Investigation of the regulatory mechanism of lijie capsules on gut microbiota in rheumatoid arthritis

Yanqiang Chen; Shaobin Qiu; Fei Qiu; Guoyuan Li; Lixian Gan; Binghui Huang; Lingmei Yang

doi:10.4103/cjop.CJOP-D-22-00134

ORIGINAL ARTICLE

Year : 2023 | Volume : 66 | Issue : 4 | Page : 220-227

Investigation of the regulatory mechanism of lijie capsules on gut microbiota in rheumatoid arthritis

Yanqiang Chen, Shaobin Qiu, Fei Qiu, Guoyuan Li, Lixian Gan, Binghui Huang, Lingmei Yang
Department of Rheumatology, The First Affiliated Hospital of Guangxi University of Chinese Medicine, Nanning, Guangxi, China

Date of Submission	04-Jan-2023
Date of Decision	13-Mar-2023
Date of Acceptance	29-Mar-2023
Date of Web Publication	15-Jun-2023

Correspondence Address:
Dr. Yanqiang Chen
Department of Rheumatology, The First Affiliated Hospital of Guangxi University of Chinese Medicine, No. 89-9 Dongge Road, Nanning, Guangxi 530023
China

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/cjop.CJOP-D-22-00134

Abstract

Lijie Capsules (LJJN) are a classical Chinese herbal formula adopted to treat rheumatoid arthritis (RA) clinically, yet the regulatory mechanism underlying the protection of LJJN against RA has not been fully elucidated. Here, the animal model of RA was established by complete Freund's adjuvant administration in mice. About 60 mg/ml of LJJN was used for treatment. The histological change of ankle joint was measured by hematoxylin and eosin staining. The inflammatory cytokines were detected using ELISA kits. The protein associated with inflammation and GLUD2 was detected using Western blot. The mice feces were analyzed by 16S rRNA sequencing. The levels of glutamate (Glu) and α-ketoglutarate (α-KG) were detected using their detection kits. In addition, fibroblast-like synoviocytes (FLSs) were stimulated by Glu to induce an injured synoviocytes model in vitro, with or without LJJN treatment for 48 h. It was demonstrated that LJJN alleviated ankle joint swelling and synovial injury in RA mice. Meanwhile, LJJN inactivated nuclear factor kappa B signaling and suppressed inflammation of RA mice. The disordered gut microbiota composition in RA mice was partly restored by LJJN. Bacteroides-mediated Glu metabolism was impacted in RA mice, and LJJN contributed to the conversion of Glu to α-KG in RA mice. In addition, the in vitro results revealed that LJJN could block Glu-induced inflammation in FLSs but had no direct influence on α-KG and GLUD2 levels. In summary, LJJN exerted a protective role against ankle joint injury and inflammation in RA, which might be partly associated with gut microbiota-mediated Glu metabolism.

Keywords: Glutamate, gut microbiota, inflammation, Lijie capsules, rheumatoid arthritis

How to cite this article:
Chen Y, Qiu S, Qiu F, Li G, Gan L, Huang B, Yang L. Investigation of the regulatory mechanism of lijie capsules on gut microbiota in rheumatoid arthritis. Chin J Physiol 2023;66:220-7

How to cite this URL:
Chen Y, Qiu S, Qiu F, Li G, Gan L, Huang B, Yang L. Investigation of the regulatory mechanism of lijie capsules on gut microbiota in rheumatoid arthritis. Chin J Physiol [serial online] 2023 [cited 2023 Dec 4];66:220-7. Available from: https://www.cjphysiology.org/text.asp?2023/66/4/220/378758

Introduction

Rheumatoid arthritis (RA) is a chronic, systemic inflammatory autoimmune disease of unknown etiology, primarily damaging both joints, accompanied with extra-articular manifestations.^[1] The main clinical symptoms of RA include pain, stiffness, and swollen joints. RA is estimated as one of the most prevalent chronic inflammatory diseases with a prevalence ranging from 0.4% to 1.3%.^[2] In particular, RA is more common in females and may develop at any age, but the peak of the disease occurs at the age of 50–60 years.^[3] Currently, the frequent therapeutic strategy of RA includes glucocorticoids, non-steroidal anti-inflammatory drugs, and disease-modifying anti-rheumatic drug therapy; however, despite the great improvement in drug development, the deleterious side effects, and exorbitant cost limited the application of these drugs in clinical.^[4],[5] Thus, it is essential to develop reliable therapeutic drugs to RA.

Lijie Capsules (LJJN) is a classical Chinese herbal formula that has favorably therapeutic effects on treating RA. It is composed of more than twenty crude herbs and mainly includes Atractylodes macrocephala Koidz., Astragalus membranaceus, Glycyrrhiza uralensis Fisch., Atractylodes lancea, Ligusticum chuanxiong Hort, Paeonia lactiflora Pall, Saposhnikovia divaricate, and so on. Currently, LJJN has been widely applied in treating patients with RA through strengthening the spleen, attenuating dampness, phlegm, pain, and joint function in clinical.^[6] In addition, experimental studies disclosed that LJJN not only activated the Fas/Fas-L apoptotic signaling and promoted apoptosis of T-lymphocytes and synoviocytes but also regulated the levels of inflammatory mediators IL-2 and IL-6 in peripheral blood and reduced cellular inflammation.^[7],[8] Nevertheless, the regulatory mechanism underlying the protection of LJJN against RA has not been fully elucidated.

In recent decades, a wide spectrum of documents has developed in support of a potential relationship between RA and gut microbiota. According to previous research, dysbiosis occurs in the gut microbiota of RA patients but was partly normalized after treatment.^[9] Meanwhile, Scher et al. analyzed the gut microbiota between RA patients and the healthy volunteers adopting 16S rRNA sequencing, and they found that, compared to the healthy control, Prevotella copri was aberrantly abundant in RA patients, whereas Bacteroides and Clostridium, which were important to maintain intestinal homeostasis, were lessened, indicating that intestinal Prevotella copri was beneficial to enhancing the susceptibility of RA.^[10],[11] Therefore, the alterations of the composition of gut microbiota got involvement in the occurrence of RA. In addition, gut microbiota has been thought to be a critical environment agent influencing the development of RA.^[12] Taken the close association between RA and gut microbiota into consideration, whether LJJN modulated the gut microbiota during RA treatment raised our interest.

Therefore, this study aims to explore the impacts of LJJN on gut microbiota in RA mice, as well as elucidating its potential mechanism. The present study will provide new insights into the protective role of LJJN against RA.

Materials and Methods

Animals

Male Balb/c mice aged 6–8 weeks were obtained from Shanghai SLAC Laboratory Animals Co., Ltd. (Shanghai, China). The animals were housed in a standard environment including controlled temperature and humidity and a 12-h light/dark cycle and allowed standard access to water and food. All the experiments were abidance by the Ethical Committee of First Affiliated Hospital of Guangxi University of Chinese Medicine (Approval number: DW20191011-087).

Induction of rheumatoid arthritis and treatment

After acclimation for 1 week, mice were randomly assigned into three groups (n = 6 per group): control group, model group, and LJJN group. The animal model of RA was established by complete Freund's adjuvant (CFA) administration into mice as previously described.^[13] In brief, a 1:1 emulsion was prepared by mixing CFA (Chondrex, Redmond, WA, USA) and bovine Type II collagen (Chondrex). Mice were subcutaneously injected with 200 μg of the emulsion to the right hind footpad. Seven days after the primary injection (d7), CFA alone was given to the mice to boost immunization. Mice in model group and LJJN group were administrated with CFA, whereas mice in control group were injected with 0.1 mL saline. Two weeks after the primary injection (d14), mice in the LJJN group were intragastrically administrated with 0.1 ml LJJN suspension which was prediluted by saline to 60 mg/ml and was provided by the First Affiliated Hospital of Guangxi University of Chinese Medicine. The treatment was continued for consecutive 21 days. Mice in control and model groups received 0.1 ml saline by gavage. At the end of the experiment (d35), all mice were sacrificed.

Measurement of foot circumference

The right hind foot circumference of the mice was measured using a flexible rule before primary immunization and on the 7^th, 14^th, 28^th, and 35^th days after primary immunization. The foot swelling rate was calculated by the following formula: swelling rate = (foot circumference after primary immunization − foot circumference before primary immunization)/foot circumference before primary immunization.

Histological analysis

After sacrifice, the ankle joint was harvested and fixed in 4% paraformaldehyde for 24 h. Ankle joints were decalcified in 20% EDTA for 6 weeks. Afterward, ankle joints were embedded in paraffin, sliced into 4-μm thickness sections, and then stained with hematoxylin and eosin. All slices were observed under an optical microscope (Olympus Corporation, Tokyo, Japan).

ELISA

Blood was collected from the rats by cardiac puncture. After centrifugation at 3000 rpm at 4°C for 10 min, the serum samples were collected. Serum levels of pro-inflammatory cytokines, including tumor necrosis factor-alpha (TNF-α), IL-6, and IL-1β, were detected using their corresponding ELISA kits (R&D Systems, Minneapolis, MN, USA) following the guidelines of the manufacturer. In addition, the levels of glutamate (Glu) and α-ketoglutarate (α-KG) in serum and synovial tissues of the ankle joint were also measured by Glu Assay kit (Abcam) and alpha KG Assay kit (Abcam), respectively.

Gut microbiota analysis

At the end of the experiments, the feces of the mice in each group were collected. The total microbial genomic DNA was exacted from the feces using a DNA Extraction Kit following the manufacturer's instructions (QIAGEN, Germany). The quality and the quantity of exacted DNA were assessed by agarose gel electrophoresis and NanoDrop 3000, respectively (Thermo Fisher Scientific, Waltham, MA, USA). The V3-V4 region of the bacterial 16S rRNA gene was amplified by polymerase chain reaction (PCR) with the universal primers (forward, 5'-ACTCCTACGGGAGGCAGCA-3' and reverse, 5'-GGACTACHVGGGTWTCTAAT-3'). Eventually, high-throughput sequencing was carried out using the Illumina Miseq system with TruSeq Nano DNA LT Library Prep Kit (Illumina MiSeq, USA). The relative abundances (%) were analyzed at the phylum, class, order, family, and genus levels. α-diversity (including Chao1, Simpson, and Shannon analysis) and β-diversity (principal component analysis) between groups were then evaluated.

Quantitative real-time polymerase chain reaction

The total RNA from synoviocytes or synovial tissue was exacted by TRIzol reagent (Invitrogen). After quantification and purity identification using spectrophotometry (Bio-Rad Laboratories, Inc., Hercules, CA, USA) at 260/280 nm, 1 μg of RNA were reverse transcribed into cDNA using the ThermoScript RT-qPCR system (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Quantitative real-time PCR (qRT-PCR) was executed using SYBR Green qPCR Mix (Aidlab Biotechnologies Co., Ltd.). The relative gene expression of glutamate dehydrogenase 2 (GLUD2) was calculated using 2^−ΔΔCt method and normalized to GAPDH.

Western blot

The total protein was exacted from synoviocytes or synovial tissue using RIPA buffer (Beyotime Institute of Biotechnology, Shanghai, China), followed by its concentration determination adopting the Bradford Assay Kit (Beyotime Institute of Biotechnology). Equal amounts of protein were loaded on 12% SDS-PAGE gels for separation and then transferred to PVDF membranes. The membranes were blocked with 5% skimmed milk and incubated with primary antibodies against p-p65 (1:1000, ab131100, Abcam), p65 (1:1000, ab207297, Abcam), p-IκBα (1:10000, ab133462, Abcam), IκBα (1:5000, ab32518, Abcam), GLUD2 (1:1000, 14462-1-AP, Abcam), and GAPDH (1:2500, ab9485, Abcam) at 4°C overnight and then probed with HRP-conjugated goat anti-rabbit IgG secondary antibody (1:3000, ab6721, Abcam) at room temperature for 2 h. Finally, immune reactivities were visualized using an ECL-plus Western blotting detection reagent (GE Healthcare, Milwaukee, WI, USA).

Cell culture and treatment

Mouse fibroblast-like synoviocytes (FLSs) were obtained from Procell (Cat. No. CP-M323; Wuhan, China) and maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (FBS; Gibco, Thermo Fisher Scientific, Inc., USA) and 1% penicillin/streptomycin (Thermo Fisher Scientific, Inc.) at 37°C in 5% CO₂.

To induce an injured synoviocytes model, FLSs were stimulated by 100 μM of Glu for 48 h. Meanwhile, FLSs were treated with increasing concentrations (from 5% to 20%) of LJJN (dissolved in DMSO) for 48 h for treatment.

Cell counting kit-8 assay

Cell viability was determined using CCK-8 assay. In brief, FLSs were plated into 96-well plates (1 × 10⁵ cells/well) and treated with LJJN for 48 h. Subsequently, 10 μl of CCK-8 reagent was added to each well, and the plates were incubated for another 3 h. Finally, the absorbance of each well at 450 nm was detected using a microplate reader.

Statistical analysis

All data were analyzed with GraphPad Prism (GraphPad Software, San Diego, CA, USA) and presented as mean ± standard deviation. Difference comparison was conducted using one-way ANOVA followed by Tukey's post hoc test. A P < 0.05 was considered as statistically significant.

Results

Lijie Capsules alleviate ankle joint swelling and synovial injury of rheumatoid arthritis mice

First, the RA mice were established following immunization and presented as ankle joint inflammation and swollen. In detail, we detected the right hind foot circumference of each mouse in different groups to reflect the degree of foot swelling, and a rapid rise of the foot swelling was observable in model and LJJN group from the data in [Figure 1]a on the 7^th day after primary immunization. In the following days, the swelling gradually declined in model and LJJN groups but was still higher than control group. Meanwhile, the occurrence of the significant difference between model group and LJJN group revealed that the LJJN treatment remarkably reduced foot swelling in RA mice. At the end of the experiments, representative photographs of mice in different groups directly revealed the severely swollen ankle joint of mice in the model group, which was partly alleviated by LJJN treatment [Figure 1]b. In addition, according to histopathological evaluation, the synovial epithelial cells were neatly arranged and clearly visible in the control group; however, great pathological changes in the ankle joint were observed in model group, exhibited as proliferated synovial epithelial cells and the infiltration of inflammatory cells. These pathological changes were also weakened in LJJN group to a certain extent [Figure 1]c. Taken together, LJJN could effectively alleviate ankle joint swelling and inflammation infiltration of synovial tissues in RA mice.

Figure 1: LJJN alleviates ankle joint swelling and synovial injury of RA mice. (a) The animal model of RA was established by complete Freund's adjuvant (CFA) administration into mice. The right hind foot circumference of each mouse in different groups was measured. ***P < 0.001 versus Control, ^###P < 0.001 versus Model. (b) Representative photographs of mice in different groups. (c) Representative photographs of the histological examination of synovial tissues in the ankle joint (Magnification ×200). LJJN: Lijie Capsules.

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Lijie Capsules inactivate nuclear factor-kappa B signaling and suppresses inflammation of rheumatoid arthritis mice

Subsequently, we further detected the production of inflammatory cytokines in serum of mice in each group. As shown in [Figure 2]a, [Figure 2]b, [Figure 2]c, the high level of TNF-α, IL-1β, and IL-6 in serum of model group proved that RA was a systemic inflammatory disease, and administration of LJJN could effectively reduce the inflammatory response to some content. Moreover, we also measured the NF-κB signaling, the classical inflammation-related signaling. The results from Western blot assay showed that the protein level of phosphorylated (p)-p65 was hugely elevated in the model group, as well as an elevated p-IκBα expression and a reduced IκBα expression level, indicating that NF-κB signaling was activated in RA mice. Nevertheless, the alternation of these protein expressions was partially retarded after LJJN treatment [Figure 2]d. Taken together, LJJN might inactivate NF-κB signaling to suppress inflammation of RA mice.

Figure 2: LJJN inactivates NF-κB signaling and suppresses inflammation of RA mice. (a-c) The levels of TNF-α, IL-1β, and IL-6 in serum of different groups were determined using ELISA kits. (d) The protein level of p-p65, p65, p-IκBα, and IκBα was detected using Western blot. ***P < 0.001 versus Control, ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 versus Model. LJJN: Lijie Capsules, NF-κB: Nuclear factor kappa B, RA: Rheumatoid arthritis, TNF-α: Tumor necrosis factor alpha, IL-1β: Interleukin-1 beta.

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Lijie Capsules modulates gut microbiota in rheumatoid arthritis mice

To investigate whether LJJN could modulate the gut microbiota, the mice feces from nine samples (n = 3 in each group) were analyzed by 16S rRNA sequencing. Although the differences in microbial diversity and richness (Chao1 index, Shannon index, and Simpson index) among groups were not statistically significant, it could be observed from the obtained data in [Figure 3]a that the uniformity of the flora was improved following modeling. Meanwhile, the β-diversity in [Figure 3]b revealed an obvious separation of the microbial community, and the divergence in the distribution was well distinguished, especially between the model group and the other groups, suggesting that the microbial community might be hugely disordered in model group but was partly restored in LJJN group. In addition, the microbial diversity analysis revealed that the gut microbiota included the following twenty main phyla: Lactobacillus, Bacteroides and Oscillospira, Odoribacter, Alistipes, Bifidobacterium, (Prevotella), Parabacteroides, Prevotella, Enterococcus, Coprococcus, Ruminococcus, AF12, (Ruminococcus), Akkermansia, Desulfovibrio, Mucispirillum, Staphylococcus, Helicobacter, and Anaeroplasma. Among these, Lactobacillus, Bacteroides, and Oscillospira were the dominant phyla across all subjects at the phylum level, the abundance of which were remarkably reduced in RA mice compared to the control, while were partly restored following LJJN administration [Figure 3]c and [Figure 3]d. Bacteroides are important to maintain intestinal homeostasis as aforementioned, which is also verified in this study, as the relative abundance of Bacteroides in model group is lower than that in control group or LJJN group, suggesting that the abundance of Bacteroides might be positively associated with RA development [Figure 3]e.

Figure 3: LJJN modulates gut microbiota in RA mice. (a) The mice feces of each group were analyzed by 16S rRNA sequencing. α-Diversity (including Chao1, Simpson, and Shannon analysis) was assessed. (b) β-diversity (PCA) was assessed. (c) The relative abundance of top 20 dominate phyla of each three mice in different groups was presented. (d) The abundance changes of top 10 dominate phyla of each group. (e) The relative abundance of Bacteroides of each three mice in different groups. LJJN: Lijie Capsules, PCA: Principal component analysis.

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Lijie Capsules regulates Glu/α-ketoglutarate pathway of rheumatoid arthritis mice

Bacteroides is an important source affecting Glu metabolism,^[14] where GLUD2 serves as an important mediator to catalyze the reversible interconversion of Glu to α-KG. Thus, we subsequently examined Glu/α-KG axis in RA mice. As exhibited in [Figure 4]a and [Figure 4]b, compared to the control, Glu levels in blood and synovial tissues were greatly upregulated in RA mice but were partly decreased after LJJN treatment, whereas α-KG level in blood and synovial tissues exhibited an opposite trend. Meanwhile, both the mRNA level and protein expression of GLUD2 were downregulated in RA mice, which was partly restored in LJJN-treated RA mice [Figure 4]c and [Figure 4]d, suggesting that Glu metabolism was impacted in RA mice, and LJJN contributed to the conversion of Glu to α-KG in RA mice.

Figure 4: LJJN regulates Glu/α-KG pathway of RA mice. (a) Glu and α-KG level in blood was detected using their corresponding detection kit. (b) Glu and α-KG level in synovial tissues was detected using their corresponding detection kit. (c) The mRNA level of GLUD2 was detected using qRT-PCR. (d) The protein expression of GLUD2 was detected by Western blot. ***P < 0.001 versus Control, ^###P < 0.001 versus Model. LJJN: Lijie Capsules, α-KG: α-ketoglutarate, qRT-PCR: Quantitative real-time polymerase chain reaction.

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Lijie Capsules blocks inflammation in Glu-induced fibroblast-like synoviocytes but not affects Glu metabolism

Next, to further explore the critical role of Glu metabolism in RA, FLSs were stimulated by Glu to induce an injured synoviocytes model in vitro, with or without treatment with LJJN for 48 h. As 20% LJJN caused a significant reduction of cell viability, 15% LJJN was selected for the following treatment [Figure 5]a. It was observed from [Figure 5]b that Glu caused a marked reduction of cell viability, which was partly restored by LJJN. In addition, Glu resulted in a remarkable reduction of α-KG level and GLUD2 expression, but LJJN treatment did not affect these changes [Figure 5]c, [Figure 5]d, [Figure 5]e. These data suggested that LJJN did not direct influenced Glu metabolism, indicating that the restored Glu metabolism following LJJN treatment in vivo might be indirectly mediated by gut microbiota. Furthermore, consistent with the in vivo findings, Glu induced a great elevation of pro-inflammatory cytokines, including TNF-α, IL-1β, and IL-6, which was then partially retarded by LJJN treatment [Figure 6]a, [Figure 6]b, [Figure 6]c. Meanwhile, LJJN treatment also exerted significant inhibitory effect on the activation of NF-κB signaling in Glu-induced FLSs, evidenced by the reduced p-p65 and p-IκBα protein levels and the increased IκBα protein level [Figure 6]d, suggesting that LJJN might block inflammation in Glu-induced FLSs through inhibiting the activation of NF-κB signaling.

Figure 5: LJJN does not affect Glu metabolism in Glu-induced FLSs. (a) FLSs were treated with increasing concentrations (from 5% to 20%) of LJJN (dissolved in DMSO) for 48 h. Cell viability was detected using CCK-8 assay. *P < 0.05 versus DMSO. (b) FLSs were stimulated by Glu to induce an injured synoviocytes model in vitro, with or without treatment with 15% LJJN for 48 h. Cell viability was detected using CCK-8 assay. (c) α-KG level was detected using its detection kit. (d) The mRNA level of GLUD2 was detected using qRT-PCR. (e) The protein expression of GLUD2 was detected by Western blot. **P < 0.01, ***P < 0.001 versus Control, ^#P < 0.05 versus Glu. LJJN: Lijie Capsules, FLSs: Fibroblast-like synoviocytes, qRT-PCR: Quantitative real-time polymerase chain reaction.

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Figure 6: LJJN blocks inflammation in Glu-induced FLSs. (a-c) The levels of TNF-α, IL-1β, and IL-6 in different groups were determined using ELISA kits. (d) The protein level of p-p65, p65, p-IκBα, and IκBα was detected using Western blot. ***P < 0.001 versus Control, ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 versus Glu. TNF-α: Tumor necrosis factor alpha, IL-1β: Interleukin-1 beta.

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Discussion

RA is chronic, systemic, autoimmune disorder that primarily targets synovial joints. To date, mass attentions have been paid to discover effective and unharmful drugs for treating RA due to the limitation of side effects or high cost of current therapies. Traditional Chinese Medicine has a long history in China attributed to their abroad pharmacological activities. LJJN, developed by the First Affiliated Hospital of Guangxi University of Chinese Medicine, has been specialized to treat patients with RA. In the present study, an in-depth research of the potential mechanism by which LJJN prevented from RA was carried out in vitro and in vivo. Here, we demonstrated the therapeutic effects of LJJN on RA in vivo by alleviating ankle joint swelling and inflammation. In addition, LJJN was beneficial to maintain gut microbiota homeostasis and modulated Glu metabolism. Moreover, in vitro experiments uncovered that LJJN could directly alleviate the inflammatory injury of synoviocytes but did not influence Glu metabolism.

Recently, with the advancements in genome sequencing technologies and bioinformatics, the crucial role of gut microbiota in host health and disease gets great breakthrough. Notably, gut microbiota has been perceived as a novel avenue to understanding RA treatment and Traditional Chinese Medicine.^[15],[16] On the one hand, alternation of gut microbiota was suggested to be correlated with RA-related clinical indicators, and sustained drug treatment was demonstrated to alter the gut microbiota in RA patients, indicating that gut microbiota might be a potential target for treating RA.^[16],[17] On the other hand, more and more Traditional Chinese Medicine was found to impact the diversity or composition of gut microbiota, so as to regulate intestinal microflora balance and promote the healthy functioning of the intestinal system. For instance, Aralia echinocaulis extracts exhibited good effects on the RA rat model, which was related to modulating gut microbiota composition and its metabolic pathways.^[18] An integrated study of microbiome and fecal metabolomics analysis revealed a potential antiarthritis effect of Yaobitong capsules, which could alter the community structure and alleviate the microecological imbalance resulted from RA, as well as improving microbiota-mediated metabolites.^[19] Consistently, in this study, LJJN made a great restoration of the disordered gut microbiota composition, especially focusing on Lactobacillus, Bacteroides, and Oscillospira.

Another important finding of this study is the modulation of LJJN on Glu and α-KG in RA mice. Glu, an excitatory neurotransmitter, is not only expressed in nervous system, but also widely expressed in many peripheral and nonnervous tissues, such as bone, lymphocytes, and microphages, serving as an important mediator in regulating inflammatory response and inflammation-related diseases.^[20],[21] It has been reported that Glu is elevated in the synovial fluid of RA patients and arthritis rats and correlates with the pro-inflammatory cytokine profiles, disclosing the critical role of Glu in the pathological process of RA.^[21],[22] Consistently, we also found an elevated level of Glu no matter in serum or in synovial tissues of RA mice, as well as the upregulated production of TNF-α, IL-6, and IL-1β. Mechanically speaking, gut microbiota, especially Bacteroides, is an important source affecting Glu metabolism.^[14] During this process, GLUD serves as an important mediator to catalyze the reversible interconversion of Glu to α-KG. α-KG exerts various roles in different metabolic and cellular pathways. In particular, α-KG can directly bind to IKKβ to suppress the activation of NF-κB signaling, leading to an inactivation of inflammatory macrophages and the downregulation of inflammatory cytokines.^[23],[24] In our study, the results demonstrated that LJJN not only affected gut microbiota composition but also modulated the following Glu metabolism. In addition, the inhibitory effect on the production of Glu might be beneficial to the conversion of Glu to α-KG, evidenced by the upregulated GLUD2 and α-KG by LJJN treatment. Interestingly, GLUD2 is confirmed to be a potential target to alleviate RA by Tripterygium hypoglaucum (levl.) Hutch.^[25] Here, to validate whether LJJN could directly influence GLUD2 to treat RA, we used a single Glu to induce FLS. The data suggested that the cell viability loss, overproduction of pro-inflammatory cytokines, and NF-κB-signaling activation caused by Glu were alleviated by LJJN treatment, but LJJN treatment had no influence on α-KG and GLUD2 expression, indicating that LJJN had no direct impact on Glu metabolism and GLUD2, and this impact might be dependent on gut microbiota in vivo.

However, there are some limitations in the present study. First, sequencing with more samples is deserved to validate the current data; second, utilizing antibiotics or germ-free mice is beneficial to determine the significance of gut microbiota in RA, which is deserved to be conducted in our future work.

Conclusion

LJJN exerted a protective role against ankle joint injury and inflammation in RA, as well as restoring gut microbiota disorder. Furthermore, LJJN indirectly regulated Glu metabolism in vivo, which might be mediated by gut microbiota.

Data availability statement

All data in this study were included in the present article.

Financial support and sponsorship

This study was supported by the National Natural Science Foundation of China (No. 81860825).

Conflicts of interest

There are no conflicts of interest.

References

1.	Conforti A, Di Cola I, Pavlych V, Ruscitti P, Berardicurti O, Ursini F, et al. Beyond the joints, the extra-articular manifestations in rheumatoid arthritis. Autoimmun Rev 2021;20:102735.
2.	Smolen JS, Aletaha D, McInnes IB. Rheumatoid arthritis. Lancet 2016;388:2023-38.
3.	Sparks JA. Rheumatoid arthritis. Ann Intern Med 2019;170:C1-16.
4.	Burmester GR, Pope JE. Novel treatment strategies in rheumatoid arthritis. Lancet 2017;389:2338-48.
5.	Lin YJ, Anzaghe M, Schülke S. Update on the pathomechanism, diagnosis, and treatment options for rheumatoid arthritis. Cells 2020;9:880.
6.	Shi W, Tang N, Xiao J, Zhang L, Chai L, Wu J. Clinical study of Lijie capsule in the treatment of active rheumatoid arthritis. Lishizhen Med Mater Med Res 2010;21:3230-1.
7.	Zhang L, Tang N, Chai L, Huang X, Lin G. Influences of Lijie capsule on IL-2, IL-6 levels and T lymphocyte apoptosis in AA rats. Pharmacol Clin Chin Mater Med 2005;21:58-9.
8.	Tang N, Chai L, Zhang L. Influences of Lijie Capsules on expressions of Fas and FasL genes in lymphocytes in rats with adjuvant arthritis. J Beijing Univ Tradit Chin Med 2006;29:471.
9.	Zhang X, Zhang D, Jia H, Feng Q, Wang D, Liang D, et al. The oral and gut microbiomes are perturbed in rheumatoid arthritis and partly normalized after treatment. Nat Med 2015;21:895-905.
10.	Scher JU, Sczesnak A, Longman RS, Segata N, Ubeda C, Bielski C, et al. Expansion of intestinal Prevotella copri correlates with enhanced susceptibility to arthritis. Elife 2013;2:e01202.
11.	Vaahtovuo J, Munukka E, Korkeamäki M, Luukkainen R, Toivanen P. Fecal microbiota in early rheumatoid arthritis. J Rheumatol 2008;35:1500-5.
12.	Maeda Y, Takeda K. Role of gut microbiota in rheumatoid arthritis. J Clin Med 2017;6:60.
13.	Shan L, Tong L, Hang L, Fan H. Fangchinoline supplementation attenuates inflammatory markers in experimental rheumatoid arthritis-induced rats. Biomed Pharmacother 2019;111:142-50.
14.	Chang CH, Lin CH, Lane HY. D-glutamate and gut microbiota in Alzheimer's disease. Int J Mol Sci 2020;21:2676.
15.	Yue SJ, Wang WX, Yu JG, Chen YY, Shi XQ, Yan D, et al. Gut microbiota modulation with traditional Chinese medicine: A system biology-driven approach. Pharmacol Res 2019;148:104453.
16.	Mei L, Yang Z, Zhang X, Liu Z, Wang M, Wu X, et al. Sustained drug treatment alters the gut microbiota in rheumatoid arthritis. Front Immunol 2021;12:704089.
17.	Wang Z, Yu Y, Liao J, Hu W, Bian X, Wu J, et al. S-Propargyl-Cysteine remodels the gut microbiota to alleviate rheumatoid arthritis by regulating bile acid metabolism. Front Cell Infect Microbiol 2021;11:670593.
18.	Li Y, Dai M, Wang L, Wang G. Polysaccharides and glycosides from aralia echinocaulis protect rats from arthritis by modulating the gut microbiota composition. J Ethnopharmacol 2021;269:113749.
19.	Shi W, Ye H, Deng Y, Chen S, Xiao W, Wang Z, et al. Yaobitong capsules reshape and rebalance the gut microbiota and metabolites of arthritic rats: An integrated study of microbiome and fecal metabolomics analysis. J Chromatogr B Analyt Technol Biomed Life Sci 2022;1190:123096.
20.	Lawand NB, McNearney T, Westlund KN. Amino acid release into the knee joint: Key role in nociception and inflammation. Pain 2000;86:69-74.
21.	McNearney T, Baethge BA, Cao S, Alam R, Lisse JR, Westlund KN. Excitatory amino acids, TNF-alpha, and chemokine levels in synovial fluids of patients with active arthropathies. Clin Exp Immunol 2004;137:621-7.
22.	McNearney T, Speegle D, Lawand N, Lisse J, Westlund KN. Excitatory amino acid profiles of synovial fluid from patients with arthritis. J Rheumatol 2000;27:739-45.
23.	Liu PS, Wang H, Li X, Chao T, Teav T, Christen S, et al. α-Ketoglutarate orchestrates macrophage activation through metabolic and epigenetic reprogramming. Nat Immunol 2017;18:985-94.
24.	Wang X, Liu R, Qu X, Yu H, Chu H, Zhang Y, et al. α-ketoglutarate-activated NF-κB signaling promotes compensatory glucose uptake and brain tumor development. Mol Cell 2019;76:148-62.e7.
25.	Long C, Yang Y, Wang Y, Zhang X, Zhang L, Huang S, et al. Role of glutamine-glutamate/GABA cycle and potential target GLUD2 in alleviation of rheumatoid arthritis by Tripterygium hypoglaucum (levl.) Hutch based on metabolomics and molecular pharmacology. J Ethnopharmacol 2021;281:114561.