mRNA Expression Levels Of Caspase-3 Research Articles

Effect of Boron on Thymic Cytokine Expression, Hormone Secretion, Antioxidant Functions, Cell Proliferation, and Apoptosis Potential via the Extracellular Signal-Regulated Kinases 1 and 2 Signaling Pathway.

Boron is an essential trace element in animals. Appropriate boron supplementation can promote thymus development; however, a high dose of boron can lead to adverse effects and cause toxicity. The influencing mechanism of boron on the animal body remains unclear. In this study, we examined the effect of boron on cytokine expression, thymosin and thymopoietin secretion, antioxidant function, cell proliferation and apoptosis, and extracellular signal-regulated kinases 1 and 2 (ERK1/2) pathway in the thymus of rats. We found that supplementation with 10 and 20 mg/L boron to the drinking water significantly elevated levels of interleukin 2 (IL-2), interferon γ (IFN-γ), interleukin 4 (IL-4), and thymosin α1 in the thymus of rats (p < 0.05), increased the number of positive proliferating cell nuclear antigen (PCNA+) cells and concentrations of glutathione peroxidase (GSH-Px) and phosphorylated extracellular signal-regulated kinase (p-ERK) (p < 0.05), and promoted mRNA expression of PCNA and ERK1/2 in thymocytes (p < 0.05). However, the number of caspase-3+ cells and the expression level of caspase-3 mRNA were reduced (p < 0.05). Supplementation with 40, 80, and 160 mg/L boron had no apparent effect on many of the above indicators. In contrast, supplementation with 480 and 640 mg/L boron had the opposite effect on the above indicators in rats and elevated levels of pro-inflammatory cytokines, such as interleukin 6 (IL-6), interleukin 1β (IL-1β), and tumor necrosis factor α (TNF-α) (p < 0.05). Our study showed that supplementation of various doses of boron to the drinking water had a U-shaped dose-effect relationship with thymic cytokine expression, hormone secretion, antioxidant function, cell proliferation, and apoptosis. Specifically, supplementation with 10 and 20 mg/L boron promoted thymocyte proliferation and enhanced thymic functions. However, supplementation with 480 and 640 mg/L boron inhibited thymic functions and increased the number of apoptotic thymocytes, suggesting that the effects of boron on thymic functions may be caused via the ERK1/2 signaling pathway.

Protective effect of caffeine citrate on neonatal rat brain white matter damage induced by infection

Objective To investigate the protective effect of caffeine citrate on white matter damage in 2-day-old neonatal rats induced by postnatal infection. Methods Forty-eight 2-day-old SD rats were randomly divided into the control group (group A, n=16), lipopolysaccharide(LPS) infection group (group B, n=16), and caffeine citrate intervention group (group C, n=16) according to the random table method.The newborn rats of group B and C were continuously injected LPS 0.6 mg/kg intraperitoneally for 5 days from 2 days old, and the newborn rats of group A were continuously injected by an equal volume of 9 g/L saline intraperitoneally.Group C was continuously injected by caffeine citrate 10 mg/kg intraperitoneally for 7 days from 4 days old; equal volume of 9 g/L saline was injected into group A and B for 7 days continuously.At 12 days old, 8 rats of each group were sacrificed randomly to evaluate the expression of myelin basic protein(MBP) subcortical white matter by immunohistochemical method.Both sides of hippocampus of the rest 8 rats of each group were taken out in ice surface rapidly.The left hippocampus was used to detect the expression of MBP and Caspase-3 by Western blot method, and the right hippocampus was used to evaluate the MBP and Caspase-3 protein level by quantitative real-time PCR(qPCR) method. Results The mean integral optical density (IOD) of subcortical MBP positive expression in group A, group B and group C were 132.64±1.94, 102.43±2.12, 114.25±2.04, and the difference was statistically significant among 3 groups (F=22.912, P<0.05). The relative expression levels of MBP mRNA of 3 groups in hippocampus were 0.79±0.01, 0.39±0.03, 0.55±0.02, and the difference was statistically significant among 3 groups (F=18.584, P<0.05). The relative expression levels of MBP protein of 3 groups in hippocampus were 0.64±0.03, 0.31±0.03, 0.51±0.05, and the difference was statistically significant among 3 groups (F=25.780, P<0.05). The relative expression levels of Caspase-3 mRNA of 3 groups in hippocampus were 0.34±0.02, 0.74±0.03, 0.57±0.04, and the difference was statistically significant among 3 groups (F=6.105, P<0.05). The relative expression of Caspase-3 protein of 3 groups in hippocampus were 0.11±0.03, 0.36±0.02, 0.23±0.03, and the difference was statistically significant among 3 groups (F=40.541, P<0.05). Conclusions Caffeine citrate has showed protective effect on white matter damage in neonatal rats of 2 days old induced by postnatal infection.The mechanism may be related to inhibition of inflammatory response and reducing apoptosis. Key words: Caffeine citrate; Lipopolysaccharide; Brain damage; SD rats

Mechanisms of autophagy and apoptosis mediated by JAK2 signaling pathway after spinal cord injury of rats.

The aim of this study was to investigate the pathogenesis of autophagy and apoptosis mediated by Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) signal pathway after the onset of acute spinal cord injury (ASCI). A total of 45 Sprague-Dawley adult rats of either sex were selected for this study. The age of rats ranged from 8 to 10 weeks, and the average weight was 245 g. These rats were randomly divided into three groups, i.e. sham-operated group, model group, and the AG-490 intervention group (AG-490 is an inhibitor of JAK2). Each group contained 15 rats. Models were prepared using the modified Allen method. Five rats in each group were sacrificed at 6, 12 and 24 h, respectively, and the expression levels of p-JAK2 and p-STAT3 were detected in spinal cord tissue via western blot analysis. The levels of proinflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were detected via ELISA, positive expression of light chain 3 (LC3)-II of microtubule-associated protein 1 via immunofluorescence labeling method, and mRNA expression levels of caspase-3 and Bax/Bcl-2 via RT-PCR. In the model group, the expression levels of p-JAK2, p-STAT3, IL-6, TNF-α and LC3-II, and the mRNA expression levels of caspase-3 and Bax/Bcl-2 at all time-points were significantly higher than those in the AG-490 intervention group, and the levels in the sham-operated group were the lowest (p<0.05). In the model group, peak levels of p-JAK2 and p-STAT3 were attained at 12 h, but a decline was seen at 24 h; while increasing trend was seen in other indicators. In conclusion, JAK2/STAT3 signal pathway can mediate the activity of autophagy and apoptosis in an early stage after the onset of ASCI of rat.

Association between myocardial cell apoptosis and calpain-1/caspase-3 expression in rats with hypoxic-ischemic brain damage.

The present study aimed to investigate the association between myocardial cell apoptosis and calpain-1/caspase-3 expression in a rat model of hypoxic-ischemic brain damage (HIBD). A total of 64 newborn rats were divided into control (n=8; sacrificed on day 7) and HIBD groups (n=56). HIBD group rats were sacrificed 2, 12 or 24h, or 2, 3, 5 or 7days following HIBD (n=8/group). A terminal deoxynucleotidyl transferase dUTP nick-end labeling assay was performed to detect myocardial apoptotic cells and calculate the apoptosis index (AI), reverse transcription-polymerase chain reaction was performed to detect myocardial calpain-1/caspase-3 mRNA expression levels and a western blot analysis was conducted to detect calpain‑1 protein expression levels. The correlations between calpain‑1 and caspase‑3 expression levels and AI were analyzed. The results demonstrated that apoptotic myocardial cells in the HIBD groups were markedly increased compared with the control group, with AI peaking in the day 3 group. Caspase‑3 and calpain‑1 mRNA expression levels were increased from 2 and 12h following HIBD, respectively, with the most elevated levels in the day 2 group. Compared with the control group, calpain‑1 protein expression levels were increased from 2h, with the greatest expression levels in the day 3 group (P<0.05). Calpain‑1 mRNA and protein (76/80kDa) expression levels demonstrated positive linear correlations with AI (r=0.786, P=0.001; and r=0.853, P=0.001, respectively) Caspase-3 mRNA expression levels were positively correlated with AI (r=0.894; P=0.001). In conclusion, the present study demonstrated that in rats with HIBD, there is a positive correlation between increased apoptosis of myocardial cells and expression levels of calpain-1 and caspase-3.

The proliferative inhibition and apoptosis promotion of Smac on human lens epithelial cells

Background Posterior capsular opacification (PCO) is a primary complication after extracapsular cataract extraction.The mechanism of PCO is associated with proliferation, migration and epithelial-mesenchymal transition (EMT) of human lens epithelial cells (LECs). To explore the target treatment of PCO is very important. Objective This study was to investigate the biological effects of second mitochondria-derived activator of caspases (Smac) on the proliferation and apoptosis of LECs. Methods Human LECs line (HLE-B3) and Smac-overexpressed LECs line were cultured, and the cells were transfected using small interfering RNA (siRNA)-Smac3 plasmid with green fluorescent protein (GFP) for 24 hours.Different concentration of transforming growth factor -β2 (TGF-β2) (5, 10, 20 and 50 μg/ml) or 200 μmol/L H2O2 were added respectively into the culture medium to establish PCO model and oxidative stress model.Cell counting kit-8 (CCK-8) assay was used to compare the cell proliferative activity among PBS group, TGF-β2 group and Smac-hyperexpression+ TGF-β2 group.Flow cytometry was used to evaluate the apoptotic rate of the PBS group, H2O2 group and siRNA-Smac+ H2O2 group.The expressions of Smac, caspase-3 and proliferating cell nuclear antigen (PCNA) mRNA and their proteins in the cells were detected by real-time quantitative PCR (RT-PCR) and Western blot. Results The GFP+ cells were≥ 80% 12 hours after siRNA-Smac3 transfection, with the optimal plasmid of siRNA-Smac3.GFP+ cell rate was (72.32±2.31)% in the siRNA-Smac3 transfection group, which was significantly higher than that in the blank plasmid group([4.91±0.24]%) (t=116.342, P<0.001). The relevant expression levels of Smac was 35.21±4.11 in the Smac-hyperexpression group, and that in the blank plasmid group was 15.24±2.48, with a significant difference between them (t=215.47, P<0.05). The cell viability of 20 ng/ml TGF-β2 affected PBS group, TGF-β2 group and Smac-hyperepression+ TGF-β2 group was (98.4±1.7)%, (98.9±0.1)% and (64.2±3.1)%, and the cell viability of Smac-hyperepression+ TGF-β2 group was significantly lower in the Smac-hyperepression+ TGF-β2 group than that in the TGF-β2 group (P<0.05). The apoptotic rate in the PBS group, H2O2 group and siRNA-Smac+ H2O2 group were (2.9±1.2) %, (45.1±4.5)% and (27.5±1.8)%, and the apoptotic rate was evidently lower in the siRNA-Smac+ H2O2 group than that in the H2O2 group (P<0.05). RT-PCR results showed that the expression levels of caspase-3 mRNA in PBS group, H2O2 group and siRNA-Smac+ H2O2 group were 0.321±0.103, 0.715±0.112 and 0.479±0.209, respectively.Compared with the H2O2 group, the relative expression level of caspase-3 mRNA in siRNA-Smac+ H2O2 group was significantly decreased, the difference was statistically significant (P<0.05). The PCNA mRNA expression levels in PBS group, TGF-β2 group and Smac-hyperepression+ TGF-β2 group were 0.299±0.013, 0.645±0.102 and 0.490±0.209, respectively.Western blot results showed that the relative expression of caspase-3 protein in siRNA-Smac+ H2O2 group and H2O2 group was 0.712±0.012 and 0.973±0.051, with significant difference between the two groups (t=132.52, P<0.05). The relative expression of PCNA protein in Smac-hyperepression+ TGF-β2 group was 0.782±0.212, which was lower than 1.126±0.251 in the TGF-β2 group (P<0.05). Conclusions Smac may prevent and treat PCO by inhibiting the proliferation and promoting apoptosis of human LECs. Key words: Smac; Apoptsis; Proliferation; After-cataract; Human; Lens epithlial cells; Small interfering RNA

The metastasis suppressor gene KISS-1 regulates osteosarcoma apoptosis and autophagy processes.

The expression of the metastasis suppressor gene KISS-1 in osteosarcoma cells during apoptosis and autophagy was evaluated. MG-63 osteosarcoma cells were transfected with either KISS-1 overexpression or KISS-1 knockdown expression vector in vitro, and compared with cell lines transfected with empty vector. After 12, 24, 48 and 72 h of cell culture, the cell proliferation was examined. The MTT method was used to detect apoptosis by flow cytometry, and the mRNA levels of apoptosis and autophagy markers caspase-3, Bcl-2, Bax, LC3 and Beclin1 were assessed by RT-PCR. Our results showed that cells in the control and low expression group kept proliferating during the cell culture period of 72 h, while the cells in the overexpression group progressively decreased in number. Also, the proliferation rate of the low expression group was significantly higher than that of the control group. The relative mRNA expression levels of caspase-3 and Bax mRNA in the control and low expression group showed no change (the expression was lowest in the low expression group). Moreover, the mRNA level of Bcl-2 increased in both cell groups. The mRNA expression levels of caspase-3 and Bax in the overexpression group were increased, and the level of Bcl-2 was reduced significantly. At the same time, the relative expression level of LC3 and Beclin1 mRNA in the control and low expression groups remained the same, and that of the overexpression group increased. The mRNA levels of LC3 and Beclin1 in the overexpression group were the highest, and that of the low expression group the lowest. The differences were statistically significant (P<0.05). Based on these results, we showed that KISS-1 inhibited the proliferation of osteosarcoma in vitro, probably by accelerating the processes of apoptosis and autophagy in the cells.

Effects of Hypobaric Hypoxia on Rat Retina and Protective Response of Resveratrol to the Stress.

High-altitude retinopathy represents retinal functional changes associated with environmental challenges imposed by hypobaric hypoxia, but the detailed cellular and molecular mechanism underlying this process remains unclear. Our current investigation was to explore the effect of hypobaric hypoxia on the rat retina and determine whether resveratrol has a protective efficacy on the hypoxic damage to the retina. Experiment rats were randomly grouped as the control group, hypoxia group and resveratrol intervention group. The hypoxia group and the resveratrol intervention group were maintained in a low-pressure oxygen cabin, and the resveratrol intervention group was given daily intraperitoneal injections with resveratrol. We found that hypobaric hypoxia increased thioredoxin 1 (Trx1) and thioredoxin 2 (Trx2) expression in retinas, and resveratrol treatment significantly reversed these changes (P < 0.05, P < 0.05 respectively). In comparison with controls, hypoxia upregulated the mRNA expression levels of caspase3 (P < 0.001), caspase9 (P < 0.01), heat shock protein 70 (Hsp70) (P < 0.05), heat shock protein 90 (Hsp90) (P < 0.001) and hypoxia-inducible factor-1 (HIF-1) (P < 0.05). Resveratrol administration caused a significant decrease in the gene expression of caspase3 (P< 0.001), HSP90 (P < 0.05) and HIF-1 mRNA (P < 0.01) as well as an increase in HSP70 mRNA when compared with the hypoxia group. These findings indicated that resveratrol exerted an anti-oxidative role by modulating hypoxia stress- associated genes and an anti-apoptosis role by regulating apoptosis-related cytokines. In conclusion, hypobaric hypoxia may have a pathological impact on rat retinas. The intervention of resveratrol reverses the effect induced by hypobaric hypoxia and elicits a protective response to the stress.

Effect of Circular ANRIL on the Inflammatory Response of Vascular Endothelial Cells in a Rat Model of Coronary Atherosclerosis

Background/Aims: This study aims to investigate the role of circular antisense non-coding RNA at the INK4 locus (cANRIL) in the inflammatory response of vascular endothelial cells (ECs) in a rat model of coronary atherosclerosis (AS). A rat model of AS was established with rats that were injected with a large dose of vitamin D₃ and fed a high-fat diet. Methods: Sixty Wistar rats were randomly assigned into control, model, empty vector, over-expressed cANRIL and low-expressed cANRIL groups (12 rats in each group). Sixteen weeks later, the ultrastructure of their coronary arteries was observed via transmission electron microscopy. Rat serum lipid levels were analyzed using an automatic biochemical analyzer, and their atherogenic index (AI) values were calculated. Hematoxylin and eosin staining was used to observe the endothelial morphology of rats. Additionally, rat EC apoptosis was tested via a TUNEL assay. Enzyme-linked immunosorbent assays (ELISAs) were applied to measure serum levels of interleukin-1 (IL-1), IL-6, matrix metalloproteinase-9 (MMP-9) and C-reactive protein (CRP). The cANRIL, Bax, bcl-2 and caspase-3 mRNA expression levels were measured with a quantitative real-time polymerase chain reaction (qRT-PCR). The protein expression levels of Bax, bcl-2 and caspase-3 were detected using immunohistochemistry. Results: In the control group, ECs were closely arranged with normal structures, and there was no proliferation. In the model, empty vector and over-expressed cANRIL groups, some cells were not present, and atherosclerotic plaques and thrombi appeared. However, in the under-expressed cANRIL group, the cells had a normal structure. Compared with the model and empty vector groups, the levels of total cholesterol (CHOL), triglycerides (TGs), low density lipoprotein (LDL), IL-1, IL-6, MMP-9, CRP, cANRIL, Bax, and caspase-3, AI values, and rates of EC apoptosis decreased in the low-expressed cANRIL group, while HDL (high density lipoprotein) levels and mRNA and protein expression levels of bcl-2 were increased. The changes in expression levels in the over-expressed cANRIL group were the opposite of those in the low-expressed cANRIL group. Conclusions: Our study provides evidence that reduced cANRIL expression could prevent coronary AS by reducing vascular EC apoptosis and inflammatory factor expression.

Expression Changes of Apoptotic Genes in Tissues from Mice Exposed to Nicotine

Objective:Smoking is the leading preventable cause of various diseases such as lung cancer, chronic obstructive pulmonary disease and cardiovascular disease. Nicotine, one of the major toxic components of tobacco, contributes to the pathogenesis of different diseases.Methods:Given the controversy about nicotine toxicity, the present study was conducted to determine apoptotic effects of nicotine on the heart, kidney, lung and liver of male mice. Real-time PCR was performed to identify mRNA expression changes in apoptotic-related genes between nicotine treated and control mice.Result:In the heart and lung, nicotine caused significant decrease in P53, Bax and Caspase-3 mRNA expression levels compared to the control group. However, in the kidney and liver, the result was significant increase in Bax, Caspase-2, Caspase-3 and a significant decrease in P53 mRNA expression (p<0.01). DNA fragmentation assays indicated no fragmentation in the heart and lung, but in the kidney and liver of nicotine treated mice, isolated DNA was fragmented.Conclusion:Our study provided insight into the molecular mechanisms of nicotine anti-apoptotic effects on the heart and lung as well as pro-apoptotic effects on kidney and liver via a P53-independent pathway.

Neuroprotective Effect of Electric Conduction Treatment on Hippocampus Cell Apoptosis in KA Induced Acute Temporal Lobe Epileptic Rats.

Electronic conduction, a new treatment approach for epilepsy, has been confirmed to reduce epileptiform discharge on EEG and convulsive behaviors, particularly epileptic discharge propagation and serious behavioral seizures, in rats with kainic acid (KA)-induced acute temporal lobe epilepsy (TLE). Hippocampal cell apoptosis was examined to confirm the neuroprotective effect of electronic conduction therapy in rats with KA-induced acute TLE. Rats were divided into four groups: control group (right CA3 injection of saline), KA group (right CA3 injection of KA), sham conduction group (KA rats with sham conduction), and conduction group (KA rats with electric conduction). Apoptotic cells were evaluated by flow cytometry, TUNEL staining, and mRNA expression levels of caspase-3, tumor necrosis factor-alpha, and glial fibrillary acidic protein measured by real-time quantitative PCR (qRT-PCR). The frequency of convulsive behaviors in the conduction group decreased significantly compared with the KA group and the sham conduction group. Significantly fewer apoptotic cells were detected in rats with conduction based on flow cytometry and TUNEL staining results. The qRT-PCR results indicated that KA-induced up-regulation of hippocampal caspase-3 mRNA expression was reduced 24 hours after KA injection in rats that received conduction treatment. Electronic conduction treatment can reduce seizure frequency and hippocampal cell apoptosis in rats with KA-induced acute TLE.

Regulatory effect of Bcl-2 in ultraviolet radiation-induced apoptosis of the mouse crystalline lens.

The aim of the present study was to analyze the role of Bcl-2 during the process of apoptosis in the mouse crystalline lens. In total, 12 normal mice served as the control group and 12 Bcl-2 knockout (K.O) mice served as the experimental group. The mouse crystalline lens was sampled for the detection of Bcl-2 and caspase-3 expression following exposure to ultraviolet (UV) radiation. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to determine Bcl-2 expression in the groups of normal mice receiving UV radiation or not receiving UV radiation. Samples of the murine crystalline lens were microscopically harvested and analyzed using western blotting. Apoptosis was detected using terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay. Furthermore, caspase 3 activity was examined using enzyme-linked immunosorbent assay kits, and RT-qPCR was used to analyze caspase-3 expression levels. The results of the present study demonstrated that there was no statistically significant difference in the level of Bcl-2 gene transcription between the two groups. In addition, UV radiation did not change the macrostructure of the crystalline lens in the group of normal mice or the group of Bcl-2 K.O mice. The results of the TUNEL assay indicated that the normal-UV group exhibited a more significant apoptosis level compared with the Bcl-2 K.O-UV group. Furthermore, the mRNA expression level of caspase-3 in the normal-UV group was significantly higher compared with the normal-nonUV group (P<0.05), while the levels in the Bcl-2 K.O-UV group were significantly higher compared with the Bcl-2 K.O and normal-nonUV groups (P<0.05). In addition, the mRNA expression level of caspase-3 was significantly higher in the normal-UV, as compared with the Bcl-2 K.O-UV group (P<0.05), and the variation trends in caspase-3 activity were consistent. In conclusion, the results of the present study demonstrated that Bcl-2 may have an important role in the promotion of UV-induced apoptosis in the crystalline lens.

Effects of sevoflurane exposure during late pregnancy on brain development of offspring mice.

Exposure to some anesthetic agents during the fetal period has been shown to induce neurodegeneration or learning deficits in animal models. Sevoflurane is one of the most prevalent general anesthetics; however, the influence of sevoflurane at a clinically relevant concentration on the developing fetal brain remains unknown. We investigated whether a single sevoflurane exposure during the fetal period would affect neuronal development and learning/memory ability in mice. Pregnant mice at gestational day 17 were anesthetized with 1.5% sevoflurane in 50% oxygen for 6 h. Mice in the control group were exposed in 50% oxygen without sevoflurane. Pups of some mice in both groups subsequently were delivered early by cesarean section and whole fetal brains were excised. The rest of the pups were delivered naturally at gestational day 20 and were maintained for 8 weeks. The mRNA expression levels of caspase-3, brain derived neurotrophic factor (BDNF), nerve growth factor (NGF), and LIM kinase-1 (LIMK-1) were measured in fetal whole brain and 8-week-old hippocampus sections. Synaptophysin protein in adult hippocampus was assessed immunochemically. In addition, 8-week-old mice were subjected to the radial maze test. No significant difference between sevoflurane and control groups regarding mRNA expression levels of all targets was seen, nor was there an obvious change in synaptophysin protein expression. The results of the maze test revealed that the each-day performance ratios (the rate of errors) of the sevoflurane group were not altered as compared with the control group. These results suggest that the exposure during late pregnancy to a clinically relevant concentration of sevoflurane does not affect neuronal development and learning/memory ability of offspring mice.

Protective effect of extract of ginkgo biloba on oxidative damage of human Müller cells

Background Oxidative stress is a pathophysiological process of retina, so it is very important to explore a protective way against retinal oxidative stress. Studies determined that extract of ginkgo biloba (EGb) has antioxidant, anti-apoptosis, anti-thrombosis and anti-inflammatory effects, however, the effect of EGb on human Muller cells in oxidative stress is still below understood. Objective This study was to investigate the protection of EGb against oxidative stress of human retinal Muller cells induced by As2O3in vitro. Methods Human retinal Muller cell line was cultured in DMEM/F12 with 10% fetal bovine serum (FBS), 2 μmol/L glutamine and 1% antibiotics. As2O3 solution at the final concentration 5 mg/L was added in the medium for 24 hours to establish oxidative models, and then the EGb with the final concentrations of 5, 10 and 20 mg/L was used to cell models for 24 hours, respectively. Cell viability was detected by MTT assay, and reactive oxygen species (ROS) levels in the cells were detected with CM-H2DCFDA fluorescent probe. The relative expression levels of caspase-3 mRNA in cytoplasm and cell nuclei were assayed by reverse-transcription PCR (RT-PCR), and the expressions of Nrf2 protein were quantitatively detected by Western blot. Results Muller cells adhered well 24 hours after cultured. At 6-7 days after culture, Muller cell body was large with abundant cytoplasm and mosaic-like arrangement. However, floating cells were seen after As2O3 treatment. Cell viability (absorbance) was significantly different among the normal culture group, As2O3-treated group, As2O3+ 5 mg/L EGb group, As2O3+ 10 mg/L EGb group and As2O3+ 20 mg/L EGb group, with the strongest viability in the normal culture group and the weakest viability in the As2O3-treated groups (F=163. 57, P=0. 00). The fluorescence intensity of ROS was the weakest in the normal culture group and the strongest in the As2O3-treated group and was gradually weakened with the increase of EGb doses, showing a remarkable difference among the groups (F=4 013. 61, P=0. 00). The relative expression level of caspase-3 mRNA in the cells was gradually reduced with the increase of EGb doses, with a statistically significant difference among the groups (F=2 199. 72, P=0. 00). In addition, no considerable difference was seen in the expression level of Nrf2 protein (grey scale) in cytoplasm among the groups (F=15. 42, P=0. 40); while in the nuclei, the expression levels of Nrf2 protein were 100. 01±0. 04, 46. 59±0. 63, 54. 51±0. 62, 59. 93±0. 17 and 67. 60±0. 24 in the normal culture group, As2O3-treated group and As2O3+ 5 mg/L EGb group, As2O3+ 10 mg/L EGb group, As2O3+ 20 mg/L EGb group respectively, with a significant difference among them (F=7 271. 72, P=0. 00). Conclusions EGb can protect human retinal Muller cells against As2O3-induced damage in a dose-dependant manner by antioxidant and antiapoptotic effects in vitro, and the activities occur primarily in cell nucleus. Key words: Ginkgo biloba; Plant extracts/pharmacology; Cell line; Muller cells/drug effects; Cell hypoxia; Oxidative stress; Cell survival/drug effects; Apoptosis/drug effects; Humans

Compound Radix Sophorae Flavescentis exerts antitumor effects by inhibiting the proliferation and inducing the apoptosis of esophageal carcinoma TE-8 cells.

The aim of this study was to examine the effects of compound Radix Sophorae Flavescentis on the proliferation of esophageal carcinoma TE-8 cells and to elucidate the mechanisms involved. For this purpose, we incubated TE-8 cells in medium containing various concentrations (0, 0.0125, 0.025, 0.05, 0.1, 0.2, 0.4 and 0.8 mg/ml) of the compound Radix Sophorae Flavescentis injection and its effects on the proliferation of TE-8 cells were examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. In addition, we observed the morphological changes and measured the expression levels of apoptosis-related genes (caspase-3, Bcl-2 and Bax) in the cells treated with different doses of the compound (low-dose group, 0.05 mg/ml; medium-dose group, 0.2 mg/ml; and high-dose group, 0.8 ng/ml) by reverse transcription-quantitative PCR (RT-qPCR). The apoptotic index of the cancer cells treated with different doses of the compound was determined by TUNEL assay. Our results revealed that compared with the control group (untreated cells), the proliferation of the cancer cells treated with the compound was significantly inhibited (P≤0.05); the inhibition of the proliferation of the cancer cells occured in a dose-dependent manner. Compared with the control group, the apoptotic rate of the cells in the low-dose, medium-dose and high-dose groups increased significantly (P<0.05) in a dose-dependent manner. In addition, compared with the control group, the mRNA expression of caspase-3 and Bax increased significantly in the cells treated with the compound. However, the mRNA expression of Bcl-2 markedly decreased (P<0.05). With the gradual increase in the drug concentration, the mRNA expression levels of caspase-3, Bcl-2 and Bax in the cancer cells were altered in a dose-dependent manner. In conclusion, our data demonstrate that compound Radix Sophorae Flavescentis injection significantly enhances the expression of pro-apoptotic genes in esophageal carcinoma TE-8 cells by increasing apoptosis and inhibiting cell proliferation. Thus, this study provides a theoretical basis for the clinical treatment of esophageal carcinoma.

Effect of the WWOX gene on the regulation of the cell cycle and apoptosis in human ovarian cancer stem cells.

In order to examine new ideas for gene therapy in ovarian cancer, the specific mechanism underlying the effects of the WW domain containing oxidoreductase (WWOX) gene on cell cycle regulation and apoptosis in human ovarian cancer stem cells was investigated. Ovarian cancer stem cells were transfected with a eukaryotic expression vector carrying the WWOX gene in vitro (recombinant plasmid) and cells transfected with the empty plasmid (empty plasmid) or untransfected cells were used as controls. Stably transfected cells were screened and amplified in culture and the WWOX protein was detected by western blot analysis in the three groups of cells. Western blot analysis was performed to detect the expression of cell cycle regulatory proteins cyclin E, cyclin-dependent kinase (CDK) 2, cyclin D1, CDK4 and apoptosis-related protein Wnt-5α and c-Jun N-terminal kinase (JNK), while polymerase chain reaction (PCR) was used to detect alterations in the mRNA expression levels of caspase-3. The results demonstrated that the WWOX protein was stably expressed in cells of the recombinant plasmid group, but was not detected in cells of the empty plasmid group and the control group. Cell proliferation at each time point decreased significantly in the recombinant plasmid group compared with the empty plasmid group and the control group. Flow cytometric analysis demonstrated that the proportion of cells in the G0/G1 phase in the recombinant plasmid group was significantly higher than that of cells in the empty plasmid group and the control group. The rate of apoptosis in the recombinant plasmid group was significantly higher than that of cells in the empty plasmid group and the control group. Western blot analysis demonstrated that the expression levels of cyclin E, CDK2, cyclin D1 and CDK4 in the recombinant plasmid group were significantly lower than those in the empty plasmid group and the control group; however, the expression levels of Wnt-5α and JNK were significantly higher than those in the empty plasmid group and the control group. PCR results demonstrated that the mRNA expression level of caspase-3 in the recombinant plasmid group was significantly higher than that in the empty plasmid group and the control group. In conclusion, the present study demonstrated that the WWOX gene can be stably expressed in ovarian cancer stem cells and that it inhibits the proliferation of ovarian cancer stem cells. The WWOX gene can downregulate the expression levels of cell cycle proteins cyclin E-CDK2 and cyclin D1-CDK4, which affects the cell cycle of ovarian cancer stem cells. Furthermore, the WWOX gene can upregulate the mRNA expression levels of Wnt-5α, JNK and caspase-3, thus contributing to apoptosis of ovarian cancer stem cells. The present study demonstrated that the WWOX gene may be an important molecular target for the treatment of ovarian cancer in the future.

Effect of Glutamine on Caspase-3 mRNA and Protein Expression in the Myocardium of Rats With Sepsis

Apoptosis and caspase-3 play an important role in the pathogenesis of sepsis. In this study, the authors monitored myocardial apoptosis and investigated caspase-3 protein expression change in rats with sepsis. in addition, we investigated the protective effect of glutamine (Gln) on myocardial injury in septic rats. A rat model of sepsis was established by intraperitoneal injection of lipopolysaccharide (LPs). Rats were divided into control group, endotoxin (LPs) group and LPS + Gln group, which were further divided into 4 subset groups (0, 6, 12 and 24 hour subgroups; n = 6). The rate of myocardial apoptosis, caspase-3 mRNA expression and caspase-3 protein expression were examined. Data were analyzed using the F-test or linear correlation test. The results revealed that the rate of myocardial apoptosis in the LPs group was significantly higher than that in the control group (P < 0.05). compared with the control group, LPs group has an upregulated caspase-3 mRNA expression level. However, the caspase-3 protein was low expressed (P < 0.05). The LPS + Gln group has significant lower myocardial apoptosis rate compared with the LPs group (P < 0.05). In addition, caspase-3 mRNA expression levels and caspase-3 protein expression levels were lower in the LPS + Gln group (P < 0.05). We found that Gln reduces the extent of myocardial apoptotic cell death by decreasing the gene and protein expression of caspase-3. Therefore, Gln may be used to prevent the onset of sepsis at an early stage.

Cytotoxic and apoptosis-inducing activity of triterpene glycosides from Holothuria scabra and Cucumaria frondosa against HepG2 cells.

The cytotoxic effects of thirteen triterpene glycosides from Holothuria scabra Jaeger and Cucumaria frondosa Gunnerus (Holothuroidea) against four human cell lines were detected and their cytotoxicity-structure relationships were established. The apoptosis-inducing activity of a more potent glycoside echinoside A (1) in HepG2 cells was further investigated by determining its effect on the morphology, mitochondrial transmembrane potential (Δψm) and mRNA expression levels of the apoptosis-related genes. The results showed that the number of glycosyl residues in sugar chains and the side chain of aglycone could affect their cytotoxicity towards tumor cells and selective cytotoxicity. 1 significantly inhibited cell viability and induced apoptosis in HepG2 cells. 1 also markedly decreased the Δψm and Bcl-2/Bax mRNA express ratio, and up-regulated the mRNA expression levels of Caspase-3, Caspase-8 and Caspase-9 in HepG2 cells. Therefore, 1 induced apoptosis in HepG2 cells through both intrinsic and extrinsic pathway. These findings could potentially promote the usage of these glycosides as leading compounds for developing new antitumor drugs.

Soy isoflavones administered to rats from weaning until sexual maturity affect ovarian follicle development by inducing apoptosis

Twenty-one-day-old female Wistar rats were treated daily with orally administered soy isoflavones (SIFs) at concentrations of 50, 100, or 200mg/kg body weight from weaning until sexual maturity (3mo.), and ovarian follicle development was evaluated. At the end of the treatment period, the ultrastructure of the ovarian granulosa cells was examined by transmission electron microscopy. The apoptotic cell death of ovarian granulosa cells was detected using TUNEL staining. The mRNA expression levels of caspase-3, caspase-8, caspase-9, Bcl2, Bax, and Fas were determined by real-time quantitative PCR. The protein expression levels of caspase-3, Bcl2, Bax, and Fas were determined by western blotting. Our data showed that exposure to SIFs resulted in morphological changes consistent with ovarian granulosa cell apoptosis. The percentage of TUNEL-positive granulosa cells was increased. The mRNA expression levels of the apoptosis-related genes caspase-3, caspase-8, caspase-9, Bax, and Fas increased significantly. The protein levels of Bax, Fas, and cleaved caspase-3 were also increased. These results indicate that the exposure of rats to modest doses of SIFs from weaning until sexual maturity can affect ovarian follicle development by inducing apoptosis. The mechanism of SIF-induced alterations in ovarian follicle development may involve the activation of Fas-mediated and Bcl2/Bax-mediated apoptotic signaling pathways.

Dracorhodin perchlorate induces apoptosis in primary fibroblasts from human skin hypertrophic scars via participation of caspase-3

Hypertrophic scar (HS) is an abnormally proliferative disorder characterized by excessive proliferation of fibroblasts and redundant deposition of extracellular matrix. An unbalance between fibroblast proliferation and apoptosis has been assumed to play an important role in HS formation. To explore the regulative effects of dracorhodin perchlorate (Dp), one of the derivants of dracorhodin that is a major constituent in the traditional Chinese medicine, on primary fibroblasts from human skin hypertrophic scars, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometric analysis were respectively used to evaluate the inhibitory effect of Dp on the cells and to determine cell cycle distribution. Additionally, cellular apoptosis was separately detected with Hoechst 33258 staining and terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay. The expression levels of caspase-3 mRNA and protein were respectively measured with reverse transcription-polymerase chain reaction and western blot analysis, and caspase-3 activity were determined using a colorimetric assay kit. The results showed that Dp significantly inhibited cell growth, and induced apoptosis in fibroblasts in a dose-and time-dependent manner, arresting cell cycle at G1 phase. Additionally, Dp slightly up-regulated caspase-3 mRNA expression in fibroblasts, but significantly down-regulated caspase-3 protein expression in a dose- and time-dependent manner, and concurrently elevated caspase-3 activity. Taken together, these data indicated that Dp could effectively inhibit cell proliferation, and induced cell cycle arrest and apoptosis in fibroblasts, at least partially via modulation of caspase-3 expression and its activity, which suggests that Dp is an effective and potential candidate to develop for HS treatment.

Molecular mechanism of SHI-1 cell apoptosis induced by puerariae radix flavones in vitro

This study was purposed to explore the effects induced by puerariae radix flavones (PRF) on human acute myeloid leukemia SHI-1 cells, apoptosis induced by PRF in vitro and its molecular mechanism. SHI-1 cells were treated with PRF in various concentration, then the inhibitory effect of cell proliferation were detected by MTT method, the cell cycle was analyzed by flow cytometry, the mRNA expression levels of Caspase-3, Caspase-8, Caspase-9, Bcl-2 and MLL-AF6 were detected by real-time polymerase chain reaction (R-T PCR), the protein expression levels of MAPK, p-MAPK and NF-κB were assayed by Western blot, and the activity of MMP was analyzed by Gelatin zymography. The results indicated that the PRF could inhibit the proliferation of SHI-1 cells in a time-and dose-dependent manner, and the cell cycle was arrested in S phase. When SHI-1 cells were treated with 25, 50 and 75 µg/ml PRF respectively, mRNA levels of Caspase-3, Caspase-8 and Caspase-9 increased in a dose-dependent manner (P < 0.05), Bcl-2 mRNA decreased in a concentration-dependent manner (P > 0.05), and the mRNA level of fusion gene MLL-AF6 did not changed as compared with the control group. Different concentration of PRF was used to treat SHI-1 cells, the expression levels of intracellular JNK, p-JNK, P38 MAPK and p-P38 MAPK increased in the concentration-dependent manner (P < 0.01); the expression of p-ERK1/2 and NF-κB decreased in the concentration-dependent manner, and the activity of MMP-2 and MMP-9 in the cell supernatant did not change in each groups. It is concluded that a certain concentration of PRF can induce the apoptosis of SHI-1 cells in vitro, its molecular mechanism may be related to the activation of Caspase hydrolase, activation of MAPK, downregulation of NF-κB, Bcl-2 and other signal molecules. However, it seemed that all these effects are not relate with the MLL-AF6 fusion gene.