Uncovering the characteristics of the gut microbiota in patients with acute ischemic stroke and phlegm-heat syndrome.

Growing evidence has indicated that the characteristics of gut microbiota are associated with acute ischemic stroke (AIS). Phlegm-heat syndrome (PHS), a specific pathological state of the AIS, is one of the common traditional Chinese syndromes of stroke. The long duration of PHS in patients with AIS could lead to poor clinical outcomes. Gut microbiota characteristics in patients with both AIS and PHS, and their relationship remains unknown. This study was designed to investigate the alterations in gut microbiota in patients with AIS and PHS through a cross-sectional study. Fecal samples were collected from 10 patients with AIS and non-PHS (ntAIS), 7 patients with AIS and PHS (tAIS), and 10 healthy controls (HC). Samples were profiled via Illumina sequencing of the 16S rRNA V3-V4. Stroke severity was assessed at admission by the National Institutes of Health Stroke Scale (NIHSS) and modified Rankin scale (mRS); their correlation with gut microbiota was investigated. The alpha-diversity of the bacterial communities was significantly higher in the fecal samples of patients with tAIS than in patients with ntAIS (Shannon index, P = 0.037). In addition, the combined tAIS and ntAIS group (tntAIS) exhibited higher microbiotic diversity when compared with HC (chao1, P = 0.019). The structure of intestinal microbiota was effectively distinguished between the tAIS and ntAIS group (ANOSIM, r = 0.337, P = 0.007). Additionally, the gut microbiota structure was significantly different between the tntAIS and HC groups (ANOSIM, r = 0.217, P = 0.005). The genera, Ruminococcaceae_ UCG_002 and Christensenellaceae_R-7_group, were implicated in the discrimination of PHS from non-PHS. The order Lactobacillales and family Lachnospiraceae were significantly negatively correlated with NIHSS and mRS at admission (P < 0.05). By contrast, the order Desulfovibrionales, families Christensenellaceae and Desulfovibrionaceae, and genera Ruminococcaceae UCG-014 and Ruminococcaceae UCG-002 were significantly positively correlated with NIHSS and mRS at admission (P < 0.05). This study is the first to profile the characteristics of gut microbiota in patients with AIS and PHS, compared with those with non-PHS. The genera, Ruminococcaceae_ UCG_002 and Christensenellaceae_R-7_group, may be objective indicators of this traditional Chinese medicine (TCM) syndrome in AIS. Furthermore, it provides a microbe-inspired biological basis for TCM syndrome differentiation.

Uncovering the Mechanism of the Xingnaojing Injection against Ischemic Stroke Using a Combined Network Pharmacology Approach and Gut Microbiota Analysis.

Objective: To explore the brain protection mechanism of Xingnaojing injection (XNJ) against ischemic stroke (IS) by the network pharmacology approach and gut microbiota analysis. Methods: We used network pharmacology analysis to identify the active components of XNJ and its potential targets against IS and inflammatory bowel disease (IBD) and carried out network analysis, functional annotation, and pathway enrichment analysis. Then, transient middle cerebral artery occlusion (tMCAO) mice model was used to verify the molecular mechanism of XNJ. Results: 36 active compounds were identified from XNJ, and the effect of XNJ against IS was related to the VEGF signaling pathway, NF-kappa B signaling pathway, and gap junction. The effect of XNJ against IBD was related to the T cell receptor signaling pathway, NF-kappa B signaling pathway, and gap junction. In vitro experiments showed that XNJ significantly improved the neurological function of tMCAO mice, reduced the size of cerebral infarction, decreased the permeability of blood-brain barrier (BBB), downregulated the expressions of TLR4, MyD88, and NF-kappa B in the ischemic site, and upregulated the expressions of occludin and ZO-1 in the colon. High-throughput 16S rDNA gene sequencing showed that XNJ upregulated the levels of Akkermansia and downregulated the levels of Flavobacteriaceae, Deferribacteraceae, and Deferribacteres. XNJ increased the concentrations of the short-chain fatty acids (SCFAs) PA (propionate), VA (valerate), IBA (isobutyrate), and IVA (isovalerate) in the feces of the sham germ-free experiment group (SGFEG) mice. Conclusion: IS causes dysbiosis of some specific bacteria in the gut microbiota. XNJ is an effective treatment for IS, and its mechanism was related to improving intestinal barrier function and regulating intestinal flora and SCFAs. Network pharmacology revealed that XNJ acts through multiple targets and multiple pathways.

Network pharmacology and in vitro studies reveal the pharmacological effects and molecular mechanisms of Shenzhi Jiannao prescription against vascular dementia.

BACKGROUND: Shenzhi Jiannao (SZJN) prescription is a type of herbal formula adopted in the management of cognitive impairment and related disorders. However, its effects and related regulatory mechanisms on vascular dementia (VD) are elusive. Herein, network pharmacology prediction was employed to explore the pharmacological effects and molecular mechanisms of SZJN prescription on VD using network pharmacology prediction, and validated the results through in vitro experiments. METHODS: Through a search in the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) database, chemical composition and targets for SZJN prescription were retrieved. The potential targets for VD were then obtained from the GeneCards and DisGeNET databases. The network was constructed that depicted the interactions between putative SZJN prescription and known therapeutic targets for VD using Cytoscape 3.7.1. Analysis of protein-protein interaction was achieved via STRING 11.0 software, followed by Gene Ontology (GO) functional enrichment and Kyoto Gene and Genome Encyclopedia (KEGG) pathway analyses. To validate the computer-predicted results, in vitro experiments based on an excitotoxic injury model were designed using glutamate-exposed PC12 cells, and treated with varying concentrations (low, 0.05; medium, 0.1 and high, 0.2 mg/mL) of SZJN prescription. Cell viability and cell death were detected using the IncuCyte imaging system. Moreover, the expression profiles of Caspase-3 were analyzed through qRT-PCR. RESULTS: Twenty-eight potentially active ingredients for SZJN prescription, including stigmasterol, beta-sitosterol, and kaempferol, plus 21 therapeutic targets for VD, including PTGS2, PTGS1, and PGR were revealed. The protein-protein interaction network was employed for the analysis of 20 target proteins, including CASP3, JUN, and AChE. The enrichment analysis demonstrated candidate targets of SZJN prescription were more frequently involved in neuroactive ligand-receptor interaction, calcium, apoptosis, and cholinergic synaptic signaling pathways. In vitro experiments revealed that SZJN prescription could significantly reverse glutamate-induced cell viability loss and cell death, and lower the levels of Caspase-3 mRNA in glutamate-induced PC12 cells. CONCLUSIONS: Collectively, this study demonstrated that SZJN prescription exerted the effect of treating VD by regulating multi-targets and multi-channels with multi-components through the method of network pharmacology. Furthermore, in vitro results confirmed that SZJN prescription attenuated glutamate-induced neurotoxicity.

Xinglou Chengqi Decoction improves neurological function in experimental stroke mice as evidenced by gut microbiota analysis and network pharmacology.

The current study was designed to explore the brain protection mechanism of Xinglou Chengqi Decoction (XCD) based on gut microbiota analysis and network pharmacology. A transient middle cerebral artery occlusion (MCAO) model of mice was established, followed by behavioral evaluation, TTC and TUNEL staining. Additionally, to investigate the effects of gut microbiota on neurological function after stroke, C57BL/6 mice were treated with anti-biotic cocktails 14 days prior to ischemic stroke (IS) to deplete the gut microbiota. High-throughput 16S rDNA gene sequencing, metabonomics technique, and flow multifactor technology were used to analyze bacterial communities, SCFAs and inflammatory cytokines respectively. Finally, as a supplement, network pharmacology and molecular docking were applied to fully explore the multicomponent-multitarget-multichannel mechanism of XCD in treating IS, implicated in ADME screening, target identification, network analysis, functional annotation, and pathway enrichment analysis. We found that XCD effectively improved neurological function, relieved cerebral infarction and decreased the neuronal apoptosis. Moreover, XCD promoted the release of anti-inflammatory factor like IL-10, while down-regulating pro-inflammatory factors such as TNF-α, IL-17A, and IL-22. Furthermore, XCD significantly increased the levels of short chain fatty acids (SCFAs), especially butyric acid. The mechanism might be related to the regulation of SCFAs-producing bacteria like Verrucomicrobia and Akkermansia, and bacteria that regulate inflammation like Paraprevotella, Roseburia, Streptophyta and Enterococcu. Finally, in the network pharmacological analysis, 51 active compounds in XCD and 44 intersection targets of IS and XCD were selected. As a validation, components in XCD docked well with key targets. It was obviously that biological processes were mainly involved in the regulation of apoptotic process, inflammatory response, response to fatty acid, and regulation of establishment of endothelial barrier in GO enrichment. XCD can improve neurological function in experimental stroke mice, partly due to the regulation of gut microbiota. Besises, XCD has the characteristic of "multi-component, multi-target and multi-channel" in the treatment of IS revealed by network pharmacology and molecular docking.

Uncovering the mechanism of the Shenzhi Jiannao formula against vascular dementia using a combined network pharmacology approach and molecular biology.

BACKGROUND: Shenzhi Jiannao formula (SZJNF) is a herbal prescription which is used for detoxification, dredging collaterals, and activating blood circulation and Qi flow in traditional Chinese medicine. SZJNF is a clinical effective prescription for the treatment of vascular dementia (VD) first formulated based on the classical theory of traditional Chinese medicine, but its anti-VD mechanism remains ambiguous. PURPOSE: The aim of this study was to elucidate the multi-target mechanisms of SZJNF against VD using a network pharmacology approach and verify its effects through biological experiments. STUDY DESIGN AND METHODS: We utilized network pharmacology-based prediction and molecular docking techniques to uncover the potential micro-mechanism of SZJNF against VD. We identified active components and potential targets, and performed network analysis, functional annotation, and pathway enrichment analysis. Subsequently, glutamate-induced PC12 cells and VD rats were used to verify the molecular mechanisms of SZJNF. RESULTS: Seventeen active compounds were identified in SZJNF rat plasma; moreover, 773 predicted targets and 1544 VD-related targets were found. Various networks, including the PPI, herb-compound-target, and compound-target-pathway network were constructed. A total of 188 shared targets were identified by network topological analysis, which were closely associated to the anti-VD effects of SZJNF. They were also enriched in various biological processes through hypoxia reaction, promotion of cell proliferation, inhibition of apoptosis, neuroactive ligand-receptor interaction, and calcium signaling pathway, as evaluated by the analysis of advanced functions and pathways. SZJNF components docked well with the key targets. Treatment with SZJNF promoted cell proliferation, ameliorated apoptosis and oxidative stress injury, and improved neurological and cognitive abilities. CONCLUSION: This study comprehensively demonstrated the multi-target mechanisms of SZJNF in VD using network pharmacology and molecular biology experiments. This provides evidence for further mechanistic studies and for the development of SZJNF as a potential treatment for patients with VD.

Network Pharmacology and Molecular Docking Analysis on Molecular Targets and Mechanisms of Buyang Huanwu Decoction in the Treatment of Ischemic Stroke.

METHODS: The bioactive components and potential targets of BHD were screened by TCMSP, BATMAN-TCM, ETCM, and SymMap databases. Besides, compounds that failed to find the targets from the above databases were predicted through STITCH, SwissTargetPrediction, and SEA. Moreover, six databases were searched to mine targets of IS. The intersection targets were obtained and analyzed by GO and KEGG enrichment. Furthermore, BHD-IS PPI network, compound-target network, and herb-target-pathway network were constructed by Cytoscape 3.6.0. Finally, AutoDock was used for molecular docking verification. RESULTS: A total of 235 putative targets were obtained from 59 active compounds in BHD. Among them, 62 targets were related to IS. PPI network showed that the top ten key targets were IL6, TNF, VEGFA, AKT1, etc. The enrichment analysis demonstrated candidate BHD targets were more frequently involved in TNF, PI3K-Akt, and NF-kappa B signaling pathway. Network topology analysis showed that Radix Astragali was the main herb in BHD, and the key components were quercetin, beta-sitosterol, kaempferol, stigmasterol, etc. The results of molecular docking showed the active components in BHD had a good binding ability with the key targets. CONCLUSIONS: Our study demonstrated that BHD exerted the effect of treating IS by regulating multitargets and multichannels with multicomponents through the method of network pharmacology and molecular docking.

Efficacy of Modified Wuzhuyu Decoction Granule ( ) for Migraine Patients with Cold and Stasis Obstructing Meridian Syndrome: A Randomized, Double-Blind, Placebo-Controlled Trial.

OBJECTIVE: To study the efficacy of modified Wuzhuyu Decoction Granule (, MWDG) in the treatment of migraine patients with cold and stasis obstructing meridian syndrome. METHODS: This study was a randomized, double-blind, placebo-controlled trial. A total of 78 migraine patients with cold and stasis obstructing meridian syndrome were recruited and randomly assigned by a ratio of 2:1 into a treatment group (51 cases) and a placebo group (27 cases). Patients in the treatment group were treated with MWDG while placebo granules were applied in the control group. The treatment course lasted for 12 weeks with a follow-up of 4 weeks. The primary outcome measures included frequency and days of migraine attacks and the secondary outcome measures were analgesics consumption and visual analogue scale (VAS) scores. All outcome assessments were conducted respectively at baseline, the 4th, 8th and 12th week, and the end of follow-up. RESULTS: In the treatment group, significant decrease in frequency of migraine attacks were observed since the 4th week and that of analgesics consumption since the 8th week (both P<0.05). While, in the placebo group, significant decrease in frequency of migraine attacks were observed since the 8th week and that of analgesics consumption since the 12th week (both P<0.05). No significant decrease in days of migraine attacks and VAS scores of migraine pain were observed in both groups. Between the two groups, there were significant differences in VAS scores and intensity of pain appeared in the 8th week (P<0.05). However, no significant differences were found in days and frequency of migraine attacks and analgesics consumption (P>0.05). CONCLUSIONS: MWDG was probably effective in the treatment of migraine especially for alleviating pain intensity. Furthermore, MWDG could reduce the frequency of migraine attacks and analgesics consumption sooner than the placebo.

Efficacy of Integrated Rehabilitation Techniques of Traditional Chinese Medicine for ischemic stroke: a randomized controlled trial.

This study aimed to determine the efficacy of Integrated Rehabilitation Techniques of Traditional Chinese Medicine (IRT-TCM) on patients with ischemic stroke as an alternative therapy to conventional rehabilitation techniques. Sixty-nine patients with ischemic stroke were randomly assigned to receive either IRT-TCM (intervention group, n = 46) or conventional rehabilitation techniques (control group, n = 23). The IRT-TCM consisted of a sequential combination of acupuncture and massage techniques. The Fugl-Meyer Assessment (FMA), National Institutes of Health Stroke Scale (NIHSS), Barthel index (BI) and modified Rankin Scale (mRS) were measured on day 0 (baseline, before treatment), day 21, and day 90. We observed that the scores in FMA and BI were increased, and NIHSS were decreased in both groups on day 21 and 90, compared with the baseline (day 0). Furthermore, significantly better scores in FMA of lower limbs and NIHSS were found in patients treated with IRT-TCM on day 21 and 90. For mRS, the percentage of patients ranking 0 and 1 in the intervention group presented a striking contrast to the control group on day 90 but with no significant difference. The results indicated that, as a feasible alternative therapy, IRT-TCM is beneficial for patients with ischemic stroke. Further research with larger sample size, long-term observation, and strict blinding are still in need to confirm the efficacy of IRT-TCM.