Epigenetic Regulatory Axis MIR22-TET3-MTRNR2L2 Represses Fibroblast-Like Synoviocyte-Mediated Inflammation in Rheumatoid Arthritis.

OBJECTIVE: The specific role of fibroblast-like synoviocytes (FLSs) in the pathogenesis of rheumatoid arthritis (RA) is still not fully elucidated. This study aimed to explore the molecular mechanisms of epigenetic pathways, including three epigenetic factors, microRNA (miRNA)-22 (MIR22), ten-eleven translocation methylcytosine dioxygenase 3 (TET3), and MT-RNR2 like 2 (MTRNR2L2), in RA-FLSs. METHODS: The expression of MIR22, TET3, and MTRNR2L2 in the synovium of patients with RA and arthritic mice were determined by fluorescence in situ hybridization, quantitative polymerase chain reaction (qPCR), immunohistochemistry, and Western blot. Mir22-/- and Tet3+/- mice were used to establish a collagen antibody-induced arthritis (CAIA) model. Mir22 angomir and Tet3 small interfering RNA (siRNA) were used to illustrate the therapeutic effects on arthritis using a collagen-induced (CIA) model. Bioinformatics, luciferase reporter assay, 5-hydroxymethylcytosine (5hmC) dot blotting, chromatin immunoprecipitation-qPCR, and hydroxymethylated DNA immunoprecipitation were conducted to show the direct repression of MIR22 on the TET3 and transcriptional activation of TET3 on MTRNR2L2. RESULTS: The Mir22-/- CAIA model and RA-FLS-related in vitro experiments demonstrated the inhibitory effect of MIR22 on inflammation. MIR22 can directly inhibit the translation of TET3 in RA-FLSs by binding to its 3' untranslated region in TET3. The Tet3+/- mice-established CAIA model showed less severe symptoms of arthritis in vivo. In vitro experiments further confirmed the proinflammatory effect of TET3 in RA. In addition, the CIA model was used to validate the therapeutic effects of Mir22 angomir and Tet3 siRNA. Finally, TET3 exerts its proinflammatory effect by promoting 5hmC production in the promoter of its target MTRNR2L2 in RA-FLSs. CONCLUSION: The key role of the MIR22-TET3-MTRNR2L2 pathway in RA-FLSs provided an experimental basis for further studies into the pathogenesis and related targets of RA from the perspective of FLSs.

Attenuation of experimental osteoarthritis with human adipose-derived mesenchymal stem cell therapy: inhibition of the pyroptosis in chondrocytes.

AIM: To explore the role and mechanism of human adipose-derived mesenchymal stem cells (hAD-MSCs) in the treatment of osteoarthritis (OA). METHODS: OA hulth model of Sprague Dawley (SD) rats and 20 ng/ml TNF-α treated chondrocytes were used as models of OA in vivo and in vitro, respectively. hAD-MSCs were administrated in the articular cavity by injection in vivo and co-cultured with chondrocytes using transwell in vitro. Haematoxylin and eosin staining and Safranin-O/Fast green staining were performed to detect tissue destruction and histopathology. Scanning electron microscopy and transmission electron microscopy were used to observe the ultrastructure of chondrocytes. The pyroptosis signaling pathway-related proteins were detected by immunohistochemistry, immunofluorescence, qRT-PCR and Western blot. And small interference technology was used to study the mechanism in depth. RESULTS: hAD-MSCs could delay the development of rat OA, improve the pathological changes of joints, inhibit the expression of NLRP3, Caspase1, GSDMD and TNFR1. In vitro, the expression of pyroptosis signal proteins in chondrocytes was significantly elevated when stimulated with TNF-α, the level of inflammatory factors such as IL-1ß, IL-18 was increased, and the cell morphology was significantly destroyed. While co-cultured with hAD-MSCs, these syndromes were reversed. Knockout of TNFR1 also returned the upregulation of pyroptosis signals which caused by TNF-α. CONCLUSION: These results demonstrated that hAD-MSCs could inhibit pyroptosis signaling pathway of chondrocytes induced by TNF-α, which have raised our understanding of the role of hAD-MSCs as promising therapy for the management of OA.

Ziyuglycoside I attenuates collagen-induced arthritis through inhibiting plasma cell expansion.

ETHNOBOTANICAL RELEVANCE: With most of the anti-rheumatic drugs having severe adverse drug reactions and poor tolerance, the active components from natural herbs provides a repository for novel, safe, and effective drug development. Sanguisorba officinalis L. exhibits definite anti-inflammatory capacity, however, whether it has anti-rheumatic effects has not been revealed. AIM OF THE STUDY: In the present study, the effect of Ziyuglycoside I (Ziyu I), one of the most important active components in Sanguisorba officinalis L., was investigated in treating collagen-induced arthritis (CIA), illuminating its potential pharmacological mechanisms. MATERIAL AND METHODS: CIA mice were treated with 5, 10, or 20 mg/kg of Ziyu I or 2 mg/kg of MTX, and clinical manifestations as well as pathological changes were observed. T and B cell viability was determined using cell counting kit-8, plasma autoantibodies and cytokines were tested with ELISA, T and B cell subsets were identified by flow cytometry, Blimp1 expression was detected by RT-qPCR and in situ immunofluorescence. The expression of activation-induced cytidine deaminase (AID) was detected by immunohistochemistry. ERK activation in B cells was verified through western blotting and immunofluorescence. Meanwhile, bioinformatics retrieval and molecular docking/molecular dynamics were used to predict the relationship between Blimp1, ERK and Ziyu I with the pharmacokinetics and toxicity of Ziyu I being evaluated in the ADMETlab Web platform. RESULTS: Ziyu I treatment effectively alleviated the joint inflammatory manifestation including arthritis index, global scores, swollen joint count and body weight of CIA mice. It improved the pathological changes of joint and spleen of arthritic mice, especially in germinal center formation. Ziyu I displayed a moderate regulatory effect on T cell activation, the percentage of total T and helper T cells, and tumor necrosis factor-α, but transforming growth factor-ß was not restored. Increased spleen index, B cell viability and plasma auto-antibody production in CIA mice were significantly reduced by Ziyu I therapy. Of note, we found that Ziyu I administration substantially inhibited the excessive expansion of plasma cells in spleen through preventing the expression of B lymphocyte induced maturation protein 1 (Blimp1) and AID in B cells. Ziyu I was predicted in silico to directly interact with ERK2, and reduce ERK2 activation, contributing to the depressed expression of Blimp1. Moreover, Ziyu I was predicted to have a favorable pharmacokinetic profile and low toxicity. CONCLUSION: Ziyu I effectively ameliorates CIA in mice by inhibiting plasma cell generation through prevention of ERK2-mediated Blimp1 expression in B cells. Therefore, Ziyu I is a promising candidate for anti-arthritic drug development.

Antitumor efficacy of CHMFL-KIT-110 solid dispersion in mouse xenograft models of human gastrointestinal stromal tumors.

PURPOSE: CHMFL-KIT-110, a selective c-KIT kinase inhibitor for gastrointestinal stromal tumors (GISTs), possesses a poorly water-soluble, limiting the further development of the drug. This study was to investigate the antitumor efficacy of CHMFL-KIT-110 and CHMFL-KIT-110 solid dispersion (laboratory code: HYGT-110 SD) in GIST tumor xenograft models and to explore the PK/PD relationship of HYGT-110 SD. METHODS: Plasma concentrations of HYGT-110 and HYGT-110 SD were determined by LC-MS/MS in KM mice. Antitumor activity was evaluated by measuring tumor volume and weight in c-KIT-dependent GIST xenograft models. PK/PD relationship was assessed by LC-MS/MS and Western Blot in the GIST-T1 xenografted mice. RESULTS: HYGT-110 exhibited a low oral bioavailability (10.91%) in KM mice. Compared with HYGT-110 treatment, the Cmax and AUC0-t of HYGT-110 SD in mice plasma were substantially increased by 18.81 and 6.76-fold, respectively. HYGT-110 SD (10, 30, and 100 mg/kg/day) also could dose-dependently decrease the tumor volume and weight in the GIST-882 cell-inoculated xenograft mouse models and show 86.35% tumor growth inhibition (TGI) at 28 days at a 25 mg/kg bid dosage in the GIST-T1 cell-inoculated xenograft mouse model. The free concentration of HYGT-110 in plasma was closely correlated with the inhibition of c-KIT phosphorylation levels in tumor tissues. CONCLUSIONS: In comparison with the HPMC formulation, both improved PK and PD characteristics of the solid dispersion formulation of CHMFL-KIT-110 were observed in in vivo animal experiments.

Deficiency of ß-arrestin2 exacerbates inflammatory arthritis by facilitating plasma cell formation.

ß-arrestin2 (ß-arr2) is, a key protein that mediates desensitization and internalization of G protein-coupled receptors and participates in inflammatory and immune responses. Deficiency of ß-arr2 has been found to exacerbate collagen antibody-induced arthritis (CAIA) through unclear mechanisms. In this study we tried to elucidate the molecular mechanisms underlying ß-arr2 depletion-induced exacerbation of CAIA. CAIA was induced in ß-arr2-/- and wild-type (WT) mice by injection of collagen antibodies and LPS. The mice were sacrificed on d 13 after the injection, spleen, thymus and left ankle joints were collected for analysis. Arthritis index (AI) was evaluated every day or every 2 days. We showed that ß-arr2-/- mice with CAIA had a further increase in the percentage of plasma cells in spleen as compared with WT mice with CAIA, which was in accordance with elevated serum IgG1 and IgG2A expression and aggravating clinical performances, pathologic changes in joints and spleen, joint effusion, and joint blood flow. Both LPS stimulation of isolated B lymphocytes in vitro and TNP-LPS challenge in vivo led to significantly higher plasma cell formation and antibodies production in ß-arr2-/- mice as compared with WT mice. LPS treatment induced membrane distribution of toll-like receptor 4 (TLR4) on B lymphocytes, accordingly promoted the nuclear translocation of NF-κB and the transcription of Blimp1. Immunofluorescence analysis confirmed that more TLR4 colocalized with ß-arr2 in B lymphocytes in response to LPS stimulation. Depletion of ß-arr2 restrained TLR4 on B lymphocyte membrane after LPS treatment and further enhanced downstream NF-κB signaling leading to additional increment in plasma cell formation. In summary, ß-arr2 depletion exacerbates CAIA and further increases plasma cell differentiation and antibody production through inhibiting TLR4 endocytosis and aggravating NF-κB signaling.

Angiotensin II and tumor necrosis factor-α stimulate the growth, migration and invasion of BEL-7402 cells via down-regulation of GRK2 expression.

PURPOSE: To investigate the effects of angiotensin II (Ang II) and tumor necrosis factor-α (TNF-α) on the biological characteristics of hepatocellular carcinoma (HCC) cells and the associated changes in G protein-coupled receptor kinase 2 (GRK2) expression. METHODS: The mean serum levels of Ang II and TNF-α in normal subjects and patients with benign liver tumors (BLTs) and HCC were evaluated by enzyme-linked immunosorbent assay (ELISA), and liver samples from the patients with HCC and HCC mice were used to assess the protein levels of both cytokines, their major receptors and GRK2. In addition, the dynamics of Bel-7402 cells were determined with cell counting kit-8 (CCK-8) and Transwell experiments, while the levels of the primary cytokine receptors Ang II type-1 receptor (AT1R) and type-2 receptor (AT2R) as well as TNF receptor 1 (TNFR1) were detected by flow cytometry (FCM). The effects of Ang II and TNF-α on the GRK2 levels in Bel-7402 cells and on the dynamics of GRK2-knockdown HCC cells were also investigated. RESULTS: Both cytokines independently enhanced Bel-7402 cell growth, migration and invasion by decreasing the GRK2 level. In contrast, down-regulating the GRK2 level in Bel-7402 cells suppressed these effects. No synergistic effects were discovered when Ang II and TNF-α were administered together. Furthermore, increased AT1R and TNFR1 levels stimulated HCC initiation and progression, whereas AT2R overexpression produced the opposite effect. CONCLUSIONS: The present results suggested that Ang II and TNF-α promote Bel-7402 cell growth, migration and invasion by down-regulating GRK2 expression, and that the associated receptors AT1R, AT2R and TNFR1 participate in HCC initiation and progression.

A Modified Compound From Paeoniflorin, CP-25, Suppressed Immune Responses and Synovium Inflammation in Collagen-Induced Arthritis Mice.

Paeoniflorin-6'-O-benzene sulfonate (CP-25) is a modified paeoniflorin, which is the main bioactive component of total glucosides of peony. This study evaluated the anti-inflammatory and immunoregulatory effects of CP-25 in mice with collagen-induced arthritis (CIA) and the potential mechanisms underlying these effects. After the onset of CIA, mice were given CP-25 (17.5, 35, or 70 mg/kg) or methotrexate (MTX, 2.0 mg/kg). The arthritis index, swollen joint count, and joint and spleen histopathology were evaluated. T and B cell subsets were assayed using flow cytometry, while the proliferation of these cells and fibroblast-like synoviocytes (FLSs) were evaluated using the Cell Counting Kit-8. ß2-adrenoceptor (ß2-AR) expression was assayed using flow cytometry, immunohistochemistry, and western blotting. FLS migration and invasion were assayed using Transwells. CP-25 (35 or 70 mg/kg) attenuated the arthritis index and swollen joint count, alleviated joint and spleen histopathology, suppressed excessive T cell activation, and attenuated humoral immunity in CIA mice. CP-25 increased ß2-AR expression on T cells, B cells, dendritic cells, and the synovium in CIA mice. CP-25 up-regulated the ß2-AR agonist response and attenuated FLS activation; these effects may reflect CP-25-mediated reduction of ß2-AR desensitization due to down-regulation of membrane G protein-coupled receptor kinase 2 expression. These results suggest that CP-25 suppressed immune responses and synovium inflammation in mice with CIA, effects that were associated with reduced ß2-AR desensitization and the promotion of ß2-AR signaling.

2K1C-activated Angiotensin II (Ang II) exacerbates vascular damage in a rat model of arthritis through the ATR/ERK1/2 signaling pathway.

OBJECTIVE: To explore the role and mechanism of the two-kidney one-clip (2K1C)-activated Angiotensin II (Ang II) in the development of vascular damage in adjuvant-induced arthritis (AA) rats. METHODS: 2K1C rats were established in normal and AA rats for 35 days. Hypertension, endothelial dysfunction, and vascular hypertrophy induced by 2K1C-activated Ang II in systemic inflammation rats were evaluated. The levels of Ang II and TNF-α in serum were observed by ELISA kits. Expressions of Ang II/ATR/ERK1/2 signaling pathway molecules in the aorta were tested by immunohistochemistry or western blot. The migration and capillary tube formation abilities of human umbilical vein endothelial cells (HUVECs) were tested by migration chamber and capillary tube formation assays. RESULTS: The level of Ang II in serum was significantly increased in 2K1C rats. Compared with AA rats, the high level of Ang II activated by 2K1C reduced the endothelium-dependent vasodilator responses to acetylcholine (ACh) in the thoracic aorta and exacerbated endothelial dysfunction and vascular hypertrophy. Expressions of ATR, GRK2, p-ERK1/2, and p-NF-κB were significantly increased in the aorta of AA combined with 2K1C rats. The migration and capillary tube formation abilities of HUVECs were significantly enhanced by Ang II and TNF-α co-stimulations in vitro through the ATR/ERK1/2 signaling pathway compared to those stimulated with TNF-α. CONCLUSIONS: 2K1C-activated Ang II is involved in aggravated vascular injury and endothelial dysfunction through the ATR/ERK1/2 signaling pathway in AA rats.

The influence of TNF-α and Ang II on the proliferation, migration and invasion of HepG2 cells by regulating the expression of GRK2.

PURPOSE: Hepatocellular carcinoma (HCC) is a common digestive system malignancy that is associated with a poor prognosis. This study researched the interaction of tumor necrosis factor-α (TNF-α) and angiotensin II (Ang II) in HCC cells proliferation, migration and invasion and examined their influence on the expression of G protein-coupled receptor kinase 2 (GRK2) and relevant receptors. METHODS: Cell Counting Kit-8 and Transwell assays were performed to evaluate the effects of TNF-α and Ang II on HepG2 cells proliferation, migration and invasion. Flow cytometry was used to investigate the expression of tumor necrosis factor receptor 1 (TNFR1), angiotensin II type 1 (AT1R) and type 2 receptors (AT2R) on the surface of HepG2 cells. Additionally, Western blot was performed to assess the modulation of GRK2 expression by TNF-α and Ang II in HepG2 cells. Meanwhile, GRK2 siRNA-transfected HepG2 cells were used to confirm the effects of GRK2, TNF-α and Ang II on the proliferation, migration and invasion of GRK2-knockdown HCC cells. Finally, the expression of TNF-α, Ang II, TNFR1, AT1R, AT2R and GRK2 proteins in HCC, tumor-adjacent and normal liver tissues were tested by immunohistochemistry. RESULTS: The data demonstrated that TNF-α and Ang II can enhance the proliferation, migration and invasion of HepG2 cells through suppressing GRK2 expression but that the two reagents combined did not have synergistic effects. Moreover,overexpression of TNFR1 and AT1R perhaps promoted the formation and progression of HCC, while high AT2R expression had the opposite effect. CONCLUSIONS: This study provides new ideas for the prevention and treatment of HCC by researching the interaction and probable mechanism of different bioactive factors associated with HCC.

JAK1-STAT3 blockade by JAK inhibitor SHR0302 attenuates inflammatory responses of adjuvant-induced arthritis rats and decreases Th17 and total B cells.

OBJECTIVE: To investigate the effects of JAK inhibitor (SHR0302) on adjuvant-induced arthritis (AA) rats and the partial mechanisms focused on T, B lymphocyte subsets through JAK1-STAT3 pathway, including Th17, Treg, total B cells and memory B cells. METHODS: Animals were divided randomly into normal control, AA, SHR0302 (0.3,1.0, 3.0mg/kg) and MTX. The effects of SHR0302 on AA rats by evaluating arthritis index, arthritis global assessment and paw swelling degree, histopathology of joint and spleen. We examined the proliferation of T, B and FLS. Th17, Treg, total B and memory B cell proportion was measured by flow cytometry. Cytokines TNF-α, IL-1ß, IL-10, IL-17 and antibody IgG1, IgG2a levels in serum were measured by Elisa. The expression of p-JAK1 and p-STAT3 was measured by western blot. RESULTS: SHR0302 suppressed the severity of AA rats by attenuating the arthritis index, arthritis global assessment and paw swelling degree, and alleviated histopathology of spleen and joint of AA rats. SHR0302 can inhibit the proliferation of T, B and FLS, and down-regulated cytokines TNF-α, IL-1ß, IL-17 and antibody IgG1, IgG2a levels, and suppressed the proportion of Th17 and total B, and inhibited JAK1-STAT3 phosphorylation. There was no significant effect on Treg function and memory B cell proportion. CONCLUSION: SHR0302 may attenuate the severity of AA rats, partially through reducing Th17 function and total B cell proportion by inhibiting JAK1-STAT3 phosphorylation.

The Elevated Secreted Immunoglobulin D Enhanced the Activation of Peripheral Blood Mononuclear Cells in Rheumatoid Arthritis.

Immunoglobulin D (IgD) is a surface immunoglobulin that is expressed as either membrane IgD (mIgD) or secreted IgD (sIgD). Researchers have shown that sIgD is often elevated in patients with autoimmune diseases. The possible roles of sIgD on the function of peripheral blood mononuclear cells (PBMCs) in rheumatoid arthritis (RA) are still unclear. In this study, we compared the expression of sIgD, mIgD and IgD receptor (IgDR) in RA patients and healthy controls, and investigated the effect of sIgD on the function of PBMCs. We found that the levels of sIgD, mIgD and IgDR were significantly higher in RA patients compared with healthy controls. The concentrations of sIgD were positively correlated with soluble receptor activator of nuclear factor-κB ligand (sRANKL), rheumatoid factor (RF) and C-reactive protein (CRP) in RA patients. Strikingly, IgD could enhance the proliferation of PBMCs and induce IL-1α, IL-1ß, TNF-α, IL-6 and IL-10 production from PBMCs. Moreover, the percentage of activated T cell subsets (CD4+CD69+, CD4+CD154+) and activated B cell subsets (CD19+CD23+, CD19+CD21+, CD19+IgD+ and CD19-CD138+) were increased by IgD. The percentage of unactivated T cell subset (CD4+CD62L+) and immature B cell subset (CD19+IgM+IgD-) were decreased by IgD in PBMCs. Furthermore, the expressions of IgDR on T and B cells were significantly increased by treatment with IgD. Our results demonstrate that IgD enhanced the activation of PBMCs, which may contribute to RA pathogenesis. Therefore, IgD could be a potential novel immunotherapeutic target for the management of RA.

Prednisone treatment inhibits the differentiation of B lymphocytes into plasma cells in MRL/MpSlac-lpr mice.

AIM: A number of evidence shows that the differentiation of B lymphocytes into plasma cells plays an important role in lupus pathogenesis. In this study we investigated how prednisone, a classical therapeutic drug for autoimmune diseases, regulated plasma cell differentiation in MRL/MpSlac-lpr mice. METHODS: MRL/lpr mice were treated with prednisone (2.5 or 5 mg·kg(-1)·d(-1), ig) for 13 weeks, and the proteinuria levels and survival times were monitored. After the mice were euthanized, blood sample, spleen and thymus were collected. The serum levels of anti-dsDNA antibody, anti-nuclear antibody, IL-21, and IL-10 were detected using ELISA kits. Subsets of splenic B and T lymphocytes were quantified with flow cytometry. Transcription factor Blimp-1 and Bcl-6 expression was determined using qPCR and Western blot. RESULTS: Prednisone treatment dose-dependently attenuated the lupus symptoms in MRL/lpr mice with decreased proteinuria levels, prolonged survival times, decreased serum anti-nuclear antibody levels, and reduced spleen and thymus indices. Prednisone treatment also significantly decreased the elevated percentages of plasma cells and plasma cell precursors, decreased the percentages of activated T cells, and increased the frequency of CD4(+)CD62L(+) cells, demonstrated that decreased anti-nuclear antibodies and improvements in lupus symptoms were associated with decreased plasma cells. Furthermore, prednisone treatment decreased serum IL-21 and IL-10 levels and reduced the expression of splenic Blimp-1 and Bcl-6 (two key regulatory factors for plasma cell differentiation) in MRL/lpr mice. CONCLUSION: Prednisone treatment restricts B lymphocyte differentiation into plasma cells in MRL/lpr mice, which may be correlated with the inhibition of IL-21 production and the restoration of the balance between Blimp-1 and Bcl-6.

IL-21 acts as a promising therapeutic target in systemic lupus erythematosus by regulating plasma cell differentiation.

Plasma cells, which secrete auto-antibodies, are considered to be the arch-criminal of autoimmune diseases such as systemic lupus erythematosus, but there are many cytokines involved in inducing the differentiation of B-cell subsets into plasma cells. Here, we emphasize IL-21, which has emerged as the most potent inducer of plasma cell differentiation. In this review, we focused on the promoting effects of IL-21 on plasma cell differentiation and discuss how these effects contribute to B cell-mediated autoimmune disease.

The effect of BLyS on the activity of peripheral B lymphocytes mediated by BLyS receptors in patients with systemic lupus erythematosus.

The study was promoted to probe into the effect of BLyS on the activity of peripheral B lymphocytes mediated by BLyS receptors in patients with systemic lupus erythematosus (SLE). Thirty new-onset patients having fulfilled the American College of Rheumatology (ACR) revised criteria for the diagnosis of SLE. Twenty age-matched healthy volunteers were recruited for this study. Peripheral blood samples were used to analyze the expression of BLyS protein and related receptors (BR3, TACI), to detect peripheral B cell subpopulations of different phases. Clinical disease activity was evaluated according to the systemic lupus erythematosus disease activity index (SLEDAI-2000). The percentage of CD19(+)CD5(+), CD19(+)CD27(+), CD19(+)CD38(+) and total CD19(+) in the peripheral blood is significantly higher in SLE patients than that in healthy controls (p < 0.01). BLyS concentrations and TACI expression are up-regulated in SLE patients (p < 0.01) while BR3 showed no differences between the two groups (p > 0.05). BLyS concentrations and TACI MFI both showed positive correlation with SLEDAI (r(2) = 0.391, p < 0.001, r(2) = 0.339, p = 0.001), whereas BR3 expression showed no relationship with SLEDAI. The disorders of peripheral B cell subsets maybe reflect the effect of BLyS on the activity of peripheral B lymphocytes mediated by BLyS receptors. The significant up-regulation of BLyS and its receptors, especially TACI may serve as a target for the treatment of SLE.

Therapeutic effects of TACI-Ig on rats with adjuvant-induced arthritis via attenuating inflammatory responses.

OBJECTIVE: To investigate the effects of TACI-Ig, a recombinant fusion protein that modulates B- and T-cell activation by binding and neutralizing B-lymphocyte stimulator (BLyS) and a proliferation-inducing ligand (APRIL), in an established adjuvant-induced arthritis (AA) rat model. METHODS: Rats with experimental arthritis were randomly separated into different groups and then treated with TACI-Ig (0.7, 2.1, 6.3 mg/kg), rhTNFR-Fc (2.8 mg/kg), MTX (0.5 mg/kg) or IgG-Fc (6.3 mg/kg), from Day 16 to Day 34 after immunization. Arthritis was evaluated by hind paw swelling, polyarthritis index and histopathological examination. Activities of BLyS, APRIL, IL-1ß, IL-2, IL-10, TGF-ß1, PGE(2), TNF-α, IFN-γ, immunoglobulin (Ig)G1, IgG2a, IgM and IgA were assessed by ELISA. Cluster of differentiation (CD)20 expression was detected by immunohistochemical analysis. RESULTS: TACI-Ig (2.1, 6.3 mg/kg) treatment significantly reduced the severity of established arthritis using the methods of clinical observation and histopathological examination. TACI-Ig treatment inhibited expression of IgM, decreased the expression of BLyS and APRIL and regulated the balance of pro-inflammatory and anti-inflammatory cytokines in serum of AA rats. Immunohistochemical analysis demonstrated that CD20 production was reduced in spleen. CONCLUSIONS: Data presented here demonstrate that administration of TACI-Ig significantly attenuates progression of experimental arthritis, with reductions in inflammatory response and bone and joint destruction.

[Pulmonary immune responses to acute lung injury following smoke inhalation and its mechanisms].

OBJECTIVE: To analyze pulmonary innate and specific immune responses following the smoke inhalation-induced acute lung injury (ALI). METHODS: Smoke inhalation-induced ALI should be replicated at high (4x10(-3)) and low (2x10(-3)) dose of carbon monoxide (CO) respectively for 24 hours. After this period, lung tissue histopathological changes and the parameters of both treatment groups were observed compared to those of control animals: the phasic variations of concentrations of pro-anti-inflammatory cytokines in bronchoalveolar lavage fluid (BALF), numbers of lymphocyte subsets in peripheral blood and BALF, CD45(+) lymphoid leukocytes, CD45(+) nonlymphoid leukocytes and CD4(+)/CD8(+) in BALF were determined by flow cytometry (FCM). RESULTS: Lung histopathological changes of smoke-exposed animals revealed obvious alveolar leakage characterizing ALI. Concentrations of tumor necrosis factor-alpha (TNF-alpha) in BALF were increased immediately after injury and reached the peak at 2 hours after injury, and more obvious changes were seen in high dose group than those in low dose group. Levels of interleukin-6 (IL-6) and interferon-gamma (IFN-gamma) were elevated following after 4 hours injury peaking at 12 hours. The changes of levels of IL-6 in low dose group were more obviously than those in high dose group, and those of IFN-gamma were reverse. Levels of IL-6 and IFN-gamma were decreased at 24 hours, still higher than those of control group (P<0.05 or P<0.01), whereas the expressions of IL-10 were increased at 6 hours, and continuing upregulation to 24 hours compared with those of control group (P<0.05 or P<0.01). Additionally, the numbers of lung-resident CD4(+) T cells, CD8(+) T cells, natural killer cells, B cells and total T-lymphocytes in peripheral blood and BALF were significantly reduced after challenge (P<0.05 or P<0.01). The number of CD45(+) lymphoid leukocytes in BALF and CD4(+)/CD8(+) were decreased; while that of CD45(+) nonlymphoid leukocytes was increased obviously compared with those of control group, and obviously changed in high dose group than in low dose group (P<0.05 or P<0.01). CONCLUSION: These data suggest that smoke inhalation induced ALI is associated with exaggerated and sustained pulmonary innate immune responses partly by activated polymorphonuclear neutrophils (PMN) and macrophage, whereas specific immunity in the lung is suppressed obviously.

Effects of total glucosides of peony on immunological hepatic fibrosis in rats.

AIM: To study the effects of total glucosides of peony (TGP) on immunological hepatic fibrosis induced by human albumin in rats. METHODS: Sixty adult male Sprague-Dawley rats were randomly divided into: Normal group, model group, TGP (60 and 120 mg/kg) treatment groups and colchicines (0.1 mg/kg) treatment group. On the day before the rats were killed, those in TGP or colchicine groups received TGP or colchicine as above from the first day of tail vein injection of human albumin. The rats in normal and model groups were only administered with the same volume of vehicle. At the end of the 16th wk, rats in each group were killed. Blood and tissue specimens were taken. Levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), nitric oxide (NO), content of malondialdehyde (MDA), activity of superoxide dismutase (SOD) and glutathione peroxidase (GSH-px), were measured by biochemical methods. Serum procollagen type III (PC III) and laminin (LN) were determined by radioimmunoassay. Liver collagen level was determined by measuring hydroxyproline content in fresh liver samples. Hepatic tissue sections were stained with hematoxylin-eosin and examined under a light microscope. RESULTS: Histological results showed that TGP improved the human albumin-induced alterations in the liver structure, alleviated lobular necrosis and significantly lowered collagen content. The antifibrotic effect of TGP was also confirmed by decreased serum content of LN and PCIII in TGP-treated group. Moreover, the treatment with TGP effectively reduced the hydroxyproline content in liver homogenates. However, the level of ALT and AST increased in fibrotic rat but had no significance compared with normal control, whereas the ratio of A/G decreased without significance. TGP had no effect on level of ALT, AST and the ratio of A/G. Furthermore, TGP treatment significantly blocked the increase in MDA and NO, associated with a partial elevation in liver total antioxidant capacity including SOD and GSH-px. CONCLUSION: TGP has beneficial effects on hepatic fibrosis in rats by inhibition of collagen synthesis and decreasing oxidative stress.

Therapeutic effects of glucosides of Cheanomeles speciosa on collagen-induced arthritis in mice.

AIM: To investigate the therapeutic effect of the glucosides of Cheanomeles speciosa (GCS) on the collagen-induced arthritis (CIA) in mice. METHODS: Mice were divided randomly into six groups, including normal, CIA, CIA+GCS (60, 120, and 240 mg/kg) and CIA plus glucosides of Tripterygium wilfordii (GTW) groups. CIA model was based on mice. The effect of GCS in CIA mice was measured by paw-swelling, arthritis scores, and histopathological assessment of synovium. Indices of thymus and spleens were measured. Thymocytes and splenocytes proliferation, activity of interleukin-1 (IL-1), and interleukin-2 (IL-2) were assayed by MTT and [(3)H]TdR method. The level of anti-collagen type II (CII) antibody in serum and prostaglandin E (PGE) in ankle were assayed by ELISA and ultraviolet spectrophotometer method, respectively. RESULTS: The onset of paw-swelling was on d 24 after injection of emulsion. The peak of secondary inflammation appeared on d 36 and then declined after d 40. GCS and GTW significantly reduced paw-swelling and arthritis scores, reduced the increase of spleen indices of CIA mice, suppressed the ConA or LPS-induced thymocyte or spleen cell proliferation, and the production of IL-1 and IL-2 in CIA mice. GCS reduced the level of anti-CII antibody and PGE. Histological pathology analysis demonstrated that the synovium of CIA mice was hyperplastic, pannus was formed, and inflammatory cells infiltrated into synovium. The pathological changes were significantly reduced by GCS. CONCLUSION: GCS had anti-inflammatory effect on CIA mice, which might be related to the modification of the abnormal immunological function of CIA mice.