Expression Of Cyt-c Research Articles

Prospective role of mitochondrial apoptotic pathway in mediating GMG-ITC to reduce cytotoxicity in H2O2-induced oxidative stress in differentiated SH-SY5Y cells

The antioxidant and neuroprotective activity of Glucomoringin isothiocyanate (GMG-ITC) have been reported in in vivo and in vitro models of neurodegenerative diseases. However, its neuroprotective role via mitochondrial-dependent pathway in a noxious environment remains unknown. The main objective of the present study was to unveil the mitochondrial apoptotic genes’ profile and prospectively link with neuroprotective activity of GMG-ITC through its ROS scavenging. The results showed that pre-treatment of differentiated SH-SY5Y cells with 1.25 μg/mL purified isolated GMG-ITC, significantly reduced reactive oxygen species (ROS) production level, compared to H2O2 control group, as evidenced by flow cytometry-based evaluation of ROS generation. Presence of GMG-ITC prior to development of oxidative stress condition, downregulated the expression of cyt-c, p53, Apaf-1, Bax, CASP3, CASP8 and CASP9 genes with concurrent upregulation of Bcl-2 gene in mitochondrial apoptotic signalling pathway. Protein Multiplex revealed significant decreased in cyt-c, p53, Apaf-1, Bax, CASP8 and CASP9 due to GMG-ITC pre-treatment in oxidative stress condition. The present findings speculated that pre-treatment with GMG-ITC may alleviate oxidative stress condition in neuronal cells by reducing ROS production level and protect the cells against apoptosis via neurodegenerative disease potential pathways.

The impact of the invasive species Vespa velutina on honeybees: A new approach based on oxidative stress

Honeybees have an essential role in ecosystems pollinating wild flowers and cultivated crops, representing an important cultural and economic benefit for humans. Honeybee populations are decreasing over the last decade, due to multifactorial causes. The aim of this field study was to investigate the effects of the presence of the invasive species Vespa velutina, a bee predator, in oxidative stress parameters of honeybee workers. To achieve this objective, positive or negative apiaries for the presence of the V. velutina were selected. Five honeybees from six hives of each apiary were sampled in spring, summer and autumn, analysing a total of 233 samples. Analysis of mRNA expression of oxidative stress-related genes, catalase enzymatic activity and lipid peroxidation were performed. An increase in sod2, tpx3, trxR1, gtpx1, gstS1, coxI, cytC and if2mt genes expression, as well as a raise in catalase activity and lipid peroxidation were observed in V. velutina positive samples. Thus, here we present a new methodology to analyze the impact of the predation pressure of the invasive species V. velutina on honeybees under field conditions. In conclusion, the results obtained in this study indicate the negative impact of the presence of the yellow-legged hornet on honeybees' health and the activation of their antioxidant system to protect them against this biotic stressor. Moreover, the redox status they present could increase the susceptibility of honeybees, essential insects that currently receive many inputs of different stresses, to another stressor.

Protective effect of carbon monoxide releasing molecules 2 on post-resuscitation myocardial dysfunction in rats

Objective: To investigate the protective effect of carbon monoxide releasing molecule 2 (CORM-2) on post-resuscitation myocardial dysfunction in rats. Methods: Forty male SD rats which were healthy were randomly divided into 5 groups: sham operated group(sham group), cardiopulmonary resuscitation(PCR) group, DMSO group, inactivated CORM-2(iCORM-2) group and CORM-2 group (n=8 each). Established the model of post-cardiac arrest myocardial dysfunction by intravenous potassium chloride (4 ℃) injection combined with asphyxiation for 4 minutes and then followed by artificial chest compression for 3 minutes. Sham group: rats were instrumented with catheter without inducing cardiac arrest and resuscitation, and intraperitoneal injection of 0.9% normal saline (4 ml/kg) was performed 12 hours before catheterization. CPR group: rats were instrumented with catheters and underwent CPR, and intraperitoneal injection of 0.9% normal saline (4 ml/kg) was performed 12 hours before surgery.CORM-2 group: rats were instrumented with catheters and underwent CPR, intraperitoneally injected the prepared CORM-2 solution (4 mg/kg) at 12 hours before surgery. DMSO group: rats were instrumented with catheters and underwent CPR, intraperitoneally injected the prepared DMSO solution (4 ml/kg) at 12 hours before surgery. iCORM-2 group: rats were instrumented with catheters and underwent CPR, iCORM-2 solution (4 mg/kg) was intraperitoneally injected at 12 hours before surgery. Hemodynamic data (MAP, +dp/dtmax, -dp/dt) were continuously monitored and recorded for 4 hours after resuscitation (or catheterization) in each group. Myocardial tissue specimen and blood samples were taken after resuscitation (or catheterization). The myocardial ultrastructure was observed by transmission electron microscope. Lactate dehydrogenase (LDH) activity was measured by lactate-pyruvate method. Serum creatine kinase isoenzyme (CK-MB) concentration was measured by ELISA. Western blot was used to detect the levels of Caspase-3, Caspase-9 and Cyt-C protein in myocardial tissue. Results: MAP, +dp/dtmax and -dp/dt at 0.5, 1, 2, 3 and 4 hours post resuscitation were significantly lower than those immediately after catheterization in CRP, DMSO, iCORM-2 groups (all P<0.05). MAP at 0.5, 1, 2, 3, and 4 hours post resuscitation were significantly lower in CRP, DMSO and iCORM-2 groups than those at respective time points in sham group (all P<0.05), while MAP was similar between CORM-2 group and Sham group at these time points (all P>0.05). +dp/dtmax and -dp/dt values at 0.5, 1, 2, 3, and 4 hours post resuscitation were lower than those at respective time points in sham group and significance was found at 0.5, 1 and 2 hours post resuscitation (both P<0.05), while +dp/dtmax and -dp/dt values were similar between CORM-2 group and sham group at various time points (all P>0.05). Myocardial ultrastructure, especially mitochondrial structural integrity was better preserved in the CORM-2 group than those in the other resuscitation groups at 4 hours after resuscitation. Serum LDH activity and CK-MB concentration were significantly elevated at 4 hours after resuscitation in the CPR group, DMSO group and iCORM-2 group than those in sham group (all P<0.01); CK-MB concentration was also higher in CORM-2 group than that in sham group,and LDH level was similar between CORM-2 group and sham group (P>0.05). Serum LDH activity and CK-MB concentrations were significantly lower in the CORM-2 group than those in the other resuscitation groups (all P<0.01). The myocardial expressions of Caspase-3, Caspase-9 and Cyt-C at 4 hours after resuscitation were significantly higher in the CPR group, DMSO group and iCORM-2 group than those in sham group; the myocardial expressions of Caspase-3 and Caspase-9 were significantly higher in CORM-2 group than those in sham group (both P<0.05), while Cyt-C expression was similar between CORM-2 group and sham group. The expressions of the above 3 proteins were significantly lower in the CORM-2 group than those in the other resuscitation groups (all P<0.05). Conclusions: CORM-2 can effectively alleviate post-resuscitation myocardial injury in rats with cardiopulmonary resuscitation and improve cardiac function. Protecting myocardial mitochondria and inhibiting mitochondrial apoptosis pathway may serve as the protective mechanisms in this model.

RETRACTED ARTICLE: Melatonin enhances TNF-α-mediated cervical cancer HeLa cells death via suppressing CaMKII/Parkin/mitophagy axis

BackgroundTumor necrosis factor-α (TNF-α) immunotherapy controls the progression of human cervical cancer. Here, we explored the detailed molecular mechanisms played by melatonin in human cervical cancer (HeLa cells) death in the presence of TNF-α injury, with a particular attention to the mitochondrial homeostasis.MethodsHeLa cells were incubated with TNFα and then cell death was determined via MTT assay, TUNEL staining, caspase ELISA assay and western blotting. Mitochondrial function was detected via analyzing mitochondrial membrane potential using JC-1 staining, mitochondrial oxidative stress using flow cytometry and mitochondrial apoptosis using western blotting.ResultsOur data exhibited that treatment with HeLa cells using melatonin in the presence of TNF-α further triggered cancer cell cellular death. Molecular investigation demonstrated that melatonin enhanced the caspase-9 mitochondrion death, repressed mitochondrial potential, increased ROS production, augmented mPTP opening rate and elevated cyt-c expression in the nucleus. Moreover, melatonin application further suppressed mitochondrial ATP generation via reducing the expression of mitochondrial respiratory complex. Mechanistically, melatonin augmented the response of HeLa cells to TNF-α-mediated cancer death via repressing mitophagy. TNF-α treatment activated mitophagy via elevating Parkin expression and excessive mitophagy blocked mitochondrial apoptosis, ultimately alleviating the lethal action of TNF-α on HeLa cell. However, melatonin supplementation could prevent TNF-α-mediated mitophagy activation via inhibiting Parkin in a CaMKII-dependent manner. Interestingly, reactivation of CaMKII abolished the melatonin-mediated mitophagy arrest and HeLa cell death.ConclusionsOverall, our data highlight that melatonin enhances TNF-α-induced human cervical cancer HeLa cells mitochondrial apoptosis via inactivating the CaMKII/Parkin/mitophagy axis.

Downregulation of intrinsic apoptosis pathway in erythroblasts contributes to excessive erythrocytosis of chronic mountain sickness

Chronic mountain sickness (CMS) has a higher incidence in the plateau region and is characterized by excessive erythrocytosis and hypoxemia. Bcl-2 family plays an important role in the process of erythropoiesis and the regulation of apoptosis. This study aimed to examine the change in apoptosis of erythroblasts in CMS patients and explore the involvement of Bcl-2 family. Bone marrow mononuclear cells (BMMNCs) were isolated by density gradient centrifugation from 18 CMS patients and 17 control participants. The apoptotic rate, mitochondrial membrane potential (MMP), the protein expression of caspase-3, TNFR, Fas, Bcl-2, Bax and Cyt-C were examined by flow cytometry, and mRNA expression was determined by real-time PCR. The results showed that apoptotic rate of erythroblasts was lower and MMP was higher in CMS group than in control group. The mRNA and protein expression levels of Bcl-2 were higher while Bax level was lower in CMS group than in control group. In CMS group, the apoptosis rate of CD71+ erythroblasts was negatively correlated with the ratio of CD71+ cells in BMMNCs and positively correlated with hemoglobin level. In conclusion, erythroblasts apoptosis is decreased due to the regulation of the expression of Bcl-2 family members in the erythroblasts of CMS patients.

Effects of Taurine on Broiler Aortic Endothelial Apoptosis Induced by Heat Stress.

Heat stress is an environmental factor that causes severe economic loss to the current intensive breeding industry and induces huge impact on the long-term growth in livestock and poultry industry. Many animal experiments confirmed that heat stress is a major cause of heat stroke death, which is due to severe damage to endothelial cells. In order to provide a theoretical basis for the treatment or mitigation of heat stress related diseases in broilers, the effect of taurine on injury and apoptosis of aortic endothelial cells in broilers under heat stress was investigated in the present study. Ten days healthy broilers were sacrificed, then aortic tissue was used to isolate and cultivate primary broiler aortic endothelial cells. The third to the fifth generations of cells were used in the experiment. The cells were randomly divided into five groups, including control group (C), heat stress group (HS), low taurine (HS+LTau) group, mild taurine (HS+MTau) group and high taurine (HS+HTau) group. Cells in all groups were cultivated for 24h in cell incubator (37°C, 5% CO2). Then the heat stress group cells were cultivated in a 43°C thermostatic water bath for 6h under heat stress, and then re-incubated under 37°C for 1h. The results showed that compared with the control group, expression levels of Bax, Caspase-9, Caspase-3, Cyt-c, P53 and other pro-apoptosis factors in HS groups were significantly increased (P<0.05), while expression levels of anti-apoptosis factor Bcl-2 showed a significant decrease (P<0.05). Compared with HS group, expression levels of Bcl-2 in endothelial cells were significantly increased by taurine administration (P<0.05), while expression of Bax, Caspase-9, Caspase-3, Cyt-c and P53 were significantly increased by taurine (P<0.05). In summary, the present data indicated that taurine could protect against injury and apoptosis of aortic endothelial cells under heat stress by inhibiting the activation of mitochondria-mediated apoptotic pathways.

Effect of dexmedetomidine on expression of hypoxia-inducible factor-1α during endotoxin-caused apoptosis in macrophages of mice

Objective To evaluate the effect of dexmedetomidine on expression of hypoxia-inducible factor-1α(HIF-1α)during endotoxin-caused apoptosis in macrophages of mice. Methods Mouse macrophage cell line RAW264.7 cultured in vitro were seeded in 6-well or 96-well plates and divided into 4 groups(n=16 each)when cell confluence reached 60%-70% using a random number table method: control group(group Con), dexmedetomidine group(group Dex), lipopolysaccharide(LPS)group, and LPS plus dexmedetomidine group(group LPS+ Dex). Phosphate buffer solution was added in group Con.Dexmedetomidine 1 μmol/L was added in group Dex.LPS 1 μg/ml was added in LPS and LPS+ Dex groups.Dexmedetomidine 1 μmol/L was added immediately after adding LPS in group LPS+ Dex.Cells were then cultured for 24 h in each group.Cell apoptosis was measured using TUNEL, mitochondrial membrane potential using JC-1, reactive oxygen species(ROS) content by ROS kit, and ATP content by ATP kit.The apoptosis rate was calculated.The expression of HIF-1α, cytochrome C(Cyt-c), caspase-9 and cleaved caspase-3 was detected by Western blot. Results Compared with group Con, the apoptosis rate and ROS content were significantly increased, ATP content and mitochondrial membrane potential were decreased, the expression of HIF-1α, Cyt-c, caspase-9 and cleaved caspase-3 was up-regulated in group LPS(P 0.05). Compared with group LPS, the apoptosis rate and ROS content were significantly decreased, ATP content and mitochondrial membrane potential were increased, the expression of HIF-1α was up-regulated, and the expression of Cyt-c, caspase-9 and cleaved caspase-3 was down-regulated in group LPS + Dex(P<0.05). Conclusion Dexmedetomidine can reduce endotoxin-caused oxidative stress injury to macrophages, improve mitochondrial function and inhibit mitochondrial apoptosis, and the mechanism may be related to up-regulating the expression of HIF-1α in mice. Key words: Dexmedetomidine; Sepsis; Apoptosis; Macrophage; Mitochondria; Apoptosis; Hypoxia-induced factor 1, αlpha subunit

Effect and mechanisms of vitamin E on early steroid-induced avascular necrosis of femoral head in rats

To investigate the possibility of mitochondria-dependent apoptosis as a mechanism of early steroid-induced avascular necrosis of femoral head (SANFH) in rats and vitamin E as a possible prevention strategy. Seventy-two male Sprague Dawley rats were randomly divided into control group, model group, and intervention group, with 24 rats in each group. The rats in control group were not treated as normal control. The rats in model group and intervention group were established early SANFH models by lipopolysaccharide combined with methylprednisolone injection. At the same time, the rats in intervention group were injected with vitamin E (40 mg/kg) every day for 7 days. At 2, 4, and 8 weeks after the final injection, the bilateral femoral heads were harvested and observed by HE staining, TUNEL assay, immunohistochemical staining, and Western blot. The rate of empty lacunae, apoptotic index, and the expressions of Caspase-9, Caspase-3, and cytochrome-c (Cyt-c) proteins were calculated. According to histological staining, there were significant differences in the rate of empty lacunae between intervention group and control group at 8 weeks ( P<0.05) and between intervention group and model group at 4 and 8 weeks ( P<0.05). The apoptotic index of intervention group was significantly lower than that of model group at each time point ( P<0.05). And there was significant difference between the intervention group and the control group at 8 weeks ( P<0.05). According to immunohistochemistry staining and Western blot, the expressions of Cyt-c, Caspase-9, and Caspase-3 all significantly decreased in intervention group than those in model group at each time point ( P<0.05); and the differences were significant between intervention group and control group at 8 weeks ( P<0.05). Vitamin E can delay the progression of early SANFH by reducing mitochondrial dependent osteocyte apoptosis.

Involvement of P38 and ERK1/2 in mitochondrial pathways independent cell apoptosis in oviduct magnum epithelial cells of layers challenged with vanadium.

Vanadium (V) can induce cell apoptosis in layers' oviduct resulting in egg quality reduction. In this study, we investigated the relationship between the mitogen-activated protein kinase (MAPK)-signaling pathway and V-induced apoptosis in poultry oviduct magnum epithelial cells (OMECs). Cultured OMECs were divided into 8 treatment groups: 0 μmol/L V (control), 100 μmol/L V (V100), V100 + P38MAPK inhibitor (SB203580), SB203580, V100 + extracellular signal-regulated kinases 1 and 2 (ERK1/2) inhibitor (U0126), U0126, V100 + c-JUN NH2 -terminal kinase (JNK) inhibitor (SP600125), and SP600125. The OMECs were pretreated with the MAPK inhibitors before their treatment with V100 for 12 h. V100 increased the apoptosis of OMECs (P < .05), while 3 MAPK inhibitors suppressed V100-induced apoptosis P < .05); V100 enhanced the depolarization of △ψm (P < .05), and SB203580 and U0126 alleviated the V100-induced △ψm decrease (P < .05); V100 downregulated B-cell lymphoma-2 (Bcl-2) and poly [Adenosine diphosphate ribose] polymerase 1 (PARP1) mRNA expression (P < .05), meanwhile it upregulated Bcl-2 associated x (Bax), Apaf1, cytochrome C (CytC) and cysteine aspartase (caspase) 3, 8, 9 mRNA expression (P < .05). All MAPKs inhibitors alleviated the up-regulation of V100 for Bax and caspase 3 mRNA expression and down-regulation of V100 for Bcl-2 expression (P < .05). SB203580 and U0126 upregulated CytC expression treated by V100 (P < .05), except SP600125, while SB203580 administration resulted in a similar upregulation of PARP1 expression (P < .05). SP600125 can alleviated V triggered p-P38MAPK (phosphor-P38), p-ERK1/2 (phosphor-ERK1/2), p-JNK (phosphor-JNK) increase on OME cells, and SB203580 and U0126 had a similar response to phosphor-P38 and p-JNK (P < .05). It concluded that V-induced apoptosis in OMECs through the activation of P38 and ERK1/2, and by increasing the ratio of Bax/Bcl-2, which resulted in △ψm decrease, CytC release into the cytosol; consequently caspase 3 is recruited and activated, PARP1 is cleaved, eventually leading to apoptosis.

Lower range of molecular weight of xanthan gum inhibits cartilage matrix destruction via intrinsic bax-mitochondria cytochrome c-caspase pathway

We have previously reported an application of lower range of molecular weight of xanthan gum (LRWXG) for inhibiting cartilage matrix destruction and preventing mitochondrial damage in rabbit osteoarthritis (OA) model. However, whether LRWXG exerts its anti-OA activity through intrinsic bax-mitochondria cytochrome c-caspase signaling pathway in OA still requires further study. To address this problem, the OA model was induced by anterior cruciate ligament transection (ACLT) in rabbit and then treated with LRWXG. The results showed that LRWXG could inhibit the loss of collagen in cartilage matrix, protect trabecular bone in subchondral, decrease the apoptosis of chondrocytes, down-regulate the expressions of active caspase-9, active caspase-3 and bax, and up-regulate the expression of bcl-2. In addition, LRWXG could up-regulate the expression of cyt-c in mitochondria, while down-regulate the expression of cyt-c in cytoplasm. These findings show that LRWXG inhibits cartilage degradation via an intrinsic bax-mitochondria cytochrome c-caspase pathway in OA.

Kaempferol protects against gamma radiation-induced mortality and damage via inhibiting oxidative stress and modulating apoptotic molecules in vivo and vitro

To investigate the potential protective effect of kaempferol, a representative flavonoid, against radiation induced mortality and injury in vivo and vitro.C57BL/6 male mice and human umbilical venous endothelial cells (HUVECs) were pretreated with kaempferol before radiation. We found that kaempferol can effectively increase 30-day survival rate after 8.5 Gy lethal total body irradiation (TBI). Mice were sacrificed at 7th day after 7 Gy TBI, we found kaempferol against radiation-induced tissues damage, by inhibiting the oxidative stress, and attenuating morphological changes and cell apoptosis. In vitro, kaempferol increased HUVECs cell viability and decrease apoptosis. It also mitigated oxidative stress and restored the abnormal expression of prx-5, Cyt-c, Caspase9 and Caspase3 in mRNA and protein level in HUVECs after radiation. Taken together, it suggests kaempferol can protect against gamma-radiation induced tissue damage and mortality. The present study is the first report of the radioprotective role of kaempferol in vivo and vitro.

Oxidative damage and mitochondrial injuries differ following pneumoperitoneum pressure in rabbit models of varying degrees of hydronephrosis.

The influence of intraabdominal pressure which is necessary to maintain the operating area during the surgery cannot be ignored especially on the kidneys. Many articles have reported the effect of intraabdominal pressure on normal kidneys. However, the influence of intraabdominal pressure on hydronephrosis kidneys is rarely studied. The aim of the present study was to clarify whether intraabdominal pressure tolerance is modified in various degrees of kidney hydronephrosis by evaluating oxidative damage and mitochondrial injuries. A total of 72 rabbits were randomly divided into three groups (groups N, M and S, which represented rabbits with no, mild and severe hydronephrosis, respectively). Rabbits in groups M (n=24) and S (n=24) underwent a surgical procedure inducing mild or severe hydronephrosis, respectively. Subsequently, rabbits in all groups were allocated to 4 subgroups (N0‑N3, M0‑M3 and S0‑S3) consisting of 6 rabbits each. Groups 0 to 3 were, respectively, subjected to intraabdominal pressures of 0, 5, 10 and 15mmHg. Oxidative damage was assessed by analyzing levels of reactive oxygen species (ROS), superoxide dismutase (SOD), malondialdehyde (MDA), glutathione peroxidase (GSH‑Px), catalase (CAT) and lactate (LD). Mitochondrial injuries were assessed based on mitochondrial membrane potential (MMP) alterations, mitochondrial structure and cytochrome c (cytc) protein expression, as measured by JC‑1 staining, electron microscopy and western blotting, respectively. Oxidative damage and mitochondrial injuries were noticeably exacerbated in group N and M with increased levels of ROS, MDA and LD, decreased levels of SOD, GSH‑Px, CAT and MMP, mitochondrial vacuolization and higher expression of cytc when the intraabdominal pressure reached 15mmHg. In group S, these alterations occurred at pressures of 10 and 15mmHg. Therefore, it was concluded that in rabbits exposed to pneumoperitoneal pressure, kidneys with severe hydronephrosis were more likely to suffer from oxidative damage and mitochondrial injuries compared with kidneys with mild hydronephrosis and normal kidneys.

Glycine Protects H9C2 Cardiomyocytes from High Glucose- and Hypoxia/Reoxygenation-Induced Injury via Inhibiting PKCβ2 Activation and Improving Mitochondrial Quality.

Background Patients with diabetes are more vulnerable to myocardial ischemia reperfusion injury (IRI), which is involved in PKCβ2 activation and mitochondrial dysfunction. Glycine has been documented as a cytoprotective agent to attenuate diabetes-related abnormalities and reduce myocardial IRI, but the underlying mechanisms are still unclear. We determined whether glycine could attenuate high glucose- (HG-) and hypoxia/reoxygenation- (H/R-) induced injury by inhibiting PKCβ2 activation and improving mitochondrial quality in cultured H9C2 cells. Methods H9C2 cells were either exposed to low glucose (LG) or HG conditions with or without treatment of glycine or CGP53353 (a selective inhibitor of PKCβ2) for 48 h, then subjected to 4 h of hypoxia followed by 2 h of reoxygenation (H/R). Cell viability, lactate dehydrogenase (LDH) release, mitochondrial membrane potential (MMP), superoxide dismutase (SOD) activity, and malondialdehyde (MDA) concentration were detected using corresponding commercial kits. Mitochondrial quality control-related proteins (LC-3II, Mfn-2, and Cyt-C) and PKCβ2 activation were detected by Western blot. Results HG stimulation significantly decreased cell viability and SOD activity and increased LDH release, MDA production, and PKCβ2 activation as compared to LG group, all of which changes were further increased by H/R insult. Glycine or CGP53353 treatment significantly reduced the increase of LDH release, MDA production, PKCβ2 activation, and Cyt-C expression and the decrease of cell viability, SOD activity, MMP, Mfn-2 expression, and LC-3II/LC-3I ratio induced by HG and H/R stimulation. Conclusions Supplementary glycine protects H9C2 cells from HG- and H/R-induced cellular injury by suppressing PKCβ2 activation and improving mitochondria quality.

Protective Effect of Rosamultin against H2O2-Induced Oxidative Stress and Apoptosis in H9c2 Cardiomyocytes.

Rosamultin is one of the main active compounds isolated from Potentilla anserina L., which belongs to a triterpene compound. Few studies have examined the effect of rosamultin on oxidative stress and its molecular mechanism. The aim of this present study was to elucidate the protective effect of rosamultin on H2O2-induced oxidative damage and apoptosis in H9c2 cardiomyocytes and its mechanism. The results showed that the pretreatment of rosamultin not only increased cell viability but also reduced the release of LDH and CK. Rosamultin inhibited a H2O2-induced decrease in SOD, CAT, and GSH-Px activities and an increase in MDA content. Meanwhile, ROS level, intracellular (Ca2+) fluorescence intensity, and apoptosis rate in the rosamultin pretreated group were markedly decreased compared with the model group. Rosamultin pretreatment significantly reversed the morphological changes and attenuated H2O2-induced apoptosis. Western blot analysis showed that rosamultin enhanced the expression of Bcl-2 and pCryAB and downregulated the expression of Bax, Cyt-c, Caspase-3, and Caspase-9 expression. Additionally, rosamultin might activate PI3K/Akt signal pathways and CryAB relative factors. Therefore, we suggest that rosamultin could have the potential for treating H2O2-induced oxidative stress injury through its antioxidant and antiapoptosis effect.

Stromal Cell-Derived Factor-1α Alleviates Calcium-Sensing Receptor Activation-Mediated Ischemia/Reperfusion Injury by Inhibiting Caspase-3/Caspase-9-Induced Cell Apoptosis in Rat Free Flaps

Surgical flaps are frequently affected by ischemia/reperfusion (I/R) injury. Calcium-sensing receptor (CaSR) and stromal cell-derived factor-1α (SDF-1α) are closely associated with myocardial I/R injury. This study was performed to evaluate the feasibility of applying SDF-1α to counteract CaSR activation-mediated I/R injury in ischemic free flaps. Free flaps that underwent ischemia for 3 h were equally randomized into five groups: CaCl2, NPS2143 + CaCl2, SDF-1α + CaCl2, AMD3100 + SDF-1α + CaCl2, and normal saline. The free flaps were harvested to evaluate flap necrosis and neovascularization after 2 h or 7 d of reperfusion. p-CaSR/CaSR was extensively expressed in vascular endothelial cells of free flaps after I/R injury, and activation of the SDF-1α/CXCR4 axis and NPS2143 could reduce the expression of cleaved caspase-3, caspase-9, FAS, Cyt-c, and Bax and increase Bcl-2 expression; the opposite was true after CaSR activation. Interestingly, initiation of the SDF-1α/CXCR4 axis might abrogate CaSR activation-induced I/R injury through enhancement of microvessel density. In conclusion, CaSR might become a novel therapeutic target of free flaps affected by I/R injury. Activation of the SDF-1α/CXCR4 axis and NPS2143 could counteract CaSR activation-mediated I/R injury and promote free flap survival through inhibition of caspase-3/caspase-9-related cell apoptosis and enhancement of neovascularization.

The Mechanism of Mitochondria-Mediated Pathway in the Apoptosis of Platelets in Immune-Induced Bone Marrow Failure.

Excessive platelet apoptosis is one of the pathogenic causes of immune-induced bone marrow failure (BMF). The aim of the present study was to explore the role of mitochondria-mediated pathway in the apoptosis of platelets in immune-induced BMF. An immune-induced BMF model was established in mice, which were randomly divided into three groups: normal control (CTL) group, BMF group and cyclosporine (CSA) group (n = 10 in each group). Mice were given 0.027 g/kg CSA daily in the CSA group. Platelet count (PLT), mitochondrial transmembrane potential (ΔΨm), cytochrome C (CytC), phosphatidylserine (PS), calcium ion (Ca²⁺) and expression of proteins of the mitochondrial apoptotic pathway, including Bak, Bax, caspase-3, caspase-8 and caspase-9, was examined and compared. Compared with the CTL group, the BMF group had significantly a lower level of PLC and ΔΨm, but higher levels of CytC, PS, Ca²⁺ and higher expression levels of Bak, Bax, cleaved caspase-9 and cleaved caspase-3 (P < 0.05). CSA restored the above changes in the BMF model (P < 0.05). Further studies showed that intravenous injection of the caspase-9 inhibitor Z-LE(OMe)HD(OMe)-fluoromethylketone (FMK) into the mice could significantly inhibit apoptosis of the platelets and the effect of CSA treatment when compared to the BMF group, and exerted a better protective effect from apoptosis if the caspase-9 inhibitor was combined with the CSA treatment. These results revealed that platelet apoptosis may play an important role in the reduction of platelet of immune-induced BMF probably through the mitochondrial pathway.

Effects of microRNA-206 and its target gene IGF-1 on sevoflurane-induced activation of hippocampal astrocytes in aged rats through the PI3K/AKT/CREB signaling pathway.

The study aims to explore the effects of microRNA-206 (miR-206) targeting IGF-1 on the activation of hippocampal astrocytes in aged rats induced by sevoflurane through the PI3K/AKT/CREB signaling pathway. Wistar rats and astrocytes were divided into the normal/blank, sham/negative control (NC), sevoflurane (sevo), miR-206 mimics+sevo, miR-206 inhibitors+sevo, miR-206 NC+sevo, IGF-1 shRNA+sevo, and miR-206 inhibitors+IGF-1 shRNA+sevo groups. The Morris water maze test was exhibited to assess the cognitive functions. Glial fibrillary acidic protein (GFAP) expression was detected by immunofluorescence assay. Western blotting and RT-qPCR were used to detect the expression of miR-206, IGF-1, PI3K, AKT, CREB, pPI3K, pAKT, pCREB, cytochrome-c (Cyt-c), and caspase-3. Cell viability and apoptosis were detected by MTT assay and annexin V/PI double staining respectively. Mitochondrial transmembrane potential (MTP) were determined by flow cytometry. The IGF-1 shRNA+sevo group showed reduced miR-206 expression. Compared with the normal/blank group, the sevo, and miR-206 NC+sevo groups showed decreased miR-206 and GFAP expressions, cell viability and MTP but increased expressions of IGF-1, PI3K, AKT, CREB, pPI3K, pAKT, pCREB, Cyt-c and caspase-3, as well as cell apoptosis. Similar trends were observed in the miR-206 inhibitors+sevo group when compared with the sevo group. The study provides evidence that miR-206 alleviates the inhibition of activation of hippocampal astrocytes in aged rats induced by sevoflurane by targeting IGT-1 through suppressing the PI3K/AKT/CREB signaling pathway.

Tonifying qi and Warming yang, a compound traditional Chinese herbal medicine, suppresses AngII-induced cardiac H9c2 cells apoptosis through regulating PI3K/Akt signaling pathway

This study aims to evaluate the effects of Tonifying qi and Warming yang on Ang II-induced cardiac apoptosis and mitochondrial dysfunction and to explore the underlying mechanism. Sprague Dawley (SD) rats were treated with Tonifying qi and Warming yang, and the drug-contained serum was used in our experiment. Ang II-treated H9c2 cells were administered with drug-contained serum in different concentrations. The cell viability was determined by MTT and cell apoptosis was analyzed by the Flow cytometry instrument. The changes of mitochondrial membrane potential (MMP) was detected by Rho123 assay. qRT-PCR, Western blot and immunocytochemical staining were used to measure the mRNA and protein expression levels, respectively. Our results showed that drug-contained serum treatment prevented AngII-induced cell viability inhibition, suppressed AngII-induced apoptosis and prevented AngII-induced MMP reduction. Mechanically, drug-contained serum treatment prevented AngII-induced apoptosis by regulating expression of apoptosis-related genes, Bcl-2, Bax, Cyt-c, caspase-3, and PI3K. Besides, we found LY29400, an inhibitor of PI3K, reversed the effects of drug-contained serum treatment on apoptosis-related genes (p-AKT, Bcl-2, and Bax) in AngII-treated H9c2 cells. Totally, Tonifying qi and Warming yang, a compound traditional Chinese herbal medicine, suppresses AngII-induced cardiac H9c2 cells apoptosis through regulating PI3K/Akt signaling pathway.

Protective Effects of Sodium (±)-5-Bromo-2-(α-Hydroxypentyl) Benzoate in a Rodent Model of Global Cerebral Ischemia

The aim of the current study was to explore the protective effects of sodium (±)-5-bromo-2-(α-hydroxypentyl) benzoate (brand name: brozopine, BZP) in a rat model of global cerebral ischemia. The rat model was established using a modified Winocur’s method; close postoperative observation was conducted at all times. Neurological function was detected through prehensile traction and beam-walking test. BZP reduced mortality and prolonged the survival time of rats with global cerebral ischemia, within 24 h. There was a decreased survival rate (60%) in the Model group, while the survival rate of the BZP (3 and 12 mg/kg) remarkably increased the survival rate (to 80 and 90%, respectively), in a dose-dependent manner. Compared with the Model group (survival time: 18.50 h), the administration of BZP (0.75, 3, and 12 mg/kg) prolonged the survival time (to 20.38, 21.85, and 23.90 h, respectively), particularly in BZP 12 mg/kg group (P < 0.05). Additionally, the BZP (12 mg/kg) group exhibited an improvement in their motor function (P < 0.05). The BZP groups (0.75, 3, and 12 mg/kg) displayed significantly reduced necrosis and the percentage of apoptotic cells (P < 0.05 and P < 0.01, respectively). Compared with Model group, BZP (0.75, 3, and 12 mg/kg) increased the NeuN optical density values (P < 0.01). Rats with global ischemia had a high expression of Cyt-c, caspase-3, and the Bax/Bcl-2 ratio compared with sham group (P < 0.01). BZP (0.75, 3, and 12 mg/kg), however, reduced the expression of Cyt-c, caspase-3, and the Bax/Bcl-2 ratio, in a dose-dependent manner (P < 0.01). There was low expression of p-Akt and PI3K in Model group, compared with the sham group (P < 0.01). Meanwhile, BZP (0.75, 3, and 12 mg/kg) increased the expression of p-Akt and PI3K in a dose-dependent manner (P < 0.01). We also found the expression of Cyt-c, caspase-3, Bax/Bcl-2 ratio, PI3K, p-Akt, and comprehensive score were directly related. In conclusion, BZP had therapeutic potential and prevented stroke in rat model of global cerebral ischemia. The underlying mechanisms may be related to the inhibition of apoptosis and activation of the survival-signaling-pathway.

Construction of a Bcl-2-shRNA expression vector and its effect on the mitochondrial apoptosis pathway in SW982 cells.

Apoptosis is considered to serve an important role in the pathogenesis of rheumatoid arthritis. The aim of the present study was to construct Bcl-2-short hairpin (sh)RNA expression vectors and transfect them into human synovial sarcoma SW982 cells, in order to screen for an effective interference sequence and analyze the effects of this interference on the expression levels of Bcl-2 and other molecules associated with the mitochondrial apoptosis pathway. Three different shRNAs (Bcl-2-sh1, 2 and 3) were designed according to the human Bcl-2 mRNA target sequence and were transformed into competent DH5α Escherichiacoli cells following the construction of an expression vector, which was then transfected into SW982 cells. SW982 cells were grouped into a control group (transfected with a negative control shRNA), and Bcl-2-sh1, Bcl-2-sh2 and Bcl-2-sh3 groups (transfected with Bcl-2-sh1, 2 and 3, respectively). The expression levels of Bcl-2 mRNA were detected using reverse transcription-quantitative PCR (RT-qPCR). Bcl-2-sh1 was identified as the most effective shRNA sequence for interference, and was used for subsequent experiments. The mRNA and protein expression levels of Bcl-2, Bax, CytC and Caspase-3 were detected in SW982 cells by RT-qPCR and western blotting at various time-points (48 and 72h) following transfection with Bcl-2-sh1, in order to observe the effectiveness of this interference. Compared with the control group, the expression levels of Bcl-2 were decreased, while those of Bax, CytC and Caspase-3 were increased in Bcl-2-sh1-transfected cells (P<0.01). The interference effect was greater at 48h than at 72h. In summary, an effective shRNA sequence (Bcl-2-sh1) targeting the Bcl-2 gene was identified from three candidates, and was demonstrated to significantly interfere with the expression of Bcl-2, Bax, CytC and Caspase-3 when transfected into SW982 cells. The interference effect of Bcl-2-sh1 was more pronounced at 48h than at 72h post-transfection.