| Objective:Chronic cerebral ischemia(CCI)refers to a group of clinical syndromes that result in cerebral ischemia and hypoxia due to chronic deficiency in cerebral blood supply.These conditions lead to impaired brain function.CCI is often implicated in the development of diseases such as Alzheimer’s disease and vascular dementia.Furthermore,CCI not only impacts the brain but also affects the intestinal flora and its metabolites through the gut-brain axis,further exacerbating brain damage.The intestinal flora comprises various bacteria and maintains a symbiotic relationship with the human host.A healthy and stable intestinal flora is crucial for maintaining body homeostasis.Increasing evidence suggests that the microbiota plays a significant role in the bidirectional communication of the microbial-gut-brain axis,influencing the progression of several central nervous system disorders,including multiple sclerosis,Parkinson’s disease,autism,Alzheimer’s disease,depression,and schizophrenia.CCI is classified as "vertigo","insomnia","dementia" and "depression" in Traditional Chinese Medicine Clinical.Professor Jiuyi Xi,a vascular disease expert in Shanghai,has summarized years of clinical experience and formulated a Chinese medicine compound called Hu-Zhang-Qing-Mai-Yin(HZQMY),which has shown significant therapeutic effects on cerebral small blood vessel injuries.This formulation has proven effective for CCI injuries.In this study,a CCI model was created using the bilateral common carotid artery lumen stenosis(BCAS)method,and the researchers investigated the effects of HZQMY on improving cognitive dysfunction in mice with chronic cerebral ischemia.The mechanism of treating chronic cerebral ischemia in mice was explored through 16 S rRNA sequencing and untargeted metabolomics.Methods:(1)Evaluation of the efficacy of HZQMY in treating CCI: A mouse model of CCI was created by wrapping microspring coils around the common carotid artery,resulting in bilateral common carotid artery lumen stenosis.After 6 weeks of regular feeding,the mice were randomly divided into five groups based on their body weight: Sham group,model group(BCAS),Ginkgo biloba extract(GBE 30 mg/kg),HZQMY-L group(13.1g/kg),and HZQMY-H group(26.2 g/kg).The mice in the Sham group underwent isolation of the bilateral common carotid arteries without microspring winding.After 12 weeks of oral administration in each group,the blood flow in the common carotid arteries(CCAs)was measured using an electromagnetic flowmeter.The learning and memory abilities of the mice were assessed through the Morris water maze test and new object recognition test.Pathological changes in the corpus callosum area were observed using transmission electron microscopy,while LFB myelin staining was used to observe pathological changes in the myelin area.Nissl staining was performed to observe neuronal damage in the hippocampal region.Additionally,CD31 and NeuN immunostaining were used to observe alterations in brain tissue neovascularization and neurons.(2)Network pharmacology was employed to predict the relevant targets and pathways of HZQMY for treating CCI.The potential targets of HZQMY were collected from the TCMSP and Pub Chem databases,while the genes associated with CCI were obtained from the Gene Cards database.By mapping the collected targets of HZQMY and the disease-related genes,potential targets for HZQMY in treating CCI were identified.These potential targets were further screened,and GO and KEGG pathway enrichment analyses were conducted to predict the signaling pathways involved in the treatment of CCI with HZQMY,providing initial validation of the mechanism.To assess the effectiveness of HZQMY in treating CCI,the levels of the inflammatory factors IL-1β,IL-6,and TNF-α in the serum and cerebral cortex of mice with CCI were measured using ELISA.Apoptosis in brain tissue was observed through TUNEL staining,and the expression of apoptosis-related proteins(BCL-2,Bax,Caspase-3 and Caspase-9)in brain tissue was measured using Western Blot.(3)To study the mechanism of action of HZQMY in the treatment of CCI in mice based on intestinal flora,the total DNA of cecum contents of each group of mice was extracted,the 16S rRNA gene V3+V4 region was amplified by PCR and sequenced,and the Operational Taxonomic Unit(OUT)was established,which was analysed by Alpha diversity,Beta diversity,PCo A principal coordinates analysis,phylum level composition and genus level composition to analyse the changes of intestinal flora in mice.Untargeted metabolomics techniques were used to detect changes in fecal metabolism in each group of mice,and the metabolomic changes were analysed by PCA,PLS-DA analysis,fecal differential metabolites,and the differential metabolic pathways were screened by KEGG enrichment.ELISA was applied to detect the protein levels of the inflammatory factors IL-1β,IL-6 and TNF-αin mouse cecum tissues.Results:(1)The blood flow changes in mice before and after surgery were measured using an electromagnetic flowmeter to assess the success of the modelling.The results demonstrated a significant decrease in blood flow in mice after surgery compared to before surgery(P < 0.01).After 12 weeks of drug administration,another test was conducted.The results revealed that the total cervical blood flow in the BCAS group was significantly reduced compared to that in the Sham group(P < 0.05),whereas the HZQMY-H group exhibited a significant increase in blood flow compared to that in the BCAS group(P < 0.01).After 6 weeks of treatment,the first water maze experiment was conducted.The results indicated that the escape latency of mice in the BCAS group was significantly higher than that of mice in the Sham group(P < 0.05).However,the escape latency of mice in the HZQMY-H group was significantly lower than that of the BCAS group(P < 0.01).After 12 weeks of treatment,the second water maze experiment was performed,the results showed that the escape latency of mice in the BCAS group was significantly higher than that in the Sham group(P < 0.05),while the escape latency of mice in the HZQMY-H group was significantly lower than that in the BCAS group(P < 0.05).the number of platform crossings and the percentage of target quadrants of mice in the BCAS group were less than those in the Sham group(P < 0.05), while the evasion latency of mice in the HZQMY-H group was significantly lower than that of the BCAS group(P < 0.05).The number of plateaus crossed and the percentage of target quadrant in the mice in the HZQMY-H group were greater than those in the BCAS group(P < 0.05).The results of the new object recognition experiment demonstrated that the memory ability(new object recognition index)of mice in the BCAS group was significantly weaker than that of mice in the Sham group after 6 and12 weeks of administration(P < 0.05).However,the memory ability of mice in the HZQMY group was significantly enhanced compared to that of mice in the BCAS group(P < 0.05).Electron microscopic analysis revealed disrupted lamellar structure and texture of the myelin sheath in the corpus callosum of mice in the BCAS group.The brain blood vessels appeared wrinkled,nerve cells exhibited signs of apoptosis,mitochondrial cristae were broken,and some cristae were dissolved and disappeared.In contrast,the myelin sheath in the HZQMY group mice remained intact,surrounded by axons in a lamellar shape,and the brain blood vessels normalized.The cytoplasm of nerve cells in the HZQMY group exhibited rich organelles with clear and abundant mitochondrial cristae.NeuN staining results indicated that HZQMY could improve the damage to hippocampal neurons in mice with chronic cerebral ischemia.LFB staining suggested that HZQMY could improve damage to the corpus callosum in mice with chronic cerebral ischemia.TUNEL staining demonstrated that HZQMY could reduce apoptosis caused by chronic cerebral ischemia.The CD31 staining results showed neovascularization in the brain tissue of mice in the HZQMY group with CCI.(2)After the network pharmacology study,696 targets related to the action of HZQMY and 3126 targets related to CCI were screened.The network construction of the chemical composition-target-chronic cerebral ischemia network was carried out by Cytoscape software,and a total of 426 relevant targets were found.The targets with high correlation with chronic cerebral ischemia were also calculated as AKT1,ALB,TNF,TP53,IL-6,INS and JUN.These targets were involved in the PI3K-Akt signaling pathway,TNF signaling pathway,IL-17 signaling pathway,apoptosis and other related pathways.The ELISA results showed that HZQMY inhibited the levels of the inflammatory factors IL-1β,IL-6 and TNF-αin serum and brain tissue(P<0.05,P<0.01).The results of TUNEL staining showed that HZQMY could reduce apoptosis caused by CCI;Western Blot analysis showed that HZQMY could inhibit the expression of Caspase-3,Caspase-9 and Bax in brain tissue and promote the expression of Bcl-2.(3)The 16 S rRNA sequencing results of the mouse gut microbiota showed that compared to the BCAS group,the relative abundances of the Verrucomicrobiota,Akkermansiaceae and Erysipelatoclostridium in the gut microbiota of the HZQMY-H group were significantly reduced(P < 0.05),while the relative abundances of the Eubacterium-xylanophilum-group and Allobaculum increased(P < 0.05).The untargeted metabolomics results showed significant differences between the metabolites in the Sham,BCAS,and HZQMY-H groups,and enrichment analysis of these differential metabolites showed that HZQMY ameliorated metabolite disorders in mice with chronic cerebral ischemia,probably through the arginine synthesis signaling pathway.ELISA results showed that HZQMY could reduce IL-1β and IL-6 levels in the intestinal tissues of mice with chronic cerebral ischemia(P < 0.05).Conclusion:HZQMY can improve cognitive impairments in mice with CCI and alleviate pathological damage to brain white matter.It repairs damage to hippocampal neurons and promotes neovascularization,thus exerting a protective effect on mice with CCI.Based on network pharmacology,the mechanism of action of HZQMY in treating CCI was predicted and validated.It was found that its protective effects may be associated with anti-inflammatory and antiapoptotic properties.The protective effects of HZQMY in mice with CCI may be related to the improvement of gut microbiota dysbiosis,regulation of metabolite generation,and attenuation of intestinal tissue inflammation.Differential changes in metabolites mainly involve the arginine synthesis signaling pathway. |
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