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Proteomic analysis reveals ginsenoside Rb1 attenuates myocardial ischemia/reperfusion injury through inhibiting ROS production from mitochondrial complex I.

ABSTRACT: Rationale: Reactive oxygen species (ROS) burst from mitochondrial complex I is considered the critical cause of ischemia/reperfusion (I/R) injury. Ginsenoside Rb1 has been reported to protect the heart against I/R injury; however, the underlying mechanism remains unclear. This work aimed to investigate if ginsenoside Rb1 attenuates cardiac I/R injury by inhibiting ROS production from mitochondrial complex I. Methods: In in vivo experiments, mice were given ginsenoside Rb1 and then subjected to I/R injury. Mitochondrial ROS levels in the heart were determined using the mitochondrial-targeted probe MitoB. Mitochondrial proteins were used for TMT-based quantitative proteomic analysis. In in vitro experiments, adult mouse cardiomyocytes were pretreated with ginsenoside Rb1 and then subjected to hypoxia and reoxygenation insult. Mitochondrial ROS, NADH dehydrogenase activity, and conformational changes of mitochondrial complex I were analyzed. Results: Ginsenoside Rb1 decreased mitochondrial ROS production, reduced myocardial infarct size, preserved cardiac function, and limited cardiac fibrosis. Proteomic analysis showed that subunits of NADH dehydrogenase in mitochondrial complex I might be the effector proteins regulated by ginsenoside Rb1. Ginsenoside Rb1 inhibited complex I- but not complex II- or IV-dependent O₂ consumption and enzyme activity. The inhibitory effects of ginsenoside Rb1 on mitochondrial I-dependent respiration and reperfusion-induced ROS production were rescued by bypassing complex I using yeast NADH dehydrogenase. Molecular docking and surface plasmon resonance experiments indicated that ginsenoside Rb1 reduced NADH dehydrogenase activity, probably via binding to the ND3 subunit to trap mitochondrial complex I in a deactive form upon reperfusion. Conclusion: Inhibition of mitochondrial complex I-mediated ROS burst elucidated the probable underlying mechanism of ginsenoside Rb1 in alleviating cardiac I/R injury.

SUBMITTER: Jiang L

PROVIDER: S-EPMC7778584 | biostudies-literature | 2021

REPOSITORIES: biostudies-literature

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Proteomic analysis reveals ginsenoside Rb1 attenuates myocardial ischemia/reperfusion injury through inhibiting ROS production from mitochondrial complex I.

Jiang Lujing L Yin Xiaojian X Chen Ya-Hui YH Chen Yan Y Jiang Wei W Zheng Hao H Huang Feng-Qing FQ Liu Baolin B Zhou Wei W Qi Lian-Wen LW Li Jia J

Theranostics 20210101 4

Rationale: Reactive oxygen species (ROS) burst from mitochondrial complex I is considered the critical cause of ischemia/reperfusion (I/R) injury. Ginsenoside Rb1 has been reported to protect the heart against I/R injury; however, the underlying mechanism remains unclear. This work aimed to investigate if ginsenoside Rb1 attenuates cardiac I/R injury by inhibiting ROS production from mitochondrial complex I. Methods: In in vivo experiments, mice were given ginsenoside Rb1 an ...[more]

PMID: 33408776

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Project description:The management of cardiac ischemic injury has been challenged by ischemia and reperfusion (I/R) injury. Reactive oxygen species (ROS) generated from mitochondrial reverse electron transport (RET) during the early phase of reperfusion is considered to be the initiating cause for ischemia and reperfusion injury. Ginsenosides and their prescriptions are widely used in the clinic for treatment of myocardial ischemia, however, the action to combat ROS remains to be elucidated. In this work, we used TMT-based proteomic approach to detect differential proteins from mitochondrial fractions of mice hearts with ischemia-reperfusion. Results indicated that the mass error of identified peptides was within 10 ppm, and most of the identified peptides were composed of 7-23 amino acids. Using these qualified data, 17,262 peptides, with a confidence level ≥ 95%, were mapped to 3,054 protein groups. PCA analysis showed that the model group was clearly separated from the blank group, while the protein pattern was partly reversed with Rb1 treatment. With a criteria of p-value < 0.05, 591 significantly changed proteins including 186 increased and 405 decreased ones in the model group were identified compared with the blank group. These proteins were divided into 4 clusters. Proteins in Cluster I highly increased in the model group, but clearly decreased in the Rb1 group. Proteins in Cluster IV clearly decreased in the model group, but markedly increased in the Rb1 group. Proteins in Cluster III and in Cluster IV were not significantly or slightly regulated by the Rb1 treatment. GO analysis of Cluster I and II indicated that the molecular function of these proteins were closely related to oxidoreductase activity and NADH dehydrogenase activity. Our result showed that Rb1 administration before ischemia markedly decreased infarct size (48 h post-I/R), and preserved cardiac function (2 weeks post-I/R), and subsequently limited tissue fibrosis (28 days post-I/R). These results indicated that targeted inhibition of mitochondrial complex I in the early stage of reperfusion by Rb1 is a potential therapeutic strategy for alleviating IR injury. This work not only indicates a potent molecular target for the precision therapy of myocardial ischemia by Rb1, but also provides novel knowledge for the management of cardiac ischemic injury by traditional Chinese medicine.

Project description:BackgroundMyocardial ischemia-reperfusion injury (MIRI) is the most common cause of death worldwide. The NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome plays an important role in the inflammatory response to MIRI. Dexmedetomidine (DEX), a specific agonist of α2-adrenergic receptor, is commonly used for sedation and analgesia in anesthesia and critically ill patients. Several studies have shown that dexmedetomidine has a strong anti-inflammatory effect in many diseases. Here, we investigated whether dexmedetomidine protects against MIRI by inhibiting the activation of the NLRP3 inflammasome in vitro.MethodsWe established an MIRI model in cardiomyocytes (CMs) alone and in coculture with cardiac fibroblasts (CFs) by hypoxia/reoxygenation (H/R) in vitro. The cells were treated with dexmedetomidine with or without MCC950 (a potent selective NLRP3 inhibitor). The beating rate and cell viability of cardiomyocytes, NLRP3 localization, the expression of inflammatory cytokines and NLRP3 inflammasome-related proteins, and the expression of apoptosis-related proteins, including Bcl2 and BAX, were determined.ResultsDexmedetomidine treatment increased the beating rates and viability of cardiomyocytes cocultured with cardiac fibroblasts. The expression of the NLRP3 protein was significantly upregulated in cardiac fibroblasts but not in cardiomyocytes after H/R and was significantly attenuated by dexmedetomidine treatment. Expression of the inflammatory cytokines IL-1β, IL-18 and TNF-α was significantly increased in cardiac fibroblasts after H/R and was attenuated by dexmedetomidine treatment. NLRP3 inflammasome activation induced the increased expression of cleaved caspase1, mature IL-1β and IL-18, while dexmedetomidine suppressed H/R-induced NLRP3 inflammasome activation in cardiac fibroblasts. In addition, dexmedetomidine reduced the expression of Bcl2 and BAX in cocultured cardiomyocytes by suppressing H/R-induced NLRP3 inflammasome activation in cardiac fibroblasts.ConclusionDexmedetomidine treatment can suppress H/R-induced NLRP3 inflammasome activation in cardiac fibroblasts, thereby alleviating MIRI by inhibiting the inflammatory response.

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Proteomic analysis reveals ginsenoside Rb1 attenuates myocardial ischemia/reperfusion injury through inhibiting ROS production from mitochondrial complex I.

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Proteomic analysis reveals ginsenoside Rb1 attenuates myocardial ischemia/reperfusion injury through inhibiting ROS production from mitochondrial complex I.

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