Ucn. Of Crh & Peripheral Inflammatory Diseases Research

Partâ… Urocortin's possible role in rat bronchial asthmaBronchial asthma is defined as a chronic airway inflammatory diseasecharacterized by various inflammatory cells' infiltration and synthesis of a variety ofproinflammatory mediators and cytokines. The present measures of prevention andtherapy for asthma are not satisfactory. Hence, it is very important to furtherinvestigate the pathogenesis of asthma and search for new targets to prevent and cureasthma.Urocortin (UCN), a recently identified 40-amino-acid mammalian peptide fromrat midbrain, is a new member of the CRH family. It exerts a variety of biologicaleffects via CRHR1 and CRHR2. By regulating hypothalamic-pituitary adrenal axis,centrally produced UCN exerts profound immunosuppressive effects. However,peripheral UCN, through activating mast cells and macrophages may participate in thepathophysiology of many inflammatory conditions.Although UCN expresses in many peripheral tissues and exerts potentproinflammatory effects, there is no report about expression of UCN in lung tissuesand its effects on asthmatic airway inflammation. This study aims to investigate theexpression of UCN in rat lung tissues and the relationship between UCN andasthmatic airway inflammation. Chapter 1 Expression of urocortin in rat lung tissuesOBJECTIVE: The aim of this study was to investigate the expression profile ofUCN in rat lung. METHODS: The expression of UCN mRNA was detected byRT-PCR. UCN peptide was measured by immunohistochemistry and western blotanalysis. RESULTS: We found that both UCN mRNA and peptide obviouslyexpressed in rat lung. Immunohistochemistry results showed that UCN peptide mainlyexpressed in bronchio epithelium mucosae and alveolar epithelium. It also expressedin lung vascular smooth muscle and endothelial cells. CONCLUSION: UCNexpressed in rat lung.Chapter 2 Enhanced expression of urocortin in lung tissues of ratswith allergic asthmaOBJECTIVE: To examine the expression profile of UCN in rat lung with allergicasthma. METHODS: Animals were actively sensitized by subcutaneous injection ofovalbumin (OVA). Airway inflammation of asthmatic rats was evaluated by HEstaining and inflammatory cell counting in BALF. The expression of UCN mRNAwas detected by semi-quantitative RT-PCR. UCN peptide was measured byimmunohistochemistry and western blot analysis. RESULTS: Rats in asthma modelgroup developed severe infiltration of inflammatory cells and inflammation in airway,together with a significantly up-regulated expression of UCN mRNA and peptide.CONCLUSION: Expression of UCN in lung tissues of asthmatic rats was morepronounced than that in normal control rats. Chapter 3 Effects of urocortin on lung vascular permeability in ratsOBJECTIVE: The enhanced pulmonary vascular permeability is an importantcharacter of asthma, The aim of this study was to investigate the effects of UCN onlung vascular permeability. METHODS: Lung vascular permeability was evaluatedby evans blue (EB) technique. Pulmonary congestion and edema were observed underlight microscope by HE staining. RESULTS: We found that rats receiving aninhalation aerosol of UCN had a significant elevation of lung vascular permeabilitycompared with rats receiving vehicle and OVA. UCN aerosol inhalation resulted inobvious pulmonary congestion and edema. Nonselective peptide CRH receptorantagonist astressin markedly reduced lung vascular permeability triggered by UCN.Enhanced pulmonary vascular permeability induced by UCN was markedly inhibitedby pretreatment with the mast cell stabilizer cromolyn and histamine-1 (H₁) receptorantagonist azelastine respectively, but not with the leukotriene receptor antagonistmontelukast. CONCLUSION: In the present study we firstly demonstrated that UCNcontributed to an increase in lung vascular permeability through activation of CRHreceptors. Mast cells and histamine might be involved in this effect of UCN.Chapter 4 Effects of urocortin on rat lung mast cells infiltrationand degranulationOBJECTIVE: UCN has proinflammatory peripheral effects possibly through mastcells. The purpose of this study was to investigate the effect of UCN on rat lung mastcell infiltration and degranulation. METHODS: Mast cell specific protease tryptase in lung tissues and expression of UCN in mast cells were examined byimmunohistochemistry. The activation and degranulation of mast cells were observedby Toluidine blue staining and transmission electron microscope. RESULTS: Theresults indicated that 10^-5 M UCN markedly promoted mast cell infiltration in rat lungtissues. This effect of UCN was attenuated by astressin and cromolyn. UCN (10^-5, 10^-6and 10^-7 M) significantly induced activation and degranulation of rat lung mast cellsin vitro. This effect was markedly inhibited by selective CRHR1 antagonistantalarmin, but not by specific CRHR2 antagonist anti-Svg-30. CONCLUSION: Thepresent study suggests that UCN promote infiltration of mast cells in rat lung throughCRHR and induced degranulation of rat lung mast cells via CRHR1.Chapter 5 Enhanced intracellular calcium induced by urocortin inrat lung mast cellsOBJECTIVE: To investigate the effect of UCN on intracellular calcium of rat lungmast cells. METHODS: The intracellular calcium was observed by confocal laserscanning microscopy and flow cytometry. RESULTS: The results showed that UCNcaused a rapid increase in [Ca²⁺]_i at the point of 300 s after UCN treatment, followedby a decrease to a sustained plateau phase. The increase in [Ca²⁺]_i induced by UCNwas significantly suppressed by antalarmin, but not by anti-Svg-30. This rerult wasconfirmed by flow cytometry. CONCLUSION: Taken together, our present studysuggested that UCN induced an increase in [Ca²⁺]_i of rat lung mast cells throughCRHR1. Chapter 6 Effect of dexamethasone on urocortin expression in ratlung with allergic asthmaOBJECTIVE: Glucocorticoid treatment markedly reduces the production of manyinflammatory mediators in asthmatic lung tissues. The purpose of this study was toexamine the effect of dexamethasone on UCN expression in rat lung with allergicasthma. METHODS: Airway inflammation of asthmatic rats was evaluated by HEstaining and inflammatory cell counting in BALF. The expression of UCN mRNAwas detected by semi-quantitative RT-PCR. UCN peptide was measured byimmunohistochemistry and western blot analysis. RESULTS: Rats in asthma modelgroup developed severe infiltration of inflammatory cells and inflammation in airway,together with a significantly up-regulated expression of UCN mRNA and peptide. Incontrast, treatment with dexamethasone resulted in markedly alleviated airwayinflammation and inflammatory cell infiltration, coupled with a significantlydecreased UCN expression. CONCLUSION: Dexamethasone treatment markedlyinhibited the enhanced expression of UCN in asthmatic lung tissues. Partâ…¡Effects of CRH & Urocortin on atherosclerosis inLDLR-/-miceAtherosclerosis is an underlying pathology of cardiovascular and cerebrovasculardisease, the leading cause of morbidity and mortality in developed countries.Atherosclerosis is influenced by multiple genetic and environmental factors thatinvolves a complex interplay between blood components and the artery wall. Since1993, the inflammation theory of Ross has been accepted by more and more people. Itis considered that atherosclerosis is a chronic inflammatory disease and involvesmonocytes, macrophages, mast cells and lymphocytes, together with a variety ofproinflammatory mediators and cytokines.By regulating hypothalamic-pituitary adrenal axis, centrally produced CRH andUCN exert profound immunosuppressive effects. However, peripheral CRH and UCN,through activating mast cells, lymphocytes and monocytes/macrophages, mayparticipate in the pathophysiology of many inflammatory conditions. Recent studiesindicate that peripherally circulating CRH, by influencing functions of monocytes andmacrophages, may lead to CRH (stress)-related endothelial dysfunction up tocardiovascular complications.This study aims to investigate the expression of CRH and UCN in atheroscleroticlesions of LDLR k/o mice, evaluate the effects of CRH and UCN on atherosclerosis. Chapter 1 Enhanced expression of CRH and urocortin in aorta ofLDLR-/-miceOBJECTIVE: To examine the expression profile of CRH and UCN inatherosclerotic aorta of LDLR-/- mice. METHODS: AS lesions were evaluated byHE staining. Serum lipids were also detected. The expression of CRH and UCNmRNA was detected by semi-quantitative RT-PCR. CRH and UCN peptides weremeasured by immunohistochemistry and ELISA analysis. RESULTS: After 8weeks of feeding on high lipid diet, LDLR-/- mice developed severe hyperlipoidemiawith TC, TG, LDL-C and HDL-C all increased and significant atherosclerotic lesionscompared to C57BL/6J mice (P＜0.01). The expression of CRH and UCN mRNA andpeptides was significantly increased in atherosclerotic aorta of LDLR-/- mice; ELISAresults indicated that CRH and UCN peptides in serum of LDLR-/- mice were alsomarkedly elevated. CONCLUSION: The expression of CRH and UCN inatherosclerotic aorta of LDLR-/- mice was significantly increased.Chapter 2 Effects of CRH and urocortin on atherosclerosis inLDLR-/- miceOBJECTIVE: To observe effects of CRH and UCN on atherosclerosis in LDLR-/-mice. METHODS: AS lesions were evaluated by HE staining. Serum lipids were alsodetected. MDA, SOD activity and TAC in serum were detected by chemicalcolorimetry. RESULTS: After 8 weeks of high lipid diet, LDLR-/- mice developedsevere hyperlipoidemia with TC, TG, LDL-C and HDL-C all increased and significant atherosclerotic lesions compared to C57BL/6J mice. Mice supplementedwith CRH developed more serious atherosclerotic lesions, while UCN had no obviousinfluence on atherosclerosis in LDLR-/- mice. Neither CRH nor UCN changed thelevels of serum lipids. In LDLR-/- mice, the body weight increased more rapidly,serum MDA was elevated and TAC markedly decreased. CRH and UCN had nosignificant influence on body weight, serum MDA, SOD activity and TAC ofLDLR-/- mice. CONCLUSION: CRH accelerated the progression of atherosclerosisin LDLR-/- mice. The proatherosclerotic effect of CRH was not dependent onincreased serum lipids and decreased antioxidative ability.Chapter 3 CRH promoted atherosclerosis in LDLR-/- mice viaCRHR1OBJECTIVE: To observe the effect of CRH on atherosclerosis in LDLR-/- mice andreceptor mechanisms. METHODS: AS lesions were evaluated by HE staining. Serumlipids were also detected. MDA, SOD activity and TAC in serum was detected bychemical colorimetry. RESULTS: After 8 weeks of feeding on high lipid diet,LDLR-/- mice developed severe hyperlipoidemia and significant atheroscleroticlesions compared to C57BL/6J mice. CRH significantly promoted the progression ofatherosclerosis in LDLR-/- mice. The proatherosclerotic effect of CRH was attenuatedby pretreatment with selective CRHR1 antagonist NBI27914. Howerer, specificCRHR2 antagonist antisauvagine-30 has no significant influence on atherosclerosisaggravated by CRH in LDLR-/- mice. CRH, NBI27914 and Anti-svg-30 did not change the levels of serum lipids in LDLR-/- mice. In LDLR-/- mice, the body weightincreased more rapidly, serum MDA was elevated and TAC markedly decreased. CRH,NBI27914 and Anti-svg-30 had no significant influence on body weight, serum MDA,SOD activity and TAC of LDLR-/- mice. CONCLUSION: CRH accelerated theprogression of atherosclerosis in LDLR-/- mice. This proatherosclerotic effect of CRHmight be mediated by CRHR1.Chapter 4 CRH increased IL-6 level & VCAM-1 expression andactivated NF-kB in LDLR-/- miceOBJECTIVE: To examine the effects of CRH on IL-6 level, VCAM-1 expressionand NF-kB activation in LDLR-/- mice. METHODS: The level of IL-6 in serum andaorta was detected by ELISA. The expression of VCAM-1 mRNA was detected bysemi-quantitative RT-PCR. VCAM-1 expression and NF-κB activation weremeasured by immunohistochemistry analysis. RESULTS: The levels of IL-6 inserum and aorta were raised by CRH subcutaneous injection. CRH significantlyup-regulated the expression of VCAM-1 and markedly increased activation of NF-κBin aortas. These effects were largely attenuated by CRHR1 antagonist NBI27914 butnot by specific CRHR2 antagonist anti-Svg-30. CONCLUSION: CRH elevated thelevels of IL-6 in serum and aorta, up-regulated the expression of VCAM-1 andincreased activation of NF-κB in aortas through CRHR1. These effects might be someimportant molecular mechanisms by which CRH accelerated the progression ofatherosclerosis in LDLR-/- mice. Chapter 5 Effects of CRH on monocyte/macrophage, lymphocyteand mast cell infiltration in aortas of LDLR-/- miceOBJECTIVE: To investigate the effects of CRH on monocyte/macrophage,lymphocyte and mast cell infiltration in aortas of LDLR-/- mice. METHODS: Theexpression of CD68,CD3 and Tryptase in aortas was measured byimmunohistochemistry. RESULTS: In LDLR-/- mice, the momocytes adhered toendothelial cells and macrophages infiltrated in intima increased markedly; the mastcells and lymphocytes infiltrated in adventitia also increased significantly. CRHmarkedly promoted the infiltration of momocytes, macrophages and mast cells inaortas in LDLR-/- mice. These effects of CRH were largely suppressed by CRHR1antagonist NBI27914 but not by CRHR2 antagonist anti-Svg-30. The number oflymphocytes in aortic adventitia was not changed by CRH. CONCLUSION: CRHpromoted the infiltration of monocytes/macrophages and mast cells in aortas ofLDLR-/- mice via CRHR1, which might be some important cell mechanisms in theproatherosclerotic effect of CRH in LDLR-/- mice.