Study on the mechanism of action of Wu Mei Pill in inhibiting rheumatoid arthritis through TLR4-NF-κB pathway

doi:10.21203/rs.3.rs-3639338/v1

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Research Article

Study on the mechanism of action of Wu Mei Pill in inhibiting rheumatoid arthritis through TLR4-NF-κB pathway

https://doi.org/10.21203/rs.3.rs-3639338/v1

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Journal Publication

published 13 Jan, 2024

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Background

Wu Mei Pills (WMP) is a traditional Chinese medication that exhibits considerable anti-inflammatory effects. While WMP has been documented for its efficacy in treating RA, its mechanism of action on the condition remains unestablished.

Methods

The chemical composition of WMP was analysed through UPLC-MS. Next, the ELISA, cell scratch, Transwell, and Western blotting techniques were used to investigate its intrinsic mechanism. Lastly, the effect of WMP in inhibiting RA was explored by applying it to CIA rats.

Result

UPLC-MS analysis detected 181 compounds in WMP. RA-FLS migration and invasion mechanisms were significantly hindered by serum containing WMP (2%, 8%). Moreover, WMP (0.5 g/kg, 2 kg/kg) restricted arthritis and immune organ indices in CIA rats with type II collagen-induced rheumatoid arthritis by blocking TLR4-NF-κB inflammatory pathway activation.

Conclusions

WMP is valuable in mitigating the course of RA through inhibiting the classical TLR4-NF-κB inflammatory pathway and reducing the secretion of inflammatory factors in the serum of RA-FLS and CIA rats. Moreover, it regulates the dynamic balance of MMP-2/TIMP-2, MMP-9/TIMP-1, modulates the mechanism of RA-FLS invasion, and safeguards articular cartilage tissues in RA.

Wu Mei pill

Rheumatoid arthritis

Fibroblastic synovial cells

Invasion and migration

TLR4-NF-κB pathway

Rheumatoid arthritis (RA) is a condition resulting from autoimmune over-activation, featuring joint inflammation, synovial tissue proliferation, and angiogenesis. Prolonged RA can cause disabilities and negatively impact an individual's life quality ¹. Approximately 1% of the world's population is affected by RA according to statistics. The pathogenesis is complex and impacted by genetic, environmental factors, and various other influences. Furthermore, research relating to the pathogenesis of RA is still in its exploratory phase ².

Research has identified pattern recognition receptors (PRRs) as a significant contributor to the pathogenesis of RA. The Toll-like receptor (TLR) family comprises the key molecule in pattern recognition receptors (PRRs) and is able to present antigens to effector cells, thereby activating them ³. As researchers delve deeper into this area, they have identified around 13 receptors within the Toll family ⁴. TLRs are protein structures that link the inner and outer membranes of a cell, regulating non-specific immunity. Research has demonstrated that when lipopolysaccharide (LPS) binds to TLR4, it leads to an increase in its expression. This, in turn, causes abnormalities in the expression of associated proteins downstream, which triggers a cascade of inflammatory pathways. The resultant effect can worsen joint inflammation in patients ^5,6. Activation of TLR4 further enhances the activation of nuclear factor-κB (NF-κB). The NF-κB pathway plays an equally influential role in promoting the development of RA. Elevated NF-κB protein expression was found in the synovium of RA, which enters the nucleus and contributes to the transcription and secretion of relevant inflammatory factors ⁷. The aforementioned inflammatory factors exacerbate inflammation and joint damage, thereby perpetuating the progression of RA ⁸. Additionally, an excessive TLRs response induces the uncontrolled release of matrix metalloproteinases (MMPs) ⁹. Matrix metalloproteinases are considered to play a significant role in the breakdown of articular cartilage within affected joints¹⁰. Overexpression of matrix metalloproteinases can lead to RA joint surface and cartilage erosion^11,12. Thus, matrix metalloproteinase imbalance may be an important factor contributing to joint destruction in RA.

According to TCM, rheumatoid arthritis is a pathological condition arising from the invasion of external factors, including wind, dampness, cold and other unfavorable environmental factors. Throughout the long history of Chinese medicine’s treatment of rheumatoid arthritis, traditional Chinese medicine has widely used herbs, whether in compound formulas or single-flavored preparations, that act to remove wind, cold and dampness from the body. Several studies have shown that when DMARDs are used in conjunction with Chinese herbal medicines, the drugs are significantly more effective in relieving RA symptoms and reducing associated side effects. In recent years, therapeutic drugs targeting immune cells and related cytokines (e.g. TNF-α, IL-6, etc.) have also achieved excellent clinical efficacy ^13,14. Although there have been advancements in treating RA, some patients experience swift joint destruction while under treatment. However, the clinical options are also limited due to drugs' side effects, such as liver and kidney damage, immunosuppression, and the high cost of biologics. As a result, the quest for new anti-rheumatoid arthritis drugs from traditional Chinese medicine could become a new approach ¹⁵.

Wu Mei Pill is a traditional Chinese compound medicine dating back to the Han Dynasty. The medicine is composed mainly of 16 traditional Chinese herbs, such as dark plum, Chinese prickly ash, Asarum and Coptic chinensis. Previous studies have shown that WMP is able to regulate the body's immunity and has an inhibitory effect on inflammation ^16,17. Relevant research has demonstrated that WMP effectively reduces serum levels of inflammatory factors and inhibits the overactivation of the TLR4-MyD88-NF-κB inflammatory pathway in mice exhibiting intestinal mucositis. Moreover, WMP has been shown to restore balance to intestinal flora and alleviate intestinal mucosal inflammation in mice ¹⁸. Additionally, WMP hinders macrophage activation through its impact on the Notch/NF-κB/NLRP3 pathway. It also has an inhibitory effect on immune overactivation and anti-inflammatory bursts within the body ¹⁹. In summary, scientific studies confirm that WMP exhibits substantial anti-inflammatory properties and shown to regulate key proteins related to inflammation, such as TLR4 and NF-κB, which are closely associated with RA pathogenesis. According to the traditional Chinese medicine theory, WMP is primarily administered to address various ailments triggered by cold. RA is classified as a Bi syndrome caused by cold, wind, and dampness, and falls under the therapeutic domain of WMP in ancient Chinese medicine. Therefore, this experiment mainly explored the specific mechanism played by WMP in alleviating the course of RA.

2.1 Identification of the chemical composition of WMP

A total of 181 compounds were detected in the WMP in both positive (Fig. 1A) and negative (Fig. 1B) ionization modes. The supplement Table 1 lists the compounds contained in each herb. Wumeirou comprises of 2 compounds, Xixin comprises of 2 compounds, Huanglian comprises of 3 compounds, Huangbai comprises of 1 compound, Ganjiang comprises of 51 compounds, Heishunpian comprises of 4 compounds, Guizhi comprises of 2 compounds, Renshen comprises of 6 compounds, Danggui comprises of 29 compounds, Zhike comprises of 4 compounds, Jiegeng comprises of 1 compound, Baishao comprises of 16 compounds, and Zhigancao comprises of 74 compounds. The entire list of 181 compounds can be found as supplementary Table S1 online. Furthermore, we have identified every herb based on its markers to verify the precision of the herbs employed. The fingerprints for specific experiments can be found as supplementary Fig. S2 online.

2.2 Effect of WMP on the Activity and Inflammatory Factors of RA-FLS Cells

The dose-dependent impact of WMP on RA-FLS activity can be seen in Fig. 2A. Our research objectives were twofold: to investigate the mechanisms behind WMP's anti-inflammatory properties and its effects on RA-FLS migration and invasion. CCK-8 tests showed no significant effect on the viability of RA-FLS when treated with 2% or 8% WMP, or 100nM MTX. Therefore, these experimental doses were used for subsequent migration and invasion assay experiments, as well as for studying mechanisms related to anti-inflammatory activity.

Based on the ELISA results, it was found that LPS had a significant impact on increasing the levels of TNF-α and IL-6 in RA-FLS cells (p < 0.01). This suggests that RA-FLS cells remained activated in an inflammatory environment. Conversely, Fig. 2B and C showed that WMP significantly inhibited the secretion of both TNF-α (p < 0.001) and IL-6 (p < 0.001). This implies that WMP has a significant anti-RA inflammatory effect.

2.3 Effect of WMP on RA-FLS Migration and Invasion

In the wound healing assay, Fig. 2D and F show that RA-FLS exhibited considerable migration in both the control and LPS groups, with scratches nearly disappearing within 12 hours. However, the horizontal migration of RA-FLS was significantly inhibited by application of MTX (100 nM) and WMP (2%, 8%) (p < 0.01). As shown in Fig. 2E and G, vertical invasion of RA-FLS was significantly inhibited by MTX (100 nM) and WMP (2%, 8%) in the transwell assay (p < 0.05 and p < 0.001, respectively).

2.4 Effect of WMP on the Expression of Inflammatory Related Proteins in RA-FLS

The TLR4/NF-κB pathway is frequently studied in the context of rheumatoid arthritis (RA). We investigated the impact of WMP on this specific pathway in RA-FLS cells using protein immunoblotting. In comparison to the control group, the LPS group showed increased levels of inflammation-related protein expression, indicating that RA-FLS inflammation was further aggravated. As demonstrated in Fig. 3 (A-D) of the experimental results, the use of WMP yielded significant inhibitory effects on TLR4 protein expression (p < 0.001), TRAF6 protein expression (p < 0.05), nuclear translocation of NF-κB proteins (p < 0.01), and the phosphorylation of IκB-α (p < 0.05), in comparison to the LPS group. Furthermore, the experimental results E, F, G and H demonstrate that WMP significantly hindered the expression of MMP-2/9 protein in RA-FLS cells (p < 0.05 or p < 0.01) and encouraged the expression of TIMP-1/2 protein in RA-FLS cells (p < 0.01) compared to the LPS group.

2.5 Effect of WMP on Immune Organ Index and Inflammatory Factors in CIA Rats

Figure 4A and B demonstrate a marked inflammatory response in rats following collagen induction, as indicated by the elevated ratio of immune organs (thymus, spleen) to body weight, which was significantly greater than that of the control group (p < 0.01). Positive drug and WMP administration began early in the model stage. After one cycle of treatment, inflammation reduced in rats. Evidence of this is shown by a decrease in immune organ ratios (thymus, spleen) to body weight in CIA rats who received WMP treatment, which was significantly different from the model group (p < 0.01 and p < 0.05, respectively). This indicates that when compared to the hyperimmunity of RA rats, WMP effectively reduces it.

In this research, we evaluated the level of inflammation in rat serum. Figure 4E and F demonstrate a substantial increase in levels of both TNF-α and IL-6 in the serum of rats with CIA, providing evidence of elevated autoinflammation. Our findings were consistent with the immune index analysis. Comparatively, rats in the WMP group had significantly reduced levels of TNF-α and IL-6 when compared with the CIA group (p < 0.001, p < 0.001 respectively). This supports our conclusion that WMP has a significant anti-inflammatory effect on RA rats in vivo.

2.6 Effect of WMP on Knee and Ankle Joint Swelling in CIA Rats

As demonstrated in Fig. 4C, the examination of the knee joint pathological sections revealed that the RA model rats induced by collagen lacked joint smoothness and, in fact, displayed cartilage erosion in comparison to the normal rats. Synovial tissue thickening and an inflammatory infiltration were also observed. Following treatment with WMP, the articular surface was noticeably intact, and the inflammatory infiltration was not prominent. The articular cartilage structure in the knee and ankle joints of rats from each group was examined using annona red O/fast green staining. Figure 4D demonstrate that the surface of the knee joints of the rats in the control group was sleek, and the cartilage (red part) bands were firmly attached to the bone surface. The rats in the CIA group displayed various degrees of joint damage, including cartilage thinning or disappearance, worm-eaten lesions in certain areas, and severe cartilage destruction. However, following WMP treatment, the cartilage bands were smooth and complete, and the bone tissue structure was intact. Meanwhile, synovial hyperplasia was also relieved and no obvious inflammatory infiltration was seen.

2.7 Effect of WMP on the Expression of Inflammatory and Invasive Proteins in the Synovium of CIA Rats

The proteins related to the synovium in rats with collagen-induced arthritis (CIA) were analyzed through Western blotting. Figure 5A-D illustrates a significant increase in the expression levels of TLR4 and TRAF6 proteins in the CIA group (p < 0.01 and p < 0.001, respectively). The CIA group also experienced marked nuclear translocation of NF-κB (p < 0.05) and phosphorylation of IκB-α (p < 0.01). The study's results indicate that WMP significantly hindered the expression of TLR4 and TRAF6 proteins compared to CIA (p < 0.05). In addition, nuclear translocation of NF-κB was significantly inhibited (p < 0.05 and p < 0.01), as was phosphorylation of IκB-α (p < 0.01). In the investigation of the mechanism of migration and invasion, the results E-H indicate that MMP-2/9 expression significantly increased in the CIA group (p < 0.01), which is associated with cell migration and invasion. In contrast, a considerable decrease in the expression of TIMP-1/2, which prohibits cell migration and invasion by binding to MMP-2/9, was observed (p < 0.001, p < 0.01 respectively). FLS cells were found to have significantly inhibited MMP-2/9 protein expression (p < 0.05) while promoting TIMP-1/2 protein expression (p < 0.05 or p < 0.001). Additionally, excess free MMP-2/9 protein was inhibited from invading the periarticular tissues and cartilage upon treatment with WMP.

WMP is best described in the Han Dynasty Chinese Treatise on Typhoid Fever. It primarily treats a variety of cold and heat-induced ailments in Traditional Chinese Medicine theory. The theory posits that RA is mainly caused by external agents like wind, cold, and dampness invading the body. Consequently, the application of WMP in treating RA aligns perfectly with Traditional Chinese Medicine theory. Additionally, recent medical research has indicated a positive anti-inflammatory effect of WMP. Furthermore, scholars have utilized WMP in the treatment of RA, although the exact mechanisms of action remain unclear. Hence, this paper aims to explore the mechanism of WMP in the prevention and management of RA. UPLC-MS analysis revealed that WMP contains 181 chemical components. Wumeirou has 2 compounds, Xixin has 2 compounds, Huanglian has 3 compounds, Huangbai has 1 compound, Ganjiang has 51 compounds, Heishunpian has 4 compounds, Guizhi has 2 compounds, Renshen has 6 compounds, Danggui has 29 compounds, Zhike has 4 compounds, Jiegeng has 1 compound, Baishao has 16 compounds, and Zhigancao has 74 compounds.

RA is a multifactorial and complex immune-mediated disease with an unclear pathogenesis. Clinical treatment aims to reduce disease activity and alleviate symptoms. Studies suggest that the immune-inflammatory response plays a crucial role in the development of RA and is continuously activated throughout the disease process ²⁰. TLR4 receptors are transmembrane proteins situated on the cell membrane, which play a critical role in non-specific immunity and can trigger inflammation. Numerous studies support the notion that activation of TLR4 can worsen RA symptoms. Thus, exploring this receptor's properties and analyzing its corresponding pathways could serve as a basis for elucidating RA's pathogenesis ²¹. When TLR4 is activated by external stimuli, it stimulates the secretion of inflammatory factors and chemokines through a MyD88-dependent pathway. Pro-inflammatory factors and chemokines regulate intracellular signaling pathways, modulating cellular inflammatory responses in terms of their nature, extent, and timing ²². Activation of the MyD88-dependent pathway through interference with TLR4 interacts with IRAK, leading to an increase in TRAF6 protein expression ²³. TRAF6 additionally enhances the nuclear translocation of NF-kB, a prominent pro-inflammatory transcription factor, and produces inflammatory factors and mediators ²⁴. The elevated expression of TLRs in activated FLS has been linked to the activation of the NF-kB pathway and transcription of inflammatory factors like TNF-α and IL-6 ²⁵. The experiment demonstrated that WMP significantly decreased the arthritis and immune organ indexes of CIA rats, as well as alleviated joint swelling and autoimmune hyperactivation. Pathological analysis showed that WMP significantly reduced inflammatory infiltration in the rats' knee joints and preserved cartilage tissue. Furthermore, WMP considerably lowered TNF-α and IL-6 levels in the CIA rat model. The Western blot analysis results indicate that WMP significantly decreased the expression of TLR4 and TRAF6 proteins. Furthermore, WMP has the ability to suppress the classical NF-κB inflammatory pathway by preventing the nuclear transportation of NF-κB and the phosphorylation of IκB-α.

FLS constitute 70–80% of synovial tissue and serve to uphold the structural integrity of the synovial lining while secreting lubricating fluid. Numerous studies indicate that RA-FLS play a crucial role in the development of RA and are the principal effector cells in the disease. It is important to note that these statements are objective and supported by scientific evidence ²⁶. The hyperproliferation of RA-FLS, which promotes the production of inflammatory factors and extracellular matrix regulators, has been identified as the primary cause of RA development, leading to joint destruction. In addition, RA-FLS has tumour-like properties and secretes large amounts of matrix metalloproteinases (MMPS) during proliferation, which can erode cartilage and destroy joints ²⁷. MMP-2 (also referred to as gelatinase-A) and MMP-9 (also known as gelatinase-B) are both capable of degrading the intercellular matrix, thus promoting the migration and invasion of RA-FLS cells. Subsequently, it is suggested that these MMPs play a role in RA pathogenesis ^28,29. Significant increases in MMP-2 and MMP-9 proteins have been found to contribute to joint opacification and irreversible cartilage erosion in RA patients, leading to impaired mobility ³⁰. Under normal circumstances, tissue inhibitors of MMPs (TIMPs) bind to MMPs in a 1:1 ratio, inhibiting the excessive production of MMPs. When rheumatoid arthritis (RA) develops, the balance is disrupted due to the concentration of specific inflammatory factors present in the joints. As a result, there is an overproduction of MMPs, which further accelerate ECM degradation. Consequently, RA-FLS migration and invasion occur ³¹. TIMP-1 and TIMP-2 bind with MMP-9 and MMP-2 proteins to create a complex and prevent their excessive production ³². Previous studies have reported elevated MMP expression in the synovial tissue, synovial fluid, and peripheral blood of patients with rheumatoid arthritis ³³. This article examines the mechanism of action of WMP on RA-FLS. Firstly, WMP significantly reduces the secretion of TNF-α and IL-6 inflammatory factors by RA-FLS. Additionally, WMP inhibits RA-FLS migration and invasion significantly through the cell scratch assay and transwell assay. The Western blotting results revealed that WMP substantially reduced the protein expression of MMP-2 and MMP-9 in RA-FLS, increased the protein expression of TIMP-1 and TIMP-2, and rectified the dynamic equilibrium of MMPs' protein expression. This intrinsic mechanism of WMP inhibits RA-FLS's migration and invasion.

In summary, WMP exhibits a noteworthy effect on impeding RA lesions and joint degradation. WMP functions by decreasing the levels of TLR4 and TRAF6 proteins, which curtails immune hyperactivity in RA, ultimately impeding the nuclear translocation of NF-κB and IκB-α phosphorylation, thereby curtailing the production of inflammatory cytokines (e.g., TNF and IL-6) in RA-α. Additionally, WMP regulates the MMP-2/TIMP-2 and MMP-9/TIMP-1 balance, ultimately hindering RA-FLS migration and invasion. Thus, WMP may hold potential as a future drug for the prevention and treatment of RA.

4.1 Drug and drug-containing serum preparation

WMP has been incorporated into the 2020 edition of the Chinese Pharmacopoeia. The experiment introduced six more Chinese herbal medicines, in addition to the ones mentioned in the Pharmacopoeia, bringing the total to 16 Chinese herbal medicines (refer to Table 1).

Table 1

Herbal constituents of WMP.
Chinese name	Latin name	Dose (g)
Wumei	Prunus mume (Siebold) Siebold & Zucc.	120
Huajiao	Zanthoxylum bungeanum Maxim.	12
Xixin	Asarum heterotropoides F.Schmidt	18
Huanglian	Coptis omeiensis (F.H.Chen) C.Y.Cheng	48
Huangbai	Phellodendron chinense C.K.Schneid.	18
Ganjiang	Zingiber officinale Roscoe	30
Heishunpian	Aconitum carmichaelii Debeaux	18
Guizhi	Neolitsea cassia (L.) Kosterm.	18
Renshen	Panax ginseng C.A.Mey.	18
Danggui	Angelica sinensis (Oliv.) Diels	12
Zhike	Citrus × aurantium L.	12
Jiegeng	Platycodon grandiflorus (Jacq.) A.DC.	12
Baishao	Paeonia lactiflora Pall.	12
Zhigancao	Glycyrrhiza glabra L.	12

Drug formulation containing WMP serum: The drug is dissolved in 0.1% sodium carboxymethylcellulose and administered by gavage to mice at a dose of 2 g/kg/day for 7 days. After 15 hours of fasting and dehydration, blood is collected and centrifuged to obtain serum. The serum was incubated in a water bath for 25 minutes to produce drug-containing serum for inactivation. The serum is then filtered through a sterile 0.22 µm microporous membrane under controlled aseptic conditions and stored at -80°C. (Note: Different sera can be used. (Note: Different concentrations of drug serum were diluted from normal mouse serum).

4.2 UPLC-MS spectrometry detection

Column: Waters UPLC HSS T3. Mobile phase: methanol, water + 0.1% formic acid. Flow rate: 0.3 mL/min. Sample injection volume: 10 µl. The mass spectrometer was a quadrupole orbital ion trap mass spectrometer (containing a thermospray ion source, Q Active ™).

Table 2

Elution Gradient Table
Time(min)	Mobile phase
Time(min)	A(v%)	B(v%)
0	2	98
1.0	2	98
41.0	100	0
50.0	100	0
50.1	2	98
52.0	2	98

4.3 Establishment of a rheumatoid arthritis model and isolation and cultivation of RA-FLS

The study utilized male Wistar rats (180 ± 200 g, Ease Animal Technology Ltd.), fed a high-fat diet with normal water intake and 12-hour cycles of day and night. The Animal Ethics Committee of Jilin Agricultural University approved all experiments.

CAI modeling: 1 ml of bovine type II collagen solution was repeatedly mixed with 1 ml of IFA to make a water-in-oil emulsion. The resulting emulsion was subcutaneously injected into the tails of rats at a dose of 0.2 ml per rat. After one week, the same dosage of emulsion was injected again to improve the immunization.

RA-FLS extraction: Synovial tissue from CIA rats was taken, cut and enzymatically digested (Type II collagenase. Sigma-Aldrich, Shanghai, China). The incompletely digested tissue was filtered. Cells were placed in DMEM complete medium (Procell Life Technologies Ltd.) and incubated in an incubator at CO₂.

Control group: without any drug treatment. LPS group: RA-FLS was treated with LPS (1µg/ml) for 12 hours. Methotrexate (MTX, 100nM, Shanghai Mellin Biochemical Technology Co., Ltd.) group: add MTX and continue incubation for 12 hours. WMP group: add WMP and continue incubation for 12 hours.

4.4 RA-FLS viability detection

Incubate RA-FLS (5 x 10³ cells/well) in a 96-well plate for twelve hours. The WMP group was then exposed to serum concentrations containing drugs at 1%, 2%, 4%, 8%, 10%, 20%, 50%, and 100%. After an additional 24 hours of incubation, 20µl of CCK-8 was added to each well and incubated for 30 minutes. The cell viability was assessed by measuring the OD value of each group at 450nm using an enzyme marker.

4.5 RA-FLS migration detection

Inoculate RA-FLS (5×10⁴ cells/well) into a 6-well plate and incubate for 12h, then use a 200µl gun tip to scratch cells perpendicular to the well plate. Gently rinse off the floating cells from each well with PBS. Re-add the appropriate drug to each group. Each group was photographed and recorded at 0h and 12h, and the migration distance of RA-FLS in each group was calculated.

4.6 RA-FLS invasion detection

The RA-FLS (1×10⁴cells/well) pretreated with LPS for 12h was inoculated into materigel-coated transwell chambers, and the corresponding dose of drug was added and incubated for 12 h. After cell culture was completed, each group of transwells was sequentially fixed, stained, and rinsed with PBS. The number of cells attached to the outer ventricular membrane was observed under the microscope. Four fields of view were randomly selected for counting.

4.7 Inflammatory Factor Testing

Cell supernatant preparation: Firstly, RA-FLS (1×105 cells/well) were seeded into a 6-well plate and incubated for 12 hours. Next, all groups except for the control group were treated with the corresponding drugs and then incubated for 24 hours. Finally, the supernatants from each group of cells were collected and placed into 1ml centrifuge tubes, which were then centrifuged at 80 × 100g for 10 minutes at 4 ℃. The supernatants were extracted and stored at -80 ℃ for later use.

Preparation of rat serum: After administering the drug treatment, we obtained blood samples from each group of rats by collecting it from the eyeball vein. Blood at 4 ℃, 80 ×100g centrifuge with force for 10 minutes, take the supernatant and store it at -80 ℃ for later use.

ELISA kits are from Biyuntian Biotechnology Co. The specific test method is strictly according to the instruction of the kit.

4.8 CIA rat modeling and grouping

The CIA rat model was replicated using the method outlined in Section 2.3. The rats were allocated randomly into five groups, each consisting of five rats, namely the control, CIA, positive (methotrexate, MTX, 30 mg/kg), WMP sub-effective dose (0.5 g/kg) and WMP effective dose (2 g/kg) groups. After successful modelling, the control and model groups were orally administered physiological saline once a day for 15 consecutive days. In the positive group, methotrexate powder was dissolved in 0.1% carboxymethyl cellulose sodium solution at a dose of 30 mg/kg/day (MTX). WMP powder was dissolved in 0.1% carboxymethyl cellulose sodium solution and then orally administered at low and high doses of 0.5g/kg/day and 2g/kg/day respectively. Fifteen days of treatment were carried out, followed by the collection of serum, synovial fluid, spleen, thymus, and knee tissue for analysis of pertinent physical and chemical indices.

4.9 CIA Rat Immune Organ Index

Extract the thymus and spleen from each group of CIA rats, weigh them, and compare their weight to calculate the immune organ index of each rat group.

4.10 Histopathology assessment

Knee joints were obtained from the hind limbs of rats and then sliced into 5 µm sections. The sections were subsequently fixed in 10% ethylenediaminetetraacetic acid (EDTA) paraffin before being stained with hematoxylin and Anna Red O/Fast Green dyes.

4.11 Western blot

Cell sample processing: RA-FLS cells were seeded at a density of 5 × 10⁵ cells per well on a 6-well plate and incubated for 12 hours. Replace the appropriate medication dosage and continue to cultivate for another 24 hours. Subsequently, remove the culture medium and add 200 µl RIPA cracking solution. Employ a centrifuge at 120 ×100g and 4 ℃ for 10 min. Lastly, extract the supernatant and store it at -80 ℃ for future use. Synovial tissue processing: The synovium was lysed using 200 µl of RIPA lysate for every 20 mg of tissue. The tissue was homogenised in an ice bath employing a glass homogeniser until it completely dissolved.

The protein concentration of each group was determined using the BCA kit and standardized accordingly. Subsequently, the samples underwent electrophoresis and were transferred via electrotransfer onto a PVDF membrane. Following a 2-hour closure in 5% skimmed milk powder, each group was subjected to overnight incubation with the relevant primary antibody, washed thrice with TBST, and then incubated for 2 hours with the secondary antibody. The PVDF membranes underwent development in a visualiser after being placed in ECL luminous reagent for 30 seconds. Technical term abbreviations were explained accordingly. The text adheres to a clear, concise, and formal writing style. The Image J software was used to analyse the grey scale values of each group of bands, and subsequent statistical analysis was conducted. The antibodies, comprising TLR4, TRAF6, NF-κBp65, IκB-α, p-IκB-α, Bax, Bcl-2, MMP-2, MMP-9, TIMP-2, TIMP-1, Lamin B, and β-actin, were procured from Abways Technology Co., Ltd.

4.12 Statistical analysis

All figures were performed using Graphpad Prism 6. Data were expressed as the mean ± standard deviation (SD). One-way analysis of variance (ANOVA) was used for statistical analysis of data, followed by Tukey’s post-hoc multiple comparison test. Statistical significance was defined as (#) or (*) P < 0.05, (##) or (**) P < 0.01, (###) or (***) P < 0.001.

WMP	Wu Mei Pill	PAMP	pathogen-associated molecular patterns
TLR4	Toll Like Receptor 4	IL-6	Interleukin-6
TRAF6	TNF receptor associated factor 6	TNF-α	Tumor necrosis factor-α
NF-κB	Nuclear factor-ĸB	CIA	Collagen-induced rheumatoid arthitis
IκB-α	Inhibitor of NF-κB-α	MTX	Methotrexate
RA	Rheumatoid arthritis	FLS	Fibroblast-like synoviocytes
PRR	pattern recognition receptors

Ethics approval and consent to participate

The experiments were approved by the Animal Ethics Committee of Jilin Agricultural University.

Data availability statement

All data generated or analyzed during this study are included in this published article and its supplementary information files.

Competing interests

The authors declare that they have no competing interests.

Funding

The authors are grateful for the Jilin Province Science and Technology Development Plan Project: Research on the Development of Classic Famous Formula Modified Wu Mei Pill Preparation (20220204009YY).

Authors' contributions

Yuheng Fu, Haibo Zhang, Yan Zhao: Conceptualization; Chunyu Gao: Methodology; Hongbo Teng: Formal analysis; Xialin Sun: Writing - Original Draft; Yaru Zhao: Data curation; Wenxin Zhang: Supervision; Siwen Li: Validation; Liang Huang, Bowen Fan: Project administration.

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No competing interests reported.

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Study on the mechanism of action of Wu Mei Pill in inhibiting rheumatoid arthritis through TLR4-NF-κB pathway

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Abstract

Background

Methods

Result

Conclusions

Figures

1. Introduction

2. Result

2.1 Identification of the chemical composition of WMP

2.2 Effect of WMP on the Activity and Inflammatory Factors of RA-FLS Cells

2.3 Effect of WMP on RA-FLS Migration and Invasion

2.4 Effect of WMP on the Expression of Inflammatory Related Proteins in RA-FLS

2.5 Effect of WMP on Immune Organ Index and Inflammatory Factors in CIA Rats

2.6 Effect of WMP on Knee and Ankle Joint Swelling in CIA Rats

3. Discussion

4. Methods

4.1 Drug and drug-containing serum preparation

4.2 UPLC-MS spectrometry detection

4.3 Establishment of a rheumatoid arthritis model and isolation and cultivation of RA-FLS

4.4 RA-FLS viability detection

4.5 RA-FLS migration detection

4.6 RA-FLS invasion detection

4.7 Inflammatory Factor Testing

4.8 CIA rat modeling and grouping

4.9 CIA Rat Immune Organ Index

4.10 Histopathology assessment

4.11 Western blot

4.12 Statistical analysis

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

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