Impact of sex differences on the clinical presentation, pathogenesis, treatment and prognosis of Sjögren's syndrome.

Sjögren's syndrome is a common chronic autoimmune disease that manifests as dry mouth, dry eyes and systemic complications. There are sex differences in the clinical manifestations between men and women, with the average age of onset being around 55 years and the majority of female patients developing the disease during the menopausal years. Understanding the impact of sex differences on SS may help in the treatment and prognosis of patients. Studies have confirmed that a number of factors are associated with the onset of SS, but the exact mechanisms are not fully understood. Sex hormones (especially oestrogens and androgens) play a very important role, and the balance of sex hormone levels in the body is crucial for maintaining homeostasis in the acinar cells of the lacrimal and salivary glands. In addition, chromosomes play a very important role in the sex differences in SS. The gut microbiota also has some influence on sex differences in SS. In this review, we focus on oestrogens and androgens, which are important in the pathogenesis of SS, and summarize the progress of non-clinical studies. Sex differences may influence differences in individualized treatment regimens and further studies are needed.

G protein-coupled receptor kinase 2 as a novel therapeutic target for gland fibrosis of Sjögren's syndrome.

Sjogren's syndrome (SS) is a chronic, progressive autoimmune disorder characterized by gland fibrosis. We previously found a close correlation between gland fibrosis and the expression of G protein-coupled receptor kinase 2 (GRK2). In this study we explored the pathological and therapeutic significance of GRK2 in SS. Submandibular gland (SMG) antigen-induced SS mouse model was established in WT and GRK2+/- mice. We showed that the expression levels of GRK2 were significantly up-regulated in glandular tissue and positively correlated with fibrotic morphology in SS patients and mice. Hemizygous knockout of GRK2 significantly inhibited the gland fibrosis. In mouse salivary gland epithelial cells (SGECs), we demonstrated that GRK2 interacted with Smad2/3 to positively regulate the activation of TGF-ß-Smad signaling with a TGF-ß-GRK2 positive feedback loop contributing to gland fibrosis. Hemizygous knockout of GRK2 attenuated TGF-ß-induced collagen I production in SGECs in vitro and hindered gland fibrosis in murine SS though preventing Smad2/3 nuclear translocation. Around 28 days post immunization with SMG antigen, WT SS mice were treated with a specific GRK2 inhibitor paroxetine (Par, 5 mg·kg-1·d-1, i.g. for 19 days). We found that Par administration significantly attenuated gland fibrosis and alleviated the progression of SS in mice. We conclude that genetic knockdown or pharmacological inhibition of GRK2 significantly attenuates gland fibrosis and alleviates the progression of SS. GRK2 binds to Smad2/3 and positively regulates the activation of TGF-ß-Smad signaling. A TGF-ß-GRK2 positive feedback loop contributes to gland fibrosis. Our research points out that GRK2 could be a promising therapeutic target for treating SS.

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.

Angiotensin II Type 2 Receptor Modulates Synovial Macrophage Polarization by Inhibiting GRK2 Membrane Translocation in a Rat Model of Collagen-Induced Arthritis.

The chronic inflammatory autoimmune disease rheumatoid arthritis (RA) is characterized by an infiltration of activated proinflammatory immune cells into the joint that is accompanied by an overproduction of various mediators, leading to destruction of cartilage and bone erosion. Angiotensin II type 2 receptor (AT2R) is involved in antioxidative, anti-inflammatory, and antifibrotic responses. Synovial macrophages (SMs) are a type of tissue macrophages that are derived from bone marrow cells. SMs plays a central role in synovial regional immunization, which is significantly increased in both collagen-induced mice with arthritis mice and RA patients. AT2R activation caused a reversal of the polarization of SMs in the joint from the proinflammatory M1 SM to the tolerogenic, benign M2 SM. In consequence, this switch resulted in an attenuated form of the joint pathology in a rat model of collagen-induced arthritis. These results were mechanistically linked to the observation that GRK2 was translocated into cytoplasm, and ERK1/2 and NF-κB activation were inhibited. These findings open the way to a new therapeutic approach using an activation of AT2R to subvert joint inflammation in RA.

CP-25 alleviates antigen-induced experimental Sjögren's syndrome in mice by inhibiting JAK1-STAT1/2-CXCL13 signaling and interfering with B-cell migration.

The etiology of primary Sjögren's syndrome (pSS) remains unknown, and there is no complete curative drug. In this study, we treated a mouse model of the submandibular gland (SG) protein-immunized experimental Sjögren's syndrome (ESS) with paeoniflorin-6'-O-benzene sulfonate (termed CP-25) to evaluate the potential therapeutic effects of CP-25. Through in vivo experiments, we found that CP-25 increased saliva flow, alleviated the salivary gland indexes, and improved tissue integrity in the ESS model. The viability of splenocytes and B-lymphocyte migration from spleen were reduced in ESS mice. Furthermore, CP-25 decreased the expression of IgG antibodies, anti-SSA and anti-SSB antibodies and modulated the levels of cytokines in the serum of SS mice. The numbers of total B lymphocytes, plasma cells (PCs), and memory B cells diminished in the salivary gland. Increased expression of the JAK1-STAT1-CXCL13 axis and IFNα was found in human tissue isolated from pSS patients. In vitro, after stimulation with IFNα, the levels of CXCL13 mRNA and CXCL13 in human salivary gland epithelial cells (HSGEC) increased, while CP-25 counteracted the secretion of CXCL13 and downregulated the expression of CXCL13. IFN-α activated the JAK1-STAT1/2-CXCL13 signaling pathway in HSGEC, which was negatively regulated by additional CP-25. As a consequence, B-cell migration was downregulated in coculture with IFN-α-stimulated HSGEC. Taken together, this study demonstrated that the therapeutic effects of CP-25 were associated with the inhibition of the JAK1-STAT1/2-CXCL13 signaling pathway in HSGEC, which impedes the migration of B cells into the salivary gland. We identified the underlying mechanisms of the therapeutic effect of CP-25 and provided an experimental foundation for CP-25 as a potential drug in the treatment of the human autoimmune disorder pSS.

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.

CD4+ T cells and TGFß1/MAPK signal pathway involved in the valvular hyperblastosis and fibrosis in patients with rheumatic heart disease.

The aim of this study was to investigate the roles of CD4+ T cells and transforming growth factor beta (TGFß1) in the pathological process of valvular hyperblastosis and fibrosis of patients with rheumatic heart disease (RHD). A total of 151 patients were enrolled, among whom, 78 patients were with RHD, and 73 were age and gender matched RHD negative patients. Blood samples and valve specimens were collected for analysis. Pathological changes and collagen fibers contents of valves were analyzed using HE and Masson staining. Percentage of peripheral blood CD4+ T cells was tested through flow cytometry. TGFß1 level in serum were identified by ELISA. CD4+ T cells infiltration and expression of TGFß1, p-p38, p-JNK, p-ERK in valves were detected by immunohistochemistry. The mRNA and protein levels of p38, JNK, ERK, TGFß1, I-collagen and α-SMA were detected by qRT-PCR and western blotting, respectively. The heart valve tissues of RHD patients showed higher degrees of fibrosis, calcification and lymphocytes infiltration, which were mainly CD4+ T cells. In addition, compared with control group, RHD patients had more total CD4+ T cells in peripheral blood and valve tissues. Expression of TGFß1, phosphorylation of JNK and p38, and synthesis of I-collagen in valve tissues of RHD patients were also significantly increased. Furthermore, we found a strong positive correlation between TGFß1 expression and phosphorylation of JNK and p38. CD4+ T cells, and fibrogenic cytokine TGFß1, which activate the intracellular MAPK signaling pathway may participate in the fibrosis of heart valve in RHD patients.

IgD-Fc-Ig fusion protein, a new biological agent, inhibits T cell function in CIA rats by inhibiting IgD-IgDR-Lck-NF-κB signaling pathways.

IgD-Fc-Ig fusion protein, a new biological agent, is constructed by linking a segment of human IgD-Fc with a segment of human IgG1-Fc, which specifically blocks the IgD-IgDR pathway and selectively inhibits the abnormal proliferation, activation, and differentiation of T cells. In this study we investigated whether IgD-Fc-Ig exerted therapeutic effects in collagen-induced arthritis (CIA) rats. CIA rats were treated with IgD-Fc-Ig (1, 3, and 9 mg/kg) or injected with biological agents etanercept (3 mg/kg) once every 3 days for 40 days. In the PBMCs and spleen lymphocytes of CIA rats, both T and B cells exhibited abnormal proliferation; the percentages of CD3+ total T cells, CD3+CD4+ Th cells, CD3+CD4+CD25+-activated Th cells, Th1(CD4+IFN-γ+), and Th17(CD4+IL-17+) were significantly increased, whereas the Treg (CD4+CD25+Foxp3+) cell percentage was decreased. IgD-Fc-Ig administration dose-dependently decreased the indicators of arthritis; alleviated the histopathology of spleen and joint; reduced serum inflammatory cytokines levels; decreased the percentages of CD3+ total T cells, CD3+CD4+ Th cells, CD3+CD4+CD25+-activated Th cells, Th1 (CD4+IFN-γ+), and Th17(CD4+IL-17+); increased Treg (CD4+CD25+Foxp3+) cell percentage; and down-regulated the expression of key molecules in IgD-IgDR-Lck-NF-κB signaling (p-Lck, p-ZAP70, p-P38, p-NF-κB65). Treatment of normal T cells with IgD (9 µg/mL) in vitro promoted their proliferation. Co-treatment with IgD-Fc-Ig (0.1-10 µg/mL) dose-dependently decreased IgD-stimulated T cell subsets percentages and down-regulated the IgD-IgDR-Lck-NF-κB signaling. In summary, this study demonstrates that IgD-Fc-Ig alleviates CIA and regulates the functions of T cells through inhibiting IgD-IgDR-Lck-NF-κB signaling.

4-Phenylbutyric Acid Protects Against Ethanol-Induced Damage in the Developing Mouse Brain.

BACKGROUND: Ethanol (EtOH) exposure during pregnancy may result in fetal alcohol spectrum disorders (FASD). One of the most deleterious consequences of EtOH exposure is neuronal loss in the developing brain. Previously, we showed that EtOH exposure induced neuroapoptosis in the brain of postnatal day 4 (PD4) mice but not PD12 mice. This differential susceptibility may result from an insufficient cellular stress response system such as unfolded protein response (also known as endoplasmic reticulum [ER] stress) in PD4 mice. In this study, we compared the effect of EtOH on ER stress in PD4 and PD12 mice and determined whether the inhibition of ER stress could protect the developing brain against EtOH damage. METHODS: We used a third-trimester equivalent mouse model of FASD. PD4 and PD12 C57BL/6 mice were subcutaneously injected with saline (control), EtOH, EtOH plus 4-phenylbutyric acid (4-PBA), a chemical chaperone known as ER stress inhibitor, and 4-PBA alone. The expression of apoptosis marker, ER stress markers, and markers for glial cell activation was examined in the cerebral cortex. RESULTS: EtOH induced neuroapoptosis and increased the expression of ER stress markers, such as activating transcription factor 6, 78-kDa glucose-regulated protein, inositol-requiring enzyme 1α, mesencephalic astrocyte-derived neurotrophic factor, and caspase-12 in PD4 but not PD12 mice. EtOH exposure also activated microglia and astrocytes. Interestingly, treatment with 4-PBA attenuated EtOH-induced neuroapoptosis. Moreover, 4-PBA inhibited the expression of the aforementioned ER stress markers and EtOH-induced glial activation in PD4 mice. CONCLUSIONS: ER stress plays an important role in EtOH-induced damage to the developing brain. Inhibition of ER stress is neuroprotective and may provide a new therapeutic strategy for treating FASD.

CP-25 reverses prostaglandin E4 receptor desensitization-induced fibroblast-like synoviocyte dysfunction via the G protein-coupled receptor kinase 2 in autoimmune arthritis.

Paeoniflorin-6'-O-benzene sulfonate (CP-25) is a novel compound derived from paeoniflorin that has been demonstrated to have therapeutic effects in a rat model of rheumatoid arthritis (RA). However, the underlying mechanism has not been elucidated to date. We explored this mechanism in the present study by treating rats with adjuvant arthritis (AA) with CP-25. We found that the membrane EP4 protein level was downregulated; whereas, GRK2 was upregulated, in fibroblast-like synoviocyte (FLS)s of AA rats. Prostaglandin (PGE)2 stimulated FLS proliferation and enhanced the membrane EP4 receptor protein level; the latter was reversed by the administration of an EP4 receptor agonist, whereas the membrane GRK2 protein level gradually increased. The changes in the EP4 receptor and GRK2 expression were enhanced by TNF-α, and the former was accompanied by an alteration in the cyclic (c)AMP level. The EP4 receptor agonist stimulation increased the association between GRK2 and the EP4 receptor. GRK2 knockdown abrogated the abnormalities in FLS proliferation. The CP-25 treatment (100 mg/kg) suppressed joint inflammation with an efficacy that was similar to that of methotrexate. This finding was associated with EP4 upregulation and GRK2 downregulation in FLSs. Thus, GRK2 plays an important role in the abnormal FLS proliferation observed in AA possibly by promoting EP4 receptor desensitization and decreasing the cAMP level. Our results demonstrate that CP-25 has therapeutic potential for the treatment of human RA via GRK2 regulation.

Paeoniflorin-6'-O-benzene sulfonate alleviates collagen-induced arthritis in mice by downregulating BAFF-TRAF2-NF-κB signaling: comparison with biological agents.

Paeoniflorin-6'-O-benzene sulfonate (CP-25) is a new ester derivative of paeoniflorin with improved lipid solubility and oral bioavailability, as well as better anti-inflammatory activity than its parent compound. In this study we explored whether CP-25 exerted therapeutic effects in collagen-induced arthritis (CIA) mice through regulating B-cell activating factor (BAFF)-BAFF receptors-mediated signaling pathways. CIA mice were given CP-25 or injected with biological agents rituximab or etanercept for 40 days. In CIA mice, we found that T cells and B cells exhibited abnormal proliferation; the percentages of CD19+ total B cells, CD19+CD27+-activated B cells, CD19+BAFFR+ and CD19+TACI+ cells were significantly increased in PBMCs and spleen lymphocytes. CP-25 suppressed the indicators of arthritis, alleviated histopathology, accompanied by reduced BAFF and BAFF receptors expressions, inhibited serum immunoglobulin levels, decreased the B-cell subsets percentages, and prevented the expressions of key molecules in NF-κB signaling. Furthermore, we showed that treatment with CP-25 reduced CD19+TRAF2+ cell expressions stimulated by BAFF and decreased TRAF2 overexpression in HEK293 cells in vitro. Thus, CP-25 restored the abnormal T cells proliferation and B-cell percentages to the normal levels, and normalized the elevated levels of IgA, IgG2a and key proteins in NF-κB signaling. In comparison, rituximab and etanercept displayed stronger anti-inflammatory activities than CP-25; they suppressed the elevated inflammatory indexes to below the normal levels in CIA mice. In summary, our results provide evidence that CP-25 alleviates CIA and regulates the functions of B cells through BAFF-TRAF2-NF-κB signaling. CP-25 would be a soft immunomodulatory drug with anti-inflammatory effect.

ß2-adrenoceptor signaling reduction is involved in the inflammatory response of fibroblast-like synoviocytes from adjuvant-induced arthritic rats.

OBJECTIVE: To investigate the effects of ß-AR signaling on fibroblast-like synoviocytes (FLS) from adjuvant-induced arthritis (AA) rats and the partial mechanisms focused on ß-AR desensitization mediated by GRK2 and ß-arrestin2. METHODS: Animals were divided into a control group and an AA model group, and FLSs were cultured. Arthritis index, histopathology of joints, epinephrine (Epi) and norepinephrine (NE) were detected in vivo. The effect of the ß-AR agonist isoprenaline (ISO) and the ß2-AR agonist salbutamol on FLS cell viability were detected by CCK8. Cytokines TNF-α, IL-1ß, OPG and RANKL were examined by ELISA. The expression of ß2-AR was detected by immunofluorescence and flow cytometry. The cytomembrane expression and desensitization of ß2-AR, GRK2, and ß-arrestin2 were measured by flow cytometry and western blot. RESULTS: The concentration of NE increased to a peak on day 21, which was consistent with the arthritis index. The levels of Epi and NE in synovial tissues were decreased. ISO inhibited FLS cell viability and TNF-α, IL-1ß, and RANKL secretion, and promoted OPG secretion. ß2-AR mediated the effects of ISO on FLS cell viability. ß2-AR signaling was weaker in AA rats compared to the controls. Elevated GRK2 and ß-arrestin2 in cytomembranes promoted ß2-AR desensitization and may decrease the anti-inflammatory effect of ß2-AR signaling. CONCLUSION: The activation of ß2-AR signaling exerts its anti-inflammatory activities on FLS. ß2-AR signaling decreased in the AA model, which might be related to the increased membrane expression of GRK2 and ß-arrestin2, and promoted the excessive desensitization of ß2-AR. Decreased ß2-AR signaling may be relevant to the exacerbation of arthritis inflammation.

Losartan suppresses the inflammatory response in collagen-induced arthritis by inhibiting the MAPK and NF-κB pathways in B and T cells.

The angiotensin II type 1 receptor (AT1R) antagonist losartan has been confirmed to have a moderate anti-inflammatory effect in vitro and in vivo. However, how it affects immune cells in Rheumatoid Arthritis (RA) is still unknown. We found that in human synovial tissues, AT1R is significantly expressed on T cells and B cells. Treatment with losartan (15 mg/kg) alone and in combination with a low dose of methotrexate (MTX 0.25 mg/kg/3 days) significantly suppressed the progression of CIA. Secondary paw swelling, joint destruction and the presence of pro-inflammatory cytokines (TNF-α and IFN-γ) in the serum were alleviated after treatment. The therapeutic effects of losartan were based on reduced T-cell and B-cell activation, specifically by decreased cell vitality and pro-inflammatory cytokine production. In addition, losartan combined with a low dose of MTX achieved a similar therapeutic effect, while protecting liver and kidney from MTX damage. Mechanistically, losartan inhibits the production of pro-inflammatory mediators, reduces the phosphorylation of p38, ERK, and p65, p50 nuclear transposition in T cells and B cells. Phosphorylation of JNK is not affected by losartan in the CIA rat model. losartan can be used as an effective RA treatment, which exhibits anti-arthritic effects potentially through down-regulating the phosphorylation of p38, ERK and signaling through NF-κB. While achieving similar anti-rheumatic effects, a combination therapy of losartan with a low dose of MTX, can protect from liver and renal damage caused by giving a high dose of MTX.

Regulatory effects of paeoniflorin-6'-O-benzene sulfonate (CP-25) on dendritic cells maturation and activation via PGE2-EP4 signaling in adjuvant-induced arthritic rats.

Rheumatoid arthritis (RA) is a chronic, systemic autoimmune disease. Dendritic cells (DCs) are one of the most powerful antigen-presenting cells, and they play an important role in RA pathogenesis. Prostaglandin E2 (PGE2) is a potent lipid mediator that can regulate the maturation and activation of DCs, but the molecular mechanisms have not been elucidated. In this study, both in vitro and in an RA rat model, we investigated the mechanisms involved by focusing on PGE2-mediated signaling and using a novel anti-inflammatory compound, paeoniflorin-6'-O-benzene sulfonate (CP-25). PGE2 combined with tumor necrosis factor-α promoted DC maturation and activation through EP4-cAMP signaling. Treatment with CP-25 increased the endocytic capacity of DCs induced by PGE2. CP-25 inhibited the potency of DCs induced by the EP4 receptor agonist, CAY10598, to stimulate allogeneic T cells. Consistent with these findings, the CAY10598-induced upregulation of DC surface activation markers and production of IL-23 was significantly inhibited by CP-25 in a concentration-dependent manner. In vivo administration of CP-25 alleviated adjuvant arthritis (AA) in rats through inhibition of DC maturation and activation. Our results indicate that PGE2-EP4-cAMP signal hyperfunction can lead to abnormal activation of DC functions, which correlates with the course of disease in AA rats and provides a possible treatment target. The inhibition of DC maturation and activation by CP-25 interference of the PGE2-EP4 pathway may significantly contribute to the immunoregulatory profile of CP-25 when used to treat RA and other immune cell-mediated disorders.

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.

BAFF upregulates CD28/B7 and CD40/CD154 expression and promotes mouse T and B cell interaction in vitro via BAFF receptor.

AIM: B cell-activating factor belonging to the TNF family (BAFF) is a member of TNF family and required for peripheral B cell survival and homeostasis. BAFF has been shown to promote the proliferation of T and B cells. In this study we examined whether and how BAFF mediated the interaction between mouse T and B cells in vitro. METHODS: BAFF-stimulated B or T cells were co-cultured with T or B cells. The interactions between T and B cells were analyzed by measuring the expression of co-stimulatory molecules (CD28/CD80 or CD40/CD154), the proliferation and secretion of T and B cells and other factors. Two siRNAs against the transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI) and BAFF receptor (BAFF-R) were used to identify the receptors responsible for the actions of BAFF. RESULTS: BAFF-stimulated B cells significantly promoted the proliferation and activity of co-cultured T cells, and increased the percentages of CD4(+)CD28(+) and CD4(+)CD154(+) T cells. Similarly, BAFF-stimulated T cells significantly promoted the proliferation and activity of co-cultured B cells, and increased CD19(+)CD80(+) and CD19(+)CD40(+)B cell subpopulations. BAFF-R siRNA-silenced B cells showed significantly lower expression of CD40 and CD80 than the control B cells. When the BAFF-R siRNA-silenced B cells were stimulated with BAFF, then co-cultured with T cells, the expression of CD28 and CD154 on T cells was not increased. TACI siRNA-silenced B cells exhibited higher expression of CD40 and CD80 than the control B cells. When the TACI siRNA-silenced B cells were stimulated with BAFF, then co-cultured with T cells, the expression of CD28 and CD154 on T cells was significantly increased. CONCLUSION: BAFF upregulates CD28/B7 and CD40/CD154 expression, and promotes the interactions between T and B cells in a BAFF-R-dependent manner.

The ginsenoside metabolite compound K exerts its anti-inflammatory activity by downregulating memory B cell in adjuvant-induced arthritis.

CONTEXT: Compound K (CK, 20-O-d-glucopyranosyl-20(S)-protopanaxadiol), a novel ginsenoside metabolite, is structurally a member of the dammarane-type triterpene saponins. Several studies have identified the anti-inflammatory activity of CK. Our previous study demonstrated that CK exerted its anti-inflammatory effect via inhibition of abnormal activation and differentiation of T cells. However, its mechanism of action on B cells remains unclear. OBJECTIVE: The objective of this study is to investigate the effect and underlying mechanisms of CK's effects on memory B cells in the setting of adjuvant-arthritis (AA). MATERIALS AND METHODS: Complete Freund's adjuvant was used to induce AA in rats. Rats were administered, either CK (10, 40, and 160 mg/kg), once daily for 15 d, or methotrexate (MTX; 0.5 mg/kg) once every 3 d, for a total of six times. To evaluate the anti-inflammatory effect of CK, a global assessment and a swollen joint count of AA rats were performed every 3 d. Spleen index and histopathology were examined. Subsets of B cells including CD45R(+)IgM(+) (total B cells) and CD45R(+)CD27(+) (memory B cells) and expression of CD40 and CD40L were assayed by flow cytometry. RESULTS: Compared with the AA rats, global assessment scores and swollen joint counts were significantly lower in the treated groups received CK (40 and 160 mg/kg; p < 0.05 and p < 0.01, respectively). CK (40 and 160 mg/kg) decreased the spleen index (p < 0.01), and alleviated hyperplasia of lymph nodes (p < 0.05 and p < 0.01, respectively) and marginal zone (p < 0.05) in the spleen. In addition, CK (40 and 160 mg/kg) suppressed memory B cell subsets (p < 0.05), and suppressed CD40L expression on T cells and CD40 expression on B cells (p < 0.05 and p < 0.01, respectively). DISCUSSION AND CONCLUSION: This study demonstrated that CK downregulated memory B cells in AA rats, and this down-regulation may be T-cell dependent.

Ginsenoside metabolite compound K suppresses T-cell priming via modulation of dendritic cell trafficking and costimulatory signals, resulting in alleviation of collagen-induced arthritis.

Ginsenoside metabolite compound K (CK; 20-O-d-glucopyranosyl-20(S)-protopanaxadiol), a novel ginsenoside metabolite, belongs to the dammarane-type triterpene saponins, according to its structure. The anti-inflammatory activity of CK has been identified in several studies. Our study demonstrated that CK exerted an anti-inflammatory effect in collagen-induced arthritis (CIA) and adjuvant-induced arthritis animal models, and this effect was due to inhibition of the abnormal activation and differentiation of T cells. However, the mechanism of CK in suppressing T-cell activation remains unclear. In this study, CK had a therapeutic effect in mice with CIA, decreased the percentage of activated T cells and dendritic cells (DCs), and increased the percentage of naive T cells in lymph nodes. The inhibitory effect on T-cell activation of CK was related to suppression of accumulation of DCs in lymph nodes. CK decreased CCL21 levels in lymph nodes and CCR7 expression in DCs and suppressed CCL21/CCR7-mediated migration of DCs, thus reducing accumulation of DCs in lymph nodes. In addition, signals for T-cell activation including major histocompatibility complex class II and costimulatory molecules, such as CD80 and CD86, were suppressed by CK, and the proliferation of T cells induced by DCs was inhibited by CK. In conclusion, this study demonstrated that CK downregulated DC priming of T-cell activation in CIA, and suppression of CCL21/CCR7-mediated DC migration and signaling between T cells and DCs might be the potential mechanism. These results provide an interesting, novel insight into the potential mechanism by which CK contributes to the anti-inflammatory effect in autoimmune conditions.

Paeoniflorin regulates the function of human peripheral blood mononuclear cells stimulated by rhIL-1ß by up-regulating Treg expression.

The aim of the present study was to investigate the effect of paeoniflorin (Pae) on recombinant human interleukin-1ß (rhIL-1ß)-stimulated human peripheral blood mononuclear cells (PBMCs) in vitro. PBMCs were collected by Ficoll density gradient centrifugation and were co-cultured with rhIL-1ß for different time periods. The proliferation response was determined by a cell counting kit-8 (CCK-8) assay. The production of IL-17 and IL-10 was measured by enzyme-linked immunosorbent assay (ELISA). The percentage of CD4(+)CD25(+)Foxp3(+) regulatory T cells (Treg) was detected by flow cytometry analysis. These results indicated that rhIL-1ß stimulation induced the proliferation of PBMCs in a concentration- and time-dependent manner; it also increased the level of IL-17 and decreased the level of IL-10 in a concentration-dependent manner. The flow cytometry analysis demonstrated that the stimulation of rhIL-1ß significantly downregulated the percentage of CD4(+)CD25(+)Foxp3(+) Treg in CD4(+) T cells. However, administration of Pae significantly suppressed the proliferation response of rhIL-1ß-induced PBMCs and regulated the secretion function of IL-17 and IL-10. Additional experiments demonstrated that Pae treatment significantly reduced rhIL-1ß-induced decreases in PBMCs CD4(+)CD25(+)Foxp3(+) subpopulation numbers. These results suggest that the anti-inflammatory action of Pae is attributable to its regulation of IL-17/IL-10 secretion and Treg expression.