Research Overview and Background

With the acceleration of global aging, the incidence of ischemic cerebrovascular diseases is also rising significantly. Elderly people over 65 are particularly susceptible to cognitive decline caused by cerebral ischemia, making Chronic Cerebral Hypoperfusion (CCH) a major cause of cognitive impairment and Vascular Dementia (VAD). According to literature, VAD has become the second most common cause of dementia in North America and Europe, accounting for about 15% to 20%; in Asia, about 30% of dementia cases are caused by VAD. Therefore, in-depth study of memory dysfunction caused by CCH and finding effective therapeutic targets is of great significance for public health.

Existing studies have shown that long-term insufficient cerebral blood flow activates inflammatory responses, oxidative stress, and neurodegeneration, leading to neuronal damage. The Endoplasmic Reticulum (ER) is one of the important organelles involved in protein synthesis, processing, and transport. ER stress is an important factor in determining the fate of neurons during cerebral ischemia. Excessive ER stress is usually caused by brain trauma, ischemia-reperfusion, and stroke, causing irreversible neuronal damage.

The purpose of this study is to explore the role and related mechanisms of the fatty acid amide hydrolase inhibitor URB597 in inhibiting ER stress and thus improving cognitive impairment caused by CCH. URB597 has previously shown antioxidant properties in various neurological diseases, but its specific mechanism is not yet fully understood.

Research Source

This article was published in the Journal of Neuroimmune Pharmacology (2024) Volume 19, Issue 1, with authors including researchers from different research institutions: Da Peng Wang, Kai Kang, Jian Hai, Qiao Li Lv, and Zhe Bao Wu.

Research Methods and Procedures

Experimental Design

Cell Experiments

The study first used mouse hippocampal HT-22 neuronal cell line to simulate cerebral ischemia environment through Oxygen-Glucose Deprivation (OGD). The cells were divided into the following groups: control group (Con), OGD group (OGD), OGD+4-phenylbutyric acid (ER stress inhibitor, 4-pba), OGD+URB597 (URB), OGD+combination treatment (URB597+4-pba).

Cells were treated with URB597 at 2µM and 4-pba at 4µM. MTT assay was used to detect cell viability, and Annexin V-FITC/PI flow cytometry was used to analyze cell apoptosis. In addition, Western Blotting was used to evaluate the expression changes of ER stress markers and apoptosis-related proteins.

Animal Experiments

One-month-old male Sprague-Dawley rats were used to establish a chronic cerebral hypoperfusion model (through bilateral common carotid artery ligation, BCCAO). After BCCAO surgery, they were divided into the following treatment groups for comparison: (1) BCCAO group (M), (2) BCCAO+URB597 group (MU), (3) BCCAO+4-pba group (MP), (4) BCCAO+URB597 combined with 4-pba group (MUP), and (5) BCCAO+URB597 combined with 4-pba and AM630 (CB2 antagonist) group (MUPA).

Morris water maze test was used to evaluate cognitive function, transmission electron microscopy (TEM) was used to observe the ultrastructural changes of mitochondria and endoplasmic reticulum in the hippocampal area, and Western Blotting was used to analyze the expression of CB2 and ERS pathway-related proteins.

Experimental Results

Cell Experiment Results

Oxygen deprivation led to significant apoptosis and decreased survival rate of HT-22 cells. After intervention with 4-pba and URB597, cell survival rate increased and apoptosis rate decreased significantly, indicating that inhibition of ER stress can improve cell survival. Meanwhile, immunofluorescence and Western Blotting detection found that OGD treatment significantly increased the expression of ER stress-related markers GRP78, ATF6, and CHOP, and this increase was inhibited after URB597 intervention.

Animal Experiment Results

Nissl staining revealed significant neuronal damage in the hippocampal tissue of BCCAO rats, with reduced Nissl body volume; after URB597 and 4-pba intervention, neuronal damage was significantly improved. Water maze test results showed that BCCAO rats performed significantly worse in spatial memory tasks than normal rats, while the cognitive function of URB597 and 4-pba treatment groups improved, indicating that inhibition of ER stress alleviated cognitive dysfunction caused by CCH.

In TEM observation, the mitochondria and endoplasmic reticulum boundaries in the hippocampal CA1 region of URB597-treated rats were clear, without obvious swelling and damage, indicating that URB597 has a protective effect on mitochondria and endoplasmic reticulum.

Role of CB2/β-arrestin1 Signaling Pathway

Western Blotting showed that URB597 upregulated the expression levels of CB2 and β-arrestin1 in rat hippocampus, and reduced the expression of ER stress and mitochondrial apoptosis-related proteins such as CHOP, Cyt-c, and Caspase-9. After intervention with CB2 antagonist AM630, these protective effects were inhibited, suggesting that URB597 exerts its protective effect through the CB2/β-arrestin1 signaling pathway.

Research Summary and Significance

This study reveals that the fatty acid amide hydrolase inhibitor URB597 significantly improves cognitive dysfunction caused by chronic cerebral hypoperfusion by inhibiting ER stress and mitochondrial dysfunction through the CB2/β-arrestin1 mediated signaling pathway. This finding further enriches our understanding of the molecular mechanisms of cognitive impairment caused by CCH and provides theoretical basis for CB2 as a potential therapeutic target.

This study not only expands our understanding of URB597 in cerebral ischemia protection but also provides reliable evidence for the development of new drug treatment strategies in the future. In addition, research on the interconnection between ER and mitochondria, especially the compensatory mechanisms of dysfunction, helps to better understand CCH and its potential prevention strategies, thereby reducing the social and economic burden of cognitive impairment globally.