that treatment with vinegar or acetic acid significantly reduced the expression levels of IL-1β/IL-18, ASC, and caspase-1 in mice with DSS-induced colitis (Figure 4). These findings demonstrated a beneficial effect of vinegar or acetic acid on DSS-induced colitis. MAPK activation has been reported to be essential in the transcriptional induction of various genes involved in inflammation, such as TNF-α, IL-1β, IFN-γ, and IL-17A.18 Previous preclinical studies with MAPK inhibitors repeatedly demonH


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Figure 7. Vinegar and acetic acid reduced the apoptotic pathways induced by DSS colitis: (A, B) representative Western blot photographs for Bax, caspase-3, caspase-8, caspase-9, and β-actin from colon (A) and spleen (B) of DSS-induced mice. Phosphorylation levels of apoptotic proteins were normalized to β-actin. Data are presented as the mean ± SEM (n = 3); (∗) p < 0.05 and (∗∗) p < 0.01 versus DSS-treated group.

Table 1. Vinegar and Acetic Acid Regulated Intestinal Bacteria of Micea strain

differential medium

control group

vinegar group

acetic acid group

Lactobacillus Bifidobacteria Escherichia coli Enterococcus faecalis

MRS medium Bifidobacterium BS medium MacConkey agar E. faecalis agar

4.9 × 108 7.8 × 107 5.8 × 107 2.7 × 105

1.6 × 109* 1.4 × 108** 0.8 × 107* 1.1 × 106*

2.9 × 109** 9.8 × 108* 4.7 × 106** 1.6 × 106**

a

Vinegar regulated intestinal bacteria of mice by analysis of variance with the one-way ANOVA test. (**) p < 0.01 and (*) p < 0.05 significant difference between vinegar/acetic acid group and control group.

In summary, the present study supports the hypothesis that vinegar or acetic acid treatment by dietetic therapy attenuates DSS-induced colitis outcomes. The protection associated with vinegar is due not only to the anti-inflammatory effects of its main component, acetic acid, but also to the prevention of apoptosis mediated by ER stress. Vinegar (acetic acid) also could regulate the intestinal bacteria of mice. Therefore, the protective effects of vinegar or acetic acid on murine colitis support its potential usefulness for the prevention of ulcerative colitis in a new dietary strategy.

induced apoptosis through various mechanisms, such as downregulation of Bcl-2 and translocation of Bax to the mitochondria.46 BiP was also demonstrated to have a role in ER stressmediated apoptosis in both in vivo and in vitro studies.48 Finally, although many factors are involved in the apoptotic program, caspases were shown to play a major role in the transduction of apoptotic signals. In line with this, the caspase-3, caspase-9, and caspase-8 activities in the colonic tissue were significantly higher in the TNBS-treated rats compared with the control group, whereas treatment with glutamine significantly decreased caspase activities compared with the TNBS-treated rats. Our result showed that acetic acid and vinegar reduced DSS-induced cell apoptosis by attenuating ER stress in DSS-induced colitis (Figures 6 and 7). In addition, it is conceivable that the maintenance of bacterial levels in mice by vinegar may have induced changes in luminal metabolism that had an anti-inflammatory effect. Acetic acid and lactate acid produced by Lactobacillus, Bif idobacteria, and Enterococcus faecalis promoted epithelial repair and also reduced mucosal inflammation through modulation of innate inflammatory pathways.

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AUTHOR INFORMATION

Corresponding Authors

*(L.Y.) Phone: +86 431 87836713. Fax: +86 431 87836160. Email: [email protected]. *(B.L.) E-mail: [email protected]. Funding

This work was funded by the National Nature Science Foundation of China (No. 31172364 and 31271951), Fund for Science & Technology Development of Jilin Province (20150101108JC), the Important National Science and Technology Specific Projects (2012ZX10003002), the Program I


Article


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for New Century Excellent Talents in University (NCET-090434; NCET-13-0245), China Postdoctoral Science Foundation (2013M530142), Fundamental Research Program of Shenzhen (No. JCYJ20130401172016183 and ZDSY20120616141302982), and Shenzhen Science and Technology Research and Development Funds (No. JCYJ20130401173155808). Notes

The authors declare no competing financial interest.

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Vinegar Treatment Prevents the Development of Murine Experimental

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