Bax Expression Ratio Research Articles

JinChan YiShen TongLuo Formula ameliorate mitochondrial dysfunction and apoptosis in diabetic nephropathy through the HIF-1α-PINK1-Parkin pathway

Ethnopharmacological relevanceThe JinChan YiShen TongLuo (JCYSTL) formula, a traditional Chinese medicine (TCM), has been used clinically for decades to treat diabetic nephropathy (DN). TCM believes that the core pathogenesis of DN is “kidney deficiency and collateral obstruction," and JCYSTL has the effect of “tonifying kidney and clearing collateral," thus alleviating the damage to kidney structure and function caused by diabetes. From the perspective of modern medicine, mitochondrial damage is an important factor in DN pathogenesis. Our study suggests that the regulation of mitophagy and mitochondrial function by JCYSTL may be one of the internal mechanisms underlying its good clinical efficacy. Aim of the studyThis study aimed to investigate the mechanisms underlying the renoprotective effects of JCYSTL. Materials and methodsUnilateral nephrectomy combined with low-dose streptozotocin intraperitoneally injected in a DN rat model and high glucose (HG) plus hypoxia-induced HK-2 cells were used to explore the effects of JCYSTL on the HIF-1α/mitophagy pathway, mitochondrial function and apoptosis. ResultsJCYSTL treatment significantly decreased albuminuria, serum creatinine, blood urea nitrogen, and uric acid levels and increased creatinine clearance levels in DN rats. In vitro, medicated serum containing JCYSTL formula increased mitochondrial membrane potential (MMP); improved activities of mitochondrial respiratory chain complexes I, III, and IV; decreased the apoptotic cell percentage and apoptotic protein Bax expression; and increased anti-apoptotic protein Bcl-2 expression in HG/hypoxia-induced HK-2 cells. The treatment group exhibited increased accumulation of PINK1, Parkin, and LC3-II and reduced P62 levels in HG/hypoxia-induced HK-2 cells, whereas in PINK1 knockdown HK-2 cells, JCYSTL did not improve the HG/hypoxia-induced changes in Parkin, LC3-II, and P62. When mitophagy was impaired by PINK1 knockdown, the inhibitory effect of JCYSTL on Bax and its promoting effect on MMP and Bcl-2 disappeared. The JCYSTL-treated group displayed significantly higher HIF-1α expression than the model group in vivo, which was comparable to the effects of FG-4592 in DN rats. PINK1 knockdown did not affect HIF-1α accumulation in JCYSTL-treated HK-2 cells exposed to HG/hypoxia. Both JCYSTL and FG-4592 ameliorated mitochondrial morphological abnormalities and reduced the mitochondrial respiratory chain complex activity in the renal tubules of DN rats. Mitochondrial apoptosis signals in DN rats, such as increased Bax and Caspase-3 expression and apoptosis ratio, were weakened by JCYSTL or FG-4592 administration. ConclusionThis study demonstrates that the JCYSTL formula activates PINK1/Parkin-mediated mitophagy by stabilizing HIF-1α to protect renal tubules from mitochondrial dysfunction and apoptosis in diabetic conditions, presenting a promising therapy for the treatment of DN.

Bisphenol-A induced cyto-genotoxicity on retinal pigment epithelial cells is differentially modulated by a multi-supplement containing guarana, selenium, and L-carnitine.

Bisphenol A (BPA) may adversely affect human health by inducing oxidative stress and irreversible damage to cells. Bioactive compounds found in some functional foods, individually or in combination, can attenuate the negative effects of BPA exposure; an example is the multi-supplement containing guarana (Gua), selenium (Se), and L-carnitine (LC) -GSC- which has already demonstrated antioxidant, genoprotective, and immunomodulatory activities. This study aimed to determine the effect of GSC and its constituents on oxidative and genotoxic alterations triggered by BPA exposure in the retinal epithelial cell line. The cells exposed to BPA (0.001, 0.01, 0.1, 1, 3, and 10 µM) to determine the lowest concentration required to induce cyto-genotoxicity. ARPE-19 cells were then concomitantly exposed to the selected BPA concentration, GSC, and its components (Gua, 1.07 mg/mL; Se, 0.178 µg/mL; and LC, 1.43 mg/mL). Flow cytometry, biochemical assays, qRT-PCR, genotoxicity, apoptosis, and cellular proliferation. Based on our results, 10 µM of BPA could induce cyto-genotoxic and oxidative alterations. BPA did not alter the Bcl-2/BAX expression ratio but induced Casp3 and Casp8 overexpression, suggesting that apoptosis was induced mainly via the extrinsic pathway. GSC partially reversed the alterations triggered by BPA in ARPE-19 cells. However, Se had unexpected negative effects on ARPE-19 cells. The multi-supplement GSC may attenuate changes in oxidative and genotoxic markers related to exposure of ARPE-19 cells to BPA. our results revealed that the antioxidant, anti-apoptotic, and genoprotective properties of GSC were not universally shared by its individual, once Se did not exhibit any positive impact.

Fisetin Attenuated Oxidative Stress-Induced Cellular Damage in ARPE-19 Human Retinal Pigment Epithelial Cells Through Nrf2-Mediated Activation of Heme Oxygenase-1.

Fisetin is a kind of bioactive flavonol, widely present in various fruits such as strawberries and apples, and is known to act as a potent free radical scavenger. However, the mechanism of action related to the antioxidant activity of this compound in human retinal pigment epithelial (RPE) cells is not precisely known. In this study, we aimed to investigate whether fisetin could attenuate oxidative stress-induced cytotoxicity on human RPE ARPE-19 cells. To mimic oxidative stress, ARPE-19 cells were treated with hydrogen peroxide (H2O2), and fisetin significantly inhibited H2O2-induced loss of cell viability and increase of intracellular reactive oxygen species (ROS) production. Fisetin also markedly attenuated DNA damage and apoptosis in H2O2-treated ARPE-19 cells. Moreover, mitochondrial dysfunction in H2O2-treated cells was alleviated in the presence of fisetin as indicated by preservation of mitochondrial membrane potential, increase of Bcl-2/Bax expression ratio, and suppression of cytochrome c release into the cytoplasm. In addition, fisetin enhanced phosphorylation and nuclear translocation of nuclear factor erythroid 2 related factor 2 (Nrf2), which was associated with increased expression and activity of heme oxygenase-1 (HO-1). However, the HO-1 inhibitor, zinc protoporphyrin, significantly reversed the protective effect of fisetin against H2O2-mediated ARPE-19 cell injury. Therefore, our results suggest that Nrf2-mediated activation of antioxidant enzyme HO-1 may play an important role in the ROS scavenging activity of fisetin in RPE cells, contributing to the amelioration of oxidative stress-induced ocular disorders.

Hypoxia-inducedoxidative stress and apoptosisin gills of scaleless carp (Gymnocypris przewalskii).

Scaleless carp (Gymnocypris przewalskii) are well adaptedtolow oxygen environment, but their specific adaptation mechanism to hypoxic condition remains unclear. The gill is an important respiratory organ that plays a crucial role in regulating hypoxic stress. Here, we established fish hypoxic stress model, as well as investigated oxidative stress, apoptotic responses, and relative enzyme activities in the gills of scaleless carp after exposure to various levels of hypoxic stress. The results demonstrated that gill lamellar height and basal length increased significantly under severe hypoxic stress, and interval lengths between lamellae increased significantly under hypoxic stress. Furthermore, lamellar epithelial cells underwent apoptosis, cytoplasmic contraction, and mitochondrial expansion, and the number of apoptotic cells increased significantly after exposure to severe hypoxic stress for 24h. Subsequently, Bcl-2 and Caspase 3 mRNA levels, as well as Bcl-2/Bax expression ratio were significantly increased after exposure to severe hypoxic stress for 24h, indicating upregulation of anti-apoptotic processes. Moreover, malondialdehyde and hydrogen peroxide levels were significantly increased after exposure to hypoxic stress for 24h. Superoxide dismutase activity increased significantly after exposure to severe hypoxia for 8h and then decreased, while glutathione peroxidase activity and total antioxidant capacity increased significantly under hypoxic stress. Taken together, the results indicated that scaleless carp gills respond to acute hypoxic conditions by undergoing lamellar morphology remodeling, enhanced apoptosis, and increased antioxidant enzymatic activity. The study findings provided new insight into the adaptation mechanisms of scaleless carp in response to hypoxic challenge.

Retinoic acid exerts neuroprotective effects against focal cerebral ischemia by preventing apoptotic cell death

Cerebral ischemia is a neurological disorder that leads to cognitive decline and high mortality. Retinoic acid is a metabolite of vitamin A that has anti-inflammatory and anti-apoptotic effects. This study investigated whether retinoic acid prevents neuronal cell damage on focal cerebral ischemia through modulating apoptosis signaling pathway. Middle cerebral artery occlusion (MCAO) was performed to induce focal cerebral ischemia in adult male rats. Retinoic acid (5 mg/kg) or vehicle was injected intraperitoneally for 4 days prior to MCAO. Neurological behavior deficit tests were performed 24 h after MCAO. Brain edema and infarct volume were measured, and TUNEL histochemistry was carried out. We also investigated the changes in apoptosis-related proteins including bcl-2 family proteins and caspases. MCAO injury induced severe neurological behavior deficits and brain edema. It also increased infarct volume, histopathological damages, and the number of TUNEL-positive cells in cerebral cortex. However, retinoic acid pretreatment attenuated MCAO-induced neurological behavior deficits, brain edema, and infarction. It also alleviated histopathological lesion and decreased the number of TUNEL-positive cells. Bcl-2 and bax proteins are representative bcl-2 family proteins. MCAO injury induced a decrease in bcl-2 expression and an increase in bax expression, and retinoic acid pretreatment alleviated these changes. MCAO injury caused a decrease in bcl-2/bax expression ratio in cerebral cortex, while retinoic acid restored this decrease by MCAO. Moreover, our result showed increases in caspase-9, caspase-3, PARP protein levels in MCAO-operated animals. Retinoic acid pretreatment prevented these increases. We identified the changes in cleaved forms of these proteins, similar to the changes in full-length protein. Activation of caspases and PARP proteins are considered to be representative apoptosis indicators. This study showed that retinoic acid regulates bcl-2 family proteins and caspase proteins in focal cerebral ischemia. Thus, our findings demonstrate that retinoic acid exhibits a neuroprotective effect against ischemic damage by modulating apoptosis signaling pathway.

운동이 골격근에서 Bcl-2, Bax 발현비율과 세포고사에 미치는 영향

이 연구는 실험용 쥐를 대상으로 운동이 골격근에서 Bcl-2와 Bax 발현비율과 세포고사에 어떠한 영향을 미치는지에 대해 알아보고자 한 연구이다. 이를 위해 ICR계 수컷 흰쥐 20마리를 일주일의 적응기간을 거쳐 통제집단(CON : n=10)과 운동집단(AEG : n=10)으로 배정하였다. 운동은 8주간 주5회 실시하였고, 중정도 운동강도인 16.4m/min(최대산소섭취량의 65～70%)의 속도와 트레드밀 경사도 4%로 설정하여 40분간 지속적인 운동을 실시하였다. 세포고사에 대한 검증 결과 Bcl-2 단백질의 발현은 운동집단이 통제집단보다 유의하게 낮은 것으로 나타났고(p .01), Bax 단백질의 발현은 운동집단이 통제집단보다 낮게 나타났으며, Bax/Bcl-2비율은 운동집단이 통제집단보다 높게 나타났다. 따라서 규칙적 중강도 유산소운동은 세포손실을 예방 하고 운동을 통해 개선 할 수 있을 것으로 사료된다.

Chrysophanol inhibits the osteoglycin/mTOR and activats NF2 signaling pathways to reduce viability and proliferation of malignant meningioma cells

ABSTRACT Chrysophanol shows promising antitumor activity, but how it may work against malignant meningioma is poorly understood. In addition, osteoglycin (OGN) may help mediate the antitumor effects of chrysophanol; thus, this study investigated the potential antitumor mechanism of chrysophanol in malignant meningioma cultures. Meningioma cell line HBL-52 were incubated with varying doses of chrysophanol (0–90 μM) for different time points, and osteoglycin (OGN) was overexpressed or inhibited in some cell cultures to assess its roles. Cell viability was quantified using the CCK8 assay and colony formation assays, while effects on cell cycle distribution and apoptotic rates were examined by flow cytometry and enzyme-linked immunosorbent assays (ELISA) to detect histone DNA levels. Caspase-3 and −9 activities were detected by related commercial kits. Protein expression was assessed using Western blotting. Chrysophanol significantly reduced HBL-52 cell viability, based on reduced colony formation, and proliferation, based on low levels of bromodeoxyuridine incorporation. Annexin V/propidium iodide staining revealed a 30% increase in apoptotic cells at 90 μM chrysophanol (33.7% vs 3.3% in control cultures). Chrysophanol treatment greatly decreased the Bcl-2/Bax expression ratio and increased the expressions of cleaved caspase-3 and −9, and the activities of caspase-3 and −9. Chrysophanol blocked cells in G1 phase and inhibited the OGN/mTOR signaling cascade, but activated neurofibromatosis 2 (NF2) cascade. OGN overexpression activated mTOR, down-regulated NF2, and partially reversed growth inhibition by chrysophanol. Chrysophanol may be useful as a treatment against malignant meningioma by inhibiting OGN/mTOR signaling and activating NF2 signaling.

Effect of Metformin on Bcl-2/Bax Expression Ratio and Endometrial Implants: A Mouse Model in Endometriosis Study

Background: Endometriosis is a gynaecological disorder characterised by the presence of endometrial tissue outside the uterine cavity. Apoptosis, regulated by the balance of Bcl-2/Bax, plays an important role in the endometrial improvement. Metformin, an insulin sensitizer that is known to have beneficial effect in the endometriosis treatment, is expected to lower Bcl-2/Bax expression ratio and reduce endometrial implants.Objective: To explore the effect of metformin on the Bcl-2/Bax expression ratio and endometrial implant area of endometriosis-induced mice.Methods: This experimental study used 3-month old 33 BALB/c mice of endometriosis that were randomly and equally divided into three groups (P0, P1, and P2). On the 15th day, the P0 group was first terminated for Bcl-2/Bax examination and the size of endometrial implants. The P1 group was given aquabidest, whereas the P2 group was given metformin 4 mg/day for 14 days. The immunohistochemistry of Bcl-2/Bax expression were performed from cavum abdomen and pelvis peritoneal tissues of the mice and measured by the Remmele Scale Index, whereas the extracted mice’ endometrial implants were analysed with computer tracing method. All data normality test was calculated with Shapiro-Wilk test. The mean difference test of all groups was analysed using the one-way ANOVA test, whereas the mean difference test between groups was completed using the Unpaired T-test (LSD/Least Significance Difference).Results: The Bcl-2/Bax expression ratio and endometrial implant area in the P2 group were significantly lower compared to P0 and P1 (p<0.001). There were no significant differences in the Bcl-2/Bax expression ratio or endometrial implant area in P0 and P1 (p>0.05)Conclusion: Metformin may be a potential effective drug treatment for endometriosis by decreasing Bcl-2/Bax expression ratio and endometrial implants.

Gynura divaricata exerts hypoglycemic effects by regulating the PI3K/AKT signaling pathway and fatty acid metabolism signaling pathway

ObjectivesThe study aimed to examine the anti-diabetic effects of Gynura divaricata (GD) and the underlying mechanism.MethodsInformation about the chemical compositions of GD was obtained from extensive literature reports. Potential target genes were predicted using PharmMapper and analyzed using Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO). To validate the results from bioinformatics analyses, an aqueous extract of GD was administered to type 2 diabetic rats established by feeding a high-fat and high-sugar diet followed by STZ injection. Key proteins of the PI3K/AKT signaling pathway and fatty acid metabolism signaling pathway were investigated by immunoblotting.ResultsThe blood glucose of the rats in the GD treatment group was significantly reduced compared with the model group without treatment. GD also showed activities in reducing the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatinine (CREA). The levels of urine sugar (U-GLU) and urine creatinine (U-CREA) were also lowered after treatment with GD. Bioinformatics analysis showed that some pathways including metabolic pathways, insulin resistance, insulin signaling pathway, PPAR signaling pathway, bile secretion, purine metabolism, etc. may be regulated by GD. Furthermore, GD significantly increased the protein expression levels of PKM1/2, p-AKT, PI3K p85, and GLUT4 in the rat liver. In addition, the expression levels of key proteins in the fatty acid metabolism signaling pathway including AMPK, p-AMPK, PPARα, and CPT1α were significantly upregulated. The anti-apoptotic protein BCL-2/BAX expression ratio in rats was significantly upregulated after GD intervention. These results were consistent with the bioinformatics analysis results.ConclusionsOur study suggests that GD can exert hypoglycemic effects in vivo by regulating the genes at the key nodes of the PI3K/AKT signaling pathway and fatty acid metabolism signaling pathway.

Impact of coadministration of apigenin and bone marrow stromal cells on damaged ovaries due to chemotherapy in rat: An experimental study.

BackgroundApigenin is a plant-derived flavonoid with antioxidative and antiapoptotic effects. Bone marrow stromal cells (BMSCs) are a type of mesenchymal stem cells (MSCs) that may recover damaged ovaries. It seems that apigenin may promote the differentiation of MSCs.ObjectiveThe aim of this study was to investigate the effect of coadministration of apigenin and BMSCs on the function, structure, and apoptosis of the damaged ovaries after creating a chemotherapy model with cyclophosphamide in rat.Materials and MethodsFor chemotherapy induction and ovary destruction, cyclophosphamide was injected intraperitoneally to 40 female Wistar rats (weighing 180–200 gr, 10 wk old) for 14 days. Then, the rats were randomly divided into four groups (n = 10/each): control, apigenin, BMSCs and coadministration of apigenin and BMSCs. Injection of apigenin was performed intraperitoneally and BMSC transplantation was performed locally in the ovaries. The level of anti-mullerian hormone serum by ELISA kit, the number of oocytes by superovulation, the number of ovarian follicles in different stages by H&E staining, and the expression of ovarian Bcl-2 and Bax proteins by western blot were assessed after four wk.ResultsThe results of serum anti-mullerian hormone level, number of oocytes and follicles, and Bcl-2/Bax expression ratio showed that coadministration of apigenin and BMSCs significantly recovered the ovarian function, structure, and apoptosis compared to the control, BMSC, and apigenin groups (p 0.001).ConclusionThe results suggest that the effect of coadministration of apigenin and BMSCs is maybe more effective than the effect of their administrations individually on the recovery of damaged ovaries following the chemotherapy with cyclophosphamide in rats.

Morphological risk assessment of cold atmospheric plasma-based therapy: bone marrow mesenchymal stem cells in treatment zone proximity

Cold atmospheric plasma (CAP), modern technology for medical purposes, has recently established its potential in cancer therapy both in vitro and in vivo, thanks to the plasma-induced formation of free radicals. Due to its growing demand and extensive use, comprehensive studies are necessary to ensure its safety, especially for healthy cells adjacent to the treatment area. To this end, in this paper and for the first time, use has been made of morphological analysis, including the cell/nucleus shape index (CSI/NSI) and fractal dimension (FD), to assess the risk of CAP therapy on normal bone marrow mesenchymal stem cells (BMMSCs). To find the best protocol of plasma treatment, BMMSCs were subjected to direct and indirect (adding plasma-activated-water (PAW) to the culture medium) treatments. Our bioassay and morphological assessments results showed that the PAW treatment was the better method for the optimal dosage equal to 80% of the culture media, provided that the maximum power was 30 W and the exposure time was 90 s. Our research showed that high-dose PAW results in significant changes in FD, e.g. an equal proportion of PAW and culture medium increases the FD 1.38-fold, with respect to the control. Furthermore, while in the low-power direct treatment (<30 W) NSI did not change significantly, in the high-power one (>30 W) it approached 1, which is an indicator of apoptosis. It was also seen that high-dose PAW elongates the cells and therefore diminishes CSI. Any change in cell/nucleus shape could results in cancer initiation. Therefore, the observed changes in shape index and FD number might indicate cancer initiation. Our measurements of the BAX and BCL2 expression ratio were in favor of our results. Taken together, all of our results showed cell damage in high powers of plasma source and high-dose PAW. Moreover, it is concluded that the probability of cancer initiation is a severe issue that should be investigated in all CAP-based cancer therapies, even in the optimized indirect methods.

Antitumor Effects of Chrysophanol in Malignant Optic Nerve Meningioma Cell Lines are Mediated via Caspase Activation, Induction of Mitochondrial Mediated Apoptosis, Mitochondrial Membrane Depolarization and Targeting the Mitogen-Activated Protein Kinase Signaling Pathway

Anthroquinones are considered remarkable anticancer agents. Chrysophanol is an important anthroquinone and it has shown to have the potential to inhibit the growth of the range of cancers. However, there are no studies regarding the anticancer effects of chrysophanol against the malignant meningioma of optic nerve. In this review, the potential of chrysophanol in the treatment of malignant -meningioma of optic nerve was explored by evaluating its anticancer activity against the malignant meningioma CH157-MN cells. The 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide assay was used for cell viability determination. The 4',6-diamidino-2-phenylindole (DAPI), acridine orange and ethidium bromide (AO/EB) and annexin V/PI assays were used to determine the induction of apoptosis. The potential of reactive oxygen species and the mitochondrial membrane was estimated by flow cytometry. Western blot analysis was performed to determine the protein expression. The results showed that chrysophanol caused significant decline in the viability of the CH157-MN cells and exhibited an IC50 of 30 µmol/L. Anticancer effects were found to be due to the induction of apoptosis as evident form the DAPI and AO/EB staining. The annexin V/PI staining revealed that the apoptotic cells increased from 1.77% in control to 37.21% at 60 µmol/L concentration of chrysophanol. The Bcl-2/Bax expression ratio was decreased and the caspases-3 and 9 were activated upon chrysophanol treatment of the CH157-MN cells. Chrysophanol also triggered the formation of reactive oxygen species and reduction of the mitochondrial membrane potential in the CH157-MN cells and also blocked the Mitogen-activated protein kinase signaling pathway. The findings of the present study suggest that chrysophanol may prove beneficial in the treatment of malignant meningioma of optic nerve. Key Message: The study revealed the anticancer potential of chrysophanol against the malignant optic nerve meningioma.

Bacillomycin D-C16 triggers apoptosis of gastric cancer cells through the PI3K/Akt and FoxO3a signaling pathways.

Bacillomycin D can inhibit the growth of Aspergillus ochraceus in food samples. In addition, it can induce apoptosis in and inhibit the proliferation of cancer cells, although the details of this mechanism are unknown. In this study, we separated bacillomycin D-C14, D-C15, D-C16 monomers from the Bacillus subtilis strain fmbJ. The bacillomycin D monomers containing longer fatty acid chains better induced apoptosis in Bgc-823, Sgc-7901, and Hgc-27 gastric cancer cells. The Bgc-823 cell line was the most sensitive. Acridine orange-ethidium bromide staining indicated that bacillomycin D-C16-induced Bgc-823 cell death by triggering apoptosis, characterized by membrane blebbing, cellular shrinkage, and DNA fragmentation. Flow cytometric analysis showed a bacillomycin D-C16 dose-dependent trigger of Bgc-823 apoptosis. Bacillomycin D-C16-induced the mitochondrial pathway, as indicated by a reduced Bcl-2/Bax expression ratio, enhanced cytochrome C release, and higher levels of cleaved caspase-3. Furthermore, bacillomycin D-C16 effectively repressed phosphorylation of the serine-threonine protein kinase Akt at Ser-473 and increased the levels of the FoxO3a protein. The combination of the PI3K/Akt-inhibitor BEZ235 with bacillomycin D-C16 enhanced the apoptosis of Bgc-823 cells. Together, these findings indicated that bacillomycin D-C16 induces apoptosis through the PI3K/Akt and FoxO3a signaling pathways.

Piperidine nitroxide Tempol enhances cisplatin-induced apoptosis in ovarian cancer cells.

A nitroxide radical, Tempol (Tempol, TPL), is usually used as an antioxidative agent clinically, whereas the mechanism underlying its pro-oxidative effect has not been thoroughly investigated. The present study investigated the pro-oxidative effect of TPL on the inhibition of cellular proliferation and its role in enhancing the effect of anticancer drug cisplatin (DDP) on the induction of apoptosis in ovarian cancer cells. Cell viability and proliferation were evaluated by MTT assay. Cell apoptosis was analyzed by flow cytometry (FCM) following staining with Annexin V/propidium iodide. Western blot analysis was performed to determine the expression levels of anti-apoptotic protein B-cell lymphoma-2 (Bcl-2) and pro-apoptotic protein Bcl-2-associated X protein (Bax), and the Bcl-2:Bax expression ratio. Cellular reactive oxygen species (ROS) were labeled with dichlorofluorescin-diacetate and analyzed by FCM. The results revealed that cell viabilities of OVCAR3 and SKOV3 cells were decreased by TPL in dose-dependent manner at concentrations of 2 to 10 mM after 48 h incubation. The cell proliferation rates of OVCAR3 and SKOV3 cells were suppressed by TPL at lower toxic concentrations of 1.5 and 1 mM, respectively, compared with the control group. The MTT assay indicated that the combination therapy significantly inhibited the cell proliferation of OVCAR3 cells compared with treatment with DDP alone. FCM demonstrated that the combination treatment increased the proportion of early apoptotic cells in OVCAR3 cells compared with single DDP treatment. Western blot analysis revealed that the combination treatment markedly decreased the Bcl-2:Bax expression ratio compared with treatment with DDP alone. Detection of cellular ROS expression levels demonstrated that the combination therapy significantly increased cellular ROS generation compared with the DDP-only therapy. These data indicated that TPL increased the effect of DDP on inducing apoptosis in OVCAR3 cells.

FoxO1-mediated autophagy plays an important role in the neuroprotective effects of hydrogen in a rat model of vascular dementia

Vascular dementia (VD) is a heterogeneous group of brain disorders in which cognitive impairment is attributed to cerebrovascular pathologies. Autophagy, a self-cannibalization mechanism, has been demonstrated to be involved in VD progression. Molecular hydrogen is known for its powerful anti-oxidative, anti-apoptotic, and anti-inflammatory activities, and it is also involved in autophagy. However, the effects of hydrogen on VD remain unclear. The current study found that hydrogen-rich water (HRW) significantly alleviated spatial learning and memory impairments. Similar to donepezil treatment, HRW also inhibited neuron loss and shrinkage in the hippocampal CA1 region. In addition, we found that HRW significantly increased the Bcl-2/Bax expression ratio and decreased cleaved caspase-3 expression levels in the hippocampus of VD rats. Moreover, electron microscopy revealed that HRW decreased the number of autophagosomes. We also observed that HRW reduced the increased ratio of LC3-II/I and Beclin 1 expression and saliently upregulated p62 expression. Furthermore, FoxO1 (a major mediator of autophagy regulation) and Atg7 levels were apparently decreased in the hippocampus of HRW-treated bilateral common carotid artery occlusion (2VO) rats. Taken together, these data show that molecular hydrogen exerts beneficial effects on cognitive impairment induced by chronic cerebral hypoperfusion. FoxO1-mediated autophagy plays an important role in the neuroprotective effects of hydrogen in a rat model of VD. Furthermore, the present findings highlight that HRW should be further investigated as a new therapeutic strategy for VD treatment in the future.

The Bioactive Substance Secreted by MSC Retards Mouse Aortic Vascular Smooth Muscle Cells Calcification.

Background Vascular calcification, which is associated with low-level chronic inflammation, is a complication that occurs during aging, atherosclerosis, chronic kidney disease, diabetes mellitus, and hyperlipaemia. In this study, we used conditioned media from mesenchymal stem cells (MSC-CM), a source of autologous cytokines, to test the hypothesis that MSC-CM inhibits vascular smooth muscle cell (VSMC) calcification by suppressing inflammation and apoptosis. Methods VSMCs were treated with β-glycerophosphate (β-GP) to induce calcification and MSC-CM was used as a treatment. Calcium deposition was evaluated using alizarin red and von Kossa staining after a 7-day induction period. Intracellular calcium contents were measured via the o-cresolphthalein complexone method, and alkaline phosphatase (ALP) activity was determined using the para-nitrophenyl phosphate method. The expressions of specific-osteogenic markers, inflammatory cytokines, and apoptosis-associated genes/proteins were examined by real-time polymerase chain reaction or western blotting. Results MSC-CM inhibited β-GP-induced calcium deposition in VSMCs and decreased intracellular calcium content and ALP activity. Additionally, MSC-CM suppressed the β-GP-induced increases in BMP2, Msx2, Runx2, and osteocalcin expression. Additionally, MSC-CM decreased the expression of TNF-α, IL-1β, and IL-6 in VSMC. MSC-CM also partly blocked β-GP-induced VSMC apoptosis, which was associated with an increase in the Bcl-2/Bax expression ratio and a decrease in caspase-3 expression. Conclusion Our study results suggest that MSC-CM can inhibit VSMC calcification. This suggests a potential novel clinical application for MSCs in the treatment of vascular calcification and associated diseases.

Dihydroartemisinin potentiates antitumor activity of 5-fluorouracil against a resistant colorectal cancer cell line

Although the combination of chemotherapy and surgical resection has effectively increased the survival rate of colorectal cancer patients in recent decades, acquired drug resistance is still a problem that leads to treatment failure. Dihydroartemisinin (DHA), a semisynthetic derivative of artemisinin, has recently been reported to show anticancer effects against numerous types of cancer, including colorectal cancer. This study showed that DHA exerted a strong anticancer effect against several colorectal cancer cell lines. We also found that p53 knockout colorectal cancer HCT116 cells (HCT116 TP53−/−) were not sensitive to 5-fluorouracil (5-FU) treatment, unlike wild-type HCT116 cells. Interestingly, co-treatment with DHA could effectively restore the anticancer effect of 5-FU against HCT116 TP53−/− cells, which manifested as the inhibition of proliferation and induction of reactive oxygen species (ROS)-mediated apoptosis and was accompanied by the upregulation of B-cell lymphoma 2 (BCL-2) and downregulation of the BCL-2-associated X protein (BAX). These findings suggested that DHA could effectively sensitize cells to 5-FU through ROS-mediated apoptosis and the alteration of the BCL-2/BAX expression ratio, which indicated that this may be one of the mechanisms of the DHA-promoted 5-FU anticancer effect.

Evidence for additionally increased apoptosis in the peripheral blood mononuclear cells of major depressive patients with a high risk for suicide.

Several studies have suggested a pathophysiological role of blood cell apoptosis in major depressive disorder (MDD). The aim of this study was to evaluate mRNA expression levels of Bcl-2, Bax, and Fas in peripheral blood mononuclear cells (PBMCs) of MDD patients with a high risk for suicide relative to those without a high risk for suicide as well as healthy subjects. The mRNA expression of Bcl-2, Bax, and Fas as well as the Bcl-2/Bax ratio was examined in the PBMCs of 30 MDD patients with a high risk for suicide, 30 MDD patients without a high risk for suicide, and 30 healthy controls. The mRNA expression of target genes was measured using real-time quantitative Polymerase Chain Reaction (PCR). FAS mRNA expression was significantly increased, and Bcl-2 mRNA expression and the Bcl-2/Bax expression ratio were significantly decreased, in the PBMCs of MDD patients with or without a high risk for suicide attempts compared to healthy controls (p < .001). However, Bax mRNA expression was significantly increased only in MDD patients with a high risk for suicide. Moreover, MDD patients with a high risk for suicide had increased Bax and FAS mRNA expression and decreased Bcl-2 and Bcl-2/Bax ratio when compared to patients without risk for suicide (p < .001). Our findings may support the role of both internal and external apoptotic pathways in the interplay between the immune system and depressive symptoms, especially in patients with a high risk for suicide.

Effect of allyl isothiocyanate on the viability and apoptosis of the human cervical cancer HeLa cell line in vitro.

The present study aimed to investigate the effect of allyl isothiocyanate (AITC) on the viability and apoptosis of the human cervical cancer HeLa cell line in vitro, and to explore the potential underlying mechanisms of this. HeLa cells were treated with varying concentrations of AITC for different durations. The cell viability was then measured using a Cell Counting kit-8 assay and the apoptosis rate of the cells was detected using flow cytometry. Additionally, the B cell lymphoma-2 (Bcl-2) and Bcl-2-associated X protein (Bax) mRNA expression levels were determined by reverse transcription-quantitative polymerase chain reaction, while the Bax and Bcl-2 protein expression levels in cells were detected by western blot analysis. AITC was revealed to inhibit the viability of HeLa cells. AITC was revealed to induce the apoptosis of HeLa cells, as the apoptosis rate increased gradually with an increase in the dose. As the concentration of AITC increased, the Bax mRNA expression level increased, whilst the Bcl-2 mRNA expression level decreased. Furthermore, the Bax protein expression intensity increased whilst Bcl-2 protein expression intensity decreased, thereby resulting in a decrease in the ratio of Bcl-2/Bax proteins. AITC may inhibit cell viability by inducing the apoptosis of HeLa cells and this may be accounted for by the imbalance in the Bcl-2/Bax expression ratio.

Effect of oocyte vitrification before and after in vitro maturation towards Bcl-2, Bax and Bcl-2/Bax ratio expression

Objectives: to compare the expression of Bcl-2, Bax and Bcl-2/Bax ratio in cumulus cell and oocyte between vitrified oocyte pre and post in vitro maturation.Materials and Methods: Maturation was operated in medium TC 100 µl for 24 hours. Vitrification begins with washing oocyte in PBS basic medium supplemented of 20% serum for 1-2 minutes, followed by equilibration medium PBS + 20% serum + 10% ethylene glycol for 10-14 minutes, then transferred to 20% serum + PBS + 0.5 M sucrose + 15% ethylene glycol + PROH 15% for 25-30 seconds. Thawing is processed by submerging the oocytes in the media: 1). PBS + 20% serum + 0.5 M sucrose, 2). PBS + 20% serum + 0.25 M sucrose, and 3). PBS + 20% serum + 0.1 M sucrose. Imunocytochemistry observed the expression of Bcl-2, bax and Bcl-2/bax ratio.Results: Bcl-2 expression on oocyte in control group differed significantly with treatment group, Bcl-2 expression on cumulus in control group differed significantly with treatment 1 group. Bax expression on oocyte in control group differed significantly with treatment group. Bax expression on cumulus in control group differed significantly with treatment group. Bcl-2/Bax expression ratio on oocyte and cumulus did not differ significantly in all groupConclusion: No difference Bcl-2/Bax expression ratio on oocyte and cumulus between vitrified oocyte pre and post in vitro maturation.