The mechanism of miR-494 negatively regulating ROCK1 and PTEN in inhibiting apoptosis of acute pancreatitis cells / 中华急诊医学杂志

Objective:To explore the mechanism of miR-494 negatively regulating ROCK1 and PTEN in inhibiting apoptosis of pancreatic cells and participating in the occurrence and development of acute pancreatitis.Methods:Pancreatic acinar cells AR42J from rats were treated by caerulein, and then the levels of amylase, tumor necrosis factor alpha (TNF-α), interleukin 1 (IL-1) and IL-6 in the supernatant of cell culture were detected by ELISA to verify the cell model of acute pancreatitis. RT-PCR was used to detect the expression of miR-494 in normal AR42J cells (control group) and acute pancreatitis cell model (model group). Flow cytometry was used to detect the apoptosis of the control group, negative control miRNA transfected acute pancreatitis cell model (negative control group) and miR-494 transfected acute pancreatitis cell model (miR-494 transfection group). Western blot was used to detect the expression of ROCK1 and PTEN in the control group, negative control group and miR-494 transfection group.Results:The levels of amylase, TNF-α, IL-1 and IL-6 in the supernatant of AR42J cells treated with caerulein for 8 h and 12 h were significantly higher than those at 0 h and the control group ( P<0.05), indicating that the model was successfully constructed. The expression levels of miR-494 at 8 h, 12 h and 24 h after the establishment of acute pancreatitis cell model were significantly higher than those at 4 h and the control group ( P < 0.05). The apoptosis rate of the model group was significantly higher than that of the control group ( P<0.05), and the apoptosis rate of the miR-494 transfection group was significantly lower than that of the model group ( P<0.05). The expression levels of ROCK1 and PTEN in the miR-494 transfection group were significantly lower than those in the model group and negative control group ( P<0.05). Conclusions:When acute pancreatitis occurs, overexpression of miR-494 can inhibit the expression of pro-apoptotic protein, thus inhibiting the apoptosis of pancreatic acinar cells and promoting the development of acute pancreatitis.

Effect of Yisui Jiedu Prescription on Apoptosis of Hippocampal Neurons Based onPI3K/Akt/Bad Signal Pathway in Rats with Vascular Dementia / 中国实验方剂学杂志

Objective:To investigate the effect of Yisui Jiedu prescription on hippocampal neuron damage in vascular dementia (VD) rats and to regulate phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/recombinant Bcl-2 associated death promoter (Bad) mechanisms of signaling pathways of neuronal apoptosis. Method:The 40 SD rats were divided into sham operation group， model group， donepezil hydrochloride group and Yisui Jiedu prescription group， with 10 rats in each group.VD animal model was prepared by bilateral carotid artery permanent ligation (2-VO) method.The sham operation group and the model group were intragastrically administered with normal saline， the donepezil hydrochloride group was intragastrically administered with donepezil hydrochloride 0.52 mg·kg-1. The Yisui Jiedu prescription group was administered with Yisui Jiedu prescription (11.11 g·kg-1)， 1 time/d . After 30 days， Morris water maze was used to test the learning and memory ability of rats， hematoxylin-eosin (HE) staining was used to observe the histomorphological structure of hippocampal CA1 region. Ultrasound of neuron in rat hippocampal CA1 region was observed by transmission electron microscopy (TEM).Real-time fluorescent quantitative（Real-time PCR） was used to detect the Akt， Bad mRNA expression.Western blot was used to detect the Akt， p-Akt and Bad protein expression in hippocampus. Result:Compared with sham operation group， the learning and memory ability of model group decreased significantly(P<0.05). The pathological structure and neuronal ultrastructure of the hippocampus were changed obviously. Hippocampal tissue Akt mRNA and the Akt，p-Akt protein expression level decreased significantly(P<0.05)， and the levels of Bad mRNA and protein were significantly increased (P<0.05). Compared with model group， Yisui Jiedu prescription group can significantly improve the learning and memory ability of rats， improve the neuronal cells and ultrastructural changes in hippocampal CA1 area，and increase the expression of Akt mRNA and Akt，p-Akt protein in hippocampus. Decreased Bad mRNA and Bad protein expression levels (P<0.05). Conclusion:Yisui Jiedu prescription can significantly improve the learning and memory ability of VD rats， improve the ultrastructural pathological changes of hippocampus and neurons， and repair damaged neurons， which may promote Akt phosphorylation and activate PI3K/Akt/Bad. The signaling pathway plays a role in the defense of neurons against apoptosis.

Proapoptotic activities of Oroxylum indicum leave extract in HeLa cells

Objective: To examine the proapoptotic properties of Oroxylum indicum methanol extract on cervical cancer cells. Methods: Methylene blue assay was used to determine the IC

Proapoptotic activities of Oroxylum indicum leave extract in HeLa cells

Objective: To examine the proapoptotic properties of Oroxylum indicum methanol extract on cervical cancer cells. Methods: Methylene blue assay was used to determine the IC50 value of the extract. Western blotting assays were done to analyze the expression of HPV oncoproteins (HPV18 E6 and E7) and apoptotic molecules (caspase-3 and caspase-8). Reverse transcriptase PCR assays were performed to determine genetic alteration of tumor suppressors p53 and pRb and apoptosis markers Fas and FasL. Enzyme-linked immunosorbent assay (ELISA) was done to determine the expression of cytokine levels (IL-6 and IL-12). Results: The determination of IC50 value indicated a higher anti-proliferative activity of the extract compared to cisplatin. After 24 hours of treatment, Western blot analysis showed that treated HeLa cells exhibited a significant down-regulation of HPV18 oncoproteins E6 and E7, and a significant induction of caspase-8 and caspase-3 activation level. Meanwhile, the mRNA expressions of p53, pRb, Fas and FasL were significantly upregulated in treated cells. Moreover, ELISA showed an increased IL-12 and decreased IL-6 production after Oroxylum indicum treatment. Conclusions: The methanol extract of Oroxylum indicum has an anti-proliferative activity and proapoptotic potential. It induces localized-immunity improvements by altering cytokine production in HPV-positive cervical cancer cells.

Analysis on the expression of Smac and Survivin in precancerous lesions of gastric ulcer / 国际检验医学杂志

Objective To investigate the expression of pro-apoptotic factor (Smac) and Survivin in gastric ulcer tissue.Methods Selected the 80 cases of gastric ulcer patients as the research object in the first people′s hospital of neijiang,in which no precancerous lesions of 40 cases of gastric ulcer (N group),40 cases of precancerous lesions of gastric ulcer(Y group),two groups of patients were Smac mRNA and Survivin mRNA were detected by using PCR method,immunohistochemical SP method of Smac and Survivin in specimens of table detect.Gastric ulcer patients were treated by triple therapy,and the apoptosis index was detected by TUNEL.Results Smac in N group(++) and (+++) in the expression accounted for 82.5％ was higher than that in Y group accounted for 47.5％,Survivin in group N (++) and (+++) in the expression accounted for 15.0％ was significantly lower than Y group accounted for 35.0％;And Smac mRNA in the N group relative expression the amount was significantly higher than that of Y group,while the expression of Survivin mRNA in the N group were significantly lower than Y group;N group the apoptosis index of the triple therapy after treatment than before treatment significantly decreased,the difference was statistically significant(P<0.05).Conclusion The clinical application of Smac and Survivin can be used as an auxiliary diagnostic index for the diagnosis of precancerous lesions in patients with gastric ulcer.Patients with gastric ulcer without precancerous lesion treated by triple therapy which can effectively control the apoptosis index of patients,improve the survival rate of patients.

Açaí Berries Inhibit Colon Tumorigenesis in Azoxymethane/Dextran Sulfate Sodium-Treated Mice

BACKGROUND/AIMS: The aim of this study was to investigate the protective effect of açaí against azoxymethane (AOM)/dextran sulfate sodium (DSS)-induced colorectal cancer development. METHODS: The effect of açaí on tumorigenesis was assessed by evaluating tumor incidence, multiplicity and invasiveness in the mouse colon. The levels of myeloperoxidase (MPO) and proinflammatory cytokines (tumor necrosis factor α [TNF-α], interleukin [IL]-1β, and IL-6) were measured via enzyme-linked immunosorbent assay. Protein levels of cyclooxygenase 2 (COX-2), proliferating cell nuclear antigen (PCNA), B-cell lymphoma 2 (Bcl-2), Bcl-2-associated death promoter (Bad) and cleaved-caspase-3 were assessed by immunoblotting. RESULTS: Administration of pellets containing 5% açaí powder reduced the incidences of both colonic adenoma and cancer (adenoma, 23.1% vs 76.9%, respectively, p=0.006; cancer, 15.4% vs 76.9%, respectively, p=0.002). In the açaí-treated mice, the MPO, TNF-α, IL-1β and IL-6 levels in the colon were significantly down-regulated. Açaí inhibited PCNA and Bcl-2 expression and increased Bad and cleaved-caspase-3 expression. In vitro studies demonstrated that açaí treatment reduced lipopolysaccharide-induced expression of TNF-α, IL-1β, IL-6 and COX-2 in murine macrophage RAW 264.7 cells. CONCLUSIONS: Açaí demonstrated protective effects against AOM/DSS-induced colon carcinogenesis, which suggests that the intake of açaí may be beneficial for the prevention of human colon cancer.

Apoptotic Effect of Macrophages against Mycobacterium tuberculosis

Mycobacterium tuberculosis (Mtb) causing tuberculosis as an intracellular pathogen initially infects alveolar macrophages following aerosol inhalation. Thus, macrophages play a critical role in the establishment of Mtb infection and macrophage cell death, a common outcome during Mtb infection, may initiate host- or pathogen-favored immune responses, resulting in facilitating protection or pathogenesis, respectively. In addition, virulent Mtb strains are known to inhibit apoptosis and consequently down-regulates immune response using a variety of strategies. In many recent studies have shown that virulent Mtb can either augment or reduce apoptosis by regulating expression of pro-apoptotic and anti-apoptotic proteins belonging to Bcl-2 family proteins. In this review, we will discuss and dissect the apoptotic pathways of Bcl-2 family proteins in Mtb-infected macrophages.

4-(Tert-butyl)-2,6-bis(1-phenylethyl)phenol induces pro-apoptotic activity

Previously, we found that KTH-13 isolated from the butanol fraction of Cordyceps bassiana (Cb-BF) displayed anti-cancer activity. To improve its antiproliferative activity and production yield, we employed a total synthetic approach and derivatized KTH-13 to obtain chemical analogs. In this study, one KTH-13 derivative, 4-(tert-butyl)-2,6-bis(1-phenylethyl)phenol (KTH-13-t-Bu), was selected to test its anti-cancer activity. KTH-13-t-Bu diminished the proliferation of C6 glioma, MDA-MB-231, LoVo, and HCT-15 cells. KTH-13-t-Bu induced morphological changes in C6 glioma cells in a dose-dependent manner. KTH-13-t-Bu also increased the level of early apoptotic cells stained with annexin V-FITC. Furthermore, KTH-13-t-Bu increased the levels of cleaved caspase-3 and -9. In contrast, KTH-13-t-Bu upregulated the levels of pro- and cleaved forms of caspase-3, -8, and -9 and Bcl-2. Phospho-STAT3, phospho-Src, and phospho-AKT levels were also diminished by KTH13-t-Bu treatment. Therefore, these results strongly suggest that KTH-13-t-Bu can be considered a novel anti-cancer drug displaying pro-apoptotic activity.

Effect of Guanxinshutong on cerebral ischemia reperfusion in rats learning and memory ability and brain Bcl-2,Bax protein expression / 中国免疫学杂志

Objective:Guanxinshutong on cerebral ischemia reperfusion ( I/R) in rats learning and memory ability and brain Bcl-2,Bax protein expression to explore the protection mechanisms of Guanxinshutong on cerebral I /R injury in brain tissue.Methods:Healthy Wistar rats for the study were randomly divided into sham operation group ,I/R model group,high dose Guanxinshutong (1 g/kg) and low dose Guanxinshutong (0.5 g/kg) group,n=15.cerebral I/R model was estabhished by using the left commom cartid artery ligation method,modeling each group were fed daily before saline ,saline,Guanxinshutong (1 g/kg) and Guanxinshutong (0.5 g/kg), continuous 7 days;3 d after water maze training while continuing administration ,continuous 7 d,the eight day ,Morris water maze test;after the test,the rats were sacrificed blood and brain tissue ,serum by ELISA of Bcl-2 and Bax protein levels change;using HE staining of rat brain pathology;using immunohistochemistry assay in rat brain organization of Bcl-2 and Bax protein levels changes.Results:Compared with the sham group ,Guanxinshutong high-dose group test latency slightly longer timers after platform and platform quadrant dwell decreased slightly ,serum and brain tissue slightly elevated levels of Bax protein ,Bcl-2 protein levels decreased slightly but not significantly different ( P>0.05 ) ,low-dose group Guanxinshutong through latency test in rats ,after the number of platforms and platform quadrant dwell time decreased , serum and brain tissue elevated levels of Bax protein , Bcl-2 protein content decreased significantly different(P<0.01),cerebral I/R model group was significantly prolonged latency test ,after the number of platforms and platform quadrant dwell time significantly reduced serum and brain tissue was significantly increased Bax protein ,Bcl-2 protein content decreased with significant differences ( P<0.01 ).Conclusion: Guanxinshutong can significantly improve spatial memory , and by reducing Bax protein content and increased Bcl-2 protein,inhibition of cerebral I/R injury of apoptosis,and thus the brain tissue I/R has a protective effect.

The Role of Adipose Tissue Vasculature in Energy Balance / 대한소아내분비학회지

The prevalence of obesity is rapidly growing throughout the developing and developed world. Given the seriousness of obesity, it critically needs to develop new therapeutic ways to defend against its growth. Persistent increase in food intake is a primary cause of the energy imbalance. The arcuate nucleus of the hypothalamus is a key region to integrate signals originating from various regions in periphery and leptin resistance in the central nervous system (CNS) contributes to the impaired regulation of food intake. It has been endeavor to treat obesity by understanding the mechanisms of CNS regulation of food intake. Adipose tissue has been regarded as a tumor because of its reversible expansibility and dependency on vasculature. There has been a challenge to starve adipose tissue by inhibiting adipose tissue vasculature. A peptide to cause apoptosis of endothelium only in white adipose tissue greatly loses body weight by reducing food intake independent of the action of leptin. This study provides convincing evidence for a previously unknown relationship between the status of adipose tissue vasculature and the regulation of food intake that may provide a novel way for decreasing body fat. However, the mechanism by which the inhibition of angiogenesis in white adipose tissue decreases food intake and body weight remains unclear. In this review, we describe the potential mechanisms of regulation of food intake induced by inhibition of angiogenesis in white adipose tissue.

Expression,Purification of PUMA-BH3 Death Domain Peptide in E.coli and Identification of Its Pro-apoptotic Activity / 中国生物工程杂志

The Bcl-2 family of proteins play a central role in the control of apoptosis, a fundamental process for both human health and disease, by mitochondrial pathway. PUMA(p53 up-regulated modulator of apoptosis protein) is one of BH3-only members of Bcl-2 family , its function is to promote cell apoptosis. To obtain BH3 death domain peptide of PUMA and detect its biological activity, the synthesized double-stranded oligomeric nucleotide encoding PUMA-BH3 peptide was cloned into expression vector pTYB2,thus generating a construct of pTYB2-PUMA-BH3 which expressed PUMA-BH3-intein-chitin binding domain fusion protein. Then the recombinant plasmid was transformed into E.coli BL-21 (DE3) and fusion protein was expressed under induction by IPTG. The soluble PUMA-BH3 peptide was purified from chitin affinity chromatography by DTT reduction. Through measuring mitochondria viability(MTT),mitochondria permeability transition(MPT) and the translocation of cytochrome c(Cyt c ) assayed by western blotting, the biological pro-apoptotic activity of PUMA-BH3 peptide was studied. The PUMA-BH3 peptide has the effects on decreasing the mitochondria viability remarkably , inducing mitochondrial swelling and promoting Cyt c releasing from isolated mitochodria . Mitochondrial swelling and the release of Cyt c induced by PUMA-BH3 peptide concerned with the opening of MPT,which can be improved by cyclosporine A(CsA).These results indicated that recombinant PUMA-BH3 peptide might possess pro-apoptosis activity and paved a reasonable way for the study of new apoptosis regulators.

Identification of Novel Genes with Proapoptotic Activity

In order to identify novel proapoptotic genes, we screened approximately 1,000 hypothetical genes whose functions are completely unknown. After these genes were transiently expressed in HeLa cells, their nuclei images were captured using automated high-speed fluorescence microscope, through which the ratio of apoptotic nuclei was estimated. We selected genes that induce greater than 3-fold increase in apoptotic nuclei compared to that of the vector control. The candidate proapoptotic genes were sequenced and their effects on cell death were further confirmed by the additional assay, DNA fragmentation ELISA. Finally, we were able to identify 4 full-length hypo-thetical genes with proapoptotic activity.

Tumor chemosensitization strategies based on apoptosis manipulations / 中国药理学通报

MDR(multidrug-resistance)is a major obstacle in the chemotherapy of cancer.Numerous mechanisms are known to contribute to MDR,alterations at the level of apoptosis control are one of those mechanisms except overexpression of drug efflux pumps.This review focuses on the research progression of alterations at the level of apoptosis inducing MDR,and some of the strategies that have been used in an attempt to chemosensitize resistant tumors by manipulating dysregulated apoptosis pathways.

Expression and significance of pro-apoptotic factors and anti-apoptotic factors in cervical intraepithelial neoplasia and cervical carcinoma / 第三军医大学学报

0.05).But the expression of Caspase-9 in AC group was significantly higher than that in CIN and squamous cell carcinoma group(P

Exogenous Nitric Oxide-Induced Apoptosis in Cultured Rat Vascular Smooth Muscle Cells

BACKGROUND: Atherosclerosis is the most important disease that may cause ischemic syndrome in many organs including heart. It is supposed that apoptosis of vascular smooth muscle cells(VSMCs) is closely related to the progression and rupture of atheromatous plaque. Recent studies have documented evidence for elevated level of nitric oxide(NO) within advanced human atheroma and evidence of regression of atheroma by NO. So this study is designed to evaluate whether exogenous NO from NO donors can induce apoptosis of cultured rat VSMCs and which proapoptotic gene(s) is involved in this type of apoptosis. METHODS: Rat VSMCs were cultured and used for experiment at passage 5 through 7. For NO donor, sodium nitroprusside (SNP) and S-nitroso-N-acetylpenicillamine(SNAP) of 0.5, 1, 2, 4mM were exposed to subconfluent VSMCs. The cells were harvested at 6, 12, 24, 48, 72hours after exposure of NO donors. Apoptosis was to be identified by 4, 6-diamidino-2-phenylindole dihydrochloride(DAPI) staining of nuclei and in-situ nick end labeling(TUNEL). The amount of fragmented DNA was analyzed semiquantitatively by diphenylamine(DPA) assay. Immunocytochemical(ICC) staining and western bolt analyses were designed to detect apoptosisrelated gene products, such as Bax-a, Fas and Bcl-2. RESULTS: 1) Decreased mitotic activity was shown after 12 hours exposure of exogenous NO donors, and condensation and margination of chromatin was identified agter 24 hours exposure, by DAPI staining. 2) Percent DNA fragmentation assessed by DPA method was 0,2,9,48,45% at 0,6,12,24,48 hours after exposure of 2mM of NO donors respectively. 3) The expression of Bax-a and Bcl-2 proteins was demonstrated in apoptotic cells by ICC staining. 4) The expression of Bax-a protein in cells under 24 hours exposure of NO donors was elevated by more than 18% of control level on densitometric analysis of western blot. The level of Bcl-2 was suppressed by 26% of control. So, Bax-a/Bcl-2 ratio in cells under exposure of NO donors was elevated to 2.0 from 1.2 of control level. CONCLUSIONS: Exogenous NO from NO donors can induce apoptosis of cultured rat VSMCs, and it is considered that bax-a and bcl-2 genes are involved in this type of apoptosis.