San-Huang-Xie-Xin-Tang protects cardiomyocytes against hypoxia/reoxygenation injury via inhibition of oxidative stress-induced apoptosis

Oxidative stress has been widely implicated in the pathogenesis of hypoxia/reoxygenation (H/R) injury. San-Huang-Xie-Xin-Tang (SHXT), a widely used traditional Chinese medication, has been shown to possess antioxidant effects. Here, we investigated whether SHXT and its main component baicalin can attenuate oxidative stress induced by H/R injury. H9c2 rat ventricular cells were exposed to SHXT or baicalin followed by hypoxia for 24 h and/or reoxygenation for 8 h. Pretreatment with SHXT and baicalin both significantly prevented cell death and production of reactive oxygen species induced by hypoxia or H/R in H9c2 cardiomyoctes. In addition, SHXT and baicalin also inhibited hypoxia- or H/R-induced apoptosis, with associated decreased Bax protein, increased Bcl-2 protein, and decreased caspase-3 activity. Furthermore, we found that hypoxia and H/R decreased endothelial nitric oxide synthase (eNOS) expression and nitrite production, and these effects were counteracted by SHXT and baicalein. Finally, SHXT inhibited H/R-induced activation of p38 mitogen activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) phosphorylation in H9c2 rat ventricular cells. The present study demonstrates for the first time that SHXT can protect cardiomyocytes from H/R injury via inhibition of oxidative stress-induced apoptosis. These cardioprotective effects are possibly mediated through eNOS enhancement and p38 MAPK and JNK-dependent signaling pathways.     

Baicalin protects H9c2 cardiomyocytes against hypoxia/reoxygenation‐induced apoptosis and oxidative stress through activation of mitochondrial aldehyde dehydrogenase 2

Baicalin, a flavonoid glycoside separated from Scutellaria baicalensis, has cardioprotection against ischaemia/reperfusion (I/R) injury. Mitochondrial aldehyde dehydrogenase 2 (ALDH2) is considered as an endogenous protective mechanism against I/R injury depending on its anti‐oxidant and anti‐apoptotic characteristics. The present study demonstrates whether ALDH2 contributes to the cardioprotection of baicalin against hypoxia/reoxygenation (H/R)‐inudced H9c2 cardiomyocytes injury. Our results observed that H/R treatment resulted in a significant decrease in cells viability and obvious increases in caspase‐3 activity and apoptosis rate in H9c2 cells, while these alterations were evidently reversed by baicalin pretreatment. Simultaneously, baicalin mitigated H/R‐induced the decreases in the levels of ALDH2 mRNA and protein as well as the activity of ALDH2 in H9c2 cells. However, we found that daidzin, an ALDH2 antagonist, remarkably attenuated baicalin‐elicited inhibitory action on H/R‐induced the downregulation of cells viability and Bcl‐2 protein expression, and the upregulations of caspase‐3 activity, apoptosis rate, cytochrome c and Bax proteins expressions in H9c2 cells. In addition, baicalin reversed H/R‐induced oxidative stress as evidenced by the downregulation of malondialdehyde (MAD) and 4‐hydroxy aldehydes (4‐HNE) levels, the inhibition of endogenous reactive oxygen species (ROS) generation, and the downregulation of superoxide dismutase (SOD) activity induced by H/R treatment, while these effects were also blocked by daidzin. Furthermore, we found that Alda‐1, an ALDH2 agonist, also abolished H/R‐induced cytotoxicity, apoptosis, and oxidative stress, indicating that ALDH2 mediated H/R‐induced H9c2 cell injury. Overall, these results suggested that baicalin prevents H/R‐induced apoptosis and oxidative stress through enhancing ALDH activity and expression in H9c2 cardiomyocytes.     

异鼠李素对多柔比星诱导的心肌细胞自噬和凋亡的影响

目的 研究异鼠李素(ISO)对多柔比星(DOX)诱导的心肌细胞自噬和凋亡的影响.方法 大鼠H9c2心肌细胞分为对照组、模型组和实验组.模型组以15.0μmol·L-1异鼠李素及生理盐水预处理2 h,实验组在模型组的基础上加入1μmol·L-1注射用盐酸多柔比星共培养24 h;对照组以等量生理盐水处理.干预48 h后,以...     

艾司洛尔对缺氧复氧诱导的心肌细胞AMPK/GSK-3β通路及凋亡的影响

目的 探讨艾司洛尔对缺氧复氧诱导的心肌细胞凋亡的影响,并初步研究其作用机制.方法 体外培养大鼠心肌细胞(H9c2),并建立缺氧/复氧(I/R)损伤模型,随机分为对照组、H/R组和艾司洛尔低、中、高剂量(0.2、5.0、25.0μmol/L)组;噻唑蓝(MTT)法检测各组H9c2细胞存活率变化情况;膜联蛋白V-异硫氰酸荧...     

Silencing of C3G increases cardiomyocyte survival inhibition and apoptosis via regulation of p‐ERK1/2 and Bax

Experimental studies have shown that overexpression of Rap guanine nucleotide exchange factor 1 (C3G) plays pro‐survival and anti‐apoptotic roles through molecule phosphorylated extracellular signal‐regulated kinase1/2 (p‐ERK1/2) in cardiomyocytes. However, it is still unclear if silencing of C3G may increase cell survival inhibition and apoptosis in cardiomyocytes, and whether C3G silence induced injuries are reduced by the overexpression of C3G through regulation of p‐ERK1/2 and pro‐apoptotic molecule Bax. In this study, the rat ‐ derived H9C2 cardiomyocytes were infected with C3G small hairpin RNA interference recombinant lentiviruses, which silenced the endogenous C3G expression in the cardiomyocytes. Then, contrary experiments were conducted using C3G overexpression. The cell proliferation and apoptosis were analyzed in the cardiomyocytes which were treated with or without hypoxia/reoxygenation (H/R). Silencing of C3G leaded to significant increase in cell survival inhibition and apoptosis, combined with aggravated the injuries induced by H/R. Overexpression of C3G reduced the injuries induced by the silencing of C3G in the cardiomyocytes via regulation of p‐ERK1/2 and Bax. In conclusion, our results provide new experimental evidence that silencing of C3G can increase cell survival inhibition and apoptosis in cardiomyocytes via regulation of p‐ERK1/2 and Bax.     

NDUFA4L2 protects against ischaemia/reperfusion‐induced cardiomyocyte apoptosis and mitochondrial dysfunction by inhibiting complex I

Myocardial ischaemia/reperfusion (I/R) injury may cause the apoptosis of cardiomyocytes as well as mitochondrial dysfunction. The aims of the present study were to investigate whether NADH dehydrogenase 1 alpha subcomplex subunit 4‐like 2 (NDUFA4L2) on myocardial ischaemia‐reperfusion (I/R) injury and the underlying molecular mechanism. The hypoxia‐reperfusion (H/R) model was established in vitro using H9c2 cells to simulate I/R injury. NDUFA4L2 and complex I expression levels were detected using RT‐PCR and western blot. The apoptosis of H9c2 cells was evaluated by flow cytometry and the expression of Bax and Bcl‐2 was detected by western blot. The mitochondrial function was assessed by ATP concentration, mPTP opening and cytochrome c (cyto C) expression. Our data indicated that NDUFA4L2 expression was significantly down‐regulated in myocardial H/R injury. Overexpression of NDUFA4L2 led to a dramatic prevention of H/R‐induced apoptosis accompanied by a decrease in the expression of Bax and an increase in the expression of Bcl‐2. Meanwhile, augmentation of NDUFA4L2 dramatically prevented mitochondrial dysfunction caused by H/R as reflecting in the increased ATP concentration, delayed mPTP opening, as well as down‐regulated cyto C expression. Moreover, complex I activation was heightened and negatively regulated by NDUFA4L2. Silencing complex I conspicuously attenuated cell apoptosis and mitochondrial dysfunction. Taken together, our findings demonstrated that NDUFA4L2 protects against H/R injury by preventing myocardium apoptosis and mitochondrial dysfunction via the complex I, and may be a potential therapeutic approach for attenuating myocardial I/R injury.     

内质网应激相关蛋白 1过表达对缺氧 /复氧诱导的 H9c2心肌细胞凋亡的影响

目的 研究内质网应激相关蛋白1(SERP1)过表达对缺氧/复氧(H/R)诱导的大鼠胚胎H9c2心肌细胞凋亡的影响.方法 2018年1—12月将中国科学院上海细胞库大鼠H9c2心肌细胞系建立H/R损伤大鼠心肌细胞模型,采用pCMV6质粒为载体构建内质pCMV6-网应激相关蛋白1(pCMV6-SERP1)过表达重组质粒,分别设H9c2心肌细胞为正常对照组(NC组),H/R损伤(缺氧4 h/复氧4 h)H9c2心肌细胞为H/R组,H/R损伤H9c2心肌细胞转染pCMV6空质粒为H/R+pCMV6组,H/R损伤H9c2心肌细胞转染pCMV6-SERP1过表达重组质粒为H/R+pCMV6-SERP1组,采用噻唑蓝(MTT)法检测各种H9c2心肌细胞存活率,采用Annexin V-FITC/PI双染技术检测各组H9c2心肌细胞凋亡率,采用实时定量PCR(RT-qPCR)和免疫印迹法分别检测各组细胞SERP1蛋白及凋亡相关蛋白的表达水平.结果 与NC组[(1.06±0.21)、(0.41±0.07)]比较,H/R组、H/R+pCMV6组H9c2心肌细胞SERP1 mRNA[(0.47±0.09)、(0.48±0.08)]及蛋白表达水平[(0.25±0.04)、(0.26±0.05)]显著降低,H/R+pCMV6-SERP1组H9c2心肌细胞SERP1 mRNA(2.94±0.52)及蛋白表达水平(0.83±0.12)显著增加(P<0.05),与H/R组、H/R+pCMV6组比较,H/R+pCMV6-SERP1组H9c2心肌细胞中内质网应激相关蛋白1微小RNA(SERP1 mRNA)及蛋白水平显著升高(P<0.05).与NC组[(100.00±00.00)％、(4.45±0.62)％]比较,H/R组、H/R+pCMV6组、H/R+pCMV6-SERP1组H9c2心肌细胞存活率[(49.86±5.14)％、(48.75±5.23)％、(89.72±8.61)％]、B淋巴细胞瘤-2(Bcl-2)蛋白水平显著降低(P<0.05),H9c2心肌细胞凋亡率[(35.84±4.13)％、(33.66±4.07)％、(10.96±1.74)％]、Bcl-2相关X蛋白(Bax)、半胱氨酸蛋白酶-3(caspase-3)蛋白表达水平显著增加(P<0.05);与H/R组、H/R+pCMV6组比较,H/R+pCMV6-SERP1组H9c2心肌细胞中存活率、Bac-l蛋白表达水平显著升高,细胞凋亡率、Bax、Caspase-3蛋白表达水平显著降低(P<0.05).结论 SERP1过表达可能通过上调Bacl-2蛋白,下调Bax、Caspase-3蛋白表达,对缺氧/复氧诱导的H9c2心肌细胞凋亡有一定保护作用.     

Salvianolic acid A protects H9C2 cardiomyocytes against hypoxia/reoxygenation injuryby regulating VDAC1

OBJECTIVE To investigate the role of voltage-dependent anion channel 1(VDAC1) in the protective effects of Salvianolic acid A(SAA) on H9C2 cardiomyocytes subjected to hypoxia/reoxygenation(H/R) injury.METHODS H9C2 cardiomyocytes were subjected to 4 h of hypoxia(1% O_2, 94% N_2, 5% CO_2, 37℃) followed by 4 h of reoxygenation(95% air, 5% CO_2, 37℃). SAA(1 umol·L~(-1))was administrated at the same time of reoxygenation. To examine whether the expression of VDAC1 is regulated by SAA, H9C2 cardiomyocytes were respectively transfected with VDAC1 si RNA and VDAC1 over expression plasmid. The viability of H9C2 cardiomyocytes was detected by Cell Counting kit-8 assay and further confirmed by detecting the level of intracel ular lactate dehydrogenase.Apoptosis were evaluated using an Annexin Ⅴ-FITC/propidiumiodide staining kit detected by flow cytometry.The level of mitochondrial ROS was measured using the fluorescent probe Mito SOX. The mitochondrial Ca~(2+) signal was monitored with a FRET-based Ca~(2+) indicator. Western blotting and q RT-PCR analysis were used to observe the protein and m RNA levels of VDAC1, and we use luciferase reporter assay to further explore whether SAA has a potential binding sites on VDAC1 promoter to perform the direct regulation on VDAC1. The oligomeric state of VDAC1 was explored by BRET2 assay. RESULTS H/R injury significantly increased the expression of VDAC1, and induced VDAC1 oligomerization. VDAC1 silencing could increase the viability of H/R-treated cardiomyocytes, and attenuate apoptosis as well as the increase on the level of mitochondrial ROS and Ca~(2+) uptake against H/R injury,which performed the same protective effects with SAA.Meanwhile, VDAC1 overexpression could aggravate cell injury. Taken together, VDAC1 was involved in the mechanisms of H/R-induced cell injury. SAA could significantly inhibit the expression of VDAC1 compared with H/R group, we also found that SAA exhibited a direct regulative effect in 3′-UTR of VDAC1 gene. Moreover, SAA could inhibit the oligomerization of VDAC1. Al these protective effects of SAA were abolished in H9C2 cardiomyocytes transfected with VDAC1 overexpression plasmid.CONCLUSION Our findings indicate that SAA can inhibit apoptosis and improve mitochondrial function by regulating the expression and oligomerization of VDAC1, which revealed VDAC1 as a novel potential target and mechanism for the cardioprotective effects of SAA.     

西格列汀通过调控自噬和凋亡减轻棕榈酸诱导的心肌细胞损伤

目的：观察西格列汀是否通过调节心肌细胞自噬来减轻棕榈酸诱导的H9c2心肌细胞凋亡。方法：棕榈酸和西格列汀处理H9c2细胞24 h后,CCK-8试剂盒检测细胞存活率;共聚焦和Western blotting检测自噬标志蛋白LC3的表达情况;TUNEL试剂盒检测细胞凋亡情况;Western blotting分析凋亡相关蛋白C-Casp3、C-PARP和Cyt C以及相关调控因子AMPK和mTOR等的表达。结果：棕榈酸抑制H9c2细胞活性,减少LC3的蛋白表达,增加TUNEL阳性的凋亡细胞比率,增加凋亡相关蛋白的表达,抑制AMPK的磷酸化;而西格列汀可抑制PA对H9c2的这些作用,促进LC3蛋白表达,减少凋亡蛋白表达和凋亡细胞比率,增加AMPK的磷酸化。结论：本研究显示西格列汀可能通过促进自噬和减轻细胞凋亡来对抗棕榈酸诱导的细胞损伤,且这种作用可能是通过AMPK信号通路介导的。     

线粒体融合素2通过Ras-PI3K-Akt信号途径抑制缺血再灌注诱导的乳鼠心肌细胞凋亡

目的：研究线粒体融合素2（Mfn2）基因对缺血再灌注诱导的乳鼠心肌细胞凋亡的影响及其相关的信号通路。方法：乳鼠心肌细胞经缺氧/复氧（H/Re）处理模拟心肌缺血再灌注损伤。用Mfn2基因的重组腺病毒（Adv-Mfn2）感染经缺氧复氧处理的乳鼠心肌细胞。采用TUNEL染色、ELISA、流式细胞术等方法检测Mfn2对缺血再灌注诱导的乳鼠心肌细胞凋亡的影响。Western blot分析线粒体凋亡路径中Bcl-2蛋白、Bax蛋白、Caspase-9以及磷酸化蛋白激酶B（p-Akt）的表达变化。结果：TUNEL染色发现Adv-Mfn2感染乳鼠心肌细胞后,细胞凋亡较H/Re组和Adv-LacZ组显著减少。ELISA和流式细胞仪检测结果表明,Adv-Mfn2组心肌细胞凋亡较H/Re组及Adv-LacZ组明显减少,且这一作用呈时间依赖性。Western blot结果显示,Adv-Mfn2组中Bcl-2蛋白表达较H/Re组和Adv-LacZ感染组上升,Bax蛋白表达下降,各组中Caspase-9的表达变化与Bax相同,Adv-Mfn2组的p-Akt蛋白表达水平则较H/Re组和Adv-LacZ感染组明显上升。结论：Mfn2基因主要通过正向调控RasPI3K-Akt信号通路,促进Akt的磷酸化水平,使Bcl-2蛋白表达量增加,Bax蛋白表达量降低,抑制Caspase-9活化,从而抑制缺血再灌注诱导的乳鼠心肌细胞凋亡。     

Cardioprotective effect of KR-33889, a novel PARP inhibitor,against oxidative stress-induced apoptosis in H9c2 cells and isolated rat hearts

Oxidative stress plays a critical role in cardiac injury during ischemia/reperfusion (I/R). Despite a potent cardioprotective activity of KR-33889, a novel poly (ADP-ribose) polymerase inhibitor, its underlying mechanism remains unresolved. This study was designed to investigate the protective effects of KR-33889 against oxidative stress-induced apoptosis in rat cardiomyocytes H9c2 cells and isolated rat hearts. H₂O₂ caused severe injury to H9c2 cells, mainly due to apoptosis, as revealed by TUNEL assay. However, KR-33889 pretreatment significantly attenuated H₂O₂-induced apoptosis of H9c2 cells, which was accompanied by decrease in expression of both cleaved caspase-3 and Bax and increase in Bcl-2 expression and the ratio of Bcl-2/Bax. KR-33889 also significantly enhanced the expression of anti-oxidant enzymes including heme oxygenase-1, Cu/Zn-superoxide dismutase (SOD), Mn-SOD, and catalase, thereby inhibiting production of intracellular ROS. Furthermore, KR-33889 reversed H₂O₂-induced decrease in phosphorylation of Akt, GSK-3β, ERK1/2, p38 MAPK, and SAPK/JNK during most H₂O₂ exposure time. In globally ischemic rat hearts, KR-33889 inhibited both I/R-induced decrease in cardiac contractility and apoptosis by increasing Bcl-2, decreasing both cleaved caspase-3 and Bax expression, and enhancing expression of anti-oxidant enzymes. Taken together, these results suggest that KR-33889 may have therapeutic potential to prevent I/R-induced heart injury in ischemic heart diseases mainly by reducing oxidative stress-mediated myocardial apoptosis.     

N-n-Butyl haloperidol iodide protects against hypoxia/reoxygenation-induced cardiomyocyte injury by modulating protein kinase C activity

N-n-Butyl haloperidol iodide (F₂), a novel compound derived from haloperidol, protects against the damaging effects of ischemia/reperfusion (I/R) injury in vitro and in vivo. We tested whether the myocardial protection of F₂ on cardiomyocyte hypoxia/reoxygenation (H/R) injury is mediated by modulating protein kinase C (PKC) activity in primary cultured cardiomyocytes. Primary cultures of ventricular cardiomyocytes underwent 2-h hypoxia and 30-min reoxygenation. Total PKC activity was measured, and the translocation pattern of PKCα, βII, δ and ? isoforms was assessed by fractionated western blot analysis. We investigated the association of PKC isoform translocation and H/R-induced injury in the presence and absence of the specific inhibitors and activator. Measurements included cell damage evaluated by creatine kinase (CK) release, and apoptosis measured by annexin V-FITC assay. In primary cultured cardiomyocytes exposed to H/R, PKCα, δ and ? were translocated, with no change in PKCβII activity. Total PKC activity, CK release and apoptosis were increased after H/R. Treatment with the conventional PKC inhibitor G?6976 reduced early growth response-1 (Egr-1) protein expression and attenuated apoptosis. The PKC? inhibitor peptide ?V1-2 increased H/R injury without influencing Egr-1 expression. Pretreatment with F₂ inhibited translocation of PKCα, increased translocation of PKC?, and relieved the CK release and apoptosis. The protection of F₂ was blocked in part by the conventional PKC activator thymeleatoxin (TXA) and ?V1-2 peptide. F₂ significantly alleviated H/R-induced injury, which might be attributed to the combined benefits of inhibiting PKCα and activating PKC?.     

Berberine protects HK-2 cells from hypoxia/reoxygenation induced apoptosis via inhibiting SPHK1 expression

Renal ischemia reperfusion injury (RIRI) refers to the irreversible damage for renal function when blood perfusion is recovered after ischemia for an extended period, which is common in clinical surgeries and has been regarded as a major risk for acute renal failures (ARF) that is accompanied with unimaginably high morbidity and mortality. Hypoxia during ischemia followed by reoxygenation via reperfusion serves as a major event contributing to cell apoptosis, which has been widely accepted as the vital pathogenesis in RIRI. Preventing apoptosis in renal tubular epithelial cell has been considered as effective method for blocking RIRI. In this paper, we established a hypoxia/reoxygenation (H/R) injury model in human proximal tubular epithelial HK-2 cells. Here, we found increased SPHK1 levels in H/R injured HK-2 cells, which could be significantly down regulated after berberine treatment. Berberine has been reported to exert a protective effect on H/R-induced apoptosis of HK-2 cells. So, in our present study, we planned to investigate whether SPHK1 participated in the anti-apoptosis process of berberine in H/R injured HK-2 cells. Our study confirmed the protective effect of berberine against H/R-induced apoptosis in HK-2 cells through promoting cells viability, inhibiting cells apoptosis, and down-regulating p-P38, caspase-3, caspase-9 as well as SPHK1, while up regulating the ratio of Bcl-2/Bax. However, SPHK1 overexpression in HK-2 cells induced severe apoptosis, which can be significantly ameliorated with additional berberine treatment. We concluded that berberine could remarkably prevent H/R-induced apoptosis in HK-2 cells through down-regulating SPHK1 expression levels, and the mechanisms included the suppression of p38 MAPK activation and mitochondrial stress pathways.     

Sodium tanshinone IIA silate inhibits oxygen-glucose deprivation/recovery-induced cardiomyocyte apoptosis via suppression of the NF-κB/TNF-α pathway

Background and Purpose

Inhibition of apoptosis may attenuate the irreversible injury associated with reperfusion. In the current study, we focused on the cytoprotective effects and the underlying mechanism of sodium tanshinone IIA silate (STS) against damage induced by oxygen-glucose deprivation/recovery (OGD/R). in H9c2 cardiomyocytes and the underlying mechanisms.

Experimental Approach

We used a model of cardiac ischaemia/reperfusion, OGD/R in H9c2 cardiomyocytes, to assess the cardioprotective effects of STS. Apoptosis of cells was measured with Hoechst 33342-based fluorescence microscopy, and annexin V-FITC-based flow cytometry. Caspase-3 and caspase-8 activities and mitochondrial membrane potential were also measured using commercial kits. TNF-α in the cell culture supernatant fractions were measured with sandwich elisa, and protein levels assayed using Western blot.

Key Results

STS inhibited OGD/R-induced apoptosis by suppressing JNK-mediated activation of NF-κB, TNF-α expression, activation of caspase-3 and caspase-8 and the Bax/Bcl-2 ratio. Additionally, positive feedback between NF-κB and TNF-α and amplification of TNF-α were inhibited, suggesting that STS plays a protective role against apoptosis in cardiomyocytes, even upon activation of pro-inflammatory cytokines. Interestingly, the cytoprotective effects of STS on OGD/R-induced apoptosis and promotion of cell survival were attenuated after inhibition of PI3K.

Conclusion and Implications

The inhibitory effects of STS on TNF-α and positive feedback signalling of the NF-κB/TNF-α pathways may play important roles in myocardial protection against ischaemia/reperfusion. These protective effects of STS are mediated by suppressing JNK activity through activation of the PI3K-Akt pathway.     

Overexpression of heat-shock protein 20 in rat heart myogenic cells confers protection against simulated ischemia/reperfusion injury

AIM: To explore whether overexpression of the small heat shock protein HSP20 in rat cardiomyocytes protects against simulated ischemia/reperfusion (SI/R) injury. METHODS: Recombinant adenovirus expressing HSP20 was used to infect rat H9c2 cardiomyocytes at high efficiency, as assessed by green fluorescent protein. H9c2 cells were subjected to SI/R stress; survival was estimated through assessment of lactate dehydrogenase and cell apoptosis through caspase-3 activity. RESULTS: Overexpression of HSP20 decreased lactate dehydrogenase release by 21.5% and caspase-3 activity by 58.8%. Pretreatment with the protein kinase C inhibitor Ro-31-8220 (0.1 micromol/L) for 30 min before SI/R canceled the protective effect of HSP20. The selective mitochondrial K+ATP channel inhibitor 5-hydroxydecanoate (100 micromol/L) had a similar effect. However, the non-selective K+ATP channel inhibitor glibenclamide (100 micromol/L) had no significant effect. CONCLUSION: These data indicate that the protective effect of HSP20 in vitro is primarily due to reduced necrotic and apoptotic death of cardiomyocytes, possibly via the protein kinase C/mitochondrial K+ATP pathway.     

碘化N-正丁基氟哌啶醇通过非外钙机制抗心肌细胞缺氧复氧损伤

目的研究碘化N-正丁基氟哌啶醇(F2)对大鼠心肌细胞无外钙缺氧/复氧(H/R)损伤的作用及其机制。方法建立无外钙(零钙液)的心肌细胞H/R模型,于缺氧液及复氧液中加入1×10-6mol/L的F2。倒置显微镜下观察细胞形态结构及搏动的变化;采用蛋白印迹法检测心肌细胞磷酸化ERK(p-ERK1/2)、总ERK1/2蛋白表达的变化。结果零钙液H/R可引起培养心肌细胞形态结构呈损伤性改变,包括胞体收缩、伪足减少和搏动无力;零钙液H/R可引起培养心肌细胞p-ERK1/2表达增加,但不影响总ERK表达,即可激活培养心肌细胞ERK1/2;F2可以改善零钙液H/R状态下心肌细胞形态结构的损伤。F2对零钙液H/R心肌细胞p-ERK1/2高表达具有抑制作用,但不影响总ERK的表达。结论 F2可通过非外钙依赖机制拮抗心肌细胞H/R损伤,这可能与其抑制零钙液H/R状态下ERK1/2的激活密切相关。     

富氢液对大鼠脑缺血/再灌注损伤后海马线粒体通透性转换孔及细胞凋亡的影响

目的观察富氢液对大鼠全脑缺血/再灌注损伤后海马线粒体通透性转换孔(mitochondrial permeability transitionpore,mPTP)及细胞凋亡的影响。方法 96只成年健康♂SD大鼠,体质量250～300 g,随机分为6组(n=16):假手术组(S)、缺血/再灌注组(IR)、生理盐水组(NS)、富氢液组(H)、苍术苷组(A)、富氢液+苍术苷组(HA)。采用四血管阻塞法建立大鼠全脑缺血/再灌注模型,缺血15 min后恢复灌注,S组仅分离椎动脉和颈总动脉,不进行阻闭。H组和HA组于再灌注即刻腹腔注射富氢液5 ml.kg-1,其余组腹腔注射等容量生理盐水。A组和HA组于再灌注前10 min侧脑室注射苍术苷15μl,NS组和H组侧脑室注射等容量生理盐水。再灌注后24 h取海马组织,分离海马细胞线粒体,通过紫外分光光度仪检测海马mPTP的开放情况,West-ern blot法测海马线粒体和胞质细胞色素C(Cyt C)水平,免疫组化法观察Bcl-2、Bax在海马CA1区表达,并计算Bcl-2和Bax表达的比值(Bcl-2/Bax),透射电镜观察海马CA1区线粒体超微结构。结果与S组比较,其余5组mPTP吸光度的改变值升高,Bcl-2和Bax表达上调,线粒体Cyt C表达下调,胞质Cyt C表达上调(P<0.05);与IR组比较,H组mPTP吸光度的改变值降低,Bcl-2表达上调,Bax表达下调,线粒体Cyt C表达上调,胞质Cyt C表达下调(P<0.05);与H组比较,HA组mPTP吸光度的改变值升高,Bcl-2表达下调,Bax表达上调,线粒体Cyt C表达下调,胞质Cyt C表达下调(P<0.05);IR组与NS组间上述指标比较差异无统计学意义(P>0.05)。透射电镜结果显示富氢液可以改善全脑缺血后线粒体超微结构的改变,苍术苷可部分取消富氢液的上述改变。结论富氢液能有效减轻大鼠全脑缺血/再灌注损伤,减少Cyt C易位至胞质,从而抑制大鼠脑缺血/再灌注后细胞凋亡,其机制与抑制海马细胞mPTP的开放有关。     

Pim-3对抗心肌细胞缺氧/复氧损伤的研究

目的研究原癌基因Pim-3对心肌细胞急性缺氧/复氧(A/R)损伤的保护作用。方法采用原代培养新生大鼠的心肌细胞A/R损伤和缺氧预适应(APC)保护模型,将已经构建好的pEGFP-N2/Pim-3质粒导入原代培养的乳鼠心肌细胞中,实验结束后测定Pim-3mRNA及蛋白表达水平(RT-PCR、Western blot法)的改变,同时检测培养液中乳酸脱氢酶(LDH)活性、四唑盐(MTT)比色试验测定细胞存活率、TUNEL法检测细胞凋亡。结果RT-PCR和Western blot结果显示,Pim-3在正常组几乎不表达,A/R组表达明显升高,缺氧预适应(APC)+A/R组表达则进一步升高;转染pEGFP-N2/Pim-3质粒后24h,荧光倒置显微镜显示转染效率达30%;在A/R损伤后,Pim-3基因转染组较未转染组的心肌细胞LDH值明显降低,细胞存活率则明显升高,心肌细胞的凋亡指数明显下降。结论在细胞水平,Pim-3参与心肌APC保护作用,导入外源性的Pim-3基因能对抗急性A/R损伤。     

甘草次酸的差向异构体对顺铂诱导H9c2心肌细胞损伤的保护机制

目的 探究甘草次酸（GA）的差向异构体18α-GA和18β-GA对顺铂（CDDP）诱导H9c2心肌细胞损伤的保护机制。方法 采用CCK-8法检测细胞活力,筛选CDDP诱导细胞损伤的浓度,18α-GA和18β-GA安全浓度以及18α-GA与18β-GA改善CDDP致心肌细胞活力下降的有效浓度;将细胞分为对照组、CDDP组、18α-GA组和18β-GA组,使用Hoechst染色检测各组细胞凋亡情况;流式细胞术检测活性氧（ROS）水平;Mito-Tracker Red CMXRos染色评估线粒体活性;Western blot法检测各组细胞剪切化胱天蛋白酶3(C-Caspase3)、B-淋巴细胞瘤基因-2(Bcl-2)、Bcl-2相关X蛋白（Bax）以及细胞色素C(Cyt-c)的蛋白表达。结果 CCK-8实验结果显示,CDDP在20μmol/L时可使H9c2心肌细胞活力显著下降（P<0.01）;18α-GA和18β-GA浓度小于100μmol/L时,对心肌细胞无明显影响,且50和100μmol/L时均可改善CDDP导致的心肌细胞活力的降低（P<0.01）。与对照组相比,CDDP组细...     

Orientin-induced cardioprotection against reperfusion is associated with attenuation of mitochondrial permeability transition

In this study, we provide new evidence that orientin from bamboo leaves (Phyllostachys nigra) protect H9c2 cardiomyocytes against ischemia/reperfusion (I/R) injury through the mitochondrial apoptotic pathway. A previous work has identified that orientin could protect myocardium against ischemia/reperfusion injury. Mitochondria are both critical determinants of cardioprotection and crucial targets of cardioprotective signaling. Their role during reperfusion is conspicuously critical because the conditions promote apoptosis through the mitochondrial pathway and necrosis though irreversible damage to mitochondria, which is in association with mitochondrial permeability transition (MPT). After myocardial ischemia, opening of the mPTP is a critical determinant of cell death. The relationship of orientin and mPTP in mediating reperfusion-induced cardiomyocytes injury is still elusive. Here, our results indicate that the protective effect of orientin in H9c2 cells subjected to I/R injury is associated with depression of the mPTP opening, resultant mitochondrial dysfunction, and apoptosis. Further investigation of cellular mechanisms revealed that these effects were associated with inhibition of reactive oxygen species (ROS) generation, repolarization of mitochondrial membrane potential (Δψ(m)), suppression of mitochondrial cytochrome C release, enhancement of the Bcl-2 level, and inhibition of Bax and Smac/DIABLO levels. Furthermore, these beneficial effects of orientin were blocked by the phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin, and orientin could enhance Akt phosphorylation. In summary, we demonstrate that orientin protects H9c2 cardiomytocytes against I/R-induced apoptosis by modulating the mPTP opening, and this role of orientin may involve the PI3K/Akt signaling pathway.