Neuroprotective effects of the immunodepressant FTY720 on caspase-3 expression and neural apoptosis in a rat model of acute spinal cord injury

BACKGROUND：Studies on the immunodepressant FTY720 have primarily focused on organ transplantation and autoimmune disease therapy.However,the effects on caspase-3 expression and neural apoptosis following acute spinal cord injury remain uncertain.OBJECTIVE：To elucidate the underlying mechanism of the immunodepressant FTY720 to alleviate spinal cord injury by inhibiting expression of caspase-3 and neural apoptosis.DESIGN,TIME AND SETTING：A randomized,controlled,animal experiment was performed at Central Laboratory of the Second Affiliated Hospital of Dalian Medical University from April to July 2009.MATERIALS：FTY720 was provided by Wuhan Yuancheng Technology Developing,China.METHODS：A total of 120 Sprague Dawley rats were randomly assigned to sham-surgery,model,and FTY720 groups.Spinal cord injury at the T9-10 segment was induced in model groups using the free-fall method.Following establishment of spinal cord injury at the T9-10 segment in the FTY720 group,rats were treated with an intragastric injection of 0.3 mL saline-diluted FTY720 （3 mg/kg）.MAIN OUTCOME MEASURES：At 6,12,24,48,and 72 hours following spinal cord injury,caspase-3 expression was detected using streptavidin-peroxidase immunohistochemistry,and neural apoptosis was detected using the TUNEL method.RESULTS：Positive caspase-3 expression and neural apoptosis was not observed in the sham-surgery group at the various time points.The number of apoptotic cells increased with time after acute spinal cord injury,peaked at 24 hours following injury,and then gradually reduced.However,neural apoptosis remained at a high level.Caspase-3 expression positively correlated with neural apoptosis （r= 0.864,P〈 0.05）.Caspase-3 expression and neural apoptosis significantly decreased following FTY720 therapy （P〈 0.05）.CONCLUSION：FTY720 significantly reduced caspase-3 expression and neural apoptosis in a rat model of acute spinal cord injury.

Naofen promotes TNF-α-mediated apoptosis of hepatocytes by activating caspase-3 in lipopolysaccharide-treated rats

AIM: To investigate whether naofen is involved in tumor necrosis factor (TNF)-α-mediated apoptosis of hepatocytes induced by lipopolysaccharide (LPS).

Oridonin induces apoptosis in gastric cancer through Apaf-1,cytochrome c and caspase-3 signaling pathway

AIM:To investigate the effect and mechanism of oridonin on the gastric cancer cell line HGC-27 in vitro.METHODS:The inhibitory effect of oridonin on HGC-27 cells was detected using the 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyl tetrazolium bromide assay.After treatment with 10 μg/mL oridonin for 24 h and 48 h,the cells were stained with acridine orange/ethidium bromide.The morphologic changes were observed under an inverted fluorescence microscope.DNA fragmen-tation(a hallmark of apoptosis) and lactate dehydrogenase activity were examined using DNA ladder assay and lactate dehydrogenase-release assay.After treated with oridonin(0,1.25,2.5,5 and 10 μg/mL),HGC-27 cells were collected for anexin V-phycoerythrin and 7-amino-actinomycin D double staining and tested by flow cytometric analysis,and oridonin-induced apoptosis in HGC-27 cells was detected.After treatment with oridonin for 24 h,the effects of oridonin on expression of Apaf-1,Bcl-2,Bax,caspase-3 and cytochrome c were also analyzed using reverse-transcript polymerase chain reaction(RT-PCR) and Western blotting.RESULTS:Oridonin significantly inhibited the proliferation of HGC-27 cells in a dose-and time-dependent manner.The inhibition rates of HGC-27 treated with four different concentrations of oridonin for 24 h(1.25,2.5,5 and 10 μg/mL) were 1.78% ± 0.36%,4.96% ± 1.59%,10.35% ± 2.76% and 41.6% ± 4.29%,respectively,which showed a significant difference(P < 0.05).The inhibition rates of HGC-27 treated with oridonin at the four concentrations for 48 h were 14.77% ± 4.21%,21.57% ± 3.75%,30.31% ± 4.91% and 61.19% ± 5.81%,with a significant difference(P < 0.05).The inhibition rates of HGC-27 treated with oridonin for 72 h at the four concentrations were 25.77% ± 4.85%,31.86% ± 3.86%,48.30% ± 4.16% and 81.80% ± 6.72%,with a significant difference(P < 0.05).Cells treated with oridonin showed typical apoptotic features with acridine orange/ethidium bromide staining.After treatment with oridonin,the cells became round,shrank,and developed small buds around the nuclear membrane while forming apoptotic bodies.Lactate dehydrogenase(LDH) release assay showed that after treated with 1.25 μg/mL and 20 μg/mL oridonin for 24 h,LDH release of HGC-27 caused by apoptosis increased from 22.94% ± 3.8% to 52.68% ± 2.4%(P < 0.001).However,the change in the release of LDH caused by necrosis was insignificant,suggesting thatthe major cause of oridonin-induced HGC-27 cell death was apoptosis.Flow cytometric analysis also revealed that oridonin induced significant apoptosis compared with the controls(P < 0.05).And the apoptosis rates of HGC-27 induced by the four different concentrations of oridonin were 5.3% ± 1.02%,12.8% ± 2.53%,28.5% ± 4.23% and 49.6% ± 3.76%,which were in a dose-dependent manner(P < 0.05).After treatment for 24 h,DNA ladder showed that oridonin induced a significant increase in DNA fragmentation in a dosedependent manner.RT-PCR revealed that mRNA expression levels were up-regulated compared with the controls in caspase-3(0.917 ± 0.103 vs 0.357 ± 0.019,P < 0.05),cytochrome c(1.429 ± 0.111 vs 1.002 ± 0.014,P < 0.05),Apaf-1(0.688 ± 0.101 vs 0.242 ± 0.037,P < 0.05) and Bax(0.856 ± 0.101 vs 0.278 ± 0.027,P < 0.05)(P < 0.05),whereas down-regulated in Bcl-2(0.085 ± 0.012 vs 0.175 ± 0.030,P < 0.05).Western blotting analysis also confirmed this result.CONCLUSION:Apoptosis of HGC-27 induced by oridonin may be associated with differential expression of Apaf-1,caspase-3 and cytochrome c,which are highly dependent upon the mitochondrial pathway.

Expression of second mitochondria-derived activator of caspases, X-linked inhibitor of apoptosis protein, and caspase-3 in pituitary adenomas

Studies concerning correlations between pituitary adenomas and cell apoptosis have mainly focused on upstream apoptosis signaling, but seldom on downstream mediators. In the present study, second mitochondria-derived activator of caspases （Smac）, X-linked inhibitor of apoptosis protein （XIAP）, and caspase-3 protein were qualitatively analyzed using immunohistochemistry, and quantified by western blot. Smac, XIAP, and caspase-3 mRNA expressions were detected by reverse transcription-PCR. Results showed that XIAP protein and mRNA expressions were greater in the invasive pituitary adenoma group compared with the noninvasive pituitary adenoma group. However, Smac and caspase-3 protein and mRNA expressions were lower in the invasive pituitary adenoma group compared with the noninvasive pituitary adenoma group. In the invasive pituitary adenomas, Smac expression was positively correlated with caspase-3 protein and mRNA expression （Protein： r = 0.55, P 0.01; mRNA： r = 0.50, P 0.01）. Smac and caspase-3 expressions were negatively correlated with XIAP protein and mRNA expression （Protein： r = -0.56, -0.64, P 0.01; mRNA： r = -0.69, -0.67, P 0.01）. However, no significant differences in correlation among Smac, XIAP, and caspase-3 were detectable in noninvasive pituitary adenomas. These data indicated that high expression of XIAP and low expression of Smac and caspase-3 suppressed cell apoptosis and led to enhanced invasiveness of pituitary adenomas. Thus, Smac, XIAP, and caspase-3 may be useful markers in determining the invasive behavior of pituitary adenomas.

Parthenolide protects human lens epithelial cells from oxidative stress-induced apoptosis via inhibition of activation of caspase-3 and caspase-9

The apoptosis of lens epithehal cells has been proposed as the common basis of cataract formation, with oxidative stress as the major cause. This study was performed to investigate the protective effect of the herbal constituent parthenolide against oxidative stress-induced apoptosis of human lens epithelial （HLE） cells and the possible molecular mechanisms involved. HLE cells （SRA01-04） were incubated with 50 μM H2O2 in the absence or presence of different doses of parthenolide （10, 20 and 50 μM）. To study apoptosis, the cells were assessed by morphologic examination and Annexin V-propidium iodide double staining flow cytometry; to investigate the underlying molecular mechanisms, the expression of caspase-3 and caspase-9 were assayed by Western blot and quantitative RT-PCR, and the activities of caspase-3 and caspase-9 were measured by a Chemicon caspase colorimetric activity assay kit. Stimulated with H202 for 18 h, a high fraction of riLE cells underwent apoptosis, while in the presence ofparthenolide of different concentrations, dose-dependent blocking of HLE cell apoptosis was observed. The expression of caspase-3 and caspase-9 induced by H202 in HLE cells was significantly reduced by parthenolide both at the protein and mRNA levels, and the activation ofcaspase-3 and caspase-9 was also suppressed by parthenolide in a dose-dependent manner. In conclusion, parthenolide prevents HLE cells from oxidative stress-induced apoptosis through inhibition of the activation ofcaspase-3 and caspase-9, suggesting a potential protective effect against cataract formation.

Effect of Bcl-2 and caspase-3 on calcium distribution in apoptosis of HL-60 cells

Apoptosis manifests in two major execution programs downstream of the death signal: the caspase pathway and organelle dysfunction. An important antiapoptosis factor, Bcl-2 protein, contributes in caspase pathway of apoptosis. Calcium, an important intracellular signal element in cells, is also observed to have changes during apoptosis, which maybe affected by Bcl-2 protein. We have previously reported that in Harringtonine (HT) induced apoptosis of HL-60 cells, there’s a change of intracellular calcium distribution, moving from cytoplast especially Golgi’s apparatus to nucleus and accumulating there with the highest concentration. We report here that caspase-3 becomes activated in HT-induced apoptosis of HL-60 cells, which can be inhibited by overexpression of Bcl-2 protein. No sign of apoptosis or intracellular calcium movement from Golgi’s apparatus to nucleus in HL-60 cells overexpressing Bcl-2 or treated with Ac-DEVD-CHO, a specific inhibitor of caspase-3. The results indicate that activated caspase-3 can promote the movement of intracellular calcium from Golgi’s apparatus to nucleus, and the process is inhibited by Ac-DEVD-CHO (inhibitor of caspase-3), and that Bcl-2 can inhibit the movement and accumulation of intracellular calcium in nucleus through its inhibition on caspase3. Calcium relocalization in apoptosis seems to be irreversible, which is different from the intracellular calcium changes caused by growth factor.

Neurocyte Apoptosis and Expressions of Caspase-3 and Fas after Spinal Cord Injury and Their Implication in Rats

To study the expression of neurocyte apoptosis and the changes of caspase-3 and Fas after spinal cord injury （SCI） in rats, improved Allen＇s method was used to make model of acute SCI at the level of T9 and T10. The animals were divided into six groups： a control group and 5 injury groups. The segments of injured spinal cords were taken 6, 24, 48 h and 7, 15 days after injury for morphological studies, including HE staining, Hoechst33258 staining and TUNEL labeling. The expression of caspase-3 was detected by immunohistochemical staining and RT-PCR. TUNEL-positive cells began to appear in the compression region 6 h after the injury, mostly located in the gray matter. TUNEL-positive cells were found in both gray and white matter, reaching a peak at the 3rd day. They began to decrease at the 7th day, distributed mostly in the white matter. Fas increased at the 6th h and peaked at the 3th day. Caspase-3 mRNA increased at the 6th h, peaking 48 h after the trauma, and decreased after 7 days. The protein expression of caspase-3, as revealed by immunohistochemical staining, was similar to TUNEL in time. It is concluded that apoptosis takes place after spinal cord injury, and caspase-3 mRNA and protein expressions were enhanced in the apoptosis. The expression of caspase-3 has a positive correlation with Fas expression.

Salvia miltiorrhiza monomer IH764-3 induces hepatic stellate cell apoptosis via caspase-3 activation

AIM: To investigate the effects of IH764-3 on HSC apoptosis,and the expression of caspase-3 protein in HSC apoptoticprocess.METHODS: HSCs were cultured in medium with differentIH764-3 doses ( 10 μg@ mL-1 , 20 μg@ mL-1 , 30 μg@ mL-1,40μg @mL-1) and without IH764-3, and HSC proliferation wasquantitatively measured by 3 H-thymidine incorporation. Themorphological changes of HSCs were observed withtransmission electron microscope after exposure to the doseof 40 μg@ mL-1 of IH764-3 for 48 hr, The apoptosis rates weredetected by annexin V/PI and TdT-mediated dUTP nick endlabeling (TUNEL). The expression of caspase-3 protein wasdetermined by flow cytometry.RESULTS: (1) HSC proliferation rates induced with differentIH764-3 doses ( 10 μg@ mL-1 , 20 μg@ mL-1 , 30 μg@ mL-1 , 40 μg@mL-1) were significantly reduced compared with that of thecontrol group ( P＜ 0.01). (2)With the doses above, IH764-3dose-dependently produced HSC apoptosis rates of 6.7 %(9.4%),9.3 %(21.6 %),15.1%(27.2 %) and 19.0 %(28.4 %)respectively, by annexin V and PI-labeled flow cytometry assay(or TUlE L), while it was only 2.3 %(6.7 %) in the control. (3)The expression of caspase-3 protein in IH764-3 groups wassignificantly higher than that of the cortrol (P＜0.05).CONCLUSION: Within the dose range used in present study,IH764-3 can inhibit HSC proliferation, as well as enhance HSCapoptosis. Furthermore, IH764-3 can significantly increasethe caspase-3 protein expression.

Effects of Losartan on Cell Apoptosis and Expression of Caspase-3 and JNK Proteins in Kidney Tissue in Renal Interstitial Fibrosis Rats

[Objectives]To investigate the effects of losartan on cell apoptosis and the expression of caspase-3 and JNK proteins in kidney tissue in the adenine-induced renal fibrosis rats.[Methods]Thirty Wistar rats were randomly divided into three groups:control group(n=10),model group(n=10)and losartan group(n=10).The rats in the control group received saline,while those in the model group and losartan group both received adenine by gavage,for 21 d.After the renal interstitial fibrosis model was established,the rats in the losartan group were treated with losartan[10 mg/(kg·d)],while the rats in the control group and the model group rats were administered with the same amount of saline.The course of treatment was 30 d.Finally,the renal function,blood urea nitrogen(BUN),serum creatinine(Scr),creatinine clearance rate(Ccr)and the pathological morphology of the rats were detected.The apoptosis of renal tubular epithelial cells was tested by TUNEL.The caspase-3 and JNK protein expression was tested by Western blotting.[Results]After administering adenine for 21 d,the BUN,24 MTP and kidney/body weight in the model group were increased,significantly higher than the control group(P<0.01),and the Ccr was remarkably decreased(P<0.01),signifying that the renal interstitial fibrosis model was successfully built.After treating with losartan for 30 d,the Scr,BUN,and 24 MTP were significantly decreased(P<0.01),and the Ccr was significantly increased in the losartan group(P<0.01).In addition,in comparison to the model group,renal tubular epithelial apoptosis was decreased and caspase-3 and JNK expression was downregulated in the losartan group(P<0.05).[Conclusions]Losartan can reduce the adenine-induced elevation of Scr,BUN and 24 hMPT,increase Ccr,improve general condition of renal interstitial fibrosis in rats and ameliorate the progression of chronic kidney failure(CKD).The effectiveness of losartan is probably due to its ability to regulate caspase-3,JNK protein expression and attenuate renal cell apoptosis.

Influence of survivin and caspase-3 on cell apoptosis and prognosis in gastric carcinoma

AIM: To evaluate the role of survivin and caspase-3 in apoptosis of gastric carcinoma, as well as in prognosis of patients with gastric carcinoma.METHODS: Expressions of survivin and caspase-3 were investigated immunohistochemically in 80 gastric carcinoma patients without a history of chemo-radiation therapy. Tumor cell apoptosis was examined by TUNEL method.RESULTS: Immunohistochemical analysis showed that survivin expression was positive in 61 of 80 patients (76%) with gastric carcinoma. In contrast, no expression of survivin in adjacent normal tissues was detected. Expression level of caspase-3 was higher in normal tissues than in carcinoma.Patients with higher expression of survivin had worse histological grades and pathological stages. Expression of caspase-3 was significantly associated with histological stages, but not with the pathological stages. Although survivin expression in carcinoma was not inversely related to caspase-3, patients with survivin (-) and caspase-3(+) had the maximum apoptosis index.CONCLUSION: Expression level of survivin was associated with histological grades and pathological stages of the tumor,indicating that survivin may be a poor prognosis factor for gastric carcinoma. Unlike caspase-3, survivin (an apoptosis inhibitor) can markedly inhibit the apoptosis of tumor cells.

Effects of ethanol extracts of scorpion on hippocampal apoptosis and caspase-3 expression in lithium chloride-pilocarpine-induced status epilepticus rats

BACKGROUND： Previous studies have demonstrated that scorpion venom in the scorpion can inhibit epilepsy and apoptosis. However, it remains unclear whether ethanol extracts of scorpion （EES） exhibit similar effects. OBJECTIVE： To investigate the effects of EES on hippocampal apoptosis and caspase-3 expression, and to compare the effects on sodium valproate （positive control drug） in a rat model of status epilepticus induced by lithium chloride-pilocarpine. DESIGN, TIME AND SETTING： This randomized, controlled study was conducted at the Drug Research and Development Center, Kanghong Pharmaceuticals Group, and the Department of Pathology, Sichuan Academy of Medical Sciences ＆ Sichuan Provincial People＇s Hospital, China from May 2007 to April 2008. MATERIALS： EES were prepared by Huashen Pharmaceutical, China. Sodium valproate （Hunan Xiangzhong Pharmaceutical, China） and lithium chloride-pilocarpine （Sigma, USA） were also used in the present study. METHODS： From a total of 156 rats, six served as normal controls. The remaining rats were intraperitoneally injected with lithium chloride-pilocarpine to establish status epileptlcus models, and then assigned to five groups （n = 30, respectively）. Animals in each group were administered drugs at 15 minutes after epileptic seizure by gavage, i.e. in the normal control and model groups, rats were treated with 1 mL/0.1 kg saline. The sodium valproate group was administered 120 mg/kg/d sodium valproate. The low-, moderate-, and high-dose EES groups received treatments of 290, 580 and 1 160 mg/kg/d EES. The dispensed concentration was 1 mL/0.1 kg. Rat seizure behavior was observed. If status epilepticus did not terminated after 1 hour, the rats were intraperitoneally administered atropine （1 mg/kg） and diazepam （10 mg/kg） to terminate seizure. These rats were continuously observed for 6 hours to ensure seizure termination. Then rats were treated with the above-mentioned drugs at 8：00 am each day until sacrifice, which took place 4 hours after drug administration. MAIN OUTCOME MEASURES： Terminal dUTP nick end labeling （TUNEL）-positive cells and caspase-3 expression were, respectively, determined by TUNEL and immunohistochemistry at 6, 24 48, and 72 hours, as well as 7 days, after status epilepticus. Behavioral changes were also measured. RESULTS： A few caspase-3-positive cells were observed. TUNEL- and caspase-3-positive ceils were mainly visible in the hippocampal CA1 and CA3 regions 6 hours following status epilepticus in the model and drug intervention groups. The number of TUNEL-positive cells reached a peak at 48 hours following status epilepticus in the sodium valproate group, as well as the moderate- and high-dose EES groups, and number of TUNEL-positive cells reached a peak at 72 hours in the model and low-dose EES groups. The number of caspase-3-positive cells reached a peak at 48 hours in each group. Following treatment of sodium valproate and EES, the number of TUNEL- and caspase-3-positive cells significantly decreased compared with the model group at various time points （P 〈 0.05）. The number of TUNEL- and caspase-3-positive cells was greatest in the low-dose EES group, followed by the moderate- and high-dose EES groups. The number of TUNEL- and caspase-3-positive cells was similar between the sodium valproate and high-dose EES groups. Epileptic seizure was significantly improved in the sodium valproate group, as well as the moderate- and high-dose EES groups, compared with the model group （P〈 0.05 or P〈 0.01）. Treatment with sodium valproate and high-dose EES resulted in the best outcome, although the results were similar （P 〉 0.05）. CONCLUSION： A dose of 1 160 mg/kg/d EES significantly inhibited status epilepticus. This outcome corresponded to a decreased number of apoptotlc cells and caspase-3-positive cells, which was similar to sodium valproate. These results suggest that it is not necessary to extract a component from the scorpion for the treatment of epilepsy. The high dose of EES significantly inhibited epilepsy, which correlated with decreased hippocampal caspase-3 expression.

Coxsackievirus B3-induced apoptosis and Caspase-3

Cell death can be classified into two categories: apoptosis and necrosis. Apoptotic pathway can beeither caspase-dependent or caspase-independent. Caspase-independent cytopathic effect (CPE) has beendescribed. In order to evaluate the pattern of HeLa cell death induced by Coxsackievirus B3 (CVB3)and whether apoptosis involves caspase activation, we co-cultivated HeLa cells with CVB3 and detectedthe cytopathic changes, the alteration of mRNA and protein expression of caspase-3 gene plus caspase-3activity, as well as analyzing DNA fragmentation before and after caspase-3 activity inhibition. Accordingto the results, we propose that CVB3 may induce apoptosis and necrosis in HeLa cells, the latter appearingmuch earlier. Caspase-3 is activated at the levels of both transcription and translation, and procaspase-3 isproteolytically cleaved, thus leading to the continuous increasing of both caspase-3 precursor protein and itssubunit. However, besides CPE, apoptosis induced by CVB3 is not a direct consequence of the activationof caspase-3, or caspase-3 is not the only effector molecule in apoptotic cell death, for caspase-3 inhibitorcan not decrease DNA fragmentation. Some other biochemical mechanisms may participate in the process,whose role weakens the effect of inhibiting caspase-3 activity.

Effects of Panax notoginseng saponins on apoptosis induced by hydrogen peroxide in cultured rabbit bone marrow stromal cells via altering the oxidative stress level and down-regulating caspase-3

Objective： To investigate the effects of Panax notoginseng saponins （PNS） on hydrogen peroxide （H2O2）-induced apoptosis in cultured rabbit bone marrow stromal cells （BMSCs）. Methods： The effects of different concentrations of PNS on proliferation and early osteoblast differentiation of BMSCs were determined by the MTT assay and an alkaline phosphatase （ALP） assay. An optimal effective concentration of PNS was determined and used in subsequent experiments. The cultured BMSCs were divided into three groups： untreated control, H2O2 treated, and PNS pretreatment of H2O2 treated. The oxidative stress level was assessed by superoxide dismutase （SOD） and malondialdehyde （MDA） assays. Flow cytometry was used to determine BMSC apoptosis by staining with annexinV-FITC/propidium iodide （PI）. The activity of caspase-3 enzyme was measured by spectrofluorometry. Results： PNS （0.1g/L） significantly increased both BMSC proliferation rate and ALP activity, while it decreased the indicators of oxidative stress, caspase-3 activity, and the apoptosis rate of BMSCs induced by H2O2.. Conclusion： PNS, acting as a biological antioxidant, had a protective effect on H2O2-induced apoptosis in cultured rabbit BMSCs by decreasing oxidative stress and down-regulating caspase-3.

Caspase-3 Mediates Apoptosis of Striatal Cells in GA Ⅰ Rat Model

In previous study,glutaric acid (GA) induced apoptosis of primary striatal neuron in vitro.In order to investigate the neurotoxic effects of GA on neonatal rat corpus striatum and the possible mechanism,34 male pups were randomly assigned to NS group,low dose GA (LGA,5 μmol GA/g body weight) group and high dose GA (HGA,10 μmol GA/g body weight) group.These pups were subcutaneously administered with three injections from postnatal day 3 to 22 at 7:30 am,15:00 pm and 22:30 pm and killed 12 h after the last injection.Microscopic pathology in corpus striatum was evalu-ated by HE staining.The apoptotic cells were identified by TUNEL staining.The transcript levels of caspase-3,8,9,Bax,Bcl-2 were detected by using real-time PCR and the protein levels of procaspase-3 and the active fraction were evaluated by Western blotting.In LGA and HGA groups,ventricle collapse,cortical atrophy by a macroscope and interstitial edema,vacuolations,widened perivascular space of bi-lateral striatum by a microscope were observed.TUNEL assay revealed that the apoptotic cells were in-creased in LGA and HGA groups.The transcript of caspase-3 was up-regulated to 2.5 fold,accompanied by the up-regulation of caspase-9,Bax and down-regulation of Bcl-2.The protein levels of procaspase-3 and the active fraction were up-regulated in LGA and HGA groups.The rat model for GA Ⅰ showed mitochondrial apoptotic pathway may be involved in the GA-induced striatal lesion.Further studies should be taken to investigate the underlying mechanisms.

Ganglion cells apoptosis in diabetic rats as early prediction of glaucoma:a study of Brn3b gene expression and association with change of quantity of NO, caspase-3, NF-κB, and TNF-α

AIM:To find a new concept to show whether or not apoptosis of retinal ganglion cells(RGCs)canbe determined in the histology of acute hyperglycemia in the role of expressed Brn3b gene related to nitric oxide(NO),caspase-3,nuclear factor kappa-B(NF-κB),and tumor necrosis factor-α(TNF-α)as an early predictor of primary open angle glaucoma(POAG)eyes and their associations.METHODS:Experimental in vivo study was carried out using adult male,white Sprague-Dawley rats aged≥2 mo,weighing 150-200 g.The animals were divided into two groups,one group receiving intraperitoneal injection of streptozotociz 50 mg/kg in 0.01 mol/L citricbuffer and p H 4.5 and a comparison made with the control group.Retinal tissue was divided into two parts(both experimental and control groups respectively):a)right retina for immunohistochemistry(IHC;caspase-3 and TNF-α);b)left retina was divided into two parts for the purpose of real-time polymerase chain reaction(PCR)test(RNA extraction for Brn3b gene expression analysis)and ELISA test(NO and NF-κB).RESULTS:The experimental group showed a decrease in Brn3b gene expression compared to the control group(1.3-fold lower in 2nd month;1.1-fold lower in 4th month and 2.5-fold lower in 6th month).However,there was a decrease of NO,caspase-3,and an increase of NF-κB and TNF-αquantity.CONCLUSION:The expression of m RNA Brn3b gene is inversely proportional to apoptosis in RGCs.The quantity of NO,caspase-3,NF-κB and TNF-αis influential in expression of Brn3b in RGCs caused by hyperglycemia in diabetic rats.

Down-modulation of heat shock protein 70 and up-modulation of Caspase-3 during schisandrin B-induced apoptosis in human hepatoma SMMC-7721 cells

AIM: To investigate the effect of schisandrin B (Sch B) on proliferation and apoptosis of human hepatoma SMMC-7721 cells in vitro and regulation of Hsp70 and Caspases-3, 7, 9 expression by Sch B. METHODS: Human hepatoma cell line SMMC-7721 was cultured and treated with Sch B at various concentrations. Growth suppression was detected with MTT colorimetric assay. Cell apoptosis was confirmed by DNA ladder detection and flow cytometric analysis. The expression of Hsp70, Caspases-3, 7, 9 were analyzed by Western blot analysis. RESULTS: Sch B inhibited the growth of hepatoma SMMC-7721 cells in a dose-dependent manner, leading to a 50% decrease in cell number (LC50) value of 23.50 mg/L. Treatment with Sch B resulted in degradation of chromosomal DNA into small internucleosomal fragments, evidenced by the formation of a 180-200 bp DNA ladder on agarose gels. FCM analysis showed the peak areas of subdiploid at the increased concentration of Sch B. The results of Western bolt analysis showed that Hsp70 was down-regulated and Caspase-3 was up-regulated, while the activity of Caspases-7,-9 had no significant change. CONCLUSION: Sch B is able to inhibit the proliferation ofhuman hepatoma SMMC-7721 cells and induce apoptosis, which goes through Caspase-3-dependent and Caspase-9-independent pathway accompanied with the down-regulation of Hsp70 protein expression at an early event.

Adenoviral vector mediated-expression of caspase-3 siRNA on apoptosis induced by lipopolysaccharide

Objective： To construct the recombinant adenovirus expressing small RNA of rats caspase-3 and observe the down-regulation effect of caspase-3 in neurons induced by lipopolysaccharide（LPS） in vitro. Methods： pShuttleHl-siCas3 containing Oligo DNA of the targeting sequences and pEGFPC1-Cas3 containing caspase-3 and EGFP sequences were constructed respectively, pShuttleH 1-siCas3 and pEGFPC 1-Cas3 were co-transfected to the 293 cells by liposomes to determine interfering efficacy by flow cytometry, pShuttleHl-siCas3 was linearized and transformed into E. coli B J5183 cells containing backbone plasmid pAdEasy-1. The recombinant plasmid was transfected into 293 cells to package the adenovirus Ad-siCas3. The titers of adenovirus were determined by the specific 50% tissue culture infection dosage method. After virus infected the cultured hippocampus neurons, LPS-induced apoptosis and caspase-3 mRNA expression were observed. Results： It was identified that the sequence of target gene was correctly inserted into the genome of virus. The expression of green fluorescence protein was reduced by pShuttleHl-siCas3 in 293 cells. The titer of recombinant adenovirus was 1.06×10^10pfu/ml. After virus infection, caspase-3 mRNA was greatly reduced and neurons apoptosis was suppressed. Conclusion： The recombinant adenovirus expressing rats caspase-3 siRNA were successfully constructed, which may probably be further used in pain therapy by its anti-apoptosis effect.

Caspase-3 activation is required for corticosteroneinduced rat Leydig cell apoptosis

Objective: Study on caspase-3 expression and its activation in CORT-mediated rat Leydig cell apoptosis. Methods: Caspase-3 protein activity, content and the timing of the onset of caspase-3 cleavage in relationship to CORT administration in rats was studied by Western blot. The timing of the onset of caspase-3 activity in Leydig cell was measured by the fluorescence. Results: Low molecular weight DNA fragments that are characteristic of apoptosis were evident in Leydig cells by 12 h of exposure to 100 nmol/L CORT in vitro and it was more pronounced at 24 h. Western blot analysis revealed that procaspase-3 was low in untreated Leydig cells and increased by 6 h of CORT administration. By 12 h, however, procaspase-3 was significantly reduced and the cleaved, active caspase-3 forms appeared and increased through 24 h. In the presence of a specific caspase inhibitor, Ac-DEVD-CHO, Leydig cell apoptosis was suppressed, corroborating the hypothesis that caspase-3 is involved in CORT-mediated cell death. Conclusion: Caspase-3 was implicated in CORT-mediated rat Leydig cell apoptosis.

Cigarette Smoke Induces Apoptosis by Activation of Caspase-3 in Isolated Fetal Rat Lung Type II Alveolar Ep-ithelial Cells <i>in Vitro</i>

Smoking during pregnancy is a major source of fetal exposure to numerous harmful agents present in tobacco smoke. Lung development involves complex biochemical processes resulting in dramatic changes which continue even after birth. In addition to type I cells which form the blood-air barrier, type II alveolar epithelial (AE) cells have important and diverse functions related to immunological protection and stabilization of the alveolus through synthesis and secretion of the pulmonary surfactant. Apoptosis or programmed cells death is an important physiological process during lung embryogenesis and for the proper maintenance of homeostasis. Caspases are proteases that play important roles in regulating apoptosis. Caspase-3 is the key executioner caspase in the cascade of events leading to cell death by apoptosis. We explored the hypothesis that cigarette smoke extract (CSE) induces apoptosis in fetal rat lung type II AE cells by activation of caspase-3. To analyze these factors, isolated fetal rat lung type II AE cells were used. The cells were exposed to different concentrations of CSE (5%, 10% or 15%) (v/v) for 60 min. The results of the present study showed that CSE induced apoptosis in fetal rat lung type II AE cells with a significant increase (p 0.05) in caspase-3 activity and decrease in cell proliferation at CSE concentrations of 10% and 15% (v/v). These observations indicate that cigarette smoke extract induces apoptosis by activation of caspase-3 in fetal rat lung type II AE cells in a dose-dependent manner and may potentially alter the regulated development of the lung and the appearance of the surfactant-producing type II alveolar cells which are critical for the establishment of adequate gas exchange at birth.

Dioscin-induced Apoptosis of Human LNCaP Prostate Carcinoma Cells through Activation of Caspase-3 and Modulation of Bcl-2 Protein Family

Dioscin is a natural steroid saponin derived from several plants, showing potent anti-cancer effect against a variety of tumor cell lines. In the present study, we investigated the anti-cancer activity of dioscin against human LNCaP cells, and evaluated the possible mechanism involved in its antineoplastic action. It was found that dioscin（1, 2 and 4 μmol/L） could significantly inhibit the viability of LNCaP cells in a time- and concentration-dependent manner. Flow cytometry revealed that the apoptosis rate was increased after treatment of LNCaP cells with dioscin for 24 h, indicating that apoptosis was an important mechanism by which dioscin inhibited cancer. Western blotting was employed to detect the expression of caspase-3, Bcl-2 and Bax in LNCaP cells. The expression of cleaved caspase-3 was significantly increased, and meanwhile procaspase-3 was markedly decreased. The expression of anti-apoptotic protein Bcl-2 was down-regulated, whereas the pro-apoptotic protein Bax was up-regulated. Moreover, the Bcl-2/Bax ratio was drastically decreased. These results suggested that dioscin possessed potential anti-tumor activity in human LNCaP cells through the apoptosis pathway, which might be associated with caspase-3 and Bcl-2 protein family.