Mechanism of artesunate on bone destruction in experimental rheumatoid arthritis based on transcriptomics and network pharmacology / 中国中药杂志

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.

Effects of moxibustion on the ultrastructure of synovial cells of knee joint and serum cytokines in adjuvant arthritis rats / 中国针灸

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.

Treatment of rheumatoid arthritis by injection of sinomenine solid lipid nanoparticles under a fluorescence endoscopic laser confocal microscope / 中国中药杂志

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.

Alcohol extract of root and root bark of Toddalia asiatica alleviates CIA in rats through anti-inflammatory and proapoptotic effects / 中国中药杂志

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.

Effect of miR-124a on collagen-induced arthritis in mice and the underlying mechanisms / 中南大学学报(医学版)

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.

Integrating 16S sequencing and metabolomics study on anti-rheumatic mechanisms against collagen-induced arthritis of Wantong Jingu Tablet / 中国天然药物

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.

Anti-inflammatory effects of aucubin in cellular and animal models of rheumatoid arthritis / 中国天然药物

Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease. It is known that aucubin (AU) exerts anti-inflammatory activity, but its effects and mechanisms in RA are unclear. This study investigated the anti-inflammatory effects and mechanisms of AU in vivo and in vitro. Human fibroblast-like synoviocyte cells from patients with RA (HFLS-RA), RAW264.7 cells, and MC3T3-E1 cells were used to evaluate the effects of AU on migration, invasion, apoptosis, osteoclast differentiation and production. Immunofluorescence was used to observe nuclear translocation of nuclear factor (NF)-κB, the double luciferase reporter gene method was used to observe NF-κB-p65 activity in AU-treated MC3T3-E1 cells. RT-qPCR was used to measure expression of bone metabolism and inflammation-related genes, and western blot was used to measure bone metabolism and NF-κB protein expression levels. Collagen-induced arthritis (CIA) rat model was used for pharmacodynamics study. Arthritis indexes were measured in the ankle and knee, histological staining and Micro-computed tomography were performed on the ankle joints. Also, inflammatory factor gene expression and the levels of NF-κB-related proteins were detected as in vitro. AU effectively inhibited HFLS-RA cell migration and invasion, promoted apoptosis, and inhibited RAW264.7 cell differentiation into osteoclasts, as well as inhibited NF-κB-p65 activity in MC3T3-E1 cells. Notably, AU significantly reduced the gene expression levels of three cell-related inflammatory factors and bone metabolism factors, effectively inhibited the expression of p-Iκκα β, p-IκBα, and p-p65 proteins. In vivo, AU relieved joint inflammation, reduced related inflammatory factors, and inhibited NF-κB signaling. It could be used to treat RA-related synovial inflammation and bone destruction through the NF-κB pathway.

Moxibustion regulates T-regulatory/T-helper 17 cell balance by modulating the microRNA-221/suppressor of cytokine signaling 3 axis in a mouse model of rheumatoid arthritis / 中西医结合学报

OBJECTIVE@#Rheumatoid arthritis (RA) progression is associated with the balance of T-regulatory (Treg) and T-helper 17 (Th17) cells, while the role of microRNAs (miRs) in regulating Treg/Th17 cell balance has not been clarified. This study aimed to assess whether moxibustion could regulate Treg/Th17 cell balance by modulating the miR-221/suppressor of cytokine signaling 3 (SOCS3) axis in the RA mouse model.@*METHODS@#A mouse model of collagen-induced arthritis (CIA) was established in male DBA/1J mice. Twenty-two days after CIA induction, the mice received daily treatment with moxibustion for 12 times. Pathological scores were assessed according to the levels of synovial hyperplasia. The expression levels of cytokines interleukin (IL)-1β, IL-6, tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), IL-17 and IL-10 were analyzed in serum by enzyme-linked immunosorbent assay. The cluster of differentiation 4 (CD4+) splenocytes was analyzed by fluorescence-activated cell sorting. The expression levels of RA-related miRs and target genes were subsequently detected, and the target of miR-221 was confirmed by the dual-luciferase reporter assay.@*RESULTS@#It was revealed that moxibustion treatment decreased the pathological scores and downregulated the expression levels of IL-1β, IL-6, TNF-α, IFN-γ and IL-17, while upregulated the expression level of IL-10. The Treg/Th17 cell balance was regulated by moxibustion treatment. The expression level of miR-221 was suppressed by moxibustion treatment. Furthermore, SOCS3 was found as the direct target of miR-221, which mediated the function of moxibustion by regulating the Treg/Th17 cell balance.@*CONCLUSION@#Moxibustion therapy regulated the Treg/Th17 cell balance by modulating the miR-221/SOCS3 axis in the RA mouse model.

Effect of Buyi Pishen acupuncture on inflammatory factor and cartilage matrix in adjuvant arthritis rats / 中国针灸

OBJECTIVE@#To observe the effect of Buyi Pishen acupuncture (acupuncture for invigorating spleen and kidney) on inflammatory factor and synovial cartilage matrix in adjuvant arthritis (AA) rats, and to explore the mechanism of acupuncture for rheumatoid arthritis (RA).@*METHODS@#A total of 60 clean male Wistar rats were randomized into a normal group, a model group, a tripterygium wilfordii polyglycoside tablet (TWP) group and an acupuncture group, 15 rats in each group. Rats in the model group, the TWP group and the acupuncture group received intradermal injection of Freund's complete adjuvant (FCA) at right hind foot pad to induce the AA model. TWP suspension of 8 mg/kg was given by gavage in the TWP group. Acupuncture was applied at "Shenshu" (BL 23), "Pishu" (BL 20) and right "Housanli" (ST 36), "Sanyinjiao" (SP 6), "Yanglingquan" (GB 34) in the acupuncture group, 15 min a time, once a day. The intervention was given 15 days in both TWP group and acupuncture group. The foot-pad swelling degree before modeling, before and after intervention and the arthritis index (AI) score before and after intervention were calculated; the serum levels of interleukin (IL)-1β, IL-4, IL-10 and tumor necrosis factor-α (TNF-α) were detected by ELISA method; the ultrastructure and histomorphological changes of synovium issue were observed by transmission electron microscope and HE staining; the positive expression of matrix metalloproteinase (MMP)-3 and MMP-9 in synovium issue was detected by immunohistochemistry method.@*RESULTS@#Before intervention, foot-pad swelling degree of the model group, the TWP group and the acupuncture group was increased compared with the normal group (P<0.01). After intervention, foot-pad swelling degree and AI score were increased compared with the normal group (P<0.01), foot-pad swelling degree and AI scores in the TWP group and the acupuncture group were lower than the model group (P<0.05), and those in the acupuncture group were decreased compared with the TWP group (P<0.05). The model group exhibited unclear nuclear membrane of synovial cells, chromatin pyknosis, massive inflammatory cell infiltration and hyperplasia in synovial tissue; the TWP group and the acupuncture group exhibited clear and smooth nuclear membrane of synovial cells, inapparent chromatin pyknosis, less inflammatory cell infiltration and hyperplasia in synovial tissue, the acupuncture group exhibited less matrix destruction as well. Compared with the normal group, serum levels of IL-1β and TNF-α and positive expression of MMP-3 and MMP-9 in synovium issue were increased (P<0.01), while serum levels of IL-4 and IL-10 were decreased (P<0.01) in the model group. Compared with the model group, serum levels of IL-1β and TNF-α and positive expression of MMP-3 and MMP-9 in synovium issue were decreased (P<0.05, P<0.01), while serum levels of IL-4 and IL-10 were increased (P<0.05) in the TWP group and the acupuncture group; compared with the TWP group, serum level of TNF-α and positive expression of MMP-3 and MMP-9 in synovium issue were decreased (P<0.05), while serum levels of IL-4 and IL-10 were increased (P<0.05) in the acupuncture group.@*CONCLUSION@#Buyi Pishen acupuncture can effectively improve the injury of articular cartilage in AA rats, its mechanism maybe related to reducing the inflammatory reaction in synovium and inhibiting the degradation of articular cartilage matrix.

Therapeutic effects of benzoylaconitine and paeoniflorin in rats with collagen-induced arthritis

Abstract To explore the effects and mechanisms of benzoylaconitine and paeoniflorin on collagen-induced arthritis (CIA) rats. Weight, paw swelling, arthritis index and joint pathologic changes were examined in each group after CIA induction. PGE2, IL-1ß, IL-6, IL-10, TNF-α, VEGF, MMP-3, IgG and anti-CII Ab were assessed by ELISA; STAT1 and STAT3 expressions were analyzed immunohistochemically, and the ultrastructure of synovial cells was observed by transmission electron microscopy. Therapeutic effects were determined in CIA rats via injecting benzoylaconitine and paeoniflorin, which could alleviate the degree of swelling and arthritis index (AI) and pathological lesions of the sacroiliac gland; decrease the levels of PGE2, IL-1ß, TNF-α, VEGF and IgG in serum; reduce STAT1 and STAT3 expression in the membrane tissue; and inhibit the secretion and proliferation of synovial cells. These results showed that benzoylaconitine and paeoniflorin could significantly palliate the arthritic symptoms of CIA rats, and better therapeutic effects could be achieved if the two components were used in combination

TDZD-8 suppresses rheumatoid arthritis induced by collagen type II in rats: potential role of apoptosis and inflammation / TDZD-8 suprime la artritis reumatoide inducida por colágeno tipo II en ratas: papel potencial de la apoptosis y la inflamación

SUMMARY: Rheumatoid arthritis (RA) is considered an autoimmune disease distinguished by chronic synovial membrane inflammation, degraded cartilage, as well as bone destruction, which lead to joints pain and stiffness. The pathogenesis of RA involved at least two mechanisms: Cellular proliferation and activation of glycogen synthase kinase-3β (GSK3β) enzyme. Thus, we tested the hypothesis that the GSK3binhibitor, TDZD-8, can treat the synovial tissue toward collagen type II (COII)-mediated RA linked to apoptosis induction and biomarker suppression of inflammation. Wistar rats were immunized with COII (the model group) for 21 days. Matched immunized rats were daily injected with TDZD-8 (1 mg/kg; i.p) for three additional weeks (COII+TDZD- 8).After 42 days of post-immunization, blood and tissues were collected. Histology (H&E) and immunohistochemistry (CD45; leukocyte common antigen) images showed that COII induced RA was demonstrated by profound damage to the synovial tissue and infiltration of the inflammatory cells, which were substantially ameliorated with TDZD-8. In addition, COII immunization caused the induction of rheumatoid factor (RF), tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and interleukin 1 beta (IL-1β) that were substantially (p<0.05) suppressed by TDZD-8. Whereas, TDZD-8 augmented the apoptotic biomarker, Bcl-2-associated X protein (Bax), which was significantly (p<0.05) ameliorated by RA. We also showed a substantial relationship (p<0.001) between the blood levels of RF and the synovial tissue levels of TNF-α (r = 0.759), IL-1b (r = 0.969), IL-6 (r = 0.749), and Bax (r = - 0.914). These results indicate effective treatment of the injured synovial tissue by TDZD-8 against COII-induced RA in rats, which also decreases inflammatory biomarkers and augmentation of apoptosis.

RESUMEN: La artritis reumatoide (AR) es una enfermedad autoinmune que se distingue por la inflamación crónica de la membrana sinovial, el cartílago degradado y la destrucción de los huesos, lo que provoca dolor y rigidez en las articulaciones. La patogenia de la AR involucra al menos dos mecanismos: la proliferación celular y la activación de la enzima glucógeno sintasa quinasa-3b (GSK3β) Por lo tanto, probamos la hipótesis de que el inhibidor de GSK3β, TDZD-8, puede tratar el tejido sinovial hacia el colágeno tipo II (COII) - AR mediada por inducción de apoptosis y supresión de biomarcadores de inflamación. Se inmunizaron ratas Wistar con COII (el grupo modelo) durante 21 días. Se inyectaron diariamente ratas emparejadas inmunizadas con TDZD-8 (1 mg / kg; i.p) durante tres semanas adicionales (COII + TDZD-8). Después de 42 días de post-inmunización, se recolectó sangre y tejidos. Las imágenes de histología (H&E) e inmunohistoquímica (CD45; antígeno común de leucocitos) mostraron que la AR inducida por COII presentaba un daño profundo en el tejido sinovial e infiltración de las células inflamatorias, las que mejoraron con TDZD-8. Además, la inmunización con COII provocó la inducción de factor reumatoide (FR), factor de necrosis tumoral alfa (TNF-α), interleucina-6 (IL-6) e interleucina 1 beta (IL-1β) que fueron suprimidos por TDZD-8 de manera significativa (p < 0.05). Considerando que TDZD-8 aumentó el biomarcador apoptótico, la proteína X asociada a Bcl-2 (Bax), que fue mejorado (p <0,05) por RA. También se observó una relación sustancial (p <0,001) entre los niveles sanguíneos de RF y los niveles de tejido sinovial de TNF-α (r = 0,759), IL-1β (r = 0,969), IL-6 (r = 0,749), y Bax (r = -0,914). Estos resultados indicaron un tratamiento eficaz del tejido sinovial lesionado por TDZD-8 contra la AR inducida por COII en ratas, que también disminuye los biomarcadores inflamatorios y el aumento de la apoptosis.

Therapeutic mechanism of Psammosilene tunicoides extract on rheumatoid arthritis based on NLRP3 inflammasome / 中国中药杂志

In this study, we investigated the mechanism of crude extract of Psammosilene tunicoides(CEPT) in the treatment of rheumatoid arthritis(RA) based on the Nod-like receptor protein 3(NLRP3) inflammasome. The collagen-induced arthritis(CIA) mouse model was established. On day 32 after the primary immunization, according to the arthritis score, the mice were randomly divided into model group, positive control(methotrexate) group, low-and high-dose CEPT groups, and normal group, with 10 mice in each group. According to the administration dose of each group, the mice were continuously administered for 21 days. Every four days during the administration, the paw edema degree, arthritis score, and spleen index of the mice were measured; histopathological examination was performed for the ankles of the mice; the contents of IL-1β and IL-18 in the serum were determined; the protein expression levels of NLRP3, caspase-1, and apoptosis-associated speck-like protein containing a CARD(ASC), as well as the mRNA expression levels of NLRP3 and caspase-1 in the ankle joints of the mice were detected. The results showed that compared with those in the model group, the mice in the positive control group and CEPT groups had significantly decreased the contents of IL-1β and IL-18 in the serum and spleen index(P<0.01), significantly lowered arthritis score and degree of paw edema(P<0.01), alleviated arthritic infiltration of the knee, and down-regulated protein and mRNA levels of NLRP3, ASC, and caspase-1 in the ankle joint(P<0.01). These results suggest that P. tunicoides may reduce the paw edema and arthritis score and alleviate the inflammatory response in CIA mice by inhibiting the expression of NLRP3. This study provides a basis for the study of immune regulation of P. tunicoides in RA.

Mechanism of oxymatrine in treatment of collagen-induced arthritis in mice / 中国中药杂志

Sophorae Flavescentis Radix, the root of Sophora flavescens Ait., has been widely applied in the medical field due to its anti-inflammatory, analgesic, bacteriostatic, antiviral, antitumor, and other pharmacological effects. The present study investigated the anti-rheumatoid arthritis effect of oxymatrine(OMT), the active component of Sophorae Flavescentis Radix by observing its effect on the function of B lymphocytes in collagen-induced arthritis(CIA) mice through the Toll-like receptor 9(TLR9)/myeloid differentiation factor 88(MyD88)/signal transducer and activator of transcription 3(STAT3) pathway. The CIA model in DBA/1 J mice was induced by bovine type Ⅱ collagen and complete Freund's adjuvant(CFA). Fifteen days after the primary immunization, mice were treated with OMT for 30 days by intraperitoneal injection. Paw swelling and arthritis index(AI) score were evaluated every 3 days. Joint histopathologic changes were observed by HE staining. Magnetic-activated cell sorting(MACS) was used to isolate B lymphocytes from the spleen of CIA mice spleen. The serum expression level of interleukin(IL)-21 was examined by the enzyme-linked immunosorbent assay(ELISA). The expression of TLR9, STAT3, p-STAT3, and IL-21 in B lymphocytes was detected by Western blot. The mRNA expression of TLR9, STAT3, and IL-21 in B lymphocytes was detected by real-time fluorescence-based quantitative PCR(qRT-PCR). The results showed that OMT could significantly alleviate the paw swelling, decrease the AI score, relieve synovial inflammatory cell infiltration and hyperplasia, reduce the level of inflammatory cytokines, and inhibit the expression of TLR9, STAT3, p-STAT3, and IL-21 of B lymphocytes in CIA mice. Therefore, OMT may alleviate rheumatoid arthritis by regulating TLR9/MyD88/STAT3 pathway in B lymphocytes, providing a valuable reference for the application of OMT in the clinical treatment of rheumatoid arthritis.

Effect of moxibustion at "Zusanli" (ST 36) and "Shenshu" (BL 23) on intestinal flora in adjuvant arthritis rats / 中国针灸

OBJECTIVE@#To observe the effect of moxibustion at "Zusanli" (ST 36) and "Shenshu" (BL 23) on inflammatory factors and intestinal flora in the rats with adjuvant arthritis.@*METHODS@#A total of 36 Wistar rats were randomized into a normal group, a model group and a moxibustion group, 12 rats in each one. In the model group and the moxibustion group, the adjuvant arthritis model was established by a compound method, including the environmental factors, i.e. wind, cold and damp, and Freund's complete adjuvant. In the moxibustion group, moxibustion intervention was exerted at "Zusanli" (ST 36) and "Shenshu" (BL 23), for 20 min at each acupoint, once daily, consecutively for 21 days. The paw swelling degree and arthritis index (AI) score were observed before and after intervention in the rats of each group. Using real-time fluorescence quantitative PCR method (real-time PCR) and Western blot method, the mRNA and protein expressions of inflammatory factors of colon tissue, i.e. interleukin (IL) 1β, tumor necrosis factor-α (TNF-α), IL-6, were detected after intervention in the rats of each group. The intestinal flora was detected with 16SrRNA sequencing technology after intervention in the rats of each group.@*RESULTS@#Compared with the normal group, the paw swelling degree and AI score were increased in the rats of the model group (@*CONCLUSION@#Moxibustion at "Zusanli" (ST 36) and "Shenshu" (BL 23) relieves the joint symptoms of adjuvant arthritis rats and inhibits the expressions of inflammatory factors, which is probably related to the regulation of the structure of intestinal flora.

Therapeutic effect of gene silencing peptidyl arginine deaminase 4 on pulmonary interstitial lesions induced by collagen-induced arthritis mice / 北京大学学报(医学版)

OBJECTIVE@#To investigate the therapeutic effect of gene silencing peptidyl arginine deaminase 4 (PAD4) on pulmonary interstitial lesions induced by collagen-induced arthritis (CIA) mice, and possible mechanisms.@*METHODS@#A CIA mouse model was established in DBA/1 mice, followed by a tail vein injection of the virus solution prepared by the PAD4-siRNA expression vector once a week for 8 times. The mice were sacrificed at the end of the experiment. The expression of PAD4 mRNA in lungs was detected by real-time quantitative PCR (qRT-PCR). The expression of PAD4 protein was detected by tissue immunohistochemistry. Cell culture was performed by spleen tissue. Flow cytometry changes in the ratio of Tfh cells to Tfr cells were examined; lung staining was performed in the lungs to observe changes in lung pathology.@*RESULTS@#(1) Compared with the blank group, the expression of PAD4 mRNA in the lung tissue of the model group increased, the difference was statistically significant (P < 0.05). PAD4 mRNA in the lung tissue of the CIA mice after PAD4-siRNA treatment. The expression level was significantly lower than that of the model group and the negative control group, and the difference was statistically significant (P < 0.05). (2) Red fluorescence was less in the lung tissue of the blank group, while more red fluorescence was observed in the inflammatory cell infiltration area and trachea around the lung tissue of the model group and the negative control group, and the red fluorescence of the three groups after PAD4-siRNA treatment was significantly reduced; (3) Compared with the blank group, the proportion of Tfh cells in the model group increased, the difference was statistically significant (P < 0.05), the proportion of Tfh cells in spleen cells of the CIA mice after PAD4-siRNA treatment was significantly lower than that of the model group and the negative control group, the difference was statistically significant (P < 0.05); compared with the blank group, in the mouse spleen cells in the model group the proportion of Tfr cells was slightly decreased, but the difference was not statistically signifi-cant. The proportion of Tfr cells in the spleen cells of the mice increased after PAD4-siRNA treatment, but the difference was statistically significant only in the PAD4-siRNA2 group compared with the model group and the negative control group (P < 0.05); (4) The proportion of Tfh/Tfr in the spleen cells of the model group was increased, compared with the blank group, the difference was statistically significant (P < 0.05); the ratio of Tfh/Tfr in the three groups after PAD4-siRNA treatment all decreased, the difference was statistically significant (P < 0.05); (5) Compared with the blank group, the alveolar wall of the lung tissue of the model group was thickened, the inflammatory cell infiltration was increased, and the lung tissue destruction and inflammatory infiltration of the CIA mice were decreased after PAD4-siRNA treatment. The degree of reduction was reduced.@*CONCLUSION@#Gene silencing of PAD4 can reduce the proportion of Tfh cells, increase the proportion of Tfr cells, reverse the proportion of Tfh/Tfr, and reduce the degree of interstitial lesions and inflammatory infiltration of lung tissue.

Evaluation of antiarthritic activity of ginkgolic acid against Freund's adjuvant induced arthritic rat model

This study aimed to analyze the antiarthritic activity of ginkgolic acid against the Complete Freund's Adjuvant (CFA)-induced arthritis in rats. Arthritis was induced through an intradermal injection of CFA (0.1 mL) at the right hind footpad of adult Wistar Albino rats. Ginkgolic acid was administered orally at doses of 25 mg/kg and 50 mg/kg, respectively, once daily via gavage for 25 days upon inducing arthritis. Indomethacin was administered orally at a dose of 3 mg/kg twice in a week which served as positive control group. The animals were sacrificed and subjected to biochemical and histopathological analysis upon completion of treatment. Ginkgolic acid was able to reverse the arthritic effect (p < 0.01) induced by CFA in a dose dependent manner. Swelling of paw, thymus and spleen index, serum biomarker levels, and pro-inflammatory cytokines were significantly reduced (p < 0.01) by the acid whereas the antioxidant enzyme activities were remarkably restored. The histopathological findings were in agreement with the biochemical results. The results indicate that the antioxidant and anti-inflammatory properties of ginkgolic acid can be credited to the antiarthritic effects, and it can be promoted as a potential agent for therapeutic use against osteoarthritis

Comparison of different techniques and stains for direct diagnosis of Trichinella spiralis

Background: The aim of Trichinella inspection is to reliably detect larvae in meat at levels which are capable of causing human trichinellosis. Trichinoscopy is a rapid, cost effective but is less sensitive in detection of low muscle larvae (ML)numbers. Artificial digestion is more convenient, and flexible, but may destroy immature larvae. Baermann technique may enhance the efficiency of diagnosis. Giemsa and Leishman stains provide identical contrasting coloration as Hematoxylin and Eosin (H-E) with the both advantages of that they can be used for staining non-histological sectionsand rapid preparation.The primary aim of the study was to compare the efficiency of digestion and Baermann techniques for detection of ML in muscles. Secondary aim was detection of ML by Geimsa and Leishman stain and their comparison with routinely used stain H-E. Methods: Muscles from 36 mice orally infected with Trichinella spiralis encysted larvae were examined. Larvae from 1 g samples of different muscles were recovered by digestion and Baermann techniques after different dose of infection. Muscle samples were prepared for staining with Giemsa, Leishman and H-E. Results: Number of larvae recovered by Baermann was higher recovered by digestion method (P ˃0.5). Leishman stain was the best stain for rapid detection of ML after 30 min followed in validity by Geimsa then H-E. Conclusion: Baermann concentration technique showed higher sensitivity than digestion method. Leishman stain is superior to Giemsa stain as it takes less time. Both stains are considered good alternative to H-E for rapid and easy diagnosis of trichinellosis in post-mortem animals

Rhoifolin regulates oxidative stress and proinflammatory cytokine levels in Freund's adjuvant-induced rheumatoid arthritis via inhibition of NF-κB

Rheumatoid arthritis (RA) is an autoimmune disease of knee joints involving pain and inflammation. Rhoifolin is a plant flavonoid known to have antioxidant and anti-inflammatory properties. This study was taken to identify the effect of rhoifolin on complete Freund's adjuvant (CFA)-induced arthritis in the rat model. Treatment with rhoifolin (10 and 20 mg/kg) showed a significant improvement in the overall health parameters such as paw edema and weight loss. This improvement in morphological parameters corroborated the findings with gross morphological changes observed in the histopathological analysis. Rhoifolin treatment also caused a significant decrease in oxidative stress, evident from changes in intracellular levels of glutathione, glutathione peroxidase, malondialdehyde, and superoxide dismutase in the articular cartilage tissue. Moreover, proinflammatory cytokines, tumor necrosis factor (TNF)-α, interleukin(IL)-1β, and IL-6 showed a significant downregulation of gene expression and intracellular protein concentration levels. The NF-κB pathway showed a significant attenuation as evident in the significant reduction in the levels of NF-κB p65 and p-IκB-α. These results indicated that rhoifolin can be a natural therapeutic alternative to the extant regimens, which include non-steroidal anti-inflammatory drugs and immunosuppressants. Additionally, the antioxidant and anti-inflammatory action of rhoifolin was probably mediated by the NF-κB pathway. However, the exact target molecules of this pathway need to be determined in further studies.

Mechanism of / 南方医科大学学报

OBJECTIVE@#To explore the mechanism of @*METHODS@#Healthy male DBA/1 mice were used for CIA modeling. Twenty-five CIA mice with successful modeling and similar arthritis index (AI) scores were randomized equally into model group (CIA), methotrexate (MTX) group, and low-, medium-, and high-dose XWGD groups (0.975, 1.95, and 3.9 g/mL, respectively), with another 5 normal mice as the normal control group. The mice in normal control and CIA groups were given saline once a day, those in MTX group were given 0.1 mg/mL MTX once a week, and those in XWGD groups were treated daily via garage of XWGD containing crude drugs of different doses for 28 consecutive days. The AI score and HE staining were used to evaluate the changes in the joints of the CIA mice. The effect of XWGD on Th1, Th17, MDSC, G-MDSC and M-MDSC cells were evaluated with flow cytometry.@*RESULTS@#Treatment with MTX and different doses of XWGD significantly decreased the AI score of the mice and relieved joint inflammation as compared with the model group (@*CONCLUSIONS@#XWGD can improve joint inflammation in CIA mice by increasing the percentages of G-MDSC cells and decreasing the percentages of M-MDSC, Th1 and Th17 cells, and a high dose of XWGD can produce an equivalent therapeutic effect to methotrexate but with better safety.

Strong inflammation is essential for expression of articular cartilage-specific citrullinated antigens / 南方医科大学学报

OBJECTIVE@#To investigate the expression of citrullinated epitopes in articular cartilage protein and whether its expression is sufficient to induce anti-citrullinated protein antibody (ACPA) response in mice.@*METHODS@#The experimental group was treated with different concentrations of lipopolysaccharide (LPS), heat-inactivated bacteria ( and ) or specific monoclonal antibody against type Ⅱ collagen to induce citrullination of articular cartilage protein, with PBS as the control. Immunohistochemistry with the monoclonal antibody ACC4 (IgG1) that specifically binds to the citrullinated epitope of cartilage protein was performed for detecting the expression of citrullinated protein, with ACC1 (IgG2a) as a positive control antibody and L243 (IgG2a) and Hy2.15 (IgG1) as the negative isotype control. In the in vivo experiment, SD rats were subjected to injection of different doses of LPS in the right knee (with PBS as the controls in the left knee), and 3 days later frozen sections were prepared for immunohistochemical detection of the expression of citrullinated protein. Models of collagen-induced arthritis (CIA) established in different mouse strains were observed for incidence and severity of CIA. Serum samples collected from these models and the sera from rheumatoid arthritis patients were examined for anti-citrullinated protein antibody, and immunohistochemistry was performed to detect the expression of citrullinated protein in the cartilage of the mouse.@*RESULTS@#The citrullinated CII epitope-specific antibody ACC4 did not bind to articular cartilage tissues with different treatments as compared with the positive control antibody ACC1. The ACC4 antibody and the antibodies from patients with rheumatoid arthritis with high titers of anti-citrullinated protein antibody were capable of binding to the synovial tissue around the articular cartilage of the CIA. Luminex analysis showed that the anti-citrullinated protein antibody was lowly expressed in mouse serum, but the anti-type Ⅱ collagen triple helix structure peptide antibody exhibited strong reactivity.@*CONCLUSIONS@#Mild acute inflammatory response is not enough to cause citrullination of articular cartilage protein, and the expression of specific epitope requires a high-intensity inflammatory response. Inflammatory articular cartilage protein can express citrullinated epitopes in type Ⅱ collagen-induced arthritis in mice, but the expression of citrullinated epitopes is not sufficient to induce an immune response to anti-citrullinated antibodies. Stronger stimulation signals are required to induce an immune response for producing anti-citrullinated protein antibodies.