RESUMO
This study aims to explore the mechanism of Linggui Zhugan Decoction(LGZGD) in inhibiting Angiotensin â ¡(Angâ ¡)-induced cardiomyocyte hypertrophy by regulating sigma-1 receptor(Sig1R). The model of H9c2 cardiomyocyte hypertrophy induced by Angâ ¡ in vitro was established by preparing LGZGD-containing serum and blank serum. H9c2 cells were divided into normal group, Angâ ¡ model group, 20% normal rat serum group(20% NSC), and 20% LGZGD-containing serum group. After the cells were incubated with Angâ ¡(1 µmol·L~(-1)) or Angâ ¡ with serum for 72 h, the surface area of cardiomyocytes was detected by phalloidine staining, and the activities of Na~+-K~+-ATPase and Ca~(2+)-Mg~(2+)-ATPase were detected by micromethod. The mitochondrial Ca~(2+) levels were detected by flow cytometry, and the expression levels of atrial natriuretic peptide(ANP), brain natriuretic peptide(BNP), Sig1R, and inositol 1,4,5-triphosphate receptor type 2(IP_3R_2) were detected by Western blot. The expression of Sig1R was down-regulated by transfecting specific siRNA for investigating the efficacy of LGZGD-containing serum on cardiomyocyte surface area, Na~+-K~+-ATPase activity, Ca~(2+)-Mg~(2+)-ATPase activity, mitochondrial Ca~(2+), as well as ANP, BNP, and IP_3R_2 protein expressions. The results showed that compared with the normal group, Angâ ¡ could significantly increase the surface area of cardiomyocytes and the expression of ANP and BNP(P<0.01), and it could decrease the activities of Na~+-K~+-ATPase and Ca~(2+)-Mg~(2+)-ATPase, the concentration of mitochondrial Ca~(2+), and the expression of Sig1R(P<0.01). In addition, IP_3R_2 protein expression was significantly increased(P<0.01). LGZGD-containing serum could significantly decrease the surface area of cardiomyocytes and the expression of ANP and BNP(P<0.05, P<0.01), and it could increase the activities of Na~+-K~+-ATPase and Ca~(2+)-Mg~(2+)-ATPase, the concentration of mitochondrial Ca~(2+ )(P<0.01), and the expression of Sig1R(P<0.05). In addition, IP_3R_2 protein expression was significantly decreased(P<0.05). However, after Sig1R was down-regulated, the effects of LGZGD-containing serum were reversed(P<0.01). These results indicated that the LGZGD-containing serum could inhibit cardiomyocyte hypertrophy induced by Angâ ¡, and its pharmacological effect was related to regulating Sig1R, promoting mitochondrial Ca~(2+ )inflow, restoring ATP synthesis, and protecting mitochondrial function.
Assuntos
Miócitos Cardíacos , ATPase Trocadora de Sódio-Potássio , Ratos , Animais , Células Cultivadas , ATPase Trocadora de Sódio-Potássio/genética , ATPase Trocadora de Sódio-Potássio/metabolismo , Fator Natriurético Atrial/genética , Fator Natriurético Atrial/metabolismo , Angiotensina II/efeitos adversos , Angiotensina II/metabolismo , Peptídeo Natriurético Encefálico/metabolismo , Hipertrofia/metabolismo , Cardiomegalia/induzido quimicamente , Cardiomegalia/tratamento farmacológico , Cardiomegalia/genéticaRESUMO
RATIONALE: Ling-Gui-Zhu-Gan decoction (LGZGD), one of the 100 herbal classic formulas, is clinically used to treat chronic heart failure with remarkable curative effect. However, LGZGD pharmacokinetic parameters in pathological model rats are poorly understood, in particular for special components. As physicochemical properties are specific to each representative component, no standard sample preparation is available for absolute quantification of representative components of LGZGD in rat plasma. METHODS: A specific, sensitive and high-throughput ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC/MS/MS) method capturing 24 representative components was developed and applied to evaluate the pharmacokinetic parameters of LGZGD in acute myocardial infarction (AMI) rat plasma after intragastric administration (2.4, 4.8 and 9.6 g/kg). Precipitation and extraction were selected and optimized for plasma preparation, and isopropanol precipitation could offer higher recovery and broader coverage. RESULTS: It was expected that AMI could cause less absorption and slower elimination of most of active components of LGZGD. Most of newly reported special components absorbed quickly and eliminated slowly. The average elimination half-life of the 24 representative components was 10.09 h, which is consistent with the dosage of LGZGD (twice daily). CONCLUSIONS: The specificity, linearity, precision and accuracy, recovery, matrix effect and stability were validated according to Food and Drug Administration guidance. The validation results demonstrated that the method could be applied to evaluate the pharmacokinetic parameters of LGZGD in AMI rats. The pharmacokinetic parameters showed substantial improvement in quality research of LGZGD, thereby laying the groundwork for preclinical and clinical trials in chronic heart failure clinical efficacy.
Assuntos
Insuficiência Cardíaca , Infarto do Miocárdio , Estados Unidos , Animais , Ratos , Cromatografia Líquida de Alta Pressão , Espectrometria de Massas em Tandem , Infarto do Miocárdio/tratamento farmacológicoRESUMO
This study aimed to explore the effect of Linggui Zhugan Decoction(LGZGD)-containing serum on mitochondrial oxidative stress in cardiomyocytes based on the NF-E2-related factor2(Nrf2)/Bcl-2/adenovirus E1 B 19 kDa interacting protein(BNIP3) signaling pathway. After the preparation of LGZGD-containing serum and blank serum, H9 c2 cardiomyocytes were exposed to H_2O_2 for inducing oxidative stress in vitro. The H9 c2 cells were divided into four groups, namely normal control group, H_2O_2 model group, 20% blank serum group, and 20% LGZGD-containing serum group. After the cells were pre-treated with different types of serum for 12 h and cultured with 100 µmol·L~(-1 )H_2O_2 for 6 h, the reactive oxygen species(ROS) level in each group was detected by DCFH-DA, and the openness of mitochondrial permeability transition pore(mPTP) was measured using a calcein AM fluorescent probe. The expression levels of cytoplasmic cytochrome C(CytC), mitochondrial CytC, cytoplasmic and nuclear Nrf2, and BNIP3 were detected by Western blot. Nrf2-silenced H9 c2 cells were prepared by transfecting them with siRNA-Nrf2 for investigating the efficacy of LGZGD-containing serum in regulating ROS, mPTP, cytoplasmic and mitochondrial CytC, and BNIP3. The results showed that compared with the normal control group, H_2O_2 significantly increased the ROS content and mPTP openness(P<0.01), and the expression of Nrf2, BNIP3, and cytoplasmic CytC(P<0.01), and decreased the expression of mitochondrial CytC(P<0.01), without causing obvious change in cytoplasmic Nrf2. LGZGD-containing serum significantly lowered ROS content(P<0.01), inhibited mPTP openness(P<0.01), down-regulated the expression of cytoplasmic CytC and BNIP3(P<0.01), up-regulated mitochondrial CytC expression(P<0.01), and promoted Nrf2 nuclear translocation(P<0.05). However, after Nrf2 silencing, the reduced ROS production, diminished BNIP3 expression, and inhibited mPTP openness and CytC release induced by LGZGD-containing serum were reversed(P<0.01). These results have suggested that LGZGD-containing serum is able to alleviate the mitochondrial oxidative stress injury of cardiomyocytes by regulating the Nrf2/BNIP3 signaling pathway.
Assuntos
Miócitos Cardíacos , Fator 2 Relacionado a NF-E2 , Apoptose , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Poro de Transição de Permeabilidade MitocondrialRESUMO
This study aims to investigate the effect of atractylenolide â ¢(ATL-â ¢) on hydrogen peroxide(H_2O_2)-induced endoplasmic reticulum stress and apoptosis of H9 c2 cells via the ROS/GRP78/caspase-12 signaling pathway.The binding activity of ATL-â ¢ to GRP78 was determined by molecular docking.The result showed that ATL-â ¢ had a good binding activity to GRP78, and the binding activity of ATL-â ¢ was stronger than that of its specific inhibitor.The endoplasmic reticulum stress model of H9 c2 was established by H_2O_2(100 µmol·L~(-1)) treatment.Five groups were designed: blank control group, model group, and ATL-â ¢(15, 30, and 60 µmol·L~(-1)) groups.Apoptosis was detected by Hoechst/PI double staining and flow cytometry.The levels of superoxide dismutase(SOD), malondialdehyde(MDA), and lactate dehydrogenase(LDH) were measured by colorimetry.The levels of reactive oxygen species(ROS) and calcium(Ca~(2+)) in cytoplasm were determined by the fluorescence probe DCFH-DA and the calcium fluorescence probe Flou-4, respectively.The protein levels of GRP78, caspase-12, and caspase-3 were determined by Western blot, and the mRNA levels of GRP78 and caspase-12 by RT-qPCR.N-acetyl-L-cysteine(NAC) and 4-phenylbutyric acid(4-PBA) were respectively used to inhibit ROS and GRP78, and then the mechanism of ATL-â ¢ in protecting the cells from endoplasmic reticulum stress induced by H_2O_2 were deduced.ATL-â ¢(15, 30, and 60 µmol·L~(-1)) decreased the apoptosis rate and ROS, MDA, and LDH levels(P<0.01), increased the SOD activity(P<0.01), and down-regulated the protein levels of GRP78, caspase-12, and caspase-3 and the mRNA levels of GRP78 and caspase-12(P<0.05).The addition of NAC decreased the apoptosis rate and ROS, MDA, GRP78, caspase-12, and caspase-3 levels(P<0.01), while it elevated the SOD level(P<0.01).The addition of 4-PBA also decreased the apoptosis rate and the levels of GRP78, caspase-12, caspase-3, and Ca~(2+)(P<0.01).The effect of inhibitors were consistent with that of ATL-â ¢.In conclusion, ATL-â ¢ can protect H9 c2 cardiomyocytes by regulating ROS/GRP78/caspase-12 signaling pathway to inhibit H_2O_2-induced endoplasmic reticulum stress and apoptosis.
Assuntos
Cálcio , Chaperona BiP do Retículo Endoplasmático , Apoptose , Cálcio/farmacologia , Caspase 12/genética , Caspase 12/metabolismo , Caspase 3/genética , Caspase 3/metabolismo , Estresse do Retículo Endoplasmático , Lactonas , Simulação de Acoplamento Molecular , RNA Mensageiro , Espécies Reativas de Oxigênio/metabolismo , Sesquiterpenos , Transdução de Sinais , Superóxido Dismutase/metabolismoRESUMO
BACKGROUND: To explore the underlying molecule mechanism of herbal medicine in preventing ventricular remodeling (VR), we take a herbal formula that is clinically effective for preventing VR as an example, which composed of Pachyma hoelen Rumph, Atractylodes macrocephala Koidz., Cassia Twig and Licorice. Due to multi-components and multi-targets in herbal medicine, it is extremely difficult to systematically explain its mechanisms of action. METHODS: An innovative systematic investigation framework which combines with pharmacokinetic screening, target fishing, network pharmacology, DeepDDI algorithm, computational chemistry, molecular thermodynamics, in vivo and in vitro experiments was performed for deciphering the underlying molecular mechanisms of herbal medicine for treating VR. RESULTS: ADME screening and SysDT algorithm determined 75 potentially active compounds and 109 corresponding targets. Then, systematic analysis of networks reveals the crucial active ingredients and key targets in herbal medicine. Additionally, transcriptomic analysis identifies 33 key regulators during VR progression. Moreover, PPI network and biological function enrichment present four crucial signaling pathways, i.e. NF-κB and TNF, PI3K-AKT and C-type lectin receptor signaling pathways involved in VR. Besides, both molecular experiments at animal and cell levels reveal the beneficial effect of herbal medicine on preventing VR. Finally, MD simulations and binding free energy validate the reliability of drug-target interactions. CONCLUSION: Our novelty is to build a systematic strategy which combines various theoretical methods combined with experimental approaches. This strategy provides a deep understanding for the study of molecular mechanisms of herbal medicine on treating diseases from systematic level, and offers a new idea for modern medicine to explore drug interventions for complex diseases as well.
Assuntos
Medicamentos de Ervas Chinesas , Plantas Medicinais , Animais , Medicamentos de Ervas Chinesas/farmacologia , Medicamentos de Ervas Chinesas/uso terapêutico , Medicamentos de Ervas Chinesas/química , Fosfatidilinositol 3-Quinases , Reprodutibilidade dos Testes , Remodelação Ventricular , Simulação de Acoplamento Molecular , Medicina Tradicional Chinesa/métodosRESUMO
Coronary heart disease (CHD) is defined by atherosclerosis, which can result in stenosis or blockage of the arterial cavity, leading to ischemic cardiac diseases such as angina and myocardial infarction. Accumulating evidence indicates that the gut microbiota plays a vital role in the beginning and progression of CHD. The gut microbial metabolite, trimethylamine-N-oxide (TMAO), is intimately linked to the pathophysiology of CHD. TMAO is formed when trimethylamine (TMA) is converted by flavin-containing monooxygenases in the hepatocytes. Therefore, inhibition of TMA production is essential to reduce TMAO levels. Flavonoids may reduce the risk of death from cardiovascular disease. In this article, we reviewed and evaluated twenty-two flavonoids for the therapy of CHD based on their inhibition of TMA-lyase by molecular docking. Docking results revealed that baicalein, fisetin, acacetin, and myricetin in flavonoid aglycones, and baicalin, naringin, and hesperidin in flavonoid glycosides had a good binding effect with TMA-lyase. This indicates that these chemicals were the most active and could be used as lead compounds for structural modification in the future. PRACTICAL APPLICATIONS: Flavonoids are a large class of polyphenolic compounds found in fruits, vegetables, flowers, tea, and herbal medicines, which are inexorably metabolized and transformed into bioactive metabolites by α-rhamnosidase, ß-glucuronidase, ß-glucosidase, and nitroreductase produced by the gut microbiota, which plays a beneficial role in the prevention and treatment of cardiovascular diseases. Because flavonoids protect the cardiovascular system and regulate the gut microbiota, and the gut microbiota is directly connected to TMAO, thus, reducing TMAO levels involves blocking the transition of TMA to TMAO, which may be performed by reducing TMA synthesis. Molecular docking results found that baicalein, fisetin, acacetin, and myricetin in flavonoid aglycones, and baicalin, naringin, and hesperidin in flavonoid glycosides had good binding effects on TMA-lyase, which were the most active and could be used as lead compounds for structural modification.
Assuntos
Doença das Coronárias , Hesperidina , Liases , Humanos , Simulação de Acoplamento Molecular , FlavonoidesRESUMO
Ventricular remodeling (VR) after acute myocardial infarction (AMI) is an important pathophysiological basis for the development of chronic heart failure (CHF). At present, Ling-Gui-Zhu-Gan decoction (LGZGD) has been widely reported in the clinical treatment and basic research of cardiovascular diseases (CVDs), such as myocardial infarction, heart failure, and angina pectoris. However, the mechanism of LGZGD against VR after AMI remains unclear. Ultra-performance liquid chromatography (UPLC) was applied to investigate the major constituents of LGZGD, and molecular docking was used to predict the targets on the NLRP3/Caspase-1/GSDMD signaling pathway. In vivo, histological changes in the myocardium were visualized using HE staining and Masson staining, and cardiomyocyte apoptosis was detected using TUNEL. IL-1ß activity in rat serum was determined by ELISA. Finally, NLRP3, Caspase-1, and GSDMD expressions were analyzed through RT-qPCR and Western blotting. The results showed that 8 authentic reference substances have been detected in LGZGD. Molecular docking showed that the major chemical constituents of LGZGD had a good binding activity with NLRP3, Caspase-1, and GSDMD. Our results showed that LGZGD treatment markedly improved cardiac pathology, decreased cardiomyocyte apoptosis, reduced IL-1ß activity, and regulated the expression of genes and proteins related to the NLRP3/Caspase-1/GSDMD signal pathway. These results suggest that LGZGD protects against VR after AMI through NLRP3/Caspase-1/GSDMD signal pathway.
RESUMO
The objective of this study was to determine the effect of atractylenolide III (ATL-III) on endoplasmic reticulum stress (ERS) injury, H9c2 cardiomyocyte apoptosis induced by tunicamycin (TM), and the GRP78/PERK/CHOP signaling pathway. Molecular docking was applied to predict the binding affinity of ATL-III to the key proteins GRP78, PERK, IREα, and ATF6 in ERS. Then, in vitro experiments were used to verify the molecular docking results. ERS injury model of H9c2 cells was established by TM. Cell viability was detected by MTT assay, and apoptosis was detected by Hoechst/PI double staining and flow cytometry. Protein expression levels of GRP78, PERK, eIF2α, ATF4, CHOP, Bax, Bcl-2, and Caspase-3 were detected by Western blot. And mRNA levels of GRP78, CHOP, PERK, eIF2α, and ATF4 were detected by RT-qPCR. Moreover, the mechanism was further studied by using GRP78 inhibitor (4-phenylbutyric acid, 4-PBA), and PERK inhibitor (GSK2656157). The results showed that ATL-III had a good binding affinity with GRP78, and the best binding affinity was with PERK. ATL-III increased the viability of H9c2 cells, decreased the apoptosis rate, downregulated Bax and Caspase-3, and increased Bcl-2 compared with the model group. Moreover, ATL-III downregulated the protein and mRNA levels of GRP78, CHOP, PERK, eIF2α, and ATF4, consistent with the inhibition of 4-PBA. ATL-III also decreased the expression levels of PERK, eIF2α, ATF4, CHOP, Bax, and Caspase-3, while increasing the expression of Bcl-2, which is consistent with GSK2656157. Taken together, ATL-III could inhibit TM-induced ERS injury and H9c2 cardiomyocyte apoptosis by regulating the GRP78/PERK/CHOP signaling pathway and has myocardial protection.
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Heart failure (HF) is a serious disease with high mortality. Oxidative stress plays a vital role in its occurrence and development. Licorice is commonly used to treat HF in traditional Chinese medicine. Liquiritin, the main ingredient of licorice, has antioxidant and anti-inflammatory properties, but the mechanism against oxidative stress in cardiomyocytes has not been reported. Establishment of oxidative damage model in H9c2 cells by hydrogen peroxide (H2 O2 ). Liquiritin (5, 10, 20 µmol/L) could significantly prevent the loss of cell viability and decrease the apoptosis rate. It can reduce the levels of reactive oxygen species (ROS), malonedialdehyde (MDA), lactate dehydrogenase (LDH), tumor necrosis factor-alpha (TNF-α), interleukin-1ß (IL-1ß), interleukin-6 (IL-6), and increase the activity of ATP, superoxidedismutase (SOD), glutathione peroxide (GSH-px), glutathione reductase (GR) and catalase (CAT) to alleviate oxidative stress and inflammation in a dose-dependent manner. Liquiritin was found to be related to AMP-Activated Protein Kinase (AMPK) pathway by molecular docking. Western blotting (WB) and quantitative reverse transcription PCR (RT-qPCR) confirmed that liquiritin could promote AMPKα phosphorylation and sirtuin 1 (SIRT1) protein expression, and inhibit phosphorylation of nuclear factor kappa B p65 (NF-κB p65). Compound C, EX 527, and PDTC can reverse the effects of liquiritin, indicating that its antioxidant effect is achieved by regulating AMPK/SIRT1/NF-κB signaling pathway. PRACTICAL APPLICATIONS: Heart failure is one of the most common cardiovascular diseases, and its treatment remains a worldwide problem. Licorice is a food and dietary supplement that has been used widely in traditional Chinese medicine (TCM). Liquiritin is one of the main active components of licorice, which has antioxidant and anti-inflammatory pharmacological effects. This study revealed the mechanism of licorice against oxidative damage of H9c2 cardiomyocytes, and provided a scientific basis for liquiritin as an antioxidant in the treatment of heart failure.
Assuntos
Insuficiência Cardíaca , NF-kappa B , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/farmacologia , Trifosfato de Adenosina/metabolismo , Anti-Inflamatórios/farmacologia , Antioxidantes/metabolismo , Antioxidantes/farmacologia , Catalase/metabolismo , Flavanonas , Glucosídeos , Glutationa/metabolismo , Glutationa Redutase/metabolismo , Humanos , Peróxido de Hidrogênio/farmacologia , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Lactato Desidrogenases/metabolismo , Simulação de Acoplamento Molecular , NF-kappa B/genética , NF-kappa B/metabolismo , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Sirtuína 1/genética , Sirtuína 1/metabolismo , Superóxido Dismutase/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
OBJECTIVES: Ling-Gui-Zhu-Gan decoction (LGZGD) is a potentially effective treatment for heart failure, and it showed significant anti-inflammatory potential in our previous studies. However, its ability to ameliorate heart failure through regulation of oxidative stress response is still unknown. This study was aimed to investigate the protective effect of LGZGD-containing serum on H2O2-induced oxidative injury in H9c2 cells and explore the underlying mechanism. METHODS: Eighteen rats were randomly divided into two groups: the blank control group and LGZGD group. The LGZGD group rats were administrated with 8.4 g/kg/d LGZGD for seven consecutive days through gavage, while the blank control group rats were given an equal volume of saline. The serum was extracted from all the rats. To investigate the efficacy and the underlying mechanism of LGZGD, we categorized the H9c2 cells into groups: the control group, model group, normal serum control (NSC) group, LGZGD group, LGZGD + all-trans-retinoic acid (ATRA) group, and ATRA group. Malonedialdehyde (MDA) and superoxide dismutase (SOD) were used as markers for oxidative stress. Dichlorodihydrofluorescin diacetate (DCFH-DA) staining was used to measure the levels of reactive oxygen species (ROS). The apoptosis rate was detected using flow cytometry. The expression levels of pro-caspase-3, cleaved-caspase-3, Bcl-2, Bax, Keap1, Nrf2, and HO-1 were measured using western blotting. The mRNA levels of Keap1, Nrf2, and HO-1 were measured using RT-qPCR. RESULTS: The LGZGD attenuated injury to H9c2 cells and reduced the apoptosis rate. It was also found to upregulate the SOD activity and suppress the formation of MDA and ROS. The expression levels of pro-caspase-3 and Bcl-2 were significantly increased, while those of cleaved-caspase-3 and Bax were decreased in the LGZGD group compared with the model group. As compared with the model group, the LGZGD group demonstrated decreased Keap1 protein expression and significantly increased Nrf2 nuclear expression and Nrf2-mediated transcriptional activity. ATRA was found to reverse the LGZGD-mediated antioxidative and antiapoptotic effect on injured H9c2 cells induced by H2O2. CONCLUSION: Our results demonstrated that LGZGD attenuated the H2O2-induced injury to H9c2 cells by inhibiting oxidative stress and apoptosis via the Nrf2/Keap1/HO-1 pathway. These observations suggest that LGZGD might prevent and treat heart failure through regulation of the oxidative stress response.