Siweixizangmaoru Decoction Ameliorated Type II Collagen-Induced Arthritis in Rats via Regulating JAK2–STAT3 and NF-κB Signaling Pathway

Abstract

Siweixizangmaoru decoction (SXD) is widely used as an anti-rheumatoid arthritis (RA) in Tibet, however, the specific anti-inflammatory mechanism of SXD is still unclear. This research attempts to examine the efficacy and possible mechanisms of SXD in treating RA. The primary chemical components of SXD were identified using UHPLC-Q-Exactive Orbitrap MS. We established a lipopolysaccharide (LPS)-induced RAW264.7 macrophage inflammatory injury model to explore the anti-inflammatory mechanism of SXD and validated it through in vivo experiments. According to our research in vitro as well as in vivo, SXD exhibits anti-inflammatory qualities. SXD can suppress nitric oxide (NO) and pro-inflammatory factor production in RAW264.7 cells activated by LPS. The mechanism underlying this effect might be connected to the janus tyrosine kinase 2–signal transducer and activator of transcription 3 (JAK2/STAT3) and nuclear factor-κB (NF-κB) signaling pathways. In vivo, SXD alleviates joint swelling, decreases the generation of inflammatory factors in the serum, lowers oxidative stress, and improves joint damage. In short, SXD improves joint degeneration and lowers symptoms associated with RA by regulating inflammation via the suppression of NF-κB and JAK2/STAT3 signaling pathway activation.

INTRODUCTION

An autoimmune illness that affects the entire body, rheumatoid arthritis (RA) is characterized by bone loss and inflammation both locally and systemically.^1,2) RA is estimated to be a major source of disability, with a prevalence of 1% in adults, and is more common in women than in men.^3,4) Without effective treatment, chronic inflammation of the lining of the joint (synovium) can lead to serious disability, and often there are associated severe systemic complications.^5,6) In comparison to other parts of China, Tibetans have a higher prevalence of RA (4.86%), which may be related to the local climate.⁷⁾ Numerous cell types, inflammatory mediators, and inflammatory cytokines are all part of the intricate pathophysiology of RA.⁸⁾ A number of cytokines’ roles in the pathogenesis of RA have been demonstrated by numerous investigations. This can lead to tissue and joint damage. Among these cytokines are interleukin-6 (IL-6), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α). Consequently, RA development can be prevented by reducing the production of these cytokines.^9,10)

The janus tyrosine kinase 2–signal transducer and activator of transcription 3 (JAK2/STAT3) pathway is an essential signaling pathway that is triggered by cytokines. It is vital for the development and advancement of inflammatory and autoimmune diseases like RA.^11,12) This activation results in the recruitment of JAK, which subsequently triggers the phosphorylation of STAT pathways. Phosphorylated STAT forms homologous or heterologous dimers, translocates to the nucleus, and promotes cell proliferation and the release of pro-inflammatory factors, leading to an inflammatory storm.^13,14) In addition, nuclear factor-κB (NF-κB) is considered a key signaling molecule in RA that regulates joint destruction and synovitis.¹⁵⁾ Inhibitor of kappaB (IκB) kinase is activated in response to several intracellular and extracellular stimuli, which results in the phosphorylation of IκB protein in cells. The release of the NF-κB dimer and its translocation to the nucleus occurs when the IκB protein is broken down. The pro-inflammatory cytokines inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) are subsequently regulated by it.^16,17) Lipopolysaccharide (LPS) is a common type of endotoxin that can activate signaling systems such as TLR4, leading to inflammatory responses.¹⁸⁾ Research has shown that LPS can induce sustained inflammatory responses in macrophages, generating a great deal of pro-inflammatory cytokines that are distinguished by persistent activation of the traditional NF-κB pathway.¹⁹⁾ The above pathways have similar activation characteristics, namely the binding of cytokines to cell surface receptors, releasing a large amount of inflammatory factors.^20,21) Oxidative stress is a significant factor in both initiating and intensifying the inflammatory process of RA, in addition to the inflammatory response. According to studies, activated T cells and macrophages release pro-inflammatory cytokines that cause reactive oxygen species (ROS) to develop, hence exacerbating synovitis.²²⁾ Thus, oxidative stress and inflammation have a positive feedback regulation connection in the pathophysiology of RA.²³⁾

Currently, nonsteroidal anti-inflammatory drugs, anti-rheumatic drugs, and biological agents are frequently utilized to treat clinical RA.^24,25) These drugs can improve RA symptoms such as pain, swelling, and joint damage. However, these drugs are far from satisfactory due to the serious adverse effects associated with their long-term use.²⁶⁾ In contrast, Chinese medicines are more readily available and relatively safer, which has made them the drug of choice for patients seeking treatment methods.²⁷⁾

The practice of traditional Tibetan medicine (TTM) has endured for hundreds of years, making it one of the major medical legacies around the globe.²⁸⁾ In TTM theory, RA is classified as a “Huang-shui disease” for treatment. The primary physical manifestation of illnesses brought on by a bad diet and unforgiving surroundings is Huang-shui sickness, which makes joint issues worse and produces an excess of Huang-shui in the body.²⁹⁾ Siweixizangmaoru decoction (SXD) is a traditional Tibetan medicine formula for treating Huang-shui disease, recorded in the “Four Volumes Medical Treatment” of the Tibetan medical classic. SXD is composed of Rhamnella gilgitica Mansf. et Melch (RG, Xizangmaoru), Berberis thunbergii (BT, Xiaobo), Gentiana macrophylla (GM, Qinjiao), and Terminalia chebula Retz (TC, Hezi). By taking SXD, joint injuries from excessive Huang-shui can be significantly improved, and dry Huang-shui can result. It has been applied in Tibetan hospitals in the Tibet Autonomous Region as a pharmaceutical pretreatment for clinical practice. Up to now, all single drug components (RG, BT, GM, and HZ) in this formula have been reported to be able to be used for the treatment of RA.^17,30–32) However, the treatment of RA with this formula has not been reported yet, and its exact mode of action is yet unknown.

The pathogenesis of RA is tightly associated with macrophages.³³⁾ Activated macrophages in RA patients enter the synovial fluid and produce cytokines that promote inflammation.³⁴⁾ Their presence is correlated with the severity of RA and causes synovial inflammation and bone deterioration.^35,36) The RAW264.7 cell line plays an important role in studying the mechanisms and potential therapeutic strategies of RA. It can simulate the inflammatory environment in RA diseases and is widely used to study various functions of macrophages, such as phagocytosis, cytokine secretion, signaling pathways, etc.³⁷⁾ These investigations are of tremendous significance for understanding the role of macrophages in RA. Therefore, in this study, we evaluated the anti-RA effect of SXD in vivo and explored the potential mechanism of SXD in RAW264.7 cell lines treated with LPS in vitro.

MATERIALS AND METHODS

Chemicals and Reagent

Standards of chebulinic acid, quercetin, naringenin, kaemperol, gentiopicroside, and berberine were purchased from Manchester Biotechnology Co., Ltd. (Chengdu, China). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco (Grand Island, NY, U.S.A.). From Wuhan Boster Biological Technology, Ltd. (Wuhan, China), enzyme-linked immunosorbent assay (ELISA) kits for TNF-α, IL-6, IL-1β, IL-4, IL-17, and IL-10 were acquired. AG490 was purchased from MedChemExpress (NJ, U.S.A.). LPS was purchased from Sigma-Aldrich (MO, U.S.A.). Methylthiazol-2-yl-2,5-diphenyl tetrazolium bromide (MTT) and bicinchoninic acid (BCA) protein assay kits were obtained from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). Malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) reagent kits were acquired from Jiancheng Biotechnology Science, Inc. (Nanjing, China). The following antibodies were bought from Affinity Biosciences (Cincinnati, OH, U.S.A.): mouse/rabbit anti-iNOS, anti-COX-2, anti-IκBα, anti-phospho IκBα, anti-p65, anti-Lamin B, anti-phospho JAK2, anti-JAK2, anti-phospho STAT3, anti-STAT3, anti-actin. Methotrexate (MTX) was obtained from SPH Sine Pharmaceutical Laboratories Co., Ltd. (Shanghai, China). Freund’s incomplete adjuvant was obtained from Sigma-Aldrich Corporation (St. Louis, MO, U.S.A.). Bovine type II collagen was acquired from Chondrex, Inc. (Woodinville, WA, U.S.A.).

Preparation of SXD

RG, GM, BT, and TC samples were verified by Professor Fei Long of the Chengdu University of Traditional Chinese Medicine (Chengdu, China). The SXD preparation method used in this study is as follows: In summary, the following crude medications were weighed: RG (12 g), GM (10 g), BT (6.5 g), and TC (3 g). After being extracted from 15 times the volume of water for 1.5 h, all herbs were filtered. After 1.5 h of re-extraction of the residue in 15 times the volume of water, the two filtrates were combined. For use in in vivo investigations, the filtrate solutions were gathered, condensed to 1 g/mL (crude medications), and stored at 4 °C. The filtered solutions were dried and concentrated in a vacuum oven (DZF-6053; Shanghai Bluepard Instruments Co., Ltd., Shanghai, China) for in vitro experiments. The dried powder was stored at −20 °C.

Chemical Composition Analysis of SXD Based on UHPLC-Q-Exactive Orbitrap MS and HPLC

The main chemical components of SXD were identified using UHPLC-Q-Exactive Orbitrap MS (Thermo Scientific). Before analysis, the samples were diluted in methanol. The solution was then centrifuged at 12000 r/min for 10 min, and the supernatant was filtered through a 0.22 µm microporous membrane. The samples were run using an Agilent ZORBAX SB-C18 (4.6 × 100 mm, 1.8 micron) at 0.3 mL/min with 0.1% formic acid solution (A) and acetonitrile (B) as the mobile phases. Elution program: 0–25 min, 95–40% A; 25–30 min, 40–5% A. Column temperature: 40 °C; the injection volume was 2 µL. The mass spectrum was conducted by positive and negative ion-mode electric spray ionization. The conditions set were as follows: Spray voltage of 3.0/+3.5 kV; the sheath gas flow rate was 35 L·h⁻¹; the scan range was m/z 100–1500 Da; the capillary temperature was 350 °C.

The primary chemical constituents of the SXD extract were determined by HPLC analysis. The samples were run using a 4.6 × 250 mm, 5 µm HypersilTM BDS C18 column at 1.0 mL/min with 1% phosphoric acid (A) and acetonitrile (B) as the mobile phases. The following was the elution process: 0–10 min, A: 97–93%, B: 3–7%; 10–25 min, A: 93–90%, B: 7–10%; 25–35 min, A: 90–83%, B: 10–17%; 35–60 min, A: 83–75%, B: 17–25%; 60–75 min, A: 75–71%, B: 25–29%; 75–95 min, A: 75–55%, B: 29–45%; 95–110 min, A: 55–40%, B: 45–60%. The wavelength at which the HPLC signals were detected was set at 245 nm.

Cell Culture and Establishment of Inflammation Model

The RAW264.7 cell line, which is generated from mice, was acquired from Wuhan Pricella Biotechnology Co., Ltd. (ATCC, Wuhan, China). One percent penicillin/streptomycin and 10% fetal bovine serum were added to DMEM for the cell culture process. The cells were cultured with 5% CO₂ at 37 °C. The cells underwent a 12-h pre-treatment with varying doses of SXD, followed by a 24-h incubation period with LPS (1 µg/mL) to produce inflammation.

Cell Viability Assay

Cell viability was determined by the MTT method. RAW264.7 cells (1 × 10⁵ cells/mL) were seeded in 96-well plates for 12 h. The cells received 12 h of treatment with SXD at different doses (25, 50, 100, and 200 µg/mL). After that, four further hours of treatment were given with the MTT solution. Using a microplate reader (Bio-Tek), each well’s absorbance was calculated at a wavelength of 490 nm. Three separate experiments were carried out.

Measurement of the ROS Level

A 24-well plate was filled with RAW 264.7 cells, seeded at 10⁵ cells per well. After 12 h, the cells were treated with varying concentrations of LPS and SXD water extract. Following a 48 h period, the cells’ nuclei were stained for 10 min with 4′,6-diamidino-2-phenylindole (DAPI) (Solarbio) and for 30 min with 2′-7′-dichlorodihydrofluorescin diacetate (DCFH-DA) (Beyotime, Shanghai, China) added at a dilution of 1 : 10000. Using an inverted fluorescent microscope (Olympus Corporation, DP74, Tokyo, Japan), cell fluorescence was visualized.

Immunofluorescence Staining

The protocol for immunofluorescence staining was followed according to the instructions from the manufacturer of the antibody. Briefly, RAW264.7 cells with a concentration of 3 × 10⁵ per well were inoculated onto sterile glass cover slips in a 6-well plate for 12 h. Then SXD extract was added to wells for 4 h, followed by LPS induction for 12 h. The cells were preserved for 20 min in 4% paraformaldehyde, following two phosphate buffered saline (PBS) rinses. After 20 min of 0.1% Triton X-100 treatment, the cells were incubated with anti-iNOS and anti-p65 primary antibodies for a whole night at 4 °C in a wet box. Following a rinse, Alexa Fluor 488-coupled anti-rabbit immunoglobulin G (IgG) secondary antibodies (ab150081, Abcam) were used to incubate the cells. In the end, DAPI (Solarbio) was added to an anti-quencher, and the cells were stained and blocked. The cells were then examined using a ZEISS fluorescent microscope (Provis AX70, Olympus Optical Co., Ltd., Tokyo, Japan).

Experimental Animals

Male Sprague-Dawley rats (180–200 g) were acquired from the Qingdao Institute for Food and Drug Control (Qingdao, China; Approval Number: SYXK (Lu) 20230410). For the first seven days, they were housed in a 12-h day/night cycle at 22 ± 2 °C and 55 ± 5% relative humidity. During this period, they can freely obtain food and water. The Qingdao University of Science and Technology Committee on the Ethics of Animal Experiments (Approval Number: ACQUST-2023-056) approved the experimental protocols, which were carried out in compliance with the Guide for the Care and Use of Laboratory Animals (https://www.ncbi.nlm.nih.gov/books/NBK54050/).

Induction of RA Rats and Experimental Design

Two groups of these rats were created, one of which received no therapy at all and was called the blank group. Another group was injected with 200 µL of emulsion prepared by mixing incomplete Freund’s adjuvant (20 mg/mL) with an equal amount of bovine type II collagen (2 mg/mL) into the tail to establish a CIA rat model, followed by enhanced immunity (100 µL) after 3 weeks. Arthritis index (AI) ≥ 4 indicates successful modeling. Five sets (n = 12) of accurately modeled rats were assigned at random: normal control group (CON); model group (CIA); positive control group treated with MTX (0.9 mg/kg, two times/week); and SXD groups treated with low, medium, or high doses of SXD (L-SXD, 1417.5 mg/kg; M-SXD, 2835 mg/kg; H-SXD, 5670 mg/kg, once/d). Rats in the CON and CIA groups were given physiological saline orally. Following the completion of the last dosage, the rats were put under anesthesia with 3% pentobarbital sodium and then put to death. The abdominal aorta was used to extract serum for use in later tests.

Arthritis Assessments

Seven days after the second immunization, the body weight, joint thickness, and paw thickness of each group of rats were recorded. In addition, every week, all rats’ AI values were determined. Each claw scores 4 points, with a maximum total score of 16 points for all four claws. After euthanasia, measure the immune organ index (liver, spleen, thymus, and kidney) of all rats. The immune organ index (%) is calculated using the following formula: viscera weight (g)/body weight (g) × 100%.

Biochemical Analysis

The Griess method was employed in accordance with earlier study techniques to ascertain the NO concentration of the RAW264.7 cell supernatant. Briefly, in a nutshell, equal volumes of Griess reagents I and II were combined with culture supernatant (50 µL/well) in 96-well plates. Finally, a microplate reader was used to evaluate the samples’ optical absorbance at 540 nm. The amounts of MDA and the activities of SOD, CAT, and GSH-Px were measured by spectrophotometry. The levels of TNF-α, IL-1β, IL-6, IL-4, IL-10, MMP-2, MMP-3, MMP-9, and MMP-13 in blood and RAW264.7 cells were measured using ELISA kits.

Histopathological Examination and Immunostaining

In order to assess the degree of articular cartilage destruction and synovial hyperplasia, paraffin-embedded synovial tissue and the left knee joint were stained with Safranin O/Fast Green and hematoxylin–eosin (H&E). Under a light microscope (Provis AX70, Olympus Optical Co., Ltd.), the pathological alterations in the synovial tissue and hind knee joints were noted.

The slices were prepared using the same technique as before for immunohistochemical staining. The sections of synovial tissue were dewaxed with xylene and then restored using sodium citrate buffer. To inhibit endogenous peroxidase activity, the sections were first cleaned with a 3% H₂O₂ solution. They were then sealed with 10% goat serum and treated with the p-p65, p-JAK2, and p-STAT3 antibodies for an entire night at 4 °C. After secondary antibodies tagged with biotin are added for incubation, a dropwise addition of a diaminobenzidine (DAB) chromogenic solution is used to produce the color. Then, light microscopy was used to examine these slices.

Western Blotting

We collected the cell and employed it for our Western blot investigation. After lysis and centrifugation, the protein concentration of the sample was determined by the BCA assay kit. To prevent non-specific binding, the complete protein of each sample was put into a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel for separation. Next, the protein was put on a nitrocellulose membrane, which was then sealed with 5% skim milk. The relevant primary antibodies against iNOS, COX-2, p65, p-IκBα, IκBα, p-JAK2, JAK2, p-STAT3, STAT3, Lamin B, and β-actin were then added to the membrane and incubated overnight at 4 °C. The membranes were incubated for 1 h with HRP-conjugated affinipure goat anti-mouse IgG (H + L), after which they were rinsed with PBST. Enhanced chemiluminescence reagents (Cytova, Marlborough, MA, U.S.A.) are used to perform color development after the nitrocellulose membrane has been cleaned with TBST. Finally, the protein bands were observed using a fully automated chemiluminescence image analysis system (Shanghai Tianneng Technology Co., Ltd., Shanghai, China).

Statistical Analysis

Perform a minimum of three separate investigations. A one-way ANOVA was used to evaluate the data, and the mean ± standard deviation (S.D.) was presented. The Tukey’s range test was used to look for any variations between pairings. The statistical analysis was carried out using the software GraphPad Prism 8.0.1. Statistical significance was defined as a p-value <0.05.

RESULTS

Identiffication of Chemical Constituents in SXD

In the early stages of the experiment, we analyzed the chemical composition of SXD. The total ion flow chromatogram of SXD positive and negative ion modes is shown in Fig. 1, and we have identified a total of 33 active ingredients. These active ingredients include flavonoids, alkaloids, phenolic acids, and other compounds, summarized in Supplementary Table S1.

Fig. 1. The Total Ion Current Chromatogram of SXD

(A) The total ion current chromatogram of SXD in negative mode. (B) The total ion current chromatogram of SXD in positive mode.

By consulting relevant literature, it is speculated that chebulinic acid, quercetin, naringenin, kaemperol, gentiopicroside, and berberine are the main active ingredients for SXD to exert anti-inflammatory effects.^17,38–46) The HPLC chromatogram of the extract showed sharp peaks for chebulinic acid, quercetin, naringenin, kaemperol, gentiopicroside, and berberine (Supplementary Fig. S1). The detected component contents in the extract were 4.61 mg/g (chebulinic acid), 0.60 mg/g (quercetin), 4.25 mg/g (naringenin), 2.15 mg/g (kaemperol), 9.2 mg/g (gentiopicroside), and 3.1 mg/g (berberine), respectively.

Effects of SXD on the Viability of RAW264.7 Macrophages

Using an MTT test, the impact of various SXD doses on the survival of RAW264.7 macrophages was investigated in order to assess the cytotoxicity. As shown in Fig. 2A, when the SXD concentration was less than 200 µg/mL, the cell viability was more than 80%, and there was no discernible change from the control. However, the drugs showed elevated cytotoxicity as the dose increased and showed significant cytotoxicity at 400 µg/mL. As a result, for the tests that followed, the SXD concentration range was kept between 25 and 200 µg/mL.

Fig. 2. SXD Inhibits the Production of Inflammatory Cytokines Induced by LPS in RAW264.7 Cells

(A) Cell viability was measured using the MTT assay. (B–E) The concentration of TNF-α, IL-1β, IL-6 and IL-17 in cell culture supernatants. Data were shown as mean ± S.D. (n = 3). ^# p < 0.05, ^## p < 0.01 vs. normal group; * p < 0.05, ** p < 0.01 vs. model group.

The Effect of SXD on LPS Induced Pro-inflammatory Cytokine Secretion

LPS can activate inflammation-related signaling pathways by binding to cell membrane surface receptors, thereby activating macrophages. Pro-inflammatory cytokines are released when macrophages are activated. To assess the possible anti-inflammatory characteristics of SXD, we measured the levels of pro-inflammatory cytokines in the cell supernatant. The results showed that in RAW264.7 cells, LPS significantly increased the levels of inflammatory cytokines. Following SXD treatment, IL-6, TNF-α, IL-1β, and IL-17 levels were significantly lower than those of the model group (Figs. 2B–E). Consequently, SXD dramatically reduced the levels of pro-inflammatory cytokines in macrophages triggered by LPS.

The Effect of SXD Treatment on LPS-Induced Oxidative Stress

For the most part, overproduction of ROS and consequent oxidative stress are caused by pro-inflammatory cytokines. Therefore, we stained RAW264.7 cells with DCFH-DA to detect intracellular ROS levels and explore whether SXD affects LPS-induced oxidative stress. The green fluorescence increased following LPS treatment, whereas intracellular ROS generation was dramatically reduced following a 6-h SXD pre-treatment. This is consistent with our expected results (Fig. 3A). Beyond its potential to mitigate excessive ROS production, we also researched the impact of SXD on antioxidant enzymes. SOD and GSH-Px activity decreased, and MDA content significantly increased after LPS treatment. In order to prevent cell damage from different forms of oxidative stress and inflammatory reactions, SXD therapy enhanced the levels of SOD and GSH-Px and suppressed the generation of MDA in a dose-dependent way (Figs. 3B–E). According to the aforementioned findings, pre-treatment with SXD reduced the oxidative stress brought on by LPS-induced cellular damage.

Fig. 3. SXD Decreases LPS-Induced Oxidative Stress

(A) The changes in ROS were detected using the DCFH-DA probe. (B) Quantification of the fluorescence intensity. (C–E) The activities of SOD, MDA and GSH-Px was measured with assay kits following treatment with SXD at different concentrations. Data were shown as mean ± S.D. (n = 3). ^# p < 0.05, ^## p < 0.01 vs. normal group; * p < 0.05, ** p < 0.01 vs. model group.

Effects of SXD on NO Generation and COX-2 and iNOS Expression

Given that oxidative stress and increased ROS production are linked to NO and COX-2, we investigated whether pre-treatment with SXD would decrease NO generation following an LPS challenge while blocking the expression of iNOS and COX-2 proteins. Therefore, the Griess assay was used to measure NO release, and the result indicated that SXD inhibited the formation of NO (Fig. 4A). According to immunofluorescence and Western blot analysis, SXD pre-treatment also decreased the expression of COX-2 and iNOS, which is in line with the NO results (Figs. 4B–E).

Fig. 4. Effects of SXD on NO Production and iNOS and COX-2 Protein Expression in the LPS-Induced RAW264.7 Macrophages

(A) Effect of SXD on the content of NO induced by LPS in RAW264.7 macrophages. (B) Representative immunofluorescence staining of iNOS in RAW264.7 cells. (C–E) Representative image of Western blot and quantitative analysis of iNOS and COX2 protein expression in RAW264.7 macrophages. Data were shown as mean ± S.D. (n = 3). ^# p < 0.05, ^##p < 0.01 vs. normal group; * p < 0.05, ** p < 0.01 vs. model group.

Effect of SXD on the NF-κB and JAK2/STAT3 Signaling Cascade

In order to learn more about how SXD prevents LPS-induced RAW264.7 macrophage activation, we determined several crucial proteins by Western blotting and immunofluorescence microscopy. These proteins are essential for controlling immune responses and message transmission. Therefore, we investigated whether SXD had an inhibitory effect on NF-κB and the JAK2/STAT3 signal upon LPS administration. The outcomes demonstrated that exposure to LPS enhanced the phosphorylation-induced activation of p65, IκBα, JAK2, and STAT3. SXD pretreatment may suppress the degradation of IκBα in a dose-dependent manner and prevent the phosphorylation of p65, JAK, and STAT3. We were able to identify the nuclear translocation of p65 by immunofluorescence. We demonstrated through confocal microscopy analysis that the accumulation of LPS-induced p65 in the nucleus is significantly controlled by SXD (Fig. 5). In addition, to further confirm the effect of SXD on the JAK2/STAT3 signaling pathway, we used JAK2 inhibitors (AG490) to compare the effects of SXD and JAK2 inhibitor treatment. The research results indicate that both AG490 and SXD can significantly reduce the expression of p-JAK2 and p-STAT3 (Supplementary Fig. S2). Based on the aforementioned research, we concluded that SXD can successfully block the activation of the JAK2/STAT3 and NF-κB pathways brought on by LPS and has an anti-inflammatory effect.

Fig. 5. Effect of SXD on the NF-κB and JAK2/STAT3 Signaling Cascade in LPS-Stimulated RAW264.7 Cells

(A) Representative Western blotting images of phosphorylated and total IκBα, JAK2, STAT3 and p65. Lamin B and actin were used as loading controls. (B) Fold-change quantification of p65, p-IκBα, and IκBα. (C) Representative immunofluorescence staining of p65 in RAW264.7 cells. (D, E) Relative expression of p-IAK2/JAK, and p-STAT3/STAT3 were quantified. Data were shown as mean ± S.D. (n = 3). ^# p < 0.05, ^## p < 0.01 vs. normal group; * p < 0.05, ** p < 0.01 vs. model group.

The Effect of Representative Compounds in SXD on LPS Induced Pro-inflammatory Cytokine Secretion

First, we evaluated the effects of representative compounds on RAW264.7 cell viability. The research results indicate that there is no significant effect on cell viability when the concentration of each representative compound is 20 µM. To further evaluate the anti-inflammatory properties of SXD, we examined the anti-inflammatory activity of the representative compounds in SXD. Following chebulinic acid, quercetin, naringenin, kaemperol, gentiopicroside, and berberine treatment, NO, IL-6, TNF-α, IL-1β, and IL-17 levels were significantly lower than those of the model group (Supplementary Fig. S3). Consequently, these representative compounds significantly reduced the levels of pro-inflammatory cytokines in macrophages triggered by LPS.

The Effect of Active Ingredients in SXD on the NF-κB and JAK2/STAT3 Signaling Cascade

To further explore the molecular mechanism of SXD treatment in RA, we investigated the effects of representative compounds on the NF-κB and JAK2/STAT3 signaling pathways in RAW264.7. As shown in Supplementary Fig. S4, compared with the control group, the LPS group showed a significant increase in expression of p65, p-IκB, p-JAK2, and p-STAT3. However, pre-treatment with chebulinic acid, quercetin, naringenin, kaemperol, gentiopicroside, and berberine may suppress the degradation of IκBα and prevent the phosphorylation of p65, JAK2, and STAT3. In summary, these representative compounds can inhibit the cascade reaction of NF-κB and JAK2/STAT3 signaling pathways in RAW264.7 cells.

SXD Promotes Arthritis Remission in CIA Rats

We investigated if orally administering SXD could facilitate the treatment of arthritis. Rats’ body weight fluctuations, joint thickness, paw thickness, and arthritis scores were all assessed during the trial and used to monitor the effectiveness of SXD treatment. The research process found that the weight of the model group rats significantly decreased, and there was some relief after SXD treatment. Compared with the model group, the joint thickness and plantar thickness of rats treated with SXD or MTX were significantly reduced. From day 42, high doses of MTX and SXD showed the beneficial benefits of reducing joint thickness and paw thickness. The high dose of SXD dramatically reduced AI starting on day 49, which was consistent with the pattern of joint thickness and paw thickness results. These RA symptoms were lessened by SXD in a dose-dependent manner (Fig. 6).

Fig. 6. Improving Effect of SXD on CIA Rats

(A) Representative pictures of hind paws were showed. (B) The body weight changes of rats. (C) The joint thickness of rats. (D) The paw thickness of rats. (E) The arthritis score of rats. Data were shown as mean ± S.D. (n = 12). ^# p < 0.05, ^## p < 0.01 vs. normal group; * p < 0.05, ** p < 0.01 vs. model group.

Effects of SXD on CIA Rats’ Organ Indexes

After SXD treatment, the main organs of the liver, spleen, thymus, and kidneys in each group of rats were collected and used to calculate organ indices. The findings demonstrated that the liver and kidney indices did not alter in response to varying SXD dosages when contrasted with the model and normal control groups. But compared to the normal group, the rats in the model group had considerably greater spleen and thymus indices. In comparison to the model group, the thymus and spleen indices in the H-SXD group were significantly lower (Fig. 7). All factors are taken into consideration, and these findings suggest that SXD significantly improves RA symptoms without having the detrimental side effects that come with other medications.

Fig. 7. Effects of SXD on CIA Rats’ Organ Indexes

(A) Liver index; (B) Kidney index; (C) Spleen index; and (D) Thymus index. Data were shown as mean ± S.D. (n = 12). ^#p < 0.05, ^##p < 0.01 vs. normal group; * p < 0.05, ** p < 0.01 vs. model group.

Effects of SXD on the Synovial Inflammation in CIA Rats

ELISA kits that are sold commercially were used to assess the biochemical indicators in the serum. Anti-inflammatory cytokines (IL-4, IL-10) were found in lower concentrations in the RA model group, while pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6) were more prevalent. Following therapy, the MTX and SXD groups’ levels of TNF-α, IL-1β, and IL-6 dropped in comparison to the model group. High doses of SXD therapy markedly elevated IL-4 and IL-10 levels (Figs. 8A–E). Furthermore, there is evidence that the development and course of RA are influenced by the expression of matrix metalloproteinases (MMPs). Therefore, we detected the secretion of MMPs, and enhanced secretion of MMPs indicates worsening of RA progression. In contrast to the model group, we observed that MTX or SXD treatment decreased previously abnormal MMP levels (MMP-2, MMP-3, MMP-9, and MMP-13) (Figs. 8F–I).

Fig. 8. SXD Inhibited Synovial Inflammation in CIA Rats

(A–E) The serum concentrations of TNF-α, IL-1β, IL-6, IL-4, and IL-10. (F–I) The serum concentrations of MMP-2, MMP-3, MMP-9, and MMP-13. (J) Histopathological images of joint tissue sections collected from CIA-induced rheumatoid arthritis rats with various treatment by H&E staining and safranin-O staining. Data were shown as mean ± S.D. (n = 12). ^# p < 0.05, ^## p < 0.01 vs. normal group; * p < 0.05, ** p < 0.01 vs. model group.

Research has shown that normal rat joint cartilage structure is normal, the joint surface is smooth, the hierarchical structure is clear, synovial cells are arranged regularly, and there is no infiltration or proliferation of inflammatory cells. When compared to the normal control group, all groups showed a significant increase in joint inflammation following CIA injection. The synovium of the model group rats showed significant proliferation, infiltration of inflammatory cells, and significant damage and erosion of articular cartilage. However, each treatment group’s degree of ankle joint damage was much better than that of the model group, exhibiting less joint damage and inflammatory cell infiltration along with a minor decrease in synovial hyperplasia (Fig. 8J). The alleviation of these symptoms suggests that SXD effectively alleviates symptoms in CIA rats. All things considered, our findings suggest that SXD usage can successfully prevent inflammation and the ensuing advancement of RA.

Effects of SXD on Serum Oxidative Stress Levels on CIA Rats

Oxidative damage is also crucial for the occurrence and development of rheumatoid arthritis, and we determined the oxidative damage-related indexes in the serum of control and treated rats. As displayed in Fig. 9, The model group of rats showed signs of oxidative stress, including increased serum MDA levels and decreased antioxidant enzyme activity (SOD, GSH-Px, and CAT). However, administration of SXD at different doses obviously decreased serum MDA levels and enhanced the activities of anti-oxidase as compared to the model group, which proved its antioxidant property. The results showed that SXD could effectively improve the imbalance of oxidative stress in the serum of CIA-induced arthritis rats, reduce the content of peroxides, and increase the activity of antioxidant enzymes.

Fig. 9. Effects of SXD on Serum Oxidative Stress Levels on CIA Rats

(A) SOD (superoxide dismutase), (B) MDA (malondialdehyde), (C) GSH-Px (glutathione peroxidases), and (D) CAT (catalase). Data were shown as mean ± S.D. (n = 12). ^# p < 0.05, ^## p < 0.01 vs. normal group; * p < 0.05, ** p < 0.01 vs. model group.

SXD Inhibited NF-κB and JAK2/STAT3 Signaling Pathway in the Synovium of CIA Rats

In order to clarify the anti-RA mechanism of SXD, we finally employed immunohistochemistry to identify protein levels in the JAK2/STAT3 and NF-κB signaling pathways. In the model group, there was a considerable rise in the expression levels of p-p65, p-JAK2, and p-STAT3 in synovial tissues, which were significantly reduced following treatment with various SXD doses. The results clearly indicated that SXD blocked the JAK2/STAT3 and NF-κB pathways, which in turn prevented the development of RA (Fig. 10).

Fig. 10. SXD Treatment Effectively Inhibited the Increase of Protein Expression of p-p65, p-JAK2, and p-STAT3 in CIA-Induced Rats by Immunohistochemical Staining Images

DISCUSSION

RA is a serious global disease with an unclear pathogenesis.⁴⁷⁾ The disability rate of this disease is high, requiring long-term treatment, and the currently used clinical treatment plans are expensive and invasive.^48,49) Traditional herbs have good application prospects in the treatment of RA, and the toxicity is minimal.⁵⁰⁾ SXD is a classic prescription commonly used in Tibetan medicine to treat RA, but the mechanism of anti-RA is still unclear. According to the basic theory of Tibetan medicine, SXD exerts anti-arthritis effects, which depend on the synergistic effect between various herbs. The main components of SXD were analyzed by UHPLC-Q-Exactive Orbitrap MS, identifying 33 compounds. The main components found were flavonoids, alkaloids, and phenolic acids. Most of the identified compounds have been reported to have anti-inflammatory activity. Research has shown that natural ingredients such as flavonoids, polyphenols, and alkaloids have made significant achievements in the treatment of RA.⁵¹⁾ Therefore, we speculate that chebulinic acid, quercetin, naringenin, kaemperol, gentiopicroside, and berberine may be representative compounds of SXD for anti-RA. Despite all this, further research is needed on the active ingredients of SXD water extract.

The inflammatory response is an important physiological mechanism that plays a crucial role in host defense and participates in the entire process of RA occurrence and development.⁵²⁾ Research has shown a strong correlation between the quantity of macrophages and the degree of joint damage and severity of RA. Macrophages are essential for the start and control of host defense.^53,54) Due to the fact that RAW264.7 macrophages are macrophages with a strong ability to swallow antigens, this study examined the effects of SXD and its representative compounds on inflammation and its molecular mechanism by creating an LPS-induced RAW2647 cell inflammation model.

Inflammatory illnesses are characterized by a pathophysiology that involves many inflammatory mediators, such as NO, TNF-α, IL-1β, IL-6, and IL-17.^55,56) There is widespread involvement of NO in inflammatory pathological and physiological processes and the immune response, and it is highly expressed in the inflammatory response. It is also considered a pro-inflammatory agent.⁵⁷⁾ TNF-α is an essential mediator of inflammation that is involved in immune system function, cell death, and inflammation.⁵⁸⁾ IL-1β induces a variety of pro-inflammatory mediators, which ultimately lead to widespread inflammation and trigger the development of RA.^15,59) The most common cytokine linked to inflammation is IL-6. Through the control of inflammatory and immunological responses, it contributes significantly to host defense.⁶⁰⁾ In addition to being an early initiator of T cell-induced inflammatory reactions, IL-17 can enhance inflammatory responses by encouraging the release of proinflammatory cytokines.⁶¹⁾ An anti-inflammatory biomarker called IL-10 has the ability to prevent immune cells from producing pro-inflammatory triggers.⁶²⁾ IL-4 is an anti-inflammatory cytokine that can balance the abnormal activity of Th1 cells and monocyte activation to alleviate RA.⁶³⁾ RA is made worse by the protracted activation of the inflammatory response brought on by the synthesis of these pro-inflammatory mediators and genes.^52,64) Therefore, inhibiting the production of pro-inflammatory mediators and pro-inflammatory factors is an effective method to inhibit the inflammatory response and block the development of RA. In this study, we observed that SXD and its representative compounds can efficiently lower NO, TNF-α, IL-1β, IL-6, and IL-17 production in LPS-induced RAW264.7 cells. This outcome is in line with the findings of in vivo experiments. Furthermore, SXD also effectively regulates the expression of key inflammatory proteins, iNOS and COX-2. LPS can activate iNOS and COX-2, which are crucial markers of the degree of the inflammatory response and are abundantly elevated during inflammation.^65,66)

Matrix metalloproteinases (MMPs), as the most important protease for degrading extracellular matrix, participate in the pathogenic mechanisms of a number of inflammatory illnesses, including rheumatoid arthritis.⁶⁷⁾ MMPs are mainly divided into five subgroups: matrix lysins, collagenases, gelatinases, membrane-type MMPs, and other MMPs.⁶⁸⁾ MMP-2 and MMP-9 belong to the gelatinase family and are two key members of the MMP family. They can cleave gelatin, type I, IV, and V collagen, and participate in the formation of pannus in RA.^69,70) MMP-3 belongs to the matrix lysin family and is a proteolytic enzyme. It is able to lyse basement membrane collagen and induce the synthesis of other MMPs, playing a crucial role in cartilage and joint erosion in RA.^71,72) MMP-13 belongs to the collagenase family and is expressed by chondrocytes and synovial cells in RA.⁷³⁾ It is believed to play a crucial role in cartilage destruction. It can not only cleave the triple helix domains of fibrous collagen types I, II, and III but also degrade proteoglycan molecules and play a dual role in matrix destruction.⁷⁴⁾ Pro-inflammatory cytokines have been demonstrated in earlier research to raise MMP expression, which in turn stimulates joint cartilage and intensifies inflammatory reactions.⁶⁸⁾ Our research results indicate that SXD can reduce the levels of MMPs in the serum of RA rats, thereby alleviating joint cartilage damage. In addition, SXD can alleviate symptoms of RA in rats, such as weight loss, decreased arthritis score, and reduced joint and paw thickness. The pathophysiological foundation of RA is the invasion of inflammatory cells and excessive synovial proliferation.⁷⁵⁾ According to the findings of the pathological section, SXD can lessen cartilage degradation, joint surface damage, and synovial hyperplasia in CIA rats, suggesting that it may have an effect on RA.

The NF-κB signaling pathway plays a crucial role in cell proliferation, differentiation, and death. It also regulates the gene expression of several cytokines, adhesion molecules, and diverse enzymes, which contributes to immune regulation and inflammatory responses.⁷⁶⁾ Several investigations have demonstrated a connection between the onset and progression of RA and the over-activation of the NF-κB signaling pathway.⁷⁷⁾ The prerequisite for NF-κB activation is the degradation of IκB, and before IκB degradation, the first occurrence is the phosphorylation of IκB.¹⁵⁾ Then, the NF-κB dimer leaves the cytoplasm and moves quickly into the nucleus, where it attaches to certain regions of the nucleus’ DNA to stimulate the transcription of genes that are linked to it.^78,79) Therefore, targeting the NF-κB pathway has always been a promising strategy in the treatment of RA. Han verified that cimifugin can lessen the inflammatory response brought on by LPS in RAW264.7 cells and prevent the NF-κB pathway from being activated.⁸⁰⁾ In addition, Linghu’s research indicates that in RA rats, the expression of p65 and IκBα significantly increased.⁸¹⁾ Based on the above research, we will focus our research on the NF-κB pathway, aiming to investigate the mechanism of SXD’s anti-RA effect. According to the research findings, SXD can considerably reduce p65 and IκBα expression in both in vitro and in vivo studies. Thus, we hypothesize that SXD inhibits the NF-κB pathway to produce its anti-RA effect.

Immune and inflammatory responses are regulated by the JAK/STAT signaling system.⁸²⁾ JAK–STAT consists of three parts: cell surface receptors, JAK, and STAT proteins.²¹⁾ When cell surface receptors are stimulated by LPS, they lead to phosphorylation of JAK, which then activates STAT through further phosphorylation, allowing it to enter the nucleus in dimeric form and bind to target genes, regulating the transcription of downstream genes.^52,55) First, the JAK/STAT signaling system controls the release of cytokines like TNF-α, IL-1β, and IL-6 in RA, which encourages the development of inflammatory responses in the synovial tissue.⁸³⁾ On the contrary, inflammation stimulates synovial tissue, and pro-inflammatory cytokines released into the bloodstream activate the JAK/STAT pathway, intensifying the inflammatory reaction.⁸⁴⁾ Furthermore, the JAK–STAT pathway can trigger immune cell proliferation, differentiation, and death in synovial tissue, as well as control the activity of immune cells, including lymphocytes and macrophages. This results in immunological regulatory effects.^20,85) In our study, SXD and its representative compounds inhibited LPS-induced phosphorylation of JAK2 and STAT3. Meanwhile, in vivo experimental results also indicate that SXD exhibits a positive effect on preventing the phosphorylation of the aforementioned proteins in RA rats. In summary, SXD may exert anti-inflammatory effects through the JAK/STAT signaling pathway.

An important component in the onset of RA is not just the inflammatory response but also oxidative stress.⁸⁶⁾ According to earlier research, RA produces a significant amount of free radicals, such as ROS, which can trigger lipid peroxidation.⁸⁷⁾ Firstly, ROS can regulate the expression of pro-inflammatory genes, leading to the degradation of articular cartilage and subsequently triggering an inflammatory response. On the contrary, the sustained production of inflammatory reactions can also exacerbate oxidative stress levels.⁸⁸⁾ Fortunately, organisms themselves have an antioxidant defense system, which can protect tissues from oxidative damage. Antioxidant enzymes are crucial throughout this process.⁸⁹⁾ Currently available research indicates that ROS generation contributes to the regulation of the JAK/STAT and NF-κB signaling pathways. Therefore, we hypothesize that ROS is essential for the inflammatory response that is controlled by the JAK/STAT and NF-κB signaling pathways.^90,91) MDA, as a product of lipid peroxidation degradation, is considered a key indicator for detecting oxidative damage.²³⁾ In this study, we found that LPS significantly raised MDA levels in RA rats and RAW 264.7 cells while causing a large drop in GSH-Px and SOD levels. However, SXD markedly lowered MDA levels while raising GSH-Px and SOD levels. Our research results indicate that the protective effect of SXD is mediated by improving the antioxidant defense system and inhibiting lipid peroxidation levels.

CONCLUSION

In summary, our findings show that SXD has anti-inflammatory properties by enhancing oxidative stress levels and notably lowering LPS-induced pro-inflammatory cytokine levels in RAW264.7 cells. Subsequent animal experiments showed that the mechanism by which SXD inhibits RA inflammation and oxidative stress is related to the activation of the NF-κB and JAK2/STAT3 pathways. These findings clarify the possible therapeutic pathways for RA while highlighting the anti-inflammatory and antioxidant properties of SXD.

Acknowledgments

This study was financially supported by the National Natural Science Foundation of China (No. 82074578 and No. 81960775), and the Shandong Provincial Natural Science Foundation (ZR2021LZY032).

Author Contributions

Authors have read and approved the manuscript. Conception and design: Yanfei Niu and Bin Li; Data acquisition and analysis: Yanfei Niu, Qianjing Feng, Mingxue Cui, Chengde Fan and Tong Wang; Review or revision of the manuscript: Ruiying Yuan, Dikye Tsering and Shan Huang.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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