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Ulinastatin Inhibit IL-4Mediated Mast Cells Activation Via Up-regulation of Heme Oxygenase-1

Author: YuLei
Tutor: WangBaoShan
School: Hebei Medical University
Course: Otorhinolaryngology
Keywords: Mast cells Ulinastatin Heme oxygenase-1 anti-inflammatory antioxidant
CLC: R96
Type: Master's thesis
Year: 2014
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Abstract


Objective: Our research group preliminary results have proved thatulinastatin which used to treat the infection toxic shock、acute lung injury andother infectious inflammatory diseases,has immune regulation and antioxidanteffects in the mouse model of allergic inflammation, and ulinastatin,sprotective effects may be related to HO-1.Ulinastatin is a kind of efficient broad-spectrum proteasome inhibitors,infective inflammation is mediated by neutrophils,and allergic inflammationis mediated by eosinophils and mast cells and other immune cells,themechanism is totally different.So further clearly definite the pharmacologicalmechanism of UTI in allergic inflammation, in order to provide laboratorybasis for the treatment of allergic inflammation. Heme oxygenase (HO)-1belongs to the microsomal enzymes,which is catalytic initial enzyme of hemedegradation and the speed limit on enzyme, is the immune regulation andanti-oxidation important regulatory factor. In preliminary experiments,wefound that ulinastatin in vivo models plays a role of immune regulation andantioxidation effects is closely related to the HO-1, Whether ulinastatin isregulated by HO-1or the signaling pathways needs to be further discussed inthe experiment in vitro cell models.Mast cells is one of the immune cells mediated allergic inflammation,allergic inflammation pathological changes are mainly mast cell degranulationactivation, and a large number of inflammatory mediators release. Manystudies have shown thatTh1/Th2cytokine imbalance is one of the mainreasons for the pathogenesis of the airway allergic inflammationdiseases.Therefore the experiment selected Th2representative factor IL-4as astimulus, it can stimulate B cell proliferation, result in a large number of IgEantibody, mast cell activation. Therefore, this topic selects IL-4to stimulate mast cell activation, establishes the vitro cell models,then explorespharmacological mechanism of immune regulation and antioxidation.Methods: Mast cell line, P815cells (ATCC, VA, USA), were cultured inRPMI Medium1640supplemented with10%FCS.Use IL-4stimulate mastcells, set up the vitro model. The mast cells cultured in vitro were randomlydivided into control group (Con), IL-4Stimulated group(IL-4), ulinastatinintervention group (UTI+IL-4) and inhibitor Znpp intervention group(Znpp+UTI+IL-4). By detecting tryptase vitality and Th1/Th2cytokines (IL-5,IL-13, TNF-α, INF-γ) levels from the perspective of immunity test whetherulinastatin can inhibit IL-4mediated mast cells activation; By detectingreactive free oxygen (ROS), protein level of hydroxyl content,malondialdehyde (MDA) and antioxidant enzymes (T-AOC, SOD, CAT, GSHand GSH-Px) levels from the perspective of oxidative stress test whetherulinastatin can regulate IL-4mediated mast cells activation induced byoxidation/antioxidant imbalance;Application of Heme Oxygenase (HO)-1inhibitors Znpp verify whether ulinastatin by adjusting HO-1to play a role ofimmune regulation, antioxidation; By electrophoretic mobility (EMSA) andWestern Blot method such as detection of HO-1upstream regulatory factorNrf2and related signaling pathway to explore ulinastatin regulatingexpression of HO-1specific mechanisms.Results:1IL-4, UTI optimal intervention time and concentrationBy determined by MTT and tryptase dynamic experiments determine themost appropriate concentration and IL-4intervention time100ng/ml and6hrespectively;By determined by MTT and UTI induces protein levels of HO-1determine the most appropriate concentration and UTI intervention100u/ml,2h respectively.2Znpp inhibits HO-1expression in mast cellsAfter added the HO-1inhibitor Znpp in mast cells,the expression of HO-1in the cell immunofluorescence level, or in the level of protein expressionsignificantly reduced, the differences were statistically significant (P<0.05). 3UTI significantly inhibits mast cell degranulationTryptase activity in mast cells was enhanced after IL-4stimulation,significantly higher than that in control group, the differences were significant(P<0.05); Ulinastatin reduced tryptase vitality, inhibited mast celldegranulation in ulinastatin intervention group, the differences weresignificant (P<0.05).4UTI regulates IL-4mediated Th1/Th2cytokines imbalanceThe expressions of Th1representative factors IL-5, IL-13, TNF-α in IL-4stimulation group significantly higher than the control group, the expression ofTh2representative factor INF-γ significantly lower than the control group, thedifferences were significant (P<0.05);UTI significantly inhibited theexpressions of IL-5, IL-13, TNF-α,increased the expression of INF-γ, whichregulates IL-4mediated mast cells activation of Th1/Th2cytokines imbalance,the differences were significant (P<0.05).5UTI regulates IL-4mediated oxidation/antioxidant imbalanceIn the control group, the activity of free oxygen (ROS), hydroxyl contentof protein and malondialdehyde (MDA) at a lower level;After addedIL-4,three expression of oxidative stress injury indexs significantly increased,the differences were significant (P<0.05), antioxidant enzymes (TAOC, SOD,CAT, GSH and GSH-Px) levels significantly decreased, the differences weresignificant (P<0.05);When giving UTI intervention, ROS, protein hydroxylcontent and MDA levels significantly decreased,the differences weresignificant (P<0.05),antioxidant enzymes (TAOC, SOD, CAT, GSH andGSH-Px) levels increased significantly,the differences were significant(P<0.05).6UTI induces HO-1protein expressions in a time and dose dependentmannerwe examined the effects of UTI on HO-1protein expressions inun-stimulated and IL-4-stimulated mast cells in culture. Consistent with ourfindings, stimulation of mast cells with UTI significantly increased basalcellular level of HO-1protein expressions in a time and dose dependent manner.7UTI stimulates Nrf2nuclear translocation.Western Blot results showed that:In the IL-4-stimulated group, IL-4cansignificantly increased numbers of Nrf2positive cells and Nrf2positive nuclei,the differences were significant (P<0.05); The numbers of Nrf2positive cellsand Nrf2positive nuclei increased further in the IL-4+UTI group, thedifferences were significant (P<0.05). The result indicated that UTIaugmented IL-4-induced up-regulation of Nrf2expression and nucleartranslocation.8UTI induces the binding of Nrf2to the AREElectrophoretic mobility shift assay (EMSA) results showed that: Afteradded UTI, UTI can induce the binding of Nrf2to the ARE in the mast cellsnucleu.9Involvement of the MAPK pathways in UTI-induced HO-1expressionUTI can activate the p38kinases pathway and increase p38kinasesphosphorylation in mast cells. Phosphorylation of p38kinases sustained up to2h after UTI treatment. In contrast, phosphorylation of JNK and ERK kinaseswere not seen at any time. Furthermore, to investigate the role of MAPK inHO-1expression, we examined the effects of specific inhibitors of p38kinases(SB203580) imidazole, JNK (SP600125), and ERK (PD98059) on expressionlevels of HO-1by western blot analysis. We found that UTI induced HO-1expression was inhibited by the p38kinases inhibitor, whereas the JNK andERK inhibitors had no effects.Conclusion:1Ulinastatin has immune regulation, antioxidation effects in the mast cellsmediated by IL-4.2Ulinastatin plays the roles of immune regulation, antioxidation through theinduction of HO-1.3Ulinastatin regulates the expression of HO-1through P38MAPK/Nrf2 signaling pathway

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