Bax Protein Ratio Research Articles

The effect of selenium on the proliferation of bovine endometrial epithelial cells in a lipopolysaccharide-induced damage model

BackgroundEndometritis is a common bovine postpartum disease. Rapid endometrial repair is beneficial for forming natural defense barriers and lets cows enter the next breeding cycle as soon as possible. Selenium (Se) is an essential trace element closely related to growth and development in animals. This study aims to observe the effect of Se on the proliferation of bovine endometrial epithelial cells (BEECs) induced by lipopolysaccharide (LPS) and to elucidate the possible underlying mechanism.ResultsIn this study, we developed a BEECs damage model using LPS. Flow cytometry, cell scratch test and EdU proliferation assay were used to evaluate the cell cycle, migration and proliferation. The mRNA transcriptions of growth factors were detected by quantitative reverse transcription-polymerase chain reaction. The activation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) and Wnt/β-catenin pathways were detected by Western blotting and immunofluorescence. The results showed that the cell viability and BCL-2/BAX protein ratio were significantly decreased, and the cell apoptosis rate was significantly increased in the LPS group. Compared with the LPS group, Se promoted cell cycle progression, increased cell migration and proliferation, and significantly increased the gene expressions of TGFB1, TGFB3 and VEGFA. Se decreased the BCL-2/BAX protein ratio, promoted β-catenin translocation from the cytoplasm to the nucleus and activated the Wnt/β-catenin and PI3K/AKT signaling pathways inhibited by LPS.ConclusionsIn conclusion, Se can attenuate LPS-induced damage to BEECs and promote cell proliferation and migration in vitro by enhancing growth factors gene expression and activating the PI3K/AKT and Wnt/β-catenin signaling pathways.

Screening of the most efficacious lactic acid bacteria strain for myocardial infarction recovery and verification and exploration of its functions and mechanisms.

Screening efficient strains by cell platform is cost-effective, but to date, no screening experiments have been performed for targeted lactic acid bacteria with hypoxic/reoxygenation (H/R)-treated cardiomyocytes, and their effects on the phosphoinositide 3-kinase (PI3K)/protein kinase b (Akt)/endothelial nitric oxide synthase (eNOS) pathway in myocardial infarction (MI) are unclear. Here we activated 102 strains of lactic acid bacteria and inoculated them into MRS medium for fermentation. The fermentation supernatants of the lactic acid bacteria were incubated with an H/R model of H9C2 cells. We found that Bifidobacterium longum ZL0210 had the greatest potential for inhibiting the apoptosis of H/R-induced H9C2 cells. Furthermore, it significantly increased the expression of heme oxygenase-1 (HO-1) and quinone oxidoreductase 1 (NQO1) in H9C2 cardiomyocytes, as well as the Bcl-2/Bax protein ratio, protecting damaged myocardial cells via an anti-apoptotic pathway. Intragastric administration of B. longum ZL0210 to mice for one week before and after establishment of an MI model drastically attenuated the myocardial cell hypertrophy and fibrosis of the MI mice. Meanwhile, B. longum ZL0210 significantly reduced the secretion of myocardial enzymes, increased the activity of antioxidant enzymes, and inhibited lipid-oxidative malondialdehyde (MDA) levels. Moreover, it upregulated the expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) protein and the phosphorylation levels of PI3K, Akt, and eNOS, resulting in increased NO contents. In summary, we screened 102 strains of lactic acid bacteria with a cell platform and determined that B. longum ZL0210 was a favorable candidate for protecting the myocardium. We are the first to reveal the protective effects of B. longum ZL0210 for MI via activation of the PI3K/Akt/eNOS pathway through TRAIL.

ALKBH5 alleviates hypoxia postconditioning injury in D-galactose-induced senescent cardiomyocytes by regulating STAT3.

Ischemic postconditioning (I/Post) reduces ischemia/reperfusion (I/R) injury by activating endogenous cardioprotection mechanisms, such as the JAK/STAT3 and PI3K/Akt pathways, which offer a traditional approach to myocardial protection. According to the previous study, cardioprotection by I/Post may lost in aged mice, and in our previous research, hypoxic postconditioning (H/Post) lacked a protective effect in senescent cardiomyocytes, which was associated with low expression of long noncoding RNA (lncRNA) H19. The N6-methyladenosine (m6A) modification is a dynamic and reversible process that has been confirmed plays a role in cardiovascular diseases. However, the mechanisms of m6A modification in myocardial I/Post remains to be explored. Neonatal cardiomyocytes were isolated from 2-day-old Sprague Dawley rats and senescence was induced by D-galactose, followed by stimulation of hypoxia-reoxygenation (H/R) and H/Post. Hypoxic injury was evaluated by cell viability and the Bcl-2/Bax protein ratio. Total m6A levels were measured using a colorimetric m6A RNA Methylation Quantification Kit and the m6A modified and differentially expressed mRNA was determined by methylated RNA immunoprecipitation (MeRIP). We found that H/Post increased m6A methylation and decreased RNA mA demethylase alkB homolog 5 (ALKBH5) expression in aged cardiomyocytes. Furthermore, ALKBH5 knockdown exacerbated injury following H/Post in senescent cardiomyocytes. Additionally, ALKBH5 regulated STAT3 expression by mediating its m6A modification and lncRNA H19/miR-124-3p. ALKBH5 also alleviated the H/Post injury induced by the low expression of STAT3 in senescent cardiomyocytes.

Emodin protects against homocysteine-induced cardiac dysfunction by inhibiting oxidative stress via MAPK and Akt/eNOS/NO signaling pathways

Elevated levels of plasma homocysteine (Hcy) causes severe cardiac dysfunction, which is closely associated with oxidative stress. Emodin, a naturally occurring anthraquinone derivative, has been shown to exert antioxidant and anti-apoptosis activities. However, whether emodin could protect against Hcy-induced cardiac dysfunction remains unknown. The current study aimed to investigate the effects of emodin on the Hcy-induced cardiac dysfunction and its molecular mechanisms. Rats were fed a methionine diet to establish the animal model of hyperhomocysteinemia (HHcy). H9C2 cells were incubated with Hcy to induce a cell model of Hcy-injured cardiomyocytes. ELISA, HE staining, carotid artery and left ventricular cannulation, MTT, fluorescence staining, flow cytometry and western blotting were used in this study. Emodin significantly alleviated the structural damage of the myocardium and cardiac dysfunction from HHcy rats. Emodin prevented apoptosis and the collapse of MMP in the Hcy-treated H9C2 cells in vitro. Further, emodin reversed the Hcy-induced apoptosis-related biochemical changes including decreased Bcl-2/Bax protein ratio, and increased protein expression of Caspase-9/3. Moreover, emodin suppressed oxidative stress in Hcy-treated H9C2 cells. Mechanistically, emodin significantly inhibited the Hcy-activated MAPK by reducing ROS generation in H9C2 cells. Furthermore, emodin upregulated NO production by promoting the protein phosphorylation of Akt and eNOS in injured cells. The present study shows that emodin protects against Hcy-induced cardiac dysfunction by inhibiting oxidative stress via MAPK and Akt/eNOS/NO signaling pathways.

Fabrication of neuroprotective silk-sericin hydrogel: potential neuronal carrier for the treatment and care of ischemic stroke

Ischemic stroke results in severe disabilities due to extensive cellular loss and the resulting impairment of brain functions. Current methods for regenerating brain tissue are ineffective. Stroke treatment requires innovative therapeutic techniques that are both safe and effective. For neuronal repair, a promising alternative is using a hydrogel-based tissue engineering technique that delivers neurotrophic cytokines and cells to injured sites. However, the limited encapsulation effectiveness, less in vivo cell survival ratio and cytokine loss make this strategy difficult to implement. We aim to design a biomaterial that can efficiently construct a matrix enriching the survival of cells and minimizing loss in vivo cytokines to overcome these constraints. We report the development of genipin conjugated sericin hydrogels (Gen-SH) with a high porous morphology and a moderate swelling rate utilizing sericin, a natural silk protein. In vitro, Gen-SH aids in the attachment and development of neurons. Our results indicate that sericin is inherently neuroprotective and neurotrophic, branching and publicizing axon extension and avoiding hypoxia-induced cell death in primary neurons. Notably, the breakdown products of Gen-SH inherit these capabilities, saving the expense of cytokines. Furthermore, we show that the Lkb1–Nuak1 pathway is required for this neurotrophic impact, whereas the Bcl-2/Bax protein ratio is necessary for the neuroprotective effect. Transplanted in vivo, Gen-SH has a high percentage of cell survival and promotes cell proliferation. Taking all this information into account, it's clear that Gen-SH can serve as a viable carrier for treatment and care for ischemic stroke healing, both in terms of delivering neuronal cells and protecting them from oxidative damage.

Deciphering the Molecular Mechanism of Red Raspberry in Apoptosis of Liver Cancer Cells.

Red raspberry contains a variety of bioactive ingredients and has high edible and medicinal value. Red raspberry extractions (RREs) have strong antioxidant capacity and anticancer ability in vivo and in vitro. This study was to explore the specific mechanism of RREs inhibiting the proliferation of liver cancer HepG2 cells and provide a theoretical basis for the prevention and treatment of liver cancer by RREs. HepG2 cells were cultured in vitro, and MTT assay was adopted to detect the effect of RREs on HepG2 cell activity. Colony formation assay was applied to detect the growth and proliferation of cells, cell apoptosis was detected by flow cytometry, and dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay was adopted to detect the effect of RREs on the production of reactive oxygen species (ROS) in cells. The effect of RREs on cell mitochondrial membrane potential was evaluated by mitochondrial membrane potential assay kit with JC-1 (JC-1 assay), and western blot was used to detect the expression of apoptosis-related proteins (B-cell lymphoma-2 (Bcl-2), Bcl-2-associated x (Bax), and Caspase-3), thus investigating the effect of RREs on the molecular mechanism of HepG2 cell apoptosis. The results showed that RREs could inhibit the proliferation activity of HepG2 cells and promote their apoptosis in a concentration-dependent manner. The level of ROS in HepG2 cells interfered by RREs increased markedly, while the cell mitochondrial membrane potential decreased sharply. As the concentration of HepG2 increased, the mitochondrial membrane potential reduced steeply. Western blot results showed that the expression of apoptosis-related protein Bcl-2 in the RREs treatment group dropped, but the expression of Bax and Caspase-3 rose. In summary, RREs could inhibit the proliferation of liver cancer HepG2 cells and promote their apoptosis. This inhibition might be executed by inducing HepG2 cells to produce ROS, a decrease in Bcl-2/Bax protein ratio, and an obvious reduction in mitochondrial membrane potential.

Effects of ODC on polyamine metabolism, hormone levels, cell proliferation and apoptosis in goose ovarian granulosa cells

Ornithine decarboxylase (ODC) plays an indispensable role in the process of polyamine biosynthesis. Polyamines are a pivotal part of living cells and have diverse roles in the regulation of cell proliferation and apoptosis, aging and reproduction. However, to date, there have been no reports about ODC regulating follicular development in goose ovaries. Here, we constructed ODC siRNA and overexpression plasmids and transfected them into goose primary granulosa cells (GCs) to elucidate the effects of ODC interference and overexpression on the polyamine metabolism, hormone levels, cell apoptosis and proliferation of granulosa cells. After interfering with ODC in GCs, the mRNA and protein levels of ODC and the content of putrescine were greatly decreased (P < 0.05). When ODC was overexpressed, ODC mRNA and protein levels and putrescine content were greatly increased (P < 0.05). The polyamine-metabolizing enzyme genes ornithine decarboxylase antizyme 1 (OAZ1) and spermidine / spermine-N1-acetyltransferase (SSAT) were significantly increased, and spermidine synthase (SPDS) was significantly decreased when ODC was downregulated (P < 0.05). OAZ1, SPDS and SSAT were significantly increased when ODC was upregulated (P < 0.05). In addition, after interference with ODC, progesterone (P4) levels in the culture medium of GCs increased greatly (P < 0.05), while the overexpression of ODC caused the P4 level to decrease significantly (P < 0.05). After ODC downregulation, granulosa cell activity was significantly reduced, the apoptosis rate was significantly increased, and the BCL-2 / BAX ratio was downregulated (P < 0.05). Under ODC overexpression, the activity of GCs was notably increased, the apoptosis rate was significantly reduced, and the BCL-2 / BAX protein ratio was upregulated (P < 0.05). Our study successfully induced ODC interference and overexpression in goose ovarian GCs, and ODC regulated mainly putrescine content in GCs with a slight influence on spermidine and spermine. Moreover, ODC participated in the adjustment of P4 levels in the culture medium of GCs, promoted granulosa cell proliferation and inhibited granulosa cell apoptosis.

Compound Dihuang Granule Inhibits Nigrostriatal Pathway Apoptosis in Parkinson's Disease by Suppressing the JNK/AP-1 Pathway.

Compound Dihuang Granule (CDG) is widely used in traditional Chinese medicine (TCM) for the treatment of Parkinson’s disease (PD). It has been shown to alleviate PD symptoms. However, the molecular mechanisms of its action have not been established. To establish the molecular mechanisms of CDG against PD, we used TCM network pharmacology methods to predict its molecular targets and signaling pathways, followed by experimental validation. The Core Protein protein interaction (PPI) network of the 150 intersections between CDG and PD-related genes, comprising 23 proteins, including CASP3 (caspase-3), MAPK8 (JNK), FOS (c-Fos), and JUN (c-Jun). KEGG and GO analyses revealed that apoptotic regulation and MAPK signaling pathways were significantly enriched. Since c-Jun and c-Fos are AP-1 subunits, an important downstream JNK effector, we investigated if the JNK/AP-1 pathway influences CDG against apoptosis through the nigrostriatal pathways in PD rat models. Molecular docking analysis found that the top three bioactive compounds exhibiting the highest Degree Centrality following online database and LC-MS analysis had high affinities for JNK. Experimental validation analysis showed that CDG decreased the number of rotating laps and suppressed the levels of phosphorylated c-Jun, c-Fos, and JNK, as well as the number of TUNEL positive cells and the cleaved caspase-3 level in the nigrostriatal pathway. Furthermore, CDG treatment elevated the number of TH neurons, TH expression level, and Bcl-2/Bax protein ratio in a 6-OHDA-induced PD rat. These findings are in tandem with those obtained using SP600125, a specific JNK inhibitor. In conclusion, CDG suppresses the apoptosis of the nigrostriatal pathway and relieves PD symptoms by suppressing the JNK/AP-1 signaling pathway.

Neuroprotective and anti-inflammatory effects of myricetin 3-glucoside in a rat model of cerebral ischemia

Purpose: To investigate the effect of myricetin 3-glucoside (M3GLS) on middle cerebral artery occlusion (MCAO)-induced cerebral ischemia in a rat model, and the mechanism of action involved.Methods: A cerebral ischemia rat model was established using MCAO under 10 % chloral hydrate anesthesia. Neurological severity score was determined by analyzing reflex, motor and sensory functions, as well as balancing potential. Infarction volume was determined using triphenyl tetrazolium chloride dye, while counting of Nissl bodies was done after toluidine blue staining. The protein expression levels of Bax and Bcl-2 were assayed using western blotting, while cytokine levels were determined by enzyme-linked immunosorbent assay (ELISA).Results: Treatment of cerebral ischemia rats with M3GLS effectively reduced infarct volume, when compared to vehicle-treated group (p &lt; 0.05). Moreover, M3GLS treatment significantly increased the population of Nissl bodies and effectively improved neurologic scores (p &lt; 0.05). In M3GLS-pretreated rats, cerebral ischemia-induced elevation of protein expressions of TNF-α, IL-6 and IL-1β were significantly suppressed. M3GL treatment significantly reversed cerebral ischemia-mediated downregulation of Bcl-2 protein level, but markedly reduced cerebral ischemia-induced upregulation of Bax protein level (p &lt; 0.05).Conclusion: M3GLS exerts protective effect against cerebral ischemia-induced brain injury in rats via downregulation of inflammatory cytokines. It reduces infarction volume in the brain of cerebral ischemia rats, and regulates Bcl-2/Bax protein ratio. Thus, M3GLS has a potential for use in the clinical management of cerebral ischemia.&#x0D; Keywords: Myricetin, Neuroprotection, Anti-inflammation, Cerebral ischemia, Cytokines, Infarction

Physcion, a tetra-substituted 9,10-anthraquinone, prevents homocysteine-induced endothelial dysfunction by activating Ca2+- and Akt-eNOS-NO signaling pathways

BackgroundHomocysteine (Hcy) induced vascular endothelial dysfunction is known to be closely associated with oxidative stress and impaired NO system. 1,8-Dihydroxy-3-methoxy-6-methylanthracene-9,10-dione (physcion) has been known to has antioxidative and anti-inflammatory properties. PurposeThe purpose of the present study was to define the protective effect of physcion on Hcy-induced endothelial dysfunction and its mechanisms involved. Study Design and MethodsHyperhomocysteinemia (HHcy) rat model was induced by feeding 3% methionine. A rat thoracic aortic ring model was used to investigate the effects of physcion on Hcy-induced impairment of endothelium-dependent relaxation. Two doses, low (L, 30 mg/kg/day) and high (H, 50 mg/kg/day) of physcion were used in the present study. To construct Hcy-injured human umbilical vein endothelial cells (HUVECs) model, the cells treated with 3 mM Hcy. The effects of physcion on Hcy-induced HUVECs cytotoxicity and apoptosis were studied using MTT and flow cytometry. Confocal analysis was used to determine the levels of intracellular Ca2+. The levels of protein expression of the apoptosis-related markers Bcl-2, Bax, caspase-9/3, and Akt and endothelial nitric oxide synthase (eNOS) were evaluated by western blot. ResultsIn the HHcy rat model, plasma levels of Hcy and malondialdehyde (MDA) were elevated (20.45 ± 2.42 vs. 4.67 ± 1.94 μM, 9.42 ± 0.48 vs. 3.47 ± 0.59 nM, p < 0.001 for both), whereas superoxide dismutase (SOD) and nitric oxide (NO) levels were decreased (77.11 ± 4.78 vs. 115.02 ± 5.63 U/ml, 44.51 ± 4.45 vs. 64.18 ± 5.34 μM, p < 0.001 and p < 0.01, respectively). However, treatment with physcion significantly reversed these changes (11.82 ± 2.02 vs. 20.45 ± 2.42 μM, 5.97 ± 0.72 vs. 9.42 ± 0.48 nM, 108.75 ± 5.65 vs. 77.11 ± 4.78 U/ml, 58.14 ± 6.02 vs. 44.51 ± 4.45 μM, p < 0.01 for all). Physcion also prevented Hcy-induced impairment of endothelium-dependent relaxation in HHcy rats (1.56 ± 0.06 vs. 15.44 ± 2.53 nM EC50 for ACh vasorelaxation, p < 0.05 vs. HHcy). In Hcy-injured HUVECs, physcion inhibited the impaired viability, apoptosis and reactive oxygen species. Hcy treatment significantly increased the protein phosphorylation levels of p38 (2.26 ± 0.20 vs. 1.00 ± 0.12, p <0.01), ERK (2.11 ± 0.21 vs. 1.00 ± 0.11, p <0.01) and JNK. Moreover, physcion reversed the Hcy-induced apoptosis related parameter changes such as decreased mitochondrial membrane potential (MMP) and Bcl-2/Bax protein ratio, and increased protein expression of caspase-9/3 in HUVECs. Furthermore, the downregulation of Ca2+, Akt, eNOS and NO caused by Hcy were recovered with physcion treatment in HUVECs. ConclusionPhyscion prevents Hcy-induced endothelial dysfunction by activating Ca2+- and Akt-eNOS-NO signaling pathways. This study provides the first evidence that physcion might be a candidate agent for the prevention of cardiovascular disease induced by Hcy.

Protective effects of oxymatrine on homocysteine-induced endothelial injury: Involvement of mitochondria-dependent apoptosis and Akt-eNOS-NO signaling pathways

Homocysteine (Hcy) is an independent risk factor in the development of cardiovascular diseases (CVD). Hyperhomocysteinemia (HHcy), induces the injury of vascular endothelial cells via oxidative stress. Oxymatrine (OMT), one of the main components of Sophora flavescens, has displayed anti-inflammatory, anti-oxidant and anti-apoptotic activity. However, the effect of OMT on the Hcy-induced endothelial injury is not clearly defined yet. The aim of this study was to determine the protective effect of OMT on the Hcy-induced endothelial injury and its mechanisms involved. Human umbilical vein endothelial cells (HUVECs) were cultured in vitro. Methyl thiazolyl tetrazolium assay (MTT), fluorescence staining, flow cytometry and western blotting were used in this study. OMT prevented the Hcy-induced toxicity and apoptosis in HUVECs. Moreover, OMT suppressed Hcy-induced increases in reactive oxygen species, lactate dehydrogenase, malondialdehyde levels and increased superoxide dismutase levels. OMT reversed the Hcy-induced decrease in the protein expression of nuclear factor erythroid-2-related factor 2 (Nrf2). In addition, OMT reversed the Hcy-induced apoptosis related biochemical changes such as decreased mitochondrial membrane potential and Bcl-2/Bax protein ratio, and increased protein expression of caspase-9 and caspase-3. Furthermore, OMT elevated the phosphorylation levels of Akt and eNOS, and the formation of nitric oxide (NO) in injured cells. These results suggest that OMT prevents Hcy-induced endothelial injury by regulating mitochondrial-dependent apoptosis and Akt-eNOS-NO signaling pathways concomitantly with accentuation of Nrf2 expression.

MicroRNA‑9‑5p downregulates Klf4 and influences the progression of hepatocellular carcinoma via the AKT signaling pathway.

Krüppel-like factor 4 (Klf4) is a transcriptional factor involved in the progression of hepatocellular carcinoma (HCC). However, the underlying regulatory mechanisms associated with the Klf4 gene as a tumor suppressor in HCC remain unclear. microRNAs (miRNAs or miRs) are a series of small non-coding RNAs that serve a vital role in regulating gene expression via their influence on protein translation and the associated degradation of mRNA. The mRNA expression levels of the miRNA, miR-9-5p, and Klf4 were measured using reverse transcription-quantitative polymerase chain reaction. The protein expression levels of Klf4, protein kinase B (AKT), phosphorylated (p-)AKT, mechanistic target of rapamycin (mTOR), p-mTOR, B cell lymphoma-2 (Bcl-2) and Bcl-2-associated X protein (Bax) were determined by western blot analysis. Dual luciferase reporter assay was used to confirm a direct interaction between miR-9-5p and the 3′-untranslated region (3′-UTR) sequence of Klf4. Cell counting kit-8 assay, wound healing assay, Transwell migration assay and flow cytometric analysis were performed to evaluate the proliferative, migratory and apoptotic capabilities of the HCC cells. In the present study, miR-9-5p was revealed to be overexpressed in HCC as a novel upstream gene of Klf4. miR-9-5p expression was inversely associated with Klf4 expression in clinical samples. Additionally, Kaplan-Meier analysis revealed a markedly poor prognosis of HCC in the miR-9-5p high-expression group. Bioinformatics analysis revealed that miR-9-5p bound directly to the 3′-UTR of Klf4, which reduced the expression levels of Klf4. The miR-9-5p/Klf4 axis promoted HCC proliferation and migration, and inhibited HCC apoptosis. Furthermore, miR-9-5p upregulated the Bcl-2/Bax protein ratio and activated AKT/mTOR signaling. Taken together, these data demonstrated that the miR-9-5p/Klf4 axis was able to promote HCC progression, which may occur via regulation of the AKT signaling pathway, highlighting a potential novel target in HCC treatment.

Essential Oil from Carpesium abrotanoides L. Induces Apoptosis via Activating Mitochondrial Pathway in Hepatocellular Carcinoma Cells.

The effects of essential oil from Carpesium abrotanoides L. (CAEO) on the proliferation and apoptosis of human hepatic cancer cells were investigated in this study. MTT assays indicated that CAEO inhibited the proliferation of HCC cells with the IC50 values ranging from 41.28±3.06 to 130.36±20.79 μg/mL. Moreover, many obviously nuclear morphological changes of apoptotic cells in CAEO-treated HepG2 cells were detected by Hoechst 33258 staining and fluorescence microscopy. Flow cytometry was used to detect cell apoptosis and cell cycle, and noticeable findings showed that CAEO arrested cell-cycle at S and G2/M phases. The decreased Bcl-2/Bax protein ratio and the activation of caspase-3, caspase-9 were also detected by Western blotting. All results suggested that CAEO is a potential agent to fight against liver cancer, and the mitochondria-mediated intrinsic apoptotic pathway could be involved in CAEO-mediated apoptosis of human liver carcinoma cells.

Cumulus-oocyte interactions and programmed cell death in bovine embryos produced in vitro

This study investigated the interactions between cumulus cells (CCs) and oocytes and programmed cell death in bovine cumulus-oocyte complexes (COCs) with different morphological characteristics. DNA fragmentation was assessed in CCs at 0 and 24 h of maturation, as well as parthenogenetic developmental competence on the 9th day post-activation, blastocyst quality and BCL-2 and BAX transcript levels in matured CCs. Most immature oocytes in the COC-A group (full cumulus and several compact layers) were in the initial germinal vesicle (iGV) stage, exhibiting minimal or no DNA damage. In contrast, after follicle removal, the COCB (partial cumulus and one or two cell layers) and C (expanded cumulus) groups presented in more advanced GV stages and exhibited DNA fragmentation. After maturation, significant increases in fragmented nuclei were noted in COCC and COCB groups. Embryos resulting from the COC-A developed more rapidly and had increased competence compared to embryos resulting from groups COCB and COCC. The COCB group exhibited the highest BAX protein levels and a reduced BCL-2/BAX protein ratio. The results show a negative correlation between nuclear fragmentation and embryonic development potential in COCs with different morphologies. In addition, a low BCL-2/BAX protein ratio might be associated with an increase in nuclear fragmentation in CCs.

Inhibition of DNA‑PK activity sensitizes A549 cells to X‑ray irradiation by inducing the ATM‑dependent DNA damage response.

Non-small cell lung cancer (NSCLC) is radioresistant to X-rays due to powerful cellular DNA damage repair mechanisms. DNA-dependent protein kinase (DNA-PK) is a key enzyme involved in DNA damage repair and the phenomenon and molecular mechanism of NSCLC radionsensitivity were investigated following inhibition of DNA-PK activity. In the present study A549 cells were treated with the DNA-PK inhibitor NU7026 and/or siRNA directed against ataxia telangiectasia mutated (ATM), followed by exposure to 4 Gy X-ray irradiation. Radiosensitivity, DNA damage, apoptosis and protein expression were measured by colony formation assay, γH2AX foci immunofluorescence, Annexin V/PI staining and western blotting, respectively. A Balb/c-nu/nu xenograft mouse model was established by subcutaneous injection of A549 cells and was used to examine the effect of administering NU7026 via intraperitoneal injection prior to 4 Gy X-ray exposure. The xenograft tumors were weighed and observed by hematoxylin and eosin staining after irradiation. NU7026 treatment followed by X-ray irradiation significantly decreased the colony formation ratio of A549 cells, and increased γH2AX foci and cell apoptosis. Furthermore, the combined treatment of NU7026 and X-rays resulted in growth inhibition and cell apoptosis in A549 xenograft tumors. Consequently, apoptosis regulators full-length transactivating (TA) p73 and an N-terminally truncated (DN) p73 were upregulated and downregulated respectively, leading to activation of glucosyltransferases and Rab-like GTPase activators and myotubularins domain-containing 4 (GRAMD4) protein to reduce the Bcl-2/Bax protein ratio. In addition, ATM siRNA efficiently prevented γH2AX foci formation, and enhanced NU7026-induced inhibition of survival and promoted apoptosis. In conclusion, inhibition of DNA-PK activity increased the radiosensitivity of A549 cells to X-ray irradiation. NU7026 treatment activated the ATM-dependent DNA damage response and induced p73 apoptosis pathway. DNA-PK inhibitor may be an effective constituent of radiosensitization products. DNA damage repair pathway could be a potential target for radiosensitization.

Neuroprotective effect of phosphocreatine on oxidative stress and mitochondrial dysfunction induced apoptosis in vitro and in vivo: Involvement of dual PI3K/Akt and Nrf2/HO-1 pathways.

Methylglyoxal (MGO), an active metabolite of glucose, is observed in high levels in the tissues and blood of diabetic patients. Phosphocreatine (PCr), a high-energy phosphate compound, exhibits a range of pharmacological actions but little is well known of its neuroprotective action. The aim of the present study was to investigate the neuroprotective effects and the possible mechanisms of PCr. Diabetes is closely associated with neurodegenerative diseases, leading not only to the peripheral nervous system (PNS) and but also to central nervous system (CNS) damage. Therefore, we established two rat models of diabetes in vivo induced by MGO and streptozocin (STZ) respectively, while utilized differentiated PC-12 cells in vitro. Treatment of PC-12 cells with PCr markedly attenuated MGO-induced change of viability, apoptosis, accompanied by decreased levels of caspase-3, casapse-9 and Bcl-2/Bax protein ratio. Determination of cellular respiratory function was performed with intact PC-12 cells and homogenized hippocampal neuron tissue of rat. Reactive oxygen species (ROS) generation was assessed by membrane permeable fluorescent probe DCFH-DA. The expressions of Akt, Nrf2 and HO-1 were examined by Western blot. PCr pretreatment significantly reduced oxidative stress-induced high LDH, MDA level, and ROS production of PC-12 cells. PCr pretreatment also significantly decreased mitochondrial dysfunction in vitro and in vivo. In addition, PCr pretreatment increased the expression of p-Akt, Nrf2 and HO-1, and reduced the apoptosis. Moreover, the expression of Cleaved caspase3 was partially increased and the p-Akt, Nrf2 and HO-1 was partially reduced by a PI3K inhibitor (LY294002). While, compared with LY294002 groups, pre-treatment with PCr at the concentrations of 20 mM significantly reduced the expression of Cleaved caspase3 and increased the expression of p-Akt, Nrf2 and HO-1. Molecular docking assay showed that PCr possessed powerful affinity towards to Akt with lower binding energy. In conclusion, the neuroprotective effects of PCr in vitro and in vivo rely on normalizing mitochondrial function and reducing oxidative stress via Akt mediated Nrf2/HO-1 pathway, suggesting that PCr may be a novel therapeutic candidate for the treatment of diabetes-associated neurodegenerative diseases.

The effect of curcumin and PI3K/Akt inhibitor on monosodium glutamate-induced rat thymocytes toxicity.

Monosodium glutamate (MSG), the sodium salt of glutamic acid, is widely used in modern nutrition as flavor enhancer. However, it has been shown that curcumin has ability to induce apoptosis in the cells of the immune system. In the present study, we evaluate the potential protective effects of curcumin in MSG-induced apoptosis and signaling pathway which may be involved. Rat thymocytes were treated with increased (1, 10, 50 mM) MSG concentrations and/or curcumin (3 μM). Cell apoptosis rate, reactive oxygen species (ROS) production, mitochondrial membrane potential (MMP), Bcl-2, Bax protein expression and caspase-3 activity were determined after 24 hours of incubation. Treatment with MSG resulted with increased apoptosis, ROS production and caspase-3 activity, followed with decreased MMP and Bcl-2/Bax protein ratio. Inhibition of caspase-3 and caspase-9 activity reduced cell apoptosis, indicating the involvement of mitochondrial apoptotic pathway. Co-treatment with curcumin markedly reduced apoptosis and ROS production, together with increased MMP and Bcl2/Bax protein ratio. Inhibition of PI3K/Akt signaling pathway abolished protective effect of curcumin in MSG-induced toxicity in rat thymocytes. Obtained findings suggest that curcumin may attenuate the MSG-induced apoptosis through PI3K/Akt signaling pathway which could be useful in preventing the potential deficiencies in T cell-mediated immunity.

Expression of Apoptosis Related and Proliferative Proteins in Malignant Lympho-Proliferative Disorders

The current study aimed to perform an immunohistochemical analysis of patterns of apoptotic and cell proliferative related protein expression in different histological grades and immune phenotypes of malignant lymphomas and other lymphoproliferative disorders. This observational study was carried on 60lymph node biopsies of lymphoproliferative disorders. The biopsies were analyzed histologically and immunohistochemically. A total of 60 lymph node biopsies were included in the study, of which 81.6% were of malignant lympho-proliferative lesions. The majority of the biopsies were B-cell (66%) and were grouped in the intermediate grade. Bax and BCL-2 protein expression was presented by percentage of immune positive neoplastic cells per 10fields and graded on a scale of 1 to4. A Bcl-2, Bax Protein Ratio (BBPR) was determined for each case by dividing the estimated Bcl-2 protein (percentage of Bcl-2 positive cells x Bcl-2 staining intensity) by the estimated Bax protein (percentage of Bax positive cells x Bax immunostaining intensity). The mean BBPR was found to be significantly higher in indolent lymphomas (2.64 ± 1.3) as compared to aggressive lymphomas (0.47 ± 0.9) (P<0.01). The expression of P53 and PCNA in 35 biopsies of Non Hodgkin Lymphomas (NHL) was found to increase from low to high grade tumors. A significant correlation was found between BBPR and predicted biological behavior of indolent and aggressive lymphomas. This indicates the important role of Bcl-2 and Bax in biological behavior of lymphomas. Furthermore, P53 and PCNA expression were found to increase from low to high-grade tumors suggesting their prognostic value in NHL.

Geniposide improves insulin production and reduces apoptosis in high glucose-induced glucotoxic insulinoma cells

Our previous work revealed that in the pancreatic β cell line, geniposide modulated ATP production and glucose-stimulated insulin secretion (GSIS) induced by the acute stimulation of high glucose concentration. However, the effects of geniposide on functional impairment and the mass of β-cells exposed to elevated levels of glucose remains unknown. In the present study, impaired GSIS and restrained proliferation were observed in the prolonged culture of insulinoma INS-1 cells with 33mM of glucose (high glucose). Our results indicate that the glucose-induced impairment of insulin release was significantly reverted by the inclusion of 1 or 10μM of geniposide. Moreover, induction of the phosphorylation of AMP-activated protein kinase (AMPK) was observed, which promoted the utilization of nutrient stores for energy production. AMPK phosphorylation was enhanced by an increased number of INS-1 cells, and the increased expression of AMPK downstream target heme oxygenase 1 (HO-1), under high glucose concentration. Furthermore, geniposide protected rat insulinoma cells from apoptosis in high-glucose concentrations. We have shown that these effects were associated with an increased apoptosis-related Bcl-2/BAX protein ratio. In conclusion, geniposide dose dependently improves β-cell function and increases the proliferation of β-cells exposed to prolonged hyperglycemia.

EGCG protects against homocysteine-induced human umbilical vein endothelial cells apoptosis by modulating mitochondrial-dependent apoptotic signaling and PI3K/Akt/eNOS signaling pathways.

Homocysteine (Hcy) induced vascular endothelial injury leads to the progression of endothelial dysfunction in atherosclerosis. Epigallocatechin gallate (EGCG), a natural dietary antioxidant, has been applied to protect against atherosclerosis. However, the underlying protective mechanism of EGCG has not been clarified. The present study investigated the mechanism of EGCG protected against Hcy-induced human umbilical vein endothelial cells (HUVECs) apoptosis. Methyl thiazolyl tetrazolium assay (MTT), transmission electron microscope, fluorescent staining, flow cytometry, western blot were used in this study. The study has demonstrated that EGCG suppressed Hcy-induced endothelial cell morphological changes and reactive oxygen species (ROS) generation. Moreover, EGCG dose-dependently prevented Hcy-induced HUVECs cytotoxicity and apoptotic biochemical changes such as reducing mitochondrial membrane potential (MMP), decreasing Bcl-2/Bax protein ratio and activating caspase-9 and 3. In addition, EGCG enhanced the protein ratio of p-Akt/Akt, endothelial nitric oxide synthase (eNOS) activation and nitric oxide (NO) formation in injured cells. In conclusion, the present study shows that EGCG prevents Hcy-induced HUVECs apoptosis via modulating mitochondrial apoptotic and PI3K/Akt/eNOS signaling pathways. Furthermore, the results indicate that EGCG is likely to represent a potential therapeutic strategy for atherosclerosis associated with Hyperhomocysteinemia (HHcy).