Study on the mechanism of Indigo naturalis and its two main compounds (indigo and indirubin) in the treatment of ulcerative colitis based on TLR4/MyD88/NF-κB pathway and intestinal microorganisms

Background: Indigo naturalis is a natural dye extracted from plants and has a good anti-inammatory effect. Clinical studies have shown that it can improve ulcerative colitis (UC), but the active constituents and the mechanism are unclear. Methods: The anti-UC activity of Indigo naturalis and its two main compounds (indigo and indirubin) were investigated in dextran sulfate sodium (DSS)-induced UC mice. Indigo naturalis, indigo and indirubin were administrated to DSS-induced UC rats by oral gavage for 1 weeks. The anti-UC effect was evaluated by pathological section, inammatory cytokine production, western blotting, and gut microbiota analysis via 16S rRNA sequencing. Results: Indigo naturalis, indigo and indirubin can improve the UC induced by DSS. Their effect intensity is Indigo naturalis > indirubin > indigo based on disease activity index, body weight, colon length and pathological section. Indigo naturalis, indigo and indirubin also decrease the expression of NF-κB and MYD88 proteins, thus reducing the level of related inammation cytokines (IL-1β, IL-6 and TNF-α) both in serum and tissue. In addition, Indigo naturalis and indigo improved symptoms of gut microbial disturbance, and decreased Firmicutes/Bacteroidetes ratio and the signicantly increased probiotics such as Lactobacillus. Indirubin has little effect on the regulation of gut microbial. Conclusions: Indigo naturalis could attenuate the DSS-induced UC in mice, by means of ameliorating intestinal inammation, improving intestinal mucosa, and regulating the disturbed gut microbiota. Indigo and indirubin could also attenuate the DSS-induced UC in mice, but their comprehensive effect is not as good as Indigo naturalis.


Background
Ulcerative colitis (UC) is an in ammatory bowel disease (IBD) of unknown etiology, probably caused by a combination of genetic and environmental factors. Its morbidity is about 0.03%-0.1% worldwide, and is increasing in recent years. The precise etiology of UC is not known and cure of this disease is not yet possible so that the primary therapeutic goal is to relieve clinical symptoms and promote mucosal healing [1]. The classical drug for treating UC is 5-aminosalicylic acid, followed by short-term use of corticosteroids, immunomodulators and biologicals [2][3][4]. However, long term use of these drugs has some potential side effects, such as allergies, infections and anti-antibody reaction, etc [5]. It is thus of great signi cance and urgency to investigate the effective anti-UC drugs with a higher safety.
Complementary and alternative drugs are widely concerned by researchers due to their high safety and activity. Indigo Naturalis, a traditional Chinese medicine, is obtained from the extract of plants, such as Indigofera tinctoria, Isatis tinctoria, and Polygonum tinctorium. It has long been used to treat in ammatory diseases, including acute promyelocytic leukemia and psoriasis [6,7]. Researches showed that indigo naturalis can improve the health of colitis model mice by regulating pro-in ammatory factors and improving the damage of colonic mucosa [8,9]. A randomized, placebo-controlled trial has also been proved that indigo naturalis has an anti-UC activity [10]. This suggests that indigo naturalis may be a potential anti-UC drug. However, it is not clear for indigo naturalis what the mechanism and main active components in the treatment of UC.
The interaction of microorganisms in vivo determines the health of the body, especially in the colon where microorganisms are abundant [11]. Studies have shown that the intestinal mucosal in ammatory response caused by various factors is accompanied by the change of intestinal microecology and the imbalance of ora [12]. With the development of intestinal microecology research, intestinal ora regulators have become a hot research object in the treatment of UC. In this manuscript, the anti-UC effects and mechanisms of Indigo naturalis and its two main components (indigo and indirubin) were evaluated. Then their gut microbiota regulation was explored based on sequencing of 16S rDNA. The disease activity index (DAI) was recorded as the mean value of the following parameters: normal stool (0), soft stools (1), soft stools and slight bleeding (2), loose stools and slight bleeding (3), gross bleeding (4). At day 10, mice were sacri ced, the colons were measured and collected for histological analysis and protein extraction.

Materials
After 14 days of administration, all mice were taken blood from eyeballs. Then they were sacri ced by iso urane inhalation, and their colon and feces samples were collected and exampled. Part of the colon segments were xed by 4% paraformaldehyde, embedded in para n and then cut into 4 mm thick sections. These sections were stained with hematoxylin and eosin (HE) and periodic acid-Schiff (PAS), respectively, in accordance with the standard procedures for histopathological analysis. Additionally, part of colon segments was weighted and homogenized with 0.1Mphosphate buffer (pH 7.4). The homogenate suspension was collected by centrifugation at 5,000 rpm for 15 min. The amounts of in ammation-associated cytokines including IL-1β, IL-6, TNF-α and MPO were determined using ELISA kits according to the manufacturers' instructions.

Western Blot Analysis
The remaining colon segments were weighted and lysed in RIPA buffer with protease and phosphatase inhibitors on ice for 30 min. The lysates were centrifuged at12,000 g for 10 min at 4•C and the supernatant was collected as the total lysate protein. The protein concentration was determined with BCA Protein Assay Kit according to the manufacturers' instructions. The samples were boiled at 98 °C for 7 min, chilled on ice and separated using 10% SDS-PAGE electrophoresis and then transferred to nitrocellulose membrane (Millipore Corporation, USA) at 25 V for overnight. The membrane was blocked in 5% no fat milk/TBST and incubated with primary antibody overnight at 4 °C. The membrane was washed with TBST and subsequently incubated with an HRP conjugated secondary antibody that was detected using a chemiluminescent substrate.
The Illumina reads were sorted into different samples according to their barcoded index sequences. The paired-end reads were merged by FLASH and then assigned to each sample according to the unique barcodes, so as to get rid of the low-quality tags (length < 200 bp, more than two ambiguous bases 'N,' or the average base quality score < 30). High-quality tags were clustered into operational taxonomic units (OTUs) using UPARSE algorithm in QIIME software based on a 97% sequence similarity, and these OTUs were further subjected to analysis using database of Greengenes by PyNAST algorithm [13]. Alpha and Beta diversities and principal coordinate analysis (PCoA) were analyzed by QIIME, Mothur, and R software. LEfSe analysis were done using Python LEfSe package [14].

Statistical Analysis
The experimental data were analyzed by GraphPad Prism 6.0 software and presented as mean ± SD. The values of various groups were evaluated by one-way ANOVA and difference test. P < 0.05 and P < 0.01, calculated using SPSS software (version 21), were considered statistically signi cant.

Results the Healthy Condition of DSS-Induced UC in mice
Preliminary experiments have shown that continuous administration of 3% DSS in drinking water for more than three consecutive days could induce various symptoms, such as diarrhea and even hematochezia, which exhibit similarities with human UC. The body weight changes of the rats throughout the experiment clearly re ected their physiological status. Figure.1B shows a signi cant body weight loss of DSS-treated UC mice at day 10, in comparison with the normal group. However, rats in INN, IND, INB and SSZ groups exhibited a less distinct weight loss compared with the model group. In view of these representative clinical features, the DAI was used to evaluate the therapeutic activity of these drugs. As shown in Figure. Figure.5A showed that the number of OTUs in the sample is stable, and the amount of sequencing data can re ect the total number of OTUs in the sample. Figure.5B shows that the sample curve tends to be gentle and the distribution is relatively centralized, which shows that the amount of sequencing data is large enough to re ect the vast majority of microbial information in the sample. As shown in the Venn diagram ( Figure. Histograms were used to re ect the differences among various groups on species and relative abundance of intestinal microbiota. At the phyla level ( Figure. Lefse (LDA effect size) was used to determine the bacterial genera with signi cant differences among groups. Figure.

Discussion
In ammatory bowel disease (IBD), including UC and Crohn's disease, is a chronic in ammatory bowel disease. In recent years, many studies have shown that UC is associated with intestinal mucosal barrier damage, genetic susceptibility and environmental factors [16,17]. Clinical treatment UC is usually treated with anti-in ammatory or immunosuppressive drugs that have the disadvantages of relapse after discontinuation, long-term adverse drug reactions, and unsatisfactory results in some refractory cases [18]. Other studies have proved that INN, IND and INB can improve UC, but their therapeutic mechanism is not clear [19][20][21].
TLR4 / MyD88 / NF -κ B pathway is one of the most important signaling pathways activated or inhibited in the pathogenesis of UC. TLR4, the rst member of TLR family, belongs to type I transmembrane receptor of TLR interleukin superfamily. It can promote in ammation and regulate immune response. What's more, it can recognize and combine pathogen related molecular pattern, activate NF -κB-related protein pathway, and promote in ammation [22]. MyD88 is an important linker protein in the TLR4 signal transduction process. TIR region of its C-terminal binds to the TIR region of TLR4 [23], while its N-terminal acts as a death region and can bind to NF-κB, which promotes the in ammatory factor Released in large quantities, causing an immune response in the intestinal mucosa [24]. NF-κB is closely related to the transcription of in ammatory transmitters, cytokines and other genes. Binding to the κB site, it initiates an in ammatory response, which breaks the immune balance in the intestine and nally leads to the occurrence of UC [25]. Recently, a large number of studies have focused on the relationship between gut microbiota disorders and ulcerative and the intestinal microbiota has been demonstrated as one of the key factors to in uence nutrient metabolism and immune response, and keep the host healthy in various intestinal diseases [26,27]. The gut microbiota mainly consists of Firmicutes, Bacteroidetes, Actinobacteria and Proteobacteria, which account for more than 98% of the total gut micro ora [28]. The reduction of intestinal micro ora diversity and low abundance of Firmicutes were observed in IBD patients compared with healthy man [29,30]. There are reports both in increase and decrease of the abundance of Proteobacteria and Bacteroidetes, which may be related to the actual situation of IBD patients [31]. In this study based on the 16S rRNA gene sequence analysis of samples from the normal group, the DSS-induced UC group respective INN IND and INB treatment groups, the predominant intestinal bacteria pro les were greatly diversi ed. Figure.6 show that the DSS-induced UC is often accompanied by shifts in gut microbiota structure, with a signi cant decrease of intestinal bacterial diversity, a reduction of Firmicutes and an increase of Bacteroidetes amounts. Further analysis found that Anaerotruncus,is a probiotic and dominant microorganism in DSS-induced group. This result is contrary to the previous reports, which may be caused by the stress reaction of the body [32,33] This study was conducted in strict accordance with the recommendations of the Guidelines for the Care and Use of Laboratory Animals of the Ministry of Science and Technology of China. The protocol and experimental designs were approved by the Ethical Committee of Chengdu University of Traditional Chinese Medicine (Approval ID:2017BL-003). All possible steps were taken to avoid the animals' suffering at any stage of the experiment. At the end of study, the animals were sacri ced following anesthesia with iso urane inhalation.

Consent for publication
The manuscript is approved by all authors for publication.

Availability of data and material
The datasets used in this study are available from the corresponding author upon reasonable request.