Objective To determine whether aquaporin-4 (AQP4) regulates acute lesions, delayed lesions, and the associated microglial activation after cryoinjury to the brain. Methods Brain cryoinjury was applied to AQP4 knockout (KO) and wild-type mice. At 24 h and on days 7 and 14 after cryoinjury, lesion volume, neuronal loss, and densities of microglia and astrocytes were determined, and their changes were compared between AQP4 KO and wild-type mice. Results Lesion volume and neuronal loss in AQP4 KO mice were milder at 24 h following cryoinjury, but worsened on days 7 and 14, compared to those in wild-type mice. Besides, microglial density increased more, and astrocyte proliferation and glial scar formation were attenuated on days 7 and 14 in AQP4 KO mice. Conclusion AQP4 deficiency ameliorates acute lesions, but worsens delayed lesions, perhaps due to the microgliosis in the late phase.

G protein-coupled receptor 17 (GPR17), the new P2Y-like receptor, is phylogenetically related to the P2Y and cysteinyl leukotriene receptors, and responds to both uracil nucleotides and cysteinyl leukotrienes. GPR17 has been proposed to be a damage sensor in ischemic stroke; however, its role in brain inflammation needs further detailed investigation. Here, we extended previous studies on the spatiotemporal profiles of GPR17 expression and localization, and their implications for brain injury after focal cerebral ischemia. We found that in the ischemic core, GPR17 mRNA and protein levels were upregulated at both 12-24 h and 7-14 days, but in the boundary zone the levels increased 7-14 days after reperfusion. The spatiotemporal pattern of GPR17 expression well matched the acute and late (subacute/chronic) responses in the ischemic brain. According to previous findings, in the acute phase, after ischemia (24 h), upregulated GPR17 was localized in injured neurons in the ischemic core and in a few microglia in the ischemic core and boundary zone. In the late phase (14 days), it was localized in microglia, especially in activated (ED1-positive) microglia in the ischemic core, but weakly in most microglia in the boundary zone. No GPR17 was detectable in astrocytes. GPR17 knockdown by a small interfering RNA attenuated the neurological dysfunction, infarction, and neuron loss at 24 h, and brain atrophy, neuron loss, and microglial activation at 14 days after reperfusion. Thus, GPR17 might mediate acute neuronal injury and late microgliosis after focal cerebral ischemia.

Cysteinyl leukotrienes (CysLTs), potent inflammatory mediators, are released from ischemic brain, and may regulate ischemic injury through activating CysLT(1) and CysLT(2) receptors. The CysLT(1) receptor is closely associated with ischemic injury and post-ischemic repair; however, the CysLT(2) receptor-mediated responses remain unknown. Here, we investigated the spatiotemporal profiles and implications of CysLT(2) receptor expression and localization in rat brain after focal cerebral ischemia. CysLT(2) receptors were normally localized in astrocytes in the cortex and around the ventricles. After focal cerebral ischemia, CysLT(2) receptor expression was up-regulated in concert with neuronal and glial responses. In the acute phase (6-24 h), up-regulated CysLT(2) receptors were restricted to injured neurons in the ischemic core; while in the late phase (3-28 days), the up-regulation was restricted to hypertrophic microglia (ischemic core) and mainly localized in hypertrophic astrocytes (boundary zone). Thus, the spatiotemporal profiles of CysLT(2) receptor expression suggest that it plays regulatory roles in acute neuron injury, and astrocytosis and microgliosis in the late phase.

OBJECTIVES Previously we demonstrated the neuroprotective effect of montelukast, a cysteinyl leukotriene receptor-1 (CysLT(1)) antagonist, on acute brain injury after focal cerebral ischaemia in mice. In this study, we have determined its effect on chronic brain injury after focal cerebral ischaemia in mice and rats. METHODS After transient focal cerebral ischaemia was induced by middle cerebral artery occlusion, montelukast was intraperitoneally injected in mice or orally administered to rats for five days. Behavioural dysfunction, brain infarct volume, brain atrophy and neuron loss were determined to evaluate brain lesions. KEY FINDINGS Montelukast (0.1 mg/kg) attenuated behavioural dysfunction, brain infarct volume, brain atrophy and neuron loss in mice, which was similar to pranlukast, another CysLT(1) receptor antagonist. Oral montelukast (0.5 mg/kg) was effective in rats and was more effective than edaravone, a free radical scavenger. CONCLUSION Montelukast protected mice and rats against chronic brain injury after focal cerebral ischaemia, supporting the therapeutic potential of CysLT(1) receptor antagonists.

HASH(0x2b47e3d04b70)

Gain-of-function mutations of JAK2 play crucial roles in the development of myeloproliferative neoplasms; however, the underlying downstream events of this activated signaling pathway are not fully understood. Our experiment was designed and performed to address one aspect of this issue. Here we report that AG490, a potent JAK2V617F kinase inhibitor, effectively inhibits the proliferation of HEL cells. Interestingly, AG490 also decreases the expression of PTTG1, a possible target gene of the aberrant signaling pathway, in a dose- and time-dependent manner. Furthermore, the promoter activity analyses reveal that the inhibition of the PTTG1 expression is affected at the transcriptional level. Thus, our results suggest that the JAK2V617F/STAT5 signaling pathway promotes cell proliferation through the transcriptional activation of PTTG1.

OBJECTIVE: To prepare and identify a polyclonal antibody against cysteinyl leukotriene receptor (CysLT(2)receptor). METHODS: Rabbits were immunized with KLH-coupled CysLT(2)receptor peptide to prepare the polyclonal antibody (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. The tissue distribution of CysLT(2) receptor was detected by Western blot and immunohistochemistry with the prepared pAb. RESULT: The pAb showed a titer higher than 1/1047296, and was specific to CysLT(2) receptor, without cross-reaction with the antigens of CysLT(1) receptor and GPR17. A higher expression of CysLT(2) receptor in kidney, brain and lung of rats and mice was detected by Western blot analysis using the prepared pAb. The molecular weight of CysLT(2) receptor protein was about 40 kD. Immunohistochemical examination showed that CysLT(2) receptor was expressed mainly in the neuron, and partly in astrocytes in rat brain. CONCLUSION: The prepared CysLT(2) receptor pAb has high sensitivity and specificity, and can be used in Western blot and immunohistochemistry.

OBJECTIVE: To prepare and identify a polyclonal antibody (pAb) against GPR17, a novel 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 1:16 364,and was not cross-reacted with the antigens of CysLT(1) and CysLT(2) receptors. A higher expression of GPR17 in the rat brain and heart was detected 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.

ETHNOPHARMACOLOGICAL RELEVANCE: Quality analysis and control of Chinese herbal medicines (CHM) or herbal medicines (HM) are being more and more investigated based on fingerprint analysis, and there are also some researches on correlating fingerprints of CHM to their efficacy. Multi-component analysis methods together with fingerprints are considered potential useful tools to select candidate herbal drugs from extracts of herbs in pharmacological/bio-prospecting investigations. AIM OF THE STUDY: To explore a strategy for evaluating efficacy strength of CHM samples based on their spectra fingerprints and validate it. METHODOLOGY: Radix bupleuri (RB), a typical Chinese medicinal herb for relieving exterior syndrome, and Flos lonicerae (FL), Fructus forsythiae (FF), and Radix isatidis (RI) that are widely applied Chinese herbs for heat clearing and detoxifying, were selected as herbal sources. The aqueous extracts, volatile oils and mixtures of the extracts and oils of the four herbs, plus Ibuprofen suspension (IS), Shuanghuanglian oral liquid (SHL), mixture of SHL and the volatile oils of FL and FF, were used for subject samples to do antipyretic experiments on rats. Ultraviolet spectra were used as the spectra fingerprints to represent chemical characteristics of the samples. Principal component analysis (PCA) and canonical correlation analysis (CCA) were adopted as evaluation tools to establish the correlation between pharmacological and spectra data, from which a spectral index for evaluating antipyretic effects of CHM samples was constructed. Furthermore, four compound samples were designed by mixing 50% volatile oils and 50% aqueous extracts of the four herbs with different ratios to validate the strategy. RESULTS: Efficacy sequence of the 15 calibrating and 4 validating CHM samples, defined by the first canonical correlative variable U(1) of their UV spectra, was consistent with that given by pharmacological experiments. CONCLUSIONS: The strategy proposed in this study could be applied to evaluate efficacy strength of CHM and helpful for screening candidate herbal drugs from different herbs or prepared by different technologies.

OBJECTIVE: To detect the level of serum and liver tissue TGF-beta1 in patients with chronic hepatitis B, to study their relation to liver fibrosis and gain the evidence for diagnosis of liver fibrosis. METHODS: The liver fibrosis grades (S0-S4) of 131 cases with chronic HBV infection were diagnosed after liver biopsy. Serum TGF-beta1 was detected by enzyme-linked immunosorbent assay, and the semiquantitative analysis was applied after detecting the expression of TGF-beta1 in liver tissue with immunohistochemistry. Their relations to liver fibrosis were analyzed. RESULTS: Serum and tissue level of TGF-beta1 increased significantly with the development of fibrosis, and the same result was obtained between themselves (P < 0.01). There was very significant difference for serum level of TGF-beta1 among the groups with different fibrosis grades (P < 0.01). Serum levels of TGF-beta1 were decreased significantly comparing the Group S0 or S1 to S4 (P < 0.005). There were significant difference for serum level of TGF-beta1 among S0 and the others (P < 0.005). And there was significant difference between S1 and S3 (P < 0.005). The expression level of TGF-beta1 in liver tissue has no significant difference between group S3 and S4 (P > 0.05). However, the differences were significantly among the other comparisons (P < 0.01). CONCLUSION: There is close relation between the level of TGF-beta1 and the different liver fibrosis grades due to chronic hepatitis B. The serum level of TGF-beta1 is a potential noninvasive maker for diagnosis of liver fibrosis.