Experimental study on anti-endotoxin activity of a tetrahydropyrimidine derivative,ZL-5015

AIM: To investigate the protective effect of 1, 3-dicyclopentyl-1, 2, 3, 6-tetrahydropyrimidine-4, 5-dicarboxylic acid diethyl ester (ZL-5015) on lethal endotoxin-challenged mice and to explore the underlying mechanism. METHODS: Mouse model of lethal endotoxin challenge and endotoxemia were established by intraperitoneal administration of lipopolysaccharide (LPS) at a dose of 70 mg/kg to the C57BL/6J mice. Mouse peritoneal macrophages stimulated with LPS (10 mg/L) were used as an in vitro inflammatory model. The levels of interleukin-1β (IL-1β), interleukin-10 (IL-10) and tumor necrosis factor-α (TNF-α) were measured by enzyme-linked immunosorbent assay (ELISA). Real-time PCR was used to evaluate the mRNA expression of the cytokines. RESULTS: Prophylactic treatment of the mice with ZL-5015 (100 and 200 mg/kg, ig) slightly increased the survival rate, extended the survival time, decreased the serum levels of IL-1β and TNF-α, and increased the serum level of IL-10 in the early stage of endotoxemia as compared with model group. The results of in vitro study demonstrated that treatment of the endotoxin-stimulated mouse peritoneal macrophages with ZL-5015 (10, 20 and 40 μmol/L) inhibited the expression of IL-1β and TNF-α at both mRNA and protein levels but promoted the expression of IL-10 at both mRNA and protein levels. CONCLUSION: The tetrahydropyrimidine derivative ZL-5015 shows a moderate anti-endotoxin effect by increasing the survival rate and extending the survival time of the mice challenged by endotoxin, which may result from inhibition of the expression of pro-inflammatory cytokines such as IL-1β and TNF-α, and promotion of the expression of anti-inflammatory cytokine IL-10.     

Effect of 4.25% peritoneal dialysis solution on CD40 expression in rat peritoneal mesothelial cells

AIM:To investigate the effect of 4.25%peritoneal dialysis solution (PDS) on CD40 expression in rat peritoneal mesothelial cells so as to reveal the potential mechanisms by which CD40-CD40 ligand (CD40L) interaction may be involved in the inflammation of peritoneal membrane. METHODS:Rat peritoneal mesothelial cells (MC) were harvested from the peritoneal cavity and maintained under defined in vitro conditions. Expression of CD40 on MC under normal culture or stimulation with 4.25%PDS or 4.25%PDS+IFN-γ was detected by RT-PCR and FACS analyses. After activation of CD40 on MC with CD40 mAb, the expression of intercellular adhesion molecule-1 (ICAM-1) on MC was analyzed by FCAS. RESULTS:MC cultured in vitro expressed CD40 constitutively. 4.25%PDS markedly up-regulated the expression of CD40 mRNA and its protein. The expression of CD40 mRNA and its protein following stimulation with 4.25%PDS+IFN-γ was significantly higher than 4.25%PDS alone. The expression of ICAM-1 on MC was significantly increased after activation of CD40 with CD40mAb.CONCLUSIONS: MC functionally express CD40.The up-regulated CD40 expression on MC fol owing stimulation with 4.25%PDS may play an important role in local peritoneal defense mechanisms and may be involved in the chronic inflammatory process of the peritoneum.     

IgE-FcεRI crosslink accelerates atherosclerosis development in allergic asthmatic mice

AIM:To investigate whether allergic asthma accelerates the development of atherosclerosis in mice related to Th2 cells and interleukin-4 (IL-4), and the roles of activation of macrophages by immunoglobulin E (IgE)-Fc ε receptor I (FcεRI) crosslink during the process. METHODS:Six-week-old ApoE^-/- mice were sensitized and challenged by ovalbumin to establish the allergic asthma model, and then assigned to 3 groups:control group, asthmatic placebo group and asthmatic IL-4 monoclonal antibody (mAb) intervention group (intervention for 8 weeks). The lesion area was measured by oil red O staining. The percentages of Th2 cells in the splenocytes of the mice were analyzed by flow cytometry. The mRNA expression of IL-4 and the macrophage-related inflammatory factors, monocyte chemotactic protein 1 (MCP-1), macrophage inflammatory protein-1α (MIP-1α) and IL-6, in the spleen was detected by real-time PCR. Local IgE and FcεRIα expression in the plaque was evaluated by immunofluorescence/immunohistochemical staining, and the circulating IL-4 and IgE were measured by ELISA. RESULTS:Accompanied by aggravated atherogenesis in asthmatic ApoE^-/- mice, the proportion of Th2 cells and IL-4 mRNA in the spleen, IgE and FcεRIα expression in the aortic root, and the mRNA expression of MCP-1, MIP-1α and IL-6 were markedly increased. After 8-week treatment with IL-4 mAb, the lesion area in the aortic root of asthmatic ApoE^-/- mice was markedly decreased, the elevated IgE and FcεRIα expression was significantly decreased, and the mRNA expression of macrophage-related inflammatory factors was also decreased. CONCLUSION:Allergic asthma accelerates the atherosclerosis in ApoE^-/- mice, which is associated with the increased Th2 cells and IL-4, and the activation of macrophages by IgE-FcεRI crosslink.     

Effect of aldosterone on rat peritoneal fibrosis induced by peritoneal dia-lysis

AIM: To investigate the pathologic role of aldosterone and protective effect of aldosterone receptor antagonist on peritoneal fibrosis in peritoneal dialysis rats. METHODS: A peritoneal fibrosis rat model was established by intraperitoneal injection of lipopolysaccharide(at 1 d, 3 d, 5 d and 7 d, 0.6 mg/kg) and dialysate(daily intraperitoneal injection of 4.25% dialysate, 100 mL/kg). At the same time, spironolactone(an aldosterone receptor antagonist, 100 mg·kg^-1·d^-1) was given to the model rats. After 4 weeks, the expression of aldosterone synthase CYP11B2, 11β-hydroxysteroid dehydrogenase type 2(11β-HSD2), mineralocorticoid receptor(MCR), and inflammatory factors were detected by immunohistochemistry, real-time PCR and Western blotting. RESULTS: The rat model of peritoneal fibrosis was successfully established. At the same time, the injury of mesothelial cells, deposition of collagen fibers and thickness of peritoneal were increased. Moreover, the infiltration of macrophages in the peritoneum/dialysate was increased. The level of aldosterone and the expression of MCR, 11β-HSD2 and CYP11B2 in fibrotic peritoneum were obviously up-regulated as compared with normal rats. The expression of NF-κB/MCP-1 was also increased. However, treatment with spironolactone alleviated peritoneal fibrosis and reduced the expression of NF-κB/MCP-1. CONCLUSION: Local aldosterone is involved in the process of peritoneal fibrosis via NF-κB/MCP-1 pathway. Spironolactone alleviates peritoneal fibrosis of peritoneal dialysis.     

Effects of zanthoxylum seed oilA2 on NF-кB signaling pathway and inflammatory factor in lung tissue of asthmatic mice

AIM: To investigate the dynamic influence of zanthoxylum seed oilA2 (ZSOA2) on NF-кB signaling pathway and inflammatory factor in pulmonary tissue of asthmatic mice. METHODS: The suspensoid (0.2 mL containing 20% albumin hydroxide and 10% ovalbumin) was administered by intraperitoneal injection to sensitize the BALB/c mice on day 1, then 0.4% ovalbumin solution (50 μL in phosphate buffer fluid) was dripped into the respiratory tract through nasal cavity to establish the asthmatic mouse model. After dripped ovalbumin for 24 h, 48 h, 3 d, 7 d and 14 d, the mice were killed at specified time points. The contents of interleukin-4 (IL-4), interleukin-5 (IL-5) and interferon-γ (IFN-γ) in bronchoalveolar lavage fluid (BALF) were determined by ELISA. The pathological changes of the lung tissues were observed with HE staining. The inflammatory cell counts were conducted by Eosin staining. The protein levels of adhesion molecule and the molecules of NF-κB signaling pathway in lung tissues were determined by Western blotting. RESULTS: In ZSOA2 treated mice, the pathological injury of the lung was significantly attenuated as compared to the model mice, the counts of eosinophils and lymphocytes were reduced obviously in lung bronchial area of asthmatic mice at all observed time points (P<0.05). The levels of IL-5 and IL-4 decreased and IFN-γ increased in BALF. The results of Western blotting showed that ZSOA2 down-regulated the protein levels of intercellular adhesion molecule-1, vascular cell adhesion molecule-1, I kappa B kinase alpha-α and phosphorylation inhibitory-κB. ZSOA2 also up-regulated the protein levels of tumor necrosis factor receptor 1 and phosphorylation nuclear factor-kappaB in lung tissue at all observed time points. CONCLUSION: ZSOA2 has therapeutic effect on asthma by down-regulating the protein expression of IκB-β and p-IκB, inhibiting the releases of cytokines and chemotactic factors, and attenuating the infiltration of inflammatory cells in the lungs of ovalbumin challenged asthma mice.     

Rosuvastatin inhibits up-regulation of inflammatory factors in endothelial outgrowth cells induced by C-reactive protein

AIM：To study the effects of rosuvastatin on C-reactive protein (CRP)-induced expression of inflammatory factors in endothelial outgrowth cells (EOCs). METHODS：Mononuclear cells (MNCs) were isolated from human umbilical cord blood by density gradient centrifugation. EOCs were treated with CRP at concentration of 5, 10, 25 and 50 mg/L and were exposed to 50 mg/L CRP for different time (0, 3, 6 and 12 h). In addition, EOCs were pre-incubated with rosuvastatin at different concentrations (10-8, 10-7 and 10-6mol/L) for 12 h and then stimulated with 50 mg/L CRP. The mRNA levels of IL-8, MCP-1, VCAM-1 and ICAM-1 in EOCs were measured by quantitative PCR. The protein levels of MCP-1 and VCAM-1 were detected by ELISA. RESULTS：CRP dose-dependently increased the mRNA levels of inflammatory factors (IL-8, MCP-1, VCAM-1 and ICAM-1) and protein levels of MCP-1 and VCAM-1. The mRNA levels of IL-8, VCAM-1, and ICAM-1 in EOCs reached to the peak at 6 h, while MCP-1 peaked at 3 h when treated with 50 mg/L CRP. The protein levels of MCP-1 and VCAM-1 increased in a time-dependent manner. The mRNA and protein levels of the inflammatory factors were significantly decreased by treatment with rosuvastatin at different concentrations. CONCLUSION：CRP is not just an inflammatory marker as CRP induces inflammation in EOCs. Rosuvastatin attenuates CRP-induced inflammatory response in EOCs, indicating a new target for the prevention of atherosclerosis.     

Role of FAT/CD36 in high-fat diet-induced adipose tissue inflammation

AIM: To investigate the role of fatty acid translocase/CD36 (FAT/CD36) in adipose tissue inflammation induced by a high-fat diet. METHODS: C57BL/6J mice were fed with a normal-chow diet (NCD) or a high-fat diet (HFD) for 14 weeks. The content of free fatty acid (FFA) in the serum was measured by ELISA. The expression of CD36, cytokines and chemokines at mRNA and protein levels in the adipose tissues was determined by real-time polymerase chain reaction and Western blotting. Immunohistochemical staining was used to examine the macrophages infiltration in the adipose tissues. The inflammatory responses in CD36 knockout mice and wild type mice with high-fat diet were analyzed. RESULTS: The levels of FAT/CD36 were higher in HFD group than that in NCD group. HFD feeding enhanced the mRNA and protein expression of IL-1β, IL-6, TNF-α, MCP-1 and MIP-1, as well as promoted macrophage infiltration in the adipose tissues. Interestingly, as fed with HFD, the expression of cytokines/chemokines and macrophage infiltration were significantly reduced in adipose tissues of the CD36 knockout mice, compared with the wild type mice. CONCLUSION: High-fat diet promotes adipose tissue inflammation in the mice in a FAT/CD36-dependent manner.     

Effects of glucocorticoid on miRNA-155 expression in CD4+T cells of asthmatic mice

AIM:To investigate the effects of glucocorticoid on the regulation of microRNA-155 (miRNA-155) expression in the CD4⁺ T cells of asthmatic mice. METHODS:The ovalbumin (OVA)-induced asthma mouse model was established and the mice were treated with glucocorticoid. The effects of glucocorticoid on the pulmpnary histopathological changes, the expression of miRNA-155 in the lung tissues and CD4⁺T cells, and the levels of cytokines in the bronchoal-veolar lavage fluid (BALF) were evaluated. RESULTS:The results of RT-qPCR showed that the expressions of miRNA-155 in the lung tissues and CD4⁺T cells from the spleen of asthmatic mice were significantly increased, and the level of miRNA-155 in the CD4⁺T cells was significantly increased with the increase in the allergen exposure time (P<0.01). HE and PAS staining showed that OVA significantly increased inflammatory cell infiltration as compared with control group, and the peribronchial and perivascular inflammation and mucus secretion of proliferative goblet cells were significantly reduced after glucocorticoid treatment. Glucocorticoid treatment inhibited the increase in the proportion of CD4⁺ CD8^- cells in the spleen and decreased the accumulation of CD4⁺ T cells in the lung tissues of asthmatic mice (P<0.01). After glucocorticoid treatment, the levels of interleukin-4 (IL-4), IL-5 and IL-13 in BALF were decreased, while the level of interferon-γ was increased significantly (P<0.01). CONCLUSION:Glucocorticoid reduces the accumulation of CD4⁺ T cells and inhibits the expression of miRNA-155 in the lung tissues and spleen CD4⁺ T cells of asthmatic mice.     

Influence of Jiaomu oil A2 on eosinophil manifold and CD34+ with marrow granule system mobilization in bronchia asthma mice

AIM: To observe the influence of Jiaomu oil A2 on eosinophil manifold and CD34+ with marrow granule system mobilization in bronchial asthma mice. METHODS: The asthmatic mouse model was established by sensitization and challenge of the animals with 20% Al(OH)3+10% ovalbumen (OVA). After the mice were excitated for 10 d and giving medical therapy at the same time, the mice were executed, the bronchial-alveolar lavage inflammatory cells and the hemocytopoiesis cells were examined using Wrish-Giemsa staining. The expressions of interleukin-5 (IL-5) and eotaxin (EON) in lung tissue and marrow were examined by in situ hybridization. The expression of IL-5 and EON albumen in lung tissue and marrow were detected by immunohistochemistry. The inflammatory cell infiltration and CD34+ cells in lung tissue were also observed by HE and immunofluorescence staining. RESULTS: Both Jiaomu oil A2 and prednisone significantly attenuated the pathological process degree of bronchial inflammatory reaction such as tissue swelling, reconstruction, hyperplasia, and epithelial cell shedding, and inhibited eosinophil count and infiltration caused by stimulation of allergic effect. Moreover, the two drugs markedly lessen the eosinophil density in tracheal surrounding tissue and marrow in asthma mice and depressed the differentiation of marrow myelocyte to eosinophil. Finally, the apparent decrement of CD34+IL-5, CD34+IL-5R, CD34+CCR-3 cells in bronchial tissue and marrow showed some relationship with the downregulation of IL-5, IL-4, GM-CSF in lung tissue. CONCLUSION: The effect of Jiaomu oil on airway inflammation in asthma mice is associated with inhibiting the mobilization of eosinophil and marrow granule system.     

Adipophilin induces expression of inflammatory factors through ERK1/2-AP-1 signaling pathway

AIM: To observe the effects of adipose differentiation-related protein (adipophilin) on the expression of inflammatory factors in RAW264.7 macrophage and to clarify the related mechanism. METHODS: The cell models with high expression and low expression of adipophilin were constructed by transfecting PA317 packaging cells with stable high or low expression adipophilin retroviral vectors into the RAW264.7 cells. The concentrations of IL-6, MCP-1 and TNF-α in the cell culture medium were detected by ELISA. The protein levels of AP-1, p-AP-1, ERK1/2 and p-ERK1/2 were measured by Western blot. The protein levels of adipophilin, p-ERK1/2 and p-AP-1 and the releases of the inflammatory factors in the RAW264.7 cells treated with or without ERK1/2 inhibitor PD98059 or AP-1 inhibitor curcumin were determined. RESULTS: The RAW264.7 cells with high expression of adipophilin had higher levels of IL-6, MCP-1 and TNF-α, and higher protein levels of p-AP-1 and p-ERK1/2 than those in the cells with low expression of adipophilin. ERK1/2 inhibitor had no significant effect on the expression of adipophilin, but the protein expression of ERK1/2 and AP-1 was significantly inhibited (P<0.05). The administration of AP-1 inhibitor curcumin had no significant effect on the protein expression of adipophilin and ERK1/2, but the protein expression of AP-1 was significantly inhibited (P<0.05). At the same time, the releases of inflammatory factors IL-6, MCP-1 and TNF-α were significantly decreased. CONCLUSION: Adipophilin may regulate the expression of inflammatory factors through ERK1/2-AP-1 pathway in RAW264.7 macrophages.     

Oxidized α1-antitrypsin induces IL-8 and MCP-1 production in HBE cells

AIM:To investigate the effect of oxidized α1-antitrypsin (Ox-AT) on interleukin 8 (IL-8) and monocyte chemotactic protein 1(MCP-1) production in cultured human bronchial epithelial (HBE) cells. METHODS:Plasma native α1-antitrypsin (N-AT) was purified from human plasma by 50% and 75% ammonium sulfate fractionation followed by glutathione and anion exchange chromatography. Ox-AT was prepared by incubating N-AT (0.5 g/L) with N-chlorosuccinimide in a 25-fold molar excess to N-AT in PBS at room temperature for 30 min. HBE cells were cultured in the presence of Ox-AT (0.5 g/L) for 4 h, 10 h and 24 h, and the levels of IL-8 and MCP-1 in the supernatant were assayed using respective DuoSet kits. The effect of NF-κB inhibitor Bay11-7082 on the inflammatory cytokine release induced by Ox-AT was also evaluated. RESULTS:Ox-AT concentration-dependently and time-dependently increased the production of IL-8 and MCP-1 in HBE cells. The concentrations of IL-8 and MCP-1 in HBE cells induced by 0.5 g/L Ox-AT at 4 h, 10 h and 24 h were significantly higher than those in blank control and N-AT groups. Ox-AT increased the activity of NF-κB in a dose-dependent manner. The proinflammatory effect of by Ox-AT was inhibited by NF-κB inhibitor Bay11-7082. CONCLUSION: Ox-AT is a strong proinflammatory factor for HBE cells. The mechanism is related to NF-κB signaling pathway activation.     

Effects of caveolin-1 scaffolding domain peptide on LPS-induced acute lung injury in mice

AIM: To investigate the effects of caveolin-1 (Cav-1) scaffolding domain peptide, cavtratin, on lipopolysaccharide (LPS)-induced mouse acute lung injury and heme oxygenase-1 (HO-1) activity. METHODS: Adult male BALB/c mice were randomly divided into 6 groups (n=8 to 10):control, Antennapedia internalization sequence (AP), LPS, LPS+hemin, LPS+ hemin+cavtratin and LPS+hemin+cavtratin+zinc protoporphyrin IX (ZnPP) groups. After LPS administration for 24 h, the lung pathological changes, the wet/dry weight (W/D) ratio of lung tissues, total cell number in bronchoalveolar lavage fluid and serum lactate dehydrogenase activity were measured. The co-localization of HO-1 and Cav-1 was displayed by immunofluorescence, and the HO-1 activity were detected. The mRNA expression of pro-inflammatory cytokines IL-1β, IL-6, TNF-α, MCP-1 and iNOS was detected by real-time PCR. RESULTS: The mice in LPS+hemin+cavtratin group had the decreased interaction between HO-1 and Cav-1, and the increased HO-1 activity compare with LPS group (P<0.05). Compared with LPS group, the pulmonary damage was attenuated in LPS+hemin+cavtratin group, and the injury indexes, including W/D ratio, total cell number in bronchoalveolar lavage fluid and lactate dehydrogenase activity in the serum, and the mRNA expression of inflammatory cytokines all decreased (P<0.05). HO-1 activity inhibitor ZnPP abolished the above protective effect of cavtratin on the lung tissues with LPS-induced acute lung injury. CONCLUSION: Cavtratin has beneficial effects on the lung with LPS-induced acute injury by restoring the HO-1 activity.     

Effects of Mcl-1 silencing on apoptosis of mouse peritoneal macrophages infected with different virulence of Mycobacterium tuberculosis

AIM: To investigate the effect of inhibiting Mcl-1 gene expression on apoptosis of mouse peritoneal macrophages infected with different virulence of Mycobacterium tuberculosis using a technique of RNA interference. METHODS: The BALB/c mice were infected with prepared bacterium of the virulence strains of Xinjiang, H37Rv, H37Ra and BCG. Mcl-1-shRNA was applied to the mouse model of infection, and the control groups were set up. On 1 d, 3 d, 5 d and 7 d, the mouse peritoneal macrophages were collected. The expression of Mcl-1 at mRNA and protein levels was determined by real-time PCR and Western blot. The apoptotic rate of peritoneal macrophages was analyzed by flow cytometry. RESULTS: The expression of Mcl-1 at mRNA and protein levels was up-regulated in the peritoneal macrophages from the mice infected with different virulence of Mycobacterium tuberculosis, and the cells from the mice infected with virulence strains of Xinjiang and H37Rv expressed higher level of Mcl-1 than the uninfected control cells (P<0.05). The expression of Mcl-1 at mRNA and protein levels was reduced by RNA interference as compared with control group (P<0.05). Inhibition of Mcl-1 expression induced apoptosis of peritoneal macrophages in the mice. CONCLUSION: The Mcl-1 expression at mRNA and protein levels in mouse peritoneal macrophages infected with different virulence of Mycobacterium tuberculosis was effectively suppressed by Mcl-1-shRNA, which can induce macrophage apoptosis.     

Effects of chronic Clonorchis sinensis infestation on inflammatory reaction of macrophages

AIM：To evaluate the immune state in rats with chronic Clonorchis sinesis (Cs) infestation by investigating the effects of Cs on macrophage polarization and inflammatory reactions. METHODS：Sprague-Dawley rats were used in the study. Chronic Cs infestation model was reproduced by intragastric perfusion with Cs eggs. Twenty rats were randomly divided into normal group (n=10) and Cs infestation group (n=10). The serum levels of interleukin (IL-4) and IL-10, tumor necrosis factor α(TNF-α) and interferon γ (IFN-γ) were detected by ELISA. The macrophages were harvested by peritoneal lavage. The differentiation proportion of M1 and M2 macrophages were detected by flow cytometry. The macrophages were divided into control group, normal group and chronic Cs infestation group according to the sources of macrophages. The levels of TNF-α and IL-10 in the culture supernatants were detected by ELISA at 0, 2, 12 and 24 h after lipopolysaccharide (LPS, 10 μg/L) stimulation in vitro. RESULTS：Compared with normal group, chronic Cs infestation increased the serum levels of TNF-α, IFN-γ, IL-4 and IL-10. The differentiation proportion of M1 detected by flow cytometry was 92.1% in normal group and that of M2 macrophages was 93.8% in Cs infestation group. The levels TNF-α and IL-10 in culture supernatants were increased at 2~24 h after LPS stimulation both in normal group and Cs infestation group, but the levels of TNF-α were lower in chronic Cs infestation group than that in normal group at 2 h,12 h and 24 h after LPS stimulation. The level of anti-inflammatory cytokine IL-10 was higher in Cs infestation group than that in normal group at 2 h, 12 h and 24 h after LPS stimulation. CONCLUSION：Chronic Cs infestation increases the serum levels of both pro-inflammatory cytokines and anti-inflammatory cytokines, thus inducing the polarization of M2 macrophages. The macrophages derived from chronic Cs-infected rats produce tolerance in the inflammatory process against LPS in vitro.     

Effect of cell cycle regulator p21WAF1 on hypertrophy of peritoneal mesothelial cells

AIM: To investigate the effect of cell cycle regulator p231^WAF1 on hypertrophy of peritoneal mesothelial cells affected by high concentrated glucose. METHODS: RT-PCR and Western Blot method were used to detect p21^WAF1 expression of rat peritoneal mesothelial cells in high glucose concentration medium (containing 1.86%, 3.86% glucose) after 24 hours. Flow cytometer technique was used to analyze the cell cycle distribution. RESULTS: In high glucose medium, most of the cells became hypertrophy, and were arrested in G1 phase of the cell cycle, which was obvious in 3.86% glucose group. Glucose increased p21 mRNA and protein expression in a dose-dependent manner, and the levels of p21^WAF1 mRNA and protein in 3.86% glucose group were higher than those in 1.38% glucose group (P＜0.05). p21 ^WAF1 mRNA and protein expression were absent in the serum-free normal medium and D-mannitol groups which had the same osmolarity as the glucose groups. CONCLUSION: p21^WAF1 may be pivotal in the hypertrophy and arrest in the G1 phase of mesothelial cells induced by high concentrated glucose.     

Role of IL-23/IL-17 inflammatory axis in the pathogenesis of psoriasis-like lesions induced by imiquimod in mice

AIM: To observe the dynamic changes of IL-23/IL-17 inflammatory axis in psoriasis-like lesions of mice induced by imiquimod (IMQ).METHODS: BALB/c female mice were randomly divided into control group and IMQ group. The morphological changes of lesional skin in mice were evaluated according to the psoriasis area and severity index (PASI) and HE staining. cytokine antibody chips were used to determine the cytokine changes in serum and lesions. The mRNA and protein expression of cytokines were analyzed by cytometric bead array, real-time PCR and Western blotting. Moreover, the changes of cellular constituents in the peripheral blood and splenic cells of mice were detected by flow cytometry.RESULTS: Typical psoriasis-like skin lesions, such as red scaly skin plaques, caused by topical IMQ showed a parabolic dynamic change. There was a dynamic increase in proinflammatory cytokines of the IL-23/IL-17 axis in IMQ-treated skin. IMQ application resulted in elevated expression of cytokines related with IL-23/IL-17 inflammatory axis,Th1-type cytokines,Th2-type cytokines and Treg-type cytokines at day 4. IMQ-treated BALB/c mice showed an increased pericentage of dentric cells in peripheral blood and spleen compared with control animals. Percentages of Th17 and Treg in IMQ-treated mice were increased by 3~4 times and twice as compared with control mice, respectively.CONCLUSION: The skin lesions, histopathological features and cytokine changes in mice induced by IMQ are similar to human psoriasis, which are suitable for investigating the pathogenesis of psoriasis as a psoriasis-like model. IL-23/IL-17 axis is involved in the formation of psoriasis-like skin lesions in mice induced by IMQ and presents a dynamic change. Besides, Th1 cell-mediated inflammatory response is also activated in the formation of lesional skin, accompanied by the increase expression of Th2 and Treg cytokines in a feedback mechanism.     

Role of 17β-estradiol in regulation of MIF expression in endometrial stromal cells of endometriosis

AIM: To investigate the effects of 17β-estradiol on the expression of macrophage migration inhibitory factor (MIF) in cultured endometrial stromal cells from endometriosis. METHODS: Immunohistochemistry was used to identify the endometrial stromal cells. The expression of MIF at mRNA and protein levels was assessed by RT-PCR and Western blotting. RESULTS: Elevated expression of MIF at mRNA and protein levels was observed in the cultured endometrial stromal cells treated with 17β-estradiol. In endometrial stromal cells from the women with endometriosis, the level of MIF up-regulation by 17β-estradiol was significantly higher than that in the cells from the women without endometriosis. CONCLUSION: Endometrial stromal cells from endometriosis are more sensitive to 17β-estradiol, which up-regulates the expression of MIF and contribute to the pathogenesis and progression of endometriosis.     

Effects of miR-155 over-expression on expression of inflammatory factors and indolamine 2, 3-dioxygenase in microglial BV-2 cells

AIM:To explore the effect of microRNA-155(miR-155)over-expression on the expression of inflammatory factors and indolamine 2, 3- dioxygenase (IDO) in the microglial BV-2 cells. METHODS:For over-expression of miR-155, the BV-2 cells were transfected with lentiviral vector carrying mmu-miR-155. The expression of inflammatory factors was detected by cytometric bead array system (CBA). The mRNA expression of inflammatory factors and IDO was analyzed by real-time PCR. The protein levels of suppressor of cytokine signalling 1 (SOCS1), p-p38 MAPK and IDO were determined by Western blot. RESULTS:The expression of miR-155 was up-regulated in the BV-2 cells transfected with lentiviral vector carrying mmu-miR-155 compared with LPS treatment group (P<0.01). The miR-155 over-expression promoted the secretion of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), monocyte chemotactic protein-1 (MCP-1) and IL-10, and inhibited the secretion of IL-12. The miR-155 over-expression increased the mRNA expression of IL-6, TNF-α, IL-10 and IDO, also increased the protein levels of IDO and p-p38 MAPK, but decreased the protein expression of SOCS1 (P<0.01). LPS promoted the secretion of IL-6, TNF-α, MCP-1 and IL-12, also increased the mRNA expression of IL-6, TNF-α and IDO, meanwhile, increased the protein levels of IDO, p-p38 MAPK and SOCS1 (P<0.01). CONCLUSION:Over-expression of miR-155 promotes the secretion of related imflammatory factors and protein expression of IDO in microglial BV-2 cells mediated with SOCS1 and p38 MAPK signaling pathway.