Rat Model Of Rheumatoid Arthritis Research Articles

Associations between Gut Microbiota and Microbial Metabolites in Adjuvant- induced Arthritis Rats with Moist Heat Arthralgia Spasm Syndrome.

Rheumatoid Arthritis (RA) is a chronic systemic autoimmune disease. According to Traditional Chinese Medicine (TCM) syndromes theory, moist heat arthralgia spasm syndrome is the most prevalent syndrome of RA patients in the active period. However, the mechanism of alteration of gut microbiota in RA with moist heat arthralgia spasm syndrome has not been reported until now. This study focused on the alteration of gut microbiota in adjuvant-induced arthritis rats with moist heat arthralgia spasm syndrome, elaborated its regulation mechanism, and analyzed the associations between gut microbiota and microbial metabolites. The disease-syndrome combination rat model of RA with moist heat arthralgia spasm syndrome was constructed with Adjuvant-Induced Arthritis (AIA) under damp-heat stimulating. Enzyme-Linked Immunosorbent Assay (ELISA) was used to measure serum biochemical indicators. Damages of ankle joints were observed using hematoxylin and eosin (H&E). 16 small ribosomal subunit RNA (16S rRNA) gene sequencing was conducted to assess the gut microbiota composition and function on feces from rats. Alterations in fecal metabolites profiling were evaluated by fecal metabolomics through Liquid Chromatography-Mass Spectrometry (LC-MS) and Gas Chromatography-Mass Spectrometry (GC-MS). Pearson correlation analysis was performed to explore the associations of altered gut microbiota and microbial metabolites in Model rats. The imbalance of gut microbiota in Model rats was accompanied by metabolic disorders. Lactobacillus, Prevotellaceae_NK3B31_group, Allobaculum, Prevotellaceae_UCG_001, Alloprevotella, and Dubosiella were found to be dominant genera in Model rats. In total, 357 metabolites were significantly altered in Model rats and predominantly enriched into fatty acid degradation and glycerophospholipid metabolism. Pearson correlation analysis showed that TNF-α and IL-1β were associated with Prevotellaceae_Ga6A1_group and 3R-hydroxy-docosan-5S-olide, alpha-N-(3-hydroxy-14-methyl-pentadecanoyl)-ornithine, 17-methyl-trans-4,5- methylenenona-decanoic acid, Semiplenamide F. The key differential microbiota genera and differential microbial metabolites may become important targets for the treatment of RA and provide the theoretical basis for exploring the pathogenesis of RA.

Visnagin alleviates rheumatoid arthritis via its potential inhibitory impact on malate dehydrogenase enzyme: in silico, in vitro, and in vivo studies

Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disorder. The present study aimed to evaluate the in silico, in vitro, and in vivo inhibitory effect of visnagin on malate dehydrogenase activity and elucidate its inflammatory efficacy when combined with methotrexate in the RA rat model. The molecular docking, ADMET simulations, MDH activity, expression, and X-ray imaging were detected. Moreover, CRP, RF, (anti-CCP) antibody, (TNF-α), (IL-6), (IL-17), and (IL-10) were evaluated. The expression levels of MMP3 and FOXP3 genes and CD4, CD25, and CD127 protein levels were assessed. Histological assessment of ankle joints was evaluated. The results revealed that visnagin showed reversible competitive inhibition on MDH with inhibitory constant (Ki) equal to 141 mM with theoretical IC50 equal to 1202.7 mM, LD50 equal to 155.39 mg/kg, and LD25 equal to 77.69 mg/kg. In vivo studies indicated that visnagin exhibited anti-inflammatory effects through decreasing MDH1 activity and expression and induced proliferation of anti-inflammatory CD4+CD25+FOXP3 regulatory T cells with increasing the anti-inflammatory cytokine IL-10 levels. Moreover, visnagin reduced the levels of inflammatory cytokines and the immuno-markers. Our findings elucidate that visnagin exhibits an anti-inflammatory impact against RA through its ability to inhibit the MDH1 enzyme, improve methotrexate efficacy, and reduce oxidative stress.Graphical

Polydatin and chitosan-silver co-loaded nanocomplexes for synergistic treatment of rheumatoid arthritis via repolarizing macrophages and inducing apoptosis of fibroblast-like synoviocytes

Rheumatoid arthritis (RA) is a chronic and refractory autoimmune disease that primarily affects the synovium of diarthrodial joints. Inflammatory macrophages and fibroblast-like synoviocytes (FLS) in the synovial microenvironment produce pathogenic mediators such as cytokines and proteases that perpetuate immune-mediated inflammation and contribute to the destruction of cartilage and bone. Polydatin (PD), a natural active compound, has demonstrated potential anti-inflammatory and anti-arthritic effects. However, drug development and delivery of PD is still a great challenge owing to its low solubility, short half-life, and high dose requirement. In order to overcome these drawbacks, we developed a novel nanodrug system named HA-M@PB@Ag@PD NPs. This system is composed of hybrid membrane (M), hyaluronic acid (HA), Prussian blue nanoparticles (PB NPs), PD, and chitosan-silver (Chi-Ag). In vitro experiments demonstrated that HA-M@PB@Ag@PD NPs effectively cleared ROS, promoted the repolarization of inflammatory macrophages, and induced apoptosis of RA-FLS. Using a rat model of RA, HA-M@PB@Ag@PD NPs markedly suppressed joint inflammation, inhibited synovial hyperplasia, and protected joints against destruction of cartilage and bone. Moreover, HA-M@PB@Ag@PD NPs significantly improved the synovial microenvironment of arthritic rats by reducing the number of RA-FLS and inflammatory macrophages, and facilitating the repolarization of inflammatory macrophages.

Study on the anti-inflammatory mechanism of moxibustion in rheumatoid arthritis in rats based on phospholipaseA2 signaling inhibition by Annexin 1.

To determine whether moxibustion had an anti-inflammatory effect on rheumatoid arthritis (RA) by regulating Annexin 1 expression and interfering with the phospholipaseA2 signaling pathway. Thirty male Sprague-Dawley rats were randomly categorized into five groups (six rats per group): blank control (CON) group, RA model (RA) group, moxibustion (MOX) group, Annexin 1 lentiviral intervention (RNAi-Anxa1) group, and Annexin 1 lentiviral intervention + moxibustion (RNAi-Anxa1 + MOX) group. The rats in the RNAi-Anxa1 and the RNAi-Anxa1 + MOX groups were injected with the lentiviral vector-mediated RNAi-Anxa1 into the rat foot pad. An experimental RA rat model was established by injecting Freund's complete adjuvant (FCA) into the RA, MOX, RNAi-Anxa1, and RNAi-Anxa1 + MOX groups. Rats in the MOX and RNAi-Anxa1 + MOX groups received moxibustion treatment. After modeling, using moxibustion "Shenshu (BL23)" and "Zusanli (ST36)", each point is 5 times, bilateral alternating, once a day, 6 times for a course of treatment, between the courses of rest for a one day. A total of three treatment courses were conducted. Both bilateral pad thicknesses were measured using Vernier calipers on experimental days 1, 7, 14, 21, and 28. The expression of cPLA2α signaling in the synovium of diseased joints was observed using Western blot. The pathology of the rat ankle synovium was observed using hematoxylin-eosin (HE) staining. Interleukin (IL)-1β, IL-10, prostaglandin E2 (PGE2), and leukotriene B4 (LTB4) were detected using enzyme-linked immunosorbent assay. Moxibustion increased the levels of Annexin 1 and decreased the inflammatory response in rats with RA. After increasing the expression of Annexin 1, the phosphorylated expression of cPLA2α was inhibited, the serum levels of IL-1β, PGE2, and LTB4 decreased, and the level of IL-10 increased. In moxibustion treated RA rats after the Annexin 1 lentiviral intervention, the serum levels of IL-1β, PGE2, LTB4, and IL-10 were almost unchanged. Moxibustion enhanced the negative regulation of the cPLA2α signaling pathway, increased the synovial Annexin 1 expression, inhibited the cPLA2α signaling pathway, indirectly inhibited the expression of downstream inflammatory factors, and played a role in reducing inflammation.

Joint Inflammation Correlates with Joint GPR30 Expression in Males and Hippocampal GPR30 Expression in Females in a Rat Model of Rheumatoid Arthritis.

It is not entirely clear how the interaction between joint inflammation and the central nervous system (CNS) response in rheumatoid arthritis (RA) works, and what pathophysiology underlies the sex differences in coexisting neuropsychiatric comorbidities. It is known that estrogen hormones reduce inflammation in RA and that this occurs mainly via the stimulation of G protein-coupled receptor-30 (GPR30), also known as G protein-coupled estrogen receptor (GPER) 1. However, changes in GPR30 expression and sex differences induced by local and systemic inflammation in RA are not yet known. Our aim was to reveal sex differences in the expression and association of joint GPR30 with local and systemic inflammation, clinical course and furthermore with hippocampal GPR30 expression during pristane-induced arthritis (PIA) in Dark Agouti (DA) rats, an animal model of RA. Furthermore, we demonstrated sex-specific differences in the association between joint and systemic inflammation and hippocampal microglia during PIA. Our results suggest sex-specific differences not only in the clinical course and serum levels of pro-inflammatory cytokines but also in the expression of GPR30. Female rats show greater synovial inflammation and greater damage to the articular cartilage compared to males during PIA attack. Male rats express higher levels of synovial and cartilaginous GPR30 than females during PIA, which correlates with a less severe clinical course. The correlation between synovial and cartilaginous GPR30 and joint inflammation scores (Krenn and Mankin) in male rats suggests that the more severe the joint inflammation, the higher the GPR30 expression. At the same time, there is no particular upregulation of hippocampal GPR30 in males. On the other hand, female rats express higher levels of neuroprotective GPR30 in the hippocampus than male rats at the basic level and during PIA attack. In addition, females have a higher number of Iba-1+ cells in the hippocampus during PIA attack that strongly correlates with the clinical score, serum levels of IL-17A, and Krenn and Mankin scores. These results suggest that male rats are better protected from inflammation in the joints and female rats are better protected from the inflammation in the hippocampus during a PIA attack, independently of microglia proliferation. However, in the remission phase, synovial GPR30 expression suddenly increases in female rats, as does hippocampal GPR30 expression in males. Further experiments with a longer remission period are needed to investigate the molecular background of these sex differences, as well as microglia phenotype profiling.

Inflammation-Responsive Mesoporous Silica Nanoparticles with Synergistic Anti-inflammatory and Joint Protection Effects forRheumatoid Arthritis Treatment.

Joint destruction is a major burden and an unsolved problem in rheumatoid arthritis (RA) patients. We designed an intra-articular mesoporous silica nanosystem (MSN-TP@PDA-GlcN) with anti-inflammatory and joint protection effects. The nanosystem was synthesized by encapsulating triptolide (TP) in mesoporous silica nanoparticles and coating it with pH-sensitive polydopamine (PDA) and glucosamine (GlcN) grafting on the PDA. The nano-drug delivery system with anti-inflammatory and joint protection effects should have good potency against RA. A template method was used to synthesize mesoporous silica (MSN). MSN-TP@PDA-GlcN was synthesized via MSN loading with TP, coating with PDA and grafting of GlcN on PDA. The drug release behavior was tested. A cellular inflammatory model and a rat RA model were used to evaluate the effects on RA. In vivo imaging and microdialysis (MD) system were used to analyze the sustained release and pharmacokinetics in RA rats. TMSN-TP@PDA-GlcN was stable, had good biocompatibility, and exhibited sustained release of drugs in acidic environments. It had excellent anti-inflammatory effects in vitro and in vivo. It also effectively repaired joint destruction in vivo without causing any tissue toxicity. In vivo imaging and pharmacokinetics experiments showed that the nanosystem prolonged the residence time, lowered the Cmax value and enhanced the relative bioavailability of TP. These results demonstrated that MSN-TP@PDA-GlcN sustained the release of drugs in inflammatory joints and produced effective anti-inflammatory and joint protection effects on RA. This study provides a new strategy for the treatment of RA.

Deciphering the therapeutic potential of trimetazidine in rheumatoid arthritis via targeting mi-RNA128a, TLR4 signaling pathway, and adenosine-induced FADD-microvesicular shedding: In vivo and in silico study.

Rheumatoid arthritis (RA) is a debilitating autoimmune condition characterized by chronic synovitis, joint damage, and inflammation, leading to impaired joint functionality. Existing RA treatments, although effective to some extent, are not without side effects, prompting a search for more potent therapies. Recent research has revealed the critical role of FAS-associated death domain protein (FADD) microvesicular shedding in RA pathogenesis, expanding its scope beyond apoptosis to include inflammatory and immune pathways. This study aimed to investigate the intricate relationship between mi-RNA 128a, autoimmune and inflammatory pathways, and adenosine levels in modulating FADD expression and microvesicular shedding in a Freund's complete adjuvant (FCA) induced RA rat model and further explore the antirheumatoid potency of trimetazidine (TMZ). The FCA treated model exhibited significantly elevated levels of serum fibrogenic, inflammatory, immunological and rheumatological diagnostic markers, confirming successful RA induction. Our results revealed that the FCA-induced RA model showed a significant reduction in the expression of FADD in paw tissue and increased microvesicular FADD shedding in synovial fluid, which was attributed to the significant increase in the expression of the epigenetic miRNA 128a gene in addition to the downregulation of adenosine levels. These findings were further supported by the significant activation of the TLR4/MYD88 pathway and its downstream inflammatory IkB/NFB markers. Interestingly, TMZ administration significantly improved, with a potency similar to methotrexate (MTX), the deterioration effect of FCA treatment, as evidenced by a significant attenuation of fibrogenic, inflammatory, immunological, and rheumatological markers. Our investigations indicated that TMZ uniquely acted by targeting epigenetic miRNA128a expression and elevating adenosine levels in paw tissue, leading to increased expression of FADD of paw tissue and mitigated FADD microvesicular shedding in synovial fluid. Furthermore, the group treated with TMZ showed significant downregulation of TLR4/MYD88 and their downstream TRAF6, IRAK and NF-kB. Together, our study unveils the significant potential of TMZ as an antirheumatoid candidate, offering anti-inflammatory effects through various mechanisms, including modulation of the FADD-epigenetic regulator mi-RNA 128a, adenosine levels, and the TLR4 signaling pathway in joint tissue, but also attenuation of FADD microvesicular shedding in synovial fluid. These findings further highlight the synergistic administration of TMZ and MTX as a potential approach to reduce adverse effects of MTX while improving therapeutic efficacy.

Exploring the mechanism of Sendeng-4 against rheumacid arthritis through integrated serum pharmacochemistry, transcriptomics, and network pharmacology.

Mongolian medicine Sendeng-4 (SD-4) has demonstrated satisfactory clinical treatment outcomes for rheumatoid arthritis (RA); nevertheless, its bioactive components and the related mechanisms have not yet been clearly elucidated. To explore the bioactive chemical components of SD-4 in the treatment of RA and its possible mechanisms, an High Performance Liquid Chromatography-tandem mass spectrometry (HPLC-MS/MS) method was established to simultaneously quantify the main components in SD-4, and ultraperformance LC-Q-Exactive-MS/MS (UPLC-Q-Exactive-MS/MS) was used to identify the phytochemicals absorbed in the serum. Then, using network pharmacology methods, these components were constructed into a compound-target network of RA to predict possible biological targets of SD-4 as well as potential signaling pathways. Transcriptomics analysis and molecular docking were used to validate the results of network pharmacology. Subsequently, we established a complete Freund's adjuvant-induced RA rat model and observed the anti-RA effects of SD-4 through assessments of foot swelling, ankle diameter, arthritis score, morphology, serum inflammatory factors, and histopathological analysis of synovial tissue. Specifically, reverse transcription-quantitative polymerase chain reaction, Western blot, and immunohistochemical analysis were used in animal experiments to validate the pathways of serum phytochemistry, network pharmacology, and transcriptomics. Tannic acid, gallic acid, corilagin, crocin I, gardenoside, ferulic acid, quercetin, limonin, rutin, chlorogenic acid, verbascoside, catechin, epicatechin, myricetin, and dihydromyricetin in SD-4 showed good linearity within their respective concentration ranges (r ≥ 0.9991); the average recovery rate was 93.77%-109.17% (relative standard deviation < 2%). A total of 37 compounds were identified in serum samples. Based on this, network pharmacology methods collected 739 genes related to these identified compounds in SD-4 and 3807 genes related to RA. Network pharmacology and transcriptomic analysis demonstrated that the phosphatidylinositol 3-kinase (PI3K)-protein kinase B (Akt) signaling pathway is the most relevant pathway affected by SD-4 in RA. In the experiments, SD-4 treatment reduced ankle swelling and arthritis scores in RA rats, improved symptoms, and reduced the production of inflammatory factors. Compared with the RA model group, SD-4 treatment significantly reduced the expression of PI3K-Akt pathway-related messenger RNA and proteins. In addition, immunohistochemical analysis confirmed these results. This study combined serum phytochemistry, network pharmacology, and transcriptomics to demonstrate that SD-4 can alleviate RA by regulating the PI3K-Akt signaling pathway. This research provides a theoretical basis for the clinical application of SD-4 and offers an effective strategy for the identification of bioactive substances in traditional Chinese medicine formulas and the study of their potential mechanisms.

Resveratrol and prednisolone loaded into human serum albumin nanoparticles for the alleviation of rheumatoid arthritis symptoms: an in vitro and in vivo study

Abstract Rheumatoid arthritis is a chronic autoimmune-disease-causing inflammation, joint pain, and joint destruction, severely affecting the quality of life of millions worldwide. In the current research, a nanocarrier system was developed for the delivery of resveratrol and prednisolone to treat rheumatoid arthritis. The drug delivery system was characterized in vitro using scanning electron microscopy and various cell culture studies. Finally, the alleviative symptoms of the developed treatment strategy were investigated in a rat model of rheumatoid arthritis. In vitro studies showed that the carrier system released the drugs in a sustained manner and possessed strong immunomodulatory functions. Nanocarriers loaded with prednisolone, resveratrol, and drug-free carriers had 396.88 ± 76.41 nm, 392.49 ± 97.31 nm, and 338.02 ± 77.75 nm of mean particle size, respectively. In vivo studies revealed that local injection of the carrier system could alleviate the degenerative effects of rheumatoid arthritis. ELISA assays showed that the co-injection of resveratrol and prednisolone-loaded albumin nanoparticles could significantly modulate inflammatory responses. The developed treatment modality may potentially be used to treat rheumatoid arthritis.

Myricetin reduces neutrophil extracellular trap release in a rat model of rheumatoid arthritis, which is associated with a decrease in disease severity.

Rheumatoid arthritis (RA) is a chronic disease characterized by joint inflammation and severe disability. However, there is a lack of safe and effective drugs for treating RA. In our previous study, we discovered that myricetin (MC) and celecoxib have a synergistic effect in the treatment of RA. We conducted in vitro and in vivo experiments to further investigate the effects and mechanisms of action of MC. Our findings demonstrated that MC treatment effectively reduced the release of neutrophil extracellular traps (NETs) and alleviated the inflammatory response in RA. Mechanistic studies showed that MC prevents the entry of PADI4 and MPO into the cell nucleus, thereby protecting DNA from decondensation. In a rat arthritis model, MC improved histological changes in ankle joints and suppressed NET-related signaling factors. In conclusion, MC protects the ankle joints against arthritis by inhibiting MPO and PADI4, thereby reducing NET release. The pharmacological mechanism of MC in RA involves the inhibition of NET release.

Evaluating the Efficacy of a Polyherbal Formulation in Ameliorating Arthritis Induced by Complete Freund’s Adjuvant

Background: This study investigates the antiarthritic potential of a polyherbal formulation (PHF) comprising extracts from Tinospora cordifolia, Rosa damescena, and Acacia leucoploea in a rat model of rheumatoid arthritis (RA). RA is a chronic autoimmune disorder with debilitating consequences. PHF's impact on joint inflammation, bone degradation, and cartilage preservation were evaluated. Methods: Arthritis was induced using complete Freund’s adjuvant (CFA), and animals were treated with PHF (200 and 400 mg/kg), prednisolone, or control treatments for 28 days. Parameters including body weight, paw volume, arthritis severity score, hematological parameters, serum markers (creatinine, ALP, total proteins), cytokine levels (IL-1β, IL-6, TNF-α, IL-10), and radiographic changes were assessed. Results: CFA-treated rats exhibited significant body weight loss, paw edema, increased arthritis severity scores, altered hematological parameters, and elevated serum markers compared to normal controls. PHF treatment at both doses mitigated body weight loss, reduced paw edema, and improved arthritis severity scores. Hematological changes induced by CFA were also attenuated by PHF treatment. Serum creatinine, ALP, and total protein levels, elevated in CFA-treated rats, were significantly improved by PHF. Furthermore, PHF modulated cytokine levels, decreasing IL-6, IL-1β, and TNF-α while increasing IL-10. Radiographic analysis displayed reduced joint damage in PHF-treated rats compared to CFA controls. Conclusion: This comprehensive investigation highlights PHF's potential to mitigate the inflammatory processes associated with RA, as evidenced by improved clinical, hematological, and biochemical parameters. The study underscores the promise of traditional botanical compounds in managing RA and suggests PHFs as novel therapeutic options. Further mechanistic studies are warranted to elucidate the exact pathways involved.

A dual dynamically cross-linked hydrogel promotes rheumatoid arthritis repair through ROS initiative regulation and microenvironment modulation-independent triptolide release

High oxidative stress and inflammatory cell infiltration are major causes of the persistent bone erosion and difficult tissue regeneration in rheumatoid arthritis (RA). Triptolide (TPL) has become a highly anticipated anti-rheumatic drug due to its excellent immunomodulatory and anti-inflammatory effects. However, the sudden drug accumulation caused by the binding of “stimulus-response” and “drug release” in a general smart delivery system is difficult to meet the shortcoming of extreme toxicity and the demand for long-term administration of TPL. Herein, we developed a dual dynamically cross-linked hydrogel (SPT@TPL), which demonstrated sensitive RA microenvironment regulation and microenvironment modulation-independent TPL release for 30 days. The abundant borate ester/tea polyphenol units in SPT@TPL possessed the capability to respond and regulate high reactive oxygen species (ROS) levels on-demand. Meanwhile, based on its dense dual crosslinked structure as well as the spontaneous healing behavior of numerous intermolecular hydrogen bonds formed after the breakage of borate ester, TPL could remain stable and slowly release under high ROS environments of RA, which dramatically reduced the risk of TPL exerting toxicity while maximized its long-term efficacy. Through the dual effects of ROS regulation and TPL sustained-release, SPT@TPL alleviated oxidative stress and reprogrammed macrophages into M2 phenotype, showing marked inhibition of inflammation and optimal regeneration of articular cartilage in RA rat model. In conclusion, this hydrogel platform with both microenvironment initiative regulation and TPL long-term sustained release provides a potential scheme for rheumatoid arthritis.

Moxibustion of Zusanli (ST36) and Shenshu (BL23) alleviates the inflammation of rheumatoid arthritis in rats through regulating macrophage migration inhibitory factor/glucocorticoids signaling.

To test the hypothesis that moxibustion may inhibit rheumatoid arthritis (RA) synovial inflammation by regulating the expression of macrophage migration inhibitory factor (MIF)/glucocorticoids (GCs). Fifty male Sprague-Dawley rats were randomly divided into five groups (n = 10 each): blank Control (CON) group, RA Model (RA) group, Moxibustion (MOX) group, MIF inhibitor (S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester (ISO-1) group, and Moxibustion + MIF inhibitor ISO-1 (MOX + ISO-1) group. Rats in the ISO-1 group and ISO-1 + MOX group were intraperitoneally injected with the inhibitor ISO-1. The rats in the RA group, ISO-1 group, MOX group, and ISO-1 + MOX group were injected with Freund's complete adjuvant (FCA) in the right hind footpad to establish an experimental RA rat model. In the MOX group and MOX + ISO-1 group, rats were treated with Moxa. The thickness of the footpads of the rats in each group was measured at three-time points before, after modeling and after moxibustion treatment. The contents of serum MIF, corticosterone (CORT), tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) were detected by enzyme-linked immunosorbent assay; and the contents of synovial MIF were detected by Western blot. Hematoxylin-eosin (HE) staining method was used to observe the pathological changes of synovial tissue under a section light microscope, and pathological scoring was performed according to the grading standard of the degree of synovial tissue disease. Moxibustion was found to reduce the level of MIF and alleviate inflammation in RA rats in this study. In addition, after inhibiting the expression of MIF, the level of CORT increased, and the level of TNF-α decreased. Treating RA rats with inhibited MIF by moxibustion, the level of CORT was almost unchanged, but the level of TNF-α further decreased. The correlation analysis data suggested that MIF was positively related to the expression of TNF-α and negatively correlated with the expression of CORT. Reducing MIF to increase CORT and decrease TNF-α by moxibustion treatment in RA. MIF may be a factor for moxibustion to regulate the expression of CORT, but the expression of TNF-α is due to the incomplete regulation of the MIF. This study added to the body of evidence pointing to moxibustion's anti-inflammatory mechanism in the treatment of RA.

Preparation and anti-inflammatory effect of mercury sulphide nanoparticle-loaded hydrogels

We successfully prepared mercury sulphide nanoparticle hydrogels by physical encapsulation method. The successfully prepared mercuric sulphide nanoparticle hydrogel was a zinc folate hydrogel, which showed an obvious porous structure with interconnected and uniformly distributed pores and a pore size range of about 20 μm. The maximum drug loading of the hydrogels was 3%, and the in vitro cumulative release degree was in accordance with the first-order kinetic equation Mt = 149.529 (1 − e−0.026 t ). The particles in mercuric sulphide nanoparticle hydrogels significantly down-regulated the expression of the cell surface co-stimulatory molecule CD86 (p < .0001). Meanwhile, the inflammatory response was regulated through the NF-κB pathway in LPS-induced inflammatory cells. Later, it was observed that mercuric sulphide nanoparticle hydrogels could significantly counteract the inflammatory and immune models through a mouse ear swelling model, a rat foot-plantar swelling model and a rheumatoid arthritis model. This design targets the immunomodulatory, and anti-inflammatory effects through nanocomposite hydrogel technology. It reduces the drawbacks of low mercury utilisation and susceptibility to accumulation of toxicity. It aims to provide an experimental basis for the development of mercuric sulphide and the treatment of inflammatory and immune-related diseases. Highlights Mercury sulphide nanoparticle hydrogel has an optimal mercury sulphide nanoparticle content of 2%, is structurally homogeneous and stable, and does not exhibit significant liver or kidney toxicity. Mercuric sulphide nanoparticle hydrogel exerts anti-inflammatory effects in cells and rats, and regulates the expression of macrophage surface molecules and factors related to the NF-κB pathway. Mercuric sulphide nanoparticle hydrogel improves the condition of ankle synovial joints in a rat model of rheumatoid arthritis.

EIDD-1931 Treatment Tweaks CYP3A4 and CYP2C8 in Arthritic Rats to Expedite Drug Interaction: Implication in Oral Therapy of Molnupiravir.

EIDD-1931 is the active form of molnupiravir, an orally effective drug approved by the United States Food and Drug Administration (USFDA) against COVID-19. Pharmacokinetic alteration can cause untoward drug interaction (drug-drug/disease-drug), but hardly any information is known about this recently approved drug. Therefore, we first investigated the impact of the arthritis state on the oral pharmacokinetics of EIDD-1931 using a widely accepted complete Freund's adjuvant (CFA)-induced rat model of rheumatoid arthritis (RA) after ascertaining the disease occurrence by paw swelling measurement and X-ray examination. Comparative oral pharmacokinetic assessment of EIDD-1931 (normal state vs arthritis state) showed that overall plasma exposure was augmented (1.7-fold) with reduced clearance (0.54-fold), suggesting its likelihood of dose adjustment in arthritis conditions. In order to elucidate the effect of EIDD-1931 treatment at a therapeutic regime (normal state vs arthritis state) on USFDA-recommended panel of cytochrome P450 (CYP) enzymes (CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4) for drug interaction using the same disease model, we monitored protein and mRNA expressions (rat homologs) in liver tissue by western blotting (WB) and real time-polymerase chain reaction (RT-PCR), respectively. Results reveal that EIDD-1931 treatment could strongly influence CYP3A4 and CYP2C8 among experimental proteins/mRNAs. Although CYP2C8 regulation upon EIDD-1931 treatment resembles similar behavior under the arthritis state, results dictate a potentially reverse phenomenon for CYP3A4. Moreover, the lack of any CYP inhibitory effect by EIDD-1931 in human/rat liver microsomes (HLM/RLM) helps to ascertain EIDD-1931 treatment-mediated disease-drug interaction and the possibility of drug-drug interaction with disease-modifying antirheumatic drugs (DMARDs) upon coadministration. As elevated proinflammatory cytokine levels are prevalent in RA and nuclear factor-kappa B (NF-kB) and nuclear receptors control CYP expressions, further studies should focus on understanding the regulation of affected CYPs to subside unexpected drug interaction.

Mechanism of aqueous extract of Strychni Semen in improving pain in rats with rheumatoid arthritis based on TLR4/TNF-α/MMP-9 signaling pathway

This study aims to explore the mechanism of aqueous extract of Strychni Semen(SA) in relieving pain in the rat model of rheumatoid arthritis(RA) via Toll-like receptor 4(TLR4)/tumor necrosis factor-α(TNF-α)/matrix metalloproteinase-9(MMP-9) signaling pathway. Firstly, the main chemical components of Strychni Semen were searched against TCMSP, TCMID, ETCM, and related literature, and the main targets of the chemical components were retrieved from TargetNet and SwissTargetPrediction. The main targets of RA and pain were searched against GeneCards, Online Mendelian Inheritance in Man(OMIM), and Therapeutic Target Database(TTD). Venny 2.1.0 was used to obtain the common targets shared by Strychni Semen, RA, and pain, and STRING and Cytoscape 3.6.1 were used to build the protein-protein interaction network. Then, molecular docking was carried out in AutoDock Vina. Finally, the rat model of type Ⅱ collagen-induced arthritis(CIA) was established. The up-down method and acetone method were employed to examine the mechanical pain threshold and cold pain threshold of rats, and the pain-relieving effect of SA on CIA rats was evaluated comprehensively. Hematoxylin-eosin(HE) staining was employed to evaluate the histopathological changes of joints in CIA rats. The expression levels of key target proteins was determined by immunohistochemistry and Western blot, and the mRNA levels of key targets were determined by real-time fluorescence quantitative polymerase chain reaction(real-time PCR). The results of network prediction showed that Strychni Semen may act on the TLR4/TNF-α/MMP-9 signaling pathway to exert the pain-relieving effect. The results of molecular docking showed that brucine, the main active component of SA, had strong binding ability to TLR4, TNF-α, and MMP-9. The results of animal experiments showed that SA improved the mechanical and cold pain sensitivity(P&lt;0.05, P&lt;0.01) and reduced the joint histopathological score of CIA rats(P&lt;0.01). In addition, medium and high doses of SA down-regulated the protein and mRNA levels of TNF-α, TLR4, and MMP-9(P&lt;0.05,P&lt;0.01). In conclusion, SA alleviated the mechanical pain sensitivity, cold pain sensitivity, and joint histopathological changes in CIA rats by inhibiting the over activation of TLR4/TNF-α/MMP-9 signaling pathway.

Isoliquiritigenin inhibits apoptosis and ameliorates oxidative stress in rheumatoid arthritis chondrocytes through the Nrf2/HO-1-mediated pathway

Rheumatoid arthritis (RA) is a chronic inflammatory condition known for its irreversible destructive impact on the joints. Chondrocytes play a pivotal role in the production and maintenance of the cartilage matrix. However, the presence of inflammatory cytokines can hinder chondrocyte proliferation and promote apoptosis. Isoliquiritigenin (ISL), a flavonoid, potentially exerts protective effects against various inflammatory diseases. However, its specific role in regulating the nuclear factor E2-associated factor 2 (Nrf2)/heme oxygenase-1 (HO-1) pathway in chondrocytes in RA remains unclear. To investigate this, this study used human chondrocytes and Sprague-Dawley rats to construct in vitro and in vivo RA models, respectively. The study findings reveal that cytokines markedly induced oxidative stress, the activation of matrix metalloproteinases, and apoptosis both in vitro and in vivo. Notably, ISL treatment significantly mitigated these effects. Moreover, Nrf2 or HO-1 inhibitors reversed the protective effects of ISL, attenuated the expression of Nrf2/HO-1 and peroxisome proliferator-activated receptor gamma-coactivator-1α, and promoted chondrocyte apoptosis. This finding indicates that ISL primarily targets the Nrf2/HO-1 pathway in RA chondrocytes. Moreover, ISL treatment led to improved behavior scores, reduced paw thickness, and mitigated joint damage as well as ameliorated oxidative stress in skeletal muscles in an RA rat model. In conclusion, this study highlights the pivotal role of the Nrf2/HO-1 pathway in the protective effects of ISL and demonstrates the potential of ISL as a treatment option for RA.

Prolonged administration of total glucosides of paeony improves intestinal immune imbalance and epithelial barrier damage in collagen-induced arthritis rats based on metabolomics-network pharmacology integrated analysis.

Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease characterized by synovial inflammation and joint damage with complex pathological mechanisms. In recent years, many studies have shown that the dysregulation of intestinal mucosal immunity and the damage of the epithelial barrier are closely related to the occurrence of RA. Total glucosides of paeony (TGP) have been used clinically for the treatment of RA in China for decades, while the pharmacological mechanism is still uncertain. The purpose of this study was to investigate the regulatory effect and mechanism of TGP on intestinal immunity and epithelial barrier in RA model rats. The results showed that TGP alleviated immune hyperfunction by regulating the ratio of CD3+, CD4+ and CD8+ in different lymphocyte synthesis sites of the small intestine, including Peyer's patches (PPs), intraepithelial lymphocytes (IELs), and lamina propria lymphocytes (LPLs). Specially, TGP first exhibited immunomodulatory effects on sites close to the intestinal lumen (IELs and LPLs), and then on PPs far away from the intestinal lumen as the administration time prolonged. Meanwhile, TGP restores the intestinal epithelial barrier by upregulating the ratio of villi height (V)/crypt depth (C) and expression of tight junction proteins (ZO-1, occludin). Finally, the integrated analysis of metabolomics-network pharmacology was also used to explore the possible regulation mechanism of TGP on the intestinal tract. Metabolomics analysis revealed that TGP reversed the intestinal metabolic profile disturbance in CIA rats, and identified 32 biomarkers and 163 corresponding targets; network pharmacology analysis identified 111 potential targets for TGP to treat RA. By intersecting the results of the two, three key targets such as ADA, PNP and TYR were determined. Pharmacological verification experiments showed that the levels of ADA and PNP in the small intestine of CIA rats were significantly increased, while TGP significantly decreased their ADA and PNP levels. In conclusion, purine metabolism may play an important role in the process of TGP improving RA-induced intestinal immune imbalance and impaired epithelial barrier.

Identification of HHT-9041P1: A novel potent and selective JAK1 inhibitor in a rat model of rheumatoid arthritis

Rheumatoid arthritis (RA) is a chronic systemic disease associated with long-term disability and premature mortality. If left untreated, it can seriously affect patients’ quality of life. The JAK-STAT signal transduction process is known to affect the occurrence and development of RA, and small molecule JAK inhibitors, such as tofacitinib, have been identified as treatments for RA. However, tofacitinib is a non-selective JAK inhibitor that was found to be associated with dose-limiting tolerability and safety issues, such as anemia in phase 2 dose-ranging studies. Therefore, we developed a selective JAK1 inhibitor, HHT-9041P1, to overcome target-related adverse reactions. We used enzyme and cytokine potency assays in vitro as well as the collagen-induced arthritis (CIA) model in vivo to explore the efficacy and mechanism. In vitro, HHT-9041P1 was diluted (0.017 nM–1 mM) in DMSO) and mixed with JAK1, JAK2, JAK3 or TYK2 kinases for use in the respective assays for inhibitory activity and selectivity evaluation. Fresh human PBMCs were activated and incubated with 100 ng/mL cytokine IL-6 or 20 ng/mL GM-CSF for use in the investigation of the immune mechanism. In vivo, HHT-9041P1 (1 mg/kg, 3 mg/kg and 10 mg/kg) was administered by oral gavage twice daily to CIA model Lewis rats from Day 8 to Day 29 for paw swelling and arthritis score evaluation. At the end of the experiment, the rats were sacrificed before collection of the hind ankle joint, spleen and blood for analysis of inflammation, arthritis phenotypes, inflammatory cytokine expression and Th1 cell proportions. As expected, HHT-9041P1 showed 10-fold greater selectivity for JAK1 over JAK2, and 23-fold greater selectivity over JAK3 in cellular assays. The high selectivity of HHT-9041P1 was also validated by in vivo safety studies. HHT-9041P1 demonstrated significant efficacy in a rat model of collagen-induced arthritis (CIA) and was associated with reduced helper T Cell 1 (Th1) cell differentiation. HHT-9041P1 also exhibited excellent pharmacokinetics properties. Thus, HHT-9041P1 was identified as a candidate for clinical development with many options for the treatment of RA.

Enhanced intra-articular therapy for rheumatoid arthritis using click-crosslinked hyaluronic acid hydrogels loaded with toll-like receptor antagonizing peptides

Rheumatoid arthritis (RA) is a chronic inflammatory disorder that results in the deterioration of joint cartilage and bone. Toll-like receptor 4 (TLR4) has been pinpointed as a key factor in RA-related inflammation. While Toll-like receptor antagonizing peptide 2 (TAP2) holds potential as an anti-inflammatory agent, its in vivo degradation rate hinders its efficacy. We engineered depots of TAP2 encapsulated in click-crosslinked hyaluronic acid (TAP2+Cx-HA) for intra-articular administration, aiming to enhance the effectiveness of TAP2 as an anti-inflammatory agent within the joint cavity. Our data demonstrated that FI-TAP2+Cx-HA achieves a longer retention time in the joint cavity compared to FI-TAP2 alone. Mechanistically, we found that TAP2 interacts with TLR4 on the cell membranes of inflammatory cells, thereby inhibiting the nuclear translocation of NF-κB and maintaining it in an inactive cytoplasmic state. In a rat model of RA, the TAP2+Cx-HA formulation effectively downregulated the inflammatory cytokines TNF-α and IL-6, while upregulating the anti-inflammatory cytokine IL-10 and the therapeutic protein 14-3-3ζ. This led to a more rapid restoration of cartilage thickness, increased deposition of glycosaminoglycans, and new bone tissue formation in the regenerated cartilage, in comparison to a single TAP2 treatment after a six-week period. Our results suggest that TAP2+Cx-HA could serve as a potent intra-articular treatment for RA, offering both symptomatic relief and promoting cartilage regeneration. This innovative delivery system holds significant promise for improving the management of RA and other inflammatory joint conditions. Statement of significanceIn this study, we developed a therapy by creating toll-like receptor 4 (TLR4)-antagonizing peptide (TAP2)-loaded click-crosslinked hyaluronic acid (TAP2+Cx-HA) depots for direct intra-articular injection. Our study demonstrates that FI-TAP2+Cx-HA exhibits a more than threefold longer lifetime in the joint cavity compared to FI-TAP2 alone. Furthermore, we found that TAP2 binds to TLR4 and masks the nuclear localization signals of NF-κB, leading to its sequestration in an inactive state in the cytoplasm. In a rat model of RA, TAP2+Cx-HA effectively suppresses inflammatory molecules, specifically TNF–α and IL-6, while upregulating the anti-inflammatory cytokine IL-10 and the therapeutic protein 14-3-3ζ. This resulted in faster regeneration of cartilage thickness, increased glycosaminoglycan deposits in the regenerated cartilage, and a twofold increase in new bone tissue formation compared to a single TAP2 treatment.