Rheumatoid Arthritis Rats Research Articles

Metabolic changes of lymphocytes in a rat model of autoimmunity

Autoimmune diseases are highly prevalent in humans, being characterized by early onset and high risks of disability, thus determining the relevance of the present work and its aim, i.e., studying metabolic characteristics of lymphocytes upon the adjuvant-induced autoimmune disorder in rats. Modeling of the autoimmune process was performed in Wistar rats by subcutaneous administration of a Freund’s complete adjuvant, i.e., water-oil emulsion with heat-killed M. tuberculosis. Hematology testing (complete blood counts), biochemical markers (hydroperoxides, malondialdehyde (MDA), catalase), and cytobiochemical changes in lymphocytes (lactate dehydrogenase, succinate dehydrogenase; LDH, SDH) were followed in dynamics. X-ray examination was performed at the end of the experiment. At the initial stage of autoimmune arthritis (2 weeks), leukocytosis was registered (26.12±2.30 × 109/L, i.e., 65% over the controls, p < 0.01), thrombocytosis (675±30 × 109/L, compared with 536±27 × 109/L in controls, p < 0.01), and oxidative stress were also observed (hydroperoxides increased by 7%, and MDA, by 32%, p < 0.001); energy levels of the lymphocytes increased due to activation of LDH by 6.5%, and SDH, by 49% against the controls. At chronic stage of the disorder (7 weeks), the systemic inflammation was milder (total WBC counts of 19.6±1.40 × 109/L, compared with 13.68±0.86 × 109 /L in controls, p < 0.01, associated with shift to the right in differential conuts), along with persisting oxidative stress (MDA exceeds the control levels by 37%; decrease in catalase activity), and lower LDH activity in lymphocytes (by 43%, p < 0.01) associated by their decrease in size (the correlation quotient between the lymphocyte radius and LDH activity is rxy= 0.87). Profound molecular changes were observed in the cell energy supply: the respiratory quotient for control animals (LDH/SDH ratio) varied within 4.6-5.0. Meanwhile, in autoimmune animals, metabolic contribution of glycolysis showed a significant decrease (the quotient of 3.2 by the 2ndweek, and 2.4 by the 7thweek). On the radiograph by 7thweek, the experimental animals show uneven joint space narrowing, cyst-like formations and subchondral sclerosis of the bone heads. Autoimmune rheumatoid arthritis in rats is characterized by metabolic disorders of lymphocytes manifesting as general energy deficiency, and imbalance between glycolysis and oxidative phosphorylation pathways. These findings allow of deeper insight into pathogenesis and suggesting further search for molecular targeted therapy and prevention of the disease.

Cytokine Regulation and Fast Inflammation Resolution in Early Rheumatoid Arthritis by Cerium-Modified Gold Nanoclusters.

Rheumatoid arthritis (RA) is an incurable chronic disorder that may induce autoinflammation and serious pain in the joints. Early diagnosis and treatment are important for RA prognosis. However, there is a lack of effective and objective diagnostic approaches. Levels of several immunity cytokines were found to change for patients with early RA, including IL-6, TNF-α, and IL-17 in serum. We assumed a combined change of these cytokines could predict early RA, and a total of 37 outpatients were found. After these patients with early symptoms had been followed for more than one year, 32 clinical cases of RA were diagnosed. The accuracy rate of the current method is >86%. We assumed the symptom relief could be achieved by regulating these cytokines and serum lipid-associated indicators. Thereafter, (R)-dihydrolipoic acid (R-DHLA)-stabilized gold nanoclusters (AuNCs) without (R-DHLA-AuNCs) and with cerium modification (R-DHLA-AuNCs-Ce) were employed for treatment of the RA rat model in vitro and in vivo. R-DHLA-AuNCs-Ce exhibited extraordinary reactive oxygen species-scavenging and anti-inflammation effects by regulating macrophage polarization, which was found to be more effective than methotrexate. The inflammation response of the joint microenvironment was also reduced with an exciting efficiency. By complex analysis of the pro-inflammatory cytokines and activity period indicators in vivo and in vitro, we concluded that macrophage-mediated inflammation exacerbated autoimmunity, which fully relieved the symptoms after administration of R-DHLA-AuNCs-Ce to RA rats.

Metabolic characteristics and pharmacokinetic differences of Qiwei Tongbi oral liquid in rheumatoid arthritis rats.

Qiwei Tongbi oral liquid (QWTB), a classical traditional Chinese medicine formula, is widely used to treat arthritis-related diseases in clinical practice. Currently, in vivo metabolic characteristics and pharmacokinetic studies are lacking. This study analyzed the prototype components of QWTB absorbed in the blood and their metabolic transformation process after intragastric administration and compared the differences in pharmacokinetic properties between healthy and rheumatoid arthritis model rats. In sum, 17 prototype components and 21 related metabolites were identified in the plasma and urine of the treated rats. Metabolites were derived from sinomenine and magnoflorine. Through systematic methodology verification, an accurate and stable detection method for sinomenine and magnoflorine in plasma samples was established and applied to pharmacokinetic research of QWTB. At the three dose levels, the AUC0-∞ (area under the curve) of the two components showed a good positive correlation with the dose (R2 > 0.9). Compared with healthy rats, the Tmax , t1/2z , and AUC of sinomenine were markedly increased, and Cmax was decreased in rheumatoid arthritis model rats, indicating that the rate of absorption and elimination rate decreased, but the body exposure increased. However, there were no significant differences in the pharmacokinetic parameters of magnoflorine under healthy and pathological conditions. In summary, the main active ingredients of QWTB are sinomenine and magnoflorine, which exhibit linear kinetic characteristics within a set dose range, and the rheumatoid arthritis pathological state is more conducive to the absorption and efficacy of sinomenine. The results of this study demonstrate the rationality of the clinical application of the QWTB.

High-Throughput Chinmedomics Strategy Discovers the Quality Markers and Mechanisms of Wutou Decoction Therapeutic for Rheumatoid Arthritis.

Wutou decoction (WTD) is a traditional Chinese medicine prescription for the treatment of rheumatoid arthritis (RA), and this study systematically analyzed the metabolic mechanism and key pharmacodynamic components of WTD in RA rats by combining untargeted metabolomics and serum pharmacochemistry of traditional Chinese medicine to enrich the evidence of WTD quality markers (Q-markers) studies. WTD prevented synovial edema in RA rats and reduced tumor necrosis factor-alpha and interleukin 6 levels in rat serum, according to the results of an enzyme-linked immunosorbent examination and histopathological inspection. In model rats, pattern recognition and multivariate statistical analysis revealed 24 aberrant metabolites that disrupted linoleic acid metabolism, arachidonic acid metabolism, arginine and proline metabolism, etc. However, continued dosing of WTD for 28 days reversed 13 abnormal metabolites, which may be an important therapeutic mechanism from a metabolomic perspective. Importantly, 12 prototypical components and 16 metabolites from WTD were characterized in RA rat serum. The results of Pearson correlation analysis showed that aconitine, L-ephedrine, L-methylephedrine, quercetin, albiflorin, paeoniflorigenone, astragaline A, astragaloside II, glycyrrhetic acid, glycyrrhizic acid, licurazide, and isoliquiritigenin are the key pharmacological components that regulate the metabolism of RA rats, and they are identified as Q-markers. In sum, utilizing metabolomics and serum pharmacochemistry of traditional Chinese medicine, the metabolic mechanisms and Q-markers of WTD therapy in RA rats were revealed, providing a theoretical basis for the quality control investigation of WTD.

Combined 16S rRNA gene sequencing and metabolomics to investigate the protective effects of Wu-tou decoction on rheumatoid arthritis in rats

Wu-tou decoction (WTD) is a traditional Chinese medicine (TCM) formula which has been used for treating rheumatoid arthritis (RA) for a thousand years. However, the underlying mechanism of WTD in treating RA is still unclear. In recent years, more and more attention has been paid to the role of gut microbiota and microbiota-derived metabolites in the treatment of RA. Hence, this study aims to investigate the roles of microbiota and microbial metabolites in the treatment of RA with WTD. Firstly, the therapeutic effects of WTD on adjuvant-induced arthritis (AIA) rats were evaluated. Then, the 16S rRNA sequencing analysis was used to clarify the changes of the intestinal microbiota and obtain the key microbiota affected by WTD. The important microbial metabolites were quantitated to explore the metabolic characteristics of WTD against RA by targeted metabolomics method. Finally, correlation analysis was performed to investigate the functional correlation among the gut microbiota, metabolites and RA-related serum indexes. The results indicated that WTD could relieve arthritis and reverse gut microbiota dysbiosis. The variation of short-chain fatty acids, bile acids, tryptophan metabolites and amino acids, which are important microbial metabolites, were reversed by WTD intervention. The correlation studies proved that WTD could regulate inflammation and intestinal barrier function partially by modulating Bacteroides, Prevotella, Akkermansia and their associated acetic acid, butyric acid, cholic acid and indole propionic acid. The anti-RA effects of WTD were partially mediated by gut microbiota and microbial metabolites. This study provides a new insight for treating RA and highlights the importance of gut microbiota in the treatment of diseases.

Effect of Acupuncture Stimulation on Improving the Immunity of Synovial Tissue in Rheumatoid Joints via MAPK Pathway

Purpose: Acupuncture treatment of rheumatoid arthritis (RA) has a long history and has been widely recognized in the medical community. However, its mechanism of action still is rarely uncovered. In the present study, the effects of acupuncture on the immune function and MAPK pathway in synovial tissue of rats with RA were investigated; it aimed to explore the effect and mechanism of acupuncture on alleviating RA.<br/> Methods: Rats were randomly divided into control, model and acupuncture groups, 15 rats each group, and RA rat model was induced via bovine type II collagen in complete Freund's adjuvant. The acupuncture group received "Zusanli" and "Shenshu" warm acupuncture treatment on the first day after modeling, acupuncture stimulation once a day for 21 days. The rat weight and degree of joint swelling were recorded and calculated. The ankle joint histopathology was observed by H&E staining. Serum inflammatory cytokines were detected using ELISA. An automatic biochemical analyzer was used to test serum IgG, IgM and IgA levels. Transcription factors T-bet, GATA3, RORγT, Foxp3 mRNA were monitored through RT-qPCR technology. Further, the expression of key targets of MAPK signal or phosphorylation in synovial tissue were determined using Western blotting.<br/> Results: Acupuncture relieved the joint swelling and weight loss of RA rats, ameliorated the pathological damage of the ankle joint, significantly increased the serum IgG, IgM, IgA and T-bet, and Foxp3 levels, and reduced the levels of IL-1β, IL-6, TNF-α, GATA3, and RORγT; down-regulated synovial tissue p-p38MAPK, p-JNK, and p-ERK1/2 levels and protein phosphorylation ratio (P <0.05).<br/> Conclusion: Acupuncture stimulation can improve the joint damage and promote the immune function of RA rats, possibly via blocking the expression of MAPK pathway in synovial tissue. This study provides a scientific and promising reference for acupuncture further in the clinical strategy of RA.

Galangin alleviates rheumatoid arthritis in rats by downregulating the phosphatidylinositol 3-kinase/protein kinase B signaling pathway

ABSTRACT Rheumatoid arthritis (RA) is a chronic autoimmune disease that greatly affect patients’ quality of life. Galangin extract is renowned for its anti-proliferative and anti-oxidative characteristics. However, galangin cytotoxicity studies are presently inadequate. We aimed to investigate the therapeutic potential of galangin on RA by investigating the PI3K/AKT signaling pathway.Fibroblast-like synovial cells (FLSs) were exposed to lipopolysaccharide (LPS) to establish an RA model in vitro. An ELISA assay was used to detect the levels of IL-1β, TNF-α, and IL-6. Cell viability and apoptosis were determined by CCK8/EdU and flow cytometry assays. A western blot assay was used to analyze the protein expression levels. An RA rat model was established to evaluate the function of galangin through histopathological examination. Our results found that galangin induced apoptosis, inhibited cell proliferation, and increased cell invasion of rheumatoid arthritis fibroblast-like synovial cells (RAFLSs). Galangin inactivated the PI3K/AKT signaling pathway and the inflammatory response. An agonist of PI3K signaling, 740Y‐P, restored the cellular functions of RAFLSs. Moreover, galangin suppressed the development of RA in vivo. Galangin effected its anti-arthritic influence through the PI3K/AKT signaling pathway. Galangin has potential as an alternative treatment for RA.

Effects of Muscone on Cartilage Morphology and Matrix Metalloproteinases-3 (MMP-3h) and Matrix Metalloproteinases-9 (MMP-9) Expression in Rheumatoid Arthritis Rat Models

Aim: To discuss Muscone treatment in Rheumatoid Arthritis Rat Models and relative mechanisms. Materials and methods: Dividing 36 rats as 4 groups as Normal, Model, DMSO and Muscone groups (n = 9). Rats of Model, DMSO and Muscone groups were made Rheumatoid Arthritis model. Muscone group were treated with 2 mg/kg Muscone after modeling. HE staining and Masson staining were used to observe the morphological changes of cartilage tissue, measuring MMP-3 and MMP-9 expression by RT-PCR, Western Blotting (WB) and Immunohistochemistry (IHC). Results: Compared with Model group, the pathological changes of Muscone group was significantly improved and average optical density of collagen fibers was significantly depressed (P < 0.001, respectively) via MMP-3 and MMP-9 proteins significantly depressing (P < 0.001, respectively). Conclusion: Muscone improved Rheumatoid Arthritis by depressing MMP-3 and MMP-9 proteins in vivo study.

Dimethylamino group modified polydopamine nanoparticles with positive charges to scavenge cell-free DNA for rheumatoid arthritis therapy.

Excessive cell-free DNA (cfDNA) released by damaged or apoptotic cells can cause inflammation, impacting the progression of rheumatoid arthritis (RA). cfDNA scavengers, such as cationic nanoparticles (NPs), have been demonstrated as an efficient strategy for treating RA. However, most scavengers are limited by unfavorable biocompatibility and poor scavenging efficacy. Herein, by exploiting the favorable biocompatibility, biodegradability and bioadhesion of polydopamine (P), we modified P with dimethylamino groups to form altered charged DPs to bind negatively charged cfDNA for RA therapy. Results showed that DPs endowed with superior binding affinity of cfDNA and little cytotoxicity, which effectively inhibited lipopolysaccharide (LPS) stimulated inflammation in vitro, resulting in the relief of joint swelling, synovial hyperplasia and cartilage destruction in RA rats. Significantly, DPs with higher DS of bis dimethylamino group exhibited higher positive charge density and stronger cfDNA binding affinity, leading to excellent RA therapeutic effect among all of the treated groups, which was even close to normal rats. These finding provides a novel strategy for the treatment of cfDNA-associated diseases.

Efficient and automatic synthesis of TSPO PET ligand [18F]-GE-180 and its application in rheumatoid arthritis model

Rheumatoid arthritis (RA) is a common autoimmune disease characterized by chronic synovial inflammation, which ultimately leads to joint deformity and dysfunction. [18F]-GE-180 is a specific PET tracer of the 18 kDa translocator protein (TSPO), which is overexpressed on activated macrophages, and proposed as a biomarker of inflammation. Our study addresses the need to streamline the automatic synthesis of [18F]-GE-180 to make it more accessible for routine production and widespread clinical evaluation and application. The nucleophilic radiofluorination was performed on an AllinOne synthesis module by SPE purification method, and the formulated [18F]-GE-180 was obtained in non-decay corrected radiochemical yields of 69 ± 1.8% in 32 min. PET/CT imaging in animal model showed that [18F]-GE-180 highly concentrated in joints from RA rats. This methodology facilitates efficient synthesis of [18F]-GE-180 in a commercially available synthesis module and shows potential diagnosis performance in RA models.

Therapeutic Effect of Nile Tilapia Type II Collagen on Rigidity in CD8+ Cells by Alleviating Inflammation and Rheumatoid Arthritis in Rats by Oral Tolerance.

Fibrillins are microfibril-associated macro glycoproteins found in connective tissues and structurally related to latent TGF-β-binding proteins (LTBPs). The special cellular immunity and blocking glycoprotein receptors IIb and IIIa of fibrillins are emerging topics in recent years. In this study, Nile Tilapia type IIcollagen (NTCII) was extracted and purified from the skull cartilages by a pepsin-soluble method. Amino acid analysis indicated that NTCII consisted of 315/1000 glycine residues, 72/1000 hydroxyproline residues and 108/1000 proline residues. SDS-PAGE analysis showed that NTCII was composed of three identical 130 kDa α-chains. The results of glycoprotein/carbohydrate assay indicated that the total polysaccharide content of NTCII was 5.6–19.0%. The IR spectrum of NTCII displayed five characteristic peaks of amide I, II, III, A, B. NTCII at 10–100 μg/mL concentration downregulated the content of cytokines in the presence or absence of LPS, especially the secretion of cytokines IL-6, IL-1β and TNF-α. Interestingly, NTCII promoted the secretion of Fas/Apo-1 compared to the control group and 25 μg/mL of NTCII resulted in a higher Fas/Apo-1 secretion level in CD8+ T cells. FITC-TCII fluorescence images confirmed that NTCII could bind to the membrane surface of CD8+ T cells, leading to the induction of rigidity. NTCII could bind to the membrane surface of CD8+ T cells that leads to the induction of rigidity, as evidenced by the FITC-NTCII fluorescence images. The qRT-PCR gene expression analysis of caspase-8 collected with Fas/Apo-1 was upregulated significantly in the 1 and 50 μg/mL NTCII-treated groups compared with the control group. Overall, the results conclude that the rigidity did not lead to an increase in inflammatory factors in CD8+ T cells treated with NTCII. The oral administration of NTCII 3 mg/kg dosage caused more prominent repair of damaged ankle cartilage than the 1 mg/kg dosage in Freund’s adjuvant-induced model of arthritis in rats. Therefore, this study disclosed the immunological and anti-arthritic effect of fibrillar collagen, which could be a potential biomaterial for practical applications with lower toxicity.

Exosomal microRNA-140-3p from human umbilical cord mesenchymal stem cells attenuates joint injury of rats with rheumatoid arthritis by silencing SGK1

ObjectiveOver the years, microRNAs (miRNAs) have been involved in the pathogenesis of rheumatoid arthritis (RA). We aim to investigate the role of human umbilical cord mesenchymal stem cells (HUCMSCs)-derived exosomal miR-140-3p in RA development.MethodsExosomes(exo) were isolated from human umbilical cord-derived mesenchymal stem cells (HUCMSCs), and this isolation was followed by the transfer of miR-140-3p. RA rat models were constructed by collagen II adjuvant and respectively treated with HUCMSCs-exo or HUCMSCs-exo carrying miR-140-3p mimic/inhibitor, and expression of miR-140-3p and serum- and glucocorticoid-inducible kinase 1 (SGK1) was assessed. Then, RA score and inflammation scoring, fibrosis degree and apoptosis, serum inflammatory response and oxidative stress in joint tissues were determined. The RA synovial fibroblasts (RASFs) were extracted from rats and identified. Conducted with relative treatment, the migration, proliferation and apoptosis in RASFs were determined.ResultsMiR-140-3p was decreased while SGK1 was increased in RA rats. HUCMSCs-exo or upregulated exosomal miR-140-3p improved pathological changes and suppressed inflammation, oxidative stress and fibrosis in RA rats, and also constrained and RASF growth. Overexpression of SGK1 reversed the inhibition of RASF growth caused by overexpression of miR-140-3p.ConclusionUpregulated exosomal miR-140-3p attenuated joint injury of RA rats by silencing SGK1. This research provided further understanding of the role of exosomal miR-140-3p in RA development.

Lactiplantibacillus plantarum HG20 attenuates II type collagen-induced rheumatoid arthritis in rats via anti-inflammatory and inhibition of apoptosis.

This study aimed to explore the therapeutic effects of Lactiplantibacillus plantarum HG20 (HG20) on collagen-induced arthritis (CIA) rats and its mechanism. CIA rats were established by injecting bovine type II collagen for 7days, and treated by intragastric administration HG20 for 21days. The foot palm temperature and arthritis score were measured once a week. The pathological changes in the knee joint were observed by hematoxylin and eosin staining. The levels of cytokines were detected by enzyme linked immunosorbent assay, and the effects of HG20 on inflammatory and apoptosis pathway of spleen cells were detected by western blot analysis. The results indicated that HG20 reduced the joint swelling degree and foot palm temperature, inhibited the development of joint histopathology, decreased the levels of pro-inflammatory cytokines, down-regulate the expression of pro-inflammatory cytokines by nuclear factor kappa-B pathway, and inhibited the apoptosis of spleen cells by inhibiting phosphatidylinositol 3-kinase/protein kinase B pathway and regulating apoptosis pathways. HG20 had an adjuvant therapeutic effect on arthritis in CIA rats, and its mechanism might be related to the inflammatory and apoptosis pathway. These results revealed that HG20 could be used as a functional probiotic in the field of food and medical, and which played a potential role in the prevention and treatment of arthritis.

Quercetin mitigates rheumatoid arthritis by inhibiting adenosine deaminase in rats

Rheumatoid arthritis (RA) is a chronic inflammatory joint disease characterized by synovial proliferation and bone destruction. Adenosine deaminase (ADA) is a key inflammatory enzyme that increases joint stiffness and pain in RA. In this study, we evaluated the in-silico, and in vivo inhibitory effect of quercetin isolated from Egyptian Fenugreek on ADA enzyme activity. We also determined the combinatorial effect of quercetin on methotrexate mediated anti-inflammatory efficacy and toxicity. In-silico molecular docking was conducted and confirmed in an in vivo RA rat model. The results showed that the inhibition constant of quercetin on joint ADA by docking and in-vitro was 61.9 and 55.5 mM, respectively. Therefore, quercetin exhibits anti-inflammatory effect in a rat RA model as evidenced by reducing the specific activity of ADA in joint tissues, lower jaw volume, enhance body weight, downregulate ADA gene expression, reduce levels of RA cytokines interleukin-1β, interleukin-6, tumor necrosis factor-α, also, rheumatoid factor, C-reactive protein, and anti-cyclic citrullinated peptide RA biomarker levels. These findings demonstrate that the purified quercetin has a promising anti-inflammatory effect against RA disease through its inhibitory effects on the ADA enzyme. Furthermore, isolated quercetin improved the anti-inflammatory efficacy of methotrexate, reduced its toxic effects by increasing antioxidant enzymes and reducing oxidative stress.

Ameliorative effect of cerium oxide nanoparticles against Freund's complete adjuvant-induced arthritis.

Aim: To assess the mechanistic effects of cerium oxide nanoparticles (CONPs) on Freund's complete adjuvant (FCA)-induced rheumatoid arthritis in rats. Methods: CONPs were characterized and evaluated in vitro (RAW 264.7 macrophages) and in vivo (FCA-induced rheumatoid arthritis model). Results:In vitro treatment with CONPs significantly reduced lipopolysaccharide-induced oxidative stress (as evident from dichlorodihydrofluorescein diacetate staining), diminished mitochondrial stress (as observed with tetraethylbenzimidazolylcarbocyanine iodide staining) and reduced superoxide radicals. In vivo, CONPs exhibited anti-rheumatoid arthritis activity, as evident from results of paw volume, x-ray, clinical scoring, levels of cytokines (IL-17, IL-1β, TNF-α and TGF-β1) and histology. Conclusion: We provide preclinical proof that CONPs may be a novel futuristic nanoparticle-based approach for therapy of rheumatoid arthritis.

Yunnan Baiyao Ameliorates Rheumatoid Arthritis in Rats by Shifting the Th17/Treg Cell Balance and Preventing Osteoclast Differentiation.

Yunnan Baiyao (YNB) is a traditional Chinese medicine that possesses anti-inflammatory effects. Previously, we have demonstrated the effects of YNB in rheumatoid arthritis (RA) animal models; however, the underlying mechanisms are unclear. In the present study, we aimed to investigate the effects of YNB on the T-helper (Th)17/T-regulatory (Treg) cell balance in a collagen-induced arthritis rat model orally administrated YNB or methotrexate, a widely used therapeutic agent for treating RA. Our results showed that YNB treatment significantly decreased the voix pedis thickness and joint functionality scores and alleviated joint histopathology in these rats. These YNB-induced effects were achieved by decreasing the number of Th17 cells and increasing that of Treg cells in the spleen. Moreover, the interleukin- (IL-) 17 level considerably decreased in the serum of YNB-treated rats, whereas the IL-10 level significantly increased. Furthermore, YNB could inhibit RANKL-induced osteoclast formation by regulating the tumor necrosis factor receptor-associated factor 6/NF-κB/nuclear factor of the activated T-cell pathway. In summary, our study shows that YNB exhibits antiarthritic activity by decreasing the ratio of Th17/Treg cells, regulating the cytokine balance, and inhibiting osteoclast activation, providing an experimental basis that supports the use of this traditional Chinese medicine for the clinical treatment of RA.

Effect of moxibustion on autophagy and the inflammatory response of synovial cells in rheumatoid arthritis model rat.

To investigate the effect of moxibustion on synovitis and the autophagy of synoviocytes in rheumatoid arthritis (RA). Forty Sprague-Dawley rats were randomly divided into a normal group, model group, moxibustion group, cigarette moxibustion group, and medicine group, with eight rats included in each group. The RA model was established by subcutaneous injection of complete Freund's adjuvant into the left posterior toe. Rats in the model group were not interfered with. In the moxibustion group, rats were treated by moxibustion, where a 1-cm diameter moxa stick was applied at the left Zusanli (ST 36) point. The distance of the moxa stick to the skin was 2 cm and moxibustion was completed for 20 min daily for 15 d total. In the cigarette moxibustion group, the moxa stick was replaced by a common cigarette. In the medicine group, rats were treated with a tripterygium glycoside suspension (8 mg/kg) once a day for 15 d total. In each group, the left hind limb toe volume was measured with a toe volume meter; the synovial cells were observed by hematoxylin and eosin staining; the interleukin (IL)-4, IL-6, IL-10, IL-1β, IL-23, IL-17, and tumor necrosis factor (TNF)-α levels in serum were measured by enzyme-linked immunosorbent assay; the erythrocyte sedimentation rate (ESR) were detected by Westergren sedimentation rate testing; the C-reactive protein (CRP) and rheumatoid factor (RF) levels in serum were detected by rate nephelometry; the expression levels of ULK1, autophagy-associated protein (Atg)3, Atg5, and Atg12 messenger RNA (mRNA) in synovium were detected by real time-quantitative polymerase chain reaction (RT-qPCR); and the protein expression levels of phosphatidylinositol-3-kinase (PI3K), protein kinase B (Akt), mammalian target of rapamycin (mTOR), LC3-II, beclin-1, phosphorylated-PI3K (p-PI3K), p-Akt, p-mTOR in synovium were detected by Western blotting. Among the RA model rats, joint swelling, an inflammatory reaction, and the proliferation of synovial tissue were obvious and the signal of the PI3K/Akt/mTOR pathway was active, while autophagy was inhibited. Moxibustion at Zusanli (ST36) or intragastric administration of Tripterygium wilfordii glycosides could alleviate the inflammatory reaction of RA rats; relieve the swelling of the toes; downregulate the levels of ESR, CRF, RF; lower the levels of IL-6, IL-1β, TNF-α, and IL-17; and increase the IL-4 and IL-10. At the same time, the mRNA expression levels of ULK1, Atg3, Atg5, and Atg12 and those of LC3-Ⅱ and beclin-1 were increased, while the PI3K, Akt, mTOR, p-PI3K, p-Akt, p-mTOR were decreased. Cigarette moxibustion did not significantly reduce the swelling of the toe joint in RA rats, and was not as good as that of moxibustion or Tripterygium wilfordii polyglycosides in the effects of inflammation relief and the influences of the levels of ESR, CRF, RF. While cigarette moxibustion has a weak effect to affect the expression of corresponding molecules in autophages and the expression level of the autophagy biomaker in synovial tissue. Moxibustion and tripterygium glycosides can significantly reduce the joint swelling, relieve synovitis and synovial hyperplasia, and inhibit the PI3K/Akt/mTOR signaling pathway to increase autophagy in a manner superior to cigarette moxibustion. Moxibustion can limit the proliferation of synoviocytes in RA rats by inhibiting the PI3K/Akt/mTOR signaling pathway, promoting autophagy, effectively reducing synovitis, and alleviating joint swelling.

The method of Yiqi Yangyin Tongluo can attenuate the pyroptosis of rheumatoid arthritis chondrocytes through the ASIC1a/NLRP3 signaling pathway.

BackgroundBased on the ASIC1a/NLRP3 signaling pathway, we explored the specific molecular mechanism of the pyroptosis of rheumatoid arthritis (RA) chondrocytes by the method of nourishing qi, nourishing yin, and dredging collaterals to provide new ideas for the treatment of this disease.MethodsA total of 50 rats were divided into a normal group, model group, methotrexate group, Yiqi Yangyin Tongluo group, and combined group. Except for the normal control group, the other groups used Freund's complete adjuvant (FCA) to make RA rat model. The arthritis index and ankle joint swelling of rats in each group were recorded. HE staining and ELISA were used to assess the pathology of the ankle joint of each group of rats and the content of IL-1β and IL-18 in rat serum. Furthermore, immunofluorescence and qPCR methods were used to detect the protein and mRNA expression levels of NLRP3, caspase 1, ACS, and ASIC1a in the cartilage tissue of each group of rats.ResultsCompared with the normal group, the right hind foot joint of the model group was significantly swollen, the levels of IL-18 and IL-1β in the serum of rats increased significantly, and the mRNA and protein levels of NLRP3, caspase 1, ACS, and ASIC1a in the chondrocytes also increased significantly. Compared with the model group, the degree of ankle joint swelling and IL-18 and IL-1β content in rat serum in each medication group was significantly reduced, and the combined group showed the greatest reduction compared with the other groups. After 8 weeks of treatment, compared with the model group, the mRNA and protein levels of NLRP3, caspase 1, ACS, and ASIC1a in the chondrocytes of each medication group were down-regulated. HE staining found that there were large numbers of infiltrating inflammatory cells and pannus in the joint tissue of the model group, while only a small amount of inflammatory cell infiltration and pannus was seen in the joint tissue of the rats in each treatment group.ConclusionsThe method of Yiqi Yangyin Tongluo can attenuate the pyroptosis of RA chondrocytes through the ASIC1a/NLRP3 signaling pathway.

Thermal and wine processing enhanced Clematidis Radix et Rhizoma ameliorate collagen Ⅱ induced rheumatoid arthritis in rats

Ethnopharmacological relevanceClematidis Radix et Rhizoma, a kind of traditional Chinese medicine, is derived from Clematis chinensis Osbeck, Clematis hexapetala Pall. and Clematis manshurica Rupr. This herb shows great effects on expelling wind and dispelling dampness in ancient and it has anti-inflammatory and analgesic activity in modern clinical application. Aim of the studyThis experiment aimed to research anti-rheumatoid arthritis effect of crude and wine processed RC based on glycolysis metabolism to provide new ideas treating RA. Materials and methodsNetwork pharmacology was applied to preliminarily forecast the potential pathways of common targets of RC and RA. RAW264.7 macrophages were induced by LPS, NO production, glucose uptake, lactate production, ROS and MMP were detected as instructions in vitro. ELISA was used to measure the content of HK2, PKM2 and LDHA involving in glycolysis process. Gut microbiota was analyzed by 16S rRNA gene amplicon sequencing in CIA rats. ResultsCrude and wine processed RC had good anti-inflammatory effect by reducing NO in RAW264.7 macrophages and ameliorating inflammatory infiltration and cartilage surface erosion in CIA rats. Whether in LPS-induced macrophages or CIA rats, crude and wine processed RC could inhibit glycolysis by down-regulating the expression of PKM2, causing less glucose uptake and lactic acid, which lead to less ROS and higher MMP to normal. PI3K-AKT and HIF-1α pathways were deduced to possibly play a crucial part in controlling glycolysis metabolism by network pharmacology analysis. Besides, it was displayed that Firmicutes and Bacteroidetes were prominent gut microbiota in CIA rats feces. CC-H and PZ-H groups could both increase the relative abundance of Firmicutes and decrease Bacteroidetes. These microbiota also played a role in RA pathological process via involving in energy metabolism, carbohydrate metabolism and immune system. ConclusionCrude and wine processed RC have a good influence in ameliorating rheumatoid arthritis by inhibiting glycolysis and modulating gut microbiota together.

System pharmacology analysis to decipher the effect and mechanism of active ingredients combination from herb couple on rheumatoid arthritis in rats

Ethnopharmacological relevanceTraditional herb couple Angelicae pubescentis radix (APR) and Notopterygii rhizoma et radix (NRR), composition of two traditional Chinese medicinal herbs, has been used clinically in China for the treatment of rheumatoid arthritis (RA) over years. APR and NRR contain coumarins and phenolic acids, which have been reported to have analgesic and anti-inflammatory activities. Aim of the studyThe active ingredients combination (AIC) and potential therapeutic mechanism of APR and NRR (AN) herb couple remain unclear. Therefore, the present study aimed to identify the AIC and elucidate the underlying mechanism of AIC on RA. Materials and methodsFirstly, a novel strategy of in vitro experiments, computational analysis, UPLC-QTOF-MS and UPLC-QQQ-MS was established to confirm the optimum ratio of AN herb couple samples and identified the AIC. Then, the anti-arthritis effects of the optimal herb couple and AIC were studied with Collagen II induced rheumatoid arthritis (CIA) rats in vivo. Finally, an integrated model of network pharmacology, metabolomics, gut microbiota analysis and biological techniques were applied to clarify the underlying mechanism through a comprehensive perspective. ResultsAN7:3 herb couple was regarded as the optimal ratio of AN herbal samples, and AIC was screened as osthole, columbianadin, notopterol, isoimperatorin, psoralen, xanthotoxin, bergapten, nodakenin and bergaptol respectively. Additionally, AIC exerted similar therapeutic effects as AN 7:3 in CIA rats. Moreover, AIC ameliorated RA might via regulating MAPK signaling pathway, altering metabolic disorders and gut microbiome involved autoimmunity. Conclusionsour findings provided scientific evidence to support that AIC of AN herb couple could be used as a prebiotic agent for RA. Importantly, this research provided a systematic and feasible strategy to optimize the proportion of medicinal materials and screen AIC from multi-component traditional Chinese herb couples or Chinese medicine formulae. Moreover, it provided a comprehensive perspective to discover AIC, clarify the overall effects and understand the mechanisms for natural products through the perspective of database and multi-omics integration.