Effect of sodium fluoride on myocardial damage by regulating apoptosis through JNK signaling pathway / 环境与职业医学

Background It has been found that fluoride may cause cardiomyocyte damage. c-Jun N-terminal kinases (JNK) signaling pathway plays an important role in apoptosis, but its role in fluorosis-induced cardiomyocyte damage is still unknown yet. Objective To explore the toxic effect of sodium fluoride (NaF) on H9c2 cardiomyocytes of rats and whether NaF affects cardiomyocyte apoptosis through the JNK signaling pathway. Methods According to the concentrations of sodium fluoride and whether sp600125 (JNK inhibitor) was added, cardiomyocytes of rats were divided into six groups, including control group, SP600125 group (SP group), 0.24, 0.48, and 0.96 mmol·L−1 NaF groups, and 0.96 mmol·L−1 NaF+SP600125 group (NaF+SP group). Cardiomyocytes exposed to NaF for 24 h were observed using a fluorescence inverted microscope. The changes of cell viability at 24, 48, and 72 h after the treatment were detected by CCK-8 method. The levels of reactive oxygen species (ROS) at 24 h after the treatment in H9c2 cardiomyocytes were determined by fluorescent probe method. The expression levels of Bcl-2, Bax, Caspase-3, and JNK mRNA at 24 h after the treatment were detected by real-time PCR. The protein expression levels of Bcl-2, Bax, Caspase-3, and p-JNK at 24 h after the treatment were detected by Western blotting. Results Compared with the control group, after being exposed to 0.48 and 0.96 mmol·L−1 NaF for 24 h, the cell growth density decreased. With the increase of NaF concentration, rounded cells and some suspended dead cells appeared. At 24h after exposure to NaF, the cell viability of the 0.48 and 0.96 mmol·L−1 NaF groups decreased compared with the control group (P<0.05). At 48h and 72h after exposure to NaF, the cell viability levels of the NaF treated groups were significantly lower than that of the control group (P<0.05). After NaF exposure for 24 h, compared with the control group, the intracellular ROS levels were increased (P<0.05); the mRNA expression levels of Bcl-2 were decreased to varying degrees, especially in the 0.48 and 0.96 mmol·L−1 NaF groups (P<0.05); the mRNA expression levels of Bax, Caspase-3, and JNK were increased (P<0.05); the protein expression levels of Bcl-2 were reduced (P<0.05); the protein expression levels of Bax, Caspase-3, and p-JNK were elevated (P<0.05). Compared with the 0.96 mmol·L−1 NaF group, the cell viability of the NaF+SP group was increased, the intracellular ROS level was decreased, the mRNA expression levels of Bax, Caspase-3, and JNK were decreased, the protein expression level of Bcl-2 was increased, and the protein expression levels of Bax, Caspase-3, and p-JNK were decreased (P<0.05); the expression level of Bcl-2 mRNA had a rising trend but showed no statistical significance (P>0.05). Conclusion Cardiomyocyte damage after excessive fluoride exposure may result from fluoride inducing excessive ROS production in cardiomyocytes, which may activate the JNK signaling pathway and induce cardiomyocyte apoptosis.

Protective effects of mangiferin against myocardial cell damage and its effects on the expression of NFATc4 / 中国药房

OBJECTIVE To study the protective effects of mangiferin against oxidative stress injury of myocardial cells induced by hydrogen peroxide （H2O2）， and its effects on the expression of nuclear factor of activated T cell cytoplasmic 4（NFATc4）. METHODS H9c2 myocardial cells were cultured in vitro and divided into blank group， H2O2 group， and 50， 100， 150 μmol/L mangiferin groups. Mangiferin groups were treated with different concentrations of mangiferin for 12 h， and then were subjected to H2O2 （200 μmol/L） stimulation for 12 hours together with the H2O2 group； relative survival rate was detected in each group， and the levels of superoxide dismutase （SOD）， catalase （CAT） and malondialdehyde （MDA） in cell supernatant and reactive oxygen species （ROS） in H9c2 cells were measured. Meanwhile， the expressions of apoptosis-related proteins [B cell lymphoma 2 （Bcl- 2）， Bcl-2 associated X protein （Bax）， cleaved caspase-3] and nuclear protein NFATc4 were determined. Furthermore， the NFATc4 interference sequence was transfected， and the effects of NFATc4 on oxidant stress indexes and apoptosis-related proteins in H2O2- induced myocardial cells were investigated. RESULTS Compared with blank group， relative cell viability， the levels of SOD and CAT， relative expression of Bcl-2 were decreased significantly， while the levels of MDA and ROS， relative expressions of Bax， cleaved caspase-3 and nuclear protein NFATc4 were increased significantly （P＜0.05 or P＜0.01）. Compared with the H2O2 group， the above indexes of 100 and 150 μmol/L mangiferin groups were reversed significantly （P＜0.05）. After the transfection of the NFATc4 interference sequence， the expression of nuclear protein NFATc4 was down-regulated significantly； the levels of MDA and SOD， the protein expressions of Bax and cleaved caspase-3 were all decreased/down-regulated significantly， while the levels of SOD and CAT， and the protein expression of Bcl-2 were all increased/up-regulated significantly， compared with H2O2 group （P＜ 0.05）. CONCLUSIONS Mangiferin can relieve H2O2-induced oxidative stress of H9c2 cells， reduce the apoptosis and inhibit the nuclear translocation of NFATc4， thereby alleviating myocardial cell damage； reducing the nuclear level of NFATc4 protein is related to reducing H2O2-induced oxidative stress and cell apoptosis.

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.

The role of Drp1 mediated mitochondrial dynamics balance in sepsis induced cardiomyopathy / 中华急诊医学杂志

Objective:To analyze the effect of inhibiting excessive mitochondrial fission mediated by dynamic related protein 1 (Drp1) on the function of injured cells and mitochondria in the septic myocardium, and to explore the protective effect of maintaining mitochondrial dynamic balance in the pathogenesis of sepsis induced cardiomyopathy(SIC).Methods:Rat H9C2 cardiomyocytes were cultured and stimulated with lipopolysaccharide (LPS) to establish a model of SIC. Mitochondrial division inhibitor 1 (Mdivi-1) was given 30 min before LPS stimulation. They were divided into the control group, LPS stimulated group (LPS), Mdivi-1 control group (Mdivi-1), and LPS+Mdivi-1 intervention group (LPS+Mdivi-1). CCK-8 was used to detect the cell viability, and lactate dehydrogenase (LDH) was used to detect cellular damage. A MitoTracker probe was used to observe mitochondrial morphology by laser scanning confocal microscopy, JC-1 staining was used to detect mitochondrial membrane potential level, a DCFH-DA probe was used to detect total ROS level, and an AnnexinV-FITC/PI probe was used to detect the cell apoptosis ratio. The expression levels of mitochondrial fission protein Drp1 and fusion proteins Optic Atrophy 1(Opa1) and Mitofusin2 (Mfn2) were detected by real-time PCR and Western blot. One-way ANOVA was used to compare the differences between groups, and the LSD- t test was used for pairwise comparisons between groups. Results:Compared with the control group, cell viability, the average length of mitochondria and the mitochondrial membrane potential were decreased, and ROS production, the cell apoptosis rate and LDH were increased in the LPS group (all P<0.05). After Mdivi-1 intervention, compared with the LPS-stimulated group, the cell viability was increased, myocardial cell damage was reduced, the average length of mitochondria was prolonged, mitochondrial dysfunction was alleviated, and the cell apoptosis rate was inhibited in the LPS+Mdivi-1 group (all P<0.05). Conclusions:Mdivi-1 might inhibit mitochondrial fission mediated by Drp1, maintain mitochondrial dynamic balance, alleviate mitochondrial dysfunction and protect myocardial cells from LPS-induced injury.

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.

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.

Effects of acteoside on hypoxia/reoxygenation-induced cardiomyocyte damage by regulating the Rnd 3/NF-κB pathway / 中国药房

OBJECTIVE To explore the effects of acteoside on hypoxia/reoxygena tion（H/R）-induced cardiomyocyte damage by regulating Rho family GTPase 3（Rnd3）/nuclear factor κB（NF-κB）pathway. METHODS The H 9c2 cardiomyocyte were divided into control group （no administration ，no modeling ），H/R group （only modeling ），H/R+AS-L group ，H/R+AS-M group ， H/R+AS-H group （10，30，90 μmol/L acteoside for above 3 groups firstly ，and then modeling ），H/R+pcDNA group [transfecting pcDNA （empty vector ） firstly，and then modeling] ，H/R + pcDNA-Rnd 3 group [overexpression of Rnd 3 by transfecting pcDNA-Rnd3（Rnd3 overexpression vector ）firstly，and then modeling] ，H/R+AS-H+si-NC group [transfecting si-NC （negative control）firstly，and then giving 90 μmol/L acteoside and modeling]，H/R+AS-H+si-Rnd3 group [inhibiting overexpression of Rnd 3 by transfecting si-Rnd 3 （Rnd3 small interfering RNA ） firstly，and then giving 90 μ mol/L acteoside and modeling]. After corresponding treatment ，the apoptotic rate ，release of lactate dehydrogenase （LDH），malondialdehyde（MDA）level，the activity of superoxide dismutase （SOD），the level of tumor necrosis factor α（TNF-α），interleukin 1β（IL-1β）and interleukin- 6（IL-6）， mRNA and protein expression of Rnd 3 and NF-κB subunit p65（NF-κB p65），the expression of aspartate proteolytic enzyme 3 （Cleaved Caspase- 3）protein and Cleaved Caspase- 9 protein were detected. RESULTS Different concentrations of acteoside could reduce the apoptotic rate of H/R-induced H 9c2 cardiomyocyte，the protein expressions of Cleaved Caspase- 3 and Cleaved Caspase-9，mRNA and protein expressions of NF-κB p65，the levels of LDH release and MDA ，TNF-α，IL-1β and IL-6，while increase the activity of SOD and mRNA and protein expressions of Rnd 3（P＜0.05），in a dose-dependent manner. Overexpression of Rnd 3 could decrease the apoptotic rate of H 9c2 cardiomyocyte，protein expressions of NF-κB p65，Cleaved Caspase- 3 and Cleaved Caspase- 9， the levels of LDH release ， MDA， TNF-α，IL-1β and IL-6，while increase the protein expression of Rnd 3 and the activity of SOD （P＜0.05）. The inhibition overexpression of Rnd 3 could weaken the inhibitory effects of acteoside on H/R-induced apoptosis of H 9c2 cardiomyocyte， oxidative stress and inflammatory reaction （P＜0.05）. CONCLUSIONS Acteoside could regulate Rnd 3/NF-κ B pathway by promoting the expression of Rnd 3 and inhibiting the expression of NF-κB p65，inhibit cardiomyocyte apoptosis ，oxidative stress and inflammation reaction so as to relieve the H/R-induced cardiomyocyte damage.

Pharmacokinetic study of Polygonum orientale extract in H9c2 cells by UPLC-MS/MS / 中国中药杂志

A detection method of ultra-performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS) was established to detect concentrations of isoorientin, orientin, quercetin, vitexin and kaempferol-3-O-β-D-glucoside in H9 c2 cells and applied to the pharmacokinetic study of Polygonum orientale extract in the cells. H9 c2 cells were treated with 100 μg·mL~(-1) P. orientale extract and then they and the corresponding nuclei, mitochondria and Golgi bodies were collected at the set time. After protein precipitation, UPLC-MS/MS was used to determine concentrations of isoorientin, orientin, quercetin, vitexin and kaempferol-3-O-β-D-glucoside in the whole cells and subcellular structures. Also, related pharmacokinetic parameters were calculated. The results showed that the peak time was 8 h for all these components. Orientin, vitexin, quercetin and isoorientin have high affinities to nuclei and mitochondria, while the affinity of kaempferol-3-O-β-D-glucoside is higher with mitochondria compared to nuclei. It is suggested that these chemical components of P. orientale may mainly act on nuclei or mitochondria to exert pharmacological effects of protecting cardiomyocytes.

Improvement Effect and Its Mechanism of Active Components of Averrhoa carambola on High Glucose-induced Injury of H 9c2 Myocardial Cell / 中国药房

OBJECTIVE：To study the imp rovement effects of 2-dodecyl-6-methoxycyclohexa-2，5-diene-1，4-dione（DMDD） from Averrhoa carambola on H 9c2 myocardial cell injury induced by high glucose and its mechanism. METHODS ：H9c2 myocardial cells were divided into normal group ，high glucose group ，osmotic pressure control group ，DMDD high ，medium and low concentration groups （8，4，2 μmol/L）. Normal group and high glucose group were treated with low glucose DMEM medium （containing 5.5 mmol/L glucose ，similarly hereinafter ） and high glucose DMEM medium （containing 33.3 mmol/L glucose ， similarly hereinafter ）for 48 h，respectively. The cells in osmotic pressure control group were cultured in low glucose DMEM medium containing 27.5 mmol/L mannitol for 48 h. In DMDD groups ，cells were cultured in high glucose DMEM medium for 24 h， and then in high glucose DMEM medium containing corresponding concentration of DMDD for 24 h. At the end of cell culture ，MTT metho d was used to detect the cell survival rate. The activities of ROS ， GSH-Px and LDH in cellsupernatant were detected by using related kits. ELISA assay was used to detect the levels of TNF-α and IL-6 in cell supernatant. Cell apoptosis was d etected by acridine orange/ethidium bromide （AO/EB）staining. Western blot assay was used to detect the expression of apoptosis related proteins （cleaved caspase- 3，Bcl-2，Bax）and the phosphorylation level of nuclear factor κB （NF-κB）/NF-κB suppressor protein α（IκBα）signaling pathway related proteins （NF-κB p65，IκBα）. RESULTS ：Compared with the normal group ，survival rate ，the activity of GSH-Px and protein expression of Bcl- 2 in high glucose groups were decreased significantly（P＜0.01）；the activities of ROS and LDH ，the levels of TNF-α and IL-6，the protein expression of Bax and cleaved caspase-3，and the phosphorylation level of NF-κB p65 and IκBα were increased significantly（P＜0.01）；the cells showed orange yellow fluorescence ，and the number of cells with fuzzy morphology increased significantly ，showing an obvious apoptotic state. There was no statistical significance in above indexes of osmotic pressure control group compared with normal group. Compared with high glucose group ，the activities or levels of above indexes （except for cell survival rate an LDH activity in low concentration group ）were reversed significantly in DMDD groups （P＜0.05 or P＜0.01）；the orange yellow fluorescence in the cells decreased significantly ，and the cell morphology was relatively complete. CONCLUSIONS ：DMDD can significantly improve H9c2 myocardial cell injury induced by high glucose ；the mechanism of which may be associated with suppressing oxidative stress and inflammatory response ，regulating the expression of apoptosis related protein and NF-κB/IκBα pathway related protein.

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.

Study on Improvement Effects of Zingiber officinale Decoction on Mitochondrial Function Injury of Cardiomyo-cytes / 中国药房

OBJECTIVE：To investigate the improvement e ffects of Zingiber officinale decoction （ZOD） on doxorubicin （DOX）-induced mitochondrial function injury of H 9c2 cardiomyocytes. METHODS ：Taking H 9c2 cardiomyocytes as research object，the effects of different concentrations of ZOD （0.125，0.25，0.5，1，2，4，8 mg/mL，by crude drug ，the same below ）on its survival rate were investigated by CCK- 8 assay. The effects of low ，medium and high concentrations of ZOD （0.125，0.25，0.5 mg/mL）on the morphology of H 9c2 cardiomyocytes after DOX （5 μmol/L）induced mitochondrial dysfunction were detected by high content living cell imaging system. The relative number of cells ，the relative fluorescence intensity of living cells and the relative fluorescence intensity of dead cells were analyzed quantitatively. The effects of ZOD （0.5 mg/mL）on related indexes of mitochondrial respiratory function （oxygen consumption rate ，extracellular acidification rate ，baseline oxygen consumption rate ， baseline extracellular acidification rate ，stress oxygen consumption rate and stress extracellular acidification rate ） and energy metabolism（basic respiration level ，maximum respiration level ，ATP production level ，H+ proton leakage level ，spare respiration level and non-mitochondrial respiration level ）were detected by bioenergy analyzer. RESULTS ：After treated with 0.125，0.25，0.5 mg/mL ZOD ，the survival rate of H 9c2 cardiomyocytes were increased significantly （P＜0.01）or had no statistical significance （P＞0.05）. After DOX induced mitochondrial dysfunction of H 9c2 cardiomyocytes，pretreated with 0.125，0.25，0.5 mg/mL（or 0.5 mg/mL）ZOD，the morphology of H 9c2 cardiomyocytes returned to normal and showed regular fibrous adherent distribution. The relative cell number ，fluorescence intensity of living cells ，oxygen consumption rate ，extracellular acidification rate ，baseline oxygen consumption rate ，baseline extracellular acidification rate ，stress oxygen consumption rate ，stress extracellular acidification rate，basic respiration level ，maximal respiration level ，ATP production level ，spare respiration level and non-mitochondrial respiration level were all significantly increased （P＜0.05 or P＜0.01），while relative dead cell fluorescence intensity and H + proton leakage level were significantly decreased （P＜0.01）. CONCLUSIONS ： ZOD can improve the respiratory function and mitochondrial energy metabolism of H 9c2 cardiomyocytes，so as to improve mitochon drial function injury.

Molecular mechanism of luteolin regulating lipoxygenase pathway against oxygen-glucose deprivation/reperfusion injury in H9c2 cardiomyocytes based on molecular docking / 中国中药杂志

The aim of this study was to investigate the mechanism of luteolin regulating lipoxygenase pathway against oxygen-glucose deprivation/reperfusion(OGD/R) injury in H9 c2 cardiomyocytes. First, Discovery Studio 2019 was used for the molecular docking of luteolin with three key enzymes including lipoxygenase 5(ALOX5), lipoxygenase 12(ALOX12), and lipoxygenase 15(ALOX15) in lipoxygenase pathway. The docking results showed that luteolin had high docking score and similar functional groups with the original ligand. From this, H9 c2 cardiomyocytes were cultured in vitro, and then the injury model of H9 c2 cardiomyocytes was induced by deprivation of oxygen-glucose for 8 h, and rehabilitation of oxygen-glucose for 12 h. Cell viability was detected by tetrazolium(MTT) colorimetry. H9 c2 cardiomyocytes were observed with a fluorescence inverted microscope, and colorimetry was used to detect the level of lactate dehydrogenase(LDH) in cell supernatant. The results showed that luteolin could significantly protect the morphology of H9 c2 cells, significantly improve the survival rate of H9 c2 cardiomyocytes in OGD/R injury model, reduce the level of LDH in cell supernatant, inhibit cytotoxicity, and maintain the integrity of cell membrane. The inflammatory cytokines interleukin-6(IL-6) and tumor necrosis factor-α(TNF-α) were detected by enzyme-linked immunosorbent assay. Compared with the model group, luteolin can significantly reduce the release of IL-6 and TNF-α. Western blot was employed to detect the protein levels of ALOX5, ALOX12, and ALOX15 in lipoxygenase pathway. After luteolin intervention, the protein levels of ALOX5, ALOX12, and ALOX15 were significantly down-regulated compared with those in model group. These results indicate that luteolin can inhibit the release of IL-6 and TNF-α by restraining the activation of lipoxygenase pathway, thereby playing a protective role in the cardiomyocyte injury model induced by OGD/R.

Investigation of Host-guest Complexation Between 4-Sulfonylcalix 6 Arene and 15 Alkaloids from Traditional Chinese Medicines and Inhibitory Effect of Binding System on Proliferation of HepG2 and H9c2 Cells / 中国实验方剂学杂志

Objective:To investigate bonding ability between 4-sulfonylcalix ［6］ arene （SCA6） and 15 alkaloids （matrine， allomatrine， dauricine， daurisoline， quinidine， quinine， crotaline， vincristine， gelsemine， koumine， tetrandrine， aloperine， oxymatrine， sophocarpine and sinomenine）， and to evaluate viability<italic> in vitro</italic> of HepG2 and H9c2 cells with 12 alkaloids/SCA6 bonding systems （except allomatrine， oxymatrine， sinomenine）. Method:Fluorescence competitive titration was used to determine the binding constants of alkaloids and SCA6， the inhibitory effect of alkaloid/SCA6 complex on proliferation of HepG2 and H9c2 cells was investigated by cell counting kit-8 （CCK-8）. Result:All the 15 alkaloids had good bonding with SCA6 at the ratio of 1∶1 （the binding constants >1×10⁵ mol·L^-1， <italic>R</italic>²>0.98）， the aloperine （quinolizidine alkaloids） and SCA6 had the biggest binding constant （20.55×10⁶ mol·L^-1）. In addition to gelsemine， crotaline， matrine and sophocarpine， 8 alkaloids （including aloperine， tetrandrine， dauricine， daurisoline， quinidine， quinine， vincristine and koumine） exhibited significant anti-tumor effects on HepG2 cells. Except for daurisoline， the anti-proliferation effect of the other 11 alkaloids before and after binding with SCA6 had no difference in HepG2 cells. In addition to gelsemine， crotaline， matrine and sophocarpine， the anti-proliferation effect of the other 8 alkaloids before and after binding with SCA6 had no difference in H9c2 cells. Conclusion:SCA6 shows intense binding ability with bisbenzylisoquinoline， quinolizidine and indole alkaloids. It can improve the solubility of alkaloids without affecting their anti-tumor activity， which provides a reference for subsequent related applications of SCA6 as a drug delivery carrier.

CLEC3B protects H9c2 cardiomyocytes from apoptosis caused by hypoxia via the PI3K/Akt pathway

Ischemic heart disease (IHD) is one of the leading causes of death worldwide. C-type lectin domain family 3 member B (CLEC3B) is a C-type lectin superfamily member and is reported to promote tissue remodeling. The serum levels of CLEC3B are downregulated in patients with cardiovascular disease. However, the molecular mechanisms of CLEC3B in IHD is not well-characterized. Therefore, we overexpressed CLEC3B and silenced CLEC3B in H9c2 rat cardiomyocytes for the first time. We then constructed a model of IHD in vitro through culturing H9c2 cardiomyocytes in serum-free medium under oxygen-deficit conditions. Then, Cell Counting Kit-8 (CCK-8), flow cytometry, qRT-PCR, and western blot assays were performed to investigate cell viability, apoptosis, and expression levels of CLEC3B, phosphatidylinositol 3-kinase (PI3K), phosphorylated PI3K (p-PI3K), protein kinase B (Akt), phosphorylated Akt (p-Akt), and cleaved-caspase 3. We observed that the mRNA expression of CLEC3B was decreased in hypoxic H9c2 cardiomyocytes (P<0.05). Overexpression of CLEC3B increased cell viability (P<0.01), inhibited cell apoptosis (P<0.05), upregulated the levels of p-PI3K/PI3K and p-Akt/Akt (P<0.01 or P<0.05), and downregulated expression of cleaved-caspase 3 (P<0.001) in hypoxic H9c2 cardiomyocytes while silencing of CLEC3B caused the opposite results. Inhibition of the PI3K/Akt pathway reversed the protective effect of CLEC3B on hypoxic H9c2 cardiomyocytes. Our study demonstrated that CLEC3B alleviated the injury of hypoxic H9c2 cardiomyocytes via the PI3K/Akt pathway.

Effect of Trans-oleic Acid on Proliferation Inhibition and Apoptosis Induction in H9c2 Cardiomyocyte / 中国药学杂志

OBJECTIVE: To investigate the effects and possible mechanism of trans-oleic acid (9t-C18:1) on proliferation inhibition and induction apoptosis in H9c2 cardiomyocyte. METHODS: H9c2 rat cardiomyocytes were cultured in vitro. High, medium and low (600, 300, 150 μmol•L-1) dose of 9t-C18:1 groups and the negative control (NC) group were administered to H9c2 cardiomyocytes. The effect of 9t-C18:1 on cell proliferation was tested using cell counting kit-8 (CCK-8) assay. Morphological changes of cells were observed by AO-EB staining. Intracellular reactive oxygen species (ROS) and apoptosis were detected by flow cytometry. The expression of Bcl-2 and Bax genes was detected by quantitative real time- polymerase chain reaction (QRT-PCR). The expression of Bcl-2 and Bax protein was determined by flow cytometry after immunofluorescence staining. RESULTS: The typical morphological characteristics of apoptosis were observed by fluorescence microscope. The result of CCK-8 assay indicated that 9t-C18:1 have an certainly inhibitory effect on the proliferation of H9c2 cells. ROS level and apoptosis rate were significantly increased. Bcl-2 gene and protein expression were down-regulated, and Bax gene and protein expression were up-regulated, compared with NC group(P<0.05, P<0.01). CONCLUSION: 9t-C18:1 can inhibit the proliferation and induce the apoptosis of H9c2 cardiomyocyte, and its mechanism may be related to promoting the mitochondrial apoptotic pathway.

Study on the Anti-apoptotic Effect of Curcumin on H 2O2-induced H 9c2 Cardiomyocyte Injury and the Regulation of NF-κB Signaling Pathway / 中国药房

OBJECTIVE：To investigate the anti-apoptotic effect of curcumin on H 2O2-induced H 9c2 cardiomyocyte injury and the regulation of NF-κB signaling pathway. METHODS：H9c2 cardiomyocyte were randomly divided into normal control group ， injury model group ，curcumin low-dose ，medium-dose and high-dose groups （25，50，100 μmol/L）. Normal control group didn ’t received any intervention. The cells in injury model group were induced with 50 μmol/L H2O2 for 12 h to establish the injury model. The cells in curcumin groups were treated with relevant concentration of drugs for 24 h，and then induced with 50 μmol/L H2O2 for 12 h. After cultured for 24 h，survival rate and apoptotic rate of cells were measured by MTT method and TUNEL method ；SOD activity and MDA content were determined by WTS- 8 assay and color test ；relative fluorescence intensity of LC 3 positive expression was detected by immunofluorescence method ；mRNA expression of NF-κB p65 in cells was detected by real-time PCR ； Western blotting assay was used to detect the protein expression of NF-κB p65 and p-NF-κB p65 in cells. RESULTS ：Compared with normal control group ，survival rate and SOD activity were decreased significantly in injury model group ，while apoptotic rate ， MDA content ，relative fluorescence intensity of LC 3 positive expression ，mRNA expression of NF-κB，protein expression of NF-κ B p 65 and p-NF-κB p65 as well as p-NF-κB/NF-κB were increased significantly（P＜0.05）. Compared with injury model group ， survival rates and SOD activities were increased significantly in curcumin groups ，while apoptotic rates ，MDA contents ，relative fluorescence intensities of LC 3 positive expression ，mRNA expression of LC 3 positive cells ，protein expression of NF-κB p65 and p-NF-κ B p65 as well as p-NF-κ B p65/NF-κ B p65 were decreased significantly （P＜0.05）. CONCLUSIONS ：Curcumin can increase the survival rate of H 2O2-induced H 9c2 cardiomyocyte injury ，decrease its apoptotic rate ，increase SOD activity and decrease MDA content in cardiomyocytes. Above effects may be related to the regulation of NF-κB signaling pathway.

Study on Regulation Effects of Volatile Oil from Angelica sinensis on Autophagy of Myocardial Cell H 9C2 in Rats with Hypoxia/reoxygenation Injury / 中国药房

OBJECTIVE：To investigate the regulation effects of volatile oil from Angelica sinensis on autophagy of myocardial cell H 9C2 in rats with hypoxia and reoxygenation （H/R）injury. METHODS ：Using myocardial cell H 9C2 as subject ，CCK-8 method was used to screen the optimal concentration and administration time of volatile oil from A. sinensis. The acitivity of LDH in cell supernatant was determined after treated with volatile oil from A. sinensis by ELISA. Using autophagy inhibitors （3-methyladenine，5 mmol/L） as positive control ，MDC method and Western blotting assay were used to detect average fluorescence intensity of MDC and the expression of autophagy related proteins [Beclin- 1，microtubule associated protein light chain 3Ⅱ（LC3Ⅱ），LC3Ⅰ] in H 9C2 cells after treated with medicines. RESULTS ：After treated with 0.6 μmol/L violate oil from A. sinensis for 6 h，compared with blank group ，LDH activity in cell supernatant ，average fluorescence intensity of MDC ，the expression of Beclin- 1，LC3Ⅱ/LC3Ⅰ ratio in cells were increased significantly in H/R group ，while the expression of p 62 was decreased significantly （P＜0.05 or P＜0.01）. Compared with H/R group ，the activity of LDH in cell supernatant of H/R+drug group as well as average fluorescence intensity of MDC ，the expression of Beclin- 1，LC3Ⅱ/LC3Ⅰratio in cells in H/R+drug group and H/R+autophagy inhibitor group were decreased significantly ，while the expression of p 62 were increased significantly （P＜0.05 or P＜0.01）. CONCLUSIONS ：The volatile oil from A. sinensis can reduce the autophagy level of H/R injury myocardial cells by regulating the expression of autophagy related proteins.

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.

Study on the Spectrum-effect Relationship of Different Solvent Extracts from Trollius chinensis against Hypoxia/ reoxygenation Injury of Cardiomyocyte / 中国药房

OBJECTIVE：To study spectrum-effect relationship of 11 different solvent extracts from Trollius chinensis against hypoxia/reoxygenation injury of cardiomyocyte . METHODS ：HPLC-MS/MS method was used to establish the fingerprints of 11 different solvent extracts from T. chinensis ，the compounds corresponding to the common peaks were identified by comparing with the substance control and literature information. MTT assay was used to detect the effects of 11 different solvent extracts from T. chinensis on the survival rate of rat myocardial H 9c2 cells injured by hypoxia/reoxygenation. The MDA content ，ROS level in cells and LDH content in the supernatant were detected by ELISA. GRA and PLS method were used to analyze the spectrum-effect relationship between the compounds corresponding to common peak and anti-hypoxia/reoxygenation injury of cardiomyocyte （drug effect）. RESULTS ：There were 22 common peaks in 11 different solvent extracts from T. chinensis ，and 22 compounds were identified. Compared with hypoxia/reoxygenation injury group ，survival rate of hypoxia/reoxygenation injury+S 1-S6，S9 and S 10 groups were increased significantly ，while MDA content ，ROS level and LDH content were decreased significantly （P＜0.05）； ROS level and LDH content of hypoxia/reoxygenation injury+S 8 group w ere decreased significantly （P＜0.05）. The r of GRA analysis of 22 compounds with drug effects were all higher than 0.8. Except for peaks 1，2，7，13，14 and 21，r of PLS analysis of rest peaks with drug effects were higher than 0 发。电话：0431-86058683。E-mial：nml2000@163.com （being positive correlation ）. Top 9 common peaks in the list of contribution rate were peak 6＞11＞4＞5＞8＞9＞12＞10＞15. CONCLUSIONS ：Orientin（peak 6），vitexin（peak 11）， orientin-2″-O-β-L-galacto- pyranosl （peak 4），orientin-2″-O-β-D-Pyrine xylosides （peak 5），quercetin-3-O-glucopyranoside（peak 8），vitexin-2″-O-β-L-galactoside（peak 9），hyperoside（peak 12），vitexin-2″-O-β-D-pyrine xylosides （peak 10），2″-O-（2″′- methylbutyry-loxy）-orientin（peak 15）may be the main components of anti-hypoxia/reoxygenation injury of cardiomyocytes.

In-vitro cardiovascular protective activity of a new achillinoside from Achillea alpina

Abstract Achillinoside was isolated from methanol extract of Achillea alpina L., Asteraceae. The structure of the compound was characterized based on various spectrum data, including IR, HR-ESI-MS, 1D and 2D NMR. The cardiovascular protective effect of achillinoside was tested on H2O2-induced H9c2 cells. In our research, achillinoside could increase the cell viability dose-dependently in H2O2-induced H9c2 cells. In addition, the levels of caspase-3/9 cells were significantly decreased in H2O2 and achillinoside incubated H9c2 cells.