Effect of "Zhibian" (BL 54)-to-"Shuidao" (ST 28) needle insertion on the expression of Fas/FADD/Caspase-8 of death receptor pathway in rats with primary ovarian insufficiency / 中国针灸

OBJECTIVE@#To explore the effect of "Zhibian" (BL 54)-to-"Shuidao" (ST 28) needle insertion on the ovarian function in the rats with primary ovarian insufficiency (POI) and the potential effect mechanism based on the Fas/FADD/Caspase-8 of death receptor pathway.@*METHODS@#Forty-eight female SD rats were randomly divided into a blank group, a model group, a medication group and an acupuncture group, with 12 rats in each group. Except in the blank group, the rats in the other groups were intraperitoneally injected with cyclophosphamide to establish the POI model. In the acupuncture group, after successful modeling, the intervention was given with "Zhibian" (BL 54)-to- "Shuidao" (ST 28) needle insertion, once daily, 30 min in each intervention; and the duration of intervention was 4 weeks. In the medication group, estradiol valerate tablets were administered intragastrically, 0.09 mg•kg-1•d-1, for 4 weeks. The general situation and the estrous cycle of the rats were compared among groups. Using ELISA, the levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH) and estradiol (E2) in the serum were detected. HE staining was adopted to observe the morphological changes of ovarian tissue of rats. The protein expression of Fas, FADD and Caspase-8 in ovarian tissue was detected with immunohistochemistry and Western blot.@*RESULTS@#After modeling, except the rats of the blank group, the rats of the other groups had dry fur, lost hair, low spirits, reduced food intake, increased urination and loose stool. After intervention, the stool became regular gradually in the acupuncture group and the medication group. The percentage of estrous cycle disturbance was increased in the rats of the model group when compared with the blank group (P<0.01); in comparison with the model group, the percentages of estrous cycle disturbance were reduced in the acupuncture group and the medication group after intervention (P<0.01). When compared with the blank group, the body mass and E2 content in the serum were lower (P<0.01), the levels of FSH and LH in the serum and the protein expression levels of Fas, FADD and Caspase-8 were increased (P<0.01) in the model group. Compared with the model group, the body mass and E2 contents in the serum were higher (P<0.01), the levels of FSH and LH in the serum and the protein expression levels of Fas, FADD and Caspase-8 were reduced (P<0.01) in the acupuncture group and the medication group.@*CONCLUSION@#"Zhibian" (BL 54)-to-"Shuidao" (ST 28) needle insertion can effectively improve the ovarian function of POI rats, and its effect mechanism may be related to regulating the serum sex hormone levels, reducing the expression of Fas, FADD and Caspase-8 in ovarian tissue and retarding apoptosis of ovarian cells.

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.

FLASH Binds p53 and Enhances its Transcriptional Activity / 中国生物化学与分子生物学报

As an important transcription factor, the tumor suppressor protein p53 is involved in multiple biological processes such as cell cycle regulation, cell division, cell senescence and DNA repair The functional roles of p53 under various physiological conditions are inseparable from the assistance of many cofactors, which can regulate the protein modification, cellular sub-localization, and protein stability of p53 Therefore, the identification of p53-binding protein(s) has important biological significance for further understanding the signal transduction network of p53 in vivo. In the present study, a novel p53-binding protein, FADD-like interleukin-1β-converting enzyme associated huge protein (FLASH) was identified through a yeast two-hybrid screen The protein-protein interaction and the structural basis of the interaction between FLASH and p53 was also confirmed by co-immunoprecipitation analysis These studies have shown that p53 can simultaneously interact with both FLASH-N1 (aa 1 ~ 200) and FLASH-C1 (aa 1 534 ~ 1 780) In addition, both of FLASH-N and FLASH-C can interact with the same region of p53 (aa 293 ~ 393) Transcription analysis has revealed that the full length of FLASH and FLASH-M (aa 921 ~ 1533) can enhance the transcription activity of p53 In summary, FLASH can bind to p53 and enhance its transcriptional activity

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.

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.

Hepato-protective effect of rutin via IL-6/STAT3 pathway in CCl4-induced hepatotoxicity in rats

BACKGROUND: Carbon tetrachloride (CCl4) induces hepatotoxicity in animal models, including the increased blood flow and cytokine accumulation that are characteristic of tissue inflammation. The present study investigates the hepato-protective effect of rutin on CCl4-induced hepatotoxicity in rats. RESULTS: Forty male Wistar rats were divided into four groups. Group I (control group) received 1 mL/kg of dimethyl sulfoxide intragastrically and 3 mL/kg olive oil intraperitoneally twice a week for 4 weeks. Group II received 70 mg/ kg rutin intragastrically. Groups III and IV received CCl4 (3 mL/kg, 30 % in olive oil) intraperitoneally twice a week for 4 weeks. Group IV received 70 mg/kg rutin intragastrically after 48 h of CCl4 treatment. Liver enzyme levels were determined in all studied groups. Expression of the following genes were monitored with real-time PCR: interleukin-6 (IL-6), dual-specificity protein kinase 5 (MEK5), Fas-associated death domain protein (FADD), epidermal growth factor (EGF), signal transducer and activator of transcription 3 (STAT3), Janus kinase (JAK), B-cell lymphoma 2 (Bcl2) and B-cell lymphoma-extra-large (Bcl-XL). The CCl4 groups showed significant increases in biochemical markers of hepatotoxicity and up-regulation of expression levels of IL-6, Bcl-XL, MEK5, FADD, EGF, STAT3 and JAK compared with the control group. However, CCl4 administration resulted in significant down-regulation of Bcl2 expression compared with the control group. Interestingly, rutin supplementation completely reversed the biochemical markers of hepatotoxicity and the gene expression alterations induced by CCl4. CONCLUSION: CCl4 administration causes alteration in expression of IL-6/STAT3 pathway genes, resulting in hepatotoxicity. Rutin protects against CCl4-induced hepatotoxicity by reversing these expression changes.

Kurarinone promotes TRAIL-induced apoptosis by inhibiting NF-kappaB-dependent cFLIP expression in HeLa cells

This study was designed to investigate the effects of the prenylated flavonoid kurarinone on TNF-related apoptosis inducing ligand (TRAIL)-induced apoptosis and its underlying mechanism. A low dose of kurarinone had no significant effect on apoptosis, but this compound markedly promoted tumor cell death through elevation of Bid cleavage, cytochrome c release and caspase activation in HeLa cells treated with TRAIL. Caspase inhibitors inhibited kurarinone-mediated cell death, which indicates that the cytotoxic effect of this compound is mediated by caspase-dependent apoptosis. The cytotoxic effect of kurarinone was not associated with expression levels of Bcl-2 and IAP family proteins, such as Bcl-2, Bcl-xL, Bid, Bad, Bax, XIAP, cIAP-1 and cIAP-2. In addition, this compound did not regulate the death-inducing receptors DR4 and DR5. On the other hand, kurarinone significantly inhibited TRAIL-induced IKK activation, IkappaB degradation and nuclear translocation of NF-kappaB, as well as effectively suppressed cellular FLICE-inhibitory protein long form (cFLIPL) expression. The synergistic effects of kurarinone on TRAIL-induced apoptosis were mimicked when kurarinone was replaced by the NF-kappaB inhibitor withaferin A or following siRNA-mediated knockdown of cFLIPL. Moreover, cFLIP overexpression effectively antagonized kurarinone-mediated TRAIL sensitization. These data suggest that kurarinone sensitizes TRAIL-induced tumor cell apoptosis via suppression of NF-kappaB-dependent cFLIP expression, indicating that this compound can be used as an anti-tumor agent in combination with TRAIL.

Expression of cellular FLICE inhibitory proteins in peripheral blood B lymphocytes in patients with systemic lupus erythematosus / 中华皮肤科杂志

Objective To study the expression of cellular FLICE inhibitory proteins(c-FLIP)in peripheral blood B lymphocytes in patients with systemic lupus erythematosus (SLE)and its correlation with clinical features.Methods Blood samples were obtained from 53 patients with SLE and 30 normal human controls.Flow cytometry and ELISA were performed to measure the expression of c-FLIP in pefipheral blood B lymphocytes and serum levels of IL-4 and IL-10,respectively.Relevant laboratory examinations were carried out for these patients.SLE disease activity index(SLEDAI)score was calculated for patients.Results The positivity rate of c-FLIP in B lymphocytes was 3.11%±0.70%in 18 patients with active SLE.significantly higher than that in 35 patients with inactive SLE (0.78%±0.28%)and normaI controls(0.68%±0.12%),while no statistical difierence was found between inactive patients and controls(t=1.56,P＞0.05).In SLE patients,the expression of c-FLIP showed a positive correlation with SLEDAl score(r=0.96.P＜0.05),erythrocyte sedimentation rate(r=0.96,P＜0.01),serum level of C reactive protein(r=0.92.P＜0.01)and the titer of antinuclear antibodies(r=0.86,P＜0.01),whereas in 36 patients with leucopenia.a negative correlation was noticed between white blood cell count and the expression level of c-FLIP(r=-0.94,P＜0.0 1).The 23 patients with lupus nephritis had a higher level of c-FLIP than those without lupus nephritis(3.04%±1.09%vs 1.76%±1.09%,t=4.23,P＜0.05).Additionally.the expression of c-FLIP positively correlated with the serum level of IL-4 and IL-10(r=0.80,0.89.respectively,both P＜0.01).Conclusions In patients with active SLE,the expression of c-FLIP is upregulated in peripheral blood B lymphocytes,and positively correlated with the severity of SLE as well as the serum level of IL-4 and IL-10.The upregulation of c-FLIP in B cells might play a certain role in deficient apoptosis or clearance of activated B cells in SLE.

Effect of FADDDEL-GFP modification in murine islet cell transplantion / 基础医学与临床

Objective To study the effect of fusion protein FADDDEL-GFP in the treatment of type 1 diabetes by murine islet cell transplantion.Methods After transfecting the recombinate FADDDEL-GFP into mouse insulinoma cells NIT-1,insulin level secreted by the cells was examined.Type 1 diabetes was induced by STZ as animal model.NIT-fg cells,the NIT-1 cells modified by FADDDEL-GFP,were transplanted into the diabetic mice.Then the effects of islet cell transplantation on diabetic mice were checked.Results GFP was expressed in NIT-1 cells transfected with FADDDEL-GFP.Diabetic mice reached normoglycemia and prolonged the life span,which was obviously different from the control group.Conclusion FADDDEL-GFP may enhance the capability of resisting allogeneic transplantation rejection.

Expressions in FAS、FASL、FADD、 caspase-8 and their significant in esophageal carsinogenesis / 基础医学与临床

Objective To investigate the effects of the gene expressions of FAS、FASL、FADD and caspase-8 in esophageal carsinogenesis. Methods Immunohistochemical method was applied to detect the expression of FAS、FASL、FADD and caspase-8 proteins in esophageal epithelium. In situ hybridization method was applied to detect the expression of FADD and caspase-8 mRNA in esophageal epithelium. Results The positive rates of FAS, FADD and caspase-8 proteins and FADD、caspase-8 mRNA were decreased from normal epithelium to dysplasia and carcinoma tissues gradually. The positive rates of FASL protein were increased from normal epithelium to dysplasia and carcinoma tissues gradually. There was very significant statistical difference between carcinoma and normal epithelium(P

Conditioned Medium from Dying Smooth Muscle Cell Induced Apoptotic Death

In this study, the authors investigated whether death of vascular smooth muscle cell (VSMC) had a pathological pertinence. Conditioned media obtained from rat aorta smooth muscle cell (SMC) that were induced death by expressing FADD in the absence of tetracycline (FADD-SMC) triggered death of normal SMC. DNA fragmentation and caspase-3 activation were observed in dying SMC by conditioned media. FADD-SMC showed transcriptional activation of tumor necrosis factor (TNF)-alpha. Conditioned medium contained TNF-alpha, indicating secretion of the cytokine from dying FADD-SMC. It was investigated if secreted TNF-alpha was functional. Conditioned medium activated ERK and p38 MAPK pathways and induced MMP-9 expression, whereas depletion of the cytokine with its soluble receptor (sTNFR) remarkably inhibited induction of MMP-9 by conditioned medium. These findings suggest that TNF-alpha in conditioned medium seems to be active. Then, contribution of TNF-alpha on death-inducing activity of conditioned medium was examined. Depletion of TNF-alpha with soluble TNF-alpha receptor decreased the death activity of conditioned medium by 35%, suggesting that TNF-alpha play a partial role in the death activity. Boiling of medium almost completely abolished the death-inducing activity, suggesting that other heat labile death inducing proteins existed in conditioned medium. Taken together, these results indicate that SMC undergoing death could contribute to inflammation by expressing inflammatory cytokines and pathological complications by inducing death of neighboring cells.

Expression and clinical value of FLIP,FADD in carcinoma of larynx / 重庆医科大学学报

Objective:To study the expression of apoptosis related gene FLIP and FADD in 56 cases of human laryngeal squamous cell carcinoma.Methods:Immunohistochemical method(SP) was used to detect the expressions of FLIP and FADD in larynx carcinoma.PCNA was detected to evaluate the proliferation of carcinoma cell.Results:FLIP expression showed a positive correlation with the proliferation of larynx carcinoma cells.FADD displayed a reverse results.No associations were observed in the expressions of FLIP and FADD.The expressions of FLIP and FADD were related to tumor's clinical stage,pathological grade,but not to age,sex of patients and lymphatic metastasis.Conclusions:The abnormal expressions of FLIP and FADD may play an important role in the development of larynx carcinoma.

Immunohistochemical Analysis of Fas-associated Death Domain Protein Expression in Stomach Cancers

PURPOSE: Evidence exists that dysregulation of apoptosis is involved in the pathogenesis of cancer development. Fas- associated death domain (FADD) protein, an adaptor protein of death receptors, is a critical regulatory component of the extrinsic cell- death pathway that exerts its pro-apoptotic effect upon binding with death receptors. Expression of the FADD protein has not been reported in stomach cancer. The aim of this study was to explore the expression status of the FADD protein in stomach cancers. MATERIALS AND METHODS: In the current study, we analyzed the expression of the FADD protein in 60 advanced stomach cancer by using immunohistochemistry and a tissue microarray approach. RESULTS: Immunopositivity (defined as > or =30%) was observed for the FADD protein in 23 (38%) of the 60 cancers. Normal gastric mucosal cells showed expression of the FADD protein. CONCLUSION: Taken together, these results indicate that decreased expression of the FADD protein is a frequent event in stomach cancers and suggest that to avoid apoptosis, stomach cancer cells in vivo may need loss of FADD expression, which might contribute to tumor development.

Apoptosis of Renal Cell Carcinoma Cells by Expression of FADD (Fas-Associated Death Domain) / 대한비뇨기과학회지

PURPOSE: The Fas-associated death domain (FADD) constitutes a novel protein that specifically associates with the cytoplasmic death domain of Fas, and induces apoptosis. FADD is composed of a death effector domain (DED) and a death domain. In this study, we evaluated the in vivo antitumor effect of the FADD, or FADD-DED, gene in renal cell carcinoma cells, using a plasmid vector expressing the human FADD and FADD-DED genes. MATERIALS AND METHODS: The cDNA of the human FADD and FADD-DED genes were amplified by RT-PCR, and cloned to the pCR(R) 3.1. The expressions of the cloned FADD and FADD-DED (pCR(R)3.1-FADD and pCR3.1-FADD-DED) were observed by Western blot analysis. The efficacy of the growth inhibition by the cloned FADD and FADD-DED genes was tested, in vitro, on A498 and Caki-1 human renal cell carcinoma cell lines using the MTT assay. To evaluate the apoptosis, DNA fragmentation and caspase-3 assays were performed. RESULTS: Expressions of the FADD protein, and the FADD-DED, of the transfected A498 and Caki-1 cells had increased by 48 and 24 hours, respectively, compared with the control cell lines. The cytotoxicity of the pCR3.1-FADD and pCR3.1-FADD-DED on the A498 and Caki-1 cells significantly increased compared to the empty vector. The increased cytotoxicity of the FADD- or FADD-DED-transfected cell lines was associated with enhanced apoptosis, as assessed by DNA fragmentation and caspase-3 assays. CONCLUSIONS: Our results showed that the cloned FADD or FADD-DED expression plasmid vector efficiently inhibited the growth of A498 and Caki-1 human renal cell carcinoma cell lines. These data suggest that the exogenous FADD or FADD-DED expressions may have therapeutic applications in renal cell carcinomas.

Expressions of cFLIP and FADD in cervical carcinoma and their significances / 重庆医科大学学报

Objective:To investigate the expressions of cFLIP and FADD in cervical carcinoma and explore their associations with the carcinogenesis and development of cervical carcinoma,and to evaluate their clinical significances.Methods:The expressions of cFLIP and FADD proteins were detected in cervical tissue samples including 64 cervical carcinoma tissues,15CIN,12 chronic cervicitis tissues and 10 normal cervix tissues by immunohistochemistry(SP).And PCNA-LI in 64 cervical carcinoma samples was determined.Results:cFLIP and FADD expressions were significantly different among cervical carcinoma,CIN,chronic cervicitis tissue and normal cervix.The high expression of cFLIP and low expression of FADD were simultaneously found in cervical carcinoma.Statistical analysis showed that there was a positive correlation between the level of cFLIP expression and PCNA-LI(r1=0.7738,P