Network pharmacology identification and in vivo validation of key pharmacological pathways of Qin Jiao for gout and arthritis

Abstract

Context: Gout is a chronic disease that imposes a huge financial and health burden on patients, which might diminish quality of life. Qin Jiao, a perennial herb found in northwestern China and Japan, is commonly used for treating various ailments.

Objective: This study investigates the effects of Qin Jiao on gout and joint inflammation and elucidates its potential mechanism for gouty arthritis.

Materials and methods: Study 1, a literature review was conducted using PubMed, Web of Science, and CNKI to assess the applications of Qin Jiao in arthritis treatment. Study 2 was performed to discover the component targets and gouty disease targets via TCMSP, OMIM, GeneCards and DRUGBANK, and network pharmacology analysis. Study 3, male Sprague-Dawley (SD) rats were divided into normal, model, colchicine, Qin Jiao low-dose (QJL), and Qin Jiao high-dose group (QJH), oral gavage for 40 d. Serum, synovial fluid, and synovial membrane tissue were collected to measure the expression levels of IL-1β, IL-6, and STAT3.

Results: The research also identified potential targets and pharmacological pathways of Qin Jiao for gout treatment. In vivo study demonstrated Qin Jiao can reduce IL-1β levels in serum and ankle flushing fluid. ELISA analysis confirmed that Qin Jiao significantly reduces the protein expression of IL-6 and STAT3.

Discussion and conclusion: Qin Jiao exerts anti-inflammatory effects on gouty arthritis by modulating the IL-6/STAT3 pathway. This study provides a biological basis for the use of Qin Jiao in treating arthritis-related diseases and offers experimental evidence for potential future drug development.

Keywords:

• Joint inflammation
• Chinese herbs
• Kampo medicine
• IL-6/STAT3 pathway
• joint arthritis

Introduction

Gout is the nucleation and aggregation of monosodium urate crystals (MSUCs) in tissues, mostly cartilage, synovial structures, bone, and skin, regardless of the presence or absence of clinical manifestations (Perez-Ruiz et al. 2014). The incidence and prevalence of gout are increasing worldwide. In the United States (USA), gout is the most popular type of inflammatory arthritis, affecting over 8 million Americans (Aslam and Michet 2017). Gout, the highest prevalent inflammatory arthritis, affects 2.5% of the general population in the United Kingdom (UK) (Abhishek et al. 2017). At the same time, an investigation showed the overall prevalence has increased from 1.4% in 1999 to 2.49% in 2012 in the UK (Hui et al. 2017). A cohort study in the USA indicated 73% participants with more than once recurrence of gout in seven years (McAdams et al. 2011). Pre-menopausal women have a lower prevalence of gout compared to men, since female sex hormone might increase urinary urate excretion. Blacks have a higher gout risk than whites (Lee et al. 2009). Clinical manifestations of gout resulting from monosodium urate crystal deposition include tophi, chronic arthritis, urolithiasis, and renal disease as well as recurrent acute arthritis, bursitis, and cellulitis (Hui et al. 2017). In Chinese medicine, gout arthritis is classified Bi syndrome (Arthralgia Syndrome), with the symptoms of sudden, severe attacks of pain, swelling, redness and tenderness in one or more joints.

Qin Jiao (Gentain Root in English), commonly Chinese medicine (known as Qin Jiao 秦艽 in Chinese) and Kampo medicine (じんぎょう in Japanese), is a perennial herb native to the northwestern China. It was first documented in Shennong Bencao Jing, and described as “Qin Jiao is an herb with cool nature, and bitter taste, which used to release either acute or chronic pain, arthritis in cold or hot wind-damp conditions, reverse Yin deficiency”. It was used as a kind of Chinese herb for thousand years. The major biological component named as gentiopicroside accounts, with about 8% content in the dry roots. It is used for the treatment of rheumatism, osteoarthritis, inflammation, or ulceration (Chen and Xu 2007). Qin Jiao is always used to treat Bi syndrome, commonly referred to as rheumatic arthritis, rheumatoid arthritis, muscular spasms, joint pain, stroke, hemiplegia, facial distortion, numbness of limbs, proliferative arthritis, and spinal osteoarthritis, as well (Xu et al. 2022).

The diagnosis of gout mainly includes two parts: clinic diagnosis and laboratory diagnosis. Gout is typically diagnosed clinically based on the rapid development of monoarticular arthritis marked by swelling and redness, usually involving the first metatarsophalangeal joint. Examination of joint fluid by microscopy is also commonly used to diagnose suspected cases of gout. In these situations, the diagnosis is established by aspiration of a joint or tophus and identification of needle-shaped monosodium urate crystals, preferably intracellular, with bright, negative birefringence on compensated polarized light microscopy (Hainer et al. 2014). Joint aspiration may be technically difficult to perform and painful for the patient, particularly in smaller joints (Newberry et al. 2017).

Acute gout may be treated with nonsteroidal anti-inflammatory drugs, corticosteroids, or colchicine. To reduce the likelihood of recurrent flares, reduction of uric acid levels is the key to avoiding gout flares. Allopurinol and febuxostat, the xanthine oxidase inhibitor, are first line medications for the prevention of recurrent gout. The other frequently used drugs are colchicine and probenecid. However, these drugs may cause adverse events. Allopurinol may potentially cause life-threatening adverse reactions, such as toxic epidermal necrolysis, hypersensitivity drug reactions with rash, eosinophilia, and systemic symptoms in addition to, liver and renal toxicity (Hainer et al. 2014).

Medicinal herbs, commonly used in developing countries, are potential inhibitors of xanthine oxidase and inflammatory factors. Herbal drugs are widely used due to their potent efficacy and high safety. The plant species of Gentiana macrophylla Pall, Gentiana straminea Maxim, Gentiana dahurica Fisch, and Gentiana crassicaulis Duthie ex Burk (Gentianaceae) are listed in The Chinese Pharmacopoeia as the sources of Qin Jiao. It is also a common Tibetan medicinal herb used for the treatment of tonsillitis, urticaria, and RA, while the flowers have been used as a Mongolian herb for curing cough and sore throat and eliminating phlegm due to its anti-inflammatory effects (Wang et al. 2013). This article focused on the effects of Qin Jiao on gout and joint inflammation, by conducting a literature review and network pharmacology analysis.

Thus, this study combines scope review of the components and clinical use of Qin Jiao, network pharmacology analysis and animal experimental validation to clarify the potential mechanism of Qin Jiao against gout arthritis. This research suggests that Qin Jiao is a potential therapeutic herb for the treatment of gout arthritis.

Materials and methods

Study 1 scope review of the components and medicinal effects of Qin Jiao

We conducted a systematic search of several public scientific databases, including PubMed, Web of Science, and CNKI, using relevant keywords such as Qin Jiao, Gentiana macrophylla Pall, Gentiana straminea Maxim, Gentiana dahurica Fisch, Gentianacrassicaulis Duthie ex Burk, Gentiana straminea Maxim, iridoid glycosides, gentiopicroside, sweroside, swerooside, 6′-O-β-d-glucosylgentiopicroside and various types of arthritis such as gout, osteoarthritis, acute gouty arthritis, collagen-induced arthritis, adjuvant arthritis, and rheumatoid arthritis. The retrieved literature was divided into three parts: the effects of Qin Jiao on gouty arthritis, the effects of Qin Jiao on other types of arthritis, and the other effects of Qin Jiao and its active components.

Study 2 network pharmacology analysis of Qin Jiao for the treatment of gout

Collection of Qin Jiao component targets and disease targets

The chemical compounds and corresponding targets of the Qin Jiao were found by using Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) and imported into the Excel sheet. Then, the Simplified Molecular Input Line Entry System (SMILES) formula of the compound is input into the Swiss Target Prediction online platform, and the target is predicted based on the structural similarity of the compound, and the predicted target information with Probability > 0 in the prediction result is selected. Targets obtained from the above two databases were deduplicated and merged, and the gene names were standardized in the UniProt database, and all targets were set to human origin.

The key word "gout arthritis" was searched in the human Mendelian gene database OMIM (https://omim.org/), GeneCards (https://www.genecards.org/) and DRUGBANK (https://go.drugbank.com/) for their related gene targets respectively. Then the duplicate targets were deleted. The screened component targets and disease targets were overlapped with Venn diagram to obtain the potential targets of Qin Jiao in the treatment of gouty arthritis. And then the "component-target" action network diagram was drawn.

Protein-Protein Interaction Networks

The functional protein association networks (string-db.org) were utilized for network topology analysis. The identified potential targets were input into the string database, employing the "multiple proteins" tool while restricting the species to "Homo sapiens". Isolated nodes within the network were concealed, ensuring a confidence interval greater than 0.4. Consequently, a protein-protein interaction network (PPI) was acquired. The PPI network data was imported into Cytoscape 3.9.0 to construct the target-protein interaction network of Qin Jiao in the treatment of gouty arthritis, and the important targets were screened according to Degree Centrality.

Biological pathway enrichment analysis

The DAVID Bioinformatics Resources (https://david.ncifcrf.gov/) were utilized to perform Gene Ontology (GO) biological enrichment and Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway enrichment analyses (Huang da et al. 2009). GO biological enrichment was conducted for Molecular Function, Biological Process, and Cellular Component, while KEGG pathway enrichment analysis was performed for various cellular biochemical pathways, to provide a comprehensive analysis of the metabolic pathways, biochemical processes, and biological functions of gene products in cells. The species category was set to "human" while all other parameter options were kept as default values. The results were visualized in the form of a bubble chart to show the degree of enrichment in the signalling pathway.

Study 3 experimental validation

Medication and preparation of the extract

Qin Jiao, Beijing Bencao Fangyuan Pharmaceutical Co., Ltd. lot # 20210415. Uric acid sodium salt, manufactured by Sigma (Switzerland) lot # BCCF5862. Colchicine tablets, produced by KPC Pharmaceuticals, Inc. Specifications: 0.5 mg × 100 tablets; Cat #210103-02, shielded and sealed, valid for 24 months. IL-1β Elisa kit, Jiang Lai Biological Co. Ltd. lot # 031706HLP200840324.IL-6 Elisa kit, Jiangsu Mei mian Industrial Co. Ltd. (Jiang su, China). Lot # MM-0190R1.STAT3 Elisa kit: From Jiangsu Mei mian Industrial Co. Ltd. (Jiang su, China). Lot # MM-70093R1. d-Fructose, VWR life science. Lot # 21H1356337.

Qin Jiao decoction was prepared by the following procedure. Qin Jiao (100 g) was soaked in 10 times amount of water and decocted twice for 1.5 h. Then the decoction was concentrated by a rotary evaporator under 80 °C, in the concentration of 0.5 mg/mL.

Grouping and feeding of animals

A total of 60 SPF-grade Sprague Dawley (SD) male rats (180 ± 10 g) were purchased from Beijing SPefu Laboratory Animal Centre [Animal qualification licence No.: SCXK(Beijing)-2019-0010] and raised in Beijing Standard Qualified Animal house with air-conditioned. [Licence No.: SYXK(Beijing)2011-0024]. The temperature of animal house was controlled at 25 ± 2 °C, 40%-60% humidity and 12 h light/dark cycle. The experimental protocol and ethical authorizing were approved by Beijing University of Chinese Medicine [Approval No.: BUCM-4-2021100803-4004].

After 3 days of adaptive feeding (average weighing more than 200 g), the animals were randomly divided into normal, model, colchicine (0.8 mg/kg/d), QJL (0.5 g/kg/d) and QJH (1 g/kg/d) groups (n = 12) (Xiangxiang et al. 2015). At the end of this experiment, all the rats were sacrificed with pentobarbital.

Modelling and drug administration

In the experiment, 10% D-Fructose water was used to establish the hyperuricaemia model of rats (Yu et al. 2016). Using an injection needle, 200 μL of a 25 mg/mL suspension of sodium urate was injected into the tibiotalar joint space of the right hind paw of rats, with the distension of the joint capsule on the opposite side used as a standard for injection success. Physiological saline solution (0.2 mL) was substituted for the normal control group (Coderre and Wall 1987). The peri-diameter of the ankle joint of rats was measured at 4, 8, 12, 24, and 48 h of the experiment, respectively. The degree of joint swelling was calculated = (after modelling - before modelling)/before modelling ×100%.

The colchicine group was given 0.8 mg/kg/d colchicine tablet aqueous solution, while the QJL and QJH groups were given 0.5 g/kg/d and 1 g/kg/d Qin Jiao respectively. The control group and the model group were given the same volume of water. The researchers were aware of the group allocation, but the researchers who detected the biochemistry were not aware of the samples collected from which group.

Measurement of IL-1β in ankle joint washing fluid and serum

The synovium of the ankle joint was cut and rinsed with normal saline, and the ankle joint flushing fluid was collected, after 48 h modelling. Blood was collected from the abdominal aorta, left for 2 h, centrifuged at 3500 rpm for 10 min at room temperature, and detected of IL-1β levels.

Enzyme-linked immunosorbent assay (ELISA) for key proteins detection

The ELISA kits were purchased to detect STAT3, IL-6 in the serum and synovial tissue of joints. All assays were performed according to the manufacturer’s instructions.

The data processing

SPSS 20.0 statistical software was used for data analysis. One-way analysis of variance was performed among different groups to compare the differences between groups, with normal data distribution, equal collection, and independence test. Bonferroni method was used for pairwise comparison under the condition of homogeneity of variance. Dunnett’s T 3 method was used for pairwise comparison under the condition of unequal variances in each group, p ≤ 0.05 was considered as significant difference, p ≤ 0.01 was considered as a very significant difference.

Results

Study 1

The effects of Qin Jiao on gout

The use of traditional medicine, such as Chinese herbs or Kampo medicines, is a popular worldwide approach for treating gout and arthritis due to their effectiveness and affordability. Clinical and experimental studies have explored the potential of Qin Jiao and its extract sweroside, iridoid glycoside, in gout treatment. A study on Qin Geng Li San Oral Liquid, containing Qin Jiao, demonstrated significant reduction in glacial acid-induced body twisting frequency and egg-white-induced footpad swelling, highlighting Qin Jiao’s potential efficacy in gout therapy (Guo et al. 2018). Another study observed that Gentiana root ethanol extraction (1.0 g/mg) inhibited ankle swelling in rats induced by injecting sodium urate into the right joint cavity and downregulated the levels of TNF-a, IL-1β, IL-6, PGE2, and MMP-3 in serum (Xiangxiang et al. 2015). Clinical studies have also shown significant relief of gouty arthritis symptoms in patients after taking preparations containing Qin Jiao (Dahui and Ji 2013; Youhan et al. 2013).

A systematic review of randomized clinical trials found that some Chinese herbal medicines, such as Tongfengting capsule containing Qin Jiao, can improve functional recovery for gouty patients, and with good safety propriety (Li et al. 2013). These findings were also supported by other report (Kodithuwakku et al. 2013).

The effects of Qin Jiao on other arthritis

There are some studies showing that Qin Jiao can inhibit other arthritis, such as rheumatoid arthritis (RA), osteoarthritis (OA), collagen-induced rheumatoid arthritis (CIA), osteoporosis, adjuvant arthritis and so on.

Rheumatoid arthritis (RA)

Rheumatoid arthritis (RA) is a chronic, systemic, autoimmune, and inflammatory disease that attacks the joints, but also the skin, lungs, and muscles of human beings. RA is a major cause of disability and affects up to 0.5%–1.0% of the adult population worldwide (Choy and Panayi 2001). There are some reports about the effects of Qin Jiao or iridoid glycosides on RA. A study has reported that the component called iridoid glycosides separated by from Qin Jiao showed protective effects against RA. They found iridoid glycosides as major constituents, showing potential COXs-2/1 inhibitory activity, demonstrating that Qin Jiao fighting RA should be due to the inhibition on the production of COXs-2/1 (Wang et al. 2013). Another study treated the rats with iridoid glycosides orally at a daily dose of 100 mg/kg body weight, founding the prostaglandin E₂ (PEG2) levels in the inflammatory tissues, sole thickness, and ankle circumference of the feet were significantly decreased (Yu et al. 2004). In vitro study indicated abnormal proliferation, the NO content increase, and the downregulated Sirtuin (SIRT1) expression In RAW264.7 cells induced by lipopolysaccharide (LPS) (Wang et al. 2019).

Osteoarthritis (OA)

Osteoarthritis (OA) is a progressive degeneration of joint tissues, loss of articular cartilage, and inflammation of the synovium (Pelletier et al. 2001). It is widely accepted that Interleukin-1β (IL-1β) exerts a vital role in the pathogenesis of OA (Feng et al. 2017). A study demonstrated that sweroside attenuates inflammation by IL-1β, including inhibiting NO and PEG₂ production, as well as iNOS and COX-2 expression in the protein level, MMP-1, MMP-3, MMP-3, and ADAMTS-5 at the mRNA level in rat articular chondrocytes (Zhang et al. 2019). These results shown a promising drug target for OA therapy. Another study conclude that human cartilage homogenates increase MSU crystal formation and promote the formation of smaller crystal which has greater inflammatory potential (Chhana et al. 2019). The above result indicated the process may contribute to the predilection of gout to osteoarthritic joints, showing the potential relationship between gout and OA.

Collagen-induced rheumatoid arthritis (CIA)

Iridoid glycosides, the activating component extracted from Qin Jiao, can inhibit the severity of paw edoema, arthritis scores, and index of spleen and thymus in the complete Bovine CII-induced Collagen-induced rheumatoid arthritis (CIA) model from day 7 to 21 after CIA. This study also demonstrated that the component iridoid glycosides can inhibit proinflammatory cytokines, such as TNF-α, IL-1β, and IL-6, and regulate the expression of iNOS and COX-2 (Jia et al. 2016).

Osteoporosis

Sweroside significantly increased the proliferation of human MG-63 cells and rat osteoblasts (p ≤ 0.01), increased the activity of ALP and the response between osteocalcin and sweroside (p ≤ 0.05), using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) test (Sun et al. 2013). This study also showed that sweroside can attenuate and inhibit apoptosis by flow cytometer assay (Sun et al. 2013).

Adjuvant arthritis

Bone erosion is a central feature of rheumatoid arthritis. A study reported that swertiamarin regulates the expression of RANKL/RANK/OPG to control the release of pro-inflammatory and pro-angiogenic markers (Hairul-Islam et al. 2017). This suggests that swertiamarin has anti-osteoclastogenic activity and has a role in preventing bone erosion (Hairul-Islam et al. 2017). Another study indicated swertiamarin can significantly inhibit inflammation by controlling lysosomal enzymes, free radicals, bone destructive enzymes, and reliving paw thickness of arthritic animals (Saravanan et al. 2014).

The other effects of Qin Jiao and its active components

A study indicated that Gentiana root significantly reduced B19-NS1-exacerbated liver injury. The mechanism is that Gentiana root inhibits lymphocyte infiltration by downregulating COX-2, IL-1β, nitric oxide synthase protein, serum aspartate transaminase (AST) and alanine transaminase (ALT), and decreasing lymphocyte infiltration (Sheu et al. 2017). Some studies indicated that Qin Jiao can increase the cardiac insulin-like growth factor (IGF)-1 survival signalling and antiapoptotic proteins in LV tissues significantly (Huang et al. 2015).

Sweroside, isolated and identified from many plants that belong to Gentianaceae family and Gentiana genus, is a typical iridoid that exhibits some biological activities, such as hepatoprotective, antidiabetic and anti-inflammatory effects. Gentiopicroside has protective effects on ethanol-induced gastric mucosal injury in mice through the improvements of antioxidative and anti-inflammatory effects, as well as up-regulation of heat shock protein-70 level and normalisation of epidermal growth factor and vascular endothelial growth factor levels (Yang et al. 2018). Another study showed that sweroside has the potential to be an effective agent against human leukaemia (Han et al. 2017). According to Han et al. (2017) study, the treatment of sweroside can induce S and G2/M cell cycle arrest, which is associated with the downregulation of cyclin D1, cyclin-dependent kinase 4 (CDK4), CDC2 and CDC25, as well as the upregulation of p53 and p21 (Han et al. 2017). Sweroside has the effect on α-naphthylisothiocyanate (ANIT)-induced cholesteric liver injury in mice as evidenced by significantly reduced serum biochemical and attenuated pathological changes in liver tissues as well (Yang et al. 2016).

Swertiamarin is a main active component of Qin Jiao. This iridoid glycoside has anti-nociceptive, anti-edematogenic, anti-cholinergic and COX-2, TNF inhibiting (Hairul-Islam et al. 2017). A report demonstrated Qin Jiao having a potential protective effect against stroke, and showed that swertiamarin pre-treatment markedly decreased infarct volume, apoptotic neurons, and oxidative damage and promoted neurologic recovery in vivo and that swertiamarin decreased reactive oxygen species (ROS) and increased cell viability in vitro (Han et al. 2015). The pre-treatment with swertiamarin (150,450 mg/kg) and valproate sodium (200 mg/kg) delayed the onset of the first convulsion and reduced the incidence of status epilepticus and mortality (Deng et al. 2017). Moreover, the literature indicated swertiamarin has pharmacological effects of antidiabetic, anti-hyperlipidemia, and anti-adipogenic (Vaidya et al. 2014; Patel et al. 2018).

Study 2

Chemical components in Qin Jiao

The components of Qin Jiao mainly include iridoid glycosides, triterpenes, flavones, sterols, benzene derivatives and so on. The pharmacological studies demonstrate that Qin Jiao plants display a wide range of bioactivities, e.g., anti-inflammatory, antioxidant, hepato-protective, cardio- and neuro-protective, insecticidal, and anti-influenza effects (Zhang et al. 2018).

Collection of Qin Jiao component targets and gout disease targets

A total of 27 components of Qin Jiao were obtained by searching Qin Jiao in TCMSP, listed in Table 1. The potential targets of action of 27 chemical components were predicted in Swiss target prediction database by using SMILES formula. a total of 641 targets were obtained, and 222 targets were obtained by removing duplicates.

Table 1. Information table of chemical constituents in Qin Jiao.

MOL ID	MOLECULE NAME	Molecular Weight	Oral Bioavailability
MOL000263	Oleanolic acid	456.78	29.02
MOL000357	Sitogluside	576.95	20.63
MOL000358	beta-Sitosterol	414.79	36.91
MOL000359	Sitosterol	414.79	36.91
MOL000520	alpha-Amyrin	426.8	10.28
MOL000636	Gentiopicroside	368.37	5.9
MOL000637	Loganic, acid	312.4	6.7
MOL000638	Macrophylloside A	874.87	3.13
MOL000639	Macrophylloside A_qt	712.71	2.9
MOL000640	Macrophylloside B	890.87	3.04
MOL000641	Macrophylloside B_qt	728.71	3.2
MOL000642	Roburic acid	428.77	9.26
MOL000644	Swertiamarin_qt	212.22	2.58
MOL000645	Gentianidine	163.19	54.45
MOL000646	Gentiopicrin	356.36	22.98
MOL000647	Scabrans G5_qt	194.2	66.67
MOL000648	Gentianine	175.2	54.67
MOL000649	Gentiatibetine	165.21	62.59
MOL000650	Sweroside	358.38	4.96
MOL000651	Sweroside aglycone	196.22	68.68
MOL000652	Venoterpine	149.21	68.97
MOL000653	Gentianal	193.22	73.34
MOL000654	Methyl montanate	438.87	13.2
MOL000655	Loganic acid	376.4	4.92
MOL000656	Loganic acid_qt	214.24	114.65
MOL000659	Montanic acid	424.84	13.69
MOL003166	Swertiamarin	374.38	21.9

The relevant gene targets were searched in three disease target databases, OMIM, Genecards and DRUGBANK by using "gouty arthritis" as the keyword. The search results were then normalized and combined to obtain 1114 disease targets for gouty arthritis. Finally, the disease targets and the components were imported into venny2.1.0 (https://bioinfogp.cnb.csic.es/tools/venny/index.html), and 76 potential action targets of Gentiana macrophylla for the treatment of gouty arthritis were obtained (Figures 1 and 2).

Figure 1. Venn diagram of Qin Jiao compound-disease intersection gene.

Figure 2. The network diagram of the components and targets of Qin Jiao.

Construction of protein-protein Interaction Networks, and the anti-gouty arthritis targets of Qin Jiao

The 76 potential targets found above were imported into the string, and the confidence level of 0.400 was selected to obtain the PPI network graph. The result contained 76 genes, 419 connections, and the average node degree value was 0.558. Use Cytoscape to calculate the Degree Centrality of each node, and the top 10 are used as the core target genes. In descending order, they are glyceraldehyde-3-phosphate dehydrogenase (GAPDH), peroxisome proliferator-activated receptor gamma (PPARG), mitogen-activated protein kinase 3(MAPK3), epidermal growth factor receptor (EGFR), prostaglandin G/H synthase 2(PTGS2), interleukin-2(IL2), signal transducer and activator of transcription 3(STAT3), peroxisome proliferator-activated receptor alpha (PPARA), receptor-type tyrosine-protein phosphatase C(PTPRC), receptor tyrosine-protein kinase erbB-2(ERBB2) (Figure 3 and Table 2).

Figure 3. PPI network diagram of the anti-gouty arthritis target of Qin Jiao.

Table 2. Topological parameters of Qin Jiao anti-gouty arthritis.

Gene	Protein	Degree Centrality
GAPDH	Glyceraldehyde-3-phosphate dehydrogenase	84.0
PPARG	Peroxisome proliferator-activated receptor gamma	74.0
MAPK3	Mitogen-activated protein kinase 3	66.0
EGFR	Epidermal growth factor receptor	64.0
PTGS2	Prostaglandin G/H synthase 2	60.0
IL2	Interleukin-2	58.0
STAT3	Signal transducer and activator of transcription 3	54.0
PPARA	Peroxisome proliferator-activated receptor alpha	50.0
PTPRC	Receptor-type tyrosine-protein phosphatase C	46.0
ERBB2	Receptor tyrosine-protein kinase erbB-2	42.0

Gene ontology biological enrichment analysis and genome encyclopaedia pathway enrichment analysis

Using the DAVID platform to analyze the GO biological function process of the potential targets of Qin Jiao in the treatment of gouty arthritis. Firstly, there are 250 potential targets of Qin Jiao in the treatment of gouty arthritis in the biological process. It involves intracellular receptor signalling pathway, negative regulation of inflammatory response, and inflammatory response. Secondly, there are 34 entries related to cellular composition, mainly involving the cytosol, cytoplasm, extracellular exosomes, and intracellular membrane-bounded organelles. Thirdly, in terms of molecular function, it involves a total of 68 entries. For example, enzyme binding, identity protein binding, and steroid binding.

Using the KEGG pathway enrichment analysis function of the DAVID platform, 76 potential targets of Qin Jiao in the treatment of gouty arthritis were analyzed, and a total of 85 signalling pathways were obtained. The top ten signal transduction pathways are listed in the figure, such as PPAR signalling pathway, T cell receptor signalling pathway, and HIF-1 signalling pathway: it also including arachidonic acid metabolism, NF-kappa B signalling pathway, TNF signalling pathway, and IL-17 signalling pathway. These pathways are all related to inflammation, which suggests that the treatment of gouty arthritis by Qin Jiao may be related to the regulation of inflammatory by its components (Figures 4 and 5).

Figure 4. Gene ontology enrichment analysis of the anti-gouty arthritis targets of Qin Jiao.

Figure 5. KEGG enrichment analysis of the anti-gouty arthritis target of Qin Jiao.

Study 3

The ankle swelling degree in model rats

Compared with the control group, the degree of joint swelling in the model group was significantly increased at 4, 8, 12, 24, and 48 h after modelling (p ≤ 0.05 or p ≤ 0.01). Compared with the model group, there was a difference in joint swelling at 12h in the colchicine group, and there was a decreasing trend in joint swelling in the QJL group (p = 0.093). At 24h after modelling, there was a difference or significant difference in joint swelling in colchicine, QJL and QJH groups. At 48h after modeling there was a significant difference in joint swelling between the colchicine group and the QJL group, and there was a tendency for joint swelling to decrease in the QJH group (p = 0.063) (Table 3).

Table 3. Joint swelling degree of rats in each group at each time point.

	4 h	8 h	12 h	24 h	48 h
Control	3.72 ± 1.75	5.43 ± 3.11	3.58 ± 1.59	2.67 ± 1.16	2.56 ± 0.83
Model	23.27 ± 10.93**	36.94 ± 14.43**	47.44 ± 10.71**	48.59 ± 8.78**	36.26 ± 20.55**
Colchicine	19.29 ± 7.32	32.16 ± 11.28	34.37 ± 10.99^#	30.52 ± 10.69^##	15.91 ± 8.41^##
QJL	22.81 ± 12.04	31.60 ± 14.95	38.42 ± 17.83	34.94 ± 13.54^##	21.40 ± 12.76^##
QJH	27.07 ± 5.70	42.38 ± 9.49	45.67 ± 10.22	36.56 ± 6.22^#	25.52 ± 4.16

All data are presented as mean ± SD (n = 10), *p < 0.05, **p < 0.01, versus the control group. ^#p < 0.05, ^##p < 0.01, versus the model group.

The changes of serum IL-1β and ankle washing fluid in rats

Compared with the control group, the serum IL-1β in the model group was significantly increased (p ≤ 0.05 or p ≤ 0.01). Compared with the model group, serum IL-1β of rats in the colchicine group, QJL and QJH groups was significantly decreased (p ≤ 0.05 or p ≤ 0.01).

Compared with the control group, the IL-1β in the joint washing fluid of the model group had a tendency to increase (p = 0.119). Compared with the model group, the IL-1β of the joint washing fluid in the QJL group was significantly decreased (p ≤ 0.05). There was a decreasing trend of IL-1β in the joint washing fluid in colchicine group and QJH group (p = 0.072, p = 0.097) (Figure 6 and Table 4).

Figure 6. The changes of IL-1β in mouse model after treatment.

Table 4. Changes of IL-1β levels in serum and washing fluid of rats in each group.

	Serum IL-1β(pg/mL)	Washing fluid IL-1β(pg/mL)
Control	22.74 ± 5.88	86.54 ± 10.20
Model	37.41 ± 10.79**	107.46 ± 72.20
Colchicine	13.83 ± 4.52^##	83.73 ± 4.05
QJL	7.61 ± 2.40^##	78.88 ± 3.48^#
QJH	9.43 ± 3.71^##	84.01 ± 6.28

All data are presented as mean ± SD (n = 10), *p < 0.05, **p < 0.01, versus the control group; ^#p < 0.05, ^##p < 0.01, versus the model group.

ELISA validation of biomarkers of Qin Jiao for gouty arthritis

Compared with the control group, the serum and synovial tissue of joints IL-6 in the model group was significantly increased (p ≤ 0.05 or p ≤ 0.01). Compared with the model group, serum and Synovial tissue of joints IL-6 of rats in the colchicine group, QJL and QJH groups was significantly decreased (p ≤ 0.05 or p ≤ 0.01).

Compared with the control group, the serum and Synovial tissue of joints STAT3 in the model group was significantly increased (p ≤ 0.05 or p ≤ 0.01). Compared with the model group, serum and Synovial tissue of joints STAT3 of rats in the colchicine group, QJL and QJH groups was significantly decreased (p ≤ 0.05 or p ≤ 0.01) (Figure 7 and Table 5).

Figure 7. Changes of IL-6 and STAT3 levels in mouse model after treatment.

Table 5. Changes of IL-6 and STAT3 levels in serum and synovial tissue of joints of rats in each group.

	Serum IL-6(pg/mL)	Synovial tissue of joints IL-6(pg/mL)	Serum STAT3(pg/mL)	Synovial tissue of joints STAT3(pg/mL)
Control	139.48 + 5.84	119.55 + 7.99	86.29 + 4.36	81.33 + 4.91
Model	232.39 + 8.16**	136.56 + 6.47**	117.79 + 4.368**	97.39 + 4.60**
Colchicine	158.35 + 8.34^##	121.22 + 6.64^##	93.36 + 4.84^##	82.60 + 5.00^##
QJL	181.39 + 8.20^##	125.91 + 6.35^##	105.54 + 4.66^##	88.15 + 4.29^##
QJH	187.72 + 8.02^##	128.21 + 6.05^##	103.84 + 4.29^##	92.23 + 4.11^#

All data are presented as mean ± SD (n = 10), *p < 0.05, **p < 0.01, versus the control group; ^#p < 0.05, ^##p < 0.01, versus the model group.

Discussion

Bi syndrome is caused by obstruction of the meridians of the limbs, and the clinical manifestations are mainly pain, soreness, numbness, stress, unfavourable flexion, and extension, or swelling and deformation of joints in muscles, joints, tendons, and bone (Xu et al. 2022). Qin Jiao has the properties of pungent, bitter and slightly cold, with indications of rheumatism, soothes tendons and collaterals, clears heat and dampness, and removes jaundice. It is commonly used in the treatment of rheumatoid arthralgia, such as osteoarthritis, gout, rheumatoid arthritis and various types of arthritis and inflammatory diseases. The Pharmacopoeia of the People’s Republic of China (2020) mentions that it can also be used to treat hand and foot insufficiency, hot flashes due to steaming of the bones, chance accumulation fever, damp-heat jaundice and so on.

Modern chemical and pharmacological studies have found that Qin Jiao has anti-inflammatory and analgesic, hepatoprotective, antiviral, and immunosuppressive effects. And the gentiopicroside and stelepicroside contained in Qin Jiao are the main components that exert anti-inflammatory activity. Gentiopicroside can reduce xylene-induced ear swelling in mice; carrageenan and zymosan A-induced paw swelling in rats may involve the inhibition of various inflammatory mediators. Administration of ropicroside to rats can improve the symptoms of adjuvant arthritis rats, and the mechanism of action may be related to the decreased release of interleukin-1β and the accelerated release of IL-10, IL-4, and other anti-inflammatory factors (Yu et al. 2004). In addition, studies have shown that the alcohol extract of P. chinensis can also reduce the levels of Th1 cytokine interferon gamma (INF-γ), anti-cyclic citrullinated peptide antibody and tumour necrosis factor-α (TNF-α) in serum (Jia et al. 2015).

In this paper, the databases were searched to obtain the action targets of the chemical constituents of Qin Jiao, as well as the disease targets of gouty arthritis. Take the intersection of the two to obtain potential targets. Key targets such as PPARG, MAPK, STAT and so on. were obtained by computer stimulation, and key pathways such as PPAR signalling pathway, T cell receptor signalling pathway, and HIF-1 signalling pathway were enriched; the above-obtained targets and enriched pathways were related to associated with inflammatory responses. The results of the Elisa test indicate that Qin Jiao can exert its anti-gouty arthritis effects by influencing the IL-6/STAT3 signalling pathway, which is consistent with the anti-inflammatory activity of Qin Jiao in modern pharmacological research. Qin Jiao has both traditional Chinese medicine theoretical support and modern pharmacological research, which makes the application of Qin Jiao more reasonable and credible and provides a basis for subsequent research on Qin Jiao.

Gout, joint inflammation, and their consequences are progressively increasing in the population. Although so many listed drugs, such as Febuxostat, Allopurinol, and Colchicine have an effect against these diseases, there are still many unexpected side effects. Therefore, new interventions and treatments are necessary (Hainer et al. 2014). Herbal medicine can be a useful approach to preventing gout or improving joint inflammation. In this work, Qin Jiao and its main active ingredients, iridoid glycosides, gentiopicroside showed positive effects in the prevention and (or) curing gout and joint inflammation, through various therapeutic mechanisms such as anti-inflammatory and anti- xanthine oxidase effects (Fei-Xia et al. 2020). Qin Jiao is a promising plant in this regard. The evidence from experimental data and clinical trials about its effects on these situations are rarely documented. It is more activities that need to be required.

Conclusions

Qin Jiao and its active ingredients have been shown to have beneficial effects in the prevention and treatment of gout and joint inflammation. These effects are attributed to Qin Jiao’s anti-inflammatory and analgesic properties, as well as its hepatoprotective, antiviral, and immunosuppressive effects. The mechanism underlying its anti-gouty arthritis effects may involve the regulation of inflammatory pathways. Literature reviews and network pharmacology analyses have highlighted gentiopicroside and stelepicroside, two components of Qin Jiao, as the main contributors to its anti-inflammatory activity. These findings offer potential for the development of new anti-gout agents using Qin Jiao, which could help alleviate the burden on patients suffering from gout and joint inflammation. The research model of this paper ranges from traditional literature mining to computer simulation analysis and combined with biological experimental validation to form a more complete chain of evidence system, which is useful for the research and development of traditional herbal medicine.

Authors’ contributions

Conception and design: Xiaoxiong Yang, Bing Zhang and Zhijian Lin. Conduct and assembly of data: Xiaoxiong Yang, Yu Wang. Data analyses and interpretation: Shanshan Ju, Xueli Ding and Xiaoye An. Manuscript writing and revision: all authors. Final approval of manuscript: all authors

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work was supported by the Beijing Municipal Natural Science Foundation (7212178), the China Postdoctoral Science Foundation (2021M690475), and the Innovation Team and Talents Cultivation Program of National Administration of Traditional Chinese Medicine (ZYYCXTD-C-2020005-11).