Journal of Experimental and Clinical Application of Chinese Medicine
Original Research

Effect of Jinhuahecha—A Fermented Green Tea on DSS-Induced Ulcerative Colitis in Mice and Its Preliminary Pharmacological Mechanism

Ke Li 1, Yingying Wang 1, Weizhi Lai 1, Xunjie Zheng 1, Guangshun Zhang 1, Xinyu Qiu 1,2, Jianli Gao 1,*, Zhaohuan Lou 1,*

1 School of Pharmaceutical Sciences, Zhejiang Chinese Medical University, 310053 Hangzhou, Zhejiang, China
2 Dispensary of Traditional Chinese Medicine, Ningbo Women and Children's Hospital, 315012 Ningbo, Zhejiang, China

* Author to whom correspondence should be addressed; E-mail: ✉(J.G.); ✉(Z.L.).


Copyright © 2024 The Author(s). Published by Exploration and Verfication Publishing

This is an open access article under the CC BY 4.0 license

Journal of Experimental and Clinical Application of Chinese Medicine 2024; 5(3): 23-36.

Received: 5 June 2024 | Revised: 19 June 2024 | Accepted: 26 June 2024 | Published: 10 July 2024

Background: Ulcerative colitis (UC) is an inflammatory bowel disease. Jinhuahecha (JHHC)-a fermented green tea is obtained by micro-fermentation of Anji white tea (AJWT) inoculated with high-purity "E. cristatum". Purpose: This study is aimed to unravel the protective effect of JHHC to UC in mice. Methods: High performance liquid chromatography (HPLC) was applied to determine the effective components and their contents in JHHC and AJWT. UC mouse model was constructed by ingestion of 2.5% dextran sulfate sodium (DSS) aqueous solution for 9 days. Disease activity index (DAI) was evaluated with the daily weight, fecal characters, and bloody stool condition, the length of colon and the spleen coefficients in mice was measured. Hematoxylin and eosin and Alcian blue-periodic acid Schiff staining was performed to observe the histopathological changes of the colon tissue. The positive expression of MyD88 and TNF-α were observed by immunofluorescence and immunohistochemistry. The possible mechanism was speculated by via transcriptomics. Results: HPLC results indicated that compared with AJWT, in JHHC, the content of gallic acid was significantly increased, the caffeine content was slightly decreased, and the polyphenolic components were notably declined. Compared to normal control group, in model control group, DAI score and spleen coefficient were elevated, the length of colon was shortened, and the colon tissue was severely damaged. In contrast to model control group, the above indexes were improved in treatment group, especially in JHHC group, which was possibly associated with regulation of inflammatory response. Conclusions: JHHC, as a natural plant product, has anti-inflammatory effects that can alleviate UC in mice.

Keywords: Jinhuahecha; fermented green tea; ulcerative colitis; dextran sulfate sodium; anti-inflammation

Main Text

Graphic Abstract

Graphic Abstract

1 Introduction

Ulcerative colitis (UC) is a chronic, non-specific and lifelong inflammatory bowel disease (IBD) [1], mainly involving the colon and extending from rectum, which affects the colon to varying degrees [2]. The clinical manifestations include severe abdominal pain, persistent or intermittent diarrhea, bloody purulent stool, etc. The etiology of UC is primarily related to genetic factors and environmental changes [3,4], but there is a lack of knowledge about pathogenesis of UC and most scholars consider UC as an autoimmune disease [5]. Since the 21st century, the incidence rate of IBDs, such as UC, has increased year by year, which greatly affects the normal life of patients owing to repeated condition and high recurrence rates after anti-inflammatory treatment [6]. Currently, the drug treatment of UC mainly depends on salicylic acid preparations (e.g., sulfasalazine), hormones (e.g., methylprednisolone), immunosuppressants (e.g., methotrexate) and biological agents (e.g., infliximab) that can effectively suppress intestinal inflammatory reaction; however, these drugs incur adverse reactions including gastrointestinal reactions, abnormal liver function, and decreased immune function, and may even increase the risk of malignant tumor [7,8].

Tea, a natural product, is one of the most popular drinks secondary to water because of its unique taste and rich nutrients [9,10]. According to the processing method and fermentation degree, tea can be divided into three types: unfermented green or white tea, semi-fermented oolong tea, and fully fermented black tea [9]. Anji white tea (AJWT) is a kind of special green tea [11] that is mainly produced in Anji County, Zhejiang province, China, and is a national geographic indication product [12], characterized by more amino acids and fewer tea polyphenols.

"Jinhua" bacteria, also known as Eurotium cristatum (E. cristatum), is a kind of fungus belonging to Eurotiales, Trichocomaceae, and Eurotium, and constituted by ascocarp and mycelium [13], which was first found in Fuzhuan brick tea [14] and is an enzymatic bacteria that is beneficial to human body. Inoculation of E. cristatum to tea can cause the covering of tea with golden yellow granular "E. cristatum" during the fermentation process, thereby forming a unique taste and flavor, and various physiological functions [15,16]. Jinhuahecha (JHHC)—a fermented green tea used in this study was obtained by microbial transformation of AJWT as raw material using high-purity E. cristatum. AJWT contains various active ingredients such as theanine, tea polyphenols (mainly Epicatechin, Epigallocatechin, Epigallocatechin gallate, Epicatechin gallate), gallic acid, caffeine, tea polysaccharides, etc. [17,18]. These active ingredients have been confirmed to have functions of anti-oxidant, anti-inflammation, anti-bacteria, reducing blood glucose, improving cardiovascular and cerebrovascular health, enhancing immunity, and maintaining digestive system health [18-20]. Based on the above findings, this study was aimed to further observe the changes in the composition of AJWT after intervention with E. cristatum and the protective effect of E. cristatum on dextran sulfate sodium (DSS)-induced UC in mice, to promote the development and utilization of AJWT.

2 Materials and methods

2.1 Reagents and drugs

Anji white tea, 'Jinhuahecha' and Eurotium cristatum conidial powder were provided by Anji Hecha Biotechnology Co., Ltd (Anji, China). Gallic acid (GA, A0110), Caffeine (Caf, A0470), (-)-Epigallocatechin Gallate (EGCG, A0159), and (-)-Epicatechin gallate (ECG, A0160) were procured from Chengdu Must Bio-technology Co., Ltd (Chengdu, China). Dextran sulfate sodium (DSS, MW 36000-50000) was purchased from MP Biomedicals (Southern California, USA). Sulfasalazine (SASP) enteric coated tablets were obtained from Shanghai Sine Tianping Pharmaceutical Co., Ltd (Shanghai, China). Methanol and acetonitrile were of chromatographic grade, and other reagents were of analytical grade.

2.2 Sample preparation

100 g of Anji white tea and 'Jinhuahecha' was soaked in 2000 mL of pure water for 30 min, after which the filtrate was collected by reflux extraction for 1 h. After adding another 1800 mL of pure water for 45 min reflux extraction, the two filtrates were collected and concentrated with a rotary evaporator. 3 g of E. cristatum conidial powder was added to 50 mL of water and sonicated to make a suspension.

2.3 Analysis of characteristic components of AJWT and JHHC

High performance liquid chromatograph (HPLC) with a 2489 UV-Vis Detector (Waters Technology, New York, NY, USA) was applied to analyze effective components. The main effective components of AJWT were tea polyphenols, caffeine, theanine, tea polysaccharides, and vitamins. This study mainly observed the changes in the contents of gallic acid, caffeine, epigallocatechin gallate, and epicatechin gallate in AJWT and its JHHC fermented by E. cristatum. To acquire test solution, an appropriate amount of 2.5 mg/mL AJWT, JHHC dry powder were added to methanol solution and weighed, followed by ultrasonic extraction for 30 min. The extract was placed at room temperature and weighed, and the lost weight was made up and shaken evenly. After filtration through a 0.45 μm filter membrane, the filtrate was collected for later use. To prepare control solution, an appropriate amount of the four control substances mentioned above was added with methanol to prepare the corresponding concentration of control solution mother liquor. 0.2 mL of each standard stock liquor was added with methanol to a constant volume of 1 mL, and shaken evenly to obtain a mixed control solution (including 0.0120 mg/mL of GA, 0.0200 mg/mL of Caf, 0.0208 mg/mL of EGCG, and 0.0248 mg/mL of ECG). The content of GA, Caf, EGCG, and ECG was measured with a UV spectrophotometer detector (VWD) at the wavelength of 220 nm. The column (XB-C18, 4.6 × 250 nm, 5 μm) temperature was maintained at 30 ˚C. The mobile phase consisted of acetonitrile-0.2% phosphoric acid aqueous solution, the flow rate was 1.0 ml/min, and the elution procedure was as follows: 0-5 min, 1 %-5 % A; 5-15 min, 5 %-10 % A; 15-30 min, 10 %-15 % A; 40-45 min, 20 %-23 % A; 45-50 min, 23 %-28 % A; 50-55 min, 28 %-38 % B.

2.4 Animals

Specific pathogen-free (SPF) C57BL/6 male mice (6-8 weeks old) were purchased from Hangzhou Qizhen experimental animal Technology Co., Ltd (Hangzhou, China) with license number: SCXK(Hangzhou)2022-0005SCXK. All animal experiments were approved by the Medical Ethics Committee of Zhejiang Chinese Medical University (IACUC-20230828-14). Mice were housed in the animal laboratory center under the conditions of 20-25 ℃, 50-60% humidity, 12 h light-dark cycle, and free access to food.

2.5 UC mouse modeling and drug administration

After one week of habituation, 18 mice were randomly assigned into 6 groups (n = 3) as normal control (NC) group, model control (MC) group, SASP group, JHHC group, AJWT group, and E. cristatum conidial powder (E. cristatum) group. Except for the NC group, mice in the other groups were given 2.5% DSS aqueous solution instead of drinking water for 9 days to establish a UC model [21,22], with the DSS aqueous solution refreshed daily. Meanwhile, 0.45 g/kg SASP, 2.3 g/kg JHHC water extract, 2.3 g/kg AJWT water extract, and 0.6 g/kg E. cristatum conidial powder suspension were administered by gavage. The remaining water weight was measured daily to calculate the amount of water drinked. Isoflurane (Veterinary Medicine Standard 031217015, RWD, Shenzhen, China) was installed on the RWD small animal inhalation anesthesia gas anesthesia machine (R510-11, RWD) for euthanasia of mice on the 10th day.

2.6 Disease activity index (DAI) score

During the experiments, the general physical sign, body weight, fecal viscosity, and fecal occult blood of mice were observed and recorded to assess DAI score (Table 1) [23].

Table 1 DAI score standard.

Decline of weightFecal morphologyFecal occult blood/gross blood stoolScore
0NormalNormal0
1~5%IncompactOccult blood positive1
5~10%2
10~15%LooseGross blood stool3
>15%4

2.7 Measurement of the length of colon and the coefficients of spleen and liver

At the end of experiment, the mice were fasted but had water ad libitum for 12 h, and then the weight of the mice was measured. After euthanasia, the spleen, liver, and complete colon tissues were harvested at low temperature. The length of the colon was measured, and the wet weight of the spleen and liver was detected to calculate the organ coefficient [organ wet weight (mg)/fasting body mass (g)].

2.8 Histopathological examination

The colon tissue was washed with ice 0.9% sodium chloride solution, and the same section was selected to be fixed in 10% formalin for 48 h. The samples were dehydrated with gradient ethanol solution, and then embedded in paraffin. Paraffin-embedded tissues were sliced into 4 μm sections, and stained with hematoxylin and eosin (H&E), after which histological changes of the colon were observed under a biological microscope (MshOt, Guangzhou, China).

2.9 Alcian blue-periodic acid Schiff (AB-PAS) staining

The colon mucus secretion capacity of mice in each group was analyzed by AB-PAS (G1285, Solarbio, Beijing, China) staining. After dewaxing and hydration, the slices were soaked in Schiff reagent, periodic acid solution, and Alcian blue dye successively. Followed dehydration with gradient ethanol for 5 min, the slices were transparentized by xylene for 5 min, and then sealed with neutral gum. The changes of mucin secretion by goblet cells were observed under an optical microscope (C13210-01, HAMAMATSU, Japan).

2.10 Immunohistochemistry (IHC)

Colon tissue samples were fixed in 10% formaldehyde solution, followed by embedding in paraffin and sectioning. Subsequently, routine immunohistochemistry (IHC) was conducted to assess the expression of TNF alpha Rabbit pAb (TNF-α, 346654, Zenbio, Chengdu, China). Each slide was monitored with a light microscope, and images were captured at random fields of vision.

2.11 Immunofluorescent staining (IF)

Paraffin sections of colon tissue were dewaxed, rehydrated, and than used EDTA for antigen repair. The slides were blocked with QuickBlock Immunol Staining Blocking Buffer dilution (P0260, Beyotime, Shanghai, China) for 10 min. Then sections were incubated overnight with MyD88 Rabbit Polyclonal Antibody (MyD88, AF7524, Beyotime) at 4 ℃. Then incubated with FITC-labeled Goat Anti-Rabbit IgG (H+L) (A0562, Beyotime). Finally, the slices were stained with DAPI and subsequently sealed, observed under a Zeiss positive fluorescence microscope (AXIO SCOPE.A1, Carl Zeiss AG, Jena, Germany).

2.12 Transcriptome analysis of colon tissue

Transcriptome sequencing of colon tissues from each group was performed using a 2 × 150 bp paired-end sequencing (PE150) on an Illumina Novaseq™ 6000 (LC-Bio Technology CO., Ltd., Hangzhou, China) as per the recommended protocol. The sequencing data were analyzed on the Omicsmart platform (www.omicsmart.com).

2.13 Statistical analysis

SPSS 25.0 statistical software (International Business Machines Corporation, New York, USA) was adopted to conduct statistical analyses, and GraphPad Prism 9.0 software (San Diego, CA, USA) was used for drawing diagrams. All quantitative results were expressed as mean ± standard deviations (SD), and statistical differences were evaluated through a two-tailed Student’s t-test or one-way analysis of variance (ANOVA). p-value less than 0.05 was defined to be statistically significant.

3 Results

3.1 E. cristatum fermentation changed the contents of active components

In line with the chromatographic conditions in 2.3, the four active components in the HPLC spectra of JHHC and AJWT (Figure 1) achieved baseline separation from adjacent peaks (R ≥ 1.5) and met the requirements for quantitative analysis (S/N ≥ 10). The changes in the contents of components from JHHC that were obtained by microbial fermenting AJWT with E. cristatum were displayed in Table 2. It can be noticed that gallic acid level was signally augmented, caffeine level was slightly diminished, and the levels of epigallocatechin gallate and epicatechin gallate were greatly dwindled.

Figure 1

Figure 1 HPLC spectra of characteristic components in standard and tea samples. (A) Spectra of characteristic components blended with standard samples (a. Gallic acid; b. Caffeine; c. (-)-Epigallocatechin gallate; d. (-)-Epicatechin gallate). (B) Finger-print of JHHC. (C) Finger-print of AJWT.

Table 2 The contents of effective components in JHHC and AJWT.

GroupEffective components
GA mg/gCaf mg/gEGCG mg/gECG mg/g
JHHC17.7970.4613.988.8
AJWT2.2973.5643.9923.61

3.2 JHHC improved the symptoms of DSS-induced UC mice

The body weight, daily weight loss percentage, fecal morphology, and occult blood of each group of mice were observed (Figure 2A,B). By contrast, drinking 2.5% DSS aqueous solution for 9 days resulted in significant weight loss, increased DAI score, severe hematochezia, and even anal prolapse in model mice. Administered with AJWT group, JHHC group, and E. cristatum group effectively improved weight loss in UC mice (Figure 2A), reduced DAI score and alleviated fecal occult blood (Figure 2B).

The colon tissue of the normal mice was intact and horseshoe-shaped, with obvious folds. However, in UC model mice, the colon length was markedly shortened (p < 0.01) (Figure 2C), the intestinal wall became thinner, the congestion and edema of the colon mucosa were visible, and black bloody stools were present in the intestines (Figure 2D). Compared with the MC group, in the JHHC group and E. cristatum group, the colon length was remarkably increased (p < 0.05), and the thickness of the intestinal wall as well as the congestion and edema of the colon mucosa were improved, while in AJWT group, we noted an increase in colon length and a recovery in colon atrophy (Figure 2C,D). The fecal alterations in mice were observed before and after the induction of the UC model. Before modeling, the feces of all mice were oval-shaped, slightly brownish black, and slightly dry. After the success of UC model, fecal formless mice in MC group had severe hematochezia, while fecal characteristics and hematochezia of mice in JHHC group, AJWT group and E. cristatum group were improved (Figure 2E). Among the tested substances, E. cristatum conidial powder was more effective than JHHC and AJWT in improving the symptoms of DSS-induced UC in mice. JHHC water extract effectively reversed the shortened colon length induced by DSS in UC mice.

Figure 2

Figure 2 JHHC improved the symptoms of DSS-induced UC mice. (A) The weight of mice. (B) DAI score of mice. (C) The colon length of mice. (D) Representative colonic morphology of mice in each group. (E) Fecal morphology of mice before and after administration. (n = 3), compared with NC group, ### p < 0.01; compared with with MC group, * p < 0.05.

3.3 JHHC improved the histopathological changes of DSS-induced UC mice

According to the H&E staining results of mouse colon tissues (Figure 3A), NC group of mice had intact intestinal mucosal tissue, a large number of intestinal villi, orderly arrangement of intestinal glands, and no inflammatory cell infiltration. Compared with NC group, in the MC group, mice presented varying degrees of damage to the upper colonic mucosa, crypt structures, and intestinal villi, with a wide range of inflammatory cell infiltration and prominent damage to the intestinal epithelium. As compared with mice in MC group, treatment of AJWT, JHHC and E. cristatum conidial powder can relatively restore crypt structures in colon and relieve the intestinal injury degree, among which the therapeutic efficacy was superior in E. cristatum group and JHHC group.

Mucin is an important component of the intestinal mucosal layer, mainly existing in the mucus secreted by the epithelial cells and glandular cells of the colon mucosa, which has an intimate association with intestinal mucosal inflammation and ulcer formation [24]. Colon AB-PAS staining results (Figure 3B) revealed heaps of goblet cells, good shape and orderly arrangement in NC group. However, MC group presented decreases in goblet cells of the colon tissue and mucus, and a disorderly arrangement. Intriguingly, AJWT, JHHC, and E. cristatum conidial powder increased goblet cells in mouse colon tissue and promote the recovery of mucosal layer.

Figure 3

Figure 3 Histopathological changes in mouse colon tissue. (A) Histopathologic image of mouse colon (H&E staining, 100×). (B) Mucin staining image of goblet cells in mouse colon tissue (AB-PAS staining, 200×).

3.4 JHHC improved the spleen and the liver index

The spleen participates in regulating the response of immune system and is a critical immune organ [25]. After modeling, mice of MC group showed a significant increase in spleen weight and spleen coefficient (p < 0.01). However, the spleen coefficient was pronouncedly lessened by JHHC (p < 0.05), while being reduced to varying degrees by AJWT and E. cristatum conidial powder (Figure 4A). The liver coefficient had no obvious changes in all drug treatment groups (Figure 4B).

Figure 4

Figure 4 Spleen coefficient (A) and liver coefficient (B) of mice in each group. compared to NC group, ## p < 0.01; compared to MC group, * p < 0.05.

3.5 Effect of JHHC on gene expression in colon tissue

To fathom out the preliminary pharmacological mechanisms of JHHC improving UC in mice, we analyzed the transcriptome sequencing data of the colon tissues. In transcriptome sequencing data analysis, NC, MC and JH represent NC group, MC group and JHHC group, respectively. The results of principal component analysis (PCA) indicated a strong correlation in JH group, and there are differences in gene samples between JH group and MC group (Figure 5A). In light of the volcano plot as well as the heatmap of the top 100 differentially expressed genes of JH and MC group (Figure 5B,C), it can be seen that compared with the MC group, some genes in the JH group showed a downward trend, but there was no significant difference in the number of up- or down-regulated genes. The GO enrichment analysis of the top 20 differential genes (Figure 5D) revealed that the functions of the differential genes mainly included "synapse", "cell junction", "integral component of membrane", and "calculus ion binding". Based on this, further screening of the KEGG pathway was conducted (Figure 5E). A therapeutic effect on UC was found in JH group, which may be closely related to the MAPK signaling pathway and tight junction signal. The FPKM data analysis on the key gene in these two pathways (Figure 5F) showed compared with NC group, MAPK signaling pathway-related MAPK8IP2 (p < 0.004), MAPK10 (p < 0.005), MAPK8IP1 (p < 0.008) and MYD88 expressions in the colon of UC mice were significantly increased, while tight junction-related OCLN, CLDN1, CLDN7, and PARD3 expressions were decreased to varying degrees, with OCLN expression showing the largest decrease (p < 0.003). The changes of the above gene expressions can be reversed by JHHC water extract to different extent, hinting that the improvement of DSS-induced UC by JHHC water extract may be achieved via modulating MAPK signaling pathway to inhibit inflammatory response and mediating tight junction protein expression to protect the intestinal barrier.

Figure 5

Figure 5 Effects of JHHC on differential genes and analysis of transcriptome data in DSS-induced UC mice (n = 3). (A) Principal component analysis. (B) Volcano plot of differential gene. (C) Differential gene clustering heatmap of top 100 genes. (D) Top 20 of GO enrichment. (E) KEGG enrichment BarPlot. (F) Expression level of MAPK signaling pathway and Tight junction related genes. Compared to NC group, ## p < 0.05, # p < 0.01. NC: NC group; MC: MC group; JH: JHHC group.

3.6 JHHC reduced the expression of MyD88 and TNF-α in colon tissue

MyD88 is a protein that mediates inflammation, and mainly acts as an important medium of interleukin-1 receptor (IL-1R) signaling and participates in the activation of the MAPK signaling pathway [26,27]. To explore the mechanism by which JHHC improves DSS-induced inflammation in UC mice, immunofluorescence staining and immunohistochemical staining were used to observe the expression changes of MyD88 and TNF-α in colon tissues, respectively. The results showed that compared with NC group, the MyD88, TNF-α positive cells were significantly increased in MC group to compared with NC group. Compared with MC group, MyD88, TNF-α positive cells were significantly decreased in JHHC, AJWT, and E. cristatum group, indicated that JHHC, AJWT and E. cristatum could inhibit the inflammatory response in UC (Figure 6).

Figure 6

Figure 6 Effects of JHHC on protein expression of MyD88 and TNF-α. (A) Representative IF images of MyD88 (IF, 200×). (B) Representative IHC images of TNF-α (IHC, 200×).

4 Discussion

UC is characterized by long course of disease and relapse, and it cannot be cured at present [28]. To prevent recurrence and avoid complications, patients need long-term medication treatment to alleviate inflammation, which brings enormous economic burden to individuals and society. There are studies revealing that the pathogenesis of UC is primarily associated with intestinal mucosal barrier integrity and T lymphocyte immunity [29,30]. Anti-inflammation and immune regulation are the two important means for the treatment of UC. It has been documented that AJWT contains abundant tea polyphenols, as well as nutrients such as amino acids and gallic acid, which has anti-bacterial, anti-inflammatory, and immune enhancement effects [31,32]. The AJWT after fermentation using E. cristatum can modulate gut microbiota, improve intestinal barrier function, and subsequently regulate gastrointestinal function [18]. This experiment for the first time analyzed the changes in the composition of AJWT after fermentation, namely JHHC, and whether the kind of tea can improve DSS-induced UC in mice. The results of component analysis indicated that after microbial fermentation, the content of gallic acid was significantly increased, but that of caffeine was slightly decreased in JHHC, signifying a transformation of tea polyphenol components during fermentation [33], and the contents of EGCG and ECG were diminished. Gallic acid has been confirmed to exert anti-inflammatory, anti-bacterial, antiviral and anti-oxidant effects to assist in treating inflammatory diseases [34,35]. Caffeine plays an anti-oxidant and anti-inflammatory role, moderate intake of which contributes to physical health [36]. EGCG and ECG, as tea polyphenols, generate anti-oxidant, antiviral, and immunomodulatory effects [37,38], and can achieve anti-inflammatory effects via stimulating the MAPK pathway [39].

In this study, 2.5% DSS aqueous solution that replaces drinking water was applied to successfully establish UC mouse model [40]. Through analysis of experimental data, we proved that the fermented JHHC had decreased tea polyphenols and increased gallic acid, analogous to existing experimental reference data [9]. JHHC can also effectively improve the clinical symptoms of DSS-induced UC in mice, such as weight loss, hematochezia, rectal prolapse, shortened colon length caused by colon atrophy, pathological changes in colon tissue, and loss of intestinal barrier. Based on experimental results and transcriptome sequencing data, it was conjectured that the mechanism of JHHC improving UC in mice may be related to its rich nutrients that help strengthen immunity [41], or components such as gallic acid that inhibit inflammatory mediator production and release, reduce tissue damage caused by inflammation, and thus maintain the intestinal epithelial barrier [42,43].

MAPK signaling pathway is involved in the pathogenesis of inflammation and inflammatory response [44]. According to the transcriptome sequencing and staining results, the DSS-induced UC model may be related to IL-1R activating MAPK pathway through MyD88, leading to the release of inflammatory factors (such as interleukin-1β, tumor necrosis factor-α, etc.), and then triggering inflammatory response [45,46]. JHHC can reduce the expression of MyD88 and TNF-α in a certain extent, and regulate the level of inflammation. Nevertheless, further mechanisms and potential targets remain to be investigated.

In recent years, in the context of the growing public awareness of the health, people trust more in the efficacy of natural products and their active components [47]. Products for relieving UC made from natural ingredients such as TCM both as medicine and food have a certain market [48,49]. The JHHC and its active components transformed by microorganisms are expected to be developed into new products for the prevention and treatment of UC.

5 Conclusion

The composition of JHHC changed after fermentation. JHHC can improve DSS-induced UC by reducing intestinal inflammatory response and protecting colonic epithelial barrier function. It is a potential candidate for UC treatment.

Back Matter

Acknowledgments

This study was supported by the National Natural Science Foundation of China (No. 82374125 to ZHL) and the Key Project of Zhejing Provincial Natural Science Foundation (No. LZ24H280001 to ZHL). Guiyuan Lv supported the experimental facilities. We appreciated the great help support from the Pharmaceutical Research Center, Academy of Chinese Medical Sciences, Zhejiang Chinese Medical University.

Conflicts of Interest

All authors declare no conflict of interest.

Author Contributions

K.L., Y.W., W.L., and X.Q.: Carried out the experiment; K.L.: Formal analysis and Writing-original manuscript; X.Z. and G.Z.: Conceptualization; Z.L. and J.G.: Experimental design and manuscript revising.

Ethics Approval and Consent to Participate

All animal experiments were approved by the Medical Ethics Committee of Zhejiang Chinese Medical University (IACUC-20230828-14).

Funding

This research received no external funding.

Availability of Data and Materials

The data presented in this study are available on request from the corresponding author.

Supplementary Materials

Not applicable.

  1. Le Berre C, Honap S, Peyrin-Biroulet L. Ulcerative colitis. Lancet 2023; 402(10401): 571-584.
  2. Ananthakrishnan AN, Kaplan GG, Ng SC. Changing Global Epidemiology of Inflammatory Bowel Diseases: Sustaining Health Care Delivery Into the 21st Century. Clinical Gastroenterology and Hepatology 2020; 18(6): 1252-1260.
  3. Kuo PC, Wu YH, Hung HY, et al. Anti-inflammatory principles from Lindera aggregata. Bioorganic & Medicinal Chemistry Letters 2020; 30(13): 127224.
  4. Naganuma M, Mizuno S, Nanki K, et al. Recent trends and future directions for the medical treatment of ulcerative colitis. Clinical Journal of Gastroenterology 2016; 9(6): 329-336.
  5. Lee SH, Kwon JE, Cho ML. Immunological pathogenesis of inflammatory bowel disease. Intestinal Research 2018; 16(1): 26-42.
  6. Ng SC, Shi HY, Hamidi N, et al. Worldwide incidence and prevalence of inflammatory bowel disease in the 21st century: a systematic review of population-based studies. Lancet 2017; 390(10114): 2769-2778.
  7. Kobayashi T, Siegmund B, Le Berre C, et al. Ulcerative colitis. Nature Reviews Disease Primers 2020; 6(1): 74.
  8. Xu Z, Chu M. Advances in Immunosuppressive Agents Based on Signal Pathway. Frontiers in Pharmacology 2022; 13: 917162.
  9. Siddiqui MW, Sharangi AB, Singh JP., et al. Antimicrobial Properties of Teas and Their Extracts in vitro. Critical Reviews in Food Science & Nutrition 2016; 56(9): 1428-1439.
  10. Wang Y, Bandara HMHN, Mikkelsen D, et al. Effects of tea extracts on the colonization behaviour of Candida species: attachment inhibition and biofilm enhancement. Journal of Medical Microbiology 2017; 66(8): 1244-1252.
  11. Wang R, Yang Z, Zhang J, et al. Liver Injury Induced by Carbon Tetrachloride in Mice Is Prevented by the Antioxidant Capacity of Anji White Tea Polyphenols. Antioxidants 2019; 8(3): 64.
  12. Xu L, Shi PT, Fu XS, et al. Protected Geographical Indication Identification of a Chinese Green Tea (Anji-White) by Near-Infrared Spectroscopy and Chemometric Class Modeling Techniques. Journal of Spectroscopy 2013; 2013(1): 1-8.
  13. Li T, Liu Z, Li J, et al. Lovastatin Production by Wild Eurotium cristatum Isolated from Fuzhuan Brick Tea Produced Using Forest Resources in Auhua. Forests 2023; 14(7): 1409.
  14. Xiao Y, He C, Chen Y, et al. UPLC-QQQ-MS/MS-based widely targeted metabolomic analysis reveals the effect of solid-state fermentation with Eurotium cristatum on the dynamic changes in the metabolite profile of dark tea. Food Chemistry 2022; 378: 131999.
  15. Lin F, Wei X, Liu H, et al. State-of-the-art review of dark tea: From chemistry to health benefits. Trends in Food Science & Technology 2021; 109: 126-138.
  16. Wang X, Li X, Liu B, et al. Comparison of chemical constituents of Eurotium cristatum-mediated pure and mixed fermentation in summer-autumn tea. LWT 2021; 143: 111132.
  17. Cui L, Guan X, Ding W, et al. Scutellaria baicalensis Georgi polysaccharide ameliorates DSS-induced ulcerative colitis by improving intestinal barrier function and modulating gut microbiota. International Journal of Biological Macromolecules 2021; 166: 1035-1045.
  18. Mathur A, Gopalakrishnan D, Mehta V, et al. Efficacy of green tea-based mouthwashes on dental plaque and gingival inflammation: A systematic review and meta-analysis. Indian Journal of Dental Research 2018; 29(2): 225-232.
  19. Kim H, Wang Q, Shoemaker CF, et al. Polysaccharide gel coating of the leaves of Brasenia schreberi lowers plasma cholesterol in hamsters. Journal of Traditional and Complementary Medicine 2015; 5(1): 56-61.
  20. Prabhakar J, Senthilkumar M, Priya MS, et al. Evaluation of antimicrobial efficacy of herbal alternatives (Triphala and green tea polyphenols), MTAD, and 5% sodium hypochlorite against Enterococcus faecalis biofilm formed on tooth substrate: an in vitro study. Journal of Endodontics 2010; 36(1): 83-86.
  21. Jiang N, Wei Y, Cen Y, et al. Andrographolide derivative AL-1 reduces intestinal permeability in dextran sulfate sodium (DSS)-induced mice colitis model. Life Sciences 2020; 241: 117164.
  22. Tang Z, Xiong D, Song J, et al. Antitumor Drug Combretastatin-A4 Phosphate Aggravates the Symptoms of Dextran Sulfate Sodium-Induced Ulcerative Colitis in Mice. Frontiers in Pharmacology 2020; 11.
  23. Alex P, Zachos NC, Nguyen T, et al. Distinct Cytokine Patterns Identified from Multiplex Profiles of Murine DSS and TNBS-Induced Colitis. Inflammatory Bowel Diseases 2008; 15(3): 341-352.
  24. Yang S, Yu M. Role of Goblet Cells in Intestinal Barrier and Mucosal Immunity. Journal of Inflammation Research 2021; 14: 3171-3183.
  25. Khanfar E, Olasz K, Gajdócsi E, et al. Splenectomy modulates the immune response but does not prevent joint inflammation in a mouse model of RA. Clinical and Experimental Immunology 2022; 209(2): 201-214.
  26. Li Z, Cheng Q, Yu L, et al. Dan-Lou tablets reduces inflammatory response via suppression of the MyD88/p38 MAPK/NF-κB signaling pathway in RAW 264.7 macrophages induced by ox-LDL. Journal of Ethnopharmacology 2022; 298: 115600.
  27. Sindhu S, Kochumon S, Shenouda S, et al. The Cooperative Induction of CCL4 in Human Monocytic Cells by TNF-α and Palmitate Requires MyD88 and Involves MAPK/NF-κB Signaling Pathways. International Journal of Molecular Sciences 2019; 20(18): 4658.
  28. Lai H, Yang Z, Lou Z, et al. Root Extract of Lindera aggregata (Sims) Kosterm. Modulates the Th17/Treg Balance to Attenuate DSS-Induced Colitis in Mice by IL-6/STAT3 Signaling Pathway. Frontiers in Pharmacology 2021; 12: 615506.
  29. Liu JZ, van Sommeren S, Huang H, et al. Association analyses identify 38 susceptibility loci for inflammatory bowel disease and highlight shared genetic risk across populations. Nature Genetics 2015; 47(9): 979-986.
  30. Wan J, Yu X, Liu J, et al. A special polysaccharide hydrogel coated onBrasenia schreberi: preventive effects against ulcerative colitisviamodulation of gut microbiota. Food & Function 2023; 14(8): 3564-3575.
  31. Baba Y, Sato Y, Owada G, et al. Effectiveness of a combination denture-cleaning method versus a mechanical method: comparison of denture cleanliness, patient satisfaction, and oral health-related quality of life. Journal of Prosthodontic Research 2018; 62(3): 353-358.
  32. Wu Y, Li K, Zeng M, et al. Serum Metabolomics Analysis of the Anti-Inflammatory Effects of Gallic Acid on Rats With Acute Inflammation. Frontiers in Pharmacology 2022; 13: 830439.
  33. Jayabalan R, Marimuthu S, Swaminathan K. Changes in content of organic acids and tea polyphenols during kombucha tea fermentation. Food Chemistry 2007; 102(1): 392-398.
  34. Bai R, Jie X, Yao C, et al. Discovery of small-molecule candidates against inflammatory bowel disease. European Journal of Medicinal Chemistry 2020; 185: 111805.
  35. Mortazavi SMR, Vaezi Z, Mahdavian R, et al. A novel cerasomal gallic acid as a non-ulcerogenic agent with an improved anti-inflammatory potential. Journal of Drug Delivery Science and Technology 2023; 86: 104610.
  36. Saimaiti A, Zhou D, Li J, et al. Dietary sources, health benefits, and risks of caffeine. Critical Reviews in Food Science and Nutrition 2023; 63(29): 9648-9666.
  37. Tang H, Hao S, Chen X, et al. Epigallocatechin-3-gallate protects immunity and liver drug-metabolism function in mice loaded with restraint stress. Biomedicine & Pharmacotherapy 2020; 129: 110418.
  38. Wang S, Li Z, Ma Y, et al. Immunomodulatory Effects of Green Tea Polyphenols. Molecules 2021; 26(12): 3755.
  39. Hsu SD, Dickinson DP, Qin H, et al. Green tea polyphenols reduce autoimmune symptoms in a murine model for human Sjogren's syndrome and protect human salivary acinar cells from TNF-α-induced cytotoxicity. Autoimmunity 2007; 40(2): 138-147.
  40. Ma Y, Hu C, Yan W, et al. Lactobacillus pentosus Increases the Abundance of Akkermansia and Affects the Serum Metabolome to Alleviate DSS-Induced Colitis in a Murine Model. Frontiers in Cell and Developmental Biology 2020; 8: 591408.
  41. Sheng Y, Wu T, Dai Y, et al. The effect of 6-gingerol on inflammatory response and Th17/Treg balance in DSS-induced ulcerative colitis mice. Annals of Translational Medicine 2020; 8(7): 442-442.
  42. Vegh Z, Kurti Z, Lakatos PL. Epidemiology of inflammatory bowel diseases from west to east. Journal of Digestive Diseases 2017; 18(2): 92-98.
  43. Xu X, Wang Y, Wei Z, et al. Madecassic acid, the contributor to the anti-colitis effect of madecassoside, enhances the shift of Th17 toward Treg cells via the PPARγ/AMPK/ACC1 pathway. Cell Death & Disease 2017; 8(3): e2723.
  44. Zhou J, Gu X, Fan X, et al. Anti-inflammatory and Regulatory Effects of Huanglian Jiedu Decoction on Lipid Homeostasis and the TLR4/MyD88 Signaling Pathway in LPS-Induced Zebrafish. Frontiers in Physiology 2019; 10.
  45. Huang Q, Yang J, Lin Y, et al. Differential regulation of interleukin 1 receptor and Toll-like receptor signaling by MEKK3. Nature Immunology 2003; 5(1): 98-103.
  46. Liu Y, Shu Y, Yin L, et al. Protective roles of the TIR/BB-loop mimetic AS-1 in alkali-induced corneal neovascularization by inhibiting ERK phosphorylation. Experimental Eye Research 2021; 207: 108568.
  47. Zhang C, Guo J, Zhang Z, et al. Biochemical components and fungal community dynamics during the flowering process of Moringa-Fu brick tea, a novel microbially fermented blended tea. LWT 2021; 140: 110822.
  48. David LA, Maurice CF, Carmody RN, et al. Diet rapidly and reproducibly alters the human gut microbiome. Nature 2013; 505(7484): 559-563.
  49. Yu S, Guo H, Ji Z, et al. Sea Cucumber Peptides Ameliorate DSS-Induced Ulcerative Colitis: The Role of the Gut Microbiota, the Intestinal Barrier, and Macrophage Polarization. Nutrients 2023; 15(22): 4813.