BACKGROUND Dopamine and cyclic adenosine monophosphate(cAMP)-regulated phosphop-rotein with an apparent Mr of 32000(DARPP-32)is a protein that is involved in regulating dopamine and cAMP signaling pathways in the brai...BACKGROUND Dopamine and cyclic adenosine monophosphate(cAMP)-regulated phosphop-rotein with an apparent Mr of 32000(DARPP-32)is a protein that is involved in regulating dopamine and cAMP signaling pathways in the brain.However,recent studies have shown that DARPP-32 is also expressed in other tissues,including colorectal cancer(CRC),where its function is not well understood.AIM To explore the effect of DARPP-32 on CRC progression.METHODS The expression levels of DARPP-32 were assessed in CRC tissues using both quantitative polymerase chain reaction and immunohistochemistry assays.The proliferative capacity of CRC cell lines was evaluated with Cell Counting Kit-8 and 5-ethynyl-2’-deoxyuridine assays,while apoptosis was measured by flow cytometry.The migratory and invasive potential of CRC cell lines were deter-mined using wound healing and transwell chamber assays.In vivo studies involved monitoring the growth rate of xenograft tumors.Finally,the underlying molecular mechanism of DARPP-32 was investigated through RNA-sequencing and western blot analyses.RESULTS DARPP-32 was frequently upregulated in CRC and associated with abnormal clinicopathological features in CRC.Overexpression of DARPP-32 was shown to promote cancer cell proliferation,migration,and invasion and reduce apoptosis.DARPP-32 knockdown resulted in the opposite functional effects.Mechanistically,DARPP-32 may regulate the phosphoinositide 3-kinase(PI3K)/AKT signaling pathway in order to carry out its biological function.CONCLUSION DARPP-32 promotes CRC progression via the PI3K/AKT signaling pathway.展开更多
AIM: To investigate the inflammatory amplification effect of high-mobility group box 1(HMGB1) in Aspergillus fumigatus(A. fumigatus) keratitis and the relationship between lectin-like oxidized low-density lipoprotein ...AIM: To investigate the inflammatory amplification effect of high-mobility group box 1(HMGB1) in Aspergillus fumigatus(A. fumigatus) keratitis and the relationship between lectin-like oxidized low-density lipoprotein receptor 1(LOX-1) and HMGB1 in keratitis immune responses.METHODS: Phosphate buffer saline(PBS), and Boxb were injected into BALB/c mice subconjunctivally before the corneas were infected with A. fumigatus. RAW264.7 macrophages and neutrophils were pretreated with PBS and Boxb to determine the HMGB1 inflammatory amplification effects. Abdominal cavity extracted macrophages were pretreated with Boxb and Poly(I)(a LOX-1 inhibitor) before A. fumigatus hyphae stimulation to prove the the relationship between the two molecules. LOX-1, interleukin-1β(IL-1β), tumor necrosis factor-α(TNF-α), macrophage inflammatory protein-2(MIP-2) and IL-10 were assessed by polymerase chain reaction and Western blot.RESULTS: Pretreatment with Boxb exacerbated corneal inflammation. In macrophages and neutrophils, A. fumigatus induced LOX-1, IL-1β, TNF-α and MIP-2 expression in Boxb group was higher than those in PBS group. Poly(I) treatments before infection alleviated the proinflammatory effects of Boxb in abdominal cavity extracted macrophages. Pretreatment with Boxb did not influence Dectin-1 mRNA levels in macrophages and neutrophils.CONCLUSION: In fungal keratitis, HMGB1 is a proinflammatory factor in the first line of immune response. HMGB1 mainly stimulates neutrophils and macrophages to produce inflammatory cytokines and chemokines during the immune response. LOX-1 participates in HMGB1 induced inflammatory exacerbation in A. fumigatus keratitis.展开更多
AIM:To determine whether lectin-like ox-LDL receptor(LOX-1)regulates adhesion molecules expression and neutrophil infiltration in Aspergillus fumigatus(A.fumigatus)keratitis of C57 BL/6 mice.METHODS:C57 BL/6 mice were...AIM:To determine whether lectin-like ox-LDL receptor(LOX-1)regulates adhesion molecules expression and neutrophil infiltration in Aspergillus fumigatus(A.fumigatus)keratitis of C57 BL/6 mice.METHODS:C57 BL/6 mice were pretreated with a neutralizing antibody to LOX-1(5μg/5μL)or control nonspecific IgG(5μg/5μL),LOX-1 inhibitor Poly-I(2μg/5μL)or PBS by subconjunctival injection.Fungal keratitis(FK)mouse models of C57 BL/6 mice were established by scraping corneal central epithelium,smearing A.fumigatus on the corneal surface and covering the eye with contact lenses.The corneal response to infection was assessed via clinical score.The mRNA levels of the adhesion molecules intercellular cell adhesion molecule-1(ICAM-1),vascular cell adhesion molecule-1(VCAM-1),P-selectin and E-selectin were tested in control and infected corneas by reverse transcriptionpolymerase chain reaction(RT-PCR).The protein levels of ICAM-1 were evaluated by immunofluorescence(IF)and Western blot.Neutrophils were extracted from the abdominal cavity of C57 BL/6 mice followed by pretreatment using antibody to LOX-1(10μg/mL)or control nonspecific IgG(10μg/mL),the Poly-I(4μg/mL)or PBS.The cells were then stimulated with A.fumigatus and tested mRNA and protein levels of lymphocyte function-associated antigen-1(LFA-1)using RT-PCR and Western blot.IF and myeloperoxidase(MPO)assays were used to assess neutrophil infiltration in mice corneas.RESULTS:Pretreatment of LOX-1 antibody or the Poly-I reduced the degree of inflammation of cornea and decreased the clinical FK score compared with pretreatment of IgG or PBS(both P<0.01).And these pretreatment also displayed an obvious decline in the mRNA levels of ICAM-1,VCAM-1,P-selectin,E-selectin and LFA-1 expression compared with control groups(all P<0.01).Furthermore,pretreated with LOX-1 antibody or Poly-I,the protein levels of ICAM-1 and LFA-1 also decreased compared with control groups(all P<0.05).Neutrophil infiltration in the cornea was significantly reduced after pretreatment of LOX-1 antibody or Poly-I compared with control groups by IF and MPO assays(both P<0.01).CONCLUSION:Inhibition of LOX-1 can decrease the expression of adhesion molecules and reduce neutrophil infiltration in A.fumigatus infected corneas of C57 BL/6 mice.展开更多
AIM: To identify whether Aspergillus fumigatus(A.fumigatus) hyphae antigens induced the release of interleukin-8(IL-8) in anti-fungal innate immunity of cultured human corneal epithelial cells(HCECs) and determine the...AIM: To identify whether Aspergillus fumigatus(A.fumigatus) hyphae antigens induced the release of interleukin-8(IL-8) in anti-fungal innate immunity of cultured human corneal epithelial cells(HCECs) and determine the involvement of intracellular signalling pathways. METHODS: HCECs were treated with A. fumigatus hyphae antigens with different concentrations and time.The cytoplasmic calcium of HCECs were assessed by fluorescence imaging. Western blot was used to detect the expression of Ca2 +-dependent protein kinase C(PKC). The IL-8 levels were determined by specific human IL-8 enzyme-linked immunosorbent assay(ELISA) and reverse transcriptase polymerase chain reaction(RT-PCR). Using a series of pharmacological inhibitors, we examined the upstream signalling pathway responsible for IL-8 expression in response to A.fumigatus hyphae antigens. RESULTS: Cells exposed to A. fumigatus hyphae antigens showed higher level of IL-8 m RNA expression and protein production. We demonstrated here that stimulation of HCECs with A. fumigatus hyphae triggers an intracellular Ca2 +flux and results in the activation of Ca2 +-dependent PKC(α, βⅠ and βⅡ) which can be attenuated by pre-treatment of cells with laminarin,suggesting that Dectin-1 signals pathway induced cytoplasmic calcium and influence the activation of PKC in HCECs. Inhibitors of Ca2 +-dependent PKC(Ro-31-8220 and Go6976) significantly abolished hyphae-induced expression of IL-8.CONCLUSION: Our findings suggest that A. fumigatushyphae-induced IL-8 expression was regulated by the activation of Dectin-1-mediated Ca2 +-dependent PKC in HCECs.展开更多
AIM:To explore the effects of lectin-like ox-LDL receptor(LOX-1) on innate immunity against Aspergillus fumigatus(A.fumigatus) in mice cornea.METHODS:The mRNA levels of LOX-1 were tested in normal and A.fumigatu...AIM:To explore the effects of lectin-like ox-LDL receptor(LOX-1) on innate immunity against Aspergillus fumigatus(A.fumigatus) in mice cornea.METHODS:The mRNA levels of LOX-1 were tested in normal and A.fumigatus infected corneas of C57BL/6and BALB/c mice.The expression of LOX-1,pro-inflammatory cytokines TNF-α,CXCL1 and IL-6,antiinflammatory cytokines IL-10,and matrix metalloproteinase 9(MMP9) were tested with treatment with LOX-1 neutralizing antibody or control IgG in A.fum/gatus infected corneas of C57BL/6.Macrophages and neutrophils were extracted from susceptible C57BL/6mice,and pretreated with LOX-1 neutralizing antibody or IgG,then stimulated with A.fum/gatus.The mRNA levels of LOX-1,TNF-α,CXCL1,IL-6,IL-10 and MMP9 were evaluated by polymerase chain reaction.RESULTS:The expression of LOX-1 was significantly increased in C57BL/6 mice corneas after A.fum/gatus infection compared with BABL/c mice.After treatment with LOX-1 neutralizing antibody,the expression of LOX-1,TNF-α,CXCL1,IL-6,MMP9 and IL-10 in C57BL/6corneas were significantly decreased compared with treatment with control IgG;the expression of LOX-1,CXCL1,IL-6 and IL-10 were significantly decreased in macrophages,while TNF-α and MMP9 expressions had no change;LOX-1,TNF-α,CXCL1,IL-6,MMP9 and IL-10 expressions were significantly decreased in neutrophils.CONCLUSION:The expression of LOX-1 can affect the expression of pro-inflammatory and anti-inflammatory cytokines in fungal infected corneas,macrophages and neutrophils of C57BL/6.LOX-1 inhibition rebalances the inflammatory response of fungal keratitis in mice.展开更多
AIM: To investigate the expression of interleukin(IL)-33 in the cornea and human corneal epithelial cells(HCECs) exposed to Aspergillus fumigatus(A. fumigatus), and to determine the function of IL-33/ST2/p38 signaling...AIM: To investigate the expression of interleukin(IL)-33 in the cornea and human corneal epithelial cells(HCECs) exposed to Aspergillus fumigatus(A. fumigatus), and to determine the function of IL-33/ST2/p38 signaling pathway in the immune response of corneal epithelial cells to A. fumigatus infection.METHODS: The mRNA and protein expression of IL-33 in HCECs and mice corneas were examined by quantitative real-time reverse transcription polymerase chain reaction(q RT-PCR) and Western blot analysis, respectively. IL-33 expression was also detected in cornea samples from healthy donors and patients with fungal keratitis with immunohistochemistry. The cultured HCECs were treated with inactive A. fumigatus hyphae at various concentrations with or without recombinant human IL-33 protein, soluble recombinant ST2 protein, specific ST2 neutralizing antibody, or the mitogen-activated protein kinase(MAPK) p38 inhibitor SB203580 for evaluation of the expression and activation of IL-33/ST2/p38 signaling in the regulation of proinflammatory cytokines. The production levels of IL-6 and IL-1β were determined by qR T-PCR and enzymelinked immunosorbent assay(ELISA). The proliferation of HCECs was determined by a Cell Counting Kit-8(CCK8) assay and cell count.RESULTS: IL-33 expression levels increased in the corneal tissues of patients with fungal keratitis and in mice corneas of experimental A. fumigatus infection,as well as in HCECs with infection of A. fumigatus. A. fumigatus strongly stimulated HCECs-generated proinflammatory cytokine(IL-6 and IL-1β) production at both the mRNA and protein levels. This production of proinflammatory mediators stimulated by A. fumigatus was further stimulated by IL-33 and was prevented by soluble ST2 protein or ST2 neutralizing antibody. Moreover, IL-33 naturally promoted the p38 phosphorylation induced by A. fumigatus, which was suppressed by soluble ST2 protein. The MAPK p38 inhibitor SB203580 also inhibited the A. fumigatus-induced proinflammatory cytokine production. IL-33 administration for 48 h and 72 h promoted the proliferation of HCECs, which was attenuated by treatment with soluble recombinant human ST2 protein.CONCLUSION: A. fumigatus elevates IL-33 expression in human and mice corneas and HCECs. Thus, IL-33/ST2/p38 signaling may play an important role in amplifying the immune response of corneal epithelial cells to A. fumigatus infection. Besides, IL-33 promotes the cell proliferation of HCECs via its receptor ST2. These findings suggest a novel autocrine mechanism of amplification of the fungalinduced inflammatory response in the corneal epithelium, highlighting a potential therapeutic target for fungal keratitis.展开更多
AIM:To characterize effect of astaxanthin(ASX)in Aspergillus fumigatus(A.fumigatus)induced keratitis in mouse model.METHODS:In vivo,fungal keratitis mouse model was established in C57BL/6 mice using A.fumigatus,follow...AIM:To characterize effect of astaxanthin(ASX)in Aspergillus fumigatus(A.fumigatus)induced keratitis in mouse model.METHODS:In vivo,fungal keratitis mouse model was established in C57BL/6 mice using A.fumigatus,followed by ASX or dimethyl sulfoxide(DMSO)treatment.Clinical responses were evaluated by clinical score and myeloperoxidase(MPO)assay.Inflammatory cytokines were assessed by reverse-transcription polymerase chain reaction(RT-PCR),Western blot,immunofluorescence,and enzyme-linked immuno sorbent assay(ELISA).RESULTS:In animal model,ASX improved corneal transparency and clinical response,suppressed the expression of inflammatory cytokine like IL-1β,TNF-α,and HMGB-1.Neutrophil levels have been shown to decrease in ASX-treated cornea by immunofluorescence and MPO.TLR2 and TLR4 levels were lower in ASX-treated group than DMSO-treated.CONCLUSION:ASX can suppress inflammatory response and reduce inflammatory cytokine production in mice model with A.fumigatus keratitis.展开更多
●AIM:To observe the expression and role of aryl hydrocarbon receptor(Ah R)in the immune response of mouse cornea infected with Aspergillus fumigatus(A.fumigatus).●METHODS:Murine models of A.fumigatus keratitis were ...●AIM:To observe the expression and role of aryl hydrocarbon receptor(Ah R)in the immune response of mouse cornea infected with Aspergillus fumigatus(A.fumigatus).●METHODS:Murine models of A.fumigatus keratitis were established by scraping the central epithelium of mouse cornea,daubing A.fumigatus on the cornea and covering with a contact lens.The mice were randomly divided into the control group and the A.fumigatus-infected(A.F.)group for 1,3 and 5 d respectively,which corneas were daily monitored by a slit lamp microscope and the clinical scores were also recorded timely after infection.In this study,immunofluorescence staining was used to detect the expression and localization of Ah R in mouse corneas,and the m RNA and protein of Ah R were detected by reverse transcription-polymerase chain reaction(RT-PCR)and Western blot.In addition,mouse peritoneal macrophages were stimulated by A.fumigatus with or without the pretreatment of Ah R antagonist CH223191 and Ah R agonist FICZ,and the tumor necrosis factor alpha(TNF-α),inducible nitric oxide synthase(i NOS),interleukin-10(IL-10)and Arg-1 m RNA were detected by RT-PCR.●RESULTS:According to the results of the slit light photography,it was clearly indicated that the corneal inflammation were the most severe and the clinical score became the highest as well on the 3 rd day after the infection of A.fumigatus.Contrasted with the control group,the expression of Ah R in the corneal epithelial cells infected with A.fumigatus was significantly increased detected by immunofluorescence staining.Ah R mainly expressed in the nucleus and cytoplasm of corneal epithelial cells.Consistent with the transcriptional level of Ah R m RNA,the expression level of Ah R protein reached the peak on the 3 rd day after infection which was detected by Western blot.Furthermore,RT-PCR showed that CH223191 up-regulated the expression of TNF-αand i NOS and down-regulated the expression of IL-10 and Arg-1 in peritoneal macrophages;inversely,FICZ reduced the expression of TNF-αand i NOS while elevated the expression of IL-10 and Arg-1.●CONCLUSION:Ah R is involved in the pathogenesis of A.fumigatus keratitis and induced immune protection in anti-A.fumigatus immune response by inhibiting M1 and increasing M2 phenotype macrophage-related inflammatory factors.展开更多
Spiral groove dry gas seal(S?DGS), the most widely used DGS in the world, encounters the problem of high leakage rate and inferior film stability when used in high?speed machinery equipment, which could not be well so...Spiral groove dry gas seal(S?DGS), the most widely used DGS in the world, encounters the problem of high leakage rate and inferior film stability when used in high?speed machinery equipment, which could not be well solved by optimization of geometrical parameters and molded line of spiral groove. A new type of bionic cluster spiral groove DGS(CS?DGS) is proved to have superior film stability than S?DGS at the condition of high?speed and low?pressure numerically. A bionic CS?DGS is experimentally investigated and compared with common S?DGS in order to provide evidence for theoretical study. The film thickness and leakage rate of both bionic spiral groove and common spiral groove DGS are measured and compared with each other and with theoretical values under different closing force at the condition of static pressure, high?speed and low?pressure, and the film stiffness and stiffness?leakage ratio of these two face seals are derived by the relationship between closing force and film thickness at the steady state. Experimental results agree well with the theory that the leakage and stiffness of bionic CS?DGS are superior to that of common S?DGS under the condition of high?speed and low?pressure, with the decreasing amplitude of 20% to 40% and the growth amplitude of 20%, respectively. The opening performance and stiffness characteristics of bionic CS?DGS are inferior to that of common S?DGS when rotation speed equals to 0 r/min. The proposed research provides a new method to measure the axis film stiffness of DGS, and validates the superior performance of bionic CS?DGS at the condition of high?speed and low?pressure experimentally.展开更多
AIM: To characterize changes in the cornea nerve and pain responses in fungal keratitis(FK).METHODS: A retrospective analysis of in vivo confocal microscopy images of 11 FK corneas was performed, and the results were ...AIM: To characterize changes in the cornea nerve and pain responses in fungal keratitis(FK).METHODS: A retrospective analysis of in vivo confocal microscopy images of 11 FK corneas was performed, and the results were compared with those for 11 normal corneas. Subbasal corneal nerves were analyzed for total nerve number, main nerve trunk number, branching patterns and tortuosity. C57 BL/6 mice were infected with Aspergillus fumigatus. Disease severity was determined through clinical scoring and slit lamp photography. Corneas were harvested at 1, 3, 5, and 7 d post infection(p.i.) and assessed for β III tubulin. Corneal mechanical sensitivity thresholds were detected by von Frey test. β-endorphin(β-EP) and μ receptor protein expression was detected through Western blotting.RESULTS: Total nerve number, main nerve trunk number, and nerve branching were significantly lower in FK patients than in controls, but tortuosity was not significantly different. In infected mice, subbasal nerve density decreased from 1 d p.i., reaching a minimum at 5 d p.i. Clinical scores rose at 1 d p.i., peaked at 3 d p.i., and decreased at 5 d p.i. Mechanical sensitivity thresholds showed the same trends. β-EP and μ receptor protein expression increased after infection.CONCLUSION: Corneal nerve density is lower in FK patients and Aspergillus fumigatus-infected mice than in controls. Pain sensitivity decreases with postinfection corneal ulcer aggravation. β-EP and μ receptor proteins are both upregulated in infected mouse corneas.展开更多
AIM: To investigate the expression and role of calcitonin gene-related peptide(CGRP) in the mouse models induced by Aspergillus fumigatus(A. fumigatus). METHODS: C57 BL/6 mice were randomized into a control group and ...AIM: To investigate the expression and role of calcitonin gene-related peptide(CGRP) in the mouse models induced by Aspergillus fumigatus(A. fumigatus). METHODS: C57 BL/6 mice were randomized into a control group and A. fumigatus keratitis group. The cornea photography was assessed under the slit lamp and the clinical score was recorded after infection. Western blot, real-time polymerase chain reaction(PCR) and immunohistofluorescence analysis were applied to detect CGRP expression in cornea of both groups. In vitro, tests were conducted with C57 BL/6 mice macrophages to investigate CGRP expression after interaction with A. fumigatus. Cytokines expression induced by exogenous CGRP and the antagonist CGRP8-37 in A. fumigatus-exposed macrophages was evaluated by real-time PCR and ELISA.RESULTS: The cornea expression of CGRP was significantly elevated in C57 BL/6 mice corneas and macrophages after A. fumigatus infection. After treatment with exogenous CGRP, the levels of interleukin-1β(IL-1β), tumor necrosis factor-α(TNF-α) and IL-6 were reduced, and IL-10 level was increased in the A. fumigatus stimulatedmacrophages. However, IL-1β, TNF-α and IL-6 levels were upregulated after pretreatment of CGRP8-37. But the m RNA levels of MIP-2, TGF-β and IL-10 were not changed. CONCLUSION: This study provides evidence that A. fumigatus increased CGRP expression. CGRP may play a protective role against inflammation in A. fumigatus keratitis.展开更多
· AIM: To explore the immunomodulatory effects of curdlan on innate immune responses against Aspergillus fumigatus(A. fumigatus) in cultured human corneal epithelial cells(HCECs), and whether C-type lectin recept...· AIM: To explore the immunomodulatory effects of curdlan on innate immune responses against Aspergillus fumigatus(A. fumigatus) in cultured human corneal epithelial cells(HCECs), and whether C-type lectin receptor Dectin-1 mediates the immunomodulatory effects of curdlan.·METHODS: The HCECs were stimulated by curdlan in different concentrations(50, 100, 200, 400 μg/m L) for various time. Then HCECs pretreated with or without laminarin(Dectin-1 blocker, 0.3 mg/m L) and curdlan were stimulated by A. fumigatus hyphae. The m RNA and protein production of tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6) were determined by real-time quantitative polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. The protein level of Dectin-1 was measured by Western blot.· RESULTS: Curdlan stimulated m RNA expression of TNF-α and IL-6 in a dose and time dependent manner in HCECs. Curdlan pretreatment before A. fumigatus hyphae stimulation significantly enhanced the expression of TNF-α and IL-6 at m RNA and protein levels compared with A. fumigatus hyphae stimulation group(P 【0.05).Both curdlan and A. fumigatus hyphae up-regulated Dectin-1 protein expression in HCECs, and Dectin-1expression was elevated to 1.5- to 2-fold by curdlan pretreatment followed hyphae stimulation. The Dectin-1blocker laminarin suppressed the m RNA expression and protein production of TNF-α and IL-6 induced by curdlan and hyphae(P 【0.05).· CONCLUSION: These findings demonstrated that curdlan pretreatment enhanced the inflammatory response induced by A. fumigatus hyphae in HCECs.Dectin-1 is essential for the immunomodulatory effectsof curdlan. Curdlan may have high clinical application values in fungal keratitis treatment.展开更多
AIMTo observe the expression of vitamin D receptor (VDR) in human specimen and immortalized human corneal epithelium cells (HCEC) when challenged with fusarium solani. Moreover, we decided to discover the pathway of V...AIMTo observe the expression of vitamin D receptor (VDR) in human specimen and immortalized human corneal epithelium cells (HCEC) when challenged with fusarium solani. Moreover, we decided to discover the pathway of VDR expression. Also, we would like to detect the expression of cathelicidin antimicrobial peptide (CAMP) in the downstream pathway of VDR.METHODSImmunohistochemistry was used to examine the VDR expression in HCEC from healthy and fungal keratitis patients. Real time quantitative polymerase chain reaction (qPCR) was performed to observe the messenger ribonucleic acid (mRNA) change of VDR when immortalized HCEC were challenged with fusarium solani for different hours. CAMP was detected at both mRNA and protein levels.RESULTSWe found out that the VDR expression in fusarium solani keratitis patients' specimen was much more than that in healthy people. The mRNA and protein expression of VDR increased when we stimulated HCEC with fusarium solani antigen (P<0.01) and it could be inhibited by toll like receptor 2 (TLR2) monoclonal antibody. The CAMP expression was decreased because of fusarium solani antigen stimulation (P<0.01).CONCLUSIONThe VDR expression can be increased via TLR2/1-VDR pathway while the CAMP expression is decreased by the stimulation of fusarium solani antigen.展开更多
AIM: To observe the presence and expression of indoleamine 2,3-dioxygenase(IDO) during the corneal immunity to Aspergillus fumigatus(A. fumigatus) in the murine models.·METHODS: The murine model of fungal k...AIM: To observe the presence and expression of indoleamine 2,3-dioxygenase(IDO) during the corneal immunity to Aspergillus fumigatus(A. fumigatus) in the murine models.·METHODS: The murine model of fungal keratitis was established by smearing with colonies of A. fumigatus after scraping central epithelium of cornea and covering with contact lenses in C57BL/6 mice. The mice were randomly divided into control group, sham group and A.fumigatus keratitis group. The cornea was monitored daily using a slit lamp and recorded disease score after infection. Corneal lesion was detected by immunofluorescence staining. IDO m RNA and protein were also detected by quantitative reverse transcription-polymerase chain reaction(q RT-PCR) and Western blot.· RESULTS: The disease score and slit lamp photography indicated that disease severity was consistent with corneal inflammation in the murine models, and the disease scores in A. fumigatus keratitis group were obviously higher than those in the sham group. By immunofluorescence staining, IDO was mainly localized in corneal epithelium and stroma in the murine corneal tissues with A. fumigatus keratitis. Compared with the sham group, IDO m RNA expression was significantly enhanced in corneal epithelium infected by A. fumigatus. Furthermore, IDO protein expression detected by Western blot was in accord with transcript levels of IDO m RNA measured by q RT-PCR. IDO protein expression was enhanced after A. fumigatus infection compared with the sham group.·CONCLUSION: IDO is detected in corneal epithelium and stroma locally, which indicates IDO takes part in the pathogenesis of A. fumigatus keratitis and plays a key role in immune regulation at the early stage.展开更多
AIM: To investigate how macrophage inducible C-type lectin(Mincle) influences inflammation in mice fungal keratitis induced by Aspergillus fumigatus(A. fumigatus).METHODS: C57 BL/6 mice were infected with A. fum...AIM: To investigate how macrophage inducible C-type lectin(Mincle) influences inflammation in mice fungal keratitis induced by Aspergillus fumigatus(A. fumigatus).METHODS: C57 BL/6 mice were infected with A. fumigatus after pretreated with Mincle agonist TDB or Mincle neutralizing antibody(MincleAb), taking DMSO or IgG as control group respectively. The cornea lesions were monitored with slit-lamp microscope and evaluated by clinical score. Mincle expression was assessed using reverse transcriptionploymerase chain reaction(RT-PCR) and immunostaining. The expression of cytokines(IL-1β, TNF-α and IL-6) chemokines(CXCL-1 and MIP-2) was determined by RTPCR and ELISA. Neutrophil infiltration was observed by immunostaining. The levels of nitric oxide(NO) generated by corneas were tested by Griess reaction. RESULTS: Mincle mRNA and protein levels were higher in infected corneas than normal corneas of C57 BL/6 mice, saving clinical scores revealed differences. When pretreated with Mincle agonist TDB, the mRNA and protein levels of IL-1β, TNF-α and IL-6 in infected corneas were significantly increased compared with the control group(P〈0.01). Results of the counterpart in corneas pretreated with Mincle neutralizing antibody was decreased consistently(P〈0.01). Expression of CXCL1 and MIP-2 mRNA levels were upregulated in TDB group and down-regulated in Mincle Ab group(P〈0.01), coincide with neutrophil aggregation degree in corneas showed by immunostaining. As for the concentration of NO, it was promoted in TDB group compared with DMSO control group, and decreased in Mincle Ab group compared with Ig G control group.CONCLUSION: Mincle plays a dual role in mice fungal keratitis. It participates in the innate immune system by enhancing inflammation. What's more, Mincle can mediate cytotoxic effects by regulating the formation of NO.展开更多
AIM: To explore whether nicotinamide adenine dinucleotide phosphate(NADPH) oxidase 2(NOX2) is expressed in fungal keratitis in mice and investigate its role in this disease.METHODS: NOX2 expression was detected in C57...AIM: To explore whether nicotinamide adenine dinucleotide phosphate(NADPH) oxidase 2(NOX2) is expressed in fungal keratitis in mice and investigate its role in this disease.METHODS: NOX2 expression was detected in C57 BL/6 mice. After testing the inhibitory effect of diphenyleneiodonium chloride(DPI) on NOX2, its impact on clinical performance, myeloperoxidase levels, the number of colonies forming units, the level of H3, the generation of reactive oxygen species(ROS) and the release of cytokines [NF-κB, interleukin-17 A(IL-17 A), interleukin-6(IL-6), tumor necrosis factor-α(TNF-α), Nrf2, IL-10, and TGF-β] were compared. A one-way ANOVA and an unpaired, two-tailed Student’s t-test was used to determine the statistical significance.RESULTS: NOX2 expression was significantly increased after Aspergillus fumigatus injection in corneas and that this increase could be reduced by treatment with DPI. DPI treatment produced more severe inflammation and resulted in higher clinical scores, more neutrophils infiltration, a weakened ability to clear fungi, the release of fewer ROS and the formation of neutrophil extracellular traps. Treatment with DPI increased the expression of the proinflammatory cytokines NF-κB, IL-17 A, IL-6, and TNF-α and decreased the expression of the anti-inflammatory cytokines Nrf2, IL-10 and TGF-β compared to their expression levels without DPI treatment.CONCLUSION: NOX2 plays an important role against Aspergillus fumigatus in the mouse cornea through killing fungi and limiting the degree of inflammation.展开更多
AIM: To investigate the anti-inflammatory role of vasoactive intestinal peptide(VIP) in Aspergillus fumigatus(A. fumigatus) ketatitis.METHODS: Expression of VIP was tested by polymerase chain reaction(PCR) in ...AIM: To investigate the anti-inflammatory role of vasoactive intestinal peptide(VIP) in Aspergillus fumigatus(A. fumigatus) ketatitis.METHODS: Expression of VIP was tested by polymerase chain reaction(PCR) in C57BL/6 and BALB/c normal and A. fumigatus infected corneas. C57BL/6 mice were pretreated with recombinant(r) VIP, while BALB/c mice were pretreated with VIP antagonist, and then infected with A. fumigatus. Clinical score was recorded. Expression of pro-and anti-inflammatory cytokines, toll-like receptor 4(TLR4), lectin-like oxidized low-density lipoprotein receptor 1(LOX-1), and neutrophil infiltration were tested by PCR, enzyme-linked immunosorbent assay(ELISA), and myeloperoxidase(MPO) assay.RESULTS: VIP mR NA expression in BALB/c cornea was higher than C57BL/6 cornea at 1 and 3 d post infection(p.i.). rV IP treatment of C57BL/6 mice showed alleviated disease and down-regulated expression of interleukin-1β(IL-1β) and tumor necrosis factor-α(TNF-α), while IL-10 expression was up-regulated. Neutrophil infiltration and TLR4, IL-17 expression were decreased after rVIP treatment, while LOX-1 expression was up-regulated in C57BL/6. VIP antagonist pretreatment showed increased disease and higher IL-1β, TNF-α, TLR4, IL-17 and MPO levels, while IL-10 and LOX-1 levels were down-regulated in BALB/c mice.CONCLUSION: rVIP alleviate disease response of C57BL/6 mice. VIP antagonist resulted in worsened disease of BALB/c mice. VIP proposed anti-inflammatory role in A. fumigatus keratitis.展开更多
AIM: To determine the disparate expression of autophagy in the Aspergillus fumigatus(A. fumigatus) keratitis between susceptible C57 BL/6 mice and resistant BALB/c mice.METHODS: C57 BL/6 and BALB/c mice were used to e...AIM: To determine the disparate expression of autophagy in the Aspergillus fumigatus(A. fumigatus) keratitis between susceptible C57 BL/6 mice and resistant BALB/c mice.METHODS: C57 BL/6 and BALB/c mice were used to establish fungal keratitis models. Disease severity and inflammatory response were observed by slit lamp microscopy in A. fumigatus-infected corneas of C57 BL/6 and BALB/c mice at 1, 3 and 5 d. Hematoxylin-eosin(H&E) staining was used to detect pathological changes of corneas. The expression of autophagy-related proteins Beclin-1, LC3, SQSTM1/p62, and LAMP-1 was assessed by Western blot in C57 BL/6 and BALB/c mice at 1, 3 and 5 d post infection(p.i.). Immunofluorescent staining was used to test the expression of LC3 in corneas after A. fumigatus infection.RESULTS: Keratitis severity was higher in C57 BL/6 mice versus BALB/c mice at 1, 3 and 5 d p.i. H&E staining showed that the number of inflammatory cells was larger and the severity of ulcer was higher in C57 BL/6 mice than in BALB/c mice after stimulation with A. fumigatus. Higher expression of LAMP-1, Beclin-1, and LC3 was shown in C57 BL/6 mice corneas than in BALB/c mice corneas at 1, 3 and 5 d p.i., while the expression of p62 was lower in C57 BL/6 mice. The fluorescence of LC3 was significantly increased in corneas of C57 BL/6 mice compared with BALB/c mice after A. fumigatus infection.CONCLUSION: The expression of autophagy is higher in corneas of C57 BL/6 mice than in BALB/c mice afterA. fumigatus infection. Autophagy may be positively correlated with keratitis severity and pathological changes.展开更多
基金Supported by Chongqing Key Diseases Research and Application Demonstration Program,No.2019ZX003General Project of Chongqing Nature Science Foundation,No.cstc2021jcyj-msxmX0283.
文摘BACKGROUND Dopamine and cyclic adenosine monophosphate(cAMP)-regulated phosphop-rotein with an apparent Mr of 32000(DARPP-32)is a protein that is involved in regulating dopamine and cAMP signaling pathways in the brain.However,recent studies have shown that DARPP-32 is also expressed in other tissues,including colorectal cancer(CRC),where its function is not well understood.AIM To explore the effect of DARPP-32 on CRC progression.METHODS The expression levels of DARPP-32 were assessed in CRC tissues using both quantitative polymerase chain reaction and immunohistochemistry assays.The proliferative capacity of CRC cell lines was evaluated with Cell Counting Kit-8 and 5-ethynyl-2’-deoxyuridine assays,while apoptosis was measured by flow cytometry.The migratory and invasive potential of CRC cell lines were deter-mined using wound healing and transwell chamber assays.In vivo studies involved monitoring the growth rate of xenograft tumors.Finally,the underlying molecular mechanism of DARPP-32 was investigated through RNA-sequencing and western blot analyses.RESULTS DARPP-32 was frequently upregulated in CRC and associated with abnormal clinicopathological features in CRC.Overexpression of DARPP-32 was shown to promote cancer cell proliferation,migration,and invasion and reduce apoptosis.DARPP-32 knockdown resulted in the opposite functional effects.Mechanistically,DARPP-32 may regulate the phosphoinositide 3-kinase(PI3K)/AKT signaling pathway in order to carry out its biological function.CONCLUSION DARPP-32 promotes CRC progression via the PI3K/AKT signaling pathway.
基金Supported by the National Natural Science Foundation of China(No.81470609 No.81500695+5 种基金 No.81700800 No.81870632 No.81800800)Natural Science Foundation of Shandong Province(No.ZR2017BH025 No.ZR2017MH008 No.ZR2013HQ007)
文摘AIM: To investigate the inflammatory amplification effect of high-mobility group box 1(HMGB1) in Aspergillus fumigatus(A. fumigatus) keratitis and the relationship between lectin-like oxidized low-density lipoprotein receptor 1(LOX-1) and HMGB1 in keratitis immune responses.METHODS: Phosphate buffer saline(PBS), and Boxb were injected into BALB/c mice subconjunctivally before the corneas were infected with A. fumigatus. RAW264.7 macrophages and neutrophils were pretreated with PBS and Boxb to determine the HMGB1 inflammatory amplification effects. Abdominal cavity extracted macrophages were pretreated with Boxb and Poly(I)(a LOX-1 inhibitor) before A. fumigatus hyphae stimulation to prove the the relationship between the two molecules. LOX-1, interleukin-1β(IL-1β), tumor necrosis factor-α(TNF-α), macrophage inflammatory protein-2(MIP-2) and IL-10 were assessed by polymerase chain reaction and Western blot.RESULTS: Pretreatment with Boxb exacerbated corneal inflammation. In macrophages and neutrophils, A. fumigatus induced LOX-1, IL-1β, TNF-α and MIP-2 expression in Boxb group was higher than those in PBS group. Poly(I) treatments before infection alleviated the proinflammatory effects of Boxb in abdominal cavity extracted macrophages. Pretreatment with Boxb did not influence Dectin-1 mRNA levels in macrophages and neutrophils.CONCLUSION: In fungal keratitis, HMGB1 is a proinflammatory factor in the first line of immune response. HMGB1 mainly stimulates neutrophils and macrophages to produce inflammatory cytokines and chemokines during the immune response. LOX-1 participates in HMGB1 induced inflammatory exacerbation in A. fumigatus keratitis.
基金Supported by the National Natural Science Foundation of China(No.81870632,No.81470609,No.81700800,No.81800800)the Key Research Project Foundation of Shandong Province(No.2019GSF107022)。
文摘AIM:To determine whether lectin-like ox-LDL receptor(LOX-1)regulates adhesion molecules expression and neutrophil infiltration in Aspergillus fumigatus(A.fumigatus)keratitis of C57 BL/6 mice.METHODS:C57 BL/6 mice were pretreated with a neutralizing antibody to LOX-1(5μg/5μL)or control nonspecific IgG(5μg/5μL),LOX-1 inhibitor Poly-I(2μg/5μL)or PBS by subconjunctival injection.Fungal keratitis(FK)mouse models of C57 BL/6 mice were established by scraping corneal central epithelium,smearing A.fumigatus on the corneal surface and covering the eye with contact lenses.The corneal response to infection was assessed via clinical score.The mRNA levels of the adhesion molecules intercellular cell adhesion molecule-1(ICAM-1),vascular cell adhesion molecule-1(VCAM-1),P-selectin and E-selectin were tested in control and infected corneas by reverse transcriptionpolymerase chain reaction(RT-PCR).The protein levels of ICAM-1 were evaluated by immunofluorescence(IF)and Western blot.Neutrophils were extracted from the abdominal cavity of C57 BL/6 mice followed by pretreatment using antibody to LOX-1(10μg/mL)or control nonspecific IgG(10μg/mL),the Poly-I(4μg/mL)or PBS.The cells were then stimulated with A.fumigatus and tested mRNA and protein levels of lymphocyte function-associated antigen-1(LFA-1)using RT-PCR and Western blot.IF and myeloperoxidase(MPO)assays were used to assess neutrophil infiltration in mice corneas.RESULTS:Pretreatment of LOX-1 antibody or the Poly-I reduced the degree of inflammation of cornea and decreased the clinical FK score compared with pretreatment of IgG or PBS(both P<0.01).And these pretreatment also displayed an obvious decline in the mRNA levels of ICAM-1,VCAM-1,P-selectin,E-selectin and LFA-1 expression compared with control groups(all P<0.01).Furthermore,pretreated with LOX-1 antibody or Poly-I,the protein levels of ICAM-1 and LFA-1 also decreased compared with control groups(all P<0.05).Neutrophil infiltration in the cornea was significantly reduced after pretreatment of LOX-1 antibody or Poly-I compared with control groups by IF and MPO assays(both P<0.01).CONCLUSION:Inhibition of LOX-1 can decrease the expression of adhesion molecules and reduce neutrophil infiltration in A.fumigatus infected corneas of C57 BL/6 mice.
基金Supported by National Natural Science Foundation of China (No. 81470609 No. 81170825)+2 种基金the Specialized Research Fund for the Doctoral Program of Higher Education (No. 20123706110003)the Key Project of Natural Science Foundation of Shandong Province (No. ZR2012FZ001) the Youth Project of Natural Science Foundation of Shandong Province (No. ZR2013HQ007)
文摘AIM: To identify whether Aspergillus fumigatus(A.fumigatus) hyphae antigens induced the release of interleukin-8(IL-8) in anti-fungal innate immunity of cultured human corneal epithelial cells(HCECs) and determine the involvement of intracellular signalling pathways. METHODS: HCECs were treated with A. fumigatus hyphae antigens with different concentrations and time.The cytoplasmic calcium of HCECs were assessed by fluorescence imaging. Western blot was used to detect the expression of Ca2 +-dependent protein kinase C(PKC). The IL-8 levels were determined by specific human IL-8 enzyme-linked immunosorbent assay(ELISA) and reverse transcriptase polymerase chain reaction(RT-PCR). Using a series of pharmacological inhibitors, we examined the upstream signalling pathway responsible for IL-8 expression in response to A.fumigatus hyphae antigens. RESULTS: Cells exposed to A. fumigatus hyphae antigens showed higher level of IL-8 m RNA expression and protein production. We demonstrated here that stimulation of HCECs with A. fumigatus hyphae triggers an intracellular Ca2 +flux and results in the activation of Ca2 +-dependent PKC(α, βⅠ and βⅡ) which can be attenuated by pre-treatment of cells with laminarin,suggesting that Dectin-1 signals pathway induced cytoplasmic calcium and influence the activation of PKC in HCECs. Inhibitors of Ca2 +-dependent PKC(Ro-31-8220 and Go6976) significantly abolished hyphae-induced expression of IL-8.CONCLUSION: Our findings suggest that A. fumigatushyphae-induced IL-8 expression was regulated by the activation of Dectin-1-mediated Ca2 +-dependent PKC in HCECs.
基金Supported by the National Natural Science Foundation of China(No.81470609No.81170825)+2 种基金Key Project of Natural Science Foundation of Shandong Province(No.ZR2012HZ001)Youth Project of Natural Science Foundation of Shandong Province(No.ZR2013HQ007)Specialized Research Fund for the Doctoral Program of Higher Education(No.20123706110003)
文摘AIM:To explore the effects of lectin-like ox-LDL receptor(LOX-1) on innate immunity against Aspergillus fumigatus(A.fumigatus) in mice cornea.METHODS:The mRNA levels of LOX-1 were tested in normal and A.fumigatus infected corneas of C57BL/6and BALB/c mice.The expression of LOX-1,pro-inflammatory cytokines TNF-α,CXCL1 and IL-6,antiinflammatory cytokines IL-10,and matrix metalloproteinase 9(MMP9) were tested with treatment with LOX-1 neutralizing antibody or control IgG in A.fum/gatus infected corneas of C57BL/6.Macrophages and neutrophils were extracted from susceptible C57BL/6mice,and pretreated with LOX-1 neutralizing antibody or IgG,then stimulated with A.fum/gatus.The mRNA levels of LOX-1,TNF-α,CXCL1,IL-6,IL-10 and MMP9 were evaluated by polymerase chain reaction.RESULTS:The expression of LOX-1 was significantly increased in C57BL/6 mice corneas after A.fum/gatus infection compared with BABL/c mice.After treatment with LOX-1 neutralizing antibody,the expression of LOX-1,TNF-α,CXCL1,IL-6,MMP9 and IL-10 in C57BL/6corneas were significantly decreased compared with treatment with control IgG;the expression of LOX-1,CXCL1,IL-6 and IL-10 were significantly decreased in macrophages,while TNF-α and MMP9 expressions had no change;LOX-1,TNF-α,CXCL1,IL-6,MMP9 and IL-10 expressions were significantly decreased in neutrophils.CONCLUSION:The expression of LOX-1 can affect the expression of pro-inflammatory and anti-inflammatory cytokines in fungal infected corneas,macrophages and neutrophils of C57BL/6.LOX-1 inhibition rebalances the inflammatory response of fungal keratitis in mice.
基金Supported by the National Natural Science Foundation of China(No.81470609 No.81700800+5 种基金 No.81870632 No.81800800)Natural Science Foundation of Shandong Province(No.ZR2013HQ007 No.ZR2017MH008 No.ZR2017BH025)the Youth National Natural Science Foundation of China(No.81500695)
文摘AIM: To investigate the expression of interleukin(IL)-33 in the cornea and human corneal epithelial cells(HCECs) exposed to Aspergillus fumigatus(A. fumigatus), and to determine the function of IL-33/ST2/p38 signaling pathway in the immune response of corneal epithelial cells to A. fumigatus infection.METHODS: The mRNA and protein expression of IL-33 in HCECs and mice corneas were examined by quantitative real-time reverse transcription polymerase chain reaction(q RT-PCR) and Western blot analysis, respectively. IL-33 expression was also detected in cornea samples from healthy donors and patients with fungal keratitis with immunohistochemistry. The cultured HCECs were treated with inactive A. fumigatus hyphae at various concentrations with or without recombinant human IL-33 protein, soluble recombinant ST2 protein, specific ST2 neutralizing antibody, or the mitogen-activated protein kinase(MAPK) p38 inhibitor SB203580 for evaluation of the expression and activation of IL-33/ST2/p38 signaling in the regulation of proinflammatory cytokines. The production levels of IL-6 and IL-1β were determined by qR T-PCR and enzymelinked immunosorbent assay(ELISA). The proliferation of HCECs was determined by a Cell Counting Kit-8(CCK8) assay and cell count.RESULTS: IL-33 expression levels increased in the corneal tissues of patients with fungal keratitis and in mice corneas of experimental A. fumigatus infection,as well as in HCECs with infection of A. fumigatus. A. fumigatus strongly stimulated HCECs-generated proinflammatory cytokine(IL-6 and IL-1β) production at both the mRNA and protein levels. This production of proinflammatory mediators stimulated by A. fumigatus was further stimulated by IL-33 and was prevented by soluble ST2 protein or ST2 neutralizing antibody. Moreover, IL-33 naturally promoted the p38 phosphorylation induced by A. fumigatus, which was suppressed by soluble ST2 protein. The MAPK p38 inhibitor SB203580 also inhibited the A. fumigatus-induced proinflammatory cytokine production. IL-33 administration for 48 h and 72 h promoted the proliferation of HCECs, which was attenuated by treatment with soluble recombinant human ST2 protein.CONCLUSION: A. fumigatus elevates IL-33 expression in human and mice corneas and HCECs. Thus, IL-33/ST2/p38 signaling may play an important role in amplifying the immune response of corneal epithelial cells to A. fumigatus infection. Besides, IL-33 promotes the cell proliferation of HCECs via its receptor ST2. These findings suggest a novel autocrine mechanism of amplification of the fungalinduced inflammatory response in the corneal epithelium, highlighting a potential therapeutic target for fungal keratitis.
基金Supported by the National Natural Science Foundation of China(No.81870632)Youth Project of Natural Science Foundation of ghandong Province(No.ZR2019BH004).
文摘AIM:To characterize effect of astaxanthin(ASX)in Aspergillus fumigatus(A.fumigatus)induced keratitis in mouse model.METHODS:In vivo,fungal keratitis mouse model was established in C57BL/6 mice using A.fumigatus,followed by ASX or dimethyl sulfoxide(DMSO)treatment.Clinical responses were evaluated by clinical score and myeloperoxidase(MPO)assay.Inflammatory cytokines were assessed by reverse-transcription polymerase chain reaction(RT-PCR),Western blot,immunofluorescence,and enzyme-linked immuno sorbent assay(ELISA).RESULTS:In animal model,ASX improved corneal transparency and clinical response,suppressed the expression of inflammatory cytokine like IL-1β,TNF-α,and HMGB-1.Neutrophil levels have been shown to decrease in ASX-treated cornea by immunofluorescence and MPO.TLR2 and TLR4 levels were lower in ASX-treated group than DMSO-treated.CONCLUSION:ASX can suppress inflammatory response and reduce inflammatory cytokine production in mice model with A.fumigatus keratitis.
基金Supported by the National Natural Science Foundation of China(No.81870632)the Youth National Natural Science Foundation of China(No.81700800,No.81800800)the Natural Science Foundation of Shandong Province(No.ZR2019BH004,No.ZR2017MH008,No.ZR2017BH025).
文摘●AIM:To observe the expression and role of aryl hydrocarbon receptor(Ah R)in the immune response of mouse cornea infected with Aspergillus fumigatus(A.fumigatus).●METHODS:Murine models of A.fumigatus keratitis were established by scraping the central epithelium of mouse cornea,daubing A.fumigatus on the cornea and covering with a contact lens.The mice were randomly divided into the control group and the A.fumigatus-infected(A.F.)group for 1,3 and 5 d respectively,which corneas were daily monitored by a slit lamp microscope and the clinical scores were also recorded timely after infection.In this study,immunofluorescence staining was used to detect the expression and localization of Ah R in mouse corneas,and the m RNA and protein of Ah R were detected by reverse transcription-polymerase chain reaction(RT-PCR)and Western blot.In addition,mouse peritoneal macrophages were stimulated by A.fumigatus with or without the pretreatment of Ah R antagonist CH223191 and Ah R agonist FICZ,and the tumor necrosis factor alpha(TNF-α),inducible nitric oxide synthase(i NOS),interleukin-10(IL-10)and Arg-1 m RNA were detected by RT-PCR.●RESULTS:According to the results of the slit light photography,it was clearly indicated that the corneal inflammation were the most severe and the clinical score became the highest as well on the 3 rd day after the infection of A.fumigatus.Contrasted with the control group,the expression of Ah R in the corneal epithelial cells infected with A.fumigatus was significantly increased detected by immunofluorescence staining.Ah R mainly expressed in the nucleus and cytoplasm of corneal epithelial cells.Consistent with the transcriptional level of Ah R m RNA,the expression level of Ah R protein reached the peak on the 3 rd day after infection which was detected by Western blot.Furthermore,RT-PCR showed that CH223191 up-regulated the expression of TNF-αand i NOS and down-regulated the expression of IL-10 and Arg-1 in peritoneal macrophages;inversely,FICZ reduced the expression of TNF-αand i NOS while elevated the expression of IL-10 and Arg-1.●CONCLUSION:Ah R is involved in the pathogenesis of A.fumigatus keratitis and induced immune protection in anti-A.fumigatus immune response by inhibiting M1 and increasing M2 phenotype macrophage-related inflammatory factors.
基金Supported by National Natural Science Foundation of China(Grant No.51575490)National Key Basic Research Development Plan(973 Plan,Grant No.2014CB046404)+1 种基金Key Program of Zhejiang Provincial Natural Science Fund Project(Grant No.LZ15E050002)Zhejiang Provincial Natural Science Foundation of Youth Fund(Grant No.LQ17E050008)
文摘Spiral groove dry gas seal(S?DGS), the most widely used DGS in the world, encounters the problem of high leakage rate and inferior film stability when used in high?speed machinery equipment, which could not be well solved by optimization of geometrical parameters and molded line of spiral groove. A new type of bionic cluster spiral groove DGS(CS?DGS) is proved to have superior film stability than S?DGS at the condition of high?speed and low?pressure numerically. A bionic CS?DGS is experimentally investigated and compared with common S?DGS in order to provide evidence for theoretical study. The film thickness and leakage rate of both bionic spiral groove and common spiral groove DGS are measured and compared with each other and with theoretical values under different closing force at the condition of static pressure, high?speed and low?pressure, and the film stiffness and stiffness?leakage ratio of these two face seals are derived by the relationship between closing force and film thickness at the steady state. Experimental results agree well with the theory that the leakage and stiffness of bionic CS?DGS are superior to that of common S?DGS under the condition of high?speed and low?pressure, with the decreasing amplitude of 20% to 40% and the growth amplitude of 20%, respectively. The opening performance and stiffness characteristics of bionic CS?DGS are inferior to that of common S?DGS when rotation speed equals to 0 r/min. The proposed research provides a new method to measure the axis film stiffness of DGS, and validates the superior performance of bionic CS?DGS at the condition of high?speed and low?pressure experimentally.
基金Supported by the National Natural Science Foundation of China(No.81470609 No.81870632)+4 种基金the Youth National Natural Science Foundation of China(No.81700800 No.81800800 No.81500695)Natural Science Foundation of Shandong Province(No.ZR2017MH008 No.ZR2017BH025)
文摘AIM: To characterize changes in the cornea nerve and pain responses in fungal keratitis(FK).METHODS: A retrospective analysis of in vivo confocal microscopy images of 11 FK corneas was performed, and the results were compared with those for 11 normal corneas. Subbasal corneal nerves were analyzed for total nerve number, main nerve trunk number, branching patterns and tortuosity. C57 BL/6 mice were infected with Aspergillus fumigatus. Disease severity was determined through clinical scoring and slit lamp photography. Corneas were harvested at 1, 3, 5, and 7 d post infection(p.i.) and assessed for β III tubulin. Corneal mechanical sensitivity thresholds were detected by von Frey test. β-endorphin(β-EP) and μ receptor protein expression was detected through Western blotting.RESULTS: Total nerve number, main nerve trunk number, and nerve branching were significantly lower in FK patients than in controls, but tortuosity was not significantly different. In infected mice, subbasal nerve density decreased from 1 d p.i., reaching a minimum at 5 d p.i. Clinical scores rose at 1 d p.i., peaked at 3 d p.i., and decreased at 5 d p.i. Mechanical sensitivity thresholds showed the same trends. β-EP and μ receptor protein expression increased after infection.CONCLUSION: Corneal nerve density is lower in FK patients and Aspergillus fumigatus-infected mice than in controls. Pain sensitivity decreases with postinfection corneal ulcer aggravation. β-EP and μ receptor proteins are both upregulated in infected mouse corneas.
基金Supported by the National Natural Science Foundation of China (No.81470609 No.81700800+5 种基金 No.81870632 No.81800800)the Youth National Natural Science Foundation of China (No.81500695)Natural Science Foundation of Shandong Province (No.ZR2013HQ007 No.ZR2017MH008 No.ZR2017BH025)
文摘AIM: To investigate the expression and role of calcitonin gene-related peptide(CGRP) in the mouse models induced by Aspergillus fumigatus(A. fumigatus). METHODS: C57 BL/6 mice were randomized into a control group and A. fumigatus keratitis group. The cornea photography was assessed under the slit lamp and the clinical score was recorded after infection. Western blot, real-time polymerase chain reaction(PCR) and immunohistofluorescence analysis were applied to detect CGRP expression in cornea of both groups. In vitro, tests were conducted with C57 BL/6 mice macrophages to investigate CGRP expression after interaction with A. fumigatus. Cytokines expression induced by exogenous CGRP and the antagonist CGRP8-37 in A. fumigatus-exposed macrophages was evaluated by real-time PCR and ELISA.RESULTS: The cornea expression of CGRP was significantly elevated in C57 BL/6 mice corneas and macrophages after A. fumigatus infection. After treatment with exogenous CGRP, the levels of interleukin-1β(IL-1β), tumor necrosis factor-α(TNF-α) and IL-6 were reduced, and IL-10 level was increased in the A. fumigatus stimulatedmacrophages. However, IL-1β, TNF-α and IL-6 levels were upregulated after pretreatment of CGRP8-37. But the m RNA levels of MIP-2, TGF-β and IL-10 were not changed. CONCLUSION: This study provides evidence that A. fumigatus increased CGRP expression. CGRP may play a protective role against inflammation in A. fumigatus keratitis.
基金Supported by National Natural Science Foundation of China(No.81170825No.81470609)+2 种基金Natural Science Foundation of Shandong Province(No.ZR2013HQ007No.ZR2012HZ001)the Specialized Research Fund for the Doctoral Program of Higher Education,2012(No.20123706110003)
文摘· AIM: To explore the immunomodulatory effects of curdlan on innate immune responses against Aspergillus fumigatus(A. fumigatus) in cultured human corneal epithelial cells(HCECs), and whether C-type lectin receptor Dectin-1 mediates the immunomodulatory effects of curdlan.·METHODS: The HCECs were stimulated by curdlan in different concentrations(50, 100, 200, 400 μg/m L) for various time. Then HCECs pretreated with or without laminarin(Dectin-1 blocker, 0.3 mg/m L) and curdlan were stimulated by A. fumigatus hyphae. The m RNA and protein production of tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6) were determined by real-time quantitative polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. The protein level of Dectin-1 was measured by Western blot.· RESULTS: Curdlan stimulated m RNA expression of TNF-α and IL-6 in a dose and time dependent manner in HCECs. Curdlan pretreatment before A. fumigatus hyphae stimulation significantly enhanced the expression of TNF-α and IL-6 at m RNA and protein levels compared with A. fumigatus hyphae stimulation group(P 【0.05).Both curdlan and A. fumigatus hyphae up-regulated Dectin-1 protein expression in HCECs, and Dectin-1expression was elevated to 1.5- to 2-fold by curdlan pretreatment followed hyphae stimulation. The Dectin-1blocker laminarin suppressed the m RNA expression and protein production of TNF-α and IL-6 induced by curdlan and hyphae(P 【0.05).· CONCLUSION: These findings demonstrated that curdlan pretreatment enhanced the inflammatory response induced by A. fumigatus hyphae in HCECs.Dectin-1 is essential for the immunomodulatory effectsof curdlan. Curdlan may have high clinical application values in fungal keratitis treatment.
基金Supported by National Natural Science Foundation of China(No.81170825)
文摘AIMTo observe the expression of vitamin D receptor (VDR) in human specimen and immortalized human corneal epithelium cells (HCEC) when challenged with fusarium solani. Moreover, we decided to discover the pathway of VDR expression. Also, we would like to detect the expression of cathelicidin antimicrobial peptide (CAMP) in the downstream pathway of VDR.METHODSImmunohistochemistry was used to examine the VDR expression in HCEC from healthy and fungal keratitis patients. Real time quantitative polymerase chain reaction (qPCR) was performed to observe the messenger ribonucleic acid (mRNA) change of VDR when immortalized HCEC were challenged with fusarium solani for different hours. CAMP was detected at both mRNA and protein levels.RESULTSWe found out that the VDR expression in fusarium solani keratitis patients' specimen was much more than that in healthy people. The mRNA and protein expression of VDR increased when we stimulated HCEC with fusarium solani antigen (P<0.01) and it could be inhibited by toll like receptor 2 (TLR2) monoclonal antibody. The CAMP expression was decreased because of fusarium solani antigen stimulation (P<0.01).CONCLUSIONThe VDR expression can be increased via TLR2/1-VDR pathway while the CAMP expression is decreased by the stimulation of fusarium solani antigen.
基金Supported by the National Natural Science Foundation of China(No.81170825No.81470609)+2 种基金Specialized Research Fund for the Doctoral Program of Higher Education(No.20123706110003)The Youth Natural Science Foundation of Shandong Province(No.ZR2013HQ007)The Key Project of Natural Science Foundation of Shandong Province(No.ZR2012HZ001)
文摘AIM: To observe the presence and expression of indoleamine 2,3-dioxygenase(IDO) during the corneal immunity to Aspergillus fumigatus(A. fumigatus) in the murine models.·METHODS: The murine model of fungal keratitis was established by smearing with colonies of A. fumigatus after scraping central epithelium of cornea and covering with contact lenses in C57BL/6 mice. The mice were randomly divided into control group, sham group and A.fumigatus keratitis group. The cornea was monitored daily using a slit lamp and recorded disease score after infection. Corneal lesion was detected by immunofluorescence staining. IDO m RNA and protein were also detected by quantitative reverse transcription-polymerase chain reaction(q RT-PCR) and Western blot.· RESULTS: The disease score and slit lamp photography indicated that disease severity was consistent with corneal inflammation in the murine models, and the disease scores in A. fumigatus keratitis group were obviously higher than those in the sham group. By immunofluorescence staining, IDO was mainly localized in corneal epithelium and stroma in the murine corneal tissues with A. fumigatus keratitis. Compared with the sham group, IDO m RNA expression was significantly enhanced in corneal epithelium infected by A. fumigatus. Furthermore, IDO protein expression detected by Western blot was in accord with transcript levels of IDO m RNA measured by q RT-PCR. IDO protein expression was enhanced after A. fumigatus infection compared with the sham group.·CONCLUSION: IDO is detected in corneal epithelium and stroma locally, which indicates IDO takes part in the pathogenesis of A. fumigatus keratitis and plays a key role in immune regulation at the early stage.
基金Supported by the National Natural Science Foundation of China(No.81470609No.81500695)
文摘AIM: To investigate how macrophage inducible C-type lectin(Mincle) influences inflammation in mice fungal keratitis induced by Aspergillus fumigatus(A. fumigatus).METHODS: C57 BL/6 mice were infected with A. fumigatus after pretreated with Mincle agonist TDB or Mincle neutralizing antibody(MincleAb), taking DMSO or IgG as control group respectively. The cornea lesions were monitored with slit-lamp microscope and evaluated by clinical score. Mincle expression was assessed using reverse transcriptionploymerase chain reaction(RT-PCR) and immunostaining. The expression of cytokines(IL-1β, TNF-α and IL-6) chemokines(CXCL-1 and MIP-2) was determined by RTPCR and ELISA. Neutrophil infiltration was observed by immunostaining. The levels of nitric oxide(NO) generated by corneas were tested by Griess reaction. RESULTS: Mincle mRNA and protein levels were higher in infected corneas than normal corneas of C57 BL/6 mice, saving clinical scores revealed differences. When pretreated with Mincle agonist TDB, the mRNA and protein levels of IL-1β, TNF-α and IL-6 in infected corneas were significantly increased compared with the control group(P〈0.01). Results of the counterpart in corneas pretreated with Mincle neutralizing antibody was decreased consistently(P〈0.01). Expression of CXCL1 and MIP-2 mRNA levels were upregulated in TDB group and down-regulated in Mincle Ab group(P〈0.01), coincide with neutrophil aggregation degree in corneas showed by immunostaining. As for the concentration of NO, it was promoted in TDB group compared with DMSO control group, and decreased in Mincle Ab group compared with Ig G control group.CONCLUSION: Mincle plays a dual role in mice fungal keratitis. It participates in the innate immune system by enhancing inflammation. What's more, Mincle can mediate cytotoxic effects by regulating the formation of NO.
文摘AIM: To explore whether nicotinamide adenine dinucleotide phosphate(NADPH) oxidase 2(NOX2) is expressed in fungal keratitis in mice and investigate its role in this disease.METHODS: NOX2 expression was detected in C57 BL/6 mice. After testing the inhibitory effect of diphenyleneiodonium chloride(DPI) on NOX2, its impact on clinical performance, myeloperoxidase levels, the number of colonies forming units, the level of H3, the generation of reactive oxygen species(ROS) and the release of cytokines [NF-κB, interleukin-17 A(IL-17 A), interleukin-6(IL-6), tumor necrosis factor-α(TNF-α), Nrf2, IL-10, and TGF-β] were compared. A one-way ANOVA and an unpaired, two-tailed Student’s t-test was used to determine the statistical significance.RESULTS: NOX2 expression was significantly increased after Aspergillus fumigatus injection in corneas and that this increase could be reduced by treatment with DPI. DPI treatment produced more severe inflammation and resulted in higher clinical scores, more neutrophils infiltration, a weakened ability to clear fungi, the release of fewer ROS and the formation of neutrophil extracellular traps. Treatment with DPI increased the expression of the proinflammatory cytokines NF-κB, IL-17 A, IL-6, and TNF-α and decreased the expression of the anti-inflammatory cytokines Nrf2, IL-10 and TGF-β compared to their expression levels without DPI treatment.CONCLUSION: NOX2 plays an important role against Aspergillus fumigatus in the mouse cornea through killing fungi and limiting the degree of inflammation.
基金Supported by the National Natural Science Foundation of China(No.81470609No.81500695)+1 种基金the Natural Science Foundation of Shandong Province(No.ZR2013HQ007No.ZR2017BH025)
文摘AIM: To investigate the anti-inflammatory role of vasoactive intestinal peptide(VIP) in Aspergillus fumigatus(A. fumigatus) ketatitis.METHODS: Expression of VIP was tested by polymerase chain reaction(PCR) in C57BL/6 and BALB/c normal and A. fumigatus infected corneas. C57BL/6 mice were pretreated with recombinant(r) VIP, while BALB/c mice were pretreated with VIP antagonist, and then infected with A. fumigatus. Clinical score was recorded. Expression of pro-and anti-inflammatory cytokines, toll-like receptor 4(TLR4), lectin-like oxidized low-density lipoprotein receptor 1(LOX-1), and neutrophil infiltration were tested by PCR, enzyme-linked immunosorbent assay(ELISA), and myeloperoxidase(MPO) assay.RESULTS: VIP mR NA expression in BALB/c cornea was higher than C57BL/6 cornea at 1 and 3 d post infection(p.i.). rV IP treatment of C57BL/6 mice showed alleviated disease and down-regulated expression of interleukin-1β(IL-1β) and tumor necrosis factor-α(TNF-α), while IL-10 expression was up-regulated. Neutrophil infiltration and TLR4, IL-17 expression were decreased after rVIP treatment, while LOX-1 expression was up-regulated in C57BL/6. VIP antagonist pretreatment showed increased disease and higher IL-1β, TNF-α, TLR4, IL-17 and MPO levels, while IL-10 and LOX-1 levels were down-regulated in BALB/c mice.CONCLUSION: rVIP alleviate disease response of C57BL/6 mice. VIP antagonist resulted in worsened disease of BALB/c mice. VIP proposed anti-inflammatory role in A. fumigatus keratitis.
基金Supported by the National Natural Science Foundation of China (No.81470609 No.81500695+5 种基金 No.81700800 No.81870632 No.81800800)Natural Science Foundation of Shandong Province (No.ZR2017BH025 No.ZR2017MH008 No.ZR2013HQ007)
文摘AIM: To determine the disparate expression of autophagy in the Aspergillus fumigatus(A. fumigatus) keratitis between susceptible C57 BL/6 mice and resistant BALB/c mice.METHODS: C57 BL/6 and BALB/c mice were used to establish fungal keratitis models. Disease severity and inflammatory response were observed by slit lamp microscopy in A. fumigatus-infected corneas of C57 BL/6 and BALB/c mice at 1, 3 and 5 d. Hematoxylin-eosin(H&E) staining was used to detect pathological changes of corneas. The expression of autophagy-related proteins Beclin-1, LC3, SQSTM1/p62, and LAMP-1 was assessed by Western blot in C57 BL/6 and BALB/c mice at 1, 3 and 5 d post infection(p.i.). Immunofluorescent staining was used to test the expression of LC3 in corneas after A. fumigatus infection.RESULTS: Keratitis severity was higher in C57 BL/6 mice versus BALB/c mice at 1, 3 and 5 d p.i. H&E staining showed that the number of inflammatory cells was larger and the severity of ulcer was higher in C57 BL/6 mice than in BALB/c mice after stimulation with A. fumigatus. Higher expression of LAMP-1, Beclin-1, and LC3 was shown in C57 BL/6 mice corneas than in BALB/c mice corneas at 1, 3 and 5 d p.i., while the expression of p62 was lower in C57 BL/6 mice. The fluorescence of LC3 was significantly increased in corneas of C57 BL/6 mice compared with BALB/c mice after A. fumigatus infection.CONCLUSION: The expression of autophagy is higher in corneas of C57 BL/6 mice than in BALB/c mice afterA. fumigatus infection. Autophagy may be positively correlated with keratitis severity and pathological changes.
基金Project supported by the National Natural Science Foundation of China(Nos.U1737202 and 51775505)the National Basic Research Program(973 Program)of China(No.2014CB046404)+2 种基金the Natural Science Key Foundation of Zhejiang Province(No.LZ15E050002)the Natural Science Foundation of Zhejiang Province(No.LY17E050018)China