Effects of Different Doses of Doxorubicin on H9C2 Cells

Objective: To study the effect of different doses of doxorubicin on H9C2 cells and to provide a reference for the clinical study of doxorubicin. Methods: Doxorubicin (1, 2, 4, 6, 10 ug/ml) was co-cultured with H9C2 cells for 6, 12 and 24 hours. The morphological changes of cells were observed, and the cell inhibition rates of different time and drug concentration were calculated. Results: Doxorubicin could inhibit the activity of cardiomyocytes in a dose-dependent manner from 1 to 10 ug/ml. Conclusion: A certain dose of doxorubicin has a toxic effect on cardiomyocytes and can cause cardiomyocyte necrosis and apoptosis.

Paeoniflorin reduces cardiotoxicity of aconitine in H9c2 cells

OBJECTIVE Aconitine(ACO)as the main active component in Aconitum carmichaelii debeaux(family Ranunlaceae),has highly toxicity in heart and the mechanisms are not clear yet.Paeoniflorin(PF),the main chemical ingredient in Herbaceous peony,can protect heart hurt by antioxidant,vasodilator effect and other effects.In this study,we focused on investigating the mechanism of PF reducing the cardiotoxicity of ACO.METHODS We chose H9c2 cells as experimental subject.MTT,Western blotting and real-time PCR were used to measure cell proliferation,apoptosis,ion channels and oxidative stress.RESULTS Cell proliferation in ACO+PF group was significantly increased compared with ACO group;the ratio with Bcl-2 and Bax and the level of p53 were upregulated by PF,while the level of caspase-3 was lightly reduced.The expression of SCN5A mRNA significantly was increased in ACO+PF group,while the expres⁃sion of RyR2 and Cx43 mRNA was dropped.Compared with ACO group,extracellular LDH and intracellular MDA were highly decreased,while intracellular SOD was regulated.CONCLUSION Cardiotoxicity of ACO in H9c2 cells was signifi⁃cantly decreased by PF.

Regulation of PM_(2.5)on mitochondrial damage in H9c2 cells through miR-421/SIRT3 pathway and protective effect of miR-421 inhibitor and resveratrol

Fine particulate matter(PM_(2.5))exposure is associated with cardiovascular disease(CVD)morbidity and mortality.Mitochondria are sensitive targets of PM_(2.5),and mitochondrial dysfunction is closely related to the occurrence of CVD.The epigenetic mechanism of PM_(2.5)-triggered mitochondrial injury of cardiomyocytes is unclear.This study focused on the mi R-421/SIRT3 signaling pathway to investigate the regulatory mechanism in cardiac mitochondrial dynamics imbalance in rat H9c2 cells induced by PM_(2.5).Results illustrated that PM_(2.5)impaired mitochondrial function and caused dynamics homeostasis imbalance.Besides,PM_(2.5)up-regulated mi R-421 and down-regulated SIRT3 gene expression,along with decreasing p-FOXO3a(SIRT3 downstream target gene)and p-Parkin expression and triggering abnormal expression of fusion gene OPA1 and fission gene Drp1.Further,mi R-421 inhibitor(mi R-421i)and resveratrol significantly elevated the SIRT3 levels in H9c2 cells after PM_(2.5)exposure and mediated the expression of SOD2,OPA1 and Drp1,restoring the mitochondrial morphology and function.It suggests that mi R-421/SIRT3 pathway plays an epigenetic regulatory role in mitochondrial damage induced by PM_(2.5)and that mi R-421i and resveratrol exert protective effects against PM_(2.5)-incurred cardiotoxicity.

Nardosinone Protects H9c2 Cardiac Cells from Angiotensin Ⅱ-induced Hypertrophy

Pathological cardiac hypertrophy induced by angiotensin Ⅱ （Ang Ⅱ ） can subsequently give rise to heart failure, a leading cause of mortality. Nardosinone is a pharmacologically active compound extracted from the roots ofNardostachys chinensis, a well-known traditional Chinese medicine. In order to investigate the effects of nardosinone on Ang Ⅱ-induced cardiac cell hypertrophy and the related mechanisms, the myoblast cell line H9c2, derived from embryonic rat heart, was treated with nardosi- none （25, 50, 100, and 200μmol/L） or Ang Ⅱ （1 μmol/L）. Then cell surface area and mRNA expression of classical markers of hypertrophy were detected. The related protein levels in PI3K/Akt/mTOR and MEK/ERK signaling pathways were examined by Western blotting. It was found that pretreatment with nardosinone could significantly inhibit the enlargement of cell surface area induced by Ang Ⅱ. The mRNA expression of ANP, BNP and 13-MHC was obviously elevated in Ang Ⅱ-treated H9c2 cells, which could be effectively blocked by nardosinone at the concentration of 100μmol/L. Further study revealed that the protective effects of nardosinone might be mediated by repressing the phosphorylation of related proteins in PI3K/Akt and MEK/ERK signaling pathways. It was suggested that the inhibitory effect of nardosinone on Ang Ⅱ-induced hypertrophy in H9c2 cells might be mediated by targeting PI3K/Akt and MEK/ERK signaling pathways.

Polysaccharide extracts of Cirsium japonicum protect rat H9c2 myocardial cells from oxidative stress induced by hydrogen peroxide

Daji （Cirsium japonicum） has been applied against gastric disorders, lung diseases, and cardiovascular problems in thetraditional Chinese medicinal system. The present study was to investigate the protective effects of Daji （Cirsiumjaponicum） polysaccharide extracts （CJP） against hydrogen peroxide （H2O2） shock in rat H9c2 myocardial cells. First,CJP was isolated by hot water extraction and ethanol precipitation; it was then characterized by high performance liquidchromatography and infrared spectrum analysis. Rat H9c2 cells were subjected to H2O2 treatment to establish a cellinjury model. The 3- （4,5- dimethylthiazol- 2-yl）-2,5- diphenyltetrazolium bromide assay showed that CJP pretreatmentsignificantly ameliorated the H2O2 injury in a dose-dependent manner. Furthermore, the cell apoptosis induced by H2O2was markedly inhibited by CJP pretreatment, whereas the cleavage level of caspase-3, -8, and -9 was reduced. Inaddition, the p38 mitogen-activated protein kinase pathway might be involved in the protective effect of CJP onmyocardial cells. Therefore, we conclude that polysaccharide extracts of Daji （Cirsium japonicum） protect rat H9c2myocardial cells from oxidative stress induced by H2O2.

Hyperbaric oxygen protects against PC12 and H9C2 cell damage caused by oxygen-glucose deprivation/reperfusion via the inhibition of cell apoptosis and autophagy

In this study,we investigated the protective effect of hyperbaric oxygen(HBO)on PC12 and H9C2 cell damage caused by oxygen-glucose deprivation/reperfusion and its possible mechanism.PC12 and H9C2 cell oxygen-glucose deprivation/reperfusion model were established.Cells were divided into a control group,model group,hyperbaric air(HBA)group and HBO group.The cell viability was detected by the CCK8 assay.Hoechst 33342 and PI staining assays and mitochondrial membrane potential(MMP)assays were used to detect cell apoptosis.The ultrastructure of cells,including autophagosomes,lysosomes,and apoptosis,were examined using a transmission electron microscope.The expression of autophagy-related proteins was detected by cellular immunofluorescence and immunocytochemistry.Our results showed that HBO can significantly improve the vitality of damaged PC12 and H9C2 cells caused by oxygen–glucose deprivation/reperfusion.HBO can significantly inhibit apoptosis of PC12 and H9C2 cells caused by oxygenglucose deprivation/reperfusion.Importantly,we found that the protective mechanism of PC12 and H9C2 cell damage caused by oxygen-glucose deprivation/reperfusion may be related to the inhibition of the autophagy pathway.In this study,the results of cellular immunofluorescence and immunocytochemistry experiments showed that the 4E-BP1,p-AKt and mTOR levels of PC12 and H9C2 cells in the model group decreased,while the levels of LC3B,Atg5 and p53 increased.However,after HBO treatment,these autophagy-related indexes were reversed.In addition,observation of the cell ultrastructure with transmission electron microscopy found that in the model group,a significant increase in the number of autophagic vesicles was observed.In the HBO group,a decrease in autophagic vesicles was observed.The study demonstrated that hyperbaric oxygen protects against PC12 and H9C2 cell damage caused by oxygen-glucose deprivation/reperfusion via the inhibition of cell apoptosis and autophagy.

Sanguinarine Attenuates Lipopolysaccharide-induced Inflammation and Apoptosis by Inhibiting the TLR4/NF-KB Pathway in H9c2 Cardiomyocytes

The inflammatory response is involved in the pathogenesis of the most common types of heart disease. Sanguinarine （SAN） has various pharmacological properties such as anti-inflammatory, antioxidant, antibacterial, antitumor, and immune-enhancing properties. However, few studies have investigated the effects of SAN on lipopolysaceharide （LPS）-induced inflammatory and apoptotic responses in H9c2 cardiomyocytes. Therefore, in this study, H9c2 cells were co-treated with SAN and LPS, and the mRNA levels of pro-inflammation markers and the apoptosis rate were measured to clarify the effect of SAN on cardiac inflammation. The underlying mechanism was further investigated by detecting the activation of Toll-like receptor （TLR）4/nuclear faetor-κB （NF-κB） signaling pathways. As a result, increased mRNA expression of interleukin （IL）-1β, IL-6, and TNFα induced by LPS was attenuated after SAN treatment; LPS-induced apoptosis ofHge2 cardiomyocytes and cleaved-caspase 8, 9, 3 were all significantly reduced by SAN. Further experiments showed that the beneficial effect of SAN on blocking the inflammation and apoptosis of H9c2 cardiomyocytes induced by LPS was associated with suppression of the TLR4/NF-κB signaling pathway. It was suggested that SAN suppressed the LPS-induced inflammation and apoptosis of H9c2 cardiomyocytes, which may be mediated by inhibition of the TLR4/NF-κB signaling pathway. Thus, SAN may be a feasible therapy to treat sepsis patients with cardiac dysfunction.

CAV3 mediates IMGU and NIMGU in skeletal muscle and cardiac myocytes

Objective:To observe whether CAV3 is involved in the physiological process of IMGU and NIMGU.Down-regulation of caveolin-3(CAV3)protein expression by small interference RNAs(iRNA)has been studied.Insulin-mediated glucose uptake(IMGU)is critical in skeletal muscle and cardiac myocytes,but non-insulin-mediated glucose uptake(NIMGU)should not be neglected.Methods:CAV3 siRNAs were designed and transfected in C2C12 cells and H9c2 cells in skeletal muscle and cardiac muscle,respectively,and C2C12 and H9c2 cells were cultured in DMEM medium with and without insulin,respectively.Glucose transporter 4(GLUT4)protein expression was detected by Western blot,and the glucose uptake rate of cells was measured by biochemical kit.Results:Transfection with CAV3 siRNA successfully down-regulated CAV3 protein expression in C2C12 and H9c2 cells.In the absence of insulin stimulation,GLUT4 expression was decreased(P<0.01)and glucose uptake was reduced(P<0.05)after 48 h of transfection in C2C12 cells,and GLUT4 expression was decreased(P<0.05)and glucose uptake was reduced(P<0.01)after 48 h of transfection in H9c2 cells.In the presence of insulin stimulation,GLUT4 expression was decreased(P<0.01)and glucose uptake was reduced(P<0.01)after 48 h of transfection in C2C12 cells,and the downregulation of GLUT4 was not statistically significant and glucose uptake was reduced(P<0.01)after 48 hours of transfection in H9c2 cells.Conclusion:Two different states,IMGU and NIMGU,exist in C2C12 cells and H9c2 cells.Both in the quiet state stimulated by insulin as well as in the absence of insulin stimulation,the cellular uptake of glucose is affected by GLUT4 changes regulated by CAV3.

Roles of transient receptor potential channel 6 in glucose-induced cardiomyocyte injury

BACKGROUND Diabetic cardiomyopathy(DCM) is a serious complication of end-stage diabetes that presents symptoms such as cardiac hypertrophy and heart failure. The transient receptor potential channel 6(TRPC6) protein is a very important selective calcium channel that is closely related to the development of various cardiomyopathies.AIM To explore whether TRPC6 affects cardiomyocyte apoptosis and proliferation inhibition in DCM.METHODS We compared cardiac function and myocardial pathological changes in wild-type mice and mice injected with streptozotocin(STZ), in addition to comparing the expression of TRPC6 and P-calmodulin-dependent protein kinase Ⅱ(P-CaMKⅡ) in them. At the same time, we treated H9C2 cardiomyocytes with high glucose and then evaluated the effects of addition of SAR, a TRPC6 inhibitor, and KN-93, a CaMKⅡ inhibitor, to such H9C2 cells in a high-glucose environment.RESULTS We found that STZ-treated mice had DCM, decreased cardiac function, necrotic cardiomyocytes, and limited proliferation. Western blot and immunofluorescence were used to detect the expression levels of various appropriate proteins in the myocardial tissue of mice and H9C2 cells. Compared to those in the control group, the expression levels of the apoptosis-related proteins cleaved caspase 3 and Bax were significantly higher in the experimental group, while the expression of the proliferation-related proteins proliferating cell nuclear antigen(PCNA) and CyclinD1 was significantly lower. In vivo and in vitro, the expression of TRPC6 and P-CaMKⅡ increased in a high-glucose environment. However, addition of inhibitors to H9C2 cells in a high-glucose environment resulted in alleviation of both apoptosis and proliferation inhibition.CONCLUSION The inhibition of apoptosis and proliferation of cardiomyocytes in a high-glucose environment may be closely related to activation of the TRPC6/P-CaMKⅡ pathway.

Regulation of lovastatin on a key inflammation-related microRNA in myocardial cells

Background Advances in the understanding of cardiovascular pathogenesis have highlighted that inflammation plays a central role in athemsclemtic coronary heart disease.Therefore,exploring pharmacologically based anti-inflammatory treatments to be used in cardiovascular therapeutics is worthwhile to promote the discovery of novel ways of treating cardiovascular disorders.Methods The myocardial cell line H9c2（2-1） was exposed to lipopolysacchadde （LPS） in culture and resulted in a cellular pro-inflammation status,miR-21 microRNA levels were detected using quantitative real-time polymerase chain reaction （Q-RT-PCR）.The influence of Iovastatin on miR-21 under normal and pro-inflammatory conditions was tested after being added to the cell culture mixture for 24 hours.Conditional gene function of two predicted cardiovascular system relevant downstream targets of miR-21,protein phosphatase 1 regulatory subunit 3A （PPP1R3A） and signal transducer and activator of transcription 3 （STAT3）,were analyzed with immunoblotting.Results Forty-eight hours of LPS treatment significantly increased the miR-21 to 170.71%±34.32% of control levels （P=0.002）.Co-treatment with Iovastatin for 24 hours before harvesting attenuated the up-regulation of miR-21 （P=0.013）.Twenty-four hours of Iovastatin exposure up-regulated PPP1R3A to 143.85%±21.89% of control levels in cardiomyocytes （P=0.023）.Lovastatin up-regulated the phosphorylation level of STAT3 compared to the background LPS pretreatment （P=0.0077）,this effect was significantly （P=0.018） blunted when miR-21 was functionally inhibited.Conclusions miR-21 plays a major role in the regulation of the cellular anti-inflammation effects of Iovastatin.

Rat cardiomyocyte H9c2(2-1)-based sulforhodamine B assay as a promising in vitro method to assess the biological component of effluent toxicity

The treatment of wastewaters is crucial to maintain the ecological status of receiving waters,and thereby guarantee the protection of aquatic life and human health.Wastewater quality evaluation is conventionally based on physicochemical parameters,but increasing attention has been paid to integrate physicochemical and biological data.Nevertheless,the regulatory use of fish in biological testing methods has been subject to various ethical and cost concerns,and in vitro cell-based assays have thus become an important topic of interest.Hence,the present study intends:(a) to evaluate the efficiency of two different sample pre-concentration techniques (lyophilisation and solid phase extraction) to assess the toxicity of municipal effluents on rat cardiomyoblast H9c2(2-1) cells,and (b) maximizing the use of the effluent sample collected,to estimate the environmental condition of the receiving environment.The gathered results demonstrate that the H9c2(2-1) sulforhodamine B-based assay is an appropriate in vitro method to assess biological effluent toxicity,and the best results were attained by lyophilising the sample as pre-treatment.Due to its response,the H9c2(2-1) cell line might be a possible alternative in vitro model for fish lethal testing to assess the toxicity of municipal effluents.The physicochemical status of the sample suggests a high potential for eutrophication,and iron exceeded the permissible level for wastewater discharge,possibly due to the addition of ferric chloride for wastewater treatment.In general,the levels of carbamazepine and sulfamethoxazole are higher than those reported for other countries,and both surpassed the aquatic protective values for long-term exposure.

Protective mechanisms of hypaconitine and glycyrrhetinic acid compatibility in oxygen and glucose deprivation injury

This study investigated the protective effect of the compatibility of hypaconitine （HA） and glycyrrhetinic acid （GA） on H9c2 cells under oxygen and glucose deprivation （OGD）-induced injury, and the possible mechanisms. We found that HA＋GA significantly improved pathology and morphology of the nucleus and ultrastructure of H9c2 cells under OGD as determined by Hoechst 33342 staining and transmission electron microscopy （TEM） tests. It also reduced the releases of lactate dehydrogenase （LDH）, creatine kinase-myocardial band isoenzyme （CK-MB）, and aspartate transaminase （AST） from the cultured supernatant of H9c2 cells, which were tested by enzyme-linked immune sorbent assay （ELISA） kits. In addition, it lessened the apoptotic rate as determined by a fluorescein isothiocyanate-annexin V/propidium iodide （FITC-AV/PI） double staining assay. It was also found that HA＋GA might regulate the protein expression associated with the phosphatidylinositol 3-kinase （PI3K）/Akt signaling pathway. Overall, the study demonstrated that HA＋GA protected H9c2 cells against OGD-induced injury, and the signaling mechanism might be related to the PI3K/Akt signaling pathway.

Protection of Astragaloside Derivate on Oxidative Stress and Hypertrophy in Cardiomyocytes

Objective The astragaloside IV(ASI)has been proved to play an important role in protecting against cell death on cardiovascular diseases.This study aims to investigate the effect of the astragaloside derivate(ASId)on confronting oxidative stress and hypertrophy in myocardial cells.Methods Following exposure embryonic rat cardiac H9c2 cells to hydrogen peroxide(H2O2)and angiotensin II for developing oxidative stress and hypertrophy,ASId at final concentrations (0.1,1,and 10μmol/L)was added to study its role in protecting cardiomyocytes by biochemical detection and cell size measurement.In addition,the mitochondrial permeability transition pore(mPTP)opener atractyloside(20μmol/L)and inhibitor cyclosporin A(CSA)(1μmol/L)were employed to investigate the possible mechanisms for anti-oxidation.Results ASId at 1 and 10μmol/L in cultures suppressed oxidative stress at different degrees,which induced the decrease in LDH activity and MDA content,and also the increase in SOD activity in comparable with the model group;The mPTP opener atractyloside and inhibitor CSA weakened and strengthened the role of ASId,respectively.ASId at 10μmol/L inhibited cell hypertrophy,and the cell diameter,surface area,and protein content were all decreased in comparable of those cells in model group.Conclusion ASId is involved in the cytoprotective effects on oxidative stress through a pathway mediated by mPTP,and also has a protective effect against hypertrophy.