Synergistic sensitization of hUMSCs-derived supernatant combined with temozolomide in different glioma cell lines / 中国病理生理杂志

AIM:To explore the synergistic sensitization effect of human umbilical cord mesenchymal stem cell culture supernatant(hUMSC-CM)combined with temozolomide(TMZ)on various glioma cell lines,and to elucidate the underlying mechanisms.METHODS:The hUMSC-CM was harvested using two different serum deprivation tech-niques at 24 and 48 h,and was converted into freeze-dried powder,which was then given to rat malignant glioma cell line RG-2,human astrocytoma cell line U251 and human glioblastoma cell line LN-428 at 5 concentrations(0,1,3,6 and 9 g/L).The effectiveness and sensitivity of hUMSC-CM for inhibiting growth of glioma cells at 24,48 and 72 h were as-sessed using CCK-8 assay.Hematoxylin-eosin(HE)staining combined with CCK-8 assay was employed to evaluate the chemotherapy sensitivity of glioma cells after 48 h of treatment with TMZ at 6 concentrations(0,25,50,100,200 and 400 μmol/L).Two concentrations(3 and 9 g/L)of hUMSC-CM and 3 concentrations(50,100 and 200 μmol/L)of TMZ were chosen for concurrent treatment of glioma cells to assess the proliferation and pathological alterations.TUNEL staining was utilized to detect apoptosis.Flow cytometry was utilized to analyze cell cycle modifications.The expression alterations of apoptosis-inducing proteins,cleaved caspase-3,cleaved caspase-8 and cleaved PARP1,as well as autophagy-inducing proteins beclin-1 and LC3,were examined using Western blot to investigate the synergistic sensitization mechanism of hUMSC-CM combined with TMZ in vitro.RESULTS:The susceptibility of glioma cell lines to hUMSC-CM and TMZ varied,with RG-2 showing the highest sensitivity,followed by U251,and then LN-428.The inhibitory effect of hUMSC-CM(3 and 9 g/L)and TMZ(50,100 and 200 μmol/L)combined treatment on glioma cells was significantly greater than that that of single-agent treatments(P＜0.05),demonstrating a dose-and concentration-dependent enhancement.Notably,the combination of 9 g/L hUMSC-CM(C9)with 50 μmol/L TMZ(T50)effectively suppressed glioma cell growth.CCK-8 as-say indicated a significant reduction of cell viability in C9+T50 group compared with either C9 or T50 alone(P＜0.05).HE staining and TUNEL staining revealed pronounced morphological changes and significant apoptotic features in glioma cells treated with C9+T50.Flow cytometric analysis confirmed that C9+T50 induced cell cycle arrest in glioma cells.Fur-thermore,compared with control group,the levels of cleaved caspase-3,cleaved caspase-8,cleaved PARP1,beclin-1,and LC3-Ⅱ/LC3-Ⅰ were significantly elevated in the C9+T50-treated glioma cells(P＜0.01).CONCLUSION:(1)The concomitant administration of hUMSC-CM and TMZ exerts a broad inhibitory effect on glioma cells,with a synergistic sen-sitization observed across different cell lines.(2)The enhancement of glioma cell sensitivity to TMZ by hUMSC-CM may be attributed to the modulation of caspase-8/caspase-3/PARP1 signaling pathway and the induction of both apoptosis and autophagy in glioma cells.

Hei Xiaoyaosan Regulates Fas/FasL/Caspase-8/Caspase-3 Signaling Pathway to Inhibit Neuronal Apoptosis in AD Rats / 中国实验方剂学杂志

ObjectiveTo explore the effect and mechanism of Hei Xiaoyaosan in regulating the tumor necrosis factor receptor superfamily member 6 （Fas）/Fas ligand （FasL）/cysteine protease-8 （Caspase-8）/cysteine protease-3 （Caspase-3） signaling pathway to intervene in neuronal apoptosis and prevent Alzheimer's disease （AD）. MethodNinety SPF-grade SD male rats of 4 months old were selected and randomly grouped as follows： 10 rats in the blank group， 10 rats in the sham group （bilateral hippocampus injected with 1 μL normal saline）， and 70 rats in the modeling group ［bilater hippocampus injected with 1 μL amyloid-beta protein 1-42 （Aβ1-42） solution for the modeling of AD］. Fifty successfully modeled rats were selected and randomly assigned into model， donepezil hydrochloride （0.45 mg·kg-1）， and high-， medium-， and low-dose （15.30， 7.65， 3.82 g·kg-1） Hei Xiaoyaosan groups. Rats were administrated with corresponding agents by gavage once a day for 42 days. Terminal-deoxynucleoitidyl transferase-mediated nick end labeling （TUNEL） was employed to observe the apoptosis of neurons in the cortex and hippocampus， and immunohistochemistry （IHC） was used to detect the expression of B-cell lymphoma-2 （Bcl-2） and Bcl-2-associated X protein （Bax） in the hippocampus. Real-time fluorescence quantitative polymerase chain reaction（Real-time PCR） was employed to determine the mRNA levels of Fas， FasL， and Fas-associated protein with death domain （Fadd）. Western blot was used to determine the protein levels of Fas， FasL， Fadd， Caspase-3， cleved Caspase-3， Caspase-8， and cleved Caspase-8. ResultCompared with the blank group and sham group， the model group showed increased apoptosis rate in the cortex and hippocampus （P<0.01）， elevated Bax level （P<0.01）， lowered Bcl-2 level （P<0.01）， up-regulated mRNA levels of Fas， FasL， and Fadd in the hippocampus （P<0.01）， and up-regulated protein levels of Fas， FasL， Fadd， cleaved Caspase-3， and cleaved Caspase-8 （P<0.01）. Compared with the model group， donepezil hydrochloride and Hei Xiaoyaosan at high and medium doses decreased the apoptosis rate in the cortex and hippocampus （P<0.05， P<0.01）， lowered the Bax level （P<0.01）， elevated the Bcl-2 level （P<0.01）， and down-regulated the mRNA levels of Fas， FasL， and Fadd and the protein levels of Fas， FasL， Fadd， cleaved Caspase-3， and cleaved Caspase-8 （P<0.05， P<0.01） in the hippocampus. Low-dose Hei Xiaoyaosan decreased the apoptosis rate in the cortex and hippocampus （P<0.05， P<0.01）， lowered the Bcl-2 level （P<0.01）， and down-regulated the mRNA levels of FasL and Fadd （P<0.05） and the protein levels of Fas， FasL， Fadd， cleaved Caspase-3， and cleaved Caspase-8 （P<0.05） in the hippocampus. ConclusionHei Xiaoyaosan can protect neurons in the cortex and hippocampus of AD rats by inhibiting the apoptosis mediated by the Fas/FasL/Caspase-8/Caspase-3 signaling pathway.

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.

Compound Tinglizi Decoction intervenes COPD-associated pulmonary hypertension through regulation of HMGB1-mediated pyroptosis and immune imbalance / 中国中药杂志

This paper aimed to investigate the effects of high mobility group box 1(HMGB1)-mediated pulmonary artery smooth muscle cell pyroptosis and immune imbalance on chronic obstructive pulmonary disease-associated pulmonary hypertension(COPD-PH) in rats and the intervening mechanism of Compound Tinglizi Decoction. Ninety rats were randomly divided into a normal group, a model group, low-dose, medium-dose, and high-dose Compound Tinglizi Decoction groups, and a simvastatin group. The rat model of COPD-PH was established by fumigation combined with lipopolysaccharide(LPS) intravascular infusion, which lasted 60 days. Rats in the low, medium, and high-dose Compound Tinglizi Decoction groups were given 4.93, 9.87, and 19.74 g·kg~(-1) Compound Tinglizi Decoction by gavage, respectively. Rats in the simvastatin group were given 1.50 mg·kg~(-1) simvastatin by gavage. After 14 days, the lung function, mean pulmonary artery pressure, and arterial blood gas of rats were analyzed. Lung tissues of rats were collected for hematoxylin-eosin(HE) staining to observe the pathological changes. Real-time fluorescent quantitative polymerase chain reaction(qRT-PCR) was used to determine the expression of related mRNA in lung tissues, Western blot(WB) was used to determine the expression of related proteins in lung tissues, and enzyme linked immunosorbent assay(ELISA) was used to determine the levels of inflammatory factors in the lung tissues of rats. The ultrastructure of lung cells was observed by transmission electron microscope. The forced vital capacity(FVC), forced expiratory volume in 0.3 second(FEV_(0.3)), FEV_(0.3)/FVC, peek expiratory flow(PEF), respiratory dynamic compliance(Cdyn), arterial partial pressure of oxygen(PaO_2), and arterial oxygen saturation(SaO_2) were increased, and resistance of expiration(Re), mean pulmonary arterial pressure(mPAP), right ventricular hypertrophy index(RVHI), and arterial partial pressure of carbon dioxide(PaCO_2) were decreased by Compound Tinglizi Decoction in rats with COPD-PH. Compound Tinglizi Decoction inhibited the protein expression of HMGB1, receptor for advanced glycation end products(RAGE), pro caspase-8, cleaved caspase-8, and gasdermin D(GSDMD) in lung tissues of rats with COPD-PH, as well as the mRNA expression of HMGB1, RAGE, and caspase-8. Pulmonary artery smooth muscle cell pyroptosis was inhibited by Compound Tinglizi Decoction. Interferon-γ(IFN-γ) and interleukin-17(IL-17) were reduced, and interleukin-4(IL-4) and interleukin-10(IL-10) were incresead by Compound Tinglizi Decoction in lung tissues of rats with COPD-PH. In addition, the lesion degree of trachea, alveoli, and pulmonary artery in lung tissues of rats with COPD-PH was improved by Compound Tinglizi Decoction. Compound Tinglizi Decoction had dose-dependent effects. The lung function, pulmonary artery pressure, arterial blood gas, inflammation, trachea, alveoli, and pulmonary artery disease have been improved by Compound Tinglizi Decoction, and its mechanism is related to HMGB1-mediated pulmonary artery smooth muscle cell pyroptosis and helper T cell 1(Th1)/helper T cell 2(Th2), helper T cell 17(Th17)/regulatory T cell(Treg) imbalance.

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.

Modified Wenjingtang Affects Caspase-8， MMP-9， N-cadherin and E-cadherin in Local Microenvironment of EM Rats with Kidney Deficiency and Blood Stasis / 中国实验方剂学杂志

Objective:To investigate the therapeutic effect and mechanism of modified Wenjingtang on endometriosis （EM） rats with kidney deficiency and blood stasis. Method:Healthy non-pregnant female Sprague-Dawley （SD） rats of SPF grade were randomly divided into the blank group and experimental group. After being modeled via soaking in ice water and subcutaneous injection of epinephrine hydrochloride， the ones in the experimental group were further divided into the sham operation group and EM model group， with the former only undergoing laparotomy and the latter further receiving autologous endometrial transplant for triggering EM. The successfully modeled rats with EM due to kidney deficiency and blood stasis were randomized into the positive drug （danazol， 63 mg·kg^-1） group and low- （5 g·kg^-1）， medium- （10 g·kg^-1）， and high-dose （20 g·kg^-1） modified Wenjingtang groups. The corresponding drugs were administered by gavage， once per day， for four weeks. Then the ectopic and eutopic endometrial tissues were stained with hematoxylin-eosin （HE） to observe the histopathological changes. The protein and mRNA expression levels of cysteinyl aspartate-specific proteinase-8 （Caspase-8）， matrix metalloproteinase-9 （MMP-9）， N-cadherin， and E-cadherin were detected by immunohistochemistry （IHC）， Western blotting， and real-time polymerase chain reaction （Real-time PCR）， respectively. Result:The IHC and Western blot revealed that the protein expression levels of MMP-9 and N-cadherin in eutopic and ectopic endometrial tissues of the model group were significantly increased as compared with those in the sham operation group （<italic>P</italic><0.01）， while the levels of Caspase-8 and E-cadherin was significantly decreased （<italic>P</italic><0.01）. Compared with the model group， the danazol and low-， medium-， and high-dose modified Wenjingtang groups exhibited obviously down-regulated MMP-9 and N-cadherin protein expression in eutopic and ectopic endometrial tissues （<italic>P</italic><0.05，<italic>P</italic><0.01）， but up-regulated Caspase-8 and E-cadherin （<italic>P</italic><0.05， <italic>P</italic><0.01）. Real-time PCR uncovered that the mRNA expression levels of MMP-9 and N-cadherin in eutopic and ectopic endometrial tissues of the model group were significantly elevated as compared with those in the sham operation group （<italic>P</italic><0.01）， whereas the levels of Caspase-8 and E-cadherin significantly declined （<italic>P</italic><0.01）. The comparison with the eutopic endometrial tissue in the model group showed that the mRNA expression levels of MMP-9 and N-cadherin in the danazol group and high- and medium-dose modified Wenjingtang groups were significantly down-regulated， while those of Caspase-8 and E-cadherin were significantly up-regulated （<italic>P</italic><0.05，<italic>P</italic><0.01）. Conclusion:Modified Wenjingtang alleviates the immunosuppression and blocks the angiogenesis in EM rats with kidney deficiency and blood stasis syndrome by regulating the expression of such cytokines as Caspase-8， MMP-9， N-cadherin， and E-cadherin， thus exerting the therapeutic effect against EM. The above-mentioned micro-indicators are potential markers reflecting the disease （EM）， syndrome （kidney deficiency and blood stasis）， and pathological mechanisms （immunosuppression and angiogenesis）.

Quinacrine causes apoptosis in human cancer cell lines through caspase-mediated pathway and regulation of small-GTPase

Quinacrine (QC), an FDA-approved anti-malarial drug, has shown to have anticancer activities. Due to its‘shotgun’ nature, QC has become an inevitable candidate for combination chemotherapy. There is lack of studyof the molecular interplay between colorectal cancer (CRC) microenvironment and its metastasis. In this study,we focused on the differential anti-cancerous effect of QC on two different human cancer cell lines, HCT 116and INT 407. Results suggest that cytotoxicity increased in both the cell lines with an increase in QCconcentration. The expression patterns of small-GTPases and caspases were altered significantly in QC-treatedcells compared to non-treated cells. HSP70 and p53 showed comparable differences in the expression pattern.The wound-healing assay showed an increase in the denuded zone, with an increase in the concentration ofQC. The formation of apoptotic nuclei increased with a rise in the concentration of QC in both the cell lines.The decrease and increase in caspase 9 and caspase 3 expression respectively were studied, confirmingapoptosis by the extrinsic pathway

decoction induces apoptosis by activating Fas/caspase-8 pathway in rheumatoid arthritis fibroblast-like synoviocytes / 南方医科大学学报

OBJECTIVE@#To explore the effect of decoction (DGNTD) on cell apoptosis and TNF receptor super family 6 (Fas)/caspase-8 pathway in rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS).@*METHODS@#FLS isolated from the synovial tissue of RA patients were cultured and identified using immunofluorescence staining. The cells were treated with 10% blank serum (blank control group), 10% sera containing low, moderate or high doses of DGNTD, or 20 μmol/mL KR-33493 (a Fas inhibitor) combined with 10% serum containing high-dose DGNTD. MTT assay was used to detect the proliferation of the cells after the treatments. Apoptosis of the cells was detected at 48 h in each group using Hoechst 33342 staining and flow cytometry with annexin V-FITC/PI staining. The mRNA and protein expressions of Fas, FADD, caspase-8 and caspase-3 in the cells at 48 h were detected using qPCR and Western blotting.@*RESULTS@#Immunofluorescence staining identified the cultured cells as FLS. Treatment with DGNTD-containing sera significantly inhibited the proliferation of FLS, and the inhibitory effects were enhanced as the dose and intervention time increased ( < 0.05). Hoechst 33342 staining and flow cytometry showed that the sera containing different doses of DGNTD significantly promoted apoptosis of FLS ( < 0.05). The expression levels of Fas, FADD, caspase-8, and caspase-3 at both mRNA and protein levels were significantly increased in the cells after treatment with different doses of DGNTD-containing sera ( < 0.05). The application of KR-33493 obviously reversed the effects of DGNTD on the FLS ( < 0.05).@*CONCLUSIONS@#DGNTD can induce apoptosis of the FLS by activating Fas/caspase-8 signaling pathway.

Dracocephalum palmatum Stephan extract induces apoptosis in human prostate cancer cells via the caspase-8-mediated extrinsic pathway / 中国天然药物

Dracocephalum palmatum Stephan is a medicinal plant traditionally used by nomadic people in Eastern Russia; however, research on this plant is currently limited. Recently, although studies have been conducted on the constituents of this plant and their antioxidant effects, data on its various pharmacological activities are still lacking. Thus, this study examined the anticancer potential of the dried leaves of D. palmatum S. (DpL) using human prostate cancer PC-3 cells. The antioxidant potential of DpL was evaluated by estimating the total flavonoid and total phenolic content (TFC and TPC, respectively). Additionally, we investigated the effects of the DpL ethyl acetate fraction (DpLE) on cell proliferation, intracellular reactive oxygen species (ROS) generation, apoptosis, and cell cycle arrest in this cell line. The expression levels of superoxide dismutase (SOD)-1, SOD-2, B-cell lymphoma 2 (Bcl-2) and Bcl-2 associated X (Bax) ratio, phospho-protein kinase B (p-AKT), cleaved caspase-8, poly adenosine diphosphate (ADP) ribose polymerase (PARP), and cleaved-PARP were evaluated by western blotting. The results indicated that DpLE causes apoptosis and exerts intracellular ROS-independent anticancer effects on prostate cancer cells, associated with increased SOD-2, cleaved caspase-8, and cleaved-PARP expression and inhibited p-AKT signaling. Thus, DpLE may be a potential resource for the development of promising chemotherapeutic agents for prostate cancer.

Investigating the effect of intracameral cefuroxime on oxidative stress and apoptosis in the rat cornea / Investigando o efeito de cefuroxima intracameral sobre o estresse oxidativo e a apoptose na córnea de ratos

ABSTRACT PURPOSE: We examined the effect of intracameral administration of cefuroxime on oxidative stress and endothelial apoptosis in rat corneal tissue. METHODS: In total, 30 rats were divided into 3 groups of 10 rats each (intracameral administration of cefuroxime 0.1 mg/0.01 mL (cefuroxime group); intracameral administration of balanced salt solution 0.01 mL (control group); or absence of intracameral injection (sham group). Corneal endothelial apoptosis was assessed by immunohistochemical analysis using caspase-3 and caspase-8. Total oxidant status, total antioxidant status, oxidative stress index, and paraoxonase and arylesterase levels were examined in corneal endothelial tissue and serum. RESULTS: Paraoxonase levels in the serum were significantly different between the sham and cefuroxime groups (p=0.027). A significant difference was also observed in total oxidant status levels between the cefuroxime and balanced salt solution groups (p=0.023). In addition, there were significant differences in total antioxidant status levels in corneal tissue between the cefuroxime and sham groups (p<0.001) and between the cefuroxime and balanced salt solution groups (p<0.001). Furthermore, significant differences were also observed in oxidative stress index levels between the cefuroxime and balanced salt solution groups (p=0.001) and between the cefuroxime and sham groups (p=0.026). According to the immunohistochemical staining results, a significant association with caspase-3 activity existed between the cefuroxime and balanced salt solution groups (p=0.007), while no significant difference was found with caspase-8 activity (p=0.541). Caspase-3 activity exhibited a significant relationship between the sham and balanced salt solution groups (p=0.018), but no relationship was found with caspase-8 activity (p=0.623). CONCLUSION: Immunohistochemical examination revealed that intracameral cefuroxime increased apoptosis when compared to the sham and balanced salt solution groups. Moreover, intracameral cefuroxime increased oxidative stress in the cornea and simultaneously induced apoptosis.

RESUMO OBJETIVO: Examinamos o efeito da administração intracameral da cefuroxima sobre o estresse oxidativo e a apoptose endotelial no tecido corneano de ratos. MÉTODOS: No total, 30 ratos foram divididos em 3 grupos de 10 ratos cada (administração intracameral de cefuroxima 0,1 mg/0,01 mL (grupo cefuroxima), administração intracameral de solução salina balanceada 0,01 mL (grupo controle) ou ausência de injeção intracameral (grupo sham)). A apoptose endotelial da córnea foi avaliada por análise imuno-histoquimica usando caspase-3 e -8. O status oxidante total, o status antioxidante total, o índice de estresse oxidativo e os níveis de a paraoxonase e arilesterase foram investigados no tecido endotelial da córnea e no soro. RESULTADOS: Os níveis de paraoxonase no soro foram significativamente diferentes entre os grupos sham e cefuroxima (p=0,027). Foi também observada uma diferença significativa nos níveis de estado oxidante total entre os grupos cefuroxima e solução salina balanceada (p=0,023). Além disso, houve diferenças significativas nos níveis de status antioxidante total no tecido da córnea entre os grupos cefuroxima e sham (p<0,001) e entre os grupos cefuroxima e solução salina balanceada (p<0,001). Diferenças significativas também foram observadas nos níveis do índice de estresse oxidativo entre os grupos cefuroxima e solução salina balanceada (p=0,001) e entre os grupos cefuroxima e sham (p=0,026). De acordo com os resultados de coloração imuno-histoquimica, houve associação significativa com a atividade da caspase-3 entre os grupos cefuroxima e solução salina balanceada (p=0,007), enquanto não houve diferença significativa com a atividade da caspase-8 (p=0,541). A atividade da caspase-3 exibiu uma relação significativa entre os grupos sham e solução salina balanceada (p=0,018), mas nenhuma relação foi encontrada com a atividade da caspase-8 (p=0,623). CONCLUSÃO: O exame imuno-histoquímico revelou que a cefuroxima intracameral aumentou a apoptose quando comparada com os grupos sham e solução salina balanceada. Além disso, a cefuroxima intracameral aumentou o estresse oxidativo na córnea e induziu simultaneamente a apoptose.

Advances in the study of relationship between Caspases and innate immunity / 中国药科大学学报

@#Caspases are a group of structurally related cysteine proteases present in cytosol. One of their important common points is that the active sites contain cysteine and can specifically break the peptide bonds after the aspartic acid residues. Caspases are broadly divided into two groups based on their functions, including inflammatory Caspases and apoptotic Caspases. Inflammatory Caspases include Caspase-1, Caspase-4, Caspase-5, Caspase-11 and Caspase-12, which play important roles in the process of innate immune defense. Unlike inflammatory Caspases, apoptotic Caspases(2/3/6/7/8/910)initiate and execute an immunologically silent form of programmed cell death known as apoptosis. However, ongoing investigations have uncovered essential functions of Caspase-8 in the regulation of immunity in cells and organisms. Accumulated studies have shown that Caspases play important roles in the occurrence and development of various immunity-related diseases. In order to comprehensively elucidate the relationship between Caspases and innate immunity, and to provide some scientific basis and theoretical reference for the treatment of various diseases, this article reviews the regulation of activity and inflammation mechanism of innate immunity-related Caspase-1/4/5/11/8/12.

Effects of Acupuncture-rehabilitation Therapy on Neurological Function and Expression of Apoptosis-related Proteins in Ischemic Penumbra of Rats with Cerebral Ischemia / 中国康复理论与实践

@#Objective To investigate the effect of acupuncture-rehabilitation therapy on the neurological function and the expression of cleaved-caspase-8, cleaved-caspase-3 and cellular inhibitor of apoptosis 1 (cIAP1) in ischemic penumbra of rats with cerebral ischemia. Methods A total of 90 male Sprague-Dawley rats were randomly divided into sham group, model group, acupuncture group, rehabilitation group and acupuncture-rehabilitation group. Each group was divided into three days, seven days and 14 days subgroups (n=6). The cerebral ischemia model was established with the modified Koizumi suture method. The sham group and the model group received no treatment. The acupuncture group received cluster needling of scalp acupuncture, the rehabilitation group received treadmill training, and the acupuncture-rehabilitation group received both acupuncture and treadmill training. Three days, seven days and 14 days after modeling, their neurological function was assessed with modified Neurological Severity Score (mNSS) and Rota-rod test, and the expression of cleaved-caspase-8, cleaved-caspase-3 and cIAP1 protein in cerebral ischemic penumbra were detected with Western blotting. Results Compared with the model group, the mNSS scores decreased, the retention time of Rota-rod test increased, the expression of cleaved-caspase-8, cleaved-caspase-3 protein decreased and the expression of cIAP1 protein increased in each treatment group at each time point (P<0.05). Compared with the other two treatment groups, the mNSS scores further decreased, the retention time further increased, the expression of cleaved-caspase-8, cleaved-caspase-3 protein further decreased, and the expression of cIAP1 protein further increased (P<0.05) seven days and 14 days after modeling in the acupuncture-rehabilitation group.Conclusion Acupuncture-rehabilitation therapy can improve the neurological function in rats with cerebral ischemia, that is better than the simple acupuncture or exercise, which may relate to the inhibition of caspase-8 and caspase-3 protein activation, and promotion of cIAP1 protein expression, to inhibit the apoptotic caspases cascade reaction.

Retinoic acid affects apoptosis of neurons injured by hypoxic-ischemic brain damage via retinoic acid receptor alpha / 中华物理医学与康复杂志

Objective To explore the effect of the retinoic acid (RA) on the apoptosis of neurons caused by hypoxic ischemic brain damage (HIBD).Methods Seventy-two newborn Sprague-Dawley rats were randomly divided into an RA deficiency (RAD) group,an RA normal (RAN) group and a control group,each of 24.The HIBD model was established in the RAD and RAN groups using Rice's method.The left common carotid artery was exposed,ligated and cut,inducing hypoxia.In the control group the left common carotid artery was exposed without any other treatment.Three and 7 days after the operation,neuron apoptosis in the brain tissue was evaluated using TUNEL staining.The degree of HIBD was quantified using modified neurological severity scores (mNSS) 7,14,21 and 28 days after the operation.Primary neurons were cultured in vitro,and oxygen glucose deprivation (OGD) was induced,then control,OGD and RA+ OGD groups were formed.The gene transcription and the protein activity of retinoic acid receptor alpha (RARcα),GDNF (glial cell line-derived neurotrophic factor) and Caspase-8 were examined with polymerase chain reactions (PCR) and Western blotting.The RA+OGD group was exposed to RA and SiRNA adenovirus,and divided into a silenced group and a negative transfection group according to the infection.Results The average mNSS of the RAD group was significantly higher than that of the RAN group.TUNEL staining showed that the apoptotic cells in the cortex increased from day 3 to 7 after the operation,but significantly more in the RAD group than in the RAN group.The gene transcription and protein activity of RARα and GDNF in the RA+OGD group were significantly higher than in the OGD group,and those of Caspase-8 were significantly lower.The gene transcription and protein activity of RARα and GDNF in the silenced group were significantly lower than in the negative transfection group,while those of Caspase-8 were just the opposite.Conclusion RA can inhibit the apoptosis of primary neurons after HIBD by up-regulating the expression of GDNF and down-regulating that of Caspase-8 via RARα.

Survivin inhibitor YM155 induces apoptosis of thyroid carcinoma cell line B-CPAP and potential mechanisms / 中国免疫学杂志

Objective:To investigate the effects of survivin inhibitor YM155{1-(2-methoxyethyl)-2-methyl-4,9-dioxo-3-(pyrazin-2-ylmethyl)-4,9-dihydro-1H-naphtho[2,3-d] imidazolium bromide} on cell viability,apoptosis and Cysteinyl aspartate specific proteinase-3,Cysteinyl aspartate specific proteinase-8,Cysteinyl aspartate specific proteinase-9 of the thyroid carcinoma cell line B-CPAP in order to discuss mitochondrial mechanisms of apoptosis.Methods: B-CPAP cells were cultured in vitro and treated with YM155 at various concentrations(0,0.5,1,2,4,8 nmol/L)for 24,48 and 72h.The cell viability of B-CPAP cells were measured by CCK-8 assay.B-CPAP cells were randomly divided into 4 groups:B-CPAP cells were treated with YM155 at various concentrations(0,1,2 nmol/L)and 5 μmol/L Cisplatin(the positive control group)for 24 h.The effects of YM155 on B-CPAP cells apoptosis were evaluated by TUNEL and flow cytometry Annexin V-FITC/PI method.The expression level of Survivin and Caspase-3,Caspase-8 ,Caspase-9 were detected by Western blot analysis.Results: Compared with the 0 nmol/L group,YM155 significantly inhibited the cell viability of B-CPAP cells and induced their apoptosis (P<0.05 or P<0.01).Compared with the 0 nmol/L group,YM155 significantly reduced the expression level of Survivin and upregulated Caspase-3,Caspase-8 ,Caspase-9(P<0.05 or P<0.01).Conclusion: YM155 can inhibit the cell viability of B-CPAP cells and induce apoptosis,its possible mechanisms maybe related to upregulated expression level of Caspase-3,Caspase-8 and Caspase-9.

Expression of Caspase 8 and phospho-Akt in condyloma acuminatum lesions / 中华皮肤科杂志

Objective To determine the expression of Caspase 8 and phospho-Akt(p-Akt)in condyloma acuminatum(CA)lesions, and to evaluate their significance. Methods Skin lesion samples were collected from 30 patients with CA, cancer tissue samples from 20 with cervical cancer, and normal skin samples from 20 healthy controls. All the samples were subjected to paraffin embedding. An immunohistochemical study was conducted to determine the expression and distribution of Caspase 8 and p-Akt in the above samples. Results The expression rate of Caspase 8 was significantly lower in CA lesions (23.33%)than in normal skin samples(90%, P < 0.01)and cervical cancer lesions(80%, P < 0.001). Moreover, the expression rate of p-Akt in CA lesions(93.33%)was significantly higher than that in the normal skin samples(90%, P<0.001), but lower than that in the cervical cancer lesions(95%, P<0.001). No significant correlations were observed between the expression of Caspase 8 and p-Akt in either CA lesions or normal skin samples. However, the expression of Caspase 8 was positively correlated with the expression of p-Akt in cervical cancer lesions(r=0.369, P<0.05). Conclusion Both suppressed apoptosis initiation of Caspase 8 and anti-apoptotic effect of p-Akt may be involved in the occurrence and development of CA.

Effects of Zuogui Jiangtang Jieyu Formula on Hippocampus Neuron Apoptosis Related Proteins under Diabetes Mellitus with Depression States / 中国中医药信息杂志

Objective To investigate the regulating effects of Zuogui Jiangtang Jieyu Formula (ZJJF) on Bcl-2, Bax and Caspase-8 in hippocampus neuron damage in diabetes mellitus with depression (DD). Methods Hippocampal neurons from 18 d pregnant rats were primitively cultivated, and then combination of glucose and corticosterone was used to construct DD simulation environment. Cultivated hippocampal neurons were randomly divided into normal group, blank serum group, model group, positive medicine (metformin+fluoxetine) serum group and to-be-tested medicine (ZJJF) serum group. Normal group and model group were given same amount culture medium, while other group were given relevant amont of 10％ medicine serum or blank serum. After modeling intervention for 18 h,Hoechst staining was used to detect the apoptosis of hippocampal neurons. The expression of Bcl-2, Bax and Caspase-8 was detected by high content analysis. Results Compared with the control group, hippocampal neuron dendrites ruptured or decreased, neural network connection decreased, cells showed significant staining, broken, uneven distribution of light spots, the expression of Bcl-2 protein decreased significantly (P<0.05), but Bax and Caspase-8 were increased (P<0.05, P<0.01). Compared with the model group, hippocampal neurons in both positive medicine serum group and ZJJF serum group gradually recovered. Hoechst staining showed that the nuclei were significantly homogenized, local highlights were significantly reduced, Bcl-2 protein expression levels were significantly increased (P<0.05, P<0.01), while Bax and Caspase-8 were obviously down-regulated (P<0.05, P<0.01). Conclusion ZJJF has protective effects on hippocampal neurons in DD of model rats, and its mechanism is related to regulating the expression of Bcl-2, Bax and Caspase-8 in hippocampus neuron.

Necroptosis and tumor / 基础医学与临床

Necroptosis,it is activated by the formation of necrosome,is a way of programmed cell death that independent of the activation of caspase.Necroptosis is regulated by many factors,for example,RIPK1 can initiate necroptosis,but also inhibit necroptosis;caspase-8 is the key negative regulation factor of necroptosis;CHIP is a new regulation protein of necroptosis.Triggering necroptosis is a promising strategy to overcome apoptosis resistance in cancer.

TNF-α induced RIP1-dependent apoptosis in L929-A cells by interrupting mitochondrial respiratory chain complex Ⅲ / 军事医学

Objective To explore the mechanism by which tumor necrosis factor alpha(TNF-α) induces RIP1 kinase-dependented apoptosis in L929-A fibroblastoma cells.Methods The sub-mitochondrial localization of receptor-interacting protein 1(RIP1),caspase-8 and Bid proteins was detected by dose-gradient trypsin digestion and Western blotting.The levels of reactive oxygen species (ROS),intracellular calcium concentration,mitochondrial membrane potential (MMP),and cellular adenosine triphosphate(ATP) content were determined by fluorescent probe labeling and flow cytometry assay.The mitochondrial respiratory chain complex Ⅰ and Ⅲ activities were detected by commercial kits.Nec-1,A RIP1 kinase specific inhibitor,and RIP1-/-or Bid-/-L929-A cells were used to detect the roles of RIP1 kinase and Bid protein in cell death.Results RIP1,caspase-8 and Bid proteins were co-located in the outer membrane of mitochondrial.TNF-α exposure for 3 h could induce Bid cleavage,inhibit mitochondrial respiratory chain complex Ⅲ activity and reduce MMP.Following these changes and after TNF-α exposure for 6-12 h,the intracellular calcium concentration and ROS were increased,whereas the ATP concentration was decreased,and the cells were killed.Inhibiting RIP1 kinase or knockdown RIP1 or Bid protein could suppress all the cytotoxic effects of TNF-α.Conclusion TNF-α treatment can result in RIP1 kinase-mediated Bid cleavage and inhibit mitochondrial respiratory chains and cell energy metabolism,which ultimately leads to the death of L929-A cells.

Study of apoptosis in aveolar epithial A549 cells exposed to high-dose paraquat / 中华急诊医学杂志

Objective To We Used high-dose paraquat to induce apoptosis of A549 cells,and observed the expression of E-cadherin,α-SMA,caspase-8,caspase-3 and caspase-9.Then explored the possible mechanisms of apoptosis.Methods A549 cells were treated with various concentrations of PQ (0.1-1.0 mmol/L) for 24 and 48 hours,then observe the cell morphology and measured cell survival rate respectively determined by MTT method.A549 cells were treated with various concentrations of PQ (0.1,0.2,0.3 mmol/L) for 24 hours.The expressions of the mRNAs for α-SMA、E-cadherin,caspase-8,caspase-3 and caspase-9 were analysed by quantitative real-time PCR.Exposed to various concentrations of PQ (0.1,0.2,0.3mmol/L) for 24 hours,we analysed the protein expressions of α-SMA、E-cadherin、caspase-8、caspase-3 and caspase-9 by Western blot analysis.Results Treated with various concentrations of PQ (0.1-1.0mmol/L) for 24 hours,the A549 cells survival rate significantly reduced by MTT method.For 48 hours,the survival rate was lower.So,in subsequent experiments,we used 0.1,0.2 and 0.3 mmol/L concentration of paraquat to A549 cells,trained for 24 hours.After high-dose (0.1,0.2 and 0.3 mmol/L) exposure to PQ,a decrease in E-cadherin was observed while a decrease in α-SMA was also detected of the mRNAs.While the expressions of the mRNAs for caspase-8、caspase-3 and caspase-9 wereincrease.The protein expressions of α-SMA and E-cadherin were decrease with the increase of concen trations by Western blot analysis,and the levels of caspase-8、caspase-3 and caspase-9 were increase.Conclusions After high-dose short-time exposure to PQ,the survival rate of A549 cells is decreased obviously,cell morphology changed significantly,and the expressions of α-SMA and E-cadherin were decrease.But the expressions of caspase-8、caspase-3 and caspase-9 were increase.

Apoptosis of HepG-2 cells induced by total flavonoids of Verbena officinalis L. and possible mechanism / 国际药学研究杂志

Objective To explore the apoptosis and mechanisms of HepG-2 cells induced by total flavonoids of Verbena offici?nalis L.(TFV). Methods HepG-2 cells were cultured with different concentrations of TFV. The apoptosis of HepG-2 cells was evalu?ated by flow cytometry and DNA ladder. Reactive oxygen species(ROS)and membrane potential were measured by flow cytometry. Ex?pression of Caspase-3,Caspase-8,Caspase-9,and P53 was analyzed by Western blotting. Results TFV 50,100 and 200 mg/L in?creased the apoptosis rate(P<0.01),increased ROS levels of HepG-2 cells(P<0.01),and decreased the mitochondria membrane potential(P<0.05,P<0.01). TFV(50,100 and 200 mg/L)increased the proteins′ expression of Caspase-3,Caspase-9 and P53 (P<0.05,P<0.01). Conclusion TFV may induce the apoptosis of HepG-2 cells via increasing ROS levels,decreasing mitochon?dria membrane potential,and up-regulateing the expression of Caspase-3,Caspase-9 and P53 protein in HepG-2 cells.