IcarisideⅡ alleviates oxygen-glucose deprivation and reoxygenation-induced PC12 celloxidative injury by activating Nrf2 / SIRT3signaling pathway

OBJECTIVE To investigate icariside(ICS)Ⅱ protects against PC12 cel damage induced by oxygen-glucose deprivation and reoxygenation and explore its mechanism.METHODS The oxidative stress injury model was induced by oxygen-glucose deprivation/reoxygenation(OGD/R) 2 h/24 h in PC12 cells.N-acetyl-lcysteine(NAC),a classical anti-oxidant,was used as positive control.Pharmacodynamic experimental study groups as follows:control,control+ICS Ⅱ50 μmol·L^(-1),OGD/R,OGD/R+ICSⅡ 12.5 μmol·L^(-1),OGD/R + ICS Ⅱ 25 μmol·L^(-1),OGD/R + ICS Ⅱ50 μmol·L^(-1),and OGD/R+NAC 100 μmol·L^(-1) groups.Cell viability and lactate dehydrogenase(LDH) leakage rate were measured by MTT assay and LDH ELISA kit,respectively.Moreover,reactive oxygen species(ROS) ELISA kit was used for detection of intracellular ROS generation,Mito-SOX fluorescence staining was used for detecting production of ROS in mitochondria and mitochondrial membrane potential(MMP)was detected by rhodamine 123 dye.In addition,PC12 cells apoptosis was detected by one-step TUNEL assay.Furthermore,the expressions of nuclear factor erythroid 2-related factors(Nrf2),Keap1,HO^(-1),NQO^(-1),silent information regulator 3(SIRT3),IDH2,Bax,Bcl-2 and caspase 3 were detected by Western blotting analysis.RESULTS The results of MTT and LDH assay showed that OGD/R reduced the cell viability and improved LDH release compared with the control or ICSⅡ 50 μmol·L^(-1) alone(P<0.01).Meanwhile,OGD/R not only increased intracellular and mitochondrial ROS generation,but also elevated the fluorescence intensity of TUNEL staining,at the same time,the MMP was declined when challenged by OGD/R.Furthermore,the Western blotting results showed that OGD/R induced the increase in the expression of cytoplasm-Nrf2,Keap1,Bax and cleaved-caspase 3 level,while the decrease in the expression of nucleus-Nrf2,HO^(-1),NQO^(-1),SIRT3,IDH2 and Bcl-2(P<0.05).However,ICS Ⅱ significantly increased the viability of PC12 cells and reduced LDH leakage(P<0.01).Notably,ICS Ⅱ also suppressed ROS generation both in the intracellular and mitochondria,as well as restored MMP.It was also worthy to note that ICS Ⅱ decreased the expressions of cytoplasmNrf2,Keap1,Bax and the level of cleaved-caspase3,whereas,it increased the expressions of nucleus-Nrf2,HO^(-1),NQO^(-1),SIRT3,IDH2 and Bcl-2(P<0.05).CONCLUSION ICSⅡ reduced OGD/Rinduced oxidative damage in PC12 cells under the laboratory conditions,and its underlying mechanism may be related to the regulation of Nrf2/SIRT3 signaling pathway.

Neuroprotective effects of neural stem cells pretreated with neuregulin1β on PC12 cells exposed to oxygen-glucose deprivation/reoxygenation

Studies on ischemia/reperfusion(I/R)injury suggest that exogenous neural stem cells(NSCs)are ideal candidates for stem cell therapy reperfusion injury.However,NSCs are difficult to obtain owing to ethical limitations.In addition,the survival,differentiation,and proliferation rates of transplanted exogenous NSCs are low,which limit their clinical application.Our previous study showed that neuregulin1β(NRG1β)alleviated cerebral I/R injury in rats.In this study,we aimed to induce human umbilical cord mesenchymal stem cells into NSCs and investigate the improvement effect and mechanism of NSCs pretreated with 10 nM NRG1βon PC12 cells injured by oxygen-glucose deprivation/reoxygenation(OGD/R).Our results found that 5 and 10 nM NRG1βpromoted the generation and proliferation of NSCs.Co-culture of NSCs and PC12 cells under condition of OGD/R showed that pretreatment of NSCs with NRG1βimproved the level of reactive oxygen species,malondialdehyde,glutathione,superoxide dismutase,nicotinamide adenine dinucleotide phosphate,and nuclear factor erythroid 2-related factor 2(Nrf2)and mitochondrial damage in injured PC12 cells;these indexes are related to ferroptosis.Research has reported that p53 and solute carrier family 7 member 11(SLC7A11)play vital roles in ferroptosis caused by cerebral I/R injury.Our data show that the expression of p53 was increased and the level of glutathione peroxidase 4(GPX4)was decreased after RNA interference-mediated knockdown of SLC7A11 in PC12 cells,but this change was alleviated after co-culturing NSCs with damaged PC12 cells.These findings suggest that NSCs pretreated with NRG1βexhibited neuroprotective effects on PC12 cells subjected to OGD/R through influencing the level of ferroptosis regulated by p53/SLC7A11/GPX4 pathway.

Protective effect of icarisideⅡ on oxygen-glucose deprivation and reoxygenation-induced injury incerebral cortical neurons

OBJECTIVE To explore the effect of icariside Ⅱ(ICS Ⅱ) on oxygen-glucose deprivation and reoxygenation(OGD/R)-induced injury in cerebral cortical neuronal cels.METHODS Primary cerebral cortical neuronal cells were deprived of oxygen and glucose for 2 h to simulate ischemic stroke injury in vitro.The experiment was divided into 8 groups,which were control,control+ICSⅡ 25 μmol·L^(-1),OGD/R,OGD/R+ICSⅡ(6.25,12.5,25 μmol·L^(-1)),OGD/R+3-methyladenine(3-MA) and OGD/R+Rapamycin(Rap).The protective effect of ICS Ⅱ were detected by MTT assay and lactate dehydrogenase(LDH),respectively.Autophagic flux and autophagy related proteins expressions were detected by using adenovirus harboring tf-LC3 and Western blotting,respectively.RESULTS Compared with OGD/R group,the cell viability treated with ICSⅡwas elevated in a concentration-dependent manner,and the leakage rate of LDH was lowed.Moreover,ICSⅡ not only suppressed OGD/R-induced autophagic flux,but also inhibited the increase of LC3-Ⅱ/LC3-Ⅰ ratio and Beclin 1 after OGD/R insulted.CONCLUSION ICS Ⅱ exerts protective effects on OGD/R-induced cerebral cortical neuronal cells through inhibiting excessive autophagy.

Protective effect of ginsenoside Rg1 on 661W cells exposed to oxygen-glucose deprivation/reperfusion via keap1/nrf2 pathway

AIM:To construct an in vitro model of oxygen-glucose deprivation/reperfusion(OGD/R)induced injury to the optic nerve and to study the oxidative damage mechanism of ischemia-reperfusion(I/R)injury in 661W cells and the protective effect of ginsenoside Rg1.METHODS:The 661W cells were treated with different concentrations of Na2S2O4 to establish OGD/R model in vitro.Apoptosis,intracellular reactive oxygen species(ROS)levels and superoxide dismutase(SOD)levels were measured at different time points during the reperfusion injury process.The injury model was pretreated with graded concentrations of ginsenoside Rg1.Real-time polymerase chain reaction(PCR)was used to measure the expression levels of cytochrome C(cyt C)/B-cell lymphoma-2(Bcl2)/Bcl2 associated protein X(Bax),heme oxygenase-1(HO-1),caspase9,nuclear factor erythroid 2-related factor 2(nrf2),kelch-like ECH-associated protein 1(keap1)and other genes.Western blot was used to detect the expression of nrf2,phosphorylated nrf2(pnrf2)and keap1 protein levels.RESULTS:Compared to the untreated group,the cell activity of 661W cells treated with Na2S2O4 for 6 and 8h decreased(P<0.01).Additionally,the ROS content increased and SOD levels decreased significantly(P<0.01).In contrast,treatment with ginsenoside Rg1 reversed the cell viability and SOD levels in comparison to the Na_(2)S_(2)O_(4)treated group(P<0.01).Moreover,Rg1 reduced the levels of caspase3,caspase9,and cyt C,while increasing the Bcl2/Bax level.These differences were all statistically significant(P<0.05).Western blot analysis showed no significant difference in the protein expression levels of keap1 and nrf2 with Rg1 treatment,however,Rg1 significantly increased the ratio of pnrf2/nrf2 protein expression compared to the Na_(2)S_(2)O_(4)treated group(P<0.001).CONCLUSION:The OGD/R process is induced in 661W cells using Na_(2)S_(2)O_(4).Rg1 inhibits OGD/R-induced oxidative damage and alleviates the extent of apoptosis in 661W cells through the keap1/nrf2 pathway.These results suggest a potential protective effect of Rg1 against retinal I/R injury.

Low-Dose Ethanol Preconditioning Protects Against Oxygen-Glucose Deprivation/Reoxygenation-Induced Neuronal Injury By Activating Large Conductance,Ca2+-Activated K+Channels In Vitro

Increasing evidence suggests that low to mod- erate ethanol ingestion protects against the deleterious effects of subsequent ischemia/reperfusion; however, the underlying mechanism has not been elucidated. In the present study, we showed that expression of the neuronal large-conductance, Ca2＋-activated K＋ channel （BKca） α- subunit was upregulated in cultured neurons exposed to oxygen-glucose deprivation/reoxygenation （OGD/R） compared with controls. Preconditioning with low-dose ethanol （10 mmol/L） increased cell survival rate in neurons subjected to OGD/R, attenuated the OGD/R-induced elevation of cytosolic Ca2＋ levels, and reduced the number of apoptotic neurons. Western blots revealed that ethanol preconditioning upregulated expression of the anti-apoptotic protein Bcl-2 and downregulated the pro-apoptotic protein Bax. The protective effect of ethanol precondi- tioning was antagonized by a BKα channel inhibitor, paxilline. Inside-out patches in primary neurons also demonstrated the direct activation of the BKCa channel by 10 mmol/L ethanol. The above results indicated that low- dose ethanol preconditioning exerts its neuroprotective effects by attenuating the elevation of cytosolic Ca2＋ and preventing neuronal apoptosis, and this is mediated by BKca channel activation.

Tilianin extracted from Xiangqinglan(Herba Dracocephali Moldovicae)inhibits apoptosis induced by mitochondrial pathway and endoplasmic reticulum stress in H9c2 cells after oxygen-glucose deprivation/reoxygenation

OBJECTIVE:To investigate the efficacy of tilianin extracted from Xiangqinglan(Herba Dracocephali Moldovicae)on apoptosis of H9c2 cell after oxygenglucose deprivation/reoxygenation(OGD/R)and the mechanism.METHODS:Tilianin was obtained from Beijing Inluck Science and Technology Development Co.Ltd.,with purity≥98%.The OGD/R model was established in H9c2 cells.Flow cytometry detected the mitochondrial membrane potential,apoptosis rates,mitochondrial reactive oxygen species(ROS)and calcium ion concentration.Succinate dehydrogenase(SDH)activity,succinate content and levels of tumor necrosis factor-α(TNF-α),interleukin-6(IL-6)and interleukin-1β(IL-1β)were detected with enzyme-linked immunosorbent assay.Western blot measured protein levels.RESULTS:Tilianin significantly reduced the apoptotic rates,ROS levels,calcium ion concentration,succinate content,and,levels of TNF-α,IL-6 and IL-1βof OGD/R cells,while significantly increased the membrane potential and SDH activity in mitochondria.Western blot analysis showed that tilianin significantly up-regulated pCalmodulin-dependent protein kinaseⅡand voltagedependent anion selective channel levels in OGD/R cells,while significantly down-regulated p-protein kinase B,Bcl-2-associated X,and dynamin-related protein 1 levels related to apoptosis in the mitochondrial pathway.Moreover,tilianin significantly up-regulated B-cell lymphoma-2 and mitochondrial protein 2 related to the inhibition of apoptosis.Furthermore,tilianin downregulated phosphorylated-apoptosis signal-regulated kinase 1,phosphorylated-p38 and C/EBP homologous protein related to endoplasmic reticulum stress.CONCLUSIONS:Tilianin may inhibit OGD/R-induced H9c2 cell apoptosis mediated by mitochondrial pathway and endoplasmic reticulum stress,thus protecting cardiomyocytes.

基于pgrmc1调控的黄体酮抑制氧糖剥夺/复氧新生小鼠神经元损伤机制分析

目的 基于黄体酮膜受体组件1(pgrmc1)调控,探讨黄体酮抑制氧糖剥夺/复氧(OGD/R)新生小鼠神经元损伤的作用机制。方法 选用出生12 h内的新生小鼠分离原代皮层神经元细胞,体外培养7 d,利用微管相关蛋白2检测进行鉴定后,随机分为对照组、二甲基亚砜(DMSO)组、AG205组、黄体酮组、AG205+黄体酮组。DMSO组、AG205组、黄体酮组、AG205+黄体酮组加入制备好的缺糖D-Hanks液、置于缺氧培养箱中培养2 h,换为神经元生长培养基;DMSO组在造模前1 h给予DMSO预处理,AG205组和AG205+黄体酮组在造模前1 h给予AG205(pgrmc1拮抗剂)10μmol/L预处理,黄体酮组和AG205+黄体酮组于造模后2 h给予黄体酮20μmol/L。复氧24 h后,采用CCK-8法检测细胞活力,流式细胞术检测凋亡细胞。结果 DMSO组、AG205组细胞存活率低于对照组,AG205组低于DMSO组;黄体酮组、AG205+黄体酮组细胞存活率高于AG205组,黄体酮组高于AG205+黄体酮组(P均<0.05)。DMSO组、AG205组细胞凋亡率高于对照组,AG205组高于DMSO组,黄体酮组、AG205+黄体酮组细胞凋亡率低于AG205组(P均<0.05)。结论 黄体酮可抑制OGD/R新生小鼠神经元损伤,抑制pgrmc1可降低OGD/R神经元活力、增加细胞凋亡,黄体酮抑制OGD/R神经元损伤的作用可能与调控pgrmc1有关。

体外培养大鼠海马神经元糖氧剥夺/复氧轴突损伤模型的建立

目的建立体外培养大鼠海马神经元糖氧剥夺/复氧轴突损伤模型。方法取体外培养7d的海马神经元,分为正常对照组和糖氧剥夺/复氧组。后者分别在糖氧剥夺0.5、1.0、1.5h再复糖复氧。常氧状态下检测各组复氧后1、5、24、48、72h培养液中的LDH活性,并观察神经元形态和轴突长度的变化。结果糖氧剥夺/复氧后的神经元折光性降低,胞体肿胀,并且轴突逐渐变短,同时LDH水平随时间延长增加。其中糖氧剥夺0.5h再复氧组效果最佳,其轴突缩短明显且神经元存活率较高。结论成功建立了体外培养SD大鼠海马神经元糖氧剥夺/复氧轴突损伤模型。

Activated Drp1 regulates p62-mediated autophagic flux and aggravates inflammation in cerebral ischemia-reperfusion via the ROS-RIP1/RIP3-exosome axis

Background: Cerebral ischemia-reperfusion injury(CIRI) refers to a secondary brain injury that can occur when the blood supply to the ischemic brain tissue is restored. However, the mechanism underlying such injury remains elusive.Methods: The 150 male C57 mice underwent middle cerebral artery occlusion(MCAO) for 1 h and reperfusion for 24 h,Among them, 50 MCAO mice were further treated with Mitochondrial division inhibitor 1(Mdivi-1) and 50 MCAO mice were further treated with N-acetylcysteine(NAC). SH-SY5Y cells were cultured in a low-glucose culture medium for 4 h under hypoxic conditions and then transferred to normal conditions for 12 h. Then, cerebral blood flow, mitochondrial structure, mitochondrial DNA(mtDNA) copy number, intracellular and mitochondrial reactive oxygen species(ROS),autophagic flux, aggresome and exosome expression profiles, cardiac tissue structure, mitochondrial length and cristae density, mtDNA and ROS content, as well as the expression of Drp1-Ser616/Drp1, RIP1/RIP3, LC3 II/I, TNF-α,IL-1β, etc., were detected under normal or Drp1 interference conditions.Results: The mtDNA content, ROS levels, and Drp1-Ser616/Drp1 were elevated by 2.2, 1.7 and 2.7 times after CIRI(P<0.05). However, the high cytoplasmic LC3 II/I ratio and increased aggregation of p62 could be reversed by 44%and 88% by Drp1 short hairpin RNA(shRNA)(P<0.05). The low fluorescence intensity of autophagic flux and the increased phosphorylation of RIP3 induced by CIRI could be attenuated by ROS scavenger, NAC(P<0.05). RIP1/RIP3inhibitor Necrostatin-1(Nec-1) restored 75% to a low LC3 II/I ratio and enhanced 2 times to a high RFP-LC3 after Drp1 activation(P<0.05). In addition, although CIRI-induced ROS production caused no considerable accumulation of autophagosomes(P>0.05), it increased the packaging and extracellular secretion of exosomes containing p62 by 4–5 times, which could be decreased by Mdivi-1, Drp1 shRNA, and Nec-1(P<0.05). Furthermore, TNF-α and IL-1βincreased in CIRI-derived exosomes could increase RIP3 phosphorylation in normal or oxygen–glucose deprivation/reoxygenation(OGD/R) conditions(P<0.05).Conclusions: CIRI activated Drp1 and accelerated the p62-mediated formation of autophagosomes while inhibiting the transition of autophagosomes to autolysosomes via the RIP1/RIP3 pathway activation. Undegraded autophagosomes were secreted extracellularly in the form of exosomes, leading to inflammatory cascades that further damaged mitochondria, resulting in excessive ROS generation and the blockage of autophagosome degradation,triggering a vicious cycle.

LncRNA SNHG12 ameliorates brain microvascular endothelial cell injury by targeting miR-199a

Long non-coding RNAs regulate brain microvascular endothelial cell death, the inflammatory response and angiogenesis during and after ischemia/reperfusion and oxygen-glucose deprivation/reoxygenation（OGD/R） insults. The long non-coding RNA, SNHG12, is upregulated after ischemia/reperfusion and OGD/R in microvascular endothelial cells of the mouse brain. However, its role in ischemic stroke has not been studied. We hypothesized that SNHG12 positively regulates ischemic stroke, and therefore we investigated its mechanism of action. We established an OGD/R mouse cell model to mimic ischemic stroke by exposing brain microvascular endothelial cells to OGD for 0, 2, 4, 8, 16 or 24 hours and reoxygenation for 4 hours. Quantitative real-time polymerase chain reaction showed that SNHG12 levels in brain microvascular endothelial cells increased with respect to OGD exposure time. Brain microvascular endothelial cells were transfected with pc DNA-control, pc DNA-SNHG12, si-control, or si-SNHG12. After exposure to OGD for 16 hours, these cells were then analyzed by 3-（4,5-dimethyl-2-thiazolyl）-2,5-diphenyl-2-H-tetrazolium bromide, trypan blue exclusion, western blot, and capillary-like tube formation assays. Overexpression of SNHG12 inhibited brain microvascular endothelial cell death and the inflammatory response but promoted angiogenesis after OGD/R, while SNHG12 knockdown had the opposite effects. miR-199a was identified as a target of SNHG12, and SNHG12 overexpression reversed the effect of miR-199a on brain microvascular endothelial cell death, the inflammatory response, and angiogenesis. These findings suggest that SNHG12 suppresses endothelial cell injury induced by OGD/R by targeting miR-199a.

Vav1 promotes inflammation and neuronal apoptosis in cerebral ischemia/reperfusion injury by upregulating microglial and NLRP3 inflammasome activation

Microglia,which are the resident macrophages of the central nervous system,are an important part of the inflammatory response that occurs after cerebral ischemia.Vav guanine nucleotide exchange factor 1(Vav1) is a guanine nucleotide exchange factor that is related to microglial activation.However,how Vav1 participates in the inflammato ry response after cerebral ischemia/reperfusion inj ury remains unclea r.In this study,we subjected rats to occlusion and repe rfusion of the middle cerebral artery and subjected the BV-2 mic roglia cell line to oxygen-glucose deprivatio n/reoxygenation to mimic cerebral ischemia/repe rfusion in vivo and in vitro,respectively.We found that Vav1 levels were increased in the brain tissue of rats subjected to occlusion and reperfusion of the middle cerebral arte ry and in BV-2 cells subjected to oxygen-glucose deprivation/reoxygenation.Silencing Vav1 reduced the cerebral infarct volume and brain water content,inhibited neuronal loss and apoptosis in the ischemic penumbra,and im p roved neurological function in rats subjected to occlusion and repe rfusion of the middle cerebral artery.Further analysis showed that Vav1 was almost exclusively localized to microglia and that Vav1 downregulation inhibited microglial activation and the NOD-like receptor pyrin 3(NLRP3) inflammasome in the ischemic penumbra,as well as the expression of inflammato ry facto rs.In addition,Vov1 knoc kdown decreased the inflammatory response exhibited by BV-2 cells after oxygen-glucose deprivation/reoxyge nation.Taken together,these findings show that silencing Vav1 attenuates inflammation and neuronal apoptosis in rats subjected to cerebral ischemia/repe rfusion through inhibiting the activation of mic roglia and NLRP3 inflammasome.

Silencing miRNA-324-3p protects against cerebral ischemic injury via regulation of the GATA2/A1R axis

Previous studies have suggested that miR-324-3p is related to the pathophysiology of cerebral ischemia,but the mechanism underlying this relationship is unclea r.In this study,we found that miR-324-3p expression was decreased in patients with acute ischemic stroke and in in vitro and in vivo models of ischemic stro ke.miR-324-3p agomir potentiated ischemic brain damage in rats subjected to middle cerebral artery occlusion,as indicated by increased infarct volumes and cell apoptosis rates and greater neurological deficits.In a PC12 cell oxygen-glucose deprivation/reoxygenation model,a miR-324-3 p mimic decreased cell viability and expression of the anti-apoptotic protein BCL2 and increased expression of the pro-apoptotic protein BAX and rates of cell apoptosis,whereas treatment with a miR-324-3p inhibitor had the opposite effects.Silencing miR-324-3p increased adenosine A1 receptor(A1R)expression thro ugh regulation of GATA binding protein 2(GATA2).These findings suggest that silencing miR-324-3p reduces ischemic brain damage via the GATA2/A1R axis.

环状RNA-ZNF292对缺氧缺血性脑损伤细胞氧化及凋亡的影响

目的建立原代神经元缺氧缺血性脑损伤细胞模型,探讨环状RNA-ZNF292(cZNF292)对脑缺血损伤后神经元氧化应激及凋亡的影响。方法选取胎龄为18 d的胎鼠培养原代神经元,采用4、10 mmol/L Na_(2)S_(2)O_(4)和无糖培养基处理细胞1、2、3、4 h后复氧培养0、5、15、30 h,建立氧糖剥夺/复氧细胞模型。通过细胞骨架蛋白和β3-微管蛋白免疫荧光染色观察原代神经元的形态变化,FITC标记鬼笔环肽染色观察不同浓度Na_(2)S_(2)O_(4)对原代神经元存活率的影响。采用ELISA检测培养上清液中活性氧(ROS)、丙二醛(MDA)、超氧化物歧化酶(SOD)和乳酸脱氢酶(LDH)的表达水平。采用蛋白质印迹法检测细胞中caspase 3及细胞周期蛋白G1(CCNG1)的表达水平。结果成功建立缺氧缺血性脑损伤细胞模型和cZNF292敲减细胞模型,并发现缺氧培养4 h时复氧、10 mmol/L的Na_(2)S_(2)O_(4)对原代神经元的生长抑制率最高,且在复氧培养15 h时细胞中cZNF292表达水平最高,因此选择缺氧培养4 h、复氧培养15 h、10 mmol/L Na_(2)S_(2)O_(4)为最佳实验条件。氧糖剥夺使细胞培养基中ROS和MDA水平上升,SOD和LDH水平下降(P均<0.05);而敲减cZNF292后,细胞培养基中ROS和MDA水平下降,SOD和LDH水平上升(P均<0.05)。敲减cZNF292可使caspase 3和CCNG1表达下降(P均<0.05)。结论脑缺血可以诱导原代神经元cZNF292表达增加,而敲减cZNF292可以缓解原代神经元在缺血缺氧环境下发生的氧化损伤、抑制细胞凋亡及增殖。

Upregulation of CDGSH iron sulfur domain 2 attenuates cerebral ischemia/reperfusion injury

CDGSH iron sulfur domain 2 can inhibit ferroptosis,which has been associated with cerebral ischemia/reperfusion,in individuals with head and neck cancer.Therefore,CDGSH iron sulfur domain 2 may be implicated in cerebral ischemia/reperfusion injury.To validate this hypothesis in the present study,we established mouse models of occlusion of the middle cerebral artery and HT22 cell models of oxygen-glucose deprivation and reoxygenation to mimic cerebral ischemia/reperfusion injury in vivo and in vitro,respectively.We found remarkably decreased CDGSH iron sulfur domain 2 expression in the mouse brain tissue and HT22 cells.When we used adeno-associated virus and plasmid to up-regulate CDGSH iron sulfur domain 2 expression in the brain tissue and HT22 cell models separately,mouse neurological dysfunction was greatly improved;the cerebral infarct volume was reduced;the survival rate of HT22 cells was increased;HT22 cell injury was alleviated;the expression of ferroptosis-related glutathione peroxidase 4,cystine-glutamate antiporter,and glutathione was increased;the levels of malondialdehyde,iron ions,and the expression of transferrin receptor 1 were decreased;and the expression of nuclear-factor E2-related factor 2/heme oxygenase 1 was increased.Inhibition of CDGSH iron sulfur domain 2 upregulation via the nuclear-factor E2-related factor 2 inhibitor ML385 in oxygen-glucose deprived and reoxygenated HT22 cells blocked the neuroprotective effects of CDGSH iron sulfur domain 2 up-regulation and the activation of the nuclear-factor E2-related factor 2/heme oxygenase 1 pathway.Our data indicate that the up-regulation of CDGSH iron sulfur domain 2 can attenuate cerebral ischemia/reperfusion injury,thus providing theoretical support from the perspectives of cytology and experimental zoology for the use of this protein as a therapeutic target in patients with cerebral ischemia/reperfusion injury.

丹参酮ⅡA磺酸钠注射液对大鼠心肌细胞H9c2氧糖剥夺/复氧损伤模型的修复作用

目的评价丹参酮ⅡA磺酸钠(Sodium tanshinoneⅡA sulfonate,STS)注射液对大鼠心肌细胞H9c2氧糖剥夺/复氧(OGD/R)损伤模型的影响。方法通过CCK-8法确定药物处理细胞的适合浓度范围。从中选取3个浓度STS加入到细胞培养基中。细胞建模后通过流式分析检测凋亡率、细胞染色检测心肌细胞形态和纤维化程度以及免疫荧光检测增殖细胞核抗原(PCNA)和心肌肌钙蛋白(cTnT)表达量。结果 STS处理H9c2细胞的安全浓度为≤15μM。5μM、10μM和15μM的STS均能显著且剂量依赖性下调OGD/R模型中细胞的凋亡率。Masson染色结果表明,不同浓度STS处理后与模型组比较其着色减弱,表明STS能一定程度地减轻细胞因造模导致的纤维化。免疫荧光双染结果表明,经过中、高浓度的STS处理后,能显著回调大鼠心肌细胞H9c2 OGD/R模型中PCNA和cTnT的表达水平,且细胞密度趋近正常,细胞形态得到改善。结论 STS能有效预防和修复因OGD/R所造成的细胞损伤。

Apoptosis inhibitory effect of anti-miR-146b on hippocampal neurons of OGD/R-induced injury

Objective:To construct a cellular model of oxygen-glucose deprivation and reoxygenation (OGD/R)-induced injury in hippocampal neuronsin vitro, and to explore the inhibitory effect of anti-miR-146b on the apoptosis of hippocampal neurons of ischemic/reperfusion injury. Methods: Primary rat hippocampal neurons were cultured, and oxygen-glucose deprivation and reoxygenation (OGD/R) were constructed. The anti-miR-146b lentivirus was used to transfect the hippocampal neurons, and the cells were divided into the normal group, the OGD/R group and the OGD/R + anti-miR-146b group. Real time fluorescence quantitative PCR (qRT-PCR) was used to detect the mRNA expression of miR-146b, KLF7, Caspase-3, Bcl-2 and Bax in each group. Cell viability was detected by CCK8 assay, and apoptosis was detected by Hoechst33342/PI double staining.Results: Compared with the normal group, the cell morphology of the OGD/R group and the OGD/R + anti-miR-146b group was damaged, the cell activity was decreased, and the cell apoptosis rate was increased significantly (P<0.05). However, Compared with OGD/R group, the cell morphology of the OGD/R + KLF7 group was improved, the cell activity was increased, and the apoptosis rate was decreased significantly (P<0.05). Compared with the normal group, the mRNA expression levels of miR-146b, KLF7, Caspase-3, Bax and Bcl-2 in the OGD/R group and the OGD/R + KLF7 group was increased (P<0.05). Compared with the OGD/R group, the mRNA expression of KLF7, Bcl-2 were increased in the OGD/R + KLF7 group significantly, however, the mRNA expression of miR-146b, Caspase-3, Bax were decreased significantly (P<0.05).Conclusion:Anti-miR-146b play protective effect on hippocampal neurons of OGD/R-induced injury. The mechanism may be related to upregulation of KLF7 signaling pathway, downregulation expression of Caspase-3 and Bax, upregulation expression of Bcl-2, and inhibition of apoptosis induced by OGD/R.

大鼠心肌细胞PTEN基因的表达情况及沉默对其氧糖剥夺/复氧模型耐受能力的影响

目的研究第10号染色体同源丢失性磷酸酶张力蛋白基因(PTEN)在大鼠心肌细胞的表达情况,并观察PTEN基因核糖核酸(RNA)干扰沉默后对大鼠心肌细胞氧糖剥夺/复氧模型耐受能力的影响。为临床心肌梗死的治疗提供新思路。方法体外分离培养大鼠心肌细胞并鉴定,使用逆转录-聚合酶链反应(RT-PCR)法检测其PTEN基因表达;使用脂质体法把PTEN基因特异的小干扰RNA(siRNA)转染沉默PTEN基因;制备体外氧糖剥夺/复氧模型,观察PTEN基因的沉默对细胞的保护作用。结果成功培养大鼠心肌细胞并鉴定。RT-PCR检测显示大鼠心肌细胞高表达PTEN基因,通过siRNA成功干扰了大鼠心肌细胞PTEN基因的表达,流式细胞仪检测结果显示PTEN基因沉默后的细胞对氧糖剥夺/复氧模型有较强的耐受能力。结论 PTEN基因沉默后,大鼠心肌细胞耐受氧糖剥夺/复氧环境的能力增强,具有更强的存活能力,为临床心肌梗死区域的心肌细胞存活提供新的治疗思路。

丹酚酸B-葛根素联用对氧糖剥夺/复氧损伤的SH-SY5Y神经细胞焦亡的影响

目的:基于细胞焦亡探讨丹酚酸B和葛根素合用对氧糖剥夺/复氧(oxygen-glucose deprivation/reoxygenation,OGD/R)损伤的SH-SY5Y神经细胞的保护作用及机制。方法:利用SH-SY5Y细胞构建OGD/R损伤模型,设立正常对照组(control)、模型组(OGD/R)、10μmol·L^(-1)丹酚酸B组(salvianolic acid B,Sal B)、100μmol·L^(-1)葛根素组(puerarin,Pue)、10μmol·L^(-1)丹酚酸B和100μmol·L^(-1)葛根素合用组(SP)及10μmol·L^(-1)NOD样受体蛋白3(NLRP3)炎症小体抑制剂MCC950组。除正常组外,其余各组细胞在氧糖剥夺6 h后迅速复糖复氧12 h进行OGD/R造模。采噻唑蓝(MTT)法检测细胞存活率,光学显微镜下观测细胞形态,分光光度计法检测培养上清中乳酸脱氢酶(LDH)释放率,Hoechst/PI染色检测细胞膜损伤情况,实时荧光定量聚合酶链式反应(RT-qPCR)检测NLRP3、半胱氨酸蛋白酶1(caspase-1)、消皮素D(GSDMD)、凋亡斑点样蛋白(ASC)和白细胞介素-1β(IL-1β)的mRNA表达水平,免疫荧光检测NLRP3和caspase-1蛋白表达的变化,蛋白免疫印迹法(Western blot)检测IL-1β、ASC、NLRP3、caspase-1和活化半胱氨酸蛋白酶1(cleaved caspase-1)蛋白表达的变化。结果:与正常组比较,模型组SHSY5Y细胞存活率显著降低(P<0.01),细胞形态受损,培养上清中LDH渗漏率显著提高(P<0.01),细胞膜破损,细胞中NLRP3、caspase-1、GSDMD、ASC和IL-1β的mRNA表达水平显著提高(P<0.01),IL-1β、ASC、NLRP3、caspase-1和cleaved caspase-1蛋白的表达水平显著提高(P<0.01)。与模型组相比,丹酚酸B、葛根素与丹酚酸B和葛根素合用均能显著提高OGD/R损伤后SH-SY5Y细胞的存活率(P<0.01);与单用组相比,合用组具有更好的效果(P<0.01)。与模型组相比,丹酚酸B和葛根素合用及MCC950均能改善细胞形态,降低细胞上清液LDH的渗漏(P<0.05),减轻细胞膜损伤水平,降低SH-SY5Y细胞NLRP3、caspase-1、GSDMD、ASC和IL-1β的mRNA表达水平(P<0.05,P<0.01),降低IL-1β、ASC、NLRP3、caspase-1和cleaved caspase-1蛋白的表达水平(P<0.05,P<0.01)。结论:结果表明,丹酚酸B和葛根素合用能减轻OGD/R对SH-SY5Y细胞造成的损伤,这可能与其能够抑制细胞焦亡相关。

大鼠海马神经元体外氧糖剥夺／复氧模型的构建

目的建立体外培养的大鼠海马神经元氧糖剥夺（OGD）／复氧实验模型，并尝试确定该模型最合适的缺氧缺糖时间点。方法新生SD乳鼠海马神经元原代培养7d后，随机（随机数字法）分为OGD组和对照组。OGD组根据氧糖剥夺时间的不同又分为1h，2h，4h，6h，8h，10h亚组。OGD组细胞置于含0．5％氧气的三气培养箱，同时将培养液换成无糖Earle氏液，模拟体内脑缺血损伤。复氧复糖24h后观察对照组和OGD各组的神经元形态，测定MTF细胞光密度值（OD）和培养液LDH含量，流式细胞仪检测细胞凋亡率。所得数据采用SPSS16．0统计软件行单因素方差分析（Durmett—t检验）和Spearman等级相关分析。结果随着缺氧缺糖时间的延长，OGD各组神经元形态学损伤逐步加重，细胞OD值和存活率逐渐下降（rs=-0．961和rs=-0．966，P〈0．01），LDH值逐渐升高（rs=0．990，P〈0．01），细胞凋亡率明显增加，与对照组比较差异均有统计学意义（P〈0．05）。OGD6h时，细胞的凋亡率接近50％。结论成功建立了大鼠海马神经元体外氧糖剥夺／复氧模型，结合形态学改变和细胞凋亡率，建议将6h作为该模型合适的缺氧缺糖损伤时间。

丹参-葛根提取物对氧糖剥夺/复氧损伤人神经母细胞瘤SH-SY5Y细胞的保护作用

目的:基于氧化应激和凋亡探究丹参-葛根提取物对氧糖剥夺/复氧(OGD/R)损伤SH-SY5Y细胞的保护作用。方法:采用水提法制备不同配伍比例丹参-葛根提取物,体外培养人神经母细胞瘤SH-SY5Y细胞并建立OGD/R损伤模型,采用细胞增殖与活性检测(CCK-8)法筛选最佳配伍比例提取物用于后续实验。将SH-SY5Y细胞分为空白组、OGD/R组和丹参-葛根(SP)提取物低、中、高剂量组(10、30、100 mg·L^(-1)),除空白组外,其余各组细胞在氧糖剥夺4 h后迅速复氧12 h进行OGD/R造模。采用CCK-8法检测细胞存活率,显微条件下观察细胞形态,分光光度计法检测乳酸脱氢酶(LDH)释放率、超氧化物歧化酶(SOD)活性、谷胱甘肽(GSH)和丙二醛(MDA)含量,2,7-二氯二氢荧光素二乙酸酯荧光探针法(DCFH-DA)检测细胞活性氧(ROS)水平,JC-1法检测线粒体膜电位,Hoechst 33342染色法观察细胞核形态,流式细胞术结合异硫氰酸荧光素/碘化丙啶(Annexin V-FITC/PI)双染检测细胞凋亡。结果:丹参-葛根2∶1配伍时SH-SY5Y细胞的存活率最佳,与空白组比较,OGD/R组细胞形态破损,细胞上清液LDH释放率、细胞ROS水平和MDA含量显著升高(P<0.01),SOD活性和GSH水平显著降低(P<0.01),线粒体膜电位下降,细胞凋亡率升高(P<0.01);与OGD/R组比较,SP提取物各给药组可呈浓度依赖性地提高细胞存活率,改善细胞形态,降低细胞上清液LDH释放率(P<0.01);SP提取物30、100 mg·L^(-1)组可明显降低细胞内ROS水平,明显提高细胞中SOD活性和GSH水平(P<0.05,P<0.01),100 mg·L^(-1)可明显降低细胞内MDA含量(P<0.05);此外,丹参-葛根提取物给药后可提高线粒体膜电位,30、100 mg·L^(-1)可显著降低细胞凋亡率(P<0.01)。结论:丹参-葛根提取物2∶1配伍对OGD/R损伤的SH-SY5Y细胞有较好的保护作用,这可能与其降低细胞氧化损伤,抑制细胞凋亡相关。