OBJECTIVE: To observe the effects of moxibustion on the ultrastructure of synovial cells of knee joint and serum cytokines in adjuvant arthritis (AA) rats, and to explore the potential mechanism of moxibustion in treatment of rheumatoid arthritis. METHODS: Forty-five Wistar male rats were randomly divided into a normal group, a model group and a moxibustion group, with 15 rats in each group. In the model group and the moxibustion group, the AA model was replicated under wind, cold and humid environment and by injection with complete freund's adjuvant. In the moxibustion group, moxibustion at "Zusanli" (ST 36) and "Shenshu" (BL 23) was used, 20 min each time, once daily, for consecutive 21 days. In the normal group and the model group, no intervention was processed. The scores of the knee joint swelling degree (JSD) and arthritis index (AI) were compared among groups. The ultrastructure of synovial cells of knee joint were observed under transmission electron microscope (TEM). The levels of serum cytokines such as tumor necrosis factor-α (TNF-α), interieukin (IL)-1β, IL-6 and IL-10 were detected using ELISA method. RESULTS: Compared with the normal group, JSD and AI scores, the levels of TNF-α, IL-1β and IL-6 were increased (P<0.01), while IL-10 was reduced (P<0.01) in the model group after intervention. JSD and AI scores, and the levels of TNF-α, IL-1β and IL-6 were lower (P<0.05, P<0.01), while the level of IL-10 was higher (P<0.01) in the moxibustion group compared with the model group. Compared with the normal group, the ultrastructure of synovial cell was obviously damaged in the model group, and the damage was attenuated in the moxibustion group compared with the model group. CONCLUSION Moxibustion can reduce the symptoms of arthritis in AA rats, which may be related to the improvement of the ultrastructure of synovial cells and the regulation of cytokines.
Male ; Rats ; Animals ; Cytokines ; Interleukin-10 ; Arthritis, Experimental ; Tumor Necrosis Factor-alpha ; Interleukin-6 ; Moxibustion ; Rats, Wistar ; Knee Joint

The present study investigated the mechanism of artesunate in the treatment of bone destruction in experimental rheumatoid arthritis(RA) based on transcriptomics and network pharmacology. The transcriptome sequencing data of artesunate in the inhibition of osteoclast differentiation were analyzed to obtain differentially expressed genes(DEGs). GraphPad Prism 8 software was used to plot volcano maps and heat maps were plotted through the website of bioinformatics. GeneCards and OMIM were used to collect information on key targets of bone destruction in RA. The DEGs of artesunate in inhibiting osteoclast differentiation and key target genes of bone destruction in RA were intersected by the Venny 2.1.0 platform, and the intersection target genes were analyzed by Gene Ontology(GO)/Kyoto Encyclopedia of Genes and Genomes(KEGG) enrichment. Finally, the receptor activator of nuclear factor-κB(RANKL)-induced osteoclast differentiation model and collagen-induced arthritis(CIA) model were established. Quantitative real time polymerase chain reaction(q-PCR), immunofluorescence, and immunohistochemistry were used to verify the pharmacological effect and molecular mechanism of artesunate in the treatment of bone destruction in RA. In this study, the RANKL-induced osteoclast differentiation model in vitro was established and intervened with artesunate, and transcriptome sequencing data were analyzed to obtain 744 DEGs of artesunate in inhibiting osteoclast differentiation. A total of 1 291 major target genes of bone destruction in RA were obtained from GeneCards and OMIM. The target genes of artesunate in inhibiting osteoclast differentiation and the target genes of bone destruction in RA were intersected to obtain 61 target genes of artesunate against bone destruction in RA. The intersected target genes were analyzed by GO/KEGG enrichment. According to the results previously reported, the cytokine-cytokine receptor interaction signaling pathway was selected for experimental verification. Artesunate intervention in the RANKL-induced osteoclast differentiation model showed that artesunate inhibited CC chemokine receptor 3(CCR3), CC chemokine receptor 1(CCR1) and leukemia inhibitory factor(LIF) mRNA expression in osteoclasts in a dose-dependent manner compared with the RANKL-induced group. Meanwhile, the results of immunofluorescence and immunohistochemistry showed that artesunate could dose-dependently reduce the expression of CCR3 in osteoclasts and joint tissues of the CIA rat model in vitro. This study indicated that artesunate regulated the CCR3 in the cytokine-cytokine receptor interaction signaling pathway in the treatment of bone destruction in RA and provided a new target gene for the treatment of bone destruction in RA.
Rats ; Animals ; Arthritis, Experimental/drug therapy* ; Artesunate/therapeutic use* ; Arthritis, Rheumatoid/genetics* ; Transcriptome ; Network Pharmacology ; Osteoclasts ; Receptors, Cytokine/therapeutic use*

This study aims to investigate the therapeutic effect of alcohol extract of root and root bark of Toddalia asiatica(TAAE) on collagen-induced arthritis(CIA) in rats through phosphatidylinoinosidine-3 kinase/protein kinase B(PI3K/Akt) signaling pathway. To be specific, CIA was induced in rats, and then the rats were treated(oral, daily) with TAAE and Tripterygium Glycoside Tablets(TGT), respectively. The swelling degree of the hind leg joints was scored weekly. After 35 days of administration, the histopathological changes were observed based on hematoxylin and eosin(HE) staining. Enzyme-linked immunosorbent assay(ELISA) was employed to detect the levels of cytokines [tumor necrosis factor-α(TNF-α), interleukin(IL)-6)]. Terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL) staining was performed to detect the apoptosis of synoviocytes in rats. Western blot was used to detect the expression levels of apoptosis-related proteins B-cell lymphoma 2(Bcl-2)-associated X(Bax), Bcl-2, and caspase-3 and pathway-related proteins phosphoinositide 3-kinase(PI3K), phosphorylated(p)-PI3K, protein kinase B(Akt), and p-Akt. RT-qPCR was conducted to examine the mRNA levels of Bax, Bcl-2, caspase-3, TNF-α, IL-6, and IL-1β and pathway-related proteins PI3K, p-PI3K, Akt, and p-Akt. TAAE can alleviate the joint swelling in CIA rats, reduce serum levels of inflammatory cytokines, improve synovial histopathological changes, promote apoptosis of synoviocytes, and inhibit synovial inflammation. In addition, RT-qPCR and Western blot results showed that TAAE up-regulated the level of Bax, down-regulated the level of Bcl-2, and activated caspase-3 to promote apoptosis in synoviocytes. TAAE effectively down-regulated the protein levels of p-PI3K and p-Akt. In this study, TAAE shows therapeutic effect on CIA in rats and reduces the inflammation. The mechanism is that it suppresses PI3K/Akt signaling pathway and promotes synoviocyte apoptosis. Overall, this study provides a new clue for the research on the anti-inflammatory mechanism of TAAE and lays a theoretical basis for the better clinical application of TAAE in the treatment of inflammatory and autoimmune diseases.
Rats ; Animals ; Proto-Oncogene Proteins c-akt/metabolism* ; Phosphatidylinositol 3-Kinases/metabolism* ; Caspase 3/genetics* ; Tumor Necrosis Factor-alpha/metabolism* ; bcl-2-Associated X Protein/metabolism* ; Plant Bark ; Anti-Inflammatory Agents/therapeutic use* ; Arthritis, Experimental/chemically induced* ; Inflammation/drug therapy* ; Cytokines/metabolism* ; Proto-Oncogene Proteins c-bcl-2 ; Apoptosis

A fluorescence endoscopic laser confocal microscope(FELCM) was used to direct the injection of sinomenine solid lipid nanoparticles(Sin-SLN) into the joint, and the in vitro effectiveness of Sin-SLN in the treatment of rheumatoid arthritis(RA) was evaluated. Sin-SLN was prepared with the emulsion evaporation-low temperature curing method. The Sin-SLN prepared under the optimal conditions showed the encapsulation efficiency of 64.79%±3.12%, the drug loading of 3.84%±0.28%, the average particle size of(215.27±4.21) nm, and the Zeta potential of(-32.67±0.84) mV. Moreover, the Sin-SLN demonstrated good stability after sto-rage for 30 days. The rabbit model of RA was established by the subcutaneous injection of ovalbumin and complete Freund's adjuvant. Five groups were designed, including a control group, a model group, a Sin(1.5 mg·kg~(-1)) group, a Sin-SLN(1.5 mg·kg~(-1)) group, and a dexamethasone(positive drug, 1.0 mg·kg~(-1), ig) group. The control group and the model group only received puncture treatment without drug injection. After drug administration, the local skin temperature and knee joint diameter were monitored every day. The knee joint diameter and the local skin temperature were lower in the drug administration groups than in the model group(P<0.05, P<0.01). FELCM recorded the morphological alterations of the cartilage of knee joint. The Sin-SLN group showed compact tissue structure and smooth surface of the cartilage. Enzyme-linked immunosorbent assay(ELISA) was employed to determine the serum le-vels of interleukin-1(IL-1) and tumor necrosis factor-α(TNF-α). The findings revealed that the Sin-SLN group had lower IL-1 and TNF-α levels than the model group(P<0.05, P<0.01). Hematoxylin-eosin(HE) staining was employed to reveal the pathological changes of the synovial tissue, which were significantly mitigated in the Sin-SLN group. The prepared Sin-SLN had uniform particle size and high stability. Through joint injection administration, a drug reservoir was formed. Sin-SLN effectively alleviate joint swelling and cartilage damage of rabbit, down-regulated the expression of inflammatory cytokines, and inhibited the epithelial proliferation and inflammatory cell infiltration of the synovial tissue, demonstrating the efficacy in treating RA.
Animals ; Rabbits ; Tumor Necrosis Factor-alpha ; Fluorescence ; Arthritis, Rheumatoid/drug therapy* ; Interleukin-1 ; Arthritis, Experimental/drug therapy*

Traditionally, Tripterygium hypoglaucum (Levl.) Hutch (THH) are widely used in Chinese folk to treat rheumatoid arthritis (RA). This study aimed to investigate whether the anti-RA effect of THH is related with the gut microbiota. The main components of prepared THH extract were identified by HPLC-MS. C57BL/6 mice with adjuvant-induced arthritis (AIA) were treated with THH extract by gavage for one month. THH extract significantly alleviated swollen ankle, joint cavity exudation, and articular cartilage destruction in AIA mice. The mRNA and protein levels of inflammatory mediators in muscles and plasma indicated that THH extract attenuated inflammatory responses in the joint by blocking TLR4/MyD88/MAPK signaling pathways. THH extract remarkably restored the dysbiosis of the gut microbiota in AIA mice, featuring the increases of Bifidobacterium, Akkermansia, and Lactobacillus and the decreases of Butyricimonas, Parabacteroides, and Anaeroplasma. Furthermore, the altered bacteria were closely correlated with physiological indices and drove metabolic changes of the intestinal microbiota. In addition, antibiotic-induced pseudo germ-free mice were employed to verify the role of the intestinal flora. Strikingly, THH treatment failed to ameliorate the arthritis symptoms and signaling pathways in pseudo germ-free mice, which validates the indispensable role of the intestinal flora. For the first time, we demonstrated that THH extract protects joint inflammation by manipulating the intestinal flora and regulating the TLR4/MyD88/MAPK signaling pathway. Therefore, THH extract may serve as a microbial modulator to recover RA in clincial practice.ver RA in clincial practice.
Mice ; Animals ; Gastrointestinal Microbiome ; Tripterygium ; Myeloid Differentiation Factor 88/genetics* ; Toll-Like Receptor 4/genetics* ; Mice, Inbred C57BL ; Arthritis, Experimental/drug therapy*

OBJECTIVES: Rheumatoid arthritis (RA) is a chronic autoimmune disease. MicroRNA has been shown to play an important role in RA. MicroRNA-124a (miR-124a) has anti-proliferative and anti-inflammatory effects in RA fibroblast synovial cells. This study aims to explore the effects of miR-124a overexpression on arthritis in collagen-induced arthritis (CIA) mice and the underlying mechanisms. METHODS: Bovine type II collagen and complete Ferris adjuvant were used to induce CIA model from DBA/1 mice. Twenty-eight days after initial immunization (D28), CIA mice were randomly divided into a model group, a miR-124a treatment group, and a negative control (NC) group. Physiological saline, miR-124a agomir, and miR-124a agomir NC were injected into the skin at the tail root of mice every 3 days for 4 times, respectively. The degree of joint swelling and arthritis index of mice were recorded accordingly. Sixty-three days after initial immunization (D63), the mice were sacrificed to obtain the synovial tissue of ankle joint. HE staining was used to observe the proliferation of synovial cell, infiltration of inflammatory cell, pannus, and bone erosion of synovial tissues; TUNEL staining was used to detect cell apoptosis; qRT-PCR was used to detect the mRNA expression of miR-124a, phosphatidylinositol-3-kinase catalytic subunit alpha (PIK3CA) and its downstream genes Bcl-2 and Bax. Immunohistochemistry was used to detect the protein expression of PIK3CA, Bcl-2, and Bax protein in synovial tissues of each group. RESULTS: Different degrees of swelling presented in the paws of DBA/1 mice at D28, which indicated the CIA model was constructed successfully. Forty-eight days after initial immunization (D48), the paws of mice in the miR-124a treatment group were only slightly red and swollen, while the paws of mice in the model group and the NC group were obviously red and swollen. The arthritis index of mice in the miR-124a treatment group were decreased significantly compared to the NC group at D51, D53, D59, and D62 (51, 53, 59, 62 days after initial immunization) (all P<0.05). Sixty-three days after initial immunization (D63), HE staining indicated that the scores of synovial cell proliferation, inflammatory cell infiltration, synovial pannus, and bone erosion were significantly reduced in the miR-124a treatment group (P<0.05 or P<0.01), while cell apoptosis was increased in the miR-124a treatment group compared with the model group and NC group (P<0.01 or P<0.001). Besides, the expression of miR-124a and Bax in the synovial tissue in miR-124a treatment group was significantly higher than those in the model group and NC group (P<0.01 or P<0.001), while the expressions of PIK3CA and Bcl-2 were decreased (P<0.05 or P<0.01 or P<0.001), and the ratio of Bcl-2 to Bax was significantly decreased (P<0.01 or P<0.001). CONCLUSIONS Overexpression of miR-124a can reduce arthritis in CIA mice bacause it could promote synovial cell apoptosis and inhibit synovial cell proliferation via targeting PIK3CA and regulating its downstream pathways.
Animals ; Arthritis, Experimental/metabolism* ; Arthritis, Rheumatoid/genetics* ; Cattle ; Cell Proliferation ; Class I Phosphatidylinositol 3-Kinases/metabolism* ; Mice ; Mice, Inbred DBA ; MicroRNAs/metabolism* ; Proto-Oncogene Proteins c-bcl-2/metabolism* ; Synovial Membrane ; bcl-2-Associated X Protein/metabolism*

Wantong Jingu Tablet (WJT), a mixture of traditional Chinese medicine, was reported to relieve the symptoms of rheumatoid arthritis (RA), but its pharmacological mechanism was not completely understood. The aim of this study was to investigate the therapeutic mechanisms of WJT for RA in vivo. The effects of WJT on joint pathology, as well as the levels of Bax, Bcl-2, caspase-3, cleaved-caspase-3, ERK1/2, pERK1/2, TNF-α, IL-1β, and IL-6 were measured using collagen-induced arthritis (CIA) rats. The intestinal flora composition and the metabolites alteration were analyzed by 16S rDNA sequencing and metabolomics method, respectively. We found that WJT ameliorated the severity of the CIA rats which might be mediated by inducing apoptosis, inactivating the MEK/ERK signals and reducing the production of pro-inflammatory cytokines. WJT, in part, relieved the gut microbiota dysbiosis, especially bacterial phylum Bacteroidetes, Tenericutes and Deferribacteres, as well as bacterial genus Vibrio, Macrococcus and Vagococcus. 3'-N-debenzoyl-2'-deoxytaxol, tubulysin B, and magnoline were significantly associated with the specific genera. We identified serotonin, glutathione disulfide, N-acetylneuraminic acid, naphthalene and thromboxane B2 as targeted molecules via metabolomics. Our findings contributed to the understanding of RA pathogenesis, and WJT played essential roles in gut microbiota health and metabolite modulation in the CIA rats.
Animals ; Arthritis, Experimental/drug therapy* ; Arthritis, Rheumatoid/drug therapy* ; Dysbiosis ; Metabolomics ; Rats ; Tablets

The present study investigated the pharmacodynamic material basis of Laportea bulbifera in the treatment of rheumatoid arthritis. Firstly, human rheumatoid arthritis fibroblast-like synoviocyte line MH7A was cultured in vitro and treated with tumor necrosis factor alpha(TNF-α, 50 ng·mL~(-1)). The proliferation and the levels of inflammatory cytokines such as prostaglandin E2(PGE2), interleukin-1β(IL-1β), and interleukin-6(IL-6) of the MH7A cells exposed to the serum containing L. bulbifera were determined to evaluate the anti-rheumatoid arthritis effects of the serum. Furthermore, the ultra-performance liquid chromatography tandem mass spectrometry fingerprints of the L. bulbifera crude extract, the drug-containing serum, and the drug-free serum were compared to identify the compounds newly generated in the serum after oral administration of the extract. According to the peak areas of common peaks and the results of anti-rheumatoid arthritis effect test, the active components were identified. The serum containing L. bulbifera significantly inhibited the proliferation of the MH7A cells activated by TNF-α and the expression of PGE2, IL-6, and IL-1β. Thirty newly generated compounds were detected in the drug-containing serum. Among them, neochlorogenic acid, cryptochlorogenic acid, chlorogenic acid, rutin, isoquercitrin, luteoloside, kaempferol-3-O-rutinoside, and quercitrin were also present in the crude extract. Twelve characteristic peaks(3, 7, 8, 14, 18, 19, 21, 23, 24, m6, m7, and m15) were significantly correlated with the pharmaceutical effect. According to the correlations, neochlorogenic acid, cryptochlorogenic acid, and chlorogenic acid had great contributions to the anti-rheumatoid arthritis activity. This study preliminarily clarified the potential pharmacodynamic substances of L. bulbifera in the treatment of rheumatoid arthritis, which laid a theoretical and experimental foundation for further development and application of the medicinal plant.
Animals ; Arthritis, Experimental/drug therapy* ; Arthritis, Rheumatoid/drug therapy* ; Chlorogenic Acid/analogs & derivatives* ; Cytokines/metabolism* ; Dinoprostone ; Humans ; Interleukin-1beta/genetics* ; Interleukin-6 ; Plant Extracts/therapeutic use* ; Quinic Acid/analogs & derivatives* ; Rutin ; Tumor Necrosis Factor-alpha/metabolism* ; Urticaceae/chemistry*

This paper aims to explore the activity of Codonopsis canescens extract against rheumatoid arthritis(RA) based on the Toll-like receptors(TLRs)/mitogen-activated protein kinases(MAPKs)/nuclear factor kappa B(NF-κB) signaling pathways and its mechanism. The ultra-performance liquid chromatography-quadrupole time-of-flight/mass spectrometry(UPLC-Q-TOF-MS) was used to identify the components of C. canescens extract. Forty-eight male SD rats were randomly divided into six groups, namely the normal group, the model group, the methotrexate(MTX) tablet group, and the low, medium, and high-dose C. canescens extract(ZDS-L, ZDS-M, and ZDS-H) groups, with 8 rats in each group. The model of collagen-induced arthritis in rats was induced by injection of bovine type Ⅱ collagen emulsion. MTX(2.5 mg·kg~(-1)), ZDS-L, ZDS-M, and ZDS-H(0.3 g·kg~(-1), 0.6 g·kg~(-1), and 1.2 g·kg~(-1)) were administrated by gavage. Rats in the normal group and the model group received distilled water. MTX was given once every three days for 28 days, and the rest medicines were given once daily for 28 days. Body weight, degree of foot swelling, arthritis index, immune organ index, synovial histopathological changes, and serum levels of tumor necrosis factor-α(TNF-α), interleukin-1β(IL-1β), and interleukin-6(IL-6) were observed. Protein expressions of TLR2, TLR4, NF-κB p65, p38 MAPK, and p-p38 MAPK in rats were determined by Western blot. Thirty-four main components were identified by UPLC-Q-TOF-MS, including 15 flavonoids, 7 phenylpropanoids, 4 terpenoids, 4 organic acids, 2 esters, and 2 polyalkynes. As compared with the normal group, the body weight of the model group was significantly decreased(P<0.01), and foot swelling(P<0.05, P<0.01), arthritis index(P<0.01), and the immune organ index(P<0.01) were significantly increased. The synovial histopathological injury was obviously observed in the model group. The serum levels of inflammatory factors TNF-α, IL-1β, and IL-6 were significantly increased(P<0.01), and the protein expression levels of TLR2, TLR4, NF-κB p65, p-p38 MAPK/p38 MAPK in the synovial tissue were significantly increased(P<0.01) in the model group. As compared with the model group, the body weights of the ZDS dose groups were increased(P<0.01), and the degree of foot swelling(P<0.01) and the arthritis index were decreased(P<0.05, P<0.01). The immune organ index was decreased(P<0.01) in the ZDS dose groups, and the synovial tissue hyperplasia and inflammatory cell infiltration were alleviated. The serum levels of TNF-α, IL-1β, and IL-6 were significantly decreased(P<0.05, P<0.01), and the protein expression levels of TLR2, TLR4, NF-κB p65, p-p38 MAPK/p38 MAPK were decreased(P<0.05, P<0.01) in the ZDS dose groups. C. canescens extract containing apigenin, tricin, chlorogenic acid, aesculin, ferulic acid, caffeic acid, and oleanolic acid has a good anti-RA effect, and the mechanism may be related to the inhibition of TLRs/MAPKs/NF-κB signaling pathways.
Animals ; Cattle ; Male ; Rats ; Arthritis, Experimental/drug therapy* ; Arthritis, Rheumatoid/drug therapy* ; Body Weight ; Codonopsis/chemistry* ; Interleukin-6/blood* ; NF-kappa B/genetics* ; p38 Mitogen-Activated Protein Kinases/metabolism* ; Plant Extracts/therapeutic use* ; Rats, Sprague-Dawley ; Signal Transduction ; Toll-Like Receptor 2/metabolism* ; Toll-Like Receptor 4/metabolism* ; Tumor Necrosis Factor-alpha/pharmacology*

The ethyl acetate fraction of ethanol extract of Eucommiae Cortex can effectively inhibit joint inflammation and bone destruction in rats with collagen-induced arthritis(CIA) and has a potential therapeutic effect on rheumatoid arthritis. The triterpenoid(EU-Tid) and iridoid(EU-Idd) of Eucommiae Cortex are derivatives isolated from the ethyl acetate fraction of the ethanol extract of Eucommiae Cortex, and it is not clear whether they have inhibitory effects on joint inflammation and bone erosion in CIA rats. Therefore, based on the CIA model, the effects of EU-Tid, EU-Idd, and their combination(EU-TP) on arthritis in rats were observed, and the material basis of Eucommiae Cortex against arthritis was further clarified. The samples were collected two and four weeks after administration to observe the pathological changes in different stages of arthritis in CIA rats. For the rats in the model control group, with the prolongation of the disease course, the paw volume and arthritis score increased and histopathological lesions aggravated. Compared with the model control group, the drug administration groups showed reduced paw volumes and arthritis scores, and improved joint lesions and cartilage destruction. Additionally, the mRNA expression levels of tumor necrosis factor-α(TNF-α), interleukin-17(IL-17), and interleukin-23(IL-23) in the spleen were down-regulated in the drug administration groups. EU-TP and EU-Tid at concentrations of 160 and 320 μg·mL~(-1) could significantly inhibit the proliferation of human fibroblast-like synoviocytes-RA(HFLS-RA) and nitric oxide(NO) release in the supernatant of RAW264.7 cells induced by lipopolysaccharide(LPS) at the concentration range of 10-80 μg·mL~(-1) in vitro. EU-Idd had no effect on the proliferation of HFLS-RA but could reduce the NO release at concentrations of 40 and 80 μg·mL~(-1). The results indicated that the terpenoids of Eucommiae Cortex had great potential in the treatment of rheumatoid arthritis.
Rats ; Humans ; Animals ; Arthritis, Experimental/drug therapy* ; Iridoids/pharmacology* ; Triterpenes/therapeutic use* ; Arthritis, Rheumatoid/drug therapy* ; Tumor Necrosis Factor-alpha ; Plant Extracts/pharmacology* ; Inflammation/drug therapy* ; Ethanol ; Cytokines