RESUMO
Objective To investigate the effect of tamibarotene(Am80)on cerebral ischemia reperfusion injury. Methods Rats were randomly divided into sham operation group(Sham),model group(I/R) and tamibarotene in-tervention group (Am80, intragastric administration of Am80 6 mg/kg). The middle cerebral artery occlusion (MCAO) was established by suture method. At 24 h after reperfusion,the brain was removed. And the neurological behavioral score was assessed by double blind method.The cerebral infarction volume was measured by TTC staining. The proteins of RARα,Bcl-2,Bax and mRNA were examined by Western blot and RT-qPCR,respectively. Results Am80 significantly improved the neurological deficits of MCAO rats and effectively reduced the infarct volume. And upregulated the level of RARα and Bcl-2, decreased the expression of Bax. Conclusions Am80 has a protective effect on cerebral ischemia-reperfusion in rats,and its effect may be related to anti-apoptosis.
RESUMO
Objective To investigate the effect of tamibarotene(Am80)on cerebral ischemia reperfusion injury. Methods Rats were randomly divided into sham operation group(Sham),model group(I/R) and tamibarotene in-tervention group (Am80, intragastric administration of Am80 6 mg/kg). The middle cerebral artery occlusion (MCAO) was established by suture method. At 24 h after reperfusion,the brain was removed. And the neurological behavioral score was assessed by double blind method.The cerebral infarction volume was measured by TTC staining. The proteins of RARα,Bcl-2,Bax and mRNA were examined by Western blot and RT-qPCR,respectively. Results Am80 significantly improved the neurological deficits of MCAO rats and effectively reduced the infarct volume. And upregulated the level of RARα and Bcl-2, decreased the expression of Bax. Conclusions Am80 has a protective effect on cerebral ischemia-reperfusion in rats,and its effect may be related to anti-apoptosis.
RESUMO
AIM: The accumulation of disease-causing proteins is a common hallmark of many neurodegenerative disorders. Measuring the degradation of such proteins using high-throughput-compatible assays is highly desired for the identification of genetic and chemical modulators of degradation. For example, Huntington's disease (HD) is an incurable hereditary neurodegenerative disorder caused by the cytotoxicity of mutant huntingtin protein (mHTT). The high-throughput measurement of mHTT degradation is important in HD drug discovery and research. Existing methods for such purposes have limitations due to their dependence on protein tags or pan protein synthesis inhibitors. Here, we report a high-throughput-compatible pulse-chase method (CH-chase) for the measurement of endogenous tag-free huntingtin protein (HTT) degradation based on Click chemistry and Homogeneous Time Resolved Fluorescence (HTRF) technologies. METHODS: The pulsed-labeled proteins were conjugated with biotin using the click reaction strain-promoted alkyne-azide cycloaddition (SPAAC), and the chase signals were calculated by measuring the reduction percentage of the HTT HTRF signals after pull-down with streptavidin beads. RESULTS: We validated that the signals were within the linear detection range and were HTT-specific. We successfully measured the degradation of endogenous HTT in a high-throughput-compatible format using 96-well plates. The predicted changes of HTT degradation by known modifiers were observed, which confirmed that the assay is suitable for the identification of HTT degradation modifiers. CONCLUSION: We have established the first high-throughput-compatible assay capable of measuring endogenous, tag-free HTT degradation, providing a valuable tool for HD research and drug discovery. The method could be applied to other proteins and can facilitate research on other neurodegenerative disorders and proteinopathies.
