Effects of ginkgolide B on Caspase-3/PTEN/Akt pathway and cell proliferation and apoptosis in hypoxia/reoxygenation cardiomyocytes / 中国胸心血管外科临床杂志

@#Objective     To investigate the effect of ginkgolide B (GB) on cysteinyl aspartate specific proteinase-3 (Caspase-3)/chromosome 10 deletion phosphatase-tension protein homologue (PTEN)/protein kinase B (Akt) pathway and cell proliferation and apoptosis in hypoxia/reoxygenation cardiomyocytes. Methods     H9C2 cells were cultured in vitro. A control group was cultured in serum-free DMEM high glucose medium at 37°C and 5% CO2 for 28 hours. The remaining groups were prepared with hypoxia/reoxygenation models. A GB low-dose group and a GB high-dose group were treated with GB pretreatment with final concentration of 50 μmol/L and 200 μmol/L respectively at 1 h before hypoxia/reoxygenation. A carvedilol group was treated with carvedilol of a final concentration of 10 μmol/L at 1 h before hypoxia/reoxygenation. The proliferation and apoptosis of H9C2 cells were detected, and the levels of lactate dehydrogenase (LDH), malondialdehyde (MDA), reactive oxygen species (ROS), PTEN, Akt, phosphorylated Akt (p-Akt) and Caspase-3 in H9C2 cells were also detected. Results     Compared with the control group, the proliferation rate of H9C2 cell, and the levels of PTEN, Akt and p-Akt in other groups decreased, and the apoptosis rate, and the levels of LDH, MDA, ROS and Caspase-3 increased (P<0.05). Compared with the hypoxia/reoxygenation group, the proliferation rate of H9C2 cell, and the levels of PTEN, Akt and p-Akt in all GB dose groups and the carvedilol group increased; the apoptosis rate, and the levels of LDH, MDA, ROS and Caspase-3 decreased, and the effect of GB was in a dose dependent manner; however, the effect of GB was not as strong as carvedilol (P<0.05). Conclusion     GB can inhibit H9C2 cell apoptosis and promote H9C2 cell proliferation by activating Caspase-3/PTEN/Akt pathway.

Qili Qiangxin, a compound herbal medicine formula, alleviates hypoxia-reoxygenation-induced apoptotic and autophagic cell death via suppression of ROS/AMPK/mTOR pathway in vitro / 中西医结合学报

OBJECTIVE@#Qili Qiangxin (QLQX), a compound herbal medicine formula, is used effectively to treat congestive heart failure in China. However, the molecular mechanisms of the cardioprotective effect are still unclear. This study explores the cardioprotective effect and mechanism of QLQX using the hypoxia-reoxygenation (H/R)-induced myocardial injury model.@*METHODS@#The main chemical constituents of QLQX were analyzed using high-performance liquid chromatography-evaporative light-scattering detection. The model of H/R-induced myocardial injury in H9c2 cells was developed to simulate myocardial ischemia-reperfusion injury. Apoptosis, autophagy, and generation of reactive oxygen species (ROS) were measured to assess the protective effect of QLQX. Proteins related to autophagy, apoptosis and signalling pathways were detected using Western blotting.@*RESULTS@#Apoptosis, autophagy and the excessive production of ROS induced by H/R were significantly reduced after treating the H9c2 cells with QLQX. QLQX treatment at concentrations of 50 and 250 μg/mL caused significant reduction in the levels of LC3II and p62 degradation (P < 0.05), and also suppressed the AMPK/mTOR signalling pathway. Furthermore, the AMPK inhibitor Compound C (at 0.5 μmol/L), and QLQX (250 μg/mL) significantly inhibited H/R-induced autophagy and apoptosis (P < 0.01), while AICAR (an AMPK activator, at 0.5 mmol/L) increased cardiomyocyte apoptosis and autophagy and abolished the anti-apoptotic effect of QLQX. Similar phenomena were also observed on the expressions of apoptotic and autophagic proteins, demonstrating that QLQX reduced the apoptosis and autophagy in the H/R-induced injury model via inhibiting the AMPK/mTOR pathway. Moreover, ROS scavenger, N-Acetyl-L-cysteine (NAC, at 2.5 mmol/L), significantly reduced H/R-triggered cell apoptosis and autophagy (P < 0.01). Meanwhile, NAC treatment down-regulated the ratio of phosphorylation of AMPK/AMPK (P < 0.01), which showed a similar effect to QLQX.@*CONCLUSION@#QLQX plays a cardioprotective role by alleviating apoptotic and autophagic cell death through inhibition of the ROS/AMPK/mTOR signalling pathway.

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.

Effect of hydroxysafflor yellow A on apoptosis of human renal tubular epithelial cells under cold hypoxia and reoxygenation / 中国综合临床

Objective:To investigate the effect of hydroxysafflor yellow A(HSYA) preconditioning group on apoptosis induced by cold hypoxia/reoxygenation (cold H/R) injury in human renal tubular epithelial cells (HK2 cells).Methods:After digestion and passage, HK2 cell lines were divided into Sham group (control group), cold hypoxia and reoxygenation group (cold H/R group, cells cold hypoxia for 4 h, reoxygenation for 4 h), and HSYA preconditioning group (each HSYA subgroup was given different doses of HSYA 0.5 h before hypoxia, and the other operations were the same as the cold H/R group). The cell survival rate was measured by CCK-8 method.The expression of Bcl-2, Bax and Caspase-3 proteins in HK-2 cells were detected by immunocytochemistry and Western blotting.Results:(1) Compared with cold H/R group, different doses of HSYA could improve cell survival rate in different degrees, but only HSYA25 μmol/L group had the most significant effect (74.000±5.500 vs.59.000±3.800, P<0.05). (2) Immunocytochemistry semi-quantitative score: Compared with cold H/R group, the expression of Bax and Caspase-3 in HK2 cells of HSYA25 μmol/L group was significantly decreased(0(0, 1) vs. 8(6, 8), Z=2.041, P<0.05 and (3.400±0.548) vs.(7.800±1.095), t=11.000, P<0.01). The expression of Bcl-2 protein was increased significantly ((6.800±1.095) vs.(1.400±0.548), t=10.590, P<0.01). The ratio of Bcl-2/Bax increased significantly.(3)Western blot was used to detect protein: Compared with the cold H/R group, the protein levels of Bax, Cleaved-Caspase-3 and Pro-caspase-3 of HK2 cells in the HSYA25 μmol/L group were significantly decreased ((0.707±0.012) vs.(0.968±0.117), (0.480±0.009)vs.(0.735±0.005), (0.992±0.008)vs.(1.197±0.005), all P<0.01). The expression of Bcl-2 protein was significantly increased, and the ratio of Bcl-2/Bax was significantly increased ((0.410±0.009) vs.(0.273±0.008), (0.582±0.016) vs (0.282±0.080), all P<0.01). The experimental results were consistent with the immunocytochemistry. Conclusion:HSYA can effectively reduce the damage of HK2 cells after cold hypoxia and reoxygenation.

Effect of small nucleolar RNA host gene 5 on hypoxia/reoxygenation injury of astrocytes / 中华行为医学与脑科学杂志

Objective:To explore the effect of lncRNA SNHG5 on injury of astrocytes induced by hypoxia/reoxygenation (H/R).Methods:(1) Astrocytes were cultured in vitro. The H/R cell model was established by hypoxia culture for 6 hours and then reoxygenaion culture for 18 hours. Lipofectamine? 2000 liposome method was used to transfect lncRNA SNHG5 into astrocytes. RT-qPCR was used to detect the expression of lncRNA SNHG5 in H/R cells and after transfection. (2) Astrocytes were divided into normal control group, model group, transfection control group (pcDNA-NC was transfected first, then H/R cell model was established) and transfection group (pcDNA-lncRNA SNHG5 was transfected first, then H/R cell model was established). Then the effect of overexpression of lncRNA SNHG5 on astrocytes was observed. (3)The astrocytes transfected with lncRNA SNHG5 and H/R intervention were divided into transfection+ vehicle group (0.1% DMSO incubation) and transfection+ inhibitor group (20 μmol/L LY294002 incubation), and then observe the effect of the inhibitor of PI3K/Akt signaling pathway LY294002 on H/R astrocytes was observed. (4) CCK-8 was used to detect cell proliferation. Flow cytometry was used to detect cell apoptosis. Western blot was used to detect the expression of cell proliferation proteins (Cyclin D1 and Cyclin E), apoptotic proteins (Caspase-3 and Bax), p-PI3K and p-AKT protein. ELISA was used to detect the levels of IL-1β and TNF-α. The colorimetric method was used to detect the level of lactate dehydrogenase(LDH) in cell culture supernatants and the level of malondialdehyde(MDA) and superoxide dismutase(SOD) in cells. SPSS 22.0 software was used for independent sample t-test and one-way ANOVA, and LSD- t test was used for further pairwise comparisons. Results:(1) RT-qPCR results showed that the level of lncRNA SNHG5 in astrocytes induced by H/R was lower than that in the normal cultured cells ( t=33.28, P<0.05). (2) lncRNA SNHG5 overexpression experiment: The cell proliferation activity of the model group was lower than that in the normal control group (CCK-8 OD value: (0.64±0.02), (1.23±0.02), t=62.58, P<0.05). The levels of proliferation proteins Cyclin D1 and Cyclin E in the model group were lower than those of the normal control group ( t=33.54, 32.20, both P<0.05). The cell proliferation activity of the transfection group was higher than that of the transfection control group (CCK-8 OD value: (1.49±0.02), (0.65±0.03), t=69.89, P<0.05), the levels of cell proliferation proteins Cyclin D1 and Cyclin E in the transfection group were lower than those in the transfection control group ( t=24.96, 28.46, both P<0.05). The apoptosis rate of the model group was higher than that of the control group (flow cytometry results: (25.33±1.13)%, (9.06±0.21)%, t=42.47, P<0.05), and the levels of apoptotic proteins Caspase-3 and Bax were also higher than those of the control group ( t=57.41, 41.60, both P<0.05). The Caspase-3 rate of the transfection group was lower than that of the transfection control group((16.56±0.60)%, (25.89±1.18)%, t=21.14, P<0.05), and the levels of apoptotic proteins Caspase-3 and Bax were also higher than those of the transfection control group( t=77.79, 58.34, both P<0.05). The levels of p-PI3K and p-AKT proteins in the model group were lower than those in the control group ( t=56.35, 33.94, both P<0.05), and the levels of p-PI3K and p-AKT proteins in the transfection group were higher than those in the transfection control group ( t=130.14, 76.37, both P<0.05). The results of ELISA showed that the levels of IL-1β and TNF-α in the model group were higher than those in the control group ( t=58.04, 30.63, both P<0.05), but the levels of IL-1β and TNF-α in the transfection group were lower than those in the transfection control group ( t=33.63, 39.01, both P<0.05). The colorimetric method showed that the levels of LDH and MDA in the model group were higher than those in the control group ( t=65.51, 41.85, both P<0.05), but the level of SOD was lower than that in the control group ( t=48.82, P<0.05). The levels of LDH and MDA in the transfection group were lower than those in the transfection control group ( t=37.93, 30.72, both P<0.05), but the level of SOD was higher than that in the transfection control group ( t=30.32, P<0.05). (3) PI3K/Akt signaling pathway inhibition experiment: the cell proliferation activity of the transfection+ inhibitor group was lower than that of the transfection+ vehicle group (CCK-8 OD value: (0.97±0.02), (1.46±0.03), t=15.24, P<0.05), and the related proliferation proteins Cyclin D1 and Cyclin E were also lower ( t=11.41, 13.15, both P<0.05). The apoptosis rate of the transfection+ inhibitor group was higher than that of the transfection+ vehicle group (Flow cytometry: (26.11±0.86)%, (16.06±0.44)%, t=10.45, P<0.05). The apoptosis rate of the transfection+ inhibitor group was higher than that of the transfection+ vehicle group (Flow cytometry: (26.11±0.86)%, (16.06±0.44)%, t=10.45, P<0.05), and the related apoptosis protein Caspase-3 and Bax were also higher ( t=19.06, 13.54, both P<0.05). The expression levels of p-PI3K and p-AKT protein in the transfection+ inhibitor group were lower than those in the transfection+ vehicle group ( t=36.67, 27.34, both P<0.05). ELISA results showed that the levels of IL-1β and TNF-α in the transfection+ inhibitor group were higher than those in the transfection+ vehicle group ( t=15.17, 9.44, both P<0.05). The colorimetric method results showed that the levels of LDH and MDA in the transfection+ inhibitor group were the same as those in the transfection+ vehicle group ( t=15.33, 9.05, both P<0.05), but the level of SOD was lower than the transfection+ vehicle group ( t=11.04, P<0.05). Conclusion:Overexpression of lncRNA SNHG5 may promote the proliferation of astrocytes induced by hypoxia/reoxygenation, and inhibit cell apoptosis, inflammation and oxidative stress.

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.

Protective effect of Shengxian decoction and the decoction of single herb component against myocardial injury induced by hypoxia/reoxygenation / 药学实践杂志

Objective To study the protective effect of Shengxian decoction and the single herb decoction against myocardial injury induced by hypoxia/reoxygenation. Methods The H9c2 cells were cultured to establish hypoxia/reoxygenation model. Rats were divided into 8 groups: normal control group, hypoxia/reoxygenation group (model group) and treated groups (Shengxian decoction and the single herb decoction). The apoptotic rate of cardiomyocytes, the activity of reactive oxygen species (ROS) and intracellular calcium concentration (Ca2+) were measured. Results Compared with hypoxia/reoxygenation group, the apoptosis rate, ROS activity and intracellular Ca2+ concentration were significantly lower in all treated groups (P<0.05). The ROS activity and intracellular Ca2+ concentration was decreased by 41.37% and 15.20% in Shengxian decoction group compared to the model group. Conclusion Shengxian decoction and the single herb decoction had protective effect on myocardial injury induced by hypoxia/reoxygenation.

Effect of methyl eugenol on hypoxia/reoxygenation injury of human renal tubular epithelial cells and its mechanism / 中国中药杂志

This study aimed to investigate the effect of methyl eugenol(ME) on hypoxia/reoxygenation(H/R)-induced injury of human renal tubular epithelial HK-2 cells and its mechanism. The viability of HK-2 cells cultured with different concentrations of ME and exposed to H/R was detected by cell counting kit-8(CCK-8) assay. The effect of ME on the morphology of HK-2 cells was observed under an inverted microscope. The content of intracellular reactive oxygen species in different groups was detected after 2',7'-dichlorodihydrofluorescein diacetate(DCFH-DA) fluorescence staining. Cell apoptosis was determined by flow cytometry. Changes in mitochondrial membrane potential were monitored by JC-1 dye. The concentrations of nuclear factor erythroid 2 related factor 2(Nrf2), heme oxygenase-1(HO-1), and nicotinamide adenine dinucleotide phosphatase oxidase 4(Nox4) were measured by Western blot, followed by the assay of Nrf2 concentration changes in cytoplasm and nucleus by confocal fluorescence staining. The results showed that when the concentration of ME was 0-40 μmol·L~(-1), the activity of HK-2 cells was not affected. Compared with the model group, ME enhanced the activity of HK-2 cells and the cell morphology was normal. As revealed by further experiments, ME inhibited the release of reactive oxygen species and the decline in mitochondrial membrane potential of HK-2 cells after H/R injury, promoted Nrf2/HO-1 expression and Nrf2 translocation to the nucleus, and down-regulated the expression of Nox4, thereby significantly reducing apoptosis. This protective effect of ME could be reversed by the specific Nrf2 inhibitor ML385. These findings have preliminarily proved that ME effectively protected HK-2 cells against H/R injury, which might be related to its promotion of Nrf2/HO-1 signaling pathway and inhibition of Nox4. Such exploration on the possible mechanism of ME in the treatment of renal ischemia-reperfusion injury(IRI) and protection of organ function from the perspective of antioxidant stress has provided reference for related research on the treatment of acute kidney injury with traditional Chinese medicine.

Protective effect of ginsenoside Rg_1 on hypoxia/reoxygenation injury and its mechanism / 中国中药杂志

This project aimed to explore the protective effect of ginsenoside Rg_1 on hypoxia/reoxygenation(H/R)-induced H9 c2 cardiomyocyte injury and its underlying signaling pathway. The H/R model of H9 c2 cardiomyocytes was established and then the cells were divided into different treatment groups. CCK-8(cell counting kit-8) was used to detect the activity of cardiomyocytes; Brdu assay was used to detect the proliferation of H9 c2 cells; the caspase-3 activity was tested, and then the protein expression was assessed by Western blot. Flow cytometry was used to evaluate the apoptosis level of cardiomyocytes. Ginsenoside Rg_1 inhibited H/R-induced cardiomyocyte apoptosis and caspase-3 activity, promoted nuclear transcription of nuclear factor erythroid-2 related factor 2(Nrf2), and enhanced the expression of the downstream heme oxygenase-1(HO-1). Ginsenoside Rg_1 could increase Nrf2 nuclear transcription and HO-1 expression with the increase of concentration(10, 20, 40, 60 μmol·L~(-1)). However, the protective effect of ginsenoside Rg_1 on cardiomyocytes was significantly weakened after the transfection of Nrf2-siRNA. Ginsenoside Rg_1 could protect cardiomyocytes by activating the Nrf2/HO-1 pathway.

Mechanism of Guhong Injection against Hypoxia/Reoxygenation Injury of H9c2 Cardiomyocytes Based on PI3K/AKT Signaling Pathway / 中国药学杂志

OBJECTIVE: To explore the protectiveness of Guhong injection (GHI) on H9c2 cardiomyocytes injured by hypoxia/reoxygenation (H/R) based on PI3K/AKT signaling pathway. METHODS: H9c2 cardiomyocytes were cultured and the optimal GHI doses were screened by CCK-8. The cells were randomly subjected into 8 groups: control group, model group, low, medium and high GHI dose groups (30, 60 and 90 μL•mL-1 respectively), positive drug group (verapamil injection 7.5 μL•mL-1), GHI+LY294002 (PI3K inhibitor) group and LY294002 group. Cells were established H/R model with hypoxia for 4 h and reoxygenation for 16 h excepted the control group. The CK-MB, LDH, MDA content and SOD activities in each group were detected by Elisa, and the expression levels of p-PI3K, PI3K, p-AKT, AKT and GSK-3β in each group were detected by Western blot. RESULTS: Compared each drug-treated group with the model group, LDH and CK-MB contents were decreased (P<0.05), MDA content was decreased (P<0.01), and SOD activity was increased (P<0.01). Meanwhile, the expression levels of p-PI3K/PI3K, p-AKT/AKT, and GSK-3β protein were elevated (P<0.01). Compared with the model group, LDH, CK-MB, MDA and SOD activity in LY294002 group were no significant difference, while the expression levels of p-PI3K/PI3K, p-AKT/AKT and GSK-3β protein decreased (P<0.01). CONCLUSION: GHI could represent anti-oxidative and anti-apoptotic effect which reduced the damage of injured H9c2 myocardial cells caused by H/R. These effects might be related to the PI3K/AKT signaling pathway.

Role of aquaporin-4 in mediating protective effect of remifentanil postconditioning against adult myocardial hypoxia/reoxygenation injury / 中国药理学与毒理学杂志

OBJECTIVE: To investigate the effect of remifentanil postconditioning on myocardial hypoxia/reoxygenation (H/R) injury in adults, and to further explore the role of aquaporin-4 (AQP-4) in mediating this effect. METHODS: Trabecular muscles from the right atrial appendage of adults were treated with hypoxia for 90 min, followed by reoxygenation for 120 min. Then, remifentanil 0.01, 0.1 and 1.0 nmol·L-1was infused 10 min before the end of hypoxia until 10 min after the start of reoxygenation. The contractile tension of the trabecular muscles was monitored during the experiment. Western blotting was performed to evaluate AQP-4 expression at the end of the experiment. RESULTS: Compared with normal control group, muscle tension decreased significantly after 30 min of induction in H/R group (P<0.05), and it was reduced to the minimum at 90 min. The muscle tension at 150 min and 180 min in remifentanil 0.1 nmol·L-1group was higher than that in H/R group (P<0.05). The muscle tension in remifentanil 1.0 nmol·L-1group was higher than that in H/R group during reoxygenation (P<0.05). Compared with normal control group, the expression of AQP-4 protein in H/R group was significantly higher (P< 0.05), whereas the expression of AQP-4 in remifentanil 0.1 and 1.0 nmol·L-1groups was down-regulated compared with H/R group (P<0.05). CONCLUSION: Remifentanil can alleviate myocardial H/R injury in adults, and its effect may be related to the decreased expression of AQP-4.

Astragaloside IV ameliorates hypoxia/reoxygenation injury via regulating mitochondrial homeostasis in rat cardiomyocytes / 药学学报

This study was designed to investigate the effect and mechanism of astragaloside IV (ASIV) on mitochondrial morphology and function of rat cardiomyocytes under hypoxia/reoxygenation injury. H9c2 cells were divided into control group, hypoxia/reoxygenation (H/R) group, and H/R + ASIV group. Cell viability and lactate dehydrogenase (LDH) leakage were measured by cell counting kit-8 (CCK-8) and LDH assay kit, respectively. Oxidative stress levels, such as superoxide dismutase (SOD), glutathione (GSH), and malondialdehyde (MDA), were analyzed by commercial kits. Intracellular and mitochondrial reactive oxygen species (ROS) levels were detected by dihydroethidium (DHE) and MitoSOX. Changes of the mitochondrial membrane potential were detected using the fluorescent probe JC-1. Opening of mitochondrial permeability transition pore was examined via calcein acetoxymethyl ester (calcein-AM). Apoptosis was assessed using terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) assay kit. To detect protein expression of dynamin-related protein 1 (Drp1), mitofusin1 (Mfn1), Mfn2, Bax, B-cell lymphoma-2 (Bcl-2), and cleaved cysteine-aspartic protease (caspase)-3, Western blot analysis was carried out. Compared with the control group, ASIV (100 μmol·L-1) significantly improved H/R induced cell injury, LDH leakage, decrease of SOD activity, and GSH content, increase of MDA content and ROS content, loss of mitochondrial membrane potential, mitochondrial permeability transition pore opening, ROS production activation, mitochondrial fission/fusion imbalance, and cell apoptosis. In addition, the effect of ASIV against H/R injury was also verified on primary rat cardiomyocytes. The animal welfare and experimental process follow the rules of Animal Ethics Committee of Zhejiang Chinese Medical University. In conclusion, ASIV may play a protective role in mitochondria by regulating morphological dynamic stability and mitochondrial function, inhibiting excessive synthesis of ROS, improving the internal environment of oxidative stress, reducing cell apoptosis, and thereby protecting against cardiomyocytes’ hypoxia/reoxygenation injury.

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.

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.

Protective effect of bisoprolol against hypoxia/reoxygenation injury of cardiac microvascular endothelial cells / 医学研究生学报

Objective　To observe the protective effect of bisoprolol against hypoxia/reoxygenation injury of cardiac microvascular endothelial cells and explore the mechanism.　Methods　Left ventricular of cardiac microvascular endothelial cells (CMECs) were isolated from 8-week-old male C57BL/6N mice. CMECs were randomized into four groups: control group, vehicle group, hypoxia/reoxygenation group (H/R group), hypoxia/reoxygenation + bisoprolol group. The level of cell proliferation, apoptosis, superoxide anion, Cleaved caspase-3 and Nox2 expression were measured in each group.　Results　Compared with control group, H/R group had lower cell proliferation, higher apoptotic level, more superoxide anion level and the expression of Cleaved caspase-3 and Nox2 (P < 0.05). Furthermore, bisoprolol reversed hypoxia/reoxygenation-induced the decreased cell proliferation, the increased apoptosis, superoxide anion level, Cleaved caspase-3 and Nox2 expression (P < 0.05).　Conclusion　Bisoprolol can protect CMECs against hypoxia/reoxygenation injury by reducing the expression of Nox 2 that decreases oxidative stress.

The long non-coding RNA H19 induces hypoxia/reoxygenation injury by up-regulating autophagy in the hepatoma carcinoma cells

BACKGROUND: Long non-coding RNA H19 (H19) plays an important role by regulating protein expression in different tissues and organs of the body. However, whether H19 induces hypoxia/reoxygenation (h/R) injury via increase of autophagy in the hepatoma carcinoma cells is unknown. RESULTS: H19 was expressed in the hepatoma carcinoma cells (Hep G2 and HCCLM3 cells) and its expression was most in 8 h/24R. The knockdown of H19 and 3-MA (an autophagy inhibitor) protected against h/R-induced apoptosis, cell damage, the expression of cleaved caspase-3 and cleaved caspase-9, the release of cytochrome c (Cyt c). The knockdown of H19 and 3-MA also decreased the autophagic vesicles (AVs) and the expression of Beclin-1 and the ration of LC3-II/LC3-I, and increased cell viability, the expression of Bcl-2 and P62 and the phosphorylation of PI3K, Akt and mTOR. In addition, chloroquine (CQ, an inhibitor of autophagy flux) markedly decreased formation of autophagy flux (the ration of LC3-II/LC3-I). The results of the knockdown of H19 group were similar to those of the 3-MA (or CQ) group. Rapamycin (a mTOR inhibitor, an autophagy activator) further down-regulated h/R-induced decrease of the phosphorylated PI3K, Akt and mTOR. The knockdown of H19 cancelled the effect of rapamycin. The overexpression of H19 further expanded h/R-induced increase of the ration of LC3-II/LC3-I and decrease of the phosphorylated PI3K, Akt and mTOR. CONCLUSIONS: Our results suggest that the long non-coding RNA H19 induces h/R injury by up-regulation of autophagy via activation of PI3K-Akt-mTOR pathway in the hepatoma carcinoma cells.

Evaluation of hypoxia/reoxygenation injury of H9C2 myocardial cells and intervention effect of notoginsenoside R1 based on RTCA technology / 中国药理学通报

Aim: To establish the standard hypoxia/reoxygenation (H/R) injury model of H9c2 myocardial cells and evaluate the intervention effect of notoginsenoside R, based on RTCA technology. Methods: H9c2 myocardial cells were inoculated into the E-Plate board, 3 wells by a group, then the growth curve and cell index of different hypoxia time, different reoxygenation time and notoginsenoside R, intervention were determined respectively, with the growth status of H9c2 myocardial cells reflected by cell index. Results: Cell survival rate was (48.82 ±5.32)% after hypoxia treatment for 4h and reoxygenation treatment for 16h with replacement of serum-free, glucose-free DMEM medium before hypoxia, and no significant difference was found between the results measured with MTT method. Notoginsenoside R, could protect the hypoxia/reoxygenation injury of H9c2 myocardial cells without time dependence. Conclusions: RTCA technology is an effective means to establish the standard H/R injury model of H9c2 myocardial cells, which also has some guiding significance in discovering new drug targets and mechanisms.

Inhibition of hydroxysafflor yellow A on apoptosis of endothelial cells EA.hy926 after hypoxia-reoxygenation / 中草药

Objective: To observe the effect of hydroxysafflower yellow A (HSYA) on the apoptosis of human umbilical vein endothelial cells EA.hy926 induced by hypoxia-reoxygenation. Methods: MTT colorimetry method was used to detect the effects of different hypoxia time (8, 12 h) and different reoxygenation time (4, 8, 12 h) on the cell viability. And after the cell had been in the status of hypoxia for 12 h and reoxygenation for 8 h, this method was adapted once again to evaluate the effects of different concentrations of HSYA (0.1, 1, 10, and 100 μmol/L) on cell viability in different time stages. After the cell had been in the status of hypoxia for 12 h, reoxygenation for 8 h, Western blotting was used to test its effects on the expressions of the following proteins in different time stages, which contained Bcl-2, Bax, cleaved Caspase-3, and activated cleaved Caspase-9. This method was also used to detect whether it had an improvement effect on the above proteins after the pr-treat the cells with HSYA. Real-time PCR was used to evaluate the mRNA expressions of Bax, Bcl-2 after the cell had been in the status of hypoxia for 12 h, reoxygenation for 8 h, and this method was also used to test the effect of HSYA on the expressions of Bax, Bcl-2 after the same time stages. Hoechest staining and flow cytometry were used to detect the apoptosis situation of the cell after it was in the status of hypoxia for 12 h and reoxygenation for 8 h. And this method was also adapted to detect the effect of HSYA on apoptosis of the cell after the same time stage. Results: Compared with the control group, the EA.hy926 cell viability decreased significantly after hypoxia for 8, 12 h, reoxygenation for 4, 8, and 12 h (P < 0.01). The cell viability decreased the most significantly after hypoxia for 12 h and reoxygenation for 8 h (P < 0.01), and during this period, the expression of Bax, cleaved Caspase-9, cleaved Caspase-3 protein increased significantly, and Bcl-2 protein was decreased significantly. Compared with the H/R group, HSYA (10 μmol/L) significantly increased the cell viability (P < 0.01) after hypoxia-reoxygenation, and significantly up-regulated the protein expression of Bcl-2, and down-regulated the protein expressions of Bax, cleaved Caspase-9, and cleaved Caspase-3. Conclusion: Hydroxysafflor yellow A can effectively inhibit the apoptosis of EA.hy926 induced by hypoxia and reoxygenation. The mechanism may be related to the down-regulation of Bax, cleaved Caspase-9, cleaved Caspase-3 protein as well as the up-regulation of Bcl-2 protein.

Hypoxic preconditioning attenuates hypoxia/reoxygenation injury of cardiomyocytes in SD rats via activating AMPK / 解放军医学杂志

[Abstract] Objective To investigate the protective effects of hypoxic preconditioning (HPC) on cardiomyocytes after hypoxia/reoxygenation (H/R) by activating AMP-activated protein kinase (AMPK). Methods The primary cardiomyocytes were prepared from neonatal SD rats. The model of H/R of cardiomyocytes was established and randomly divided into control group, H/R group, HPC group, H/R+A-769662 (AMPK agonist) group and HPC+compound C (AMPK inhibitor) group. The survival rate of cardiomyocytes was detected by chromatometry with Cell Counting Kit-8 (CCK-8); the reactive oxygen species (ROS) and ATP contents in the cells were detected respectively by dihydroethidium (DHE) dyeing and ATP Detection Assay Kit measured by fluorescence microplate reader; the phosphorylation level of AMPK and activation level of caspase-3 were detected by Western blotting; the concentration of free Ca2+ in cardiomyocytes was detected by Fluo-3AM dyeing using laser scanning confocal microscopy; the apoptotic rate of cardiomyocytes was detected by TUNEL staining. Results CCK-8 assay showed that compared with H/R group, the survival rate of cardiomyocytes in HPC group and H/R+A-769662 group increased obviously (P0.05); compared with H/ R group, the AMPK phosphorylation levels elevated significantly in HPC group and H/R+A-769662 group respectively (P<0.05, P<0.01). Meanwhile, the activation levels of caspase-3 in H/R group and HPC+compound C group were increased obviously compared with that in control group (P<0.01), but in HPC group and H/R+A-769662 group were significantly lower than that in H/R group (P<0.01, P<0.05). The results of Fluo-3AM staining showed that compared with control group, the concentrations of intracellular free Ca2+ increased obviously in H/R group and HPC+compound C group (P<0.01); the concentrations of free Ca2+ in cardiomyocytes of HPC group and H/R+A-769662 group were less than that in H/R group (P<0.01), while the concentrations of free Ca2+ in HPC+compound C group much more increased than in HPC group (P<0.01). The results of TUNEL staining showed that compared with H/R group, the apoptotic rate of cardiomyocytes decreased obviously in HPC group and H/R+A-769662 group (P<0.01, P<0.05); while compared with HPC group, the apoptotic rate of cardiomyocytes increased significantly in HPC+compound C group (P<0.05). Conclusions H/R may lead to the increase of ROS production, the decrease of ATP contents, and increase the levels of free Ca2+ in cardiomyocytes, and thus activate caspase-3, lead to apoptosis eventually. While HPC may reduce the production of ROS by activating AMPK to ensure the energy supply of cardiomyocytes after H/R treatment, prevent apoptosis of cardiomyocytes, and then reduce the H/R injury to cardiomyocytes.

Roudoukou-8 San reduces the hypoxia/reoxygenation injury of cardiac myocytes through JAK2/STAT3 pathway / 中国药科大学学报

@#To study the protective effects of Roudoukou-8 San extract on hypoxia/reoxygenation injury of cardiac myocytes and its relationship with tyrosine protein kinase 2(JAK2)/signal transducer and activator 3(STAT3)signaling pathway, hypoxia/reoxygenation injury model of H9c2 cells was built by sodium dithionite(Na2S2O4). The vitality of the cells was tested by CCK-8; the contents of lactate dehydrogenase(LDH), creatine phosphate kinase(CK)and aspartate aminotransferase(AST)in cell culture medium were tested by fully automatic biochemical analyzer; the contents of catalase(CAT), superoxide dismutase(SOD)and glutathione peroxidase(GSH-Px)in cells were tested by kits; cell apoptosis degree was tested by hoechst staining. And the protein expressions of JAK2, p-JAK2, STAT3, p-STAT3, Bcl-2 and Bax in myocardial cells of each group were tested by Western blot. Hypoxia for 1 hour and reoxygenation for 3 hours of 2 mmol/L Na2S2O4 caused damage of about 50% H9c2 cells. Compared with the model group, the extracts of Roudoukou-8 San with different concentrations could increase the viability of cells. Besides, contents of CK, LDH and AST in cell culture medium were decreased significantly, while contents of CAT, SOD and GSH-Px were increased significantly. At the same time, the expression of p-JAK2, p-STAT3 and Bcl-2 were significantly increased and that of Bax was significantly decreased. The effects of Roudoukou-8 San extract could bereduced by AG490 blocker. Therefore, Roudoukou-8 San extract possesses protective effects on Na2S2O4 induced-hypoxia/reoxygenation injury of cardiac myocytes through JAK2/STAT3 signaling pathway.