Effect of Salvianolic Acid B Combined with Puerarin on Pyroptosis of SH-SY5Y Cells Injured by Oxygen-glucose Deprivation/Reoxygenation / 中国实验方剂学杂志

ObjectiveTo investigate the effect and mechanism of salvianolic acid B combined with puerarin in protecting the SH-SY5Y cells from the damage by oxygen-glucose deprivation/reoxygenation （OGD/R） based on pyroptosis. MethodSH-SY5Y cells were used to establish the model of OGD/R， and cells were classified into the control， OGD/R， 10 μmol·L-1 salvianolic acid B， 100 μmol·L-1 puerarin， 10 μmol·L-1 salvianolic acid B + 100 μmol·L-1 puerarin， and 10 μmol·L-1 NOD-like receptor protein 3 （NLRP3） inhibitor MCC950 groups. Except the control group， other groups were rapidly reoxygenated for 12 h after 6 h OGD for modeling. The cell survival rate was determined by the methyl thiazolyl tetrazolium （MTT） assay. An optical microscope was used to observe the cell morphology. A spectrophotometer was used to determine the content of lactic dehydrogenase （LDH） in culture supernatant. Cell damage was measured by Hoechst/PI staining. The mRNA levels of NLRP3， cysteinyl aspartate specific proteinase-1 （Caspase-1）， gasdermin D （GSDMD）， apoptosis-associated speck-like protein （ASC）， and interleukin-1β （IL-1β） were determined by real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）. The protein activation of Caspase-1 and NLRP3 was detected by immunofluorescence. Western blot was employed to determine the protein levels of IL-1β， ASC， NLRP3， Caspase-1， and cleaved Caspase-1. ResultCompared with the control group， the OGD/R group showed decreased cell survival rate （P<0.01）， damaged cell morphology， increased leakage rate of LDH （P<0.01）， up-regulated mRNA levels of NLRP3， Caspase-1， GSDMD， ASC， and IL-1β （P<0.01）， and up-regulated protein levels of IL-1β， ASC， NLRP3， Caspase-1， and cleaved Caspase-1 （P<0.01）. Compared with the OGD/R group， salvianolic acid B， puerarin， and salvianolic acid B combined with puerarin improved cell survival rate （P<0.01）， and the combined treatment group outperformed salvianolic acid B and puerarin used alone （P<0.01）. Salvianolic acid B combined with puerarin and MCC950 both improved cell morphology， reduced the leakage of LDH （P<0.01）， alleviated cell damage， and down-regulated the mRNA levels of NLRP3， Caspase-1， GSDMD， ASC， and IL-1β （P<0.05， P<0.01） and also the protein levels of IL-1β， ASC， NLRP3， Caspase-1， and cleaved Caspase-1 （P<0.05， P<0.01）. ConclusionThe results indicated that salvianolic acid B combined with puerarin can alleviate the OGD/R-induced damage of SH-SY5Y cells by inhibiting pyroptosis.

Effect of exogenous hydrogen sulfide on hypoxia/reoxygenation injury in glomerular mesangial cells and its mechanism / 中国药理学通报

Aim To explore the effect of exogenous hydrogen sulfide ( H2 S ) on hypoxia/reoxygenation ( H/R) injury in glomerular mesangial cells and elucidate its relevant mechanism. Methods H/R induced mouse mesangial cell line ( SV40MES13 ) to establish cell damage model. Cell viability was detected by cell proliferation kit ( CCK8 ), the content of H

Research progress on hypoxia/reoxygenation model of nerve cells in vitro / 中国药理学通报

Ischemic stroke is the second leading cause of human death and the third reason of disability. Meanwhile, the incidence is rising year after year worldwide. Ischemic stroke could cause ischemia-reperfusion injury after blood recanalization treat-ment, but the mechanism of ischemia-reperfusion injury is still not very clear, so it is necessary to build a preclinical model with specific characteristics. Up to now, animal experiments have been still complicated, and the culture of brain slices has some limitations. The cell model in vitro has become a simplified and valuable tool widely used by researchers. The paper systematically summarizes the common type of nerve cells, and further analyzes establishment methods and principle, relevant research progress on the in vitro model of ischemia-reperfusion, in order to provide reference for rationally selecting hypoxia and reoxygenation model for basic research on cerebral ischemia and reperfusion and drug screening.

Effect of boschniakia rossica polysaccharides on hypoxia/reoxygenation-induced cardiomyocyte damage by down-regulating miR-302a-3p expression / 中国药理学通报

Aim To explore the effect of boschniakia rossica polysaccharides ( BRPS ) on cardiomyocyte damage induced by hypoxia/reoxygenation (H/R) and its possible mechanism. Methods H/R was used to induce rat cardiomyocyte H9c2 to establish a cell inju¬ry model, and different doses of BRPS were used to treat H9c2 cells. ELISA method was used to detect the level of MDA and the activity of SOD and GSH-Px. Flow cytometry was used to detect the rate of apopto-sis. qRT-PCR was used to detect the expression of miR-302a-3p. anti-miR-NC and anti-miR-302a-3p were respectively transfected into H9c2 cells and then subjected to H/R treatment. miR-NC and miR-302a-3p mimics were respectively transfected into H9c2 cells and treated with 100 mg • L

Effect of different hypoxic and hypobaric interventions on blood gas and erythrocyte-related indicators in rats / 浙江大学学报·医学版

OBJECTIVES@#To explore the effects of hypoxic and hypobaric conditions on blood gas and erythrocyte-related indicators in rats.@*METHODS@#SD male rats were exposed to low-pressure hypoxic conditions simulating an altitude of 6500 m in a small or a large experimental cabin. Abdominal aortic blood samples were collected and blood gas indicators, red blood cells (RBCs) count, and hemoglobin (Hb) content were measured. The effects of exposure to different hypoxia times, different hypoxia modes, normal oxygen recovery after hypoxia, and re-hypoxia after hypoxia preconditioning on blood gas indicators, RBCs count and Hb content were investigated.@*RESULTS@#The effect of blood gas indicators was correlated with the length of exposure time of hypoxia and the reoxygenation after leaving the cabin. Hypoxia caused acid-base imbalance and its severity was associated with the duration of hypoxia; hypoxia also led to an increase in RBCs count and Hb content, and the increase was also related to the time exposed to hypoxia. The effects of reoxygenation on acid-base imbalance in rats caged in a small animal cabin were more severe that those in a large experimental cabin. Acetazolamide alleviated the effects of reoxygenation after leaving the cabin. Different hypoxia modes and administration of acetazolamide had little effect on RBCs count and Hb content. Normal oxygen recovery can alleviate the reoxygenation and acid-base imbalance of hypoxic rats after leaving the cabin and improve the increase in red blood cell and hemoglobin content caused by hypoxia. The improvement of hypoxia preconditioning on post hypoxia reoxygenation is not significant, but it can alleviate the acid-base imbalance caused by hypoxia in rats and to some extent improve the increase in red blood cell and hemoglobin content caused by hypoxia.@*CONCLUSIONS@#Due to excessive ventilation and elevated RBCs count and Hb content after hypoxia reoxygenation, oxygen partial pressure and other oxygenation indicators in hypoxic rats are prone to become abnormal, while blood gas acid-base balance indicators are relatively stable, which are more suitable for evaluating the degree of hypoxia injury and related pharmacological effects in rats.

Effect of CXC chemokine receptor 7 on cell cycle of neuronal cells in ischemic stroke / 中华危重病急救医学

Objective:To investigate the function and mechanism of CXC chemokine receptor 7 (CXCR7) in neuronal cells of ischemic stroke.Methods:The expression of CXCR7 in human neuroblastoma SH-SY5Y cells was interfered by small interfering RNA (si-RNA) technique. Oxygen-glucose deprivation/reoxygenation (OGD/R) injury model was constructed in SH-SY5Y cells. CXCR7 protein expression and cell cycle were detected by flow cytometry (FCM). The protein expression of CXCR7 and Akt signaling pathway was detected by Western blotting.Results:After 6 hours of OGD/R, the expression of CXCR7 was significantly decreased compared with OGD/R 0 hour (CXCR7/GAPDH: 0.483±0.098 vs. 1.000±0.000 by Western blotting and 0.686±0.0524 vs. 1.000±0.000 by FCM, both P < 0.01), cell cycle arrest in G0/G1 phase (1.190±0.040 vs. 1.000±0.000, P < 0.01). After CXCR7 si-RNA interference with SH-SY5Y cells, OGD/R was constructed again for 6 hours. Compared with negative control group (si-NC group) under the same environment, the expression of CXCR7 and phosphorylated Akt (p-Akt) was significantly decreased (CXCR7/GAPDH: 0.471±0.051 vs. 1.000±0.000, p-Akt/GAPDH: 0.616±0.027 vs. 1.000±0.000, both P < 0.001) and cell cycle arrest in G0/G1 phase (1.105±0.033 vs. 1.000±0.000, P < 0.05). Conclusion:The CXCR7 could regulate the cycle of neuronal cells in ischemic stroke through Akt signaling pathway, which has a protective effect on neuronal cells.

Total Saponins of Panax notoginseng Activate Akt/mTOR Pathway and Exhibit Neuroprotection in vitro and in vivo against Ischemic Damage / 中国结合医学杂志

OBJECTIVE@#To reveal the neuroprotective effect and the underlying mechanisms of a mixture of the main components of Panax notoginseng saponins (TSPN) on cerebral ischemia-reperfusion injury and oxygen-glucose deprivation/reoxygenation (OGD/R) of cultured cortical neurons.@*METHODS@#The neuroprotective effect of TSPN was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, flow cytometry and live/dead cell assays. The morphology of dendrites was detected by immunofluorescence. Middle cerebral artery occlusion (MCAO) was developed in rats as a model of cerebral ischemia-reperfusion. The neuroprotective effect of TSPN was evaluated by neurological scoring, tail suspension test, 2,3,5-triphenyltetrazolium chloride (TTC) and Nissl stainings. Western blot analysis, immunohistochemistry and immunofluorescence were used to measure the changes in the Akt/mammalian target of rapamycin (mTOR) signaling pathway.@*RESULTS@#MTT showed that TSPN (50, 25 and 12.5 µ g/mL) protected cortical neurons after OGD/R treatment (P<0.01 or P<0.05). Flow cytometry and live/dead cell assays indicated that 25 µ g/mL TSPN decreased neuronal apoptosis (P<0.05), and immunofluorescence showed that 25 µ g/mL TSPN restored the dendritic morphology of damaged neurons (P<0.05). Moreover, 12.5 µ g/mL TSPN downregulated the expression of Beclin-1, Cleaved-caspase 3 and LC3B-II/LC3B-I, and upregulated the levels of phosphorylated (p)-Akt and p-mTOR (P<0.01 or P<0.05). In the MCAO model, 50 µ g/mL TSPN improved defective neurological behavior and reduced infarct volume (P<0.05). Moreover, the expression of Beclin-1 and LC3B in cerebral ischemic penumbra was downregulated after 50 µ g/mL TSPN treatment, whereas the p-mTOR level was upregulated (P<0.05 or P<0.01).@*CONCLUSION@#TSPN promoted neuronal survival and protected dendrite integrity after OGD/R and had a potential therapeutic effect by alleviating neurological deficits and reversing neuronal loss. TSPN promoted p-mTOR and inhibited Beclin-1 to alleviate ischemic damage, which may be the mechanism that underlies the neuroprotective activity of TSPN.

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.

Protective Effect of Salviae Miltiorrhizae Radix et Rhizoma and Puerariae Lobatae Radix Extract on SH-SY5Y Cells Injured by Oxygen-glucose Deprivation/Reoxygenation / 中国实验方剂学杂志

ObjectiveTo explore the protective effect of Salviae Miltiorrhizae Radix et Rhizoma and Puerariae Lobatae Radix （SP） extract on oxygen-glucose deprivation/reoxygenation （OGD/R）-injured SH-SY5Y cells based on oxidative stress and apoptosis. MethodThe extracts of the two medicinal materials mixed in different ratios were prepared. Human neuroblastoma SH-SY5Y cells were cultured in vitro and the injury was induced by OGD/R. Cell counting kit-8 （CCK-8） assay was used to screen the optimal ratio of the two medicinals and then the extract was used for further experiment. SH-SY5Y cells were classified into normal control group， OGD/R group， and low-， medium-， and high-dose SP （2∶1） extract groups （10， 30， 100 mg·L-1， respectively）. Cells in the groups， except the normal control group， were rapidly reoxygenated for 12 h after 4 h OGD for modeling. Then cell viability was detected by CCK-8 and cell morphology was observed under the microscope. The release rate of lactate dehydrogenase （LDH）， superoxide dismutase （SOD） activity， and content of glutathione （GSH） and malondialdehyde （MDA） were determined by spectrophotometry. The level of reactive oxygen species （ROS） was detected with 2，7-dichlorodihydrofluorescein diacetate （DCFH-DA） and mitochondrial membrane potential with JC-1 assay. The nuclear morphology was observed based on Hoechst 33342 staining， and apoptosis was examined by flow cytometry combined with Annexin V-FITC/PI staining. ResultThe viability of the cells was highest in the presence of the extract of the two medicinals mixed at the ratio of 2∶1. Compared with normal control group， OGD/R group showed damaged cell morphology， high release rate of LDH and levels of ROS and MDA （P<0.01）， low SOD activity and GSH level （P<0.01）， low mitochondrial membrane potential， and high apoptosis rate （P<0.01）. Compared with OGD/R group， SP extract improved cell viability and cell morphology and reduce cell LDH release rate in a concentration-dependent manner （P<0.01）. In addition， SP extract at 30， 100 mg·L-1 reduced the level of intracellular ROS and increased SOD activity and GSH level （P<0.05， P<0.01）， and SP extract at 100 mg·L-1 decreased the content of MDA （P <0.05）. Moreover， SP extract increased mitochondrial membrane potential， and SP extract at 30， 100 mg·L-1 lowered the apoptosis rate （P<0.01）. ConclusionThe extract of Salvia miltiorrhiza Bunge and Radix Puerariae mixed at 2∶1 shows better protective effect on OGD/R-injured SH-SY5Y cells. The mechanism is the likelihood that it alleviates oxidative damage of cells and inhibits cell apoptosis.

Protective Effect of Salviae Miltiorrhizae Radix et Rhizoma and Puerariae Lobatae Radix Extract on SH-SY5Y Cells Injured by Oxygen-glucose Deprivation/Reoxygenation / 中国实验方剂学杂志

ObjectiveTo explore the protective effect of Salviae Miltiorrhizae Radix et Rhizoma and Puerariae Lobatae Radix （SP） extract on oxygen-glucose deprivation/reoxygenation （OGD/R）-injured SH-SY5Y cells based on oxidative stress and apoptosis. MethodThe extracts of the two medicinal materials mixed in different ratios were prepared. Human neuroblastoma SH-SY5Y cells were cultured in vitro and the injury was induced by OGD/R. Cell counting kit-8 （CCK-8） assay was used to screen the optimal ratio of the two medicinals and then the extract was used for further experiment. SH-SY5Y cells were classified into normal control group， OGD/R group， and low-， medium-， and high-dose SP （2∶1） extract groups （10， 30， 100 mg·L-1， respectively）. Cells in the groups， except the normal control group， were rapidly reoxygenated for 12 h after 4 h OGD for modeling. Then cell viability was detected by CCK-8 and cell morphology was observed under the microscope. The release rate of lactate dehydrogenase （LDH）， superoxide dismutase （SOD） activity， and content of glutathione （GSH） and malondialdehyde （MDA） were determined by spectrophotometry. The level of reactive oxygen species （ROS） was detected with 2，7-dichlorodihydrofluorescein diacetate （DCFH-DA） and mitochondrial membrane potential with JC-1 assay. The nuclear morphology was observed based on Hoechst 33342 staining， and apoptosis was examined by flow cytometry combined with Annexin V-FITC/PI staining. ResultThe viability of the cells was highest in the presence of the extract of the two medicinals mixed at the ratio of 2∶1. Compared with normal control group， OGD/R group showed damaged cell morphology， high release rate of LDH and levels of ROS and MDA （P<0.01）， low SOD activity and GSH level （P<0.01）， low mitochondrial membrane potential， and high apoptosis rate （P<0.01）. Compared with OGD/R group， SP extract improved cell viability and cell morphology and reduce cell LDH release rate in a concentration-dependent manner （P<0.01）. In addition， SP extract at 30， 100 mg·L-1 reduced the level of intracellular ROS and increased SOD activity and GSH level （P<0.05， P<0.01）， and SP extract at 100 mg·L-1 decreased the content of MDA （P <0.05）. Moreover， SP extract increased mitochondrial membrane potential， and SP extract at 30， 100 mg·L-1 lowered the apoptosis rate （P<0.01）. ConclusionThe extract of Salvia miltiorrhiza Bunge and Radix Puerariae mixed at 2∶1 shows better protective effect on OGD/R-injured SH-SY5Y cells. The mechanism is the likelihood that it alleviates oxidative damage of cells and inhibits cell apoptosis.

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.

Sequencing Analysis of MiRNA in the Exosomes from Normal and Oxygen and Glucose Deprivation Reperfusion Astrocytes / 中国生物化学与分子生物学报

In this study, high-throughput sequencing technology was used to analyze the differentiallyexpressed microRNA (miRNA) of astrocyte-derived exosomes in control group and oxygen and glucosedeprivation/ reoxygenation (OGD/ R) group. Ultracentrifugation was used to extract exosomes from thesupernatant of astrocyte medium in the control group and OGD/ R group. Transmission electron microscopyshowed that exosomes had a typical vesicle shape with intact membrane and low electron content density. Nanoparticle tracking technology (NTA) detected astrocyte exosomes with a size of 100. 5 ± 31. 1 nm, accounting for 96. 8%. Western blot detection showed that the exosome contained exosome-specificproteins tumor-susceptibility protein (TSG101), heat shock protein 60 (Hsp60), ALG-2-interactingprotein X (ALIX). Compared with the control group, 41 miRNAs in the exosomes of the OGD / R groupwere significantly changed, of which 20 miRNAs were increased and 21 miRNAs were decreasedsignificantly (P < 0. 05). Gene ontology function (GO) analysis showed that significantly differentiallytarget genes were mainly involved in protein glycosylation, lipid metabolism, phosphorylation, Golgiapparatus, endoplasmic reticulum, endosome, cytoplasmic vesicles and cell protrusions, etc. KyotoEncyclopedia of Genes and Genomes (KEGG) pathway analysis found that differential miRNAs weremainly related to metabolic pathways and signaling pathways such as butyrate metabolism, ß-alaninemetabolism, fatty acid degradation, mitophagy and P53 signaling pathway. Sequencing analysis of theexosomal miRNAs derived from control and OGD / R astrocytes and target gene function enrichmentanalysis can be useful for the mechanism study of astrocyte exosomes in response to oxygen and glucosedeprivation reperfusion.

Effects of levosimendan on the hypoxic / reoxygenated cardiomyocyte fibrosis by regulating long chain noncoding RNA / microRNA-641 / 解剖学报

[Abstract] Objective To investigate whether levosimendan (Lev) affects hypoxia / reoxygenation (H / R) - induced cardiomyocyte proliferation, apoptosis and fibrosis by regulating the molecular axis of long chain noncoding RNA (LncRNA) eosinophil granule ontogeny transcript (EGOT) / microRNA (miR) -641. Methods Rat cardiomyocytes H9C2 were cultured in vitro, and H / R-treated cells were used to establish cell damage models, which were randomly divided into control group, H / R group, H / R + Lev 1 μmol / L (H / R + Lev-L) group, H / R + Lev 5 μmol / L (H / R + Lev-M) group, and H / R + Lev 10 μmol / L (H / R + Lev-H) group, 9 samples per group. MTT method was used to detect cell proliferation. Flow cytometry was used to detect the apoptosis rate. Real-time P CR was used to detect the expression levels of EGOT and miR-641 mRNA. P cDNA-EGOT and EGOT small interfering RNA (si-EGOT) were transfected into H9 C2 cells respectively, and the cell proliferation and apoptosis rates were detected by the above method. The dual luciferase report experiment verified the targeting relationship between EGOT and miR-641. Western blotting was used to detect the expression levels of Bax, Bcl-2, collagen I (colI), collagen Ⅲ (col Ⅲ), tissue inhibitor of matrix metalloproteinase 2 (TIMP 2), matrix metalloproteinase-2 (MMP -2) . Results Compared with the control group, the cell survival rate of the H / R group reduced significantly (P < 0. 05), the apoptosis rate increased significantly (P < 0. 05), and the protein levels of Bax, c I, col Ⅲ, TIMP 2, and MMP -2 increased significantly (P < 0. 05), the level of Bcl-2 protein reduced significantly (P < 0. 05), the expression level of EGOT reduced significantly (P < 0. 05), the expression level of miR-641 increased significantly (P < 0. 05) . Compared with the H / R group, the cell survival rate of the H / R + Lev-L group, H / R + Lev-M group, and H / R + Lev-H group increased significantly (P < 0. 05), and the apoptosis rate decreased significant (P < 0. 05), the protein levels of Bax, colI, colⅢ, TIMP 2, MMP -2 reduced significantly (P < 0. 05), the level of Bcl-2 protein increased significantly (P < 0. 05), the expression level of EGOT increased significantly (P < 0. 05), the expression level of miR-641 reduced significantly (P < 0. 05), and each index of H / R + Lev-L group, H / R + Lev-M group, H / R + Lev-H group, the difference was statistically significant (P < 0. 05) . The dual luciferase report experiment confirmed that EGOT ccould target and bind to miR-641. The effect of transfecting pcDNA-EGOT and Lev was similar. Transfection of si-EGOT could reduce the effect of Lev on H / R-induced proliferation, apoptosis and fibrosis of H9 C2 cells. Conclusion Levosimendan may promote H / R-induced H9 C2 cell proliferation and inhibit apoptosis and fibrosis by up-regulating EGOT expression and down-regulating miR-641 expression.

Effect of long non-coding RNA nuclear-enriched abundant transcript on hypoxia-reoxygenation rat astrocyte injury by targeting microRNA-761 / 解剖学报

Objective To investigate the effect of long non-coding RNA (IncRNA) nuclear-enriched abundant transcript 1 (NEATl) on hypoxia-reoxygenation (H/R) glial astrocyte injury, and to explore whether the mechanism was related to the regulation of micro RNA (miR)-761. Methods Rat cortical astrocytes were cultured to construct a H/R injury model. Astrocytes were divided into control group, model group, model+ small interfering RNA negative control (si-NC) group, model+ si-NEATl group, model+ miR-NC group, model + miR-761 group, model + si-NEATl + anti-miR-NC group, model+si-NEATl+anti-miR-761 group. Expression of NEATl and miR-761 were detected by Real-time PCR. The experiment was repeated 3 times. The content of malonaldefryde (MDA), and the activity of superoxide dismutase (SOD) and catalase (CAT) were detected by kits. Dual luciferase reporter experiment and Real-time PCR were used to analyze the targeting relationship between NEATl and miR-761. The experiment was repeated 3 times. Results Compared with the control group, the cell apoptosis rate and MDA content of the model group increased significantly, SOD and CAT activities decreased significantly, NEATl expression increased significantly, and miR-761 expression decreased significantly (P< 0. 05). Compared with the model+si-NC group, the apoptosis rate and MDA content of the model+si-NEATl group reduced significantly, and SOD and CAT activities increased significantly (P < 0 . 0 5) . Compared with the model + miR-NC group, the apoptosis rate and MDA content of the model + miR-761 group reduced significantly, and SOD and CAT activities increased significantly (P < 0 . 0 5) . MiR-761 was the target gene of N E A T l, and NEATl negatively regulated miR-761 expression. Compared with the model+si-NEATl+anti-miR-NC group, the apoptosis rate and MDA content of the model+siNEAT1+anti-miR-761 group increased significantly, and SOD and CAT activities decreased significantly (P < 0 . 0 5) . Conclusion Interference with NEATl expression can protect astrocytes from H / R injury by up-regulating miR-761.

Dexmedetomidine attenuates ferroptosis through regulation of Nrf2 on HT22 cells with hypoxia/reoxyge nation / 中国药理学通报

Aim To investigate the effects of Dexmedetomidine(DEX)on HT22 cells with hypoxia/reoxygenation based on ferroptosis and the underlying mechanism.Methods HT22 cells were used to prepare H/R injury model.In order to investigate the optimal concentration of DEX, cells were divided into five groups: control group, H/R group, low concentration(H/R+DEX2.5, 2.5 μmol·L-1), medium concentration(H/R+DEX5, 5 μmol·L-1), high concentration(H/R+DEX10, 10 μmol·L-1)DEX intervention H/R group, and the survival rates of cells were detected by MTT assay.To investigate the mechanism of the protective effects on HT22 cells with H/R injury, HT22 cells were divided into four groups: control group, H/R group, H/R+DEX5 group, and H/R+DEX5 +ML385 group.The survival rates of cells were detected by MTT assay; the levels of Fe2+ were detected by FerroOrange fluorescent probe; the C11BODIPY581/591 was used to detect the change of lipid ROS on the cells; MDA and reduced glutathione kits were used to detect the content of MDA and GSH of cells respectively.The expressions of Nrf2, GPX4, TFR1 and SLC7A11 were detected by Western blot.Results Compared with control group, the survival rate of cells, the content of GSH, and the expression of Nrf2, GPX4 and SLC7A11 all significantly decreased(all P<0.05), the level of lipid ROS, the content of MDA, the level of Fe2+, and the expression of TFR1 all significantly increased in H/R group(all P<0.01).Compared with H/R group, the survival rate of cells, the content of GSH, and the expression of Nrf2, GPX4 and SLC7A11 significantly increased(all P<0.05), the level of lipid ROS, the content of MDA, the level of Fe2+, and the expression of TFR1 significantly decreased with the treatment of DEX(all P<0.05).Compared with H/R+DEX group, the survival rate of cells, the content of GSH, and the expression of Nrf2, GPX4 and SLC7A11 markedly decreased(all P<0.05), the lipid ROS, MDA and Fe2+, and the expression of TFR1 significantly increased in H/R+DEX+ML385 group(all P<0.05).Conclusions DEX can reduce H/R injury on HT22 cells by inhibiting ferroptosis, and the mechanism might be related to the promotive expression of Nrf2.

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.

LncRNA MALAT1 aggravates oxygen-glucose deprivation/reoxygenation-induced neuronal endoplasmic reticulum stress and apoptosis via the miR-195a-5p/HMGA1 axis

BACKGROUND: This study aimed to investigate the potential role and molecular mechanism of lncRNA metastasis associated lung adenocarcinoma transcript 1 (MALAT1) in cerebral ischemia/reperfusion injury. RESULTS: Using an oxygen-glucose deprivation/reoxygenation (OGD/R) cell model, we determined that the expression of MALAT1 was significantly increased during OGD/R. MALAT1 knockdown reversed OGD/R-induced apoptosis and ER stress. Mechanistically, MALAT1 promoted OGD/R-induced neuronal injury through sponging miR-195a-5p to upregulating high mobility group AT-hook1 (HMGA1). CONCLUSIONS: Collectively, these data demonstrate the mechanism underlying the invovlvement of MALAT1 in cerebral ischemia/reperfusion injury, thus providing translational evidence that MALAT1 may serve as a novel biomarker and therapeutic target for ischemic stroke.

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.

Buyang Huanwutang Promoted Proliferation and Differentiation of Neural Stem Cells via Regulating Autophagy Following Oxygen-glucose Deprivation/Reoxygenation Injury / 中国实验方剂学杂志

Objective:To investigate the effect of Buyang Huanwutang （BHT） on proliferation and differentiation in neural stem cells （NSCs） after oxygen-glucose deprivation/reoxygenation （OGD/R） injury. Method:NSCs isolated from the hippocampus of SD rats were cultured and randomly divided into a normoxia group， a model group， a BHT group， a rapamycin （Rapa） group， and a combination group ［autophagy inhibitor 3-methyladenine （3-MA） combined with BHT］. The 20% blank serum was used in the normoxia group， and 20% BHT-medicated serum in the BHT group. The doses of Rapa and 3-MA were 1 μmol·L^-1 and 5 mmol·L^-1， respectively. The cells were subjected to OGD/R except those in the normoxia group. The cell morphology was observed under a light microscope. NSCs were confirmed by immunofluorescence detection of nestin expression. The viability and proliferation of NSCs were assessed by cell counting kit-8 （CCK-8） assay and 5-ethynyl-2-deoxyuridine （EdU） labeling， respectively. Furthermore， Ad-mCherry-GFP-LC3B fluorescence assay was performed to investigate autophagy. The effect of BHT on autophagy-related protein expression was detected by western blot assay. Brain derived neurotrophic factor （BDNF）， <italic>β</italic>-tubulin Ⅲ， and glial fibrillary acidic protein （GFAP） were evaluated by immunofluorescence assay. Result:OGD/R significantly reduced the cell viability of rat NSCs as compared with the normoxia group. Compared with the model group， the BHT group exhibited significantly improved viability of rat NSCs （<italic>P</italic><0.01）. BHT induced the production of autophagosomes in NSCs after OGD. The BHT group showed increased expression of microtuble-associated protein 1 light chain 3Ⅱ (LC3Ⅱ） and Beclin-1 （<italic>P</italic><0.05，<italic>P</italic><0.01） and slightly changed p62 compared with the normoxia group， and significantly up-regulated LC3Ⅱ and Beclin-1 （<italic>P</italic><0.05，<italic>P</italic><0.01） and down-regulated expression of p62 （<italic>P</italic><0.01） compared with the model group. The Rapa group had similar effect as the BHT group （<italic>P</italic><0.05，<italic>P</italic><0.01）， while the combination group inhibited the activity of autophagy （<italic>P</italic><0.01）. As indicated by the results of ad-mCherry-GFP-LC3B， compared with the normoxia group， the model group showed increased fluorescence intensity （<italic>P</italic><0.01）， and the BHT and Rapa groups could further increased the fluorescence intensity of autophagy （<italic>P</italic><0.01）， while the combination group inhibited autophagy activity （<italic>P</italic><0.01）. Immunofluorescence results revealed that compared with the normoxia group， the model group displayed significantly reduced positive cells of EdU， <italic>β</italic>-tubulin Ⅲ， GFAP， and BDNF （<italic>P</italic><0.01）， and the BHT and Rapa groups exerted similar protective and promoting effects （<italic>P</italic><0.05，<italic>P</italic><0.01）， while the combination group partially blocked the neuroprotection and differentiation ability of BHT （<italic>P</italic><0.05）. Conclusion:BHT pretreatment can effectively protect rat NSCs against OGD-induced injury and promoted proliferation and differentiation by up-regulating autophagy.