The Effect Of Mizolastine On Expression Of VEGF, KC And TNF-α In Murine Mast Cells

BackgroundIn allergic inflammation,the recognized key initiator and effector cells are the mast cells.Also in allergy,as a consequence of tissue damage,coordinated sequelae of re-epithelization,angiogenesis and remodelling are evident. It has been recognized that fibrosis and angiogenesis are major pathogenic process in allergical diseases which has previously been overlooked .Meanwhile, angiogenesis might be proposed as a target for anti-inflammatory drugs in allergical diseases. Indeed, many anti-inflammation drugs have been found to be anti-angiogenic. For example, glucocorticosteroids , the most potent anti-inflammation drugs employed, have anti-angiogenic properties. Interestingly, glucocorticosteroid anti-angiogenic activity is not only due to their ability to decrease inflammatory cell numbers and infiltration intothe tissues, but also to their direct inhibition of the gene expression. Mast cells,that have the capability of surviving for extended periods of time and being activated and reactivated on a multiple basis,have been proposed to influence these processes as well.These pro-angiogenic mediators derived from mast cells may be granule-associated,lipid-derived and cytokines and chemokines.Upon stimulation through Fc e RJ or c-Kit or after challenge with phorbol myristate acetate,or calcium ionophore,mast cells can rapidly release vascular endothelial cell growth factor(VEGF) apparently from a pre-formed pool,and can then sustain release by secreting newly synthesized protein.VEGF is the essential growth factor for endothelial cells which is sufficient for the formation of new blood vessels.Acting as a true growth factor,VEGF induces an increase in the number of endothelial cells.lt can also act as a lumenizing factor by virtue of its ability to increase vascular permeability.Evidence obtained using mainly pharmacological,cell biological or genetic methods show that mast cells, when adequately stimulated by various secretagogues, are able to induce and enhance angiogenesis by releasing the angiogenic factors such as VEGF,tumor necrosis factor(TNF)-a, interleukin(IL)-8,transform-ing growth factor(TGF)-p and so on. Recent research has focused on the ability of some antihistamines to inhibit the release of mediators from mast cells . The evidences have shown that manyof the antihistamines can inhibit the release of mediators from mast cells, and perhaps mast cell activation itself ,in addition to their direct HI antagonism. The mechanisms underlying the anti-inflammatory effects of these antihistamines remain unclear. Mizolastine (MIZ) is a novel anti-allergic therapy that has been shown to exhibit potent HI-antagonist activity and may also possess non-histamine-related anti-allergic and anti-inflammation activities. However, there is little information regarding the effects of MIZ on the release and generation of cytokines acting on angiogenesis. ObjectiveIn the basic study of MIZ on the angiogensis in murine allergic model,we further observed the effect of MIZ on KC,VEGF and TNF-a in murine mast cells ,which are known to be major factors affecting angiogenesis, comparing with dexamethasone(DEX) and loratadine(LOR) . MethodsWe examined the effects of MIZ on the angiogensis with murine allergic model,and on KC,VEGF and TNF-a expression in mast cells isolated from the skins of Kunming mice by ELISA and RT-PCR, in comparison with DEX and LOR.The image densities were measured with Image-Pro Plus and data were analysed with SPSS 10.0. Then, the effect of MIZ on signaling pathways in mast cells isolated from the skins of Kunming micewas examined by Western blot analysis. Immunoblots were prepared from whole cell lysates and probed with Abs against Fyn ,Akt , ERK,p38 and phospho-Fyn, phospho- Akt, phospho-ERK and phospho-p38 respectively . ResultsThis study showed that DEX,MIZ and LOR could suppress the capillary permeability and cytokines release including KC,VEGF and TNF-a in murine allergic models induing by OVA.There were significant differences in degrees of the capillary permeability ,mount of vascular and the releases of KC,VEGF and TNF-a between DEX and MIZ or LOR .Obviously,the ability of MIZ on angiogenesis in the murine model was supior to that of LOR.In following experiment,our findings showed that MIZ was markedly effective at inhibiting KC,VEGF and TNF-a release in the mast cells induced by IgE-dependent mechanism, taking on time- and dose-dependent manners. The results by ANOVA showed significant differences in the inhibition ratios between the three drugs on KC,VEGF and TNF-a from 10'9 to 10"5 mol/L (PO.05, PO.01). The effect of DEX on KC,VEGF and TNF-a was significantly stronger than that of MIZ or LOR from 10"9 to 10'5 mol/L (P<0.01) and the effect of MIZ on VEGF and TNF-a was stronger than that of LOR(P<0.01).However, the effect of LOR on KC from 10'9 to 10*7 mol/L was stronger than that of MIZ(P<0.05),but no statistical difference was found between the effect of MIZ or LOR at concentrationsover 10'7 mol/L(/>>0.05). This inhibition occurred as early as 30min,with a maximum effect at 6h. Incubation of the cells for 4h with the three drugs at 10"7 mol/L led to a significant reduction of VEGFi65,VEGFi2o,TNF-a and KC mRNA expression. Statistical comparison between the agents showed that the product of DEX on VEGF165 mRNA was different from that of MIZ or LOR(P<0.01), and differences between the products of the three drugs on VEGFi2o,TNF-a and KC mRNA were not statistically significant (P>0.05). Our findings showed that MIZ was markedly effective at inhibiting VEGF release and synthesis induced by IgE-dependent mechanism, taking on time- and dose-dependent manners. We then investigated whether MIZ had any effect on Fyn ,Akt,ERK and p38,which were essential for signaling cascades in the release and synthesis of cytokines of mast cells . These molecules were found to be phosphorylated in nonstimulated mast cells,and Ag stimulation induced additional phosphorylation of all these molecules.Treatment with MIZ at various concentrations and Ag stimulation led to markedly suppress phosphorylation of Akt and Akt ,whereas it had not any effect on Akt without Ag.The inhibition by MIZ on phospho-Akt was dose-dependent from 10'9 to 10"5 mol/L. However, phospho- Fyn , phospho-ERK and phospho-p38 increasing with the stimulation by antigen were not affected by MIZ at various concentrations.Conclusions(1) The findings in murine allergic models raise the possibility that MIZ may mediate angiogenesis activity. MIZ may possess potent anti-angiogenesis ability,which combined to its anti-histamine and anti-inflammation properties could be valuable for the treatment of allergic inflammation or other inflammatory diseases.(2) The capacity of MIZ to inhibit cytokines release and gene expression in murine mast cells could accounts for the previous observed diminished Ag-induced the pro-angiogenic factors in MIZ -treated murine models.The effect of MIZ on angiogenesis may be largely related to the mast cells in allergical diseases,and It is uncertain whether the inhibitory effects of MIZ on the pro-angiogenic factors seen in the studies have any relevance to other inflammatory cells.(3) In this study, MIZ exerts inhibitory actions on the release of KC,VEGF and TNF-a and their gene expression in concentrations close to clinical doses(about 6.0 X 10~7 mol/L ) , of a magnitude similar to that of DEX or LOR. It maybe well explain the effect of MIZ on the angiogenesis in allergical diseases.(4) The capacity of MIZ to inhibit cytokines release and gene expression in murine mast cells may be due to multi-pathways involved and further indicate signaling pathways.(5) MIZ at concentrations within the range of therapeutic plasma levels in humans can inhibit activation of Akt but not Fyn to Ag stimulation supporting the idea that MIZ inhibits Ag-induced Akt activation via a PI3K -dependent pathway,which may further decrease activation of protein kinase C leading to degranulation of mast cells, as well as the activity of nuclear factors mediating the synthesis and secret of cytokines.Many evidences have shown that Akt is activated via at least two pathyways,one of which is PBK-dependent and other is PI3K-independent;the inhibition of Akt through a PKC pathyway also can be mediated by either a PI3K-dependent or PI3K-independent pathway.Therefore,our results indicates that the inhibition of Akt activity by MIZ probably occurs upstream of PI3K,possibly involving PKC activity.However,in the study,we have demonstrated that MIZ at various concentrations exert inhibitory ability on PKC activation in a dose-dependent manner ,without markedly effect on activation of IP3-kinase . It is possible that PKC-mediated phosphorylation of Akt play an important role in the conformation of the protein in mast cells,and can be blocked by MIZ. On other hand, PBK-dependent phosphorylation of Akt by the PDK ultimately leads to the activation of PKC during signaling transduction. Therefore, the inhibition of Akt can directly decrease the expression of PKC . The effect of MIZ on activation of PKC and...