Rat Model Of Rheumatoid Arthritis Research Articles

Toddalia asiatica extract attenuates adjuvant-induced arthritis by modulating colon Th17/Treg balance and colony homeostasis

Ethnopharmacological relevanceGiven the adverse effects of the current principal treatments, there is still a great need for effective cures for rheumatoid arthritis (RA), an immune-mediated disease. Toddalia asiatica (L.) Lam is a traditional medicinal herb that can be used for RA treatment because of its anti-inflammatory and analgesic properties. Aim of the studyTo investigate the possible effects of Toddalia asiatica extract (TAE) on intestinal immunity and the intestinal bacterial flora in a rat model of RA. Materials and methodsThe anti-arthritis effect of TAE was evaluated in arthritis rats induced by complete Freund's adjuvant-induced arthritis (AIA). Arthritis index (AI) scores, systemic inflammation scores, histopathologic changes in the colon and ankle were detected by hematoxylin and eosin staining. Western blot analysis was performed to assess the protein expression of IL-17A, RORC, IL-1β, IL-6, FOXP3, IL-10 in the colon. RT-PCR was performed to assess the expression of the colon's mRNA. Finally, changes to the gut microbiome by sequencing 16S rDNA. Microbial function prediction was performed using PICRUSt with the KEGG databases and correlation analysis was carried out by computing Spearman's rank correlations. Resultsdemonstrated that TAE administration at a dose of 3 g/kg dramatically decreased AI scores, systemic inflammation scores, and histopathologic lesions of the ankle and colon in AIA rats. TAE was found to significantly reduce the expression levels of Th17-related proteins and mRNAs (IL-17A, RORC, IL-1β and IL-6) in the colon, while increasing the expression levels of Treg-related proteins and mRNA (IL-10 and FOXP3), which helped restore the balance of Th17/Treg immune cells in the colon. Meanwhile, TAE was also found to be capable of remodeling the gut microbiota in AIA rats. Depleting RA-associated genera and thereby increasing α-diversity enriched the gut microbiota's diversity and shifted the community composition dramatically, leading to the increase of Firmicutes_unclassified, Ruminococcaceae_unclassified, Muribaculum, Subdoligranulum, Lachnospira, Marvinbryantia, and the reduction of RA-related bacteria Ligilactobacillus, Streptococcus and Eubacterium-eligens-group. Furthermore, PICRUSt analysis revealed that metabolic pathways were associated with TAE treatment, with metabolic pathways dominating. Among them, metabolic pathways were predominant. Correlation studies showed that a total of 9 microorganisms, including Ligilactobacillus, Eubacterium-eligens-group and Subdoligranulum, were significantly associated with Th17/Treg expression. ConclusionsThis study demonstrates that TAE is a low-toxicity poly alkaline drug that can rapidly and effectively improve joint symptoms in RA rats and increases beneficial intestinal bacteria and decreases harmful ones, which is associated with modulating Th17/Treg interactions in intestinal T cells and reversing microbial disorders.

Combined treatment with glucosamine and chondroitin sulfate improves rheumatoid arthritis in rats by regulating the gut microbiota

BackgroundTo investigate the ameliorative effects of glucosamine (GS), chondroitin sulphate (CS) and glucosamine plus chondroitin sulphate (GC) on rheumatoid arthritis (RA) in rats, and to explore the mechanism of GS, CS and GC in improving RA based on the gut microbiota.MethodsRA rat models were effectively developed 14 days after CFA injection, and then garaged with GS, CS and GC. Body weight and paw volume of rats were monitored at multiple time points at the beginning of CFA injection. Until D36, serum and ankle tissue specimens were used to measure levels of circulating inflammatory factors (TNF-α, IL-1β, MMP-3, NO and PGE2) and local inflammatory indicators (TLR-4 and NF-κB). On D18, D25, and D36, intergroup gut microbiota was compared using 16S rRNA gene sequencing and bioinformatics analysis. We also performed the correlation analysis of gut bacteria, joint swelling and inflammatory indicators.ResultsGC, rather than GS and CS, could reduce right paw volumes, levels of TLR-4 and NF-κB in synovial tissues. In addition, enriched genera in RA model rats screened out by LEfSe analysis could be inhibited by GC intervention, including potential LPS-producing bacteria (Enterobacter, Bacteroides, Erysipelotrichaceae_unclassified and Erysipelotrichaceae_uncultured) and some other opportunistic pathogens (Esherichia_Shigella, Nosocomiicoccus, NK4A214_group, Odoribacter, Corynebacterium and Candidatus_Saccharimonas.etc.) that positively correlated with pro-inflammatory cytokines, right paw volume, and pathology scores. Furthermore, the gut microbiota dysbiosis was observed to recover before alleviating joint swelling after interventions.ConclusionsGC could inhibit potential LPS-producing bacteria and the activation of TLR-4/NF-κB pathway in RA rats, thus alleviating RA-induced joint injury.

A Rat Model of Rheumatoid Arthritis: TDZD-8 is Associated with the Protection Against the Induction of the Synovium Knee Joint IL-17A/GSK3β/ROS/α-SMA Axis of Fibrosis

Rheumatoid arthritis (RA) that affects the synovial knee joint causes swelling of the synovial membrane and tissue damage. Interleukin-17A (IL-17A) and the enzyme glycogen synthase kinase-3β (GSK3β) are involved in the pathogenesis of RA. The link between IL-17A, GSK3β, the oxidative stress, and the profibrogenic marker alpha-smooth muscle actin (α-SMA) with and without TDZD-8, GSK3β inhibitor has not been studied before. Consequently, active immunization of rats was performed to induce RA after three weeks using collagen type II (COII) injections. The treated group received daily injection of 1 mg/kg TDZD-8 for 21 days following the immunization protocol (COII+TDZD-8). Blood and synovium tissue samples were harvested at the end of the experiment. RA development was confirmed as corroborated by a substantial increase in blood levels of the highly specific autoantibody for RA, anti-citrullinated protein antibody as well as augmentation of reactive oxidative species (ROS) levels measured as lipid peroxidation. RA induction also increased synovium tissue levels of IL-17A and the profibrogenic marker, α-SMA. All these parameters seemed to be significantly (p<0.0001) ameliorated by TDZD-8. Additionally, a significant correlation between IL-17A, ROS, and α-SMA and biomarkers of RA was observed. Thus, knee joint synovium RA induction augmented IL-17A/GSK3β/ROS/α-SMA axis mediated arthritis in a rat model of RA, which was inhibited by TDZD-8.

The mechanism of action of paeoniae radix rubra-angelicae sinensis radix drug pair in the treatment of rheumatoid arthritis through PI3K/AKT/NF-κB signaling pathway.

Objective: To investigate the effects and mechanisms of Paeoniae radix rubra-Angelicae sinensis radix (P-A) drug pair in the treatment of rheumatoid arthritis (RA). Methods: Mass spectrometry was employed to accurately characterize the main components of the P-A drug pair. Network pharmacology was used to analyze the main components and pathways of the P-A drug pair in the treatment of RA, and Discovery Studio software was used to molecularly dock the key proteins on the pathway with their corresponding compounds. The levels of serum TNF-a, IL-1β, and IL-6 were measured by enzyme linked immunosorbent assay (ELISA). The histopathology of the ankle joint was observed by hematoxylin-eosin (HE) staining, and the positive expression of p-PI3K, p-IKK, p-NF-κB, and p-AKT in the synovial tissue of the ankle joint was detected by immunohistochemical analysis. Finally, the expression of PI3K, IKK, and AKT and their phosphorylation levels were determined by western blot in each group of rats. Results: Network pharmacology combined with molecular docking analysis revealed that the pharmacodynamic mechanism of the P-A drug pair for the treatment of RA may be related to the contents of caffeic acid, quercetin, paeoniflorin, and baicalein in the regulation of the expression of the PI3K/AKT/NF-κB signaling pathway and the targets of PIK3CA, PIK3R1, AKT1, HSP90AA1 and IKBKB in the pathway. Compared with the model group, the P-A drug pair significantly improved the pathological changes of the synovial tissue and reduced feet swelling in RA model rats. Moreover, it regulated the levels of TNF-α, IL-1β, and IL-6 in serum (p < 0.05). The results of the immunohistochemical analysis and western blot showed that the expression of PI3K, IKK, NF-κB, and AKT decreased after phosphorylation in the synovial tissue (p < 0.05). Conclusion: The P-A drug pair exhibited an inhibitory effect on the hyperactivation of the PI3K/AKT/NF-κB signaling pathway in the synovial membrane of RA rats. The mechanism may be related to the downregulation of the phosphorylation levels PI3K, IKK, NF-κB, and AKT, which in turn decreased inflammatory cell infiltration and synovial membrane proliferation.

METTL14 promotes fibroblast-like synoviocytes activation via the LASP1/SRC/AKT axis in rheumatoid arthritis.

The objective of this study is to explore the specific roles of a crucial N6-methyladenosine (m6A) methyltransferase, methyltransferase-like 14 (METTL14), in fibroblast-like synoviocytes (FLSs) activation of rheumatoid arthritis (RA). RA rat model was induced by administering intraperitoneally collagen antibody alcohol. Primary fibroblast-like synoviocytes (FLSs) were isolated from joint synovium tissues in rats. shRNA transfection tools were used to downregulate METTL14 expression in vivo and vitro. The injury of joint synovium was shown by hematoxylin and eosin (HE) staining. The cell apoptosis of FLSs was determined by flow cytometry. The levels of IL-6, IL-18, and C-X-C motif chemokine ligand (CXCL)10 in serum and culture supernatants were measured by ELISA kits. The expressions of LIM and SH3 domain protein 1 (LASP1), p-SRC/SRC, and p-AKT/AKT in FLSs and joint synovium tissues were determined by Western blots. The expression of METTL14 was greatly induced in the synovium tissues of RA rats compared with normal control rats. Compared with sh-NC-treated FLSs, METTL14 knockdown significantly increased cell apoptosis, inhibited cell migration and invasion, and suppressed the production of IL-6, IL-18, and CXCL10 induced by TNF-α. METTL14 silencing suppresses the expression of LASP1 and the activation of Src/AKT axis induced by TNF-α in FLSs. METTL14 improves the mRNA stability of LASP1 through m6A modification. In contrast, these were reversed by LASP1 overexpression. Moreover, METTL14 silencing clearly alleviates FLSs activation and inflammation in a RA rat model. These results suggested that METTL14 promotes FLSs activation and related inflammatory response via the LASP1/SRC/AKT signaling pathway and identified METTL14 as a potential target for treating RA.

Identification of copper death-associated molecular clusters and immunological profiles in rheumatoid arthritis.

An analysis of the relationship between rheumatoid arthritis (RA) and copper death-related genes (CRG) was explored based on the GEO dataset. Based on the differential gene expression profiles in the GSE93272 dataset, their relationship to CRG and immune signature were analysed. Using 232 RA samples, molecular clusters with CRG were delineated and analysed for expression and immune infiltration. Genes specific to the CRGcluster were identified by the WGCNA algorithm. Four machine learning models were then built and validated after selecting the optimal model to obtain the significant predicted genes, and validated by constructing RA rat models. The location of the 13 CRGs on the chromosome was determined and, except for GCSH. LIPT1, FDX1, DLD, DBT, LIAS and ATP7A were expressed at significantly higher levels in RA samples than in non-RA, and DLST was significantly lower. RA samples were significantly expressed in immune cells such as B cells memory and differentially expressed genes such as LIPT1 were also strongly associated with the presence of immune infiltration. Two copper death-related molecular clusters were identified in RA samples. A higher level of immune infiltration and expression of CRGcluster C2 was found in the RA population. There were 314 crossover genes between the 2 molecular clusters, which were further divided into two molecular clusters. A significant difference in immune infiltration and expression levels was found between the two. Based on the five genes obtained from the RF model (AUC = 0.843), the Nomogram model, calibration curve and DCA also demonstrated their accuracy in predicting RA subtypes. The expression levels of the five genes were significantly higher in RA samples than in non-RA, and the ROC curves demonstrated their better predictive effect. Identification of predictive genes by RA animal model experiments was also confirmed. This study provides some insight into the correlation between rheumatoid arthritis and copper mortality, as well as a predictive model that is expected to support the development of targeted treatment options in the future.

Simultaneous Immunomodulation and Tissue Protection on the Rheumatoid Arthritis Models Using a Tragacanth Frankincense-Based Core–Shell Nanostructure

Rheumatoid arthritis (RA) is a chronic autoimmune disease that requires anti-inflammatory drugs, analgesics, and tissue protectors. To deal with this disease, the pain and inflammation should be relieved in the first stage, and tissue protectors should be used to restore joint damage that limits movement. Therefore, a comprehensive multifunctional core–shell nanocomposite (TFH@TA/AuNPs-HDA) with a continuous anti-inflammatory effect and a special tissue protection potential in the core was designed. Here, the hydrothermal reaction was applied to synthesize the hydrogel core from tragacanth and frankincense gums; then, the shell was self-assembled with tannic acid/gold nanoparticles (TA/AuNPs) and 10-hydroxy-2-decanoic acid (10-HDA). The biocompatibility, anti-inflammatory properties, and tissue protective effect of TFH@TA/AuNPs-HDA were investigated on RAW264.7 cells and in collagen-induced RA rat models. Moreover, the molecular docking demonstrated the interactions of 10-HDA and AuNPs with NF-κB signaling pathway proteins, confirming the anti-inflammatory effect of 10-HDA and AuNP suppression of the NF-κB pathway. The results revealed that TFH@TA/AuNPs-HDA could be an appropriate system for RA therapy due to the synergistic alleviation of inflammation and joint tissue destruction.

Prodigiosin improves acute lung injury in a rat model of rheumatoid arthritis via down-regulating the nuclear factor kappaB/nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3 signaling pathway.

Prodigiosin (PRO) is a natural pigment that possesses multiple activities, covering anti-tumor, anti-bacteria, and immunosuppression. This study is committed to an investigation into the underlying function and the certain mechanism of PRO in acute lung damage followed by rheumatoid arthritis (RA). Cecal ligation and puncture (CLP) method was implemented to trigger a rat lung injury model, and a rat RA model was constructed with the help of rheumatoid arthritis induced by collagen. Prodigiosin was administered to intervene in the rats' lung tissues post-treatment. The expressions of pro-inflammatory cytokines (interleukin-1beta, interleukin-6, tumor necrosis factor-alpha, and monocyte chemoattractant protein-1 were determined. Western blot was carried out to detect anti-surfactant protein A (SPA), anti-surfactant protein D (SPD), apoptosis-concerned proteins (Bax, cleaved-caspase-3, Bcl-2, and pro-caspase-3), the nuclear factor-kappaB (NF-κB)/nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3 (NLRP3)/apoptosis-concerned speckle-like protein (ASC)/caspase-1 signaling pathway. The apoptosis of pulmonary epithelial tissues was checked via TUNEL assay, as corresponding kits were adopted to confirm the activity of lactate dehydrogenase (LDH) and the levels of oxidative stress markers malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px). Prodigiosin ameliorated the pathological damage of CLP rats. Prodigiosin alleviated the production of inflammatory and oxidative stress mediators. In the RA rats with acute lung injury, prodigiosin hampered apoptosis in the lung. Mechanistically, prodigiosin hinders the activation of the NF-κB/NLRP3 signaling axis. In conclusion: prodigiosin relieves acute lung injury in a rat model of rheumatoid arthritis by exerting anti-inflammatory and anti-oxidative effects through downregulating the NF-κB/NLRP3 signaling axis.

Prodigiosin improves acute lung injury in a rat model of rheumatoid arthritis via down-regulating the nuclear factor kappaB/nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3 signaling pathway.

Prodigiosin (PRO) is a natural pigment that possesses multiple activities, covering anti-tumor, anti-bacteria, and immunosuppression. This study is committed to an investigation into the underlying function and the certain mechanism of PRO in acute lung damage followed by rheumatoid arthritis (RA). Cecal ligation and puncture (CLP) method was implemented to trigger a rat lung injury model, and a rat RA model was constructed with the help of rheumatoid arthritis induced by collagen. Prodigiosin was administered to intervene in the rats' lung tissues post-treatment. The expressions of pro-inflammatory cytokines (interleukin-1beta, interleukin-6, tumor necrosis factor-alpha, and monocyte chemoattractant protein-1 were determined. Western blot was carried out to detect anti-surfactant protein A (SPA), anti-surfactant protein D (SPD), apoptosis-concerned proteins (Bax, cleaved-caspase-3, Bcl-2, and pro-caspase-3), the nuclear factor-kappaB (NF-κB)/nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3 (NLRP3)/apoptosis-concerned speckle-like protein (ASC)/caspase-1 signaling pathway. The apoptosis of pulmonary epithelial tissues was checked via TUNEL assay, as corresponding kits were adopted to confirm the activity of lactate dehydrogenase (LDH) and the levels of oxidative stress markers malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px). Prodigiosin ameliorated the pathological damage of CLP rats. Prodigiosin alleviated the production of inflammatory and oxidative stress mediators. In the RA rats with acute lung injury, prodigiosin hampered apoptosis in the lung. Mechanistically, prodigiosin hinders the activation of the NF-κB/NLRP3 signaling axis. In conclusion: prodigiosin relieves acute lung injury in a rat model of rheumatoid arthritis by exerting anti-inflammatory and anti-oxidative effects through downregulating the NF-κB/NLRP3 signaling axis.

Novel Insights on CAI-CORM Hybrids: Evaluation of the CO Releasing Properties and Pain-Relieving Activity of Differently Substituted Coumarins for the Treatment of Rheumatoid Arthritis.

Pain control is among the most important healthcare services in patients affected by rheumatoid arthritis (RA), but the current therapeutic options (i.e., disease-modifying anti-rheumatic drugs) are limited by the risk of the side effects. In this context, we proposed an innovative approach based on the hybridization between carbonic anhydrase inhibitors (CAIs) and CO releasing molecules (CORMs). The resulting CAI-CORM hybrids were revealed to possess strong anti-inflammatory effects in in vitro models of diseases and to relieve ache symptoms in an in vivo RA rat model. In this work, we have deepened the study of these promising hybrids, designing a library of coumarin-based compounds, also including internal dicobalt hexacarbonyl systems. The results obtained from the CO releasing study, the CA inhibitory activity, and the in vivo pain-relief efficacy evaluation in the RA rat model confirmed the success of this strategy, allowing us to consider CAI-CORM hybrids promising anti-nociceptive agents against arthritis.

Biomimetic chitosan nanoparticles with simultaneous water lubricant and anti-inflammatory

Rheumatoid arthritis (RA) is a chronic inflammatory immune and lubrication dysfunction disease that causes great damage to the joints. Herein, inspired by the unique biochemistry structure and excellent hydration of chondroitin sulfate (CHI) existing in joint system, one kind of novel polysaccharide nanoparticle lubricant, that is chitosan nanoparticles (CS NPs) grafting CHI (CS-CHI), is synthesized by one-step surface chemistry reaction. CHI with negative charges can form hydration layers on the surface of CS NPs, thus improving the lubricity of nanoparticles. Simultaneously, CS-CHI NPs have effective loading and sustained drug release ability for anti-inflammatory drug diclofenac sodium (DS), along with good biocompatibility. Finally, based on a collagen-induced rat RA model, in vitro animals experimental results indicate that the as-synthesized CS-CHI@DS NPs has obvious inhibitory effects on inflammatory factors and can effectively prevent the damaged cartilage from further destruction.

Longitudinal 18F-VUIIS1008 PET imaging in a rat model of rheumatoid arthritis.

Macrophages have crucial roles in the pathogenesis of rheumatoid arthritis (RA). We aimed to elucidate the temporal profile of macrophage infiltration in synovitis in RA rat models using PET (positron emission tomography) imaging based a new generation of TSPO (Translocator protein, 18kDa)-PET ligand, 18F-VUIIS1008 {2-[5,7-Diethyl-2-{4-[2-(18F)fluoroethoxy]phenyl}pyrazolo(1,5-a)pyri-midin-3-yl]-N, N-diethylacetamide}. In vitro and in vivo studies were conducted using RAW264.7 macrophage cells and a rat model of RA induced by Complete Freund's Adjuvant (CFA). Our results showed 18F-VUIIS1008 showed excellent stability in vitro and binding specificity to RAW264.7 cells, and rapid accumulation in the left inflammatory ankles. PET studies revealed that 18F-VUIIS1008 could clearly identify the left inflammatory ankles with good contrast at 30-120min post-injection. The uptake of 18F-VUIIS1008 of left inflammatory ankles was a wiggle trace with two peaks on day 7 and 29, and then, the highest peak uptake was seen on day 29 (3.00% ± 0.08%ID/g) at 60min after injection. Tracer uptakes could be inhibited by PK11195 or VUIIS1008. Immunohistochemistry and immunofluorescence tests showed that elevated TSPO expression and infiltrated macrophages were found in the left inflammation ankles. 18F-VUIIS1008 as a novel PET imaging agent showed great potential to identify temporal profile of macrophage infiltration in synovitis in RA, and deliver accurate non-invasive diagnosis and real-time monitoring of RA development.

Saussurea involucrata oral liquid regulates gut microbiota and serum metabolism during alleviation of collagen-induced arthritis in rats.

Saussurea involucrata oral liquid (SIOL) can clinically relieve symptoms, such as joint pain and swelling, and morning stiffness, in patients with rheumatoid arthritis (RA). However, the mechanism of action remains unclear. This study used a combination of gut microbiota and serum metabolomics analysis to investigate the effects and potential mechanisms of SIOL intervention on rats with RA induced by type II bovine collagen and Freund's complete adjuvant. Results showed that SIOL treatment consequently improved the degree of ankle joint swelling, joint histopathological changes, joint pathological score, and expression of serum-related inflammatory cytokines (interleukin (IL)-1β, IL-4, IL-6, IL-10, and tumor necrosis factor-α) in RA model rats. 16 S rRNA sequencing results showed that SIOL increased the relative richness of the Lactobacillus and Bacteroides genus and decreased the relative richness of Romboutsia, Alloprevotella, Blautia, and Helicobacter genus. Serum nontargeted metabolomic results indicated that SIOL could regulate metabolites related to metabolic pathways, such as glycine, serine, threonine, galactose, cysteine, and methionine metabolism. Spearman correlation analysis showed that the regulatory effects of SIOL on the tricarboxylic acid (TCA) cycle, phenylalanine metabolism, phenylalanine, tyrosine, and tryptophan biosynthesis, and glyoxylate and dicarboxylate metabolism pathways were correlated with changes in the richness of the Lactobacillus, Romboutsia, Bacteroides, and Alloprevotella genus in the gut microbiome. In conclusion, this study revealed the ameliorative effects of SIOL on RA and suggested that the therapeutic effects of SIOL on RA may be related to the regulation of the community richness of the Lactobacillus, Romboutsia, Bacteroides, and Alloprevotella genus, thereby improving the TCA cycle; phenylalanine metabolism; phenylalanine, tyrosine, and tryptophan biosynthesis, and glyoxylate and dicarboxylate metabolism-related pathways.

Effect of moxibustion on AMPK/ULK1 signaling pathway in synovium tissue of toes in rats with rheumatoid arthritis

To observe the effect of moxibustion on AMP-activated protein kinase (AMPK)/UNC-51-like kinase 1 (ULK1) signaling pathway in the synovial tissue of toes in rheumatoid arthritis rats, so as to explore the mechanism of mo-xibustion in the treatment of rheumatoid arthritis (RA). A total of 45 SD rats were randomly divided into blank, model， moxibustion, methotrexate and rapamycin groups, with 9 rats in each group. RA rat model was established by injection of Freund's complete adjuvant. Moxibustion was applied to "Zusanli" (ST36) and "Guanyuan" (CV4) for 20 min, once a day for 3 weeks. Methotrexate group was given methotrexate (0.35 mg/kg) by gavage, twice a week for 3 weeks. Rapamycin group was intraperitoneally injected with rapamycin (1 mg/kg)，once every other day for 3 weeks. The toe volume of the left hind limb of rats was measured by the toe volume measuring instrument. The content of AMP in toe synovium was detected by ELISA. The expression of AMPK and VPS34 protein in toe synovium was detected by Western blot.The expression of ULK1 and Atg13 mRNA in toe synovium was detected by RT-PCR. Compared with the blank group, the volume of toe in the model group was increased (P<0.01)，while the content of AMP, the expression of AMPK and VPS34 proteins, the expression of ULK1 and Atg13 mRNAs were significantly decreased(P<0.01).Compared with the model group, the volume of toe in the moxibustion，methotre-xate and rapamycin groups was decreased (P<0.05); the content of AMP, the expression of AMPK and VPS34 proteins, the expression of ULK1 and Atg13 mRNAs were significantly increased(P<0.05) in the moxibustion group; the content of AMP, the expression of VPS34 protein, the expression of Atg13 mRNA were significantly increased(P<0.05) in the methotrexate group; the expression of AMPK and VPS34 proteins, the expression of ULK1 and Atg13 mRNAs were significantly increased (P<0.05, P<0.01) in the rapamycin group. Compared with the moxibustion group, the expression of AMPK protein in the methotrexate group and the content of AMP in the rapamycin group were significantly decreased (P<0.05). Moxibustion can improve joint swelling in RA rats, and the mechanism may be related to promoting the activity of AMPK/ULK1 signaling pathway.

Nitric Oxide Scavenging and Hydrogen Sulfide Production Synergistically Treat Rheumatoid Arthritis.

To restore the disordered endogenous gas levels is an efficient alternative for the treatment of rheumatoid arthritis (RA). Both insufficient hydrogen sulfide (H2 S) and excessive nitric oxide (NO) contribute to synovial inflammation. Herein, a new block polymer PEG10 -b-PNAPA30 -b-PEG10 composed of an NO-responsive monomer and a cysteine-triggered H2 S donor, which can simultaneously scavenge NO and release therapeutic H2 S for RA treatment, is reported. In vitro experiments demonstrate that the polymer exhibits a synergistic effect on suppressing reactive oxygen species levels and pro-inflammatory cytokine production via NF-κB signaling pathway. It leads to the polarization of macrophages from M1 to M2 phenotype. Moreover, the released H2 S further restrains NO production by suppressing the expression of iNOS. In vivo experiments with an RA rat model show that the system markedly mitigates the synovial inflammation, osteoporosis, and clinical symptoms of RA rats, which is attributed to the combination therapy of H2 S release and NO depletion. This work provides new insight into the synergistic treatment of RA and endogenous gas-related diseases.

Action of Tofacitinib in a Rat Model of Synovitis

Purpose: To evaluate effects and mechanism of tofacitinib in treatment of rheumatoid arthritis (RA) model rats. Materials and Methods: Dividing 27 rats into 3 groups: NC (normal control), Model (RA model) and Tofacitinib (RA model rats treated with tofacitinib) groups. Observation joint swelling and articular synovium pathology by HE staining, IL-1β, IL-6, IL-10 and TNF-α levels by ELISA assay, JAK2, STAT3 and NF-κB(p65) proteins by IHC and WB assay. Results: Compared with NC group, joint swelling, histopathological score IL-1β, IL-6, IL-10 and TNF-α significantly deteriorated (P &lt; 0.001, respectively); by IHC and WB assay, JAK2, STAT3 and NF-κB(p65) proteins expression were significantly up-regulation in joint synovial tissue in model group (P &lt; 0.001, respectively). With tofacitinib supplement, joint swelling, histopathological score IL-1β, IL-6, IL-10 and TNF-α significantly improved (P &lt; 0.001, respectively); by IHC and WB assay, JAK2, STAT3 and NF-κB(p65) proteins expression significantly down-regulation in joint synovial tissue in Tofacitinib group (P &lt; 0.001, respectively). Conclusion: Tofacitinib could improve RA via regulation JAK2/STAT3/NF-κB(p65) pathway in vivo study.

Disease Markers and Therapeutic Targets for Rheumatoid Arthritis Identified by Integrating Bioinformatics Analysis with Virtual Screening of Traditional Chinese Medicine.

The aim of this study was to identify potentially important Rheumatoid arthritis (RA) targets related to immune cells based on bioinformatics analysis, and to identify small molecules of traditional Chinese medicine (TCM) associated with these targets that have potential therapeutic effects on RA. Gene expression profile data related to RA were downloaded from the Gene Expression Omnibus (GSE55235, GSE55457, and GSE77298), and datasets were merged by the batch effect removal method. The RA key gene set was identified by protein-protein interaction network analysis and machine learning-based feature extraction. Furthermore, immune cell infiltration analysis was carried out on all DEGs to obtain key RA markers related to immune cells. Batch molecular docking of key RA markers was performed on our previously compiled dataset of small molecules in TCM using AutoDock Vina. Moreover, in vitro experiments were performed to examine the inhibitory effect of screened compounds on the synovial cells of an RA rat model. The PPI network and feature extraction with machine learning classifiers identified eight common key RA genes: MYH11, CFP, LY96, IGJ, LPL, CD48, RAC2, and CSK. RAC2 was significantly correlated with the infiltration and expression of five immune cells, with significant differences in these immune cells in the normal and RA samples. Molecular docking and in vitro experiments also showed that sanguinarine, sesamin, and honokiol could effectively inhibit the proliferation of RA rat synovial cells, also could all effectively inhibit the secretion of TNF-α and IL-1β in synovial cells, and had a certain inhibitory effect on expression of the target protein RAC2. The core gene set of RA was screened from a new perspective, revealing biomarkers related to immune cell infiltration. Using molecular docking, we screened out TCM small molecules for the treatment of RA, providing methods and technical support for the treatment of RA with TCM.

Therapeutic effects of shaogan fuzi decoction in rheumatoid arthritis: Network pharmacology and experimental validation

Shaogan Fuzi Decoction (SGFD), one of the classical prescriptions of Chinese Medicine, has a long history in the treatment of rheumatoid arthritis (RA), but definitive studies on its efficacy and mechanism of action are lacking. This study aims to elucidate the pharmacodynamic role of SGFD against RA and the potential mechanisms based on a combination of network pharmacology and experimental verification. The RA model in rats was induced by intradermal injection of bovine type Ⅱ collagen and incomplete Freund’s adjuvant at the tail root. SGFD was administered once a day by oral gavage for 4 weeks. After SGFD administration, rat’s arthritis index (AI) score and paw swelling decreased to some extent, and synovial inflammation, vascular hyperplasia, and cartilage destruction of the ankle joint were improved. Simultaneously, thymus and spleen index and serum levels of C-reactive protein (CRP) were lowered. Network pharmacology revealed that quercetin, kaempferol, naringenin, formononetin isorhamnetin and licochalcone A were the potentialiy active components, and IL6, TP53, TNF, PTGS2, MAPK3 and IL-1β were potential key targets for SGFD in the treatment of RA. Ingredients-targets molecular docking showed that the components had the high binding activity to these target proteins. The mechanism of SGFD for RA involves various biological functions and is closely correlated with TNF signaling pathway, Osteoclast differentiation, T cell receptor signaling pathway, mitogen-activated protein kinase (MAPK) signaling pathway, NF-κB signaling pathway, toll-like receptor signaling pathway, and so on. Western blot and ELISA showed that the expression of toll-like receptor 4 (TLR4), nuclear factor kappa-B (NF-κB) p65, phosphorylated c-Jun N-terminal kinase (p-JNK), p-p38, phosphorylated extracellular regulated kinase (p-ERK) and TNF-α was significantly upregulated in the synovium of RA rats, and the levels of serum inflammatory factors were significantly increased. SGFD inhibits the activation of the TLR4/NF-κB/MAPK pathway and the expression/production of pro-inflammatory cytokines. In summary, SGFD could improve the symptoms and inflammatory response in collagen-induced arthritis (CIA) rat model. The mechanism might be related to the regulation of TLR4/MAPKs/NF-κB signaling pathway and the reduction of inflammatory factor release, which partially confirms the results predicted by network pharmacology.

Deciphering the pharmacological mechanisms of Chaenomeles Fructus against rheumatoid arthritis by integrating network pharmacology and experimental validation.

Chaenomeles Fructus is a plant that can be used for both food and medicine. Modern studies have shown that Chaenomeles Fructus has anti‐inflammatory and immunosuppressive effects on arthritis. However, the mechanism of action of Chaenomeles Fructus on rheumatoid arthritis (RA) and its main active ingredients are still unclear. This study was aimed at devising an integrated strategy for investigating the bioactivity constituents and possible pharmacological mechanisms of Chaenomeles Fructus against RA. The components of Chaenomeles Fructus were analyzed using UPLC‐Q‐Exactive orbitrap MS techniques and applied to screen the active components of Chaenomeles Fructus according to their oral bioavailability and drug‐likeness index. Then, we speculated on the potential molecular mechanisms of Chaenomeles Fructus against RA through a network pharmacology analysis. Finally, the potential molecular mechanisms of Chaenomeles Fructus against RA were validated in a complete Freund's adjuvant (CFA)‐induced RA rat model. We identified 48 components in Chaenomeles Fructus and screened seven bioactive ingredients. The results of the network pharmacology prediction and the experimental verification results were analyzed by Venn analysis, and the experimental results concluded that Chaenomeles Fructus mainly interferes with the inflammation of RA by inhibiting arachidonic acid metabolism and the MAPK signaling pathway. This study identified the ingredients of Chaenomeles Fructus by UPLC‐Q‐Exactive orbitrap MS and explained the possible mechanisms of Chaenomeles Fructus against RA by integrating network pharmacology and experimental validation.

Preparation of meloxicam-salicylic acid co-crystal and its application in the treatment of rheumatoid arthritis

Meloxicam is a non-steroidal anti-inflammatory drug that can selectively inhibit cyclooxygenase 2 (COX-2), showing high anti-inflammatory activity, low gastrointestinal and renal side effects. However, the low solubility and slow dissolution rate limit its role in rapid pain relief. In this paper, using meloxicam as the active drug molecule and salicylic acid as the co-crystal former, the meloxicam-salicylic acid co-crystal (MLX-SLC CoC) was prepared by the reaction crystallization method. The co-crystal was characterized by differential scanning calorimetry (DSC), Fourier transform infrared spectroscopy (FT-IR) and X-ray diffraction (XRD). The dissolution curve of the co-crystal was determined, and the result showed that the formation of drug co-crystal enhanced the solubility and dissolution rate of MLX. The oil-water partition coefficient of the MLX-SLC CoC was determined, and the results showed that the oil-water partition coefficient of the co-crystal was similar to that of pure MLX. The gel prepared by MLX-SLC CoC was used to study its in vitro release and transdermal penetration characteristics were investigated, which showed that the transdermal penetration of the drug was increased without affecting its in vitro release. Preparation of gel in the form of MLX-SLC CoC increased the drug concentration in the gel. Finally, the therapeutic effect of the gel on arthritis was preliminarily studied by establishing a rheumatoid arthritis rat model.