• Journal of Ginseng Research
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• v.44 no.2
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• pp.258-266
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• 2020

Background: Oxidative stress-induced cardiomyocytes apoptosis is a key pathological process in ischemic heart disease. Glutathione reductase (GR) reduces glutathione disulfide to glutathione (GSH) to alleviate oxidative stress. Ginsenoside Rb1 (GRb1) prevents the apoptosis of cardiomyocytes; however, the role of GR in this process is unclear. Therefore, the effects of GRb1 on GR were investigated in this study. Methods: The antiapoptotic effects of GRb1 were evaluated in H9C2 cells by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, annexin V/propidium iodide staining, and Western blotting. The antioxidative effects were measured by a reactive oxygen species assay, and GSH levels and GR activity were examined in the presence and absence of the GR inhibitor 1,3-bis-(2-chloroethyl)-1-nitrosourea. Molecular docking and molecular dynamics simulations were used to investigate the binding of GRb1 to GR. The direct influence of GRb1 on GR was confirmed by recombinant human GR protein. Results: GRb1 pretreatment caused dose-dependent inhibition of tert-butyl hydroperoxide-induced cell apoptosis, at a level comparable to that of the positive control N-acetyl-L-cysteine. The binding energy between GRb1 and GR was positive (-6.426 kcal/mol), and the binding was stable. GRb1 significantl reduced reactive oxygen species production and increased GSH level and GR activity without altering GR protein expression in H9C2 cells. Moreover, GRb1 enhanced the recombinant human GR protein activity in vitro, with a half-maximal effective concentration of ≈2.317 μM. Conversely, 1,3-bis-(2-chloroethyl)-1-nitrosourea co-treatment significantly abolished the GRb1's apoptotic and antioxidative effects of GRb1 in H9C2 cells. Conclusion: GRb1 is a potential natural GR agonist that protects against oxidative stress-induced apoptosis of H9C2 cells.

• Animal Bioscience
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• v.34 no.6
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• pp.1078-1087
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• 2021

Objective: Skeletal muscle satellite cells (SMSCs) are significant for the growth, regeneration, and maintenance of skeletal muscle after birth. However, currently, few studies have been performed on the isolation, culture and inducing differentiation of goose muscle satellite cells. Previous studies have shown that C1q and tumor necrosis factor-related protein 3 (CTRP3) participated in the process of muscle growth and development, but its role in the goose skeletal muscle development is not yet clear. This study aimed to isolate, culture, and identify the goose SMSCs in vitro. Additionally, to explore the function of CTRP3 in goose SMSCs. Methods: Goose SMSCs were isolated using 0.25% trypsin from leg muscle (LM) of 15 to 20 day fertilized goose eggs. Cell differentiation was induced by transferring the cells to differentiation medium with 2% horse serum and 1% penicillin streptomycin. Immunofluorescence staining of Desmin and Pax7 was used to identify goose SMSCs. Quantitative realtime polymerase chain reaction and western blot were applied to explore developmental expression profile of CTRP3 in LM and the regulation of CTRP3 on myosin heavy chains (MyHC), myogenin (MyoG) expression and Notch signaling pathway related genes expression. Results: The goose SMSCs were successfully isolated and cultured. The expression of Pax7 and Desmin were observed in the isolated cells. The expression of CTRP3 decreased significantly during leg muscle development. Overexpression of CTRP3 could enhance the expression of two myogenic differentiation marker genes, MyHC and MyoG. But knockdown of CTRP3 suppressed their expression. Furthermore, CTRP3 could repress the mRNA level of Notch signaling pathway-related genes, notch receptor 1, notch receptor 2 and hairy/enhancer-of-split related with YRPW motif 1, which previously showed a negative regulation in myoblast differentiation. Conclusion: These findings provide a useful cell model for the future research on goose muscle development and suggest that CTRP3 may play an essential role in skeletal muscle growth of goose.

• The Korean Journal of Physiology and Pharmacology
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• v.25 no.2
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• pp.147-157
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• 2021

Coronary microembolization (CME) is associated with cardiomyocyte apoptosis and cardiac dysfunction. Puerarin confers protection against multiple cardiovascular diseases, but its effects and specific mechanisms on CME are not fully known. Hence, our study investigated whether puerarin pretreatment could alleviate cardiomyocyte apoptosis and improve cardiac function following CME. The molecular mechanism associated was also explored. A total of 48 Sprague-Dawley rats were randomly divided into CME, CME + Puerarin (CME + Pue), sham, and sham + Puerarin (sham + Pue) groups (with 12 rats per group). A CME model was established in CME and CME + Pue groups by injecting 42 ㎛ microspheres into the left ventricle of rats. Rats in the CME + Pue and sham + Pue groups were intraperitoneally injected with puerarin at 120 mg/kg daily for 7 days before operation. Cardiac function, myocardial histopathology, and cardiomyocyte apoptosis index were determined via cardiac ultrasound, hematoxylin-eosin (H&E) and hematoxylin-basic fuchsin-picric acid (HBFP) stainings, and TdT-mediated dUTP nick-end labeling (TUNEL) staining, respectively. Western blotting was used to measure protein expression related to the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt)/glycogen synthase kinase-3β (GSK-3β) pathway. We found that, puerarin significantly ameliorated cardiac dysfunction after CME, attenuated myocardial infarct size, and reduced myocardial apoptotic index. Besides, puerarin inhibited cardiomyocyte apoptosis, as revealed by decreased Bax and cleaved caspase-3, and up-regulated Bcl-2 and PI3K/Akt/GSK-3β pathway related proteins. Collectively, puerarin can inhibit cardiomyocyte apoptosis and thus attenuate myocardial injury caused by CME. Mechanistically, these effects may be achieved through activation of the PI3K/Akt/GSK-3β pathway.

• The Korean Journal of Physiology and Pharmacology
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• v.26 no.6
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• pp.541-556
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• 2022

Myocardial fibrosis is a key link in the occurrence and development of diabetic cardiomyopathy. Its etiology is complex, and the effect of drugs is not good. Cardiomyocyte apoptosis is an important cause of myocardial fibrosis. The purpose of this study was to investigate the effect of gaseous signal molecule sulfur dioxide (SO₂) on diabetic myocardial fibrosis and its internal regulatory mechanism. Masson and TUNEL staining, Western-blot, transmission electron microscopy, RT-qPCR, immunofluorescence staining, and flow cytometry were used in the study, and the interstitial collagen deposition, autophagy, apoptosis, and changes in phosphatidylinositol 3-kinase (PI3K)/AKT pathways were evaluated from in vivo and in vitro experiments. The results showed that diabetic myocardial fibrosis was accompanied by cardiomyocyte apoptosis and down-regulation of endogenous SO₂-producing enzyme aspartate aminotransferase (AAT)_1/2. However, exogenous SO₂ donors could up-regulate AAT_1/2, reduce apoptosis of cardiomyocytes induced by diabetic rats or high glucose, inhibit phosphorylation of PI3K/AKT protein, up-regulate autophagy, and reduce interstitial collagen deposition. In conclusion, the results of this study suggest that the gaseous signal molecule SO₂ can inhibit the PI3K/AKT pathway to promote cytoprotective autophagy and inhibit cardiomyocyte apoptosis to improve myocardial fibrosis in diabetic rats. The results of this study are expected to provide new targets and intervention strategies for the prevention and treatment of diabetic cardiomyopathy.

• The Korea Journal of Herbology
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• v.32 no.2
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• pp.77-85
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• 2017

Objectives : This study aimed to investigate the unknown mechanisms behind the anti- inflammatory activity of Hyeonto-dan(HT) 70% ethanol extract on LPS-stimulated RAW 264.7 cells. Methods : Cells were treated with Hyeonto-dan 1 h prior to addition of 200 ng/mL of LPS. Cell viability was measured by the MTS assay. Nitric oxide levels were determined by the Griess assay. $PGE_2$ were measured using EIA kit. Pro-inflammatory cytokine production was measured by the enzyme-linked immunosorbent assay (ELISA). The expression of COX-2, iNOS, and MAPKs was investigated by Western blot, qRT-PCR. $NF-{\kappa}B$/p65 localization and interaction of the TLR-4 receptor with LPS was examined by immunofluorescence assays. Results : Hyeonto-dan had no cytotoxicity at the measured concentration. Hyeonto-dan inhibited NO production and pro-inflammatory cytokines such as IL-6, $TNF-{\alpha}$, and PGE2 as well as the protein and mRNA expression of iNOS and COX-2. Moreover, Hyeonto-dan inhibited the interaction between LPS and TLR-4 in murine macrophages. It suppressed phosphorylation of extracellular signal-regulated kinase (ERK 1/2), c-jun N-terminal kinase (JNK 1/2) and p38. Finally, it inhibited translocation of $NF-{\kappa}B$ in response to competitive LPS. Conclusions : Based on the results of this study, Hyeonto-dan inhibited the binding of TLR-4 receptor to LPS and inhibited the phosphorylation of extracellular signaling pathway MAPKs. These inhibitory effects are thought that the amount of $NF-{\kappa}B$ delivered to the nucleus was decreased and the inflammatory reaction was prevented by decreasing the production of LPS-induced $PGE_2$, NO, IL-6 and $TNF-{\alpha}$.

• Journal of Ginseng Research
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• v.47 no.2
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• pp.199-209
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• 2023

Background: Due to the interrupted blood supply in cerebral ischemic stroke (CIS), ischemic and hypoxia results in neuronal depolarization, insufficient NAD+, excessive levels of ROS, mitochondrial damages, and energy metabolism disorders, which triggers the ischemic cascades. Currently, improvement of mitochondrial functions and energy metabolism is as a vital therapeutic target and clinical strategy. Hence, it is greatly crucial to look for neuroprotective natural agents with mitochondria protection actions and explore the mediated targets for treating CIS. In the previous study, notoginseng leaf triterpenes (PNGL) from Panax notoginseng stems and leaves was demonstrated to have neuroprotective effects against cerebral ischemia/reperfusion injury. However, the potential mechanisms have been not completely elaborate. Methods: The model of middle cerebral artery occlusion and reperfusion (MCAO/R) was adopted to verify the neuroprotective effects and potential pharmacology mechanisms of PNGL in vivo. Antioxidant markers were evaluated by kit detection. Mitochondrial function was evaluated by ATP content measurement, ATPase, NAD and NADH kits. And the transmission electron microscopy (TEM) and pathological staining (H&E and Nissl) were used to detect cerebral morphological changes and mitochondrial structural damages. Western blotting, ELISA and immunofluorescence assay were utilized to explore the mitochondrial protection effects and its related mechanisms in vivo. Results: In vivo, treatment with PNGL markedly reduced excessive oxidative stress, inhibited mitochondrial injury, alleviated energy metabolism dysfunction, decreased neuronal loss and apoptosis, and thus notedly raised neuronal survival under ischemia and hypoxia. Meanwhile, PNGL significantly increased the expression of nicotinamide phosphoribosyltransferase (NAMPT) in the ischemic regions, and regulated its related downstream SIRT1/2/3-MnSOD/PGC-1α pathways. Conclusion: The study finds that the mitochondrial protective effects of PNGL are associated with the NAMPT-SIRT1/2/3-MnSOD/PGC-1α signal pathways. PNGL, as a novel candidate drug, has great application prospects for preventing and treating ischemic stroke.

• Molecules and Cells
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• v.45 no.3
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• pp.122-133
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• 2022

The aim of this study was to investigating whether lncRNA H19 promotes myocardial fibrosis by suppressing the miR-29a-3p/miR-29b-3p-VEGFA/TGF-β axis. Patients with atrial fibrillation (AF) and healthy volunteers were included in the study, and their biochemical parameters were collected. In addition, pcDNA3.1-H19, si-H19, and miR-29a/b-3p mimic/inhibitor were transfected into cardiac fibroblasts (CFs), and proliferation of CFs was detected by MTT assay. Expression of H19 and miR-29a/b-3p were detected using real-time quantitative polymerase chain reaction, and expression of α-smooth muscle actin (α-SMA), collagen I, collagen II, matrix metalloproteinase-2 (MMP-2), and elastin were measured by western blot analysis. The dual luciferase reporter gene assay was carried out to detect the sponging relationship between H19 and miR-29a/b-3p in CFs. Compared with healthy volunteers, the level of plasma H19 was significantly elevated in patients with AF, while miR-29a-3p and miR-29b-3p were markedly depressed (P < 0.05). Serum expression of lncRNA H19 was negatively correlated with the expression of miR-29a-3p and miR-29b-3p among patients with AF (r_s = -0.337, r_s = -0.236). Moreover, up-regulation of H19 expression and down-regulation of miR-29a/b-3p expression facilitated proliferation and synthesis of extracellular matrix (ECM)-related proteins. SB431542 and si-VEGFA are able to reverse the promotion of miR-29a/b-3p on proliferation of CFs and ECM-related protein synthesis. The findings of the present study suggest that H19 promoted CF proliferation and collagen synthesis by suppressing the miR-29a-3p/miR-29b-3p-VEGFA/TGF-β axis, and provide support for a potential new direction for the treatment of AF.

• The Korean Journal of Physiology and Pharmacology
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• v.27 no.2
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• pp.143-155
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• 2023

Percutaneous coronary intervention and acute coronary syndrome are both closely tied to the frequently occurring complication of coronary microembolization (CME). Resveratrol (RES) has been shown to have a substantial cardioprotective influence in a variety of cardiac diseases, though its function and potential mechanistic involvement in CME are still unclear. The forty Sprague-Dawley rats were divided into four groups randomly: CME, CME + RES (25 mg/kg), CME + RES (50 mg/kg), and sham (10 rats per group). The CME model was developed. Echocardiography, levels of myocardial injury markers in the serum, and histopathology of the myocardium were used to assess the function of the cardiac muscle. For the detection of the signaling of TLR4/MyD88/NF-κB along with the expression of pyroptosis-related molecules, ELISA, qRT-PCR, immunofluorescence, and Western blotting were used, among other techniques. The findings revealed that myocardial injury and pyroptosis occurred in the myocardium following CME, with a decreased function of cardiac, increased levels of serum myocardial injury markers, increased area of microinfarct, as well as a rise in the expression levels of pyroptosis-related molecules. In addition to this, pretreatment with resveratrol reduced the severity of myocardial injury after CME by improving cardiac dysfunction, decreasing serum myocardial injury markers, decreasing microinfarct area, and decreasing cardiomyocyte pyroptosis, primarily by blocking the signaling of TLR4/MyD88/NF-κB and also reducing the NLRP3 inflammasome activation. Resveratrol may be able to alleviate CME-induced myocardial pyroptosis and cardiac dysfunction by impeding the activation of NLRP3 inflammasome and the signaling pathway of TLR4/MyD88/NF-κB.

• Journal of Ginseng Research
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• v.47 no.6
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• pp.743-754
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• 2023

Background: Myocardial fibrosis post-myocardial infarction (MI) can induce maladaptive cardiac remodeling as well as heart failure. Although 20(S)-ginsenoside Rg3 (Rg3) has been applied to cardiovascular diseases, its efficacy and specific molecular mechanism in myocardial fibrosis are largely unknown. Herein, we aimed to explore whether TGFBR1 signaling was involved in Rg3's anti-fibrotic effect post-MI. Methods: Left anterior descending (LAD) coronary artery ligation-induced MI mice and TGF-β1-stimulated primary cardiac fibroblasts (CFs) were adopted. Echocardiography, hematoxlin-eosin and Masson staining, Western-blot and immunohistochemistry, CCK8 and Edu were used to study the effects of Rg3 on myocardial fibrosis and TGFBR1 signaling. The combination mechanism of Rg3 and TGFBR1 was explored by surface plasmon resonance imaging (SPRi). Moreover, myocardial Tgfbr1-deficient mice and TGFBR1 adenovirus were adopted to confirm the pharmacological mechanism of Rg3. Results: In vivo experiments, Rg3 ameliorated myocardial fibrosis and hypertrophy and enhanced cardiac function. Rg3-TGFBR1 had the 1.78×10^-7 M equilibrium dissociation constant based on SPRi analysis, and Rg3 inhibited the activation of TGFBR1/Smads signaling dose-dependently. Cardiac-specific Tgfbr1 knockdown abolished Rg3's protection against myocardial fibrosis post-MI. In addition, Rg3 downregulated the TGF-β1-mediated CFs growth together with collagen production in vitro through TGFBR1 signaling. Moreover, TGFBR1 adenovirus partially blocked the inhibitory effect of Rg3. Conclusion: Rg3 improves myocardial fibrosis and cardiac function through suppressing CFs proliferation along with collagen deposition by inactivation of TGFBR1 pathway.

• Journal of Microbiology and Biotechnology
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• v.34 no.9
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• pp.1769-1777
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• 2024

Chemotherapy-induced nausea and vomiting (CINV) is a debilitating side effect related to activation of substance P (SP). SP activation can result from dysregulation of the gut-brain axis, and also from activation of protein kinase A signaling (PKA) signaling. In this study, we connected these factors in an attempt to unveil the mechanisms underlying CINV and develop new therapeutic strategies. Female rats were injected with cisplatin (Cis) to induce pica. Fecal samples were collected before/after injection, and subjected to lipid metabolomics analysis. In another portion of pica rats, the PKA inhibitor KT5720 was applied to investigate the involvement of PKA signaling in CINV, while fecal microbiota transplantation (FMT) was implemented to verify the therapeutic effect of the lipid metabolite 14(15)-EpETE. Pica symptoms were recorded, followed by ileal histological examination. The targeting relationship between 14(15)-EpETE and glucagon was determined by bioinformatics. SP and glucagon/PKA signaling in rat ileum, serum, and/or brain substantia nigra were detected by immunohistochemistry, enzyme-linked immunosorbent assay, and/or western blot. The results showed a significantly lower level of 14(15)-EpETE in rat feces after Cis injection. KT5720 treatment alleviated Cis-induced pica symptoms, ileal injury, SP content increase in the ileum, serum, and brain substantia nigra, and ileal PKA activation in rats. The ileal level of glucagon was elevated by Cis in rats. FMT exerted an effect similar to that of KT5720 treatment, relieving the Cis-induced changes, including ileal glucagon/PKA activation in rats. Our findings demonstrate that FMT restores 14(15)-EpETE production, which inhibits SP release by targeting GCG/PKA signaling, ultimately mitigating CINV.