Identification of Drug Targets and Potential Molecular Mechanisms of Wantong Jingu Tablet in Treatment of Rats with Collagen-Induced Arthritis based on 16S rDNA High-Throughput Sequencing and Metabolomic Analysis


 Background: Wantong Jingu Tablet (WJT), a mixture of traditional Chinese medicine, can reduce the symptoms of rheumatoid arthritis (RA), but its pharmacological mechanism is unclear. The aims of this study were to investigate the therapeutic mechanisms of WJT for RA in vivo.Methods: The effects of WJT on the joint pathology, and the levels of Bax, Bcl-2,caspase-3, cleaved-caspase-3, ERK1/2, pERK1/2, TNF-α, IL-1β, and IL-6 were demonstrated based on several experiments in the model of collagen-induced arthritis (CIA) in rats. 16S rDNA high-throughput sequencing was used to investigate the effect of WJT on the overall structure and composition of gut microbiota. Meanwhile, metabolite changes in faeces were analyzed by metabolomics techniques. Results: The results showed that WJT restored the joint pathology in CIA rats, upregulated Bax and cleaved-caspase-3, downregulated Bcl-2, caspase-3, and pERK1/2, and reduced the levels of pro-inflammatory cytokines. The overall gut microbial structure in CIA rats was altered after WJT treatment. Three bacterial phyla were prominently restored: Bacteroidetes,Tenericutes and Deferribacteres, and three bacterial genera were significantly reversed: Vibrio, Macrococcus and Vagococcus. Furthermore, five specific metabolites associated with these specific bacterial genera were identified by correlation analysis. In addition, WJT supplement trended to down-regulate the other five metabolites according to metabolomic analyses. Conclusions: These results revealed that WJT restored the pathological changes of RA might through activating the mitochondrial apoptosis pathway, inhibited MEK/ERK signaling, and modulating the special bacteria and the special metabolites.


Background
Rheumatoid arthritis (RA) is a chronic, systemic, autoimmune disease characterized by joint inflammation and synovial hyperplasia, and research indicates an increasing prevalence of RA worldwide [1]. In RA, a large number of immune cells, T cells, B cells and macrophages, are present in the infamed synovium [2]. Recruited T cells activate B cells, macrophages, and broblasts to create a complex network of various secreted cytokines, such as TNF-α, IL-1β and IL-6, thereby maintaining synoviocytes in an activated and infamed state [3,4]. Moreover, increased concentrations of TNF-α, IL-1β, and IL-6 in the synovium lead to varying degrees of bone resorption and joint destruction [4,5]. There are no structuremodifying drugs or optimal treatments available for RA. Thus, there is an urgent need for novel, costeffective, and safe anti-infammatory therapies for the treatment of RA.
Wantong Jingu Tablet (WJT), the prescription drug approved by the Chinese government for RA treatment (Approval Number: Z20025183), is an herbal compound containing 25 herbal medicines with many pharmaceutical effects. It exerted curative effects on treating cervical spondylotic myelopathy, a serious degenerative disease, and the total effective rate was up to 83.3% [6]. Recently, a study has found that WJT had clinical curative effect in treatment of periarthritis, signi cantly relieving shoulder pain and improving shoulder function [7]. Moreover, WJT could protect against collagen-induced arthritis in rats by disturbance of gene expression levels and induction of synoviocy tes apoptosis [8]. These studies suggest that WJT has pharmaceutically potential roles in restoring the pathogenesis and progression of RA.
Recent studies on the therapeutic mechanisms of herbal medicines have focused on alterations in gut microbial communities [9]. Some studies have demonstrated that herbal medicines effectively treat diseases and also regulate the structure and composition of intestinal flora [10]. Metabolomics is another emerging high-throughput technique that comprehensively analyzes endogenous small metabolites in organisms, and can identify novel molecular markers and potential therapeutic targets [11]. Furthermore, the rat CIA model is widely used for the study of joint in ammation because of its low mortality and characteristics by mimicing the progression and pathological phenomena of human RA [12,13].
Accordingly, the aims of this work are to determine the effect of WJT on the production of proin ammatory cytokines, namely TNF-α, IL-1β, and IL-6, in the serum and joint tissue of rats. More speci cally, we used different omics technologies to identify potential molecular mechanisms and pharmacological targets of WJT in the therapy of RA.

Preparation of samples
Samples of WJT powder were provided by Jilin Wantong Pharmacy Group Company (Tonghua, China).
The traditional Chinese medicine containing in WJT was indicated in Supplymentary Table S1. Acute toxicity in rats One hundred and twenty female speci c pathogen free (SPF) Sprague-Dawley (SD) rats weighing 160 to 180g were obtained from Changchun Yisi Experimental Animal Technology Company. The rats were randomly assigned to six groups (G1, G2, G3, G4, G5, and G6), fasted for 12 h, but with free access to drinking water, before a single administration of 1 g (G1), 2 g (G2), 3.98 g (G3), 7.94 g (G4), 15.85 g (G5), 31.62 g (G6) of WJT power per kg of body weight. Signs of toxicity were evaluated based on mortality. On day 14, survival rates were measured and the LD 50 value was calculated based on the Improved Koch's method.
The rats were anaesthetised with an intraperitoneal injection of 10% pentobarbital sodium (4 ml/kg) and subsequently sacri ced by rapid decapitation. All animal experiments were approved by the Animal Research Committee of Jilin University. The principles in the ARRIVE guidelines and the Basel declaration (http://www.basel.declaration.org) have been considered when planning the experiments.
Induction of collagen-induced arthritis (CIA) in rats Fifty female SPF SD rats were randomly divided into ve groups after acclimatization for 1 week: control (no CIA), model (CIA alone), CIA + 150 mg/kg WJT, CIA + 300 mg/kg WJT, and CIA + 600 mg/kg WJT, with 10 rats per group. CIA in rats was conducted according to previously described by Jian Zuo et al. [14]. Then, rats in three WJT groups were given oral WJT solution (150, 300, or 600 mg/kg) each day starting on day-1; rats in control and model groups were given the same volume of normal saline. All rats were sacri ced on day-28 for further study.

Assessment of arthritis severity
On day 28, paw swelling and paw thickness were measured using a plethysmometer and an electronic vernier caliper, respectively. The severity of arthritis was evaluated using an arthritis index (AI) with a CIA semi-quantitative scoring system, as previously described by Nagaraja Haleagrahara et al. [15].

Measurement of in ammatory factors
On day 28, paw and ankle tissues were collected and broken apart using a homogenizer (JinXin, Shanghai, China) at 4 °C for collection of tissue uid. The tissue uid and blood were then centrifuged, and the supernatant was analyzed for the levels of pro-in ammatory factors (TNF-α, IL-1β, and IL-6) using enzyme linked immunosorbent assay (ELISA) kits (Abcom, Cambridge, UK), following the manufacturer's protocols.

Histopathological evaluation of ankle joints
After xation, the tissue samples were processed, embedded in para n, and then cut into 4 mm slices using a microtome (Leica, Shanghai, China). Microscopic changes of the ankle joints were visualized using a light microscope (Olympus, Japan) after hematoxylin and eosin (H&E) staining. The severity of pathological changes in the ankle joints was measured according to previously described by Acharya Balkrishna et al. [16].

Immunohistochemical analyses
The para n-embedded samples of the ankle joints were blocked by 5% bovine serum albumin (BSA; Solarbio, Beijing, China) for 30 min, incubated with an appropriate primary antibody from Abcom (Cambridge, UK) overnight at 4°C, and then with a secondary antibody (Bioss, Beijing, China) at room temperature for 30 min. Enhancement was performed using a DAB dye solution and counter staining with hematoxylin. Finally, the ankle samples were observed under a light microscope (Olympus) at 400×, and typical images are presented. Image analysis software (Image Pro Picture) was used for quantitative analysis.

Sequencing and data analysis
Sequencing and data analysis were performed following the manufacturer's guidelines by Novogene bioinformation technology Co., Ltd, as previously described by Jing Wang et al. [17].

Metabolism and data analysis
On day-28, blood samples from sacri ced CIA rats were centrifuged at 4000 rpm (4°C for 5 min), and the supernatant was diluted with methanol so the nal methanol concentration was 60% by use of liquid chromatograph-mass spectrometer (LC-MS) grade water. Then, the samples were transferred to fresh Eppendorf tubes with 0.22 μm lters, and centrifuged at 15,000 g (4°C for 10 min). Finally, the ltrate was subjected to LC-MS/MS analysis using a Vanquish UHPLC system (Thermo Fisher, Waltham, USA) coupled with an Orbitrap Q Exactive series mass spectrometer (Thermo Fisher).
The raw data les generated by UHPLC-MS/MS were processed and normalized using Compound Discoverer version 3.1 (Thermo Fisher). Statistical analyses were performed using the R software package (version 3.4.3), Python (version 2.7.6), and CentOS (release 6.6). Then the normalized data were matched with the mzCloud (https://www.mzcloud.org) and ChemSpider (http://www.chemspider.com/) databases to obtain the accurate qualitative and relative quantitative metabolism results. Principal coordinates analysis (PCoA) was performed using metaX (http://metax.genomics.cn). Univariate analysis was used with the t-test to calculate statistical signi cance (P-value). All metabolites with a variable importance in projection (VIP) greater than 1, a P-value below 0.05, and a fold change (FC) of 2 or more or 0.5 or less or were considered to be differentially regulated metabolites. The screened out metabolites were nally visualized as volcano plots, heatmaps, and mapped onto KEGG pathways.

Statistical analysis
Except the 16S rDNA high-throughput sequencing and metabolomics, data are presented as means ± standard deviations (SDs). Student's t-test was used to compare groups, and all statistical analyses were conducted using SPSS version 20.0. All experiments were performed at least three independent times.
Differences were considered to be statistically signi cant at p < 0.05 and highly signi cant at p < 0.01.

Acute toxicity assay
To determine the appropriate doses of WJT for rats, we initially performed an acute toxicity assay ( Figure  1A). The median lethal dose (LD 50 ), calculated using the Improved Koch's method, was 6.24 g/kg, and the 95% con dence interval was 4.16 to 9.34 g/kg. In this work, the maximum drug dose is one-tenth of the LD 50 . Therefore, the drug doses of WJT were 600 mg/kg, 300 mg/kg, and 150 mg/kg for further study.
The H&E staining results indicated that WJT treatment exhibited no evident damage to the liver, spleen, and kidney of rats ( Figure 1B).

WJT inhibited paw swelling in CIA rats
A signi cant increase in paw volume was shown after CIA immunization ( Figure 1C). However, the paw edema was obviously reduced after oral administration of WJT at 300 and 600 mg/kg/day compared to model group. The arthritis index, paw thickness, and paw swelling decreased signi cantly in a dosedependent manner ( Figure 1D).

WJT reversed pathological changes in CIA rats
Histopathological results found that normal rats exhibited clear and complete histological architecture of the ankle joints ( Figure 1E). But CIA rats had abnormal histological architecture in the ankle joints, with manifestations of synovial hyperplasia, massive in ammatory cells in ltration, pannus formation, and cartilage and bone erosion. By comparison, WJT alleviated these pathological conditions a lot. Synovial hyperplasia, in ltration of in ammatory cells and erosion of synovial tissues were obvious suppressed after oral administration of WJT at 600 mg/kg/day ( Figure 1F).
WJT activated the mitochondrial apoptosis pathway and inhibited MEK/ERK signaling in synovial tissues Compared to the CIA rats, the protein levels of Bax and cleaved-caspase-3 were prominently increased after WJT treatment, and down-regulation of Bcl-2 and caspase-3 was observed following WJT supplement (Figure 2A). In addition, the expression levels of MEK, pMEK, ERK, and pERK were prominently up-regulated in CIA rats compared to the normal rats. However, WJT supplement (600 mg/kg) signi cantly reversed these proteins expression ( Figure 2B and C).
WJT suppressed the levels of pro-in ammatory cytokines in serum and ankle joints The levels of TNF-α, IL-1β, and IL-6 in serum and ankle joints were shown in Figure 3. Compared with the control group, the levels of these pro-in ammatory cytokines were markedly up-regulated in CIA rats. However, treatment with WJT (600 mg/kg) down-regulated TNF-α, IL-1β, and IL-6 levels compared to CIA rats.
WJT changes the overall structure of gut microbiota The intestinal ora structures of rats in control, model, and 600 mg/kg WJT treatment groups were investigated by 16S rDNA sequencing. There were 714 differential operational taxonomic units (OTUs) in all three groups, and 14, 51 and 420 unique OTUs were examined in the control, model, and WJT treatment groups, respectively (Supplymentary Figure S1). WJT signi cantly changed the Chao index and Shannon index, selected for the alpha diversity of the gut microbiota. PCoA plot and UPGMA clustering tree found that rats among the three groups had different overall structures of the bacterial communities.

WJT modulates the composition of gut microbiota
The histograms and the clustering map illustrating the community structure of gut microbiota revealed the dominant microbial species and their relative abundance at the phylum level ( Figure 4A). WJT supplement signi cantly reduced the abundances of Bacteroidetes, Tenericutes and Deferribacteres compared to the CIA rats ( Figure 4B). Firmicutes and Bacteroidetes were the two most abundant bacterial phyla in the gut microbiota community. Compared to the normal rats, the ratio of Firmicutes to Bacteroidetes was markedly increased in CIA rats, whereas WJT treatment signi cantly reversed the ratio ( Figure 4C).
At the genus level, the top 35 abundant bacterial genera among the three different groups were displayed by histograms and clustering map ( Figure 4D). The proportion of Vibrio, Macrococcus and Vagococcus was markedly up-regulated after CIA immunizations compared to thenormal rats ( Figure 4E). However, WJT treatment signi cantly reversed the trend.

WJT manipulates the metabolic patterns
Our metabolomic analysis, based on UHPLC-MS/MS, detected a total of 522 metabolites in negative mode and 707 metabolites in positive mode. Volcano plots depicting differentially abundant metabolites among groups revealed that there were 177 and 151 differentiated metabolites between control and model groups, and model and WJT treatment groups, respectively (Supplymentary Figure S2A). More details were shown in Supplymentary TableS 2. In addition, heatmaps were used to display the relative levels of the differentiated metabolites among groups(Supplymentary Figure S2B).
KEGG analysis was conducted to reveal the signi cantly enriched pathways by differentiated metabolites among different groups ( Figure 5A and B). More details were shown in Supplymentary Table S3 and S4.
The abundances of metabolites in these correspondingly enriched pathways were statistically analyzed ( Figure 5C and D). Com_643_pos (Serotonin), Com_1623_pos (Glutathione disul de), Com_5570_pos (N-Acetylneuraminic acid), Com_3187_pos (Naphthalene) and Com_520_pos (Thromboxane B2) were tended to be reversed by WJT compared to the CIA rats. These ve metabolites might be involved in the therapeutic treatment of WJT for RA, and more information about the ve metabolites was shown in Supplymentary Table S5.

Correlation analysis and ROC curve
The metabolites associated with the special bacterial genera (Vibrio, Macrococcus and Vagococcus) were identi ed by Pearson correlation analysis ( Figure 6A), and the relative abundances of these special metabolites were statistically analyzed according to metabolomic analyses ( Figure 6B). The abundances of Com_5483_pos (3'-N-debenzoyl-2'-deoxytaxol), Com_1491_pos (Tubulysin B), Com_4251_pos (Dexamethasone cipecilate), Com_3042_neg (Magnoline) and Com_1330_neg (Hydrocortisone Valerate) were signi cantly reversed by WJT treatment compared to the CIA rats. ROC analysis con rmed that these ve differentiated metabolites had AUC values ranging from 0.90 to 0.97, indicating these compounds might also be potential targets of WJT treatment for RA (Supplymentary Figure S3). In addition, more information about the ve metabolites was shown in Supplymentary Table S5.

Discussion
Rheumatoid arthritis (RA) characterized by joint swelling, synovial in ammation, and cartilage and bone destruction, is a chronic autoimmune disease that impairs joint movements [2]. An increasing number of RA patients and those with other diseases are selecting natural products to satisfy their healthcare needs [18]. WJT is a type of traditional Chinese medicine (TCM) that is a mixture of herbal compounds, and has been used for clinical treatments for many years. In this study, we mainly investigated the antiin ammatory and pharmacological effects of WJT on CIA rats to identify its pharmacological targets and potential molecular mechanisms, and the therapeutic mechanism of WJT for RA was elucidated in Supplymentary Figure S4.
The results of clinical manifestations and pathological changes demonstrated that WJT played an important role in suppressing the pathological progression of arthritis in CIA rats, which laid a foundation for the further study. In addition, The aberrant release and recruitment of pro-in ammatory cytokines (TNF-α, IL-1β, and IL-6) are essential to the pathogenesis of RA [17,29,20]. It's well known that both TNFα and IL-1β play roles in the activation and in ltration of immune cells, and contribute to production of in ammatory cytokines and chemokines, thereby resulting in a complex network that promotes in ammatory reactions, cartilage damage and autoimmune pathology [17,21]. Moreover, IL-6 acts on B lymphocytes and helps them to release many pro-in ammatory molecules around the joints [22]. It also up-regulates the sensitivity of the osteoclasts and promotes the activation of the broblast synovial cells, thereby contributing to synovial pannus formation and cartilage destruction [4]. Our results indicated that WJT down-regulated the levels of these pro-in ammatory cytokines in serum and joint tissue. Thus, the therapeutic mechanisms of WJT for RA appears related to its inhibition of pro-in ammatory cytokines.
In this study, our results showed that WJT signi cantly altered the overall structure of gut microbiota in CIA rats. Firmicutes and Bacteroidetes were the two most abundant bacterial phyla in the gut microbiota community. A common metabolite of Gram-positive Firmicutes, butyrate, is identi ed as an antiin ammatory molecule, which help to impede bacterial transport across the intestinal epithelial wall and increase the rate of tight junction formation, thereby maintaining the healthy gut environment [23,24]. The aberrant proliferation of Gram-negative Bacteroidetes contributes to the production of lipopolysaccharides (LPS) and then induces the low-grade in ammation by activating Toll-like receptor 4 (TLR-4) [25]. In addition, Bacteroidetes can increase intestinal permeability and promote a chronic in ammatory state by degrading mucin on the intestinal membrane [26]. The ratio of Bacteroidetes to Firmicutes was up-regulated in many autoimmune diseases, such as type 1 diabetes mellitus (T1DM), systemic lupus erythematosus (SLE), and so on [23]. In this study, the high ratio of Bacteroidetes to Firmicutes in CIA rats was prominently reversed by WJT treatment, which might be a therapeutic mechanism of WJT for RA.
At the genus level, the CIA rats signi cantly increased the number of Vibrio, Macrococcus, and Vagococcus. However, WJT supplement recovered these bacterial genera, Vibrio, a highly motile Gramnegative bacteria of the phylum Proteobacteria, is correlated with human diseases, such as cholera, vibriosis, wound infections, and so on [27]. Additionally, Vibrio increasing the levels of IL-6 and TNF-α induces the in ammatory responses RAW264.7 macrophages and causes in ammation in vivo [21]. From the point of view, that WJT decreased Vibrio population might be associated with the reduction of pro-in ammatory cytokines levels in serum and ankle tissue. Both Macrococcus and Vagococcus are the members of the phylum Firmicutes. It is accepted that Macrococcus is involved in the in ammatory in ltration, hemorrhages, multifocal necrosis of various organs, and pathological changes of caseous exudation in cranial cavities [28]. In addition, a recent study revealed that Vagococcus modulating the in ammatory responses induced the neuropsychiatric disorders [29]. Therefore, we hypothesize that WJT supplement is bene cial to normalize these bacteria close to the normal level, thereby recovering the pathogenesis in CIA rats.
In this work, WJT trended to restore the abundances of serotonin, glutathione disul de, Nacetylneuraminic acid, naphthalene and thromboxane B2 in CIA rats, and the signaling pathways and the functions of these ve metabolites associated with RA were illustrated in Supplymentary Figure S4, which was another therapeutic mechanism of WJT for RA.
3'-N-debenzoyl-2'-deoxytaxol, dexamethasone cipecilate, tubulysin B, magnoline, and hydrocortisone valerate were another ve metabolites identi ed by correlation analysis, which were prominently reversed by WJT in CIA rats. Dexamethasone cipecilate characterized by increasing lipophilicity and enhancing pharmacological action has been identi ed as an effective therapy for allergic rhinitis [30]. Magnoline, an anti-inflammatory compound, has ability to inhibit the proliferation of cancer cells [31,32]. Meanwhile, tubulysins also exhibits anti-proliferative effects on cancer cell [33]. And hydrocortisone valerate is another metabolite molecule correlated with low-grade in ammation [34]. 3'-N-debenzoyl-2'-deoxytaxol has not been reported in pharmacology until now. Thus, these ndings suggest that the special metabolites might be therapeutic targets of WJT for RA.

Conclusion
In this study, our results indicated that WJT restored the paw swelling, reversed the levels of in ammatory cytokines, inhibited the MEK/ERK signaling , and induced synoviocyte apoptosis. Additionally, it prominently altered the overall gut microbial structure of CIA rats. The ratio of Bacteroidetes to Firmicutes, and the abundances of bacterial genera (Vibrio, Macrococcus and Vagococcus) signi cantly reversed after WJT treatment in CIA rats. These ndings might be the therapeutic mechanisms of WJT for RA. Moreover, serotonin, glutathione disul de, N-acetylneuraminic acid, naphthalene, thromboxane B 2, 3'-N-debenzoyl-2'-deoxytaxol, dexamethasone cipecilate, tubulysin B, magnoline, and hydrocortisone valerate also might be therapeutic targets of WJT for RA. Our results provide a basis for its safe and effective administration in clinical practice. However, a systematic and comprehensive interpretation of the therapeutic effect of WJT on RA requires further investigation.

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

Availability of data and materials
The datasets generated for this study are available on request to the corresponding author.

Competing interests
The authors declare no Competing interests.

Funding
This research did not receive any speci c grant from funding agencies in the public, commercial, or not-for-pro t sectors.
Author Contributions FL and ZL conceived and designed the experiments; ZL and FQ performed the experiments; ZL and FQ carried out the statistical analysis; GW and ZL wrote the paper. All authors have read and approved the manuscript.   Effect of WJT on production of pro-in ammatory cytokines in rats with CIA. (A) Serum levels of TNF-α, IL-1β, and IL-6, measured using ELISA. (B and C) Ankle tissue levels of TNF-α, IL-1β, and IL-6, measured using ELISA and immunohistochemical staining. Values were expressed as mean ± SD (n = 10). ##p<0.01, ###p<0.001 vs. control group; *p<0.05, **p<0.01 vs. model group.   Values were expressed as mean ± SD. n = 10 per group. *p < 0.05, **p < 0.01; n.s., no signi cance.

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