Original Article the Effects of Qing Hua Chang Yin on the Epithelial Tight Junctions of Mice with Inflammatory Bowel Disease
Total Page：16
File Type：pdf, Size：1020Kb
Int J Clin Exp Med 2019;12(6):6864-6873 www.ijcem.com /ISSN:1940-5901/IJCEM0087088 Original Article The effects of Qing Hua Chang Yin on the epithelial tight junctions of mice with inflammatory bowel disease Xiao Ke1*, Liya Liu2,3,4*, Peilin Zhao1, Youqin Chen4, Jun Peng2,3, Wenyi Fang1, Jintuan Chen2, Guanghong Hu1, Youliang Gao1, Aling Shen2,3,4, Hongwei Chen2,3,4, Thomas J Sferra4, Senthilkumar Sankararaman4 1Spleen and Stomach Research Room, Second People’s Hospital Affiliated to Fujian University of Traditional Chinese Medicine, Fuzhou 350003, Fujian, China; 2Academy of Integrative Medicine, 3Fujian Key Laboratory of Integrative Medicine on Geriatric, Fujian University of Traditional Chinese Medicine, Fuzhou 350122, Fujian, China; 4Department of Pediatrics, Case Western Reserve University School of Medicine, Rainbow Babies and Children’s Hospital, Cleveland 44106, OH, USA. *Equal contributors. Received October 21, 2018; Accepted March 6, 2019; Epub June 15, 2019; Published June 30, 2019 Abstract: Intestinal barrier dysfunction is well described in inflammatory bowel disease (IBD) and has been studied as a target for IBD treatment. Qing Hua Chang Yin (QHCY) is a traditional Chinese medicine which has been used in the treatment of inflammatory bowel disease for many years. However, the underlying mechanisms of its therapeu- tic effects remain largely unknown. In the present study, the protective effects of QHCY on epithelial barrier dysfunc- tion were investigated using a DSS-induced mice colitis model. We found that treatment with QHCY significantly ameliorated the clinical manifestations of colitis including the disease activity index (DAI), colon shortening, and the histological evidence of colitis in DSS-induced acute colitis in mice. The administration of QHCY profoundly reduced the DSS-induced increase of TNF-α levels in both colonic tissue and serum and MPO expression in colonic tissue, along with a decrease in serum SAA levels. Furthermore, QHCY significantly reversed the DSS-induced downregula- tion of ZO-1, occluding, and claudin-1 inversely and reduced the DSS induced increase in the phosphorylation of Elk-1. Overall, our findings indicate that QHCY regulates the expression of tight junctions in DSS-induced colitis, so this may be one of the mechanisms associated with its therapeutic effects on IBD. Keywords: Qing Hua Chang Yin, traditional Chinese medicine, inflammatory bowel disease, intestinal barrier, tight junction Introduction ese TJ complexes are well recognized as playing a pivotal role in maintaining the epithelial barri- Inflammatory bowel disease (IBD), which incl- er. TJ disruption results in the functional distur- udes ulcerative colitis (UC) and Crohn’s disease bances of the paracellular barrier and an in- (CD), is a group of chronic gastrointestinal dis- crease in intestinal epithelial paracellular per- orders characterized by intestinal inflammation meability. Irregular and high paracellular per- and mucosal damage [1]. Both the prevalence meability increases the intestinal penetration and incidence has been increasing worldwide of potential harmful antigens and luminal bac- [2]. Although the pathogenesis of IBD remains teria, which can act as a trigger for initiating elusive, environmental factors, genetic suscep- and accelerating the mucosal inflammation in tibility, and immune dysfunction are considered IBD [8-11]. Elk-1 belongs to the ETS domain the major contributing factors. Emerging evi- transcription factor family and the ternary com- dence indicates that intestinal barrier dysfunc- plex factor subfamily and is a direct substrate tion is also fundamental to its pathogenesis [3, of ERK1/2 [12, 13]. Serine phosphorylation of 4]. The epithelial barrier consists of epithelial Elk-1 (Ser383 and Ser389) is necessary for the cells and intercellular junctional complexes [5] transcriptional regulation. ERK1/2 is known to including the tight junction (TJ) proteins, namely induce the phosphorylation and activation of occludin, claudin-1, interacting with the central Elk-1. A previous study has shown that Elk-1 pl- plague protein zona occludens (ZO)-1 [6, 7]. Th- ays a regulatory role in TJ barrier modulation The effect of Qing Hua Chang Yin on colitis in mice [14]. Therefore, ameliorating intestinal barrier role in the pathogenesis of IBD, but there is lit- dysfunction may alleviate the development of tle evidence showing whether QHCY can pro- inflammation or may accelerate the resolution tect or boost the intestinal epithelial barrier of intestinal inflammation [10, 15]. function. In order to further understand the th- erapeutic mechanism of QHCY, in this present Pharmacological intervention is regarded as study, we investigated the effects of QHCY on one of the critical strategies for IBD treatment. intestinal epithelial barrier function and the For instance, corticosteroids, 5-aminosalicyic expression of the TJ protein in mice with colitis acid (5-ASA) derivatives, immune suppressants induced by dextran sulfate sodium (DSS). and immune modulators can relieve mucosal inflammation and IBD-related symptoms [16]. Materials and methods However, the long-term use of these therapies is often limited due to the loss of response in Materials and reagents many patients and also the remarkable side effect profile, such as immune suppression DSS (molecular weight: 36-50 kDa) was obtai- [17]. Therefore, it is necessary to find new ther- ned from MP Biochemicals (Solon, OH, USA). apeutic strategies for IBD which are safe and The mouse serum amyloid A (SAA) ELISA kit and capable of sustaining prolonged clinical remis- the protein extraction and BCA assay kit were sion while improving gut mucosal healing. from Thermo Fisher Scientific (Grand Island, NY, USA). ZO-1 and Elk-1 antibodies were pur- Natural products such as baicalin and astraga- chased from GeneTex (Irvine, CA, USA), and the losides have been used in traditional Chinese phospho-Elk-1 and MPO antibodies were sup- medicine (TCM), and these compounds have plied by Abcam (Cambridge, MA, USA). Claudin-1 acquired global attention because of their and occludin antibodies were obtained from potent anti-inflammatory role [18-20]. Qing Hua Proteintech (Chicago, IL, USA). β-actin antibody Chang Yin (QHCY), a well-known traditional Chi- and horseradish peroxidase-(HRP) conjugated nese formula which is composed of Coptis chi- secondary antibodies were purchased from nensis Franch, Herba et Gemma Agrimoniae, Cell Signaling Technology (Beverly, MA, USA). Radix Sanguisorbae, Magnolia officinalis, Radix The mouse TNF-α ELISA kit was provided by Paeoniae Rubra, Elettaria cardamomum, Se- BioLegend (San Diego, CA, USA). All the other men Coicis, Artemisia capillaris Thunb, Semen chemicals were provided by Sigma-Aldrich (St. Dolichoris Album, Herba Eupaatorii Fortunei, Louis, MO, USA) unless otherwise noted. and Poria cocos. In TCM, it is believed that th- Preparation of QHCY ese products synchronously work together wh- ich confer QHCY the following functional prop- In total, 33 g dehydrated Coptis chinensis Fr- erties: elimination of heat and dampness, aug- anch, 220 g dehydrated Herba et Gemma Ag- mentation of the functions of the spleen, and rimoniae, 110 g dehydrated Radix Paeoniae thereby an overall increase in the vitality of life. Rubra, 100 g dehydrated Radix Sanguisorbae, In the TCM system, accumulation of toxic damp- 110 g dehydrated Magnolia officinalis, 56 g ness and heat is one of the critical causative dehydrated Elettaria cardamomum, 110 g de- factors in ulcerative colitis (UC) pathogenesis, hydrated Herba Eupaatorii Fortunei, 110 g de- so clearing heat and dampness is an effective hydrated Artemisia capillaris Thunb, 110 g de- strategy for the treatment of UC. Thus, QHCY hydrated Semen Dolichoris Album, 220 g dehy- has been successfully used in China for the drated Semen Coicis and 220 g dehydrated management for UC for many years [21-26]. Poria cocos were extracted with boiling water 3 times in 2 L of distilled water. The extracts were Our previous studies of the DSS-induced colitis then filtered and concentrated by boiling them mice model have indicated that QHCY success- down to a final volume of 1,000 ml. The stock fully alleviates the clinical manifestations of co- concentration of QHCY was 1.4 mg/ml. litis, such as weight loss, blood in the stool, his- tological evidence of colitis, and the release of Establishment of the mouse model of colitis inflammatory cytokines modulating via the and QHCY treatment TLR4/NF-κB and IL-6/STAT3 signaling pathways [27-29]. Previous research demonstrated that a All animal protocols used in this study were defective intestinal barrier plays a fundamental approved by the Institutional Animal Care and 6865 Int J Clin Exp Med 2019;12(6):6864-6873 The effect of Qing Hua Chang Yin on colitis in mice Table 1. Disease activity index score approximately 5 mm section of each distal Stool colon was fixed in 10% (v/v%) formalin solu- Weight loss (%) Rectal bleeding Score consistency tion and given a histological examination. The 0 Normal Normal 0 remaining ones were stored at -80°C for other analyses. 1-5 1 6-10 Loose Hemoccult-positive 2 Histopathological evaluation 11-15 3 >15 Diarrhea Gross bleeding 4 The colon tissues were fixed and processed The disease activity index was calculated as the sum of the in paraffin. Next, the colon tissues 4 µm thi- scores for weight loss, stool consistency, and rectal bleeding. ck were stained with hematoxylin and eosin (H&E). The histologic score was examined wi- th the sum of the inflammation severity, in- Use Committee of Fujian University of Tradi- flammation extent, and crypt damage divided tional Chinese Medicine. Fifteen Male BALB/cJ by the percentage involvement following the mice (6-8 weeks; 25-28 g) were purchased scoring system shown in Table 2. The histo- from the Shanghai SLAC Laboratory Animal Co., pathological score was examined in a blinded Ltd. (Shanghai, China) and acclimatized for 5 fashion according to the criteria described by days with ad libitum access to water and food Sann et al. [31]. prior to the experiment. The mice were main- tained under standardized conditions with 12-h Measuring the level of TNF-α and SAA by light/dark cycles and 5 animals per cage.