miR-92a-3p regulates ethanol-induced apoptosis in H9c2 cardiomyocytes.

The role of miR-92a-3p in the ethanol-induced apoptosis of H9c2 cardiomyocytes remains unclear. In this study, we explored the role of miR-92a-3p in the ethanol-induced apoptosis of H9c2 cardiomyocytes and identified its target genes and signaling pathways. H9c2 cells were cultured with or without 100 mM ethanol for 24 h. The differential expression of miR-92a-3p was verified in H9c2 cells through reverse transcription-quantitative polymerase chain reaction (RT-qPCR). To manipulate the expression of miR-92a-3p, both a mimic and an inhibitor were transfected into H9c2 cells. An Annexin V-fluorescein isothiocyanate/propidium iodide apoptosis detection kit and apoptosis-related antibodies were used for apoptosis detection through flow cytometry and Western blotting, respectively. Target genes were verified through RT-qPCR, Western blotting, and double luciferase reporter gene assays. miR-92a-3p was significantly overexpressed in ethanol-stimulated H9c2 cardiomyocytes (P < 0.001). After ethanol stimulation, H9c2 myocardial cells exhibited increased apoptosis. The apoptosis rate was higher in the miR-92a-3p mimic group than in the control group. However, the apoptosis rate was lower in the miR-92a-3p inhibitor group than in the control group, indicating that miR-92a-3p promotes the ethanol-induced apoptosis of H9c2 myocardial cells. RT-qPCR and Western blotting revealed that the miR-92a-3p mimic and inhibitor significantly regulated the mRNA and protein expression levels of mitogen- and stress-activated protein kinase 2 and cyclic AMP-responsive element-binding protein 3-like protein 2 (CREB3L2), suggesting that miR-92a-3p promotes the apoptosis of H9c2 cardiomyocytes by inhibiting the MSK2/CREB/Bcl-2 pathway. Therefore, the apoptosis of H9c2 cardiomyocytes increases after ethanol stimulation, and miR-92a-3p can directly target MSK2 and CREB3L2, thereby promoting the ethanol-induced apoptosis of H9c2 myocardial cells.

25-hydroxycholesterol aggravates oxygen-glucose deprivation/reoxygenation-induced pyroptosis through promoting activation of NLRP3 inflammasome in H9C2 cardiomyocytes

25-hydroxycholesterol (25-HC) plays a role in the regulation of cell survival and immunity. However, the effect of 25-HC on myocardial ischemia/reperfusion (MI/R) injury remains unknown. Our present study aimed to investigate whether 25-HC aggravated MI/R injury through NLRP3 inflammasome-mediated pyroptosis. The overlapping differentially expressed genes (DEGs) in MI/R were identified from the GSE775, GSE45818, GSE58486, and GSE46395 datasets in Gene Expression Omnibus (GEO) database. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were conducted using the database of Annotation, Visualization and Integration Discovery (DAVID). The protein-protein interaction (PPI) network of the overlapping DEGs was established using the Search Tool for the Retrieval of Interacting Genes (STRING) database. These bioinformatics analyses indicated that cholesterol 25-hydroxylase (CH25H) was one of the crucial genes in MI/R injury. The oxygen-glucose deprivation/reoxygenation (OGD/R) cell model was established to simulate MI/R injury. Western blot and RT-qPCR analysis demonstrated that CH25H was significantly upregulated in OGD/R-stimulated H9C2 cardiomyocytes. Moreover, knockdown of CH25H inhibited the OGD/R-induced pyroptosis and nod-like receptor protein 3 (NLRP3) inflammasome activation, as demonstrated by cell counting kit-8 (CCK8), lactate dehydrogenase (LDH), RT-qPCR, and western blotting assays. Conversely, 25-HC, which is synthesized by CH25H, promoted activation of NLRP3 inflammasome in OGD/R-stimulated H9C2 cardiomyocytes. In addition, the NLRP3 inhibitor BAY11-7082 attenuated 25-HC-induced H9C2 cell injury and pyroptosis under OGD/R condition. In conclusion, 25-HC could aggravate OGD/R-induced pyroptosis through promoting activation of NLRP3 inflammasome in H9C2 cells.

Organosulfur Compounds in Aged Garlic Extract Ameliorate Glucose Induced Diabetic Cardiomyopathy by Attenuating Oxidative Stress, Cardiac Fibrosis, and Cardiac Apoptosis.

BACKGROUND: Diabetic cardiomyopathy has emerged as a major cause of cardiac fibrosis, hypertrophy, diastolic dysfunction, and heart failure due to uncontrolled glucose metabolism in patients with diabetes mellitus. However, there is still no consensus on the optimal treatment to prevent or treat the cardiac burden associated with diabetes, which urges the development of dual antidiabetic and cardioprotective cardiac therapy based on natural products. This study investigates the cardiotoxic profile of glucose and the efficacy of AGE against glucose-induced cardiotoxicity in H9c2 cardiomyocytes. MATERIAL METHODS: The cellular metabolic activity of H9c2 cardiomyocytes under increasing glucose concentration and the therapeutic efficacy of AGE were investigated using the MTT cell cytotoxicity assay. The in vitro model was established in six groups known as 1. control, 2. cells treated with 25 µM glucose, 3. 100 µM glucose, 4. 25 µM glucose +35 µM AGE, 5. 100 µM glucose + 35 µM AGE, and 6. 35 µM AGE. Morphological and nuclear analyses were performed using Giemsa, HE, DAPI, and PI, respectively, whereas cell death was simultaneously assessed using the trypan blue assay. The antioxidant potential of AGE was evaluated by DCFH-DA assay, NO, and H202 scavenging assay. The activities of the antioxidant enzymes catalase and superoxide dismutase were also investigated. The antiglycative potential of AGE was examined by antiglycation assays, amylase zymography, and SDS PAGE. These results were then validated by in silico molecular docking and qRTPCR. RESULTS: Hyperglycemia significantly reduced cellular metabolic activity of H9c2 cardiomyocytes, and AGE was found to preserve cell viability approximately 2-fold by attenuating oxidative, fibrosis, and apoptotic signaling molecules. In silico and qRTPCR studies confirmed that organosulfur compounds target TNF-α, MAPK, TGF-ß, MMP-7, and caspase-9 signaling molecules to ameliorate glucose-induced cardiotoxicity. CONCLUSION: AGE was found to be an antidiabetic and cardioprotective natural product with exceptional therapeutic potential for use as a novel herb-drug therapy in the treatment of diabetic cardiomyopathy in future therapies.

Apurinic/apyrimidinic endonuclease 1 regulates palmitic acid-mediated apoptosis in cardiomyocytes via endoplasmic reticulum stress.

Cardiomyocyte apoptosis caused by fat metabolism disorder plays an essential role in the pathogenesis of diabetic cardiomyopathy (DCM). Apurinic/apyrimidinic endonuclease 1 (APE1) has multiple functions, including regulating redox and DNA repair. However, the role of APE1 in the pathogenesis of DCM remains unclear. To investigate the mechanism of APE1 on high-fat induced apoptosis in H9C2 cells, we treated H9C2 cells with palmitic acid (PA) as an apoptosis model caused by hyperlipidemia. We found that PA reduced the viability and increased apoptosis of H9C2 cells by inducing up-regulation of APE1 protein and endoplasmic reticulum (ER) stress. APE1 knockdown enhanced PA-induced apoptosis, and ER stress and overexpression of APE1 demonstrated the opposite effect. Furthermore, APE1 regulated PA-induced apoptosis via ER stress. The APE1 mutant (C65A, lack of redox regulation) loses its protective effect against ER stress and apoptosis. These findings indicate that APE1 protects PA-induced H9C2 cardiomyocyte apoptosis through ER stress via its redox-regulated function. This study provided new insights into the therapy for DCM.

Liraglutide ameliorates high glucose-induced oxidative stress injury in rat H9c2 cells through modulation of SIRT1 / 中华内分泌代谢杂志

Objective:To investigate the effect of liraglutide(LRG) on high glucose-induced oxidative stress injury in(H9c2) cardiomyocytes and its underlying mechanisms.Methods:A high glucose treatment was applied to H9c2 cells for 24 hours to establish an in vitro model of myocardial cell injury. Different concentrations of liraglutide(10, 100, 1000 nmol/L) were administered for intervention. Cell viability was evaluated using the CCK-8 assay, and changes in cell morphology were observed under an inverted microscope. After 24 hours of liraglutide(100 nmol/L) intervention following high glucose treatment, the levels of lactate dehydrogenase(LDH), superoxide dismutase(SOD), and malondialdehyde(MDA) in the cell supernatant were measured. RT-PCR and Western blotting were used to detect the mRNA and protein levels of silent information regulator factor 1(SIRT1) and forkhead box protein O1(FOXO1). Western blotting was also used to assess the acetylation level of FOXO1 protein. Small interfering RNA(siRNA) technology was employed to silence SIRT1 in H9c2 cells to confirm its role in the study. Results:Compared to the control group, the high glucose group showed decreased cell viability, cell structure damage, increased levels of LDH and MDA in the cell supernatant, decreased SOD levels, aggravated oxidative stress, decreased SIRT1 expression, and increased acetylation level of FOXO1(all P<0.05). Compared to the high glucose group, liraglutide intervention resulted in increased cell viability, improved cardiac cell morphology, reduced oxidative stress levels, increased SIRT1 expression, and decreased acetylation level of FOXO1(all P<0.05). When SIRT1 was downregulated, the protective effects of liraglutide were weakened(all P<0.05). Conclusions:Liraglutide has a protective effect against high glucose-induced oxidative stress injury in H9c2 cells, which may be associated with the upregulation of SIRT1 expression.

Yttrium chloride-induced cytotoxicity and DNA damage response via ROS generation and inhibition of Nrf2/PPARγ pathways in H9c2 cardiomyocytes.

Increasing exploration of rare-earth elements (REEs) has resulted in a high REEs' exposure risk. Owing to their persistence and accumulation of REEs in the environment, their adverse effects have caused widespread concern. However, limited toxicological data are available for the adverse effects of yttrium (Y) and its underlying mechanisms of action. In the present study, H9c2 cardiomyocytes were used in vitro model to investigate the cardiotoxicity of yttrium chloride (YCl3). Results show that YCl3 treatment resulted in reactive oxygen species (ROS) overproduction, decrease in ∆Ψm, and DNA damage. Mechanistically, we detected expression levels of protein in response to cellular DNA damage and antioxidative defense. Results indicated that the phosphorylation of histone H2AX remarkably increased in a dose-dependent manner. At a high YCl3-exposure concentration (120 µM), specific DNA damage sensors ATM/ATR-Chk1/Chk2 were significantly decreased. The protein levels of key antioxidant genes Nrf2/PPARγ/HO-1 were also remarkably inhabited. Additionally, the antioxidant N-acetyl-L-cysteine (NAC) pretreatment promoted the activation of antioxidative defense Nrf2/PPARγ signaling pathways, and prevented the production of cellular ROS, thus protecting the DNA from cleavage. Altogether, our findings suggest that YCl3 can induce DNA damage through causing intracellular ROS overproduction and inhibition of antioxidative defense, leading to cytotoxicity in H9c2 cardiomyocytes.

Mechanism of Aconiti Kusnezoffii Radix Processed with Chebulae Fructus Against H9c2 Cardiomyocyte Toxicity Based on TRPV1 Channel / 中国实验方剂学杂志

ObjectiveTo explore the role of transient receptor potential vanilloid 1 （TRPV1） channel in reducing cardiomyocyte toxicity of Aconiti Kusnezoffii Radix processed with Chebulae Fructus. MethodH9c2 cardiomyocytes cultured in vitro were used as a model to assess cell viability by methyl thiazolyl tetrazolium （MTT） assay， the expression of TRPV1 mRNA was detected by real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）， and the leakage rate of lactate dehydrogenase （LDH）， the changes of nucleus， reactive oxygen species （ROS）， mitochondrial membrane potential and Ca2+ contents were detected by enzyme linked immunosorbent assay （ELISA）. ResultCompared with the blank group， when the concentration was ≥0.5 g·L-1， the cell viability was significantly decreased （P<0.01）， the leakage rate of LDH， the release of ROS and Ca2+ were increased， the mitochondrial membrane potential was decreased， and the nucleus was pyknosis or even broken in raw Aconiti Kusnezoffii Radix and Aconiti Kusnezoffii Radix processed with Chebulae Fructus groups. When the concentration was ≥0.5 g·L-1， compared with the same mass concentration of raw Aconiti Kusnezoffii Radix group， the cell viability increased significantly （P<0.01）， the leakage rate of LDH， the release of ROS and Ca2+ decreased， the mitochondrial membrane potential increased， and the nuclear morphology improved in Aconiti Kusnezoffii Radix processed with Chebulae Fructus group. Application of the same mass concentration of raw Aconiti Kusnezoffii Radix to H9c2 cardiomyocytes pretreated with the TRPV1 inhibitor BCTC significantly increased cell viability， decreased leakage rate of LDH， ROS and Ca2+ release， increased mitochondrial membrane potential and improved nuclear pyknosis compared with untreated H9c2 cardiomyocytes. Application of the same mass concentration of Aconiti Kusnezoffii Radix processed with Chebulae Fructus to H9c2 cardiomyocytes pretreated with BCTC decreased cell viability， increased LDH leakage rate， ROS and Ca2+ release， reduced mitochondrial membrane potential compared with untreated H9c2 cardiomyocytes. Real-time PCR results showed that both raw Aconiti Kusnezoffii Radix and Chebulae Fructus decoction could increase the expression of TRPV1 mRNA in cardiomyocytes in a concentration dependent manner. ConclusionRaw Aconiti Kusnezoffii Radix can induce cardiomyocyte apoptosis and cardiotoxicity by activating TRPV1 channel， while Aconiti Kusnezoffii Radix processed with Chebulae Fructus can attenuate the toxicity through TRPV1 channel， which may be related to the synergistic effect of acid components in Chebulae Fructus and alkaloids in Aconiti Kusnezoffii Radix on TRPV1 channel.

Study on protective effects of methyl ferulate on mitochondria of H 9c2 cardiomyocytes injured by hypoxia / 中国药房

OBJECTIVE To investigate the e ffects of methyl ferulate （MF） on the mitochondrial function of H 9c2 cardiomyocytes after hypoxia-induced injury. METHODS H9c2 cardiomyocytes were divided into normal group （no administration，no modeling ），hypoxia model group （modeling alone ），MF high-dose ，medium-dose and low-dose groups （40， 20，10 μmol/L）and positive control drug group （cyclosporin A ，1 μmol/L）. After drug pretreatment and inducing hypoxia-induced injury，the levels of lactate dehydrogenase （LDH），malondialdehyde（MDA），creatine kinase （CK）and adenosine triphosphate （ATP）were tested. The intracellular reactive oxygen species （ROS），mitochondrial membrane potential （MMP），the opening of mitochondrial membrane permeability transition pore （mPTP） were detected with flow cytometry. RESULTS Compared with hypoxia model group ，the levels of LDH ，MDA，CK and ROS fluorescence intensity were decreased significantly in MF high-dose，medium-dose and low-dose groups ，while the level of ATP was increased significantly （P＜0.01 or P＜0.05）. The red/ green fluorescence intensity ratio of MMP and the green fluorescence intensity of mPTP were increased significantly （P＜0.01 or P＜0.05）. CONCLUSIONS MF can reverse the levels of biochemical indexes in H 9c2 cardiomyocyte after hypoxia-induced injury，keep MMP stable ，reduce the opening of mPTP ，and has an obvious protective effect on the mitochondrial function of H9c2 cardiomyocytes injured by hypoxia ，and this protective effect is dose-dependent.

Nuciferine Attenuates Doxorubicin-Induced Cardiotoxicity: An In Vitro and In Vivo Study.

Chemotherapeutic drugs are a known factor that impairs the system of life due to their severe side effects. A more worrying fact is that the patients administered with doxorubicin fall under the risk of cardiotoxicity. The evolution of exploring plant-derived compounds is a possible way to combat health issues in therapeutic applications. Hence, this study focuses on the protective effect of plant-based compound nuciferine (NFN) against doxorubicin-induced cardiotoxicity in both in vitro and in vivo models. In this investigation, nuciferine significantly reduces DOX-mediated cardiotoxicity by mitigating reactive oxygen species, thereby preventing DNA fragmentation, regulating apoptosis genes and reducing the caspase 3/7 levels in vitro. Besides, nuciferine has shown significant protection against DOX-induced cardiac impairment and the upregulation of cardiogenic markers in vivo. The DOX-induced oxidative stress can be mitigated via enhancing the endogenous antioxidants, thereby controlling ROS-mediated apoptosis. In virtue of these potential features, nuciferine can be a budding candidate to address therapeutic needs.

Nuclear respiratory factor 1 protects H9C2 cells against hypoxia-induced apoptosis via the death receptor pathway and mitochondrial pathway.

Hypoxia-induced cardiomyocyte apoptosis is one of the leading causes of heart failure. Nuclear respiratory factor 1 (NRF-1) was suggested as a protector against cell apoptosis; However, the mechanism is not clear. Therefore, the aim of this study was to elucidate the role of NRF-1 in hypoxia-induced H9C2 cardiomyocyte apoptosis and to explore its effect on regulating the death receptor pathway and mitochondrial pathway. NRF-1 was overexpressed or knocked down in H9C2 cells, which were then exposed to a hypoxia condition for 0, 3, 6, 12, and 24 h. Changes in cell proliferation, cell viability, reactive oxygen species (ROS) generation, and mitochondrial membrane potential (MMP) were investigated. The activities of caspase-3, -8, and -9, apoptosis rate, and the gene and protein expression levels of the death receptor pathway and mitochondrial pathway were analyzed. Under hypoxia exposure, NRF-1 overexpression improved the proliferation and viability of H9C2 cells and decreased ROS generation, MMP loss, caspase activities, and the apoptosis rate. However, the NRF-1 knockdown group showed the opposite results. Additionally, NRF-1 upregulated the expression of antiapoptotic molecules involved in the death receptor and mitochondrial pathways, such as CASP8 and FADD-like apoptosis regulator, B-cell lymphoma 2, B-cell lymphoma-extra-large, and cytochrome C. Conversely, the expression of proapoptotic molecules, such as caspase-8, BH3-interacting domain death agonist, Bcl-2-associated X protein, caspase-9, and caspase-3 was downregulated by NRF-1 overexpression in hypoxia-induced H9C2 cells. These results suggest that NRF-1 functions as an antiapoptotic factor in the death receptor and mitochondrial pathways to mitigate hypoxia-induced apoptosis in H9C2 cardiomyocytes.

Dexmedetomidine at a dose of 1 µM attenuates H9c2 cardiomyocyte injury under 3 h of hypoxia exposure and 3 h of reoxygenation through the inhibition of endoplasmic reticulum stress.

Myocardial ischemia-reperfusion injury (MIRI) has been confirmed to induce endoplasmic reticulum stress (ERS) during downstream cascade reactions after the sufficient deterioration of cardiomyocyte function. However, clinically outcomes have been inconsistent with experimental findings because the mechanism has not been entirely elucidated. Dexmedetomidine (DEX), an α2 adrenergic receptor agonist with anti-inflammatory and organ-protective activity, has been shown to attenuate IRI in the heart. The present study aimed to determine whether DEX is able to protect injured cardiomyocytes under in vitro hypoxia/reoxygenation (H/R) conditions and evaluate the conditions under which ERS is efficiently ameliorated. The cytotoxicity of DEX in H9c2 cells was evaluated 24 h after treatment with several different concentrations of DEX. The most appropriate H/R model parameters were determined by the assessment of cell viability and injury with Cell Counting Kit-8 and lactate dehydrogenase (LDH) release assays after incubation under hypoxic conditions for 3 h and reoxygenation conditions for 3, 6, 12 and 24 h. Additionally, the aforementioned methods were used to assess cardiomyocytes cultured with various concentrations of DEX under H/R conditions. Furthermore, the degree of apoptosis and the mRNA and protein expression levels of glucose-regulated protein 78 (GRP78), C/EBP homologous protein (CHOP) and caspase-12 were evaluated in all groups. The addition of 1, 5 and 10 µM DEX to the cell culture significantly increased the proliferation of H9c2 cells by >80% under normal culture conditions. In the H/R model assessment, following 3 h of anoxia exposure, H9c2 cell viability decreased to 62.67% with 3 h of reoxygenation and to 36% with 6 h of reoxygenation compared with the control. The viability of H9c2 cells subjected to hypoxia for 3 h and reoxygenation for 3 h increased by 61.3% when pretreated with 1 µM DEX, and the LDH concentration in the supernatant was effectively decreased by 13.7%. H/R significantly increased the percentage of apoptotic cells, as detected by flow cytometry, and increased the expression levels of GRP78, CHOP and caspase-12, while treatment with either DEX or 4-phenylbutyric acid (4-PBA) significantly attenuated these effects. Additionally, despite the protective effect of DEX against H/R injury, 4-PBA attenuated the changes induced by DEX and H/R. In conclusion, treatment with 1 µM DEX alleviated cell injury, apoptosis and the increases in GRP78, CHOP and caspase-12 expression levels in H9c2 cells induced by 3 h of hypoxia and 3 h of reoxygenation.

Protective effect of inhibiting TRPM7 expression on hypoxia post-treatment H9C2 cardiomyocytes.

BACKGROUND: Transient receptor potential channel 7 (TRPM7) plays an important role in maintaining intracellular ion concentration and osmotic pressure. OBJECTIVE: The purpose of this study was to investigate the role and mechanism of inhibiting the expression of TRPM7 in the treatment of distal myocardial ischemia. METHODS: H9C2 cells were treated with hypoxia post-treatment and reperfusion, respectively, detect the expression of HIF-1α and TRPM7, the concentration of Ca2+ and the degree of apoptosis in the H9C2 cells. The relevant miRNAs targeting TRPM7 were searched, the TRPM7 interference vectors were constructed, and the interference of different interference vectors on TRPM7 in H9C2 cells was detected. RESULTS: The results showed that hypoxia post-treatment treatment would lead to increased expression of miR-22-3p which directly targeting TRPM7, decreased expression of TRPM7, increased expression of HIF-α and increased intracellular Ca2+ concentration. While reperfusion can increase the expression of HIF-1α and TRPM7 in H9C2 cells and increase the degree of apoptosis. CONCLUSION: Knockdown of TRPM7 can significantly reduce reperfusion injury in H9C2 cells, reduce the degree of apoptosis, and the TRPM7 interference vector can inhibit the expression of TRPM7 and have a certain protective effect on the reperfusion injury of H9C2 cells.

Study on protective mechanism of compatibility of Huoxue Jiedu recipe on H9C2 myocardial cell autophagy induced by hypoxia/reoxygenation / 中国药理学通报

Aim To investigate the protective effect of Huoxue Jiedu recipe on autophagy injury of H9C2 cardiomyocytes induced by hypoxia/reoxygenation and its mechanism. Methods H9C2 cardiomyocytes were used to establish a hypoxia/reoxygenation injury model. The effective concentration was screened and the cell activity was detected by CCK8 assay. The apoptotic rate of myocardial cells was detected by flow cytometry. The expression of autophagy marker LC3 was observed by laser confocal microscopy. The mRNA levels of Beclin-1, LC3 and Bcl-2 were detected by real-time quantitative PCR. The expressions of Beclin-1, LC311/I, Cleaved caspase-3, β-catenin, p-p65, Bcl-2, p62, p-Akt, p-mTOR were detected by Western blot. Results Huoxue Jiedu recipe can enhance the growth activity of myocardial cells and reduce the apoptotic rate and autophagy level, and it can enhance the activation of PI3K/Akt/mTORCl pathway, decrease Beclin-1 and LC3 mRNA levels, while increase Bcl-2 mRNA levels. It also decreased the expression of Beclin-1, LC311/I, Cleaved caspase-3, β-catenin, p-p65, and increased the expression of p62, p-Akt, p-mTOR, and Bcl-2. Conclusions Huoxue Jiedu recipe can reduce the level of autophagy and apoptosis of myocardial cells by regulating the autophagy pathway of PI3K/Akt/mTORCl, thereby playing a protective role in hypoxia/reoxygenation H9C2 myocardial cells.

Study on Improvement Effects of Polygonum orientale Flower Extract on Hypoxia-reoxygenation Injury of H 9c2 Cardiomyocytes / 中国药房

OBJECTIVE：To study the improvement effects and mechanism of Polygonum orientale flower extract on hypoxia- reoxygenation injury of H 9c2 cardiomyocytes. METHODS ：H9c2 cardiomyocytes were divided into normal control group ，model group and low- ，medium- and high-concentrations groups of P. orientale flower extract （20，40，80 μg/mL）. Except for normal control group ，other groups were given 800 μmol/L CoCl2 to induce hypoxia-reoxygenation injury model. Cell apoptosis was observed. The levels of Ca 2+（in cytoplasm ），mitochondrial membrane potential （MMP），ATP enzyme （Na+-K+-ATP enzyme ，Ca2+-Mg2+-ATP enzyme） activities， the ratio of cytochrome c （Cyto c ）， protein in cytosol to mitochondria ，phosphorylation levels of reperfusion injury salvage kinase （RISK） signaling pathwayrelated protein [protein kinase B （Akt）and extracellular signal regulated kinase 1/2（ERK1/2）] as well as protein expression of HIF- 1 α were detected respectively. In addition，the cells were divided into normal control group ，model group and P. orientale flower extract group （80 μ g/mL），PI3K inhibitor LY294002+CoCl2 group（15 μmol/L LY294002+80 μmol/L ，LY294002+P. orientale flower extract group （15 μmol/L LY294002+80 μg/mL P. orientale flower extract ），MEK inhibitor PD98059+CoCl2 group（25 μmol/L PD98059+800 μmol/L CoCl2），PD98059+P. orientale flower extract group （25 μmol/L PD98059+80 μg/mL P. orientale flower extract ）. After cultured by the same method ，the phosphorylation levels of Akt protein and ERK1/2 protein in the cells were measured to verify the activation of P. orientale flower extract to RISK signaling pathway. RESULTS：Compared with model group ，nuclear pyknosis and the number of apoptotic bodies were reduced in different concentrations groups of P. orientale flower extract. ROS level ，Ca2+ level（except for low-concentration group ），MMP，ratio of Cyto c in cytoplasm to Cyto c in mitochondria ，protein expression of HIF- 1α were decreased significantly（P＜0.05 or P＜0.01）； the activity of ATP enzyme （except for the low-concentration group ），Akt protein and ERK 1/2 protein phosphorylation level were significantly increased （P＜0.01）. After treated with PI 3K inhibitor LY 294002 and MEK inhibitor PD 98059，Akt protein and ERK 1/2 protein phosphorylation level in cadiomyocyte were decreased significantly （P＜0.05 or P＜0.01）. CONCLUSIONS ：P. orientale flower extract can improve hypoxia-reoxygenation injury of H 9c2 cardiomyocytes，the mechanism of which may be associated with inhibiting cardiomyocyte apoptosis ，improving ATPase activity ，protecting mitochondria ，regulating RISK signaling pathway related proteins and HIF- 1α protein expression.

Schisandra chinensis bee pollen's chemical profiles and protective effect against H2O2-induced apoptosis in H9c2 cardiomyocytes.

BACKGROUND: Schisandra chinensis (Turcz.) Baill bee pollen extract (SCBPE) is often used as a functional food in China due to its good antioxidant property. However, its chemical compositions and effects on H9c2 cardiomyocytes against H2O2-induced cell injury still lacks of reports thus far. This study aimed to characterize the main components of SCBPE and investigate its protective effects against H2O2-induced H9c2 cardiomyocyte injury. METHODS: The main components of SCBPE were analyzed via ultraperformance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (UPLC-QTOF MS/MS). The three main nucleosides in SCBPE were quantitatively analyzed via ultraperformance liquid chromatography-diode array detection. Furthermore, the potential mechanism by which SCBPE exerts protective effects against H2O2-induced H9c2 cardiomyocyte injury was explored for the first time via cell survival rate measurements; cell morphological observation; myocardial superoxide dismutase (SOD) activity and malondialdehyde (MDA) and glutathione (GSH) level determination; flow cytometry; and quantitative polymerase chain reaction. RESULTS: Two carbohydrates, three nucleosides, and nine quinic acid nitrogen-containing derivatives in SCBPE were identified or tentatively characterized via UPLC-QTOF MS/MS. The nine quinic acid nitrogen-containing derivatives were first reported in bee pollen. The contents of uridine, guanosine, and adenosine were 2.4945 ± 0.0185, 0.1896 ± 0.0049, and 1.8418 ± 0.0157 µg/mg, respectively. Results of in vitro experiments showed that cell survival rate, myocardial SOD activity, and GSH level significantly increased and myocardial MDA level significantly decreased in SCBPE groups compared with those in H2O2 group. Cell morphology in SCBPE groups also markedly improved compared with that in H2O2 group. Results indicated that SCBPE protected H9c2 cardiomyocytes from H2O2-induced apoptosis by downregulating the mRNA expressions of Bax, cytochrome C, and caspase-3 and upregulating the Bcl-2 mRNA expression. CONCLUSIONS: This study is the first to report that SCBPE could protect against oxidative stress injury and apoptosis in H2O2-injured H9c2 cells. Results indicated that the nucleosides and quinic acid nitrogen-containing derivatives could be the main substances that exert protective effects against H2O2-induced H9c2 cardiomyocyte injury.

[Salvianolic acid B regulates mitochondrial autophagy mediated by NIX to protect H9c2 cardiomyocytes from hypoxia/reoxygenation injury].

The aim of this paper was to investigate whether the mechanism of salvianolic acid B in protecting H9 c2 cardiomyocytes from hypoxia/reoxygenation injury is related to the regulation of mitochondrial autophagy mediated by NIX. H9 c2 cardiomyocytes were cultured in vitro and divided into normal group, model group and salvianolic acid B group(50 µmol·L~(-1)). Hypoxia/reoxygenation injury model was established by hypoxia for 4 h and reoxygenation for 2 h. In normal group, high glucose DMEM medium was used for culture. Those in model group were cultured with DMEM medium without glucose and oxygen, and no drugs for hypoxia and reoxyge-nation. In salvianolic acid B group, salvianolic acid B prepared by glucose-free DMEM medium was added during hypoxia, and the other process was as same as the model group. The cell viability was evaluated by CCK-8 assay. The leakage of lactate dehydrogenase(LDH) was detected by microplate method. The levels of intracellular reactive oxygen species(ROS) and mitochondrial membrane potential(ΔΨm) were measured by chemical fluorescence method. The level of intracellular adenosine triphosphate(ATP) was mea-sured by fluorescein enzyme method. The autophagy related proteins LC3-Ⅰ, LC3-Ⅱ, apoptosis related protein cleaved caspase-3 and mitochondrial autophagy receptor protein NIX were detected by Western blot. As compared with the normal group, the activity of H9 c2 cardiomyocytes and ATP level were decreased(P<0.05); LDH leakage and ROS production were increased(P<0.01); ΔΨm was decreased(P<0.01); LC3-Ⅱ/LC3-Ⅰ ratio, cleaved caspase-3 and NIX protein expression levels were increased(all P<0.05) in the model group. As compared with the model group, the activity of cells and ΔΨm were significantly increased(P<0.01); ATP level was increased(P<0.05); LDH leakage and ROS generation were decreased(P<0.01); LC3-Ⅱ/LC3-Ⅰ ratio was decreased(P<0.01); cleaved caspase-3 and NIX expression levels were decreased(P<0.05) in the salvianolic acid B group. The protective effect of salvianolic acid B on hypoxia/reoxygenation injury of H9 c2 cardiomyocytes may be associated with inhibiting mitochondrial auto-phagy. The specific mechanism may be related to inhibiting the activation of mitochondrial autophagy mediated by NIX, increasing ΔΨm, reducing ROS production, reducing the expression of cleaved caspase-3, LC3-Ⅱ, and increasing cell viability.

Effect of miR-499a-5p on damage of cardiomyocyte induced by hypoxia-reoxygenation via downregulating CD38 protein.

The aim is to investigate the mechanism of miR-499a-5p on the damage of cardiomyocyte induced by hypoxia/reoxygenation. The activity of lactate dehydrogenase (LDH), apoptosis rate and the expression of miR-499a-5p and cluster of differentiation 38 (CD38) in hypoxia-reoxygenation model cells were detected by LDH Cytotoxicity Assay Kit, flow cytometry, real-time polymerase chain reaction, and Western blot analysis, respectively. Apoptosis, the activity of LDH was detected after overexpression of miR-499a-5p or silencing of CD38 in H9c2 cells. The target relationship between miR-499a-5p and CD38 was verified by Targetscan online prediction and dual-luciferase assay. Apoptosis, the activity of LDH was detected after overexpression of miR-499a-5p and CD38. Apoptosis, the activity of LDH and the expression of CD38 were increased (P < .05) while expression of miR-499a-5p was decreased (P < .05) in hypoxia/reoxygenation model cells. Apoptosis and the activity of LDH in H9c2 cells after overexpression of miR-499a-5p or silence of CD38 were decreased (P < .05). The results of Targetscan online prediction and dual-luciferase assay indicated that CD38 was a potential target gene of miR-499a-5p. Overexpression of CD38 could reverse the inhibition of miR-499a-5p on LDH activity and apoptosis in H9c2 cells. miR-499a-5p could relief the injury of cardiomyocytes induced by hypoxia/reoxygenation via targeting CD38.

Protective effect of Nelumbo nucifera (Gaertn.) against H2O2-induced oxidative stress on H9c2 cardiomyocytes.

Ischemic heart disease (IHD), a severe condition of myocardium facing impediment in the supply of basic needs for cellular metabolism is caused by atherosclerosis. Though statin drugs could control the use of surgery on IHD patients, the complete rehabilitation or prophylaxis can be achieved through herbal-based medicines viz. either in the form of crude extract or pure phytocompounds. In the present study, pretreatment with leaf extract of Nelumbo nucifera Gaertn. was investigated for cardioprotective activity-in vitro by mitigating H2O2-induced oxidative stress. Analysis such as estimation of antioxidants, lipid peroxidation, and DNA fragmentation assay revealed significant protective effect of plant extract on cardiomyocytes. Reactive oxygen species detection was done by using 2',7'-dichlorofluorescein diacetate, apoptosis detection with Acridine Orange/Ethidium Bromide and nuclear damage detection by diamidino-2-phenylindole which confirmed the protective effect of N. nucifera extract. In addition, gene expression studies of apoptotic regulatory genes (Bcl2 and Cas-9) resulted in significant protection of nucifera extract pretreated and maintained cells. To conclude, in vitro cardioprotective activity of N. nucifera against H2O2 induced oxidative stress was achieved at the concentration of 50 µg/ml. Therefore, major phytocompounds present in extract could be beneficial in managing cardiac complications in the future.

Salvianolic acid B regulates mitochondrial autophagy mediated by NIX to protect H9c2 cardiomyocytes from hypoxia/reoxygenation injury / 中国中药杂志

The aim of this paper was to investigate whether the mechanism of salvianolic acid B in protecting H9 c2 cardiomyocytes from hypoxia/reoxygenation injury is related to the regulation of mitochondrial autophagy mediated by NIX. H9 c2 cardiomyocytes were cultured in vitro and divided into normal group, model group and salvianolic acid B group(50 μmol·L~(-1)). Hypoxia/reoxygenation injury model was established by hypoxia for 4 h and reoxygenation for 2 h. In normal group, high glucose DMEM medium was used for culture. Those in model group were cultured with DMEM medium without glucose and oxygen, and no drugs for hypoxia and reoxyge-nation. In salvianolic acid B group, salvianolic acid B prepared by glucose-free DMEM medium was added during hypoxia, and the other process was as same as the model group. The cell viability was evaluated by CCK-8 assay. The leakage of lactate dehydrogenase(LDH) was detected by microplate method. The levels of intracellular reactive oxygen species(ROS) and mitochondrial membrane potential(ΔΨm) were measured by chemical fluorescence method. The level of intracellular adenosine triphosphate(ATP) was mea-sured by fluorescein enzyme method. The autophagy related proteins LC3-Ⅰ, LC3-Ⅱ, apoptosis related protein cleaved caspase-3 and mitochondrial autophagy receptor protein NIX were detected by Western blot. As compared with the normal group, the activity of H9 c2 cardiomyocytes and ATP level were decreased(P<0.05); LDH leakage and ROS production were increased(P<0.01); ΔΨm was decreased(P<0.01); LC3-Ⅱ/LC3-Ⅰ ratio, cleaved caspase-3 and NIX protein expression levels were increased(all P<0.05) in the model group. As compared with the model group, the activity of cells and ΔΨm were significantly increased(P<0.01); ATP level was increased(P<0.05); LDH leakage and ROS generation were decreased(P<0.01); LC3-Ⅱ/LC3-Ⅰ ratio was decreased(P<0.01); cleaved caspase-3 and NIX expression levels were decreased(P<0.05) in the salvianolic acid B group. The protective effect of salvianolic acid B on hypoxia/reoxygenation injury of H9 c2 cardiomyocytes may be associated with inhibiting mitochondrial auto-phagy. The specific mechanism may be related to inhibiting the activation of mitochondrial autophagy mediated by NIX, increasing ΔΨm, reducing ROS production, reducing the expression of cleaved caspase-3, LC3-Ⅱ, and increasing cell viability.