Study On The Protective Effect Of HSPA12A On Ischemic Stroke

Background and Purpose:Stroke is a major serious disease with high mortality and morbidity. Identification of novel prosurvival factors in the brain is paramount for developing neuroprotective therapies for stroke. Heat shock proteins form a stress protein family, which is induciable after environmental stimulation. Heat shock protein A12A (HSPA12A) is a newly discovered member of Hsp70 family protein. Evidence has shown a most abundant of HSPA12A mRNA in mammalian brains. However, it is unclear about the roles of HSPA12A. In this study, we examined whether HSPA12A is involved in ischemic stroke by using hspal2a conditional knockout mice.Methods:1. Locolization of HSPA12A in brains:The localization of HSPA12A protein was examined by immunoflueresence costaining HSPA12A with neuronal marker NeuN or β-Ⅲ Tublin.2. Expression of HSPA12A after stroke:Stroke model was induced by left middle cerebral artery occlusion (MCAO) for 1 h followed by various periods of reperfusion in mice in vivo, and oxygen-glucose deprivation for 2 h and reoxygenation for 24 h in primary neurons in vitro. HSPA12A mRNA was examined by real-time PCR and HSPA12A protein by immunoblotting.3. Creation of hspal2a conditional knockout mice:Conditional knockout mice of hspal2a was created by Cre-loxp recombinant system. To delete HSPA12A gene, conditional HSPA12A knockout mice were breeded with EⅡa-Cre transgenic mice.4. Confirmation of hspal2a gene knockout:Polymerase Chain Reaction (PCR) was used to genotype, Western Blot (WB) for the protein expression level of HSPA12A, real-time PCR for HSPA12A and Immunofluorescence (IF) for detecting the HSPA12A protein in brain tissues.5. Evaluation of brain ischemic injury:The cerebral ischemic injury was examined by infarct volumes (Triphenyltetrazolium Chloride staining), neurological scoring, neuronal damages (Hemotoxylin&Eosin Staining and Nissl staining), neuronal apoptosis (TUNEL staining and WB for the expression level of Bax and Bcl-2).6. Statistic analysis:Results are expressed as mean±standard deviation (X±SD). Comparison analysis between groups was performed using a one-way analysis of variance (ANOVA). Tukey’s procedure for multiple range tests was performed. P<0.05 was considered to be significant.Results:1. HSPA12A is expressed prominently in brains and neurons2. HSPA12A is downregulated in ischemic brains and hypoxia primary neurons3. Deficiency of hspa12a enlarges infarct volumes after stroke4. Deficiency of hspa12a exacerbates neurological deficits after stroke5. Deficiency of hspa12a aggravates hippocampal damages after stroke6. Deficiency of hspa12a aggravates apoptosis in brains after strokeConclusions:HSPA12A is expressed prominently in brains and neurons and is essential for the protection against ischemic stroke.