Part1,Cysteinyl Leukotrienes Receptor Modulates Aquaporins 4 Expression After Oxygen-glucose Deprivation-induced Injury In Rat Astrocytes Part2,Preparation And Identification Of A Polyclonal Antibody Against GPR17, A New Cysteinyl Leukotriene Receptor

Part 1 Cysteinyl leukotrienes receptor modulates aquaporins 4 expression after oxygen-glucose deprivation-induced injury in rat astrocytesObjective:To determine whether cysteinyl leukotrienes(CysLT) receptor modulates aquaporin 4(AQP4) expression and cell injury after oxygen-glucose deprivation/recovery(OGD/R) in rat astrocytes,and the possibly related signaling pathway.Methods:(1) In vitro ischemic-like injury in primary cultures of rat astrocytes was induced by various durations of oxygen-glucose deprivation(OGD) and 24-h recovery. Cell viability of astrocytes was measured by MTT reduction assay and LDH release, and a suitable OGD duration was evaluated to establish OGD/R injury model.(2) By RT-PCR and immunocytochemistry,the expression of CysLT₁ and CysLT₂ receptors after OGD/R was detected.(3) By determination of MTT reduction and LDH release, the effects of CysLT receptor antagonists on OGD/R injury were evaluated;by Western blotting and immunocytochemistry,AQP4 expression in astrocytes was determined after OGD/R.(4) Inhibition of AQP4 expression was induced by AQP4 siRNA to further confirm the role of AQP4 in OGD/R injury.(5) Finally,the MAPK signaling in OGD/R injury and AQP4 expression was examined by Western blotting determination of MAPK molecule phosphorylation,and by observation of the effects of MAPK inhibitors.Results:OGD/R induced astrocyte injury in an OGD duration-dependent manner;the injury gradually became more serious with elongation of OGD treatment.After 4-h OGD and 24-h recovery,the viability of astrocytes was reduced to 65%～70%of baseline level.This injury condition was used in the following experiments.Under normal culture,the CysLT₁ receptor was moderately expressed in control astrocytes, while the CysLT₂ receptor expression was much weaker.After OGD/R,the CysLT₂ receptor expression was increased,but the CysLT₁ receptor expression did not change. OGD/R-induced cell injury was inhibited by CysLT₂ receptor antagonist(Bay cysLT2) and CysLT receptor non-selective antagonist(Bay u9773),but not by the CysLT₁ receptor antagonist(montelukast).This indicated that CysLT₂ receptor mediated OGD/R injury in astrocytes.Meanwhile,the expression of AQP4 increased after OGD/R,and was also inhibited by CysLT₂ receptor antagonist,but not by CysLT₁ receptor antagonist.AQP4 siRNA inhibited AQP4 expression and attenuated OGD/R injury,suggesting that up-regulated AQP4 might aggravate astrocyte ischemic-like injury.The phosphorylation of ERK1/2 and p38 MAPK was increased after 4-h OGD, and ERK1/2 and p38 MAPK inhibitors relieved the cell injury and AQP4 up-regulation.Conclusion:1.The CysLT₂ receptor mediates astrocyte injury induced by OGD 4 h/R 24 h.2.After OGD 4 h/R 24 h injury,the CysLT₂ receptor mediates up-regulation of AQP4 expression,and the up-regulated AQP4 aggravates astrocyte OGD 4 h/R 24 h induced cell injury.3.Activation of ERK1/2 and p38 MAPK is involved in the regulation of CysLT₂ receptor-mediated AQP4 up-regulation and OGD 4 h/R 24 h cell injury. Part 2 Preparation and identification of a polyclonal antibody against GPR17,a new cysteinyl leukotriene receptorObjective:To prepare and identify a polyclonal antibody(pAb) against GPR17,a new cysteinyl leukotriene receptor.Methods:Rabbits were immunized with KLH-coupled GPR17 peptide to prepare the pAb.The titer of the pAb in rabbit plasma was detected by indirect ELISA,and the specificity of the pAb was tested by antigen blockade.GPR17 tissue distribution was detected by Western blot with the pAb.Results:The pAb showed a titer as high as 1:16364,and was not cross-reacted with the antigens of CysLT₁ and CysLT₂ receptors.We detected a higher expression of GPR17 in rat brain and heart using the newly prepared pAb.The molecular weigh of GPR17 protein was about 43 kD.Conclusion:The prepared GPR17 pAb has high sensitivity and specificity,and can be used in Western blot for detecting GPR17.