MiR-125b inhibits growth of gastric cancer cells by targeting Fas/FasL signaling pathway / 中国医师杂志

Objective:To investigate the regulation effect of miR-125b in the gastric cancer cell growth mediated by apoptosis related protein (Fas)/apoptosis related protein ligand (FasL) signal.Methods:Gastric cancer SGC-7901 cells were cultured in vitro. MiR-125b inhibitor sequence, NC sequence and transfection reagent were transfected into SGC-7901 cells and divided into three groups: miR-125b inhibited group, NC group and control group. The expression of miR-125b in transfected cells was detected by real-time fluorescence quantitative polymerase chain reaction (qRT-PCR), and cell proliferation was detected by cell counting kit-8 (CCK-8) method. The colony formation was detected by plate cell clone formation assay. Cell apoptosis and cycle were detected by flow cytometry. The protein expression of Fas and FasL was detected by Western blot. The targeted regulation of Fas by miR-125b was detected by luciferase activity assay. Results:The expression level of miR-125 and the number of cell colony in miR-125b inhibited group was significantly lower than those in control group and NC group, and the inhibition rate of cell proliferation and apoptosis rate were significantly higher than that in control group and NC group (all P<0.05). The DNA content in G 1 phase in miR-125b inhibited group was significantly higher than that in control group and NC group, and the DNA content in S phase in miR-125b inhibited group was significantly lower than that in control group and NC group (all P<0.05). The expression of Fas and FasL protein in miR-125b inhibited group was significantly higher than that in control group and NC group (all P<0.05). The target site of miR-125b was found in 3′-UTR of Fas mRNA, and compared with the NC+ Fas 3′UTR-Wt group, the activity of luciferase in the miR-125b inhibited group+ Fas 3′-UTR-Wt group decreased significantly ( P<0.05). Conclusions:Inhibition of miR-125b expression can activate Fas/FasL signal and inhibit SGC-7901 cell proliferation, induce G 1 phase arrest of cell cycle and promote apoptosis.

Higher-order assemblies in immune signaling: supramolecular complexes and phase separation

Signaling pathways in innate and adaptive immunity play vital roles in pathogen recognition and the functions of immune cells. Higher-order assemblies have recently emerged as a central principle that governs immune signaling and, by extension, cellular communication in general. There are mainly two types of higher-order assemblies: 1) ordered, solid-like large supramolecular complexes formed by stable and rigid protein-protein interactions, and 2) liquid-like phase-separated condensates formed by weaker and more dynamic intermolecular interactions. This review covers key examples of both types of higher-order assemblies in major immune pathways. By placing emphasis on the molecular structures of the examples provided, we discuss how their structural organization enables elegant mechanisms of signaling regulation.

Effect of Shaoyaotang on Expressions of CD14, FADD and Caspase-8 in Colonic Tissues of Rats with Large Intestinal Damp-heat Syndrome of Ulcerative Colitis / 中国实验方剂学杂志

Objective:To observe the effect of Shaoyaotang on the contents of cell adhesion molecule-1 （ICAM-1） and transforming growth factor-<italic>β</italic>₁ （TGF-<italic>β</italic>₁） in serum of large intestine damp-heat syndrome of ulcerative colitis （UC） in rats， and the gene and protein expressions of leukocyte differentiation antigen14 （CD14）， Fas-related death domain protein （FADD） and cysteinyl aspartate specific protease-8 （Caspase-8） in the focal colon tissue. Method:A total of 80 SPF Wistar rats were randomly divided into the blank group （<italic>n</italic>=10） and modeling group （<italic>n</italic>=70）. The large intestine damp-heat syndrome of UC rats was replicated by the combination of disease and syndrome， which was high-fat， high-sugar and spicy diets combined with 2， 4-dinitrobenzene sulfonic acid （DNBS） and ethanol. After successful modeling， the modeled groups were divided into model group， sulfasalazine （SASP）control group， and low， medium and high-dose Shaoyaotang groups by the method of random number table， with14 rats in each group. Low， medium and high doses of Sulfasalazine 0.2 g·kg^-1·d^-1 and Shaoyaotang （6， 12， 24 g·kg^-1·d^-1）were given by gavage. The blank group and the model group were given equal volume of normal saline for 21 days. The contents of serum ICAM-1 and TGF-<italic>β</italic>₁ were detected by enzyme-linked immunosorbent assay （ELISA）， the expressions of CD14， FADD and Caspase-8 mRNA in colon tissues were detected by Real-time quantitative polymerase chain reaction （Real-time PCR）， and the expressions of CD14， FADD and Caspase-8 protein in colon tissues were detected by Western blot. Result:Compared with the blank group， the serum ICAM-1 level in the model group were significantly increased， whereas the content of TGF-<italic>β</italic>₁ were significantly decreased （<italic>P</italic><0.05）. The relative expression levels of CD14， FADD， Caspase-8 mRNA and protein were significantly increased （<italic>P</italic><0.05）. Compared with the model group， the content of ICAM-1 in the serum of the rats in the medium， high-dose Shaoyaotang groups and the SASP group were significantly decreased， while the content of TGF-<italic>β</italic>₁ in the serum of the rats in the low， medium， high-dose Shaoyaotang groups and the SASP group were significantly increased （<italic>P</italic><0.05）. The expression levels of CD14， FADD， Caspase-8 mRNA and protein in each intervention group were significantly decreased （<italic>P</italic><0.05）， especially in the high-dose Shaoyaotang group and the SASP group. Conclusion:Shaoyaotang has a certain intervention effect on UC rats with large intestine damp-heat syndrome， and its mechanism may be related to the inhibition of CD14， FADD and Caspase-8 genes and proteins expression.

A new perspective of triptolide-associated hepatotoxicity: the relevance of NF- B and NF- B-mediated cellular FLICE-inhibitory protein

Previously, we proposed a new perspective of triptolide (TP)-associated hepatotoxicity: liver hypersensitivity upon lipopolysaccharide (LPS) stimulation. However, the mechanisms for TP/LPS-induced hepatotoxicity remained elusive. The present study aimed to clarify the role of LPS in TP/LPS-induced hepatotoxicity and the mechanism by which TP induces liver hypersensitivity upon LPS stimulation. TNF- inhibitor, etanercept, was injected intraperitoneally into mice to investigate whether induction of TNF- by LPS participated in the liver injury induced by TP/LPS co-treatment. Mice and hepatocytes pretreated with TP were stimulated with recombinant TNF- to assess the function of TNF- in TP/LPS co-treatment. Additionally, time-dependent NF-B activation and NF-B-mediated pro-survival signals were measured and . Finally, overexpression of cellular FLICE-inhibitory protein (FLIP), the most potent NF-B-mediated pro-survival protein, was measured and to assess its function in TP/LPS-induced hepatotoxicity. Etanercept counteracted the toxic reactions induced by TP/LPS. TP-treatment sensitized mice and hepatocytes to TNF-, revealing the role of TNF- in TP/LPS-induced hepatotoxicity. Mechanistic studies revealed that TP inhibited NF-B dependent pro-survival signals, especially FLIP, induced by LPS/TNF-. Moreover, overexpression of FLIP alleviated TP/LPS-induced hepatotoxicity and TP/TNF--induced apoptosis . Mice and hepatocytes treated with TP were sensitive to TNF-, which was released from LPS-stimulated immune cells. These and other results show that the TP-induced inhibition of NF-B-dependent transcriptional activity and FLIP production are responsible for liver hypersensitivity.

KLF5 suppresses TRAIL-induced cell apoptosis in PC-3 prostate cancer cells / 西安交通大学学报(医学版)

Objective: To explore the possibility of promoting tumor necrosis factor-related apoptosis inducing ligand (TRAIL)-induced apoptosis in prostate cancer PC-3 cell by inhibiting Krüppel-like factor 5 (KLF5). Methods: MTT assay, flow cytometry, Western blot assay and qRT-PCR assay were deployed to detect the cell viability, apoptosis and apoptotic markers in KLF5-inhibited and TRAIL-induced PC-3 cells. Results: After KLF5 was inhibited in TRAIL-induced PC-3 cells, cell viability reduced, apoptosis enhanced, the expressions of DR4 and DR5 increased while the expression of cellular fas-associated death domain-like interleukin-1β converting enzyme inhibitory protein (c-FLIP) decreased. Conclusion: Inhibiting KLF5 suppresses cell viability by promoting TRAIL-induced apoptosis in prostate cancer cell PC-3. It may be a potential means to treat hormone-insensitive prostate cancer.

Effect of Xiao Chaihutang on NF-κB Signaling Pathway in Synovial Tissue of CFA Rats / 中国实验方剂学杂志

Objective:To observe the expression of tumor necrosis factor receptor-associated death domain (TARDD), nuclear transcription factor-κB inhibiting protein α(IκBα)IκB kinase-α (IKKα) and nuclear transcription factor (NF)-κB p65 protein in the NF-κB signaling pathway of synovial tissues of complete Freund's adjuvant (CFA) rats after treatment with Xiao Chaihutang (XCHT). Method:In animal experiments, SPF health adult female Wistar rats were used to prepare the CFA animal model of rats with rheumatoid arthritis with Freund's complete adjuvant and cattle Ⅱ collagen type. According to the random number table, the rats were randomly divided into the normal group, the model group, the low-dose XCHT group, the medium-dose XCHT group, the high-dose XCHT group, and the Tripterygium glucosides group. The drugs were given at 7 d after the model was built. Both normal group and model group were given water for injection,and low-dose XCHT group(5.94 g·kg-1),medium-dose XCHT group(11.88 g·kg-1),high-dose XCHT group(23.76 g·kg-1),Tripterygium glucosides group(0.006 3 g·kg-1) were given corresponding drugs by gavage for three times a day, 2 mL/time. The histopathology of rat ankle joint was observed, and the protein expressions of TARDD,IKKα,IκBα,NF-κB p65 in the NF-κB signaling pathway in synovial tissue of CFA rats were detected by Western blot. Result:With the increase of the dosage of XCHT, the histopathological score of the right posterior ankle joint of the experimental rats was increased. And in the protein expressions of TARDD,IKKα,IκBα,NF-κB p65 in NF-κB signaling pathway in Synovial Tissue of CFA rats, compared with the model group, the statistical results of the low-dose XCHT group showed decreased protein expressions (PPPα, IκB α, NF-κB p65 in the NF-κB signaling pathway were significantly increased (PPα, IκBα, NF-κB p65 key protein expressions in the NF-κB signaling pathway and protein expressions in low-dose XCHT group were obviously lower (PPConclusion:This study shows that as the dose of Xiao Chaihutang increases, it could effectively improve synovitis, and suppress the expressions of key proteins in the inflammatory signaling pathway of NF-κB, thereby preventing inflammation and suppressing bone erosion.

Study on influence of Daxx on cell cycle and chemotherapy in human ovarian cancer cells / 重庆医学

Objective To explore the influence of down-regulating Daxx on cell cycle and chemotherapeutic drug resistance in human ovarian cancer cells.Methods SiRNA and NC negative control(NC) RNA of Daxx were constructed and divided into the NC group and silencing Daxx(siDaxx) group after transfecting to ovarian cancer cell line C13k.The doxorubicin concentration gradient of 0,0.15,0.30,0.60 μmol/L was set.The cellular cycle changes and apoptosis changes of these two groups were detected by using the flow cytometry.Western blot was used to detect the expression changes of apoptosis related protein cyclinB1 and cleavedparp.Results 0.30 μmol/L doxorubicin down-regulated Daxx to result in significant G2/M arrest(P＜0.05).Down-regulating Daxx resulted in doxorubicin resistance in C13k cells.Conclusion The effect of Daxx on ovarian cancer chemotherapy might be related to its regulation on cell cycle.

Garlic-derived compound -allylmercaptocysteine inhibits hepatocarcinogenesis through targeting LRP6/Wnt pathway

Whether and how garlic-derived -allylmercaptocysteine (SAMC) inhibits hepatocellular carcinoma (HCC) is largely unknown. In the current study, the role of low-density lipoprotein receptor (LDLR)-related protein 6 (LRP6) in HCC progression and the anti-HCC mechanism of SAMC was examined in clinical sample, cell model and xenograft/orthotopic mouse models. We demonstrated that SAMC inhibited cell proliferation and tumorigenesis, while induced apoptosis of human HCC cells without influencing normal hepatocytes. SAMC directly interacted with Wnt-pathway co-receptor LRP6 on the cell membrane. LRP6 was frequently over-expressed in the tumor tissue of human HCC patients (66.7% of 48 patients) and its over-expression only correlated with the over-expression of -catenin, but not with age, gender, tumor size, stage and metastasis. Deficiency or over-expression of LRP6 in hepatoma cells could partly mimic or counteract the anti-tumor properties of SAMC, respectively. administration of SAMC significantly suppressed the growth of Huh-7 xenograft/orthotopic HCC tumor without causing undesirable side effects. In addition, stable down-regulation of LRP6 in Huh-7 facilitated the anti-HCC effects of SAMC. In conclusion, LRP6 can be a potential therapeutic target of HCC. SAMC is a promising specific anti-tumor agent for treating HCC subtypes with Wnt activation at the hepatoma cell surface.

The Clinical Implications of Death Domain-Associated Protein (DAXX) Expression

BACKGROUND: Death domain-associated protein (DAXX), originally identified as a pro-apoptotic protein, is now understood to be either a pro-apoptotic or an anti-apoptotic factor with a chromatin remodeler, depending on the cell type and context. This study evaluated DAXX expression and its clinical implications in squamous cell carcinoma of the esophagus. METHODS: Paraffin-embedded tissues from 60 cases of esophageal squamous carcinoma were analyzed immunohistochemically. An immune reaction with more than 10% of tumor cells was interpreted as positive. Positive reactions were sorted into 2 groups: reactions in 11%–50% of tumor cells and reactions in more than 51% of tumor cells, and the correlations between expression and survival and clinical prognosticators were analyzed. RESULTS: Forty-three of the 60 cases (71.7%) showed strong nuclear DAXX expression, among which 19 cases showed a positive reaction (31.7%) in 11%–50% of tumor cells, and 24 cases (40.0%) showed a positive reaction in more than 51% of tumor cells. A negative reaction was found in 17 cases (28.3%). These patterns of immunostaining were significantly associated with the N stage (p=0.005) and American Joint Committee on Cancer stage (p=0.001), but overall survival showed no significant difference. There were no correlations of DAXX expression with age, gender, or T stage. However, in stage IIB (p=0.046) and stage IV (p=0.014) disease, DAXX expression was significantly correlated with survival. CONCLUSION: This investigation found upregulation of DAXX in esophageal cancer, with a 71.7% expression rate. DAXX immunostaining could be used in clinical practice to predict aggressive tumors with lymph node metastasis in advanced-stage disease, especially in stages IIB and IV.

Expression characteristics of the Daxx gene in the mouse testis during spermatogenesis / 中华男科学杂志

Objective@#To investigate the expression characteristic of the Daxx gene in the mouse testis and its role in spermatogenesis.@*METHODS@#Real-time PCR, Western blot and immunofluorescence were used in examining the expression characteristics of DAXX in the testis tissue from wild-type, Sertoli cell-specific androgen receptor knockout (SCARKO) and androgen receptor knockout (ARKO) mice at different postnatal weeks .@*RESULTS@#The Daxx gene was highly expressed in the testis tissue and mainly in the nuclei of the wild-type mice at 4 postnatal weeks. Compared with the wild-type, the ARKO mice showed a markedly decreased expression of DAXX (0.299±0.026), which displayed a polar distribution in the spermatogenic cells (0.853±0.058) and exhibited no significant difference in the SCARKO mice (1.000±0.015).@*CONCLUSIONS@#The Daxx gene expression is the highest in the middle-stage development of the mouse testis, significantly decreased in ARKO mice as compared with the wild-type, and its location influenced by specific AR knockout in Sertoli cells. DAXX may be involved in the regulation of spermatogenesis in mice.

Reproductive toxicity of bisphenol A in adult male mice and mechanism / 吉林大学学报(医学版)

Objective:To investigate the damage effect of bisphenol A (BPA)on the testis tissue of adult mice, and to reveal the reproductive toxicity of BPA in the body of animal and mechanism.Methods:40 KM mice aged 8 weeks were randomly divided into control group (according to the weight ratio of corn oil gavage), low dose of BPA group (100 mg·kg-1BPA),moderate dose of BPA group (200 mg·kg-1 BPA),and high dose of BPA group (400 mg·kg-1 BPA).4 weeks laster,the testis tissue was taken.The apoptotic rates in the testis tissue were detected by flow cytometry;the distribution and expression of Fas and FADD were measured by immunohistochemistry.Results:Compared with control group,the apoptotic rate,the expression rates of Fas and FADD in testis tissue of the mice in low dose of BAP group had no changes (P>0.05),while the apoptotic rates in the testis tissue and the positive expressions rates of Fas and FADD in moderate and high doses of BPA groups were increased (P<0.05).Compared with low dose of BPA group,the apoptotic rates and the positive expression rates of FAS and FADD in tests tissue of the mice in moderate and high doses of BPA groups were significantly increased (P<0.05).Compared with moderate dose of BPA group,the apoptotic rate and the positive expression rates of FAS and FADD in testis tissue of the mice in high dose of BPA group was significantly increased (P<0.05).The overexpression of Fas and FADD was positively correlated to the apoptotic rate in testis tissue in moderate dose of BPA group (r=0.430,P<0.05;r=0.238,P<0.01)and high dose of BPA group (r=0.637,P<0.01;r=0.359,P<0.01).Conclusion:BPA with content dose can increase the apoptotic rates of cells in testis tissue and the expressions of Fas and FADD.BPA’s reproductive toxicity may be closely associated with the activation of Fas signal pathway and resulting in massive apoptosis.

Immune -mediated liver failure / 临床肝胆病杂志

The primary causative factors of liver failure include direct damage and immune -mediated liver injury.Increasing evidence sug-gests that immune -mediated injury plays a pivotal role in the pathogenesis of liver failure.The new concepts concerning the mechanisms of immune -mediated liver injury in liver failure are reviewed with relevant basic and clinical studies in both humans and animals.The innate and adaptive immunity,particularly the interaction of various immune cells and molecules,as well as apoptosis -related molecules,are dis-cussed in detail.

Influence of ginsenoside Rg1 in expressions of FADD and FLIP in substantia nigra of Parkinson’s disease model mice / 吉林大学学报(医学版)

Objective To investegate the effect of ginsenoside Rg1 on the apoptosis related protein FLICE-inhibitory protein(FLIP),Fas-associated death domain protein (FADD)and Caspase-3 in the subatania nigra(SN)of 1-methyl-4-phenyl-1,2,3,6-tetrahyd-ropyridine (MPTP)-induced mouse models of Parkinson’s disease(PD), and to investigate the role of FADD and FLIP in the pathogenesis of PD and the protective effect of ginsenosides Rg1 on dopaminergic neurons.Methods 45 C57BL/6N mice were randomly divided into control group,model group and ginsenoside Rg1 group (n=15).The mice in model group were injected with MPTP by intraperitoneal,the mice in Rg1 group were injected with ginsenoside Rg1 before injecting MPTP,and the mice in control group were injected with normal saline by intraperitoneal. The behavioral changes of the mice in various groups were observed, and immunohistochemistry and Western blotting methods were used to observe the expressions of tyrosine hydroxylase (TH),FADD,FLIP and Caspase-3 in substantia nigra of the mice.Results Compared with control group,the mice in model group presented with typical symptoms of PD, the TH-positive neurons in the subatania nigra was significantly reduced (P<0.01 ), the number of FADD, FLIP and Caspase-3 positive cells was significantly increased(P<0.01),and the cytoplasm was deeply stained;the protein expression levels of FADD,FLIP and Caspase-3 were significantly increased (P<0.01).Compared with model group,the PD symptoms of the mice in ginsenoside Rg1 group reduced, the number of TH-positive neurons was significantly increased, the number of positive cells of FLIP,FADD and Caspase-3 were significantly reduced(P<0.01),and the cytoplasm was lightly stained;the protein expression levels of FADD, FLIP and Caspase-3 were significantly reduced (P<0.01 ). Nonlinear correlation analysis found that there was a positive relationship between the number of FADD and Caspase-3 positive cells (r=0.791,P<0.05).Conclusion Ginsenoside Rg1 may play a neural protective effect dopaminergic on neurons by modulating the FADD and FLIP expressions in SN of PD model mice.

Correlation analysis between polycystic ovary syndrome susceptibility genes and metabolic phenotypes / 中华妇产科杂志

Objective To investigate the relationship between polycystic ovary syndrome (PCOS) susceptibility single nucleotide polymorphisms (SNP) and metabolic phenotypes in glucose and lipid metabolism and explore the pathophysiological mechanism of the susceptibility genes.Methods Three of PCOS susceptibility locus 2p16.3 (rs13405728 of LHCGR gene),2p21 (rs13429458,rs12478601 of THADA gene) and 9q33.3 (rs2479106,rs10818854 of DENNDIA gene) were selected and the metabolic phenotypes were compared between different genotypes of SNP in PCOS patients (using dominant model).Results Low-density lipoprotein cholesterol was significantly increased in CC genotype group than in TC + TT groups at rs12478601 of THADA gene [(2.5 ± 0.8),(2.4 ± 0.8) mmol/L; P =0.01].Serum insulin level of 2 hours after 75 g glucose intake was significantly higher in GG + AG groups than that of AA group at rs2479106 of DENND1A gene[(71 ±65),(64 ±50) mU/L;P =0.05],and the prevalence of type Ⅱ diabetes in first-degree relatives of patients were also increased [9.9％ (66/666),6.9％ (52/751) ; P ＜ 0.05].No association was found between metabolic phenotypes and genotypes of rs13429458,rs10818854,and rs13405728.Conclusions Genetic factors probably have effect on the metabolic characteristics of PCOS.THADA gene is related to lipid metabolism,while DENND1A gene may be involved in insulin metabolism in patients with PCOS.

Desregulação da apoptose em neoplasias mieloproliferativas crônicas / Apoptosis deregulation in myeloproliferative neoplasms

As neoplasias mieloproliferativas crônicas cromossomo Filadélfia negativas são doenças hematológicas clonais que se caracterizam pela independência ou pela hipersensibilidade dos progenitores hematopoiéticos às citocinas. Os mecanismos celulares e moleculares envolvidos na fisiopatologia das neoplasias mieloproliferativas crônicas ainda não estão totalmente esclarecidos. Achados fisiopatológicos relevantes para as neoplasias mieloproliferativas crônicas estão associados às alterações genéticas como, por exemplo, a mutação somática no gene que codifica o JAK2 (JAK2V617F). A desregulação do processo de morte celular programada, denominada apoptose, parece participar da patogênese dessas desordens. Sabe-se que a desregulação da expressão dos genes pró- e antiapoptóticos promove a resistência das células à apoptose, culminando com o acúmulo das células mieloides e estabelecendo a neoplasia. Esta revisão enfocou as alterações na regulação da apoptose em neoplasias mieloproliferativas crônicas e a importância da melhor compreensão desse mecanismo para o desenvolvimento de novas terapias para essas doenças.

Philadelphia-chromosome negative chronic myeloproliferative neoplasms are clonal hematologic diseases characterized by hematopoietic progenitor independence from or hypersensitivity to cytokines. The cellular and molecular mechanisms involved in the pathophysiology of myeloproliferative neoplasms have not yet been fully clarified. Pathophysiologic findings relevant for myeloproliferative neoplasms are associated with genetic alterations, such as, somatic mutation in the gene that codifies JAK-2 (JAK V617F). Deregulation of the process of programmed cellular death, called apoptosis, seems to participate in the pathogenesis of these disorders. It is known that expression deregulation of pro- and anti-apoptotic genes promotes cell resistance to apoptosis, culminating with the accumulation of myeloid cells and establishing neoplasms. This review will focus on the alterations in apoptosis regulation in myeloproliferative neoplasms, and the importance of a better understanding of this mechanism for the development of new therapies for these diseases.

Effects of gene silencing of Fas-associated death domain on apoptosis-related proteins in rat models of parkinsonism / 中华神经科杂志

Objective To investigate the effects of gene silencing of Fas-associated death domain (FADD) with synthetic small interfering RNA (siRNA) on apomorphine-induced contralateral rotation,and the expression of Fas and caspase-8 in rat models of parkinsonism. Methods Sprague-Dawley rats were randomly divided into 5 groups:control group,Parkinson' s disease (PD) group,FADD siRNA group,FADD siRNA positive control group and FADD siRNA negative control group. Synthetic FADD siRNA sequences,siRNA positive sequences or siRNA negative sequences were infused into right substantianigra of midbrain using RNA interference and stereotactic techniques before parkinsonian rat model establishment.Apomorphine-induced contralateral rotations of the rats were observed after the injection.The protein and mRNA expression levels of FADD,Fas and caspase-8 were measured by Western blot and RT-PCR.Results In the control group,no rotation was observed after injecting apomorphine; however,in the rest groups,the number of rats respectively was 12 (12/14),3 (3/13),4 (4/15) and 11 ( 11/14 ) in apomorphine-induced contralateral rotation,which had significant statistical differences ( x2 ＝ 18.56,P =0.000).In parkinsonian substantia nigra,marked increases in the protein and mRNA levels of FADD,Fas and caspase-8 were observed,compared with control group.Furthermore,compared with PD group,FADD protein and mRNA levels were strongly suppressed by administration of FADD siRNA in FADD siRNA group. FADD siRNA administration also resulted in great attenuation of 6-hydroxydopamine-induced increases in expression and activation of caspase-8.However,no decrease in expression of Fas was observed in FADD siRNA group and FADD siRNA positive control group,compared with PD group.Conclusion Our results suggest that death receptor signaling pathway plays a critical role in the pathogenesis of PD.FADD siRNA is effective against this pathway and it may,at least in part,provide a potential neuroprotective effect.

Effect of recombinant adenovirus Ad5F35-SH2-DED on tumorigenicity of K562 cells in nude mice / 肿瘤

Objective: To investigate the effect of fusion protein SD (SH2-DED) on the K562 leukemia subcutaneous xenografts in nude mice. Methods: The models of K562 leukemia subcutaneous xenografts in nude mice based on pretreatment and treatment with recombinant adenovirus were established. In the pretreatment model, the K562 cells pretreated with recombinant adenoviruse Ad5F35-SD or its mutant Ad5F35-SmD were subcutaneously injected into the nude mice; in the treatment model, the K562 cells were firstly subcutaneously injected into the nude mice to induce the subcutaneous xenografts, and then the recombinant adenoviruse Ad5F35-SD or its mutant Ad5F35-SmD was intratumorally injected into the xenografts. The growth of the subcutaneous xenografts and the morphological changes of the tumor cells were observed. The apoptosis of the tumor cells in subcutaneous xenografts was detected by TUNEL method and observed under a transmission electron microscope. The expression levels of caspase-3 and caspase-8 proteins in the xenografts were examined by immunohistochemistry. Results: In the treatment model, the volume of subcutaneous xenografts was significantly inhibited by Ad5F35-SD treatment, and the pathological results showed nuclear condensation and deep staining of cytoplasm. The apoptosis of tumor cells was confirmed by TUNEL method and transmission electron microscopy. The expressions of apoptosis-associated proteins caspase-3 and caspase-8 were increased. In the pretreatment model, the growth of xenografts was also inhibited by pretreatment with Ad5F35-SD. Conclusion: Recombinant adenovirus Ad5F35-SD can inhibit the tumorigenicity of K562 cells and the growth of tumor xenografts in nude mice, and promote the apoptosis of tumor cells. © 2012 by Tumor.

Experimental studies of the effect of erythropoietin on fas-associated death domain protein and caslmse-8 protein in rat with intracerebral hemorrhage / 中国综合临床

Objective To study the protein expressions of Fas-associated death domain protein (FADD) and caspase-8 in rats with intracerebral hemorrhage ,and the effects of erythropoietin tp reveal the mechanism of neu-m-protection by EPO. Methods 126 male SD rats were randomly divided into three groups: Sham-operated group, intracerebral hemorrhage group, and EPO group. Each group was divided into seven subgroups according to the differ-ent time points (3,6,12,24,48,72 h and 7 d). The model of intracerebral hemorrage was established in rats by in-tracerebral injection of autogenous blood. The protein expressions of FADD and caspas-8 in rats tissue around the hemorrhagic and the normal brain tissue were detected by immunohistochemistry. Results The protein expressions of FADD and caspase-8 were increased [(4.66±0.46 ) and ( 15.89±1.81)] at 3 h after intracerebral hemorrhage, and peaked at 48 h [ (35.88±4.24 ) and (45.04±3.99)], the expressions of FADD and caspas-8 in the region around hematoma in EPO group significantly decreased compared with model group[ (3.92±0.64) and (28.24±1.90), (13.32±2.01 ) and (35.08±2.82)] at 3 h and 48 h. Conclusion The protein expressions of FADD and easpase-8 are markedly increased after intracerebral hemorrhage. EPO can protect the neurons by signifi-cantly reducing the expressions of FADD and caspase-8.

Fas-associated death domain protein enhance inhibitory effect of 5-fluorouracil on growth of human colorectal carcinoma cells / 中华消化杂志

Objective To examine the sensitivity of human colorectal carcinoma cells to 5-fluorouracil treatment by stable transfection of extrinsic Fas-associated death domain protein(FADD) gene，both in vitro and in vivo，so as to investigate the feasibility of combination therapy of FADD gene and 5-fluorouracil in human colorectal carcinoma.Methods①RT-PCR and Western blotting were used to detect the expressions of both mRNA and protein of FADD gene in SW480／FADD (stably transfected with FADD)，SW480／neo and SW480 cells.②After treatment with 5-fluorouracil as an apoptotic inducer，in vitro cell growth activities were investigated by MTT assay.Cell apoptosis and its rates were determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling(TUNEL)assay and flow cytometry of annexin V-FITC／PI staining.The expressions of caspase-8 and caspase-3 were examined by Western blotting.③To examine the inhibitory effect of FADD gene combined with 5-fluorouracil, tumor xenograft model was prepared for in vivo study.Results ① Compared with SW480 and SW480/neo cells, FADD mRNA and protein levels of SW480/FADD cells were higher (P<0.05). ② Inhibitory rate of SW480/FADD cells was remarkably higher than SW480 and SW480/neo cells (P＜0.05 ). ③ Forty-eight hours after treatment with 5-fluorouracil (10 mg/L), the apoptotic rate of SW480/FADD cells was (33.3 ± 4.5)%, which was higher than SW480 [(13. 9 ± 3. 2)%3 and SW480/neo [(14. 1 ± 3. 4)%], with significant difference (P< 0.05).④ Forty-eight hours after treatment with 5-fluorouracil (10 mg/L),procaspase-8 and procaspase-3 expressions of SW480 and SW480/neo cells were higher than SW480/FADD cells, whereas their cleaved caspase-8 and cleaved caspase-3 expressions were lower than SW480/FADD cells (P＜0. 05).⑤ In in vivo study, SW480/FADD cells increased the efficacy of fluorouracil-induced inhibition of tumor growth in nude mice. ConclusionsStable overexpression of FADD increases sensitivity of the cells to 5-fluorouracil and combination of FADD with 5-fluorouracil will he a promising alternative in colorectal cancer treatment.

Effect of Shenxiong Injection on Neuronal Apoptosis and Fas-Associated Death Domain Protein Expression After Ischemia-Reperfusion in Rats / 国际脑血管病杂志

Objective:To imestigate the effect of Shenxiong injection on neuronal aooptosis and Fasassociated death dormin protein(FADD)and its mRNA expression after ischernia-reperfusion in rats.Methods:Atotal of 100 SD rats wgre randomly allocated into normal(n=10),sham-operation(n=10),cerebral ischemia-reperfusion(n=40),and Shenxiong injection(n=40)groups.A model of middle cerebral wtery occlusion was induced by suture method.The neuronal apoptosis was detected by the TUNEL assay.The expressions of FADD protein and mRNA were detected by inmmnohistochemical staining and reverse tramcription-polymerase chain reaction(RT-PCR),respectively.Results:As compared with the normal and sham-operation groups.the numbers of apoptotic cell in the cerebral ischemia-reperfusion group were increased significantly(P＜0.01),and the expressions of FADD protein and mRNA were enhanced significantly(all P＜0.01).As compared with the ceretral ischemia-reperfusion group,the numbers of apoptotic cell were decreased significantly (P＜0.05,P＜0.001),and the expressiom of FADD protein and mRNA were reduced significantly in the Shenxiong group(P＜0.05,P＜o.001).Convlusions:Shertxiong injection may inhibit the neuronal apoptosis after ischemia-reprfusion in r-cas by down-regulaftng the expression of FADD.