Background Vascular dementia (VD) is one of the refractory neurological diseases, and its pathogenesis remains unclear. Therefore, it is crucial to explore key targets for the treatment of VD.
Objective To investigate the intervention effect of Yifei Xuanfei Jiangzhuo prescription (YFXFJZF) on VD rats based on the AMP-activated protein kinase (AMPK)/mechanistic target of rapamycin (mTOR)/Unc-51-like autophagy activating kinase 1 (ULK1) pathway.
Methods A total of 70 8-week-old SPF-grade male SD rats were used, with 10 assigned to the normal group (K group). The remaining rats were used to establish a VD rat model via bilateral common carotid artery ligation. After successful modeling, the rats were randomly divided into the VD model group (M group), YFXFJZF group (Z group), autophagy inhibitor group (Y group), autophagy activator group (D group), autophagy inhibitor+YFXFJZF group (YZ group), and VD model group+autophagy activator+YFXFJZF group (DZ group), with 10 rats in each group. The Z group was administered YFXFJZF at a dose of 1.2 g/kg via gavage. The K and M groups were given an equal volume of normal saline via gavage. Starting from day 15 of the experiment, the Y group was given intraperitoneal injections of 3-methyladenine (3-MA) at 1.5 mg/kg every other day for 14 days, and the D group was given intraperitoneal injections of rapamycin at 2 μg/kg every other day for 14 days. The intervention lasted for 28 days in total. After behavioral testing, blood samples and hippocampal tissues were collected from the rats. Hematoxylin-eosin (HE) staining was used to observe the pathological changes in the hippocampal tissue. Lead citrate staining (electron microscopy) was used to observe the condition of hippocampal neurons and autophagosomes. Western blotting was used to detect the protein expression of AMPK, p-AMPK, mTOR, p-mTOR, ULK1, phospho-Ser757-Unc-51-like autophagy activating kinase 1 (p-S757-ULK1), Microtubule-associated protein 1 light chain 3 (LC3), and P62 in the hippocampus. Real-time quantitative PCR was used to detect the mRNA expression of AMPK, mTOR, ULK1, LC3, and P62 in the hippocampus. Data conforming to a normal distribution were expressed as (±s). For comparisons among multiple groups with homogeneous variances, one-way analysis of variance was used, and the LSD method was used for pairwise comparisons between groups.
Results Behavioral testing showed that compared with the K group, the escape latency of VD rats in the M group was significantly prolonged, and the number of platform crossings was significantly reduced (P<0.05). Compared with the M group, the escape latency was shortened in the Z, Y, D, YZ, and DZ groups, and the number of platform crossings was increased (P<0.05). HE staining showed that the cells in the CA1 region of the hippocampus in the K group were neatly arranged with normal morphology and structure. The M group had severe damage, with deeper cell staining, unclear boundaries between nuclei and cytoplasm, cytoplasmic vacuolization, and irregular shapes. The D, DZ, and YZ groups showed some repair of the damaged hippocampal tissue compared with the M group, while the Z and Y groups showed more obvious recovery of the damaged hippocampal tissue. Lead citrate staining showed that the neuronal cell membranes in the CA1 region of the hippocampus in the K group were intact and smooth, with clear nucleoli, evenly distributed chromatin in the nuclei, and intact nuclear membranes. Compared with the K group, the neurons in the M group were severely damaged, with intact cell membranes, edematous cytoplasm, sparse and dissolved cytoplasmic contents, and markedly swollen organelles. Most mitochondria were significantly swollen, with intact membranes, large amounts of dissolved matrix, and broken or disappeared cristae. Compared with the M group, the neuronal damage was improved in the Z, Y, D, YZ, and DZ groups, with reduced autophagy levels. Western blotting results showed that the p-AMPK/AMPK ratio was higher in the M, Y, and D groups than in the K group, while it was lower in the Z, YZ, and DZ groups than in the M group. The p-mTOR/mTOR ratio was lower in the M, Y, D, and DZ groups than in the K group, while it was higher in the Z and YZ groups than in the M group (P<0.05). The p-S757-ULK1/ULK1 ratio was higher in the M and D groups than in the K group, while it was lower in the Z, Y, YZ, and DZ groups than in the M group (P<0.05). The LC3II/LC3I ratio was higher in the M and D groups than in the K group, while it was lower in the Z, Y, YZ, and DZ groups than in the M group (P<0.05). P62 expression was lower in the M and D groups than in the K group, while it was higher in the Z, Y, YZ, and DZ groups than in the M group (P<0.05). Real-time quantitative PCR results showed that the mRNA expression of AMPK, mTOR, LC3, P62, and ULK1 in the M group was significantly different from that in the K group (P<0.05). The mRNA expression of AMPK was lower in the Z, DZ, and YZ groups than in the M group (P<0.05). The mRNA expression level of mTOR was lower in the Z, DZ, and YZ groups than in the M group (P<0.05). The mRNA expression level of LC3 was lower in the Z, YZ, and DZ groups than in the M group (P<0.05). The mRNA expression level of LC3 was lower in the Z and YZ groups than in the M group (P<0.05).
Conclusion YFXFJZF may regulate the level of autophagy in the hippocampus by modulating the AMPK/mTOR/ULK1 signaling pathway, protect the function of hippocampal neurons, and delay the progression of VD.
