Chuanzhitongluo capsule improves cognitive impairment in mice with chronic cerebral hypoperfusion via the cholinergic anti-inflammatory pathway

Vascular


Introduction
Vascular cognitive impairment (VCI) is a clinical syndrome characterized by the impairment of at least one cognitive function domain due to damage to critical anatomical parts of the brain caused by cerebrovascular disease and its risk factors (van der Flier et al., 2018;Iadecola et al., 2019;Rundek et al., 2022).VCI encompasses a range of cognitive impairments, from mild cognitive impairment to vascular dementia (VaD) (Rundek et al., 2022).In China, the VCI has become the second most common type of cognitive deficit after Alzheimer's disease (AD) (Jia et al., 2020).However, the pathological features of VCI are different from those of AD (Lane et al., 2018), which has distinct pathological features including amyloid plaques due to β-amyloid peptide deposition and neurofibrillary tangles due to tau protein hyperphosphorylation (Lane et al., 2018;Serý et al., 2013;Eratne et al., 2018).In contrast, VCI is mainly caused by pathological changes resulting from cerebrovascular disease or its risk factors, and the diagnosis of VCI is also based on cerebrovascular factors (Skrobot et al., 2018;Zanon Zotin et al., 2021).The pathological changes in VCI include neuroinflammation, oxidative stress, mitochondrial dysfunction, and impaired neurotransmitter system function (Rajeev et al., 2022;Kalaria, 2016), which can lead to neuronal damage, white matter lesions, and other pathological features (Du et al., 2017).Chronic cerebral hypoperfusion (CCH) plays a critical role in linking cerebrovascular diseases and their risk factors with VCI (Duncombe et al., 2017).CCH leads to reduced cerebral blood flow (CBF) resulting in continuous cerebral ischemia and hypoxia, ultimately leading to the aforementioned pathological changes (Duncombe et al., 2017;Sarti et al., 2002).However, CCH is usually not easy to reverse, highlighting the importance of interventions targeting these pathological changes to improve cognitive impairment caused by cerebral ischemia and hypoxia.Neuroinflammation is a significant contributor to VCI pathology and is frequently observed in various research studies of VCI (Tian et al., 2022).Targeting neuroinflammation in the treatment of VCI may potentially lead to improved results following its onset (Poh et al., 2022).There are no approved medicines specifically for treating VCI (Zanon Zotin et al., 2021;Farooq et al., 2017).Donepezil has been reported to provide slight improvement in mild cognitive impairment, but it is not a targeted drug for VCI and often causes gastrointestinal reactions (Farooq et al., 2017).The effect of memantine on VCI is also minimal (Farooq et al., 2017;Ritter and Pillai, 2015).Therefore, it is important to understand how Traditional Chinese Medicine (TCM) can be utilized to address the increasing prevalence of VCI and its potential mechanisms of action in China.
Chuanzhitongluo capsule (CZTL) is a simplified version of Buyan-gHuanwu Decoction (BYHWD), which was used to treat cerebrovascular diseases.By employing virtual target prediction techniques and transcriptomic integration strategies, Yang et al. found that BYHWD plays a key role in the treatment of VCI by balancing oxidative stress, reducing inflammation and apoptosis, and enhancing the metabolism and immune system (Yang et al., 2021).It is suggested that NF-κB, BAX, BCL-2 and Caspase-3 are important targets of BYHWD in the treatment of VCI (Yang et al., 2021).CZTL is a compound preparation of the TCM composed of four ingredients: Leech (Shuizhi, Hirudo), Sichuan lovase rhizome (Chuanxiong, Chuanxiong Rhizoma), Milkvetch Root (Huangqi, Astragali Radix), Dan-Shen Root (Danshen, Salviae Miltiorrhizae Radix et Rhizoma).Hirudin is the main active component of leech, intracerebral hirudin injections have been reported to alleviate cognitive impairment and oxidative stress in cerebral ischemia rats and promote hippocampal neurogenesis (Xia et al., 2023).Sichuan lovase rhizome essential oil can significantly reduce the excessive production of inflammatory mediators and pro-inflammatory factors through the NF-κB signaling pathway (Zuo et al., 2024), and ferulic acid, a derivative of Sichuan lovase rhizome, has neuroprotective effects (Zhang et al., 2018).Milkvetch Root has a potential neuroprotective molecular mechanism, and astragalus polysaccharide can improve motor disorders and protect neurons in Parkinson's disease mice (Liu et al., 2018).Its potential effects on neurodegenerative diseases have also been reported, with antiinflammatory, antioxidant and immunomodulatory effects (Abd Elrahim Abd Elkader et al., 2022).A meta-analysis showed that Dan-Shen Root has a protective effect on animal models of cerebral ischemiainduced injury, the mechanism of which may be related to the reduction of inflammation and oxidative stress (Xie et al., 2023).In addition, other studies have reported that the active components of Dan-Shen Root can improve cell viability and acetylcholine level, which may be a potential chemical for treating AD (Li et al., 2022).
Our team has previously studied the therapeutic effect of CZTL on mice with photochemical stroke (Wang et al., 2022;Qi et al., 2023).However, the potential of CZTL in treating VCI remains unexplored.Therefore, this study aims to establish a mouse model of CCH using bilateral common carotid artery stenosis (BCAS) with microcoils and investigate the cognitive improvement effect and potential mechanism of CZTL in CCH-induced VCI mice.

Animals
All animal procedures in this study were approved by the Animal Care & Welfare Committee of the Affiliated Hospital of Qingdao University (AHQU-MAL20220422).Male C57BL/6 J mice aged 12 weeks (weight, 20-25 g) were utilized in this study.A total of 90 mice were obtained from Beijing Vital River Laboratory Animal Technology Co., LTD.The mice were housed in specific pathogen-free rooms at the Affiliated Hospital of Qingdao University, following a 12-h light/dark cycle and access to water and food ad libitum.

BCAS procedure
BCAS was employed to induce CCH (Nishio et al., 2010).Initially, mice were placed in an anesthesia induction box and anesthetized with 5 % isoflurane.Subsequently, anesthesia was maintained using a mask with 3 % isoflurane.The mice were then positioned supine with their limbs secured, and a vertical incision 1.5 cm was made along the neck.The common carotid triangle was dissected, revealing the common carotid artery along with the vagus nerve.The dissection of the common carotid artery was completed.Finally, a mental microcoil (internal diameter: 0.18 mm, lehgth: 2.5 mm) was gently wrapped around both the left and right common carotid arteries.Please refer to Fig. 1 for further details.A total of 90 male mice were included in this study, but 23 mice died due to intraoperative bleeding and varying degrees of brain ischemia following the operation.The drug dosage was calculated based on the ratio of body surface area between mice and adults.The geometric progression was then used to expand the dose range.For the initial phase of determining the optimal drug dose, a random number table was utilized to allocate the mice into the following groups: BCAS (7/10), 0.15 g/kg (8/10), 0.3 g/kg (8/10), 0.6 g/kg (8/10), and 1.2 g/kg (7/10).In the subsequent phase involving molecular experiments, the groups consisted of BCAS (15/20) and 0.6 g/kg CZTL (14/20).Administer the drug (intragastric administration, i.g.) for 30 days, with a dosage of 0.1 ml of the drug solution once daily per mouse.The BCAS group is given an equivalent dose of 0.9 % Normal Saline.

Morris water maze (MWM) experiment
After 30 days of continuous intragastric administration, the MWM experiment system was used to test spatial learning and memory in mice.The MWM experiment system consisted of a pool with a diameter of 120 cm and a depth of 45 cm, divided into four virtual quadrants.A platform with a diameter of 10 cm was fixed in the center of one quadrant, submerged 1 cm beneath the water surface.An animal behavior recording instrument was placed above the pool.To assist the mice in distinguishing directions, four color and shape references were hung on a curtain around the pool.The water temperature was maintained at 23 ± 2 • C. White, non-toxic pigments were added to the water to make it easier to record the mice's tracks with a camera.The experiment consisted of two stages: Place Learning and Probe Test.In the place learning phase, the mice were placed in the pool, allowed to swim freely, and watched for 60 s to see if the mice could successfully climb onto the platform.If the mice failed to find the platform, they were manually guided to it for 10 s.The time it took for a mouse to reach the platform from the starting point in the water was recorded as the escape latency.This process was repeated four times a day for five days.On the sixth day, the platform was removed for the Probe Test to evaluate the mice's spatial memory.The mice were placed in the opposite quadrant to the platform quadrant, and their swimming tracks and the number of times they passed the platform in 60 s were observed.Throughout the six-day experiment, the mice's swimming tracks and times were recorded using an animal behavior recording instrument, and the data were analyzed using the Any-Maze software (Stoelting Co., Wood Dale, IL, USA).

RNA sequencing (RNA-Seq)
The mice hippocampal tissue was analyzed by RNA-seq technique.The mice were anesthetized, underwent cardiac perfusion, took brain, and separated hippocampal tissue on the ice.Total RNA was extracted using the TRIzol reagent following the manufacturer's instructions.The NanoDrop 2000 spectrophotometer from Thermo Scientific (USA) was used to assess RNA purity and quantity.The integrity of the RNA was evaluated using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA).Libraries were constructed using the TruSeq Stranded mRNA LT Sample Prep Kit (Illumina, San Diego, CA, USA) according to the manufacturer's instructions.OE Biotech Co., Ltd. in Shanghai, China was responsible for transcriptome sequencing and analysis.
Sequencing of the libraries was performed on the Illumina HiSeq X Ten platform, generating 150 bp paired-end reads.Average 47.74 M raw reads were obtained for each sample.Trimmomatic (Bolger et al., 2014) was used to preprocess the raw FASTQ data, removing low-quality reads and generating clean reads.Approximately 46.48 M clean reads were retained for further analysis in each sample.These clean reads were then mapped to the mouse genome (GRCm38) using HISAT2 software (Kim et al., 2015).The FPKM (Roberts et al., 2011) of each gene was calculated using Cufflinks (Trapnell et al., 2010) and HTSeq count (Anders et al., 2015).Differential expression analysis was performed using the DESeq (2012) R package (Anders et al., 2013).The criteria for significantly different expressions were set as P < 0.05 and fold change ≥2.Hierarchical cluster analysis was conducted on the different expressed genes (DEGs) to visualize the variations in gene expression across different populations and datasets.Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) (Kanehisa et al., 2008) pathway enrichment analyses of the DEGs were performed using R based on the hypergeometric distribution.

Immunofluorescence (IF)
After cardiac perfusion, mice hippocampal tissue was stripped and fixed with 4 % paraformaldehyde for 6-8 h, and sucrose solution was used to gradient dehydration.Hippocampal tissue with OCT compound (4583, SAKURA, USA) embedded, which was cut into 8 um slices by the freezing microtome (CM1950, Leica, Germany) at − 20 • C. Wash 3 times with 1xPBS for 3-5 min each time and blocked with 5 % BSA (Elabsciecce, China) for 1 h.The primary antibody included rabbit anti-ChAT (1:1500, ABclonal) and rabbit anti-α7 subunit-containing nicotinic acetylcholine receptor (α7nAChR, 1:1500, ABclonal), then was incubated at 4 • C overnight.After washing with 1xPBS for 3-5 min, Incubated with the corresponding secondary antibody at room temperature and away from light for 1 h.The nuclei were labeled using DAPI and photographed using fluorescence microscopy(Olympus, Japan).Each group of 3 mice was used in this step.

Enzyme-linked immunosorbent assay (ELISA)
Each group of 6 mice was used to isolate the hippocampus (25-30 mg), cut into small fragments.A 300 ul tissue protein extraction reagent (Beijing Baiaolaibo, China) was added to the fragments, followed by homogenization using a vortex.The supernatant was obtained by centrifugation.The optical density of inflammatory factors (Interleukin-1β (IL-1β), Interleukin-6 (IL-6), and Tumor necrosis factor-α (TNF-α)) in the supernatant, which was combined with solid phase antibody and enzyme-labeled antibody, was measured using a microplate reader (TECAN, Switzerland) at a wavelength of 450 nm, following the step-bystep instructions of the ELISA kit (Jiangsu SuMeike Biotechnology Co, Ltd., China).Finally, the concentration of inflammatory factors was calculated based on the standard curve.

Statistical analysis
The escape latency was calculated using Repeated Measures ANOVA, while the number of platform crossings and residence time in the platform quadrant was calculated using one-way ANOVA.Two-sample ttests were used for the analysis of IF, ELISA, and WB results.All data were statistically analyzed using SPSS (version 26.0,IBM Corp., Armonk, NY) and presented using GraphPad Prism (version 8.0, La J, CA, USA).A significance level of P < 0.05 was considered statistically significant.

CZTL can improve spatial learning and memory in CCH mice
The MWM experiment was conducted to assess the spatial learning and memory of mice.By comparing with the BCAS group to determine the optimal dose of different concentrations of medicines (0.15 g/kg, 0.3 g/kg, 0.6 g/kg, 1.2 g/kg, as shown in Fig. 2A).The swimming trajectory of all groups in the MWM experiment as shown in Fig. 2B.The result indicates that mice in the CZTL (0.6 g/kg) group exhibited a lower escape latency compared to the BCAS group during the Place Navigation phase, and showed better performance than other groups receiving different drug doses (Fig. 2C).In the Probe Test stage, mice in the 0.6 g/ kg group exhibited significantly enhanced performance compared to the BCAS group and other groups, as indicated by both the number of platform crossings and platform quadrant residence time (Fig. 2D, E).These results from the MWM experiment suggest that CZTL may ameliorate cognitive impairment in mice with CCH, with the 0.6 g/kg dosage showing the most pronounced effect.Consequently, the drug concentration of 0.6 g/kg was used for subsequent experiments.

CZTL changes gene expression profile in hippocampal tissue of CCH mice
The CZTL (0.6 g/kg) and BCAS groups were selected for RNA-Seq to identify DEGs and potential targets of CZTL.The expression of FPKM for each sample in the two groups as shown in Fig. 3A.Following the DEGs analysis between the two groups, we identified 44 up-regulated genes and 93 down-regulated genes (Fig. 3B), with an overall distribution of all DEGs shown in Fig. 3C, D. Subsequently, GO and KEGG enrichment analyses were conducted to explore the DEGs associated signal pathways.The results of the GO analysis revealed that upregulated genes were involved in biological processes such as neuron differentiation (Fig. 3E), while down-regulated genes were associated with processes related to inflammatory response (Fig. 3F).In terms of KEGG enrichment analysis, the up-regulated pathways included cholinergic synapses (Fig. 3G), whereas the down-regulated pathways were associated with the NF-κB and TNF signaling pathways (Fig. 3H).Further investigation indicated that choline acetyltransferase (ChAT) was involved in the neuronal differentiation and the cholinergic synapse pathway (Fig. 3I,  J).Therefore, based on our experimental objectives and previous research findings (Jiang et al., 2017;Han et al., 2017;Park et al., 2020;Pavlov and Tracey, 2005), ChAT was selected as the target gene for further investigation.

CZTL increased ChAT and α7nAChR expression in the hippocampus of CCH mice
Combined with RNA-Seq results, ChAT was identified as the target gene for further investigation.We hypothesize that CZTL may exert its effects through the cholinergic anti-inflammatory pathway (CAIP) (Han et al., 2017).Thus, IF was used to detect the presence of ChAT and α7nAChR in the hippocampal tissues of mice.The IF results of frozen sections of the mice's hippocampus revealed significant differences between the two groups.The expressions of ChAT and α7nAChR were significantly increased in CCH mice following CZTL (0.6 g/kg) treatment, as compared to the BCAS group (Fig. 4A, B).

CZTL reduces CCH-induced inflammatory factors in the hippocampus
The ELISA was employed to ascertain disparities in the levels of inflammatory factors between the two groups.The CZTL (0.6 g/kg) group exhibited lower concentrations of IL-1β, IL-6, and TNF-α in comparison to the BCAS group (Fig. 4C), thereby suggesting that CZTL may have the ability to reduce inflammatory factors and mitigate inflammation levels in CCH mice.These results imply that CZTL holds the potential to ameliorate hippocampal neuroinflammation caused by CCH, and CZTL may potentially alleviate cognitive dysfunction triggered by reduced CBF via the reduction of neuroinflammation in CCH mice.

Fig. 3. A:
The horizontal axis is the sample name, the vertical axis is log10(FPKM+1), and the violin plot for each region corresponds to five statistics (maximum, third quartile, median, first quartile, and minimum, respectively, from top to bottom).B: The number of up-regulated and down-regulated differential genes between the two groups.C: Gray is the non-significant difference genes, red and blue are the significant difference genes, red: up-regulation, blue: down-regulation.D: Red indicates relatively high expression protein-coding genes, and blue indicates relatively low expression protein-coding genes.E: Biological processes involved in upregulated genes in the GO analysis.F: Biological processes involved in down-regulated genes in the GO analysis.G: The pathway involved in up-regulated genes in the KEGG analysis.H: The pathway involved in down-regulated genes in the KEGG analysis.I: In the GO analysis, genes are involved in neuron differentiation and other biological processes.J: In the KEGG analysis, genes involved in cholinergic synapses and other pathways.(For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

CZTL exerts anti-neuroinflammatory effects via the ChAT/ α7nAChR/NF-κB pathway
The protein expression of ChAT and α7nAChR was detected using WB, which was consistent with the results obtained from IF analysis (Fig. 5 A, B, C).The KEGG analysis indicated that the expression of the NF-κB pathway was down-regulated in the CZTL(0.6 g/kg) group compared to the BCAS group.Therefore, we hypothesized that CZTL may exert its anti-inflammatory effects via the ChAT/α7nAChR/NF-κB pathway, leading to a reduction in inflammatory factors in the hippocampus, improvement of neuroinflammation, and ultimately improvement in cognition.The protein expression levels of P-IκBα, cytoplasm-p65, and nuclear-p65 were significantly lower in the CZTL group than in the BCAS group (Fig. 5 D, E, F).These results suggest that CZTL may ameliorate cognitive impairment in CCH mice by inhibiting the NF-κB pathway and neuroinflammation.

Discussion
In this study, we investigated the cognitive improvement effect of CZTL on VCI mice.We constructed a BCAS model in mice by using metal microcoils with an inner diameter of 0.18 mm and a length of 2.5 mm.This model induced CCH and simulated the key pathological changes observed in VCI (Duncombe et al., 2017).The BCAS mouse model was widely regarded as a better animal model for VCI research (Washida et al., 2019;Ishikawa et al., 2023).Other commonly used animal models include rat bilateral common carotid artery occlusion (BCAO) and mice asymmetric bilateral common carotid surgery (ACAS) (Washida et al., 2019).However, BCAO is not suitable for drug experiments involving gavage due to the complete blockage of the rat's bilateral common carotid artery, ACAS can cause stable CCH but its process is not consistent in both cerebral hemispheres of mice, which can affect subsequent molecular experiments (Washida et al., 2019;Zhao and Gong, 2015).Therefore, the BCAS model was deemed more suitable for this study.We investigated the effects of CZTL on the spatial learning and memory abilities of mice with CCH using the MWM experiment.The results showed that CCH mice treated with CZTL (0.6 g/kg) had shorter escape latency, more platform crossing times, and increased platform quadrant residence time.The MWM utilizes the innate swimming ability of mice and their instinct to avoid water to detect their behavioral trajectories and assess cognitive performance in CCH mice (Othman et al., 2022).A shorter escape latency indicates better spatial learning ability, while more platform crossing times and platform quadrant residence time suggest improved memory ability.These results suggest that CZTL may enhance the cognitive ability of CCH mice.Luo et al. investigated the cognitive improvement effect of Shenmayizhi Decoction (SMYZD) on VCI rats and found that SMYZD improved spatial learning and memory deficits in BCAO rats (Sun et al., 2021).Another research team explored the cognitive improvement effect of Huoluoyinao Decoction (HLYND) on BCAS mice and found that BCAS mice treated with HLYND had shorter escape latency and higher platform crossing frequency in the MWM experiment compared to the control group (Wang et al., 2019).Based on these findings and the results of this study, it is suggested that TCM compounds may have a positive effect on improving CCH-induced VCI in mice.
RNA-seq was utilized in this study to investigate the potential targets of CZTL in CCH mice, benefiting from its high throughput, high sensitivity, and high resolution capabilities (Wang et al., 2009).The expression of total RNA in hippocampal tissues was assessed using RNAseq to compare mice in the CZTL (0.6 g/kg) group with the BCAS group, aiming to observe the impact of CZTL on gene expression in CCH mice.Analysis of DEGs between the two groups revealed that CZTL enhanced the expression of ChAT, suggesting ChAT as a potential target of CZTL.ChAT, which encodes choline acetyltransferase, is a crucial enzyme involved in acetylcholine synthesis in neurons (Gabalski et al., 2024).GO and KEGG enrichment analysis of DEGs indicated that CZTL could up-regulate genes promoting neuronal differentiation and the cholinergic synaptic pathway, with ChAT being involved in both processes.Additionally, enrichment analysis demonstrated that down-regulated genes were associated with inflammatory response and the NF-κB pathway.These results suggest that CZTL may exert a neuroprotective effect against neuroinflammation by enhancing ChAT expression.The cholinergic anti-inflammatory pathway (CAIP) refers to the pathway that regulates the inflammatory response by releasing acetylcholine when the vagus nerve acts on corresponding receptors (Pavlov and Tracey, 2005;Alen, 2022).ChAT, a key enzyme in acetylcholine synthesis, plays a significant role in the CAIP (Han et al., 2017).In the central nervous system, acetylcholine inhibits the downstream NF-κB signaling pathway by acting on α7nAChR, thereby reducing the release of inflammatory cytokines such as TNF-α, IL-1β, and IL-6, and regulating neuroinflammation (Han et al., 2017;Benfante et al., 2021;Kelly et al., 2022).Based on the results of RNA-seq, we hypothesized that CZTL may exert its effects through the CAIP, reducing levels of neuroinflammatory factors via the ChAT/α7nAChR/NF-κB signaling pathway, alleviating the pathological changes caused by CCH, and improving spatial learning and memory in VCI mice.
To investigate this, we first conducted ELISA to measure the expression levels of the inflammatory factors TNF-α, IL-1β, and IL-6 in hippocampal tissues of mice treated with CZTL (0.6 g/kg) or in the BCAS group.The results showed that TNF-α, IL-1β, and IL-6 levels were significantly lower in the CZTL (0.6 g/kg) group, suggesting a potential protective effect of CZTL against neuroinflammation.IF staining was used to assess the expression of ChAT and α7nAChR proteins in hippocampal tissue sections.The results showed higher expression levels of ChAT and α7nAChR in the hippocampus of the CZTL (0.6 g/kg) group, suggesting that CZTL may be involved in regulating neuroinflammation via the CAIP.
Furthermore, we performed WB to investigate the expression levels of key proteins in the ChAT/α7nAChR/NF-κB signaling pathway.The results showed that ChAT and α7nAChR expression levels were higher in the CZTL (0.6 g/kg) group compared to the BCAS group.In contrast, the expression levels of P-IκBα, cytoplasmic p65, and nuclear p65 were decreased.The NF-κB classical signaling pathway was known to be associated with the regulation of inflammatory signals.In the resting state, the trimeric complex IkBα-p65-p50 binds and exists in the cytoplasm.However, upon receiving a stimulus signal, IkBα was phosphorylated to form P-IκBα, leading to its separation from the NF-κB trimer and the formation of the NF-κB dimer, p65-p50.Subsequently, the p65-p50 dimer enters the nucleus and binds to specific sequences on nuclear DNA, promoting the transcription of inflammatory factors such as TNFα, IL-1β, and IL-6 (Yu et al., 2020;Lawrence, 2009).The decreased expression of P-IκBα in the hippocampus of the CZTL (0.6 g/kg) group may indicate reduced activation of the NF-κB signaling pathway.The decrease in cytoplasmic p65 expression may be due to reduced phosphorylation of IκBα protein, leading to failure in its separation from the NF-κB trimer.Similarly, a decrease in cytoplasmic p65 results in reduced p65 entering the nucleus, thereby reducing the transcription of TNF-α, IL-1β, and IL-6.These results suggest that CZTL may decrease the expression levels of inflammatory factors and reduce the pathological changes associated with neuroinflammation by increasing the expression of ChAT and α7nAChR in the hippocampus of CCH mice and inhibiting the NF-κB signaling pathway.This, in turn, may improve the spatial learning and memory abilities of CCH mice.

Conclusion
In summary, we conducted RNA-seq analysis to screen for DEGs in CCH mice treated with CZTL, and identified ChAT as the target gene.Further enrichment analysis of the DEGs led us to select CAIP for final verification.These findings suggest that CZTL may exert an antiinflammatory effect via the ChAT/α7nAChR/NF-κB pathway, thereby improving cognitive dysfunction in CCH mice.These results indicate that CZTL may as a potential treatment for VCI.However, there are certain limitations to our study.Additional experiments, such as knockout or overexpression of ChAT in transgenic mice, could provide more robust evidence.Further studies in female BCAS mouse models and CAIP are needed in the future to thoroughly elucidate the potential mechanisms by which CZTL regulates inflammation in CAIP.

Declaration of competing interest
The authors declare that they have no conflicts of interest.

Fig. 1 .
Fig. 1.A: normal right common carotid artery; B, C: right common carotid artery with microcoils.D: normal left common carotid artery; E, F: left common carotid artery with microcoils.Visually, bilateral common carotid arteries were significantly narrowed.

Fig. 2 .
Fig. 2.A: from left to right, saline solution, 0.15 g/kg, 0.3 g/kg, 0.6 g/kg, 1.2 g/kg.B: The typical swimming paths of each group of mice during learning and memory probe tests.C: From the next day, escape latency was much shorter in 0.6 g/kg and 1.2 g/kg mice than those in BCAS mice.From the fourth day, escape latency was much shorter in 0.3 g/kg than those in BCAS mice.#: 0.6 g/kg vs BCAS, △: 1.2 g/kg vs BCAS, *: 0.3 g/kg vs BCAS; #, △, *:p < 0.05.D: Number of platform crossings during Probe Test; **: p < 0.01.E: Platform quadrant residence time of each group of mice during Probe Test; **: p < 0.01.

Fig. 5 .
Fig. 5. A: The protein expression of ChAT, α7nAChR, P-IκBα, cytoplasm p65, and nuclear p65 between two groups.B: The semiquantitative analysis of the ChAT expression.C: The semiquantitative analysis of the α7nAChR expression.D: The semiquantitative analysis of the P-IκBα expression.E: The semiquantitative analysis of the cytoplasm p65 expression.F: The semiquantitative analysis of the nuclear p65 expression.*:p < 0.05; **: p < 0.01.