Lipoxin A₄Methyl Ester Alleviates Vascular Cognition Impairment In Rats By Regulating The Expression Of Autophagy And ER Stress-related Proteins In Hippocampus

Objective: There are abundant evidences that autophagy and ER stressmechanisms participate in some cerebral vascular pathological conditions (forexample, ischemia/reperfusion). As one of the critical risk factors to inducevascular cognitive impairment (VCI), whether chronic cerebral hypoperfusion(CCH) changes the expression of autophagy and ER stress-related proteins isstill unknown. The present study will discuss the mechanisms above andexplore the intervention effects of lipoxin A4methyl ester (LXA4ME).Methods: Male Spague-Dawley rats aged3mouths were subjected to thecannulae implantation operation one week before the bilateral common carotidartery ligation/two vessel occlusion (BCCAL/2VO) surgery. The animalswere randomly divided into4groups, i.e., sham+saline group,2VO+salinegroup,2VO+LXA4ME (10ng/d, i.c.v.) group and2VO+LXA4ME (100ng/d,i.c.v.) group. The drug delivery was conducted by hand for14days.The Morris water maze test was conducted at day15-20, including theplace navigation test and the spatial probe test. The perpose of the test was toevaluate the spatial learning and memory abilities of rats. After the behavioraltest, HE staining was used to observe the pathological changes in thehippocampus. Western blot and real time-PCR technology were applied todetect GRP78/Bip, CHOP, beclin1and LC3B expression in hippocampustissue. mTOR and p-mTOR were detected via western blot assay. RegularRT-PCR was performed to analyze the splicing level of XBP-1mRNA.Results:1In the Morris water maze, all the animals showed a progressive decline inthe escape latency with training. The rats in2VO+saline group exhibitedsignificant prolonged escape latencies during the5-day training period compared with the sham+saline group. After the LXA4ME treatment, however,the poor performance was mitigated. These two groups showed shorter escapelatencies compared with the2VO+saline group, with statistic significance. Onthe probe trial, with the platform removed, there were significant differencesamong the groups. The2VO+saline group spent significantly less time in thetarget quadrant than the control group. In LXA4ME10ng/d and100ng/dgroups, the deficits were significantly improved.2HE staining observation: The pyramidal cells in the hippocampal CA1andCA3areas of the sham+saline group were dense and arranged in neat rows.The morphology was integrity and clear. The nuclei in these area were largerand rounder, with clear nucleolus. Compared with the control, the2VO+salinegroup demonstrated loosely arranged pyramidal cells. Some pyramidalneurons turned karyopyknosis, with the nucleolus lost, cytoplasm pyknoticand hyperchromatic. While treatment with LXA4ME partly alleviated thehippocampus damage exhibited in the2VO+saline group.3Protein and gene expression:3.1Western blot and regular RT-PCR results: Compared with the sham+salinegroup, p-mTOR was downregulated with statistical significance (P<0.01).There was also a decline in mTOR expression, without statistical significance(P>0.05). Beclin1，the ratio of LC3-II/LC3-I and CHOP were significantlyincreased (P<0.01, P<0.01and P<0.05, respectively). GRP78/Bip protein inthe2VO+saline group slightly decreased with no statistical significance(P>0.05). The splicing level of XBP-1mRNA was not changed in2VO group(P>0.05).3.2Western blot and regular RT-PCR results: Compared with the2VO+salinegroup, treatment with10ng/d LXA4ME downregulated beclin1, reducedLC3-II/LC3-I ratio and upregulated p-mTOR statistical significantly (P<0.05).There were no statistical significance of the protein expression of GRP78/Bip,CHOP and XBP-1mRNA splincing in LXA4ME10ng/d treatment group(P>0.05).3.3Western blot and regular RT-PCR results: Compared with the2VO+saline group, treatment with100ng/d LXA4ME downregulated beclin1, reducedLC3-II/LC3-I ratio and upregulated p-mTOR statistical significantly (P<0.05,P<0.01, P<0.01, respectively). The LXA4ME100ng/d treatment groupshowed increased expression of GRP78/Bip, spliced XBP-1mRNA anddecreased CHOP (P<0.01).3.4RT-qPCR results: Real time-PCR showed similar changes of mRNA(except GRP78/Bip) to the corresponding proteins. There were not statisticaldifferences among groups (P>0.05).Conclusions: Autophagy and ER stress related mechanisms maybe involved in the process of CCH. LXA4ME can effectively improvethe cognitive functions of CCH rats and alleviate the pathological injuriesof the hippocampal region. The cerebral protective effect of LXA4ME mightbe achieved by regulating autophagy and ER stress related proteins.