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Therapeutic Effect of Buyang Huanwu Decoction on the Gut Microbiota and Hippocampal Metabolism in a Rat Model of Cerebral Ischemia

ABSTRACT:

Buyang Huanwu decoction (BHD) is a well-known Chinese herbal prescription. It has been widely used in the clinical treatment of cerebral ischemia (CI) in China. However, the mechanism underlying the treatment of CI with BHD remains to be elucidated. In this study, we combined microbiomic and metabolomic strategies to explore the therapeutic effects of BHD on middle cerebral artery occlusion (MCAO) in rats. Our results showed that BHD could effectively improve neurological severity scores and alleviate neuronal damage in rats with MCAO. BHD could also reduce the level of peripheral proinflammatory cytokines and inhibit neuroinflammation. 16S rRNA sequencing showed that BHD could increase the relative abundances of the genera Lactobacillus, Faecalibacterium, Ruminococcaceae_UCG-002, etc., while decreasing the relative abundances of the genera Escherichia-Shigella, Klebsiella, Streptococcus, Coprococcus_2, Enterococcus, etc. Untargeted metabolomic analysis of hippocampal samples showed that 17 significantly differentially abundant metabolites and 9 enriched metabolic pathways were linked with BHD treatment. We also found that the regulatory effects of BHD on metabolites were correlated with the differentially abundant microbial taxa. The predicted function of the gut microbiota and the metabolic pathway enrichment results showed that purine metabolism, glutamatergic synapses, arginine and proline metabolism, and alanine, aspartic acid and glutamate metabolism were involved in the effects of BHD. These pathways may be related to pathological processes such as excitotoxicity, neuroinflammation, and energy metabolism disorder in CI. In summary, these findings suggest that regulation of hippocampal metabolism and of the composition and function of the gut microbiota may be important mechanisms underlying the effect of BHD in the treatment of CI.

INSTRUMENT(S): Liquid Chromatography MS - negative - reverse phase, Liquid Chromatography MS - positive - reverse phase

SUBMITTER: rongmei tang

PROVIDER: MTBLS4366 | MetaboLights | 2022-06-02

REPOSITORIES: MetaboLights

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			Action	DRS
	MTBLS4366	Other
	FILES	Other
	a_MTBLS4366_LC-MS_negative_reverse-phase_metabolite_profiling.txt	Txt
	a_MTBLS4366_LC-MS_positive_reverse-phase_metabolite_profiling.txt	Txt
	files-all.json	Other

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Project description:Cerebral ischemia/reperfusion injury (CI/RI), including neurological behavior deficits, cerebral infarction, blood-brain barrier (BBB) dysfunction, and neuroinflammation, et al. is a severe challenge in treatment of ischemic stroke. Traditional Chinese medicine (TCM) with the characteristics of multi-components and multi-functions for multiple targets/pathways has been historically used in the treatment of stroke and post stroke recovery in China. QiShenYiQi (QSYQ), a representative component-based Chinese medicine, is capable of reducing organ injury caused by ischemia/reperfusion via multiple mechanisms. Recently, we have previously reported that the positive action of QSYQ against the acute phase of CI/RI is partly via modulation of neuroinflammatory response. However, the effects and underlying mechanisms of QSYQ on subacute phase of CI/RI remain unknown, which are aimed to investigate in present study. The pharmacological action of QSYQ treatment on brain damage was estimated in the experimental stroke rats underwent 90 min ischemia and 8 days reperfusion by assessing the neurological and locomotor deficits, cerebral infarction, brain edema, and BBB integrity. Furthermore, we used TMT-based quantitative proteomics technology to identify significantly differentially expressed proteins following QSYQ treatment, which could give important clues for revealing the possible mechanism underlying the positive role of QSYQ in CI/RI. Besides, immunohistochemistry, western blot analysis, RT-qPCR, and rat ELISA kits were executed to further verify the accuracy of proteomics analysis results. As a consequence, we found that treatment with QSYQ (600mg/kg) for 7 days obviously improved neurological and behavioral impairment, attenuated infarct volume and brain edema, and alleviated BBB breakdown in stroke rats. Bioinformatics analysis suggested that differentially expressed protein galectin-3 mediated inflammatory response was closely related to the beneficial effect of QSYQ. Results of immunohistochemistry, western blot analysis and RT-qPCR showed that the model rats treated with QSYQ (600mg/kg) markedly downregulated the mRNA and protein expression level of galectin-3 in brain tissues compared to the untreated stroke rats. In addition, the decrease of mRNA level in brain tissues and contents in serum of TNF-α and IL-6 following QSYQ treatment were also observed. These interesting finding manifested that the effective action of QSYQ against subacute phase of CI/RI was partly via regulating galectin-3 mediated inflammatory reaction.

Project description:<p><em>Gastrodia elata</em> Blume (GE) has been widely used to treat various central and peripheral nerve diseases, and P-Hydroxybenzaldehyde (PHBA) is one of the indicative components of GE brain protection. Since the previous study of our group found that PHBA has a good effect on protecting mitochondria against cerebral ischemia-reperfusion injury in rats (I/R). We will further explore how PHBA regulates the metabolic mechanism in blood after cerebral I/R to find an effective therapeutic target to prevent and treat IS. Firstly, using the rat model of cerebral ischemia-reperfusion injury induced by middle cerebral artery occlusion/Reperfusion (MCAO/R). The therapeutic effect of PHBA on brain I/R was evaluated by neurological function score, triphenyl tetrazolium chloride (TTC), Hematoxylin and eosin (HE), and Nissl staining. Secondly, a non-targeted metabonomic based on HPLC-QTOF-MS/MS was established to identify differential metabolites. Finally, we analyzed a targeted metabolic spectrum and verified the potential therapeutic targets. The results showed that the neurological function score, cerebral infarction area, hippocampal morphology, and the number of neurons in the PHBA group were significantly improved compared with the model group. Metabonomic analysis showed that 13 different metabolites were identified between the model and PHBA group, which may be involved in the 'tricarboxylic acid cycle', 'glutathione metabolism', 'mutual transformation of pentose and glucuronates', and so on. Among them, the most significant differential metabolite, dGMP, decreased significantly after PHBA treatment. We used WB to verify the expression of membrane-associated guanosine kinase PSD-95 and The subunit of glutamate AMPA receptor GluA1, which significantly increased after PHBA treatment. In addition, we also found that PHBA increased the expression of the light chain-3 protein (LC3) and autophagy effector protein 1 (Beline1) in WB while decreasing the expression of sequestosome-1 (p62), indicating that autophagy activity was promoted. Similarly, in TUNEL staining and detection of apoptosis-related proteins, it was found that MCAO/R up-regulated the expression of Bax and Cleaved-caspase-3 while down-regulated the expression of Bcl-2, and increased the apoptosis of hippocampal neurons, but PHBA could improve this situation. In summary, these results suggest that cerebral I/R causes postsynaptic dysfunction by disrupting the interaction between PSD-95 and AMPARs, and the inhibition of the autophagy system eventually leads to the apoptosis of hippocampal neurons.</p>

Project description:Background and Objective: Cerebral infarction (CI) is a severe neurological disorder caused by localized disruption of blood supply to brain tissue, leading to high rates of disability and mortality. Circular RNAs (circRNAs), highly expressed in the brain, have been identified as potential therapeutic targets for CI. Although acupuncture has been shown to promote neurological recovery, the precise molecular mechanisms, particularly the role of circRNAs in regulating angiogenesis, remain insufficiently understood. This study is the first to demonstrate that electroacupuncture (EA) promotes angiogenesis by regulating the circRNA_011971/miR-196c-5p/ARL13B axis, enhancing the proliferation and migration of brain microvascular endothelial cells (BMECs), and accelerating angiogenesis. These findings provide new insights into the molecular mechanisms of acupuncture treatment for CI and identify potential therapeutic targets. Methods: Male Wistar rats were used to establish a cerebral infarction model via middle cerebral artery occlusion (MCAO). The rats were divided into a model group, an EA treatment group, and a control group. Immediately after MCAO induction, the EA group received acupuncture at the GV26 acupoint. Neurological deficits were assessed using the modified Neurological Severity Score (mNSS), and infarct volumes were measured using TTC staining. Immunofluorescence double staining was performed to detect angiogenesis markers CD31 and Ki67 in the peri-infarct area. CircRNA expression profiles in brain tissue were obtained through circRNA microarray analysis, and key circRNAs were validated via qRT-PCR and fluorescence in situ hybridization (FISH). In BMECs, circRNA_011971 knockdown was achieved using shRNA, and its biological functions were assessed through CCK8, wound healing, and transwell assays. The expression levels of Arl13b, VEGFA, and VEGFR2 were measured using qRT-PCR and Western blot after circRNA_011971 knockdown or overexpression. Molecular interactions among circRNA_011971, miR-196c-5p, and ARL13B were explored using dual-luciferase reporter assays, RNA immunoprecipitation (RIP), and co-immunoprecipitation (Co-IP). Results: Compared to the untreated group, the EA group exhibited significantly smaller infarct volumes, suggesting immediate EA treatment at GV26 following infarction can effectively limit infarct progression. Immunofluorescence analysis revealed a marked increase in the co-expression of CD31 and Ki67 in the peri-infarct region of the EA-treated rats, indicating enhanced angiogenesis. CircRNA microarray analysis showed that circRNA_011971 was significantly upregulated following ischemia and downregulated after EA treatment. In BMECs, circRNA_011971 knockdown inhibited cell proliferation, migration, and invasion, whereas its overexpression promoted these processes. Further mechanistic studies revealed that EA downregulated rno_circ_011971 expression, lifting its inhibition on miR-196c-5p and activating the ARL13B/VEGF signaling pathway, thereby significantly promoting angiogenesis. This study discover a molecular mechanism by which EA regulates post-CI angiogenesis through the circRNA/miRNA axis for the first time, offering new potential targets for acupuncture therapy. Conclusion: EA improves neurological function, reduces infarct size, and promotes angiogenesis, potentially through regulating of VEGFR via the rno_circ_011971/miR-196c-5p/ARL13B axis.

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Therapeutic Effect of Buyang Huanwu Decoction on the Gut Microbiota and Hippocampal Metabolism in a Rat Model of Cerebral Ischemia

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