Andrographolide sulfonate (AS) is a sulfonated derivative of andrographolide extracted from Andrographis paniculata (Burm.f.) Nees, and has been approved for several decades in China. The present study aimed to investigate the novel therapeutic application and possible mechanisms of AS in the treatment of rheumatoid arthritis. Results indicated that administration of AS by injection or gavage significantly reduced the paw swelling, improved body weights, and attenuated pathological changes in joints of rats with adjuvant-induced arthritis. Additionally, the levels of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and IL-1β in the serum and ankle joints were reduced. Bioinformatics analysis, along with the spleen index and measurements of IL-17 and IL-10 levels, suggested a potential relationship between AS and Th17 cells under arthritic conditions. In vitro, AS was shown to block Th17 cell differentiation, as evidenced by the reduced percentages of CD4⁺ IL-17A⁺ T cells and decreased expression levels of RORγt, IL-17A, IL-17F, IL-21, and IL-22, without affecting the cell viability and apoptosis. This effect was attributed to the limited glycolysis, as indicated by metabolomics analysis, reduced glucose uptake, and pH measurements. Further investigation revealed that AS might bind to hexokinase2 (HK2) to down-regulate the protein levels of HK2 but not glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or pyruvate kinase M2 (PKM2), and overexpression of HK2 reversed the inhibition of AS on Th17 cell differentiation. Furthermore, AS impaired the activation of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signals in vivo and in vitro, which was abolished by the addition of lactate. In conclusion, AS significantly improved adjuvant-induced arthritis (AIA) in rats by inhibiting glycolysis-mediated activation of PI3K/AKT to restrain Th17 cell differentiation.
Animals ; Th17 Cells/immunology* ; Diterpenes/pharmacology* ; Arthritis, Rheumatoid/metabolism* ; Proto-Oncogene Proteins c-akt/immunology* ; Glycolysis/drug effects* ; Cell Differentiation/drug effects* ; Phosphatidylinositol 3-Kinases/genetics* ; Rats ; Male ; Rats, Sprague-Dawley ; Humans ; Andrographis paniculata/chemistry* ; Arthritis, Experimental/drug therapy* ; Interleukin-17/immunology* ; Signal Transduction/drug effects*

Animals ; Arthritis, Experimental ; Collagen Type II ; Male ; Mice ; Mice, Inbred DBA ; Receptors, Adrenergic, alpha-1 ; Signal Transduction ; T-Lymphocytes, Regulatory/immunology*

Notopterygium incisum (QH) has been used for the treatment of rheumatoid arthritis (RA), and volatile oils may be its mainly bioactive constituents. The present study was designed to analyze the volatile compounds in QH and to determine the anti-arthritic capacity of Notopterygium volatile oils and the potential mechanism of action. The volatile compounds analysis was conducted by GC-MS. The anti-arthritic capacity test of the volatile oils was conducted on adjuvant-induced arthritis (AIA) rats. The anti-inflammatory property was tested in NO release model in RAW 264.7 cells. Endothelial cells were used to evaluate the anti-proliferative and anti-tube formative effects. 70 compounds were analyzed by GC-MS in the volatile oils. Notopterygium volatile oils weakened the rat AIA in a dose-dependent manner (2, 4, and 8 g crude drug/kg). The NO production by RAW 264.7 was decreased by more than 50% in Notopterygium volatile oils (5, 15, and 45 μg·mL) pretreated groups. Notopterygium volatile oils also inhibited EAhy926 cell proliferation and further delayed EAhy926 cell capillary tube formation in a concentration-dependent manner. The anti-NO productive, anti-proliferative, and anti-tube formative effects of Notopterygium volatile oils strongly suggested that the therapeutic effect of QH in AIA might be related to the potent anti-inflammatory and anti-angiogenic capacities of the volatile oils.
Angiogenesis Inhibitors ; administration & dosage ; chemistry ; Animals ; Anti-Inflammatory Agents ; administration & dosage ; chemistry ; Apiaceae ; chemistry ; Arthritis, Experimental ; drug therapy ; immunology ; physiopathology ; Cell Proliferation ; drug effects ; Drugs, Chinese Herbal ; administration & dosage ; chemistry ; Gas Chromatography-Mass Spectrometry ; Male ; Mice ; Nitric Oxide ; immunology ; Oils, Volatile ; administration & dosage ; chemistry ; RAW 264.7 Cells ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo investigate the effects of prophylactic administration of all-trans retinoic acid (ATRA) in relieving inflammation in a rat model of collagen-induced arthritis (CIA).

METHODSFemale Wistar rats (6 to 8 weeks old) were randomly divided into normal control group, solvent control group, and prophylactic ATRA treatment (0.05, 0.5, and 5 mg/kg) groups. All the rats except for those in normal control group were subjected to subcutaneous injection of type II collagen and incomplete Freund adjuvant in the tails to induce CIA, followed by injection on the following day with saline, corn oil or different doses of ATRA 3 times a week. The arthritis index (AI) scores, histological scores, serum levels of TNF-α, IL-17A, and IL-10, and expressions of proteases related with cartilage damage were evaluated.

RESULTSOn the 15th day after the primary immunization, the AI scores increased significantly in all but the normal control groups; the scores increased progressively in all the 3 ATRA groups but remained lower than that in the solvent control group, which was stable over time. The rats in the 3 ATRA groups showed obvious pathologies in the knee and ankle joints, but the semi-quantitative scores of pathology damage showed no significance among them. Compared with those in solvent control group, the serum IL-17A and TNF-α levels decreased, serum IL-10 level increased, and the expressions of ADAMT-4 and MMP-3 proteins decreased significantly in the knees in the 3 ATRA groups.

CONCLUSIONATRA can reduce the production of TNF-α and IL-17A and increase the production of IL-10 to alleviate the inflammation in rats with CIA. ATRA may delay the progression of RA by correcting the imbalance of Th1/Th2 and Th17/Treg.

ADAMTS4 Protein ; metabolism ; Animals ; Arthritis, Experimental ; chemically induced ; drug therapy ; Collagen Type II ; Female ; Freund's Adjuvant ; Inflammation ; drug therapy ; Interleukin-10 ; blood ; Interleukin-17 ; blood ; Lipids ; Matrix Metalloproteinase 3 ; metabolism ; Rats ; Rats, Wistar ; T-Lymphocytes, Regulatory ; immunology ; Th17 Cells ; immunology ; Tretinoin ; pharmacology ; Tumor Necrosis Factor-alpha ; blood

OBJECTIVETo determine the effective dosage and formulation of agkistrodon in collagen-induced arthritis (CIA) rats.

METHODSCIA was induced by injection of collagen in complete/incomplete Freund's adjuvant. Agkistrodon decoction, agkistrodon powder, and agkistrodon wine were administered daily starting from the onset of arthritis. Paw swelling degree was measured by using a volume-measuring instrument every 7 days after primary immunization. Arthritis index was measured and calculated using the "five scoring method" every 7 days. The levels of serum interleukin-1ß (IL-1ß) and type II collagen IgG antibodies were detected by enzyme-linked immunosorbent assay. Finally, all ankles were removed, and X-ray radiography was performed with In-vivo Imaging System FX. Samples were counterstained with hematoxylin and eosin for analysis.

RESULTSAmong the various dosage formulations of agkistrodon, high-dose powder, which was equivalent to an amount of 6 g/day in adults, showed better effects on the inhibition of joint swelling and reduction of arthritis index score. The relatively low levels of serum IL-1 and anti-type II collagen IgG antibodies, as well as the X-ray radiography and pathology results, further proved the superiority of the high-dose powder over the other formulations. The effect of decoction on inhibiting joint swelling was inversely proportional to the dosage. Other effects, such as reduction of arthritis index score and the levels of serum IL-1 and anti-type II collagen IgG antibodies, were directly proportional to the dosage. While the use of large dose agkistrodon wine led to negative effects.

CONCLUSIONThese data highlight the potential function of high-dose agkistrodon powder, which was equivalent to an amount of 6 g/day in adults. The powder can quickly relieve the symptoms of rheumatoid arthritis and prevent aggravation of disease, especially during the early period.

Agkistrodon ; metabolism ; Animals ; Antibodies ; blood ; Arthritis, Experimental ; blood ; chemically induced ; drug therapy ; Collagen Type II ; immunology ; Dosage Forms ; Dose-Response Relationship, Drug ; Enzyme-Linked Immunosorbent Assay ; Extremities ; diagnostic imaging ; pathology ; Female ; Interleukin-1beta ; blood ; Medicine, Chinese Traditional ; Rats, Wistar

Eupatilin is the main active component of DA-9601, an extract from Artemisia. Recently, eupatilin was reported to have anti-inflammatory properties. We investigated the anti-arthritic effect of eupatilin in a murine arthritis model and human rheumatoid synoviocytes. DA-9601 was injected into collagen-induced arthritis (CIA) mice. Arthritis score was regularly evaluated. Mouse monocytes were differentiated into osteoclasts when eupatilin was added simultaneously. Osteoclasts were stained with tartrate-resistant acid phosphatase and then manually counted. Rheumatoid synoviocytes were stimulated with TNF-alpha and then treated with eupatilin, and the levels of IL-6 and IL-1beta mRNA expression in synoviocytes were measured by RT-PCR. Intraperitoneal injection of DA-9601 reduced arthritis scores in CIA mice. TNF-alpha treatment of synoviocytes increased the expression of IL-6 and IL-1beta mRNAs, which was inhibited by eupatilin. Eupatilin decreased the number of osteoclasts in a concentration dependent manner. These findings, showing that eupatilin and DA-9601 inhibited the expression of inflammatory cytokines and the differentiation of osteoclasts, suggest that eupatilin and DA-9601 is a candidate anti-inflammatory agent.
Animals ; Anti-Inflammatory Agents/pharmacology/*therapeutic use ; Arthritis, Experimental/chemically induced/*drug therapy ; Arthritis, Rheumatoid/drug therapy/pathology ; Cell Differentiation/*drug effects ; Cells, Cultured ; Collagen Type II ; Cytokines/biosynthesis ; Disease Models, Animal ; Drugs, Chinese Herbal/therapeutic use ; Female ; Flavonoids/pharmacology/*therapeutic use ; Humans ; Inflammation/drug therapy/immunology ; Interleukin-1beta/genetics/metabolism ; Interleukin-6/genetics/metabolism ; Lymph Nodes/cytology ; Mice ; Mice, Inbred DBA ; Monocytes/cytology ; Osteoclasts/*cytology ; Plant Extracts/pharmacology ; RNA, Messenger/biosynthesis ; Synovial Membrane/cytology ; T-Lymphocytes, Regulatory/cytology/immunology ; Tumor Necrosis Factor-alpha/pharmacology

OBJECTIVETo investigate the effect of oral administration of Zaocys type II collagen (ZCII) on the percentages of Treg/Th17 cells in mesenteric lymph node lymphocytes (MLNLs) in mice with collagen-induced arthritis (CIA).

METHODSCIA was induced in male C57BL/6 mice by immunization with chicken type II collagen. Three weeks later, ZCII, purified by pepsin digestion, was orally administered in the mice for 7 consecutive days (daily dose of 10, 20, or 40 µg/kg). The severity of arthritis in each limb was evaluated using a macroscopic scoring system, and histopathological changes of the joint were observed microscopically with HE staining. The percentages of Treg and Th17 cells in MLNLs was detected by flow cytometry, and the levels of transforming growth factor-β (TGF-β) and interleukin-17 (IL-17) in the supernatant of MLNLs were measured by enzyme-linked immunosorbent assay.

RESULTSCompared with normal control mice, the mice with CIA had significantly higher scores for arthritis and histopathological changes, with also significantly increased percentages of Treg and Th17 cells in MLNLs and elevated levels of TGF-β and IL-17 in MLNL supernatant (P<0.05). In ZCII peptide-treated mice, the scores for arthritis and histopathological changes were significantly lower than those in CIA model group (P<0.05), and Treg cell percentage in MLNLs was up-regulated while Th17 cell percentage lowered; the level of TGF-β was increased but IL-17 was decreased significantly (P<0.05).

CONCLUSIONOral administration of ZCII improves CIA in mice by regulating the percentages of Treg/Th17 cells and the cytokine levels in MLNLs, suggesting the value of ZCII as a promising candidate agent for treatment of rheumatoid arthritis.

Animals ; Arthritis, Experimental ; immunology ; Arthritis, Rheumatoid ; Collagen Type II ; pharmacology ; Enzyme-Linked Immunosorbent Assay ; Flow Cytometry ; Interleukin-17 ; metabolism ; Lymph Nodes ; cytology ; Male ; Mice ; Mice, Inbred C57BL ; T-Lymphocytes, Regulatory ; immunology ; Th17 Cells ; immunology ; Transforming Growth Factor beta ; metabolism

OBJECTIVEWe used an animal model of collagen-induced arthritis (CIA) to study changes and roles of tyrosine hydroxylase (TH) expressed by CD4+ T cell subsets, and then explore the relationship between CD4+ T cell subset-derived catecholamines and inflammatory responses in CIA.

METHODSThirty-six male DBA/1 mice were randomly divided into control group, CIA model group (day 35) and CIA model group (day 55) (n = 12). CIA model was induced by type II collagen (CII) in DBA/1 mice. On the 35th and 55th day following primary immunization, the joints of the mice were observed for clinical score of swelling and the level of anti-CII IgG antibody in serum was examined. Expression of specific transcription factors and cytokines of Th1, Th17, Th2 and Treg cells and TH in mesenteric lymph nodes was measured by means of Western blot. The changes of TH expressed by CD4+ T cell subsets in mesenteric lymph nodes were determined by flow cytometry.

RESULTSClinical score and anti-CII antibody level increased in CIA compared with that in intact mice. Specific transcription factors and cytokines expressed by Th1 and Th17 cells were upregulated and cytokines expressed by Th2 and Treg cells were downregulated in mesenteric lymph nodes in CIA mice. Expression of TH was upregulated and the increased expression of TH in CD4+ T cells was attributed to Th1 and Th17 cells in mesenteric lymph nodes of CIA.

CONCLUSIONThe increase in catecholamines from CD4+ T cell subsets in mesenteric lymph nodes of CIA may be related to inflammatory alleviation in CIA progression.

Animals ; Arthritis, Experimental ; immunology ; CD4-Positive T-Lymphocytes ; enzymology ; Collagen Type II ; adverse effects ; Cytokines ; metabolism ; Disease Models, Animal ; Lymph Nodes ; immunology ; Male ; Mice ; Mice, Inbred DBA ; T-Lymphocyte Subsets ; immunology ; Transcription Factors ; metabolism ; Tyrosine 3-Monooxygenase ; metabolism

In order to obtain the lead compound for treatment of rheumatoid arthritis (RA), in this study, therapeutic efficacy of three bispecific antibodies (BsAB-1, BsAB-2 and BsAB-3) against both hIL-1beta and hIL-17 were compared on CIA model mice. First, by ELISA method we compared the binding capacity of the three bispecific antibodies to the two antigens. The results showed that all three antibodies could simultaneously bind both antigens, among these antibodies, BsAB-1 was superior over BsAB-2 and BsAB-3. CIA model was established with chicken type II collagen (CII) and developed RA-like symptoms such as ankle swelling, skin tight, hind foot skin hyperemia. The CIA mice were treated with three antibodies once every two days for total of 29 days. Compared with the CIA model mice, the RA-like symptoms of the antibody treated-mice significantly relieved, while the BsAB-1 treated-mice were almost recovered. CII antibody level in the serum and cytokines (IL-2, IL-1beta, IL-17A and TNF-alpha) expression in the spleen were examined. Compared with the CIA model mice, all three antibodies could significantly reduce CII antibody and cytokine expression levels. BsAB-1 antibody was more potent than BsAB-2 and BsAB-3. In summary, BsAB-1 is superior over BsAB-2 and BsAB-3 in amelioration of RA symptoms and regulation of CII antibody production and pro-inflammatory cytokine expression, therefore, BsAB-1 can be chosen as a lead compound for further development of drug candidate for treatment of RA.
Animals ; Antibodies ; metabolism ; Antibodies, Bispecific ; immunology ; therapeutic use ; Antigen-Antibody Reactions ; Arthritis, Experimental ; chemically induced ; metabolism ; therapy ; Arthritis, Rheumatoid ; chemically induced ; metabolism ; therapy ; Collagen Type II ; immunology ; Interleukin-17 ; metabolism ; Interleukin-1beta ; metabolism ; Interleukin-2 ; metabolism ; Male ; Mice ; Spleen ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism

This study was purposed to observe the influence of umbilical cord mesenchymal stem cells (UC-MSC) on the peripheral blood CD4(+)CD25(+)regulatory T cells (Treg), Th17 cells and neutrophils in rats with collagen type II-induced arthritis(CIA), and to explore the regulating effect of UC-MSC transplantation on immunocyte subgroup. The rats wee divided into 3 groups: CIA group (model group), UC-MSC treated group and blank control group. The CIA rats were injected with UC-MSC via tail vein. The percentage of CD4(+)CD25(+) cells in peripheral blood and the expression of NCD11b on neutrophil surface in CIA rates was detected by flow cytometry (FCM), and the serum interleukin-17 (IL-17) was observed by enzyme-linked immunosorbent assay (ELISA). The results showed that the mean fluorescence intensity(MFI) of NCD11b and the level of IL-17 in the model group were significantly higher than those in the blank control group, and the ratio of CD4(+)CD25(+) cells were significantly lower (P < 0.05). The MIF of NCD11b and the level of IL-17 in the UC-MSC treated group were significantly lower than that in the model group (P < 0.05), while the proportion of CD4(+)CD25(+) Treg increased (P < 0.05). Since the fifth week, the above indicators in the UC-MSC group have almostly approached the control group. It is concluded that the UC-MSC can increase peripheral blood Treg proportion in CIA rat, inhibit the secretion of Th17 and the activity of neutrophils, reduce the immune inflammation reaction, decrease the release of proinflammatory factor, and induce immune reconstruction.
Animals ; Arthritis, Experimental ; immunology ; therapy ; Female ; Interleukin-17 ; metabolism ; Mesenchymal Stem Cell Transplantation ; Mesenchymal Stromal Cells ; cytology ; Neutrophils ; immunology ; Rats ; Rats, Sprague-Dawley ; Th17 Cells ; immunology ; Umbilical Cord ; cytology