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1.
Ren Fail ; 46(1): 2337287, 2024 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-38627212

RESUMO

OBJECTIVE: This study explored the molecular mechanisms by which dexmedetomidine (Dex) alleviates cisplatin (CP)-induced acute kidney injury (AKI) in rats. METHODS: CP-induced AKI models were established, and Dex was intraperitoneally injected at different concentrations into rats in the model groups. Subsequently, rats were assigned to the control, CP, CP + Dex 10 µg/kg, and CP + Dex 25 µg/kg groups. After weighing the kidneys of the rats, the kidney arterial resistive index was calculated, and CP-induced AKI was evaluated. In addition, four serum biochemical indices were measured: histopathological damage in rat kidneys was detected; levels of inflammatory factors, interleukin (IL)-1ß, IL-18, IL-6, and tumor necrosis factor alpha, in kidney tissue homogenate of rats were assessed through enzyme-linked immunosorbent assay (ELISA); and levels of NLRP-3, caspase-1, cleaved caspase-1, gasdermin D (GSDMD), and GSDMD-N in kidney tissues of rats were determined via western blotting. RESULTS: Dex treatment reduced nephromegaly and serum clinical marker upregulation caused by CP-induced AKI. In addition, hematoxylin and eosin staining revealed that Dex treatment relieved CP-induced kidney tissue injury in AKI rats. ELISA analyses demonstrated that Dex treatment reduced the upregulated levels of proinflammatory cytokines in the kidney tissue of AKI rats induced by CP, thereby alleviating kidney tissue injury. Western blotting indicated that Dex alleviated CP-induced AKI by inhibiting pyroptosis mediated by NLRP-3 and caspase-1. CONCLUSION: Dex protected rats from CP-induced AKI, and the mechanism may be related to NLRP-3/Caspase-1-mediated pyroptosis.


Assuntos
Injúria Renal Aguda , Dexmedetomidina , Ratos , Animais , Dexmedetomidina/efeitos adversos , Cisplatino/toxicidade , Injúria Renal Aguda/induzido quimicamente , Injúria Renal Aguda/prevenção & controle , Injúria Renal Aguda/patologia , Rim/patologia , Interleucina-1beta , Caspases/efeitos adversos
2.
Zhongguo Zhen Jiu ; 44(4): 441-448, 2024 Apr 12.
Artigo em Inglês, Chinês | MEDLINE | ID: mdl-38621732

RESUMO

OBJECTIVES: To observe the effects of electroacupuncture (EA) with "intestinal disease prescription" on the intestinal mucosal barrier and NLRP3 inflammasome in rats with dextran sulfate sodium (DSS)-induced acute ulcerative colitis (UC), and explore the underlying mechanism of EA with "intestinal disease prescription" for the treatment of UC. METHODS: Thirty-two healthy male SPF-grade SD rats were randomly divided into a blank group, a model group, a medication group, and an EA group, with 8 rats in each group. Except for the blank group, the UC model was established by administering 5% DSS solution for 7 days. After modeling, the rats in the medication group were treated with mesalazine suspension (200 mg/kg) by gavage, while the rats in the EA group were treated with acupuncture at bilateral "Tianshu" (ST 25), "Shangjuxu" (ST 37) and "Zhongwan" (CV 12), with the ipsilateral "Tianshu" (ST 25) and "Shangjuxu" (ST 37) connected to the electrodes of the EA instrument, using disperse-dense wave, with a frequency of 10 Hz/50 Hz, and each intervention lasted for 20 minutes. Both interventions were performed once daily for 3 days. The general conditions of rats were observed daily. After intervention, the disease activity index (DAI) score was calculated; colon tissue morphology was observed using HE staining; serum levels of pro-inflammatory cytokines (interleukin [IL]-18, IL-1ß) were measured by ELISA; protein expression of NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC), and Caspase-1 in colon tissues was detected by Western blot; positive expression of zonula occludens-1 (ZO-1) and Occludin in colon tissues was examined by immunofluorescence. RESULTS: Compared with the blank group, the rats in the model group exhibited poor general conditions, slow body weight gain, shortened colon length (P<0.01), increased DAI score and spleen index (P<0.01), elevated serum IL-18 and IL-1ß levels, and increased protein expression of NLRP3, ASC, and Caspase-1 in colon tissues (P<0.01), along with decreased positive expression of ZO-1 and Occludin in colon tissues (P<0.01). Compared with the model group, the rats in the medication group and the EA group exhibited improved general conditions, accelerated body weight gain, increased colon length (P<0.05), reduced DAI scores and spleen indexes (P<0.05), decreased serum IL-18 and IL-1ß levels, and lower protein expression of NLRP3, ASC and Caspase-1 in colon tissues (P<0.05), as well as increased positive expression of ZO-1 and Occludin in colon tissues (P<0.05). There were no significant differences in the above indexes between the medication group and the EA group (P>0.05). Compared with the blank group, the rats in the model group exhibited disrupted colon mucosal morphology, disordered gland arrangement, and atrophy of crypts, along with significant inflammatory cell infiltration. Compared with the model group, the rats in both the medication group and the EA group showed relatively intact colon mucosal morphology, with restored and improved gland and crypt structures, and reduced inflammatory cell infiltration. CONCLUSIONS: EA with "intestinal disease prescription" has a significant therapeutic effect on DSS-induced UC, possibly by regulating the expression of NLRP3 inflammasome and proteins related to the intestinal mucosal barrier, thereby alleviating symptoms of ulcerative colitis.


Assuntos
Colite Ulcerativa , Eletroacupuntura , Ratos , Masculino , Animais , Colite Ulcerativa/induzido quimicamente , Colite Ulcerativa/terapia , Inflamassomos/efeitos adversos , Interleucina-18 , Ratos Sprague-Dawley , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Ocludina , Peso Corporal , Caspases/efeitos adversos
3.
Respir Res ; 25(1): 100, 2024 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-38402153

RESUMO

BACKGROUND: Acute lung injury (ALI) and its more severe form, acute respiratory distress syndrome (ARDS) as common life-threatening lung diseases with high mortality rates are mostly associated with acute and severe inflammation in lungs. Recently, increasing evidence supports activated inflammation and gasdermin D (GSDMD)-mediated pyroptosis in macrophage are closely associated with ALI. Basic helix-loop-helix family member e40 (Bhlhe40) is a transcription factor that is comprehensively involved in inflammation. However, there is little experimental evidence connecting Bhlhe40 and GSDMD-driven pyroptosis. The study sought to verify the hypothesis that Bhlhe40 is required for GSDMD-mediated pyroptosis in lipopolysaccharide (LPS)-induced inflammatory injury. METHOD: We performed studies using Bhlhe40-knockout (Bhlhe40 -/-) mice, small interfering RNA (siRNA) targeting Bhlhe40 and pyroptosis inhibitor disulfiram to investigate the potential roles of Bhlhe40 on LPS-induced ALI and the underlying mechanisms. RESULTS: Bhlhe40 was highly expressed in total lung tissues and macrophages of LPS-induced mice. Bhlhe40-/- mice showed alleviative lung pathological injury and inflammatory response upon LPS stimulation. Meanwhile, we found that Bhlhe40 deficiency significantly suppressed GSDMD-mediated pyroptosis in macrophage in vivo and in vitro. By further mechanistic analysis, we demonstrated that Bhlhe40 deficiency inhibited GSDMD-mediated pyroptosis and subsequent ALI by repressing canonical (caspase-1-mediated) and non-canonical (caspase-11-mediated) signaling pathways in vivo and in vitro. CONCLUSION: These results indicate Bhlhe40 is required for LPS-induced ALI. Bhlhe40 deficiency can inhibit GSDMD-mediated pyroptosis and therefore alleviate ALI. Targeting Bhlhe40 may be a potential therapeutic strategy for LPS-induced ALI.


Assuntos
Lesão Pulmonar Aguda , Lipopolissacarídeos , Animais , Camundongos , Lipopolissacarídeos/toxicidade , Piroptose , Macrófagos/metabolismo , Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/prevenção & controle , Lesão Pulmonar Aguda/metabolismo , Caspases/efeitos adversos , Inflamação , RNA Interferente Pequeno , Proteínas de Homeodomínio/efeitos adversos , Fatores de Transcrição Hélice-Alça-Hélice Básicos
4.
Immunol Lett ; 264: 31-35, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-37913914

RESUMO

The second mitochondria-derived activator of caspases (SMAC) mimetic birinapant attenuated liver injury by inhibited the degradation of tumor necrosis factor receptor-associated factor 3 (TRAF3) and activation of mitogen-activated protein kinase (MAPK) signaling pathway in liver macrophage, but its role in LPS induced acute lung injury (ALI) is not understood. The present study was to investigate the effects of birinapant on ALI and its possible mechanism. A dose of birinapant (30 mg/kg) or a vehicle was administered intravenously 24 hours before LPS (100 µg) stimulation in mice. The levels of TNF-α, IL-6 and IL-1ß in bronchoalveolar lavage fluid (BALF) were measured by ELISA. The infiltrated macrophages and expression of monocyte chemoattractant protein-1 (MCP-1) was determined by immunohistochemistry staining in the lung tissues. The JNK and p38 MAPK activation, protein expression and K48-linked polyubiquitination of TRAF3 were determined in alveolar macrophage cell line (MH-S cells) after 1µg/ml LPS stimulation. The results showed that the birinapant down-regulated the levels of TNF-α, IL-6 and IL-1ß in the BALF. In addition, birinapant markedly inhibited macrophages infiltration and MCP-1 protein expression in lung tissues. At last, birinapant suppressed the MAPKsignaling pathway and K48-linked ubiquitinated degradation of TRAF3 in MH-S cells after LPS administration. In conclusion, the results proved that birinapant protected against LPS-induced ALI through inhibiting MAPK activation and K48-linked ubiquitination of TRAF3 in alveolar macrophages.


Assuntos
Lesão Pulmonar Aguda , Proteínas Quinases Ativadas por Mitógeno , Animais , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Lipopolissacarídeos/farmacologia , Fator 3 Associado a Receptor de TNF/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Caspases/efeitos adversos , Caspases/metabolismo , Interleucina-6/metabolismo , Transdução de Sinais , Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/tratamento farmacológico , Pulmão/patologia , Interleucina-1beta/metabolismo , Mitocôndrias/metabolismo
5.
BMC Nephrol ; 24(1): 25, 2023 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-36732683

RESUMO

BACKGROUND: Contrast-induced acute kidney injury (CI-AKI) is the third most common cause of hospital-acquired renal failure. However, there is no effective treatment of CI-AKI, and its mechanism is unknown. Interestingly, atorvastatin has been reported to be effective in renal injury. Therefore, the aim of this study was to explore the effect and possible molecular mechanism of atorvastatin in CI-AKI. METHODS: On the CI-AKI in vitro model, rat tubular epithelial cells (NRK-52E) were treated with 18 mg I/ml meglumine diatrizoate (MEG) and then pretreated with atorvastatin. pcDNA3.1-TLR4 treatment was performed to overexpress toll-like receptor 4 (TLR4) in NRK-52E cells. Cell Counting Kit-8 (CCK-8) and lactate dehydrogenase (LDH) kits were used to detect NRK-52E cell viability as well as LDH release in each group, respectively; qRT-PCR to determine mRNA expression of TLR4 in cells; western blot to detect protein expression levels of pyroptosis-related proteins (NLRP3, caspase-1, ASC, and GSDMD) and TLR4/MyD88/NF-κB signaling pathway-related proteins (TLR4, MyD88, NF-κBp65, and p-NF-κB p65) in cells. RESULTS: MEG treatment significantly inhibited the viability of NRK-52E cells, increased pro-inflammatory factor levels and promoted pyroptosis, representing successful establishment of a rat tubular epithelial cell (NRK-52E) CI-AKI in vitro model. Notably, atorvastatin increased the activity of MEG-treated NRK-52E cells and alleviated cell injury in a concentration-dependent manner. In addition, atorvastatin significantly down-regulated the expression of TLR4 in MEG-treated NRK-52E cells. However, overexpression of TLR4 inhibited the effects of atorvastatin on increasing cell viability, alleviating cell injury, reducing pro-inflammatory factors (IL-1ß, IL-6, and TNF-α) levels, and inhibiting apoptosis (by down-regulating the expression of NLRP3, caspase-1, ASC, and GSDMD). Furthermore, atorvastatin also inhibited the expression of TLR4/MyD88/NF-κB pathway-related proteins (TLR4, MyD88, and p-NF-κB p65). CONCLUSION: Atorvastatin can attenuate CI-AKI through increasing the activity of MEG-treated renal tubular epithelial cells, relieving cell injury, as well as inhibiting pyroptosis and inflammation. More importantly, the mechanism was achieved by inhibiting the TLR4//MyD88/NF-κB signaling pathway.


Assuntos
Injúria Renal Aguda , NF-kappa B , Ratos , Animais , NF-kappa B/metabolismo , Atorvastatina/efeitos adversos , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , Fator 88 de Diferenciação Mieloide/farmacologia , Meios de Contraste/efeitos adversos , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Piroptose , Receptor 4 Toll-Like/genética , Transdução de Sinais , Injúria Renal Aguda/induzido quimicamente , Injúria Renal Aguda/prevenção & controle , Células Epiteliais , Caspases/efeitos adversos , Caspases/metabolismo
6.
J Invest Surg ; 36(1): 1-7, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36350036

RESUMO

Background: Acute kidney injury (AKI) is a common complication in patients with severe acute pancreatitis (SAP). Caspase-11-mediated pyroptosis is essential for the progression of multiple diseases, but its role in SAP-induced AKI remains unknown.Aims: This research investigated whether caspase-11-mediated pyroptosis is involved in SAP-induced AKI and whether inhibiting caspase-11-mediated pyroptosis improves SAP-induced AKI.Methods: A rat model of SAP with AKI was established by slowly injecting 5% sodium taurocholate into the biliopancreatic duct, then wedelolactone (25 or 50 mg/kg), an inhibitor of caspase-11, was injected through the intra-peritoneum 1 and 6 h after SAP induction. Serum biochemical indexes, including serum amylase, lipase, interleukin (IL)-6, blood urea nitrogen (BUN), tumor necrosis factor (TNF)-α, and creatinine (Cr) in rats, were evaluated using biochemical test kits. Caspase-11 and gasdermin D (GSDMD) expression in the kidney tissues was evaluated by western blotting and immunohistochemical staining. IL-1ß and IL-18 levels in kidney tissues were detected by ELISA kits. Furthermore, histopathological alterations of pancreas and kidney were assessed by H&E staining.Results: The serum biochemical indexes and pyroptosis-related proteins in kidney tissues were significantly increased after SAP induction. Furthermore, wedelolactone decreased the expression of pyroptosis-linked proteins in kidney tissues, reduced serum lipase, amylase, IL-6, TNF-α, BUN, and Cr, and ameliorated the renal and pancreatic histological damage in SAP rats.Conclusion: Caspase-11-mediated pyroptosis contributes to SAP-induced AKI, and targeting caspase-11-mediated pyroptosis might be a novel treatment strategy for SAP-induced AKI.


Assuntos
Injúria Renal Aguda , Pancreatite , Ratos , Animais , Pancreatite/complicações , Pancreatite/tratamento farmacológico , Piroptose , Caspases/efeitos adversos , Doença Aguda , Injúria Renal Aguda/etiologia , Injúria Renal Aguda/prevenção & controle , Injúria Renal Aguda/metabolismo , Creatinina , Fator de Necrose Tumoral alfa , Amilases , Interleucina-6 , Lipase
7.
Exp Anim ; 72(1): 77-87, 2023 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-36184484

RESUMO

Carbon monoxide (CO) has been reported to exhibit a therapeutic effect in lipopolysaccharide (LPS)-induced acute lung injury (ALI). However, the precise mechanism by which CO confers protection against ALI remains unclear. Pyroptosis has been recently proposed to play an essential role in the initiation and progression of ALI. Thus, we investigated whether pyroptosis is involved in the protection of CO against ALI and its underlying mechanism. First, an LPS-induced ALI mouse model was established. To determine the role of pyroptosis, we evaluated histological changes and the expression levels of cleaved caspase-11, N-gasdermin D (GSDMD), and IL-1ß in lung tissues, which are the indicators of pyroptosis. Inhalation of CO exhibited protective effects on LPS-induced ALI by decreasing TNF-α and IL-10 expression and ameliorating pathological changes in lung tissue. In vitro, CO significantly reduced the expression of cleaved caspase-11, N-GSDMD, IL-1ß, and IL-18. In addition, it increased nuclear factor E2-related factor 2 (NRF-2) expression in a time-dependent manner in RAW 264.7 cells and decreased N-GSDMD expression. The expression of cleaved GSDMD and release of LDH were increased after treatment with a specific NRF-2 inhibitor, ML385, indicating that NRF-2 mediates the inhibition of pyroptosis by CO. Taken together, these results demonstrated that CO upregulated NRF-2 to inhibit pyroptosis and subsequently ameliorated LPS-induced ALI.


Assuntos
Lesão Pulmonar Aguda , Macrófagos Alveolares , Camundongos , Animais , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/patologia , Lipopolissacarídeos/efeitos adversos , Monóxido de Carbono/efeitos adversos , Piroptose , Lesão Pulmonar Aguda/induzido quimicamente , Caspases/efeitos adversos
8.
Clin Exp Nephrol ; 26(11): 1055-1066, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-35925422

RESUMO

BACKGROUND: Focal segmental glomerulosclerosis (FSGS) is characterized by podocyte damage and severe proteinuria. The exact mechanism of podocyte damage and loss remains unclear. Necroptosis, a lytic form of programmed cell death mediated by RIP3 and MLKL, has emerged as an important cell death pattern in multiple tissues and cell types. Necroptosis in FSGS has not been investigated. METHODS: Public GEO data regarding podocyte treated with vehicle or adriamycin (ADR) was identified and analyzed. Cultured human podocytes were used to explore the activation of necroptosis upon ADR stimulation. The expression of necroptosis pathway molecules, p-RIP3 and p-MLKL, was examined in the glomeruli and defoliated urinary podocytes of patients with FSGS. The effect of necroptosis inhibition was assessed in ADR-induced glomerulopathy mice using GSK872. RESULTS: Publicly available RNA-sequencing data analysis showed that both necroptosis and NLRP3 inflammasome pathway were up-regulated in ADR-injured podocyte. Immunofluorescent staining showed increased expression of p-RIP3 and p-MLKL, the active forms of RIP3 and MLKL, in podocytes of FSGS patients and ADR-induced glomerulopathy mice but not in the normal control. GSK872, an RIP3 kinase inhibitor, significantly inhibited the expression of p-RIP3, p-MLKL and activation of NLRP3 in cultured podocytes treated with ADR. GSK872 treatment of mice with ADR-induced nephropathy resulted in the reduced expression of p-RIP3, p-MLKL, NLRP3 and caspase-1 p20. GSK872 also significantly inhibited the expression of p-MLKL in the podocytes of ADR-induced nephropathy, resulting in the attenuation of proteinuria and renal histological lesions. CONCLUSION: Necroptosis pathway might be a valuable target for the treatment of FSGS.


Assuntos
Glomerulosclerose Segmentar e Focal , Nefropatias , Podócitos , Animais , Caspases/efeitos adversos , Caspases/metabolismo , Doxorrubicina/efeitos adversos , Doxorrubicina/metabolismo , Glomerulosclerose Segmentar e Focal/patologia , Humanos , Inflamassomos/efeitos adversos , Inflamassomos/metabolismo , Nefropatias/patologia , Camundongos , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Necroptose , Podócitos/metabolismo , Proteinúria/patologia , RNA/efeitos adversos , RNA/metabolismo , Esclerose/induzido quimicamente , Esclerose/metabolismo , Esclerose/patologia
9.
Arq. bras. med. vet. zootec ; 62(2): 258-264, abr. 2010. ilus
Artigo em Português | LILACS | ID: lil-551825

RESUMO

Apoptose e seus mecanismos reguladores são eventos fisiológicos cruciais para a manutenção da homeostase placentária, e o desequilíbrio desses processos pode comprometer a função placentária e, consequentemente, o sucesso da gravidez. Neste estudo, investigou-se a apoptose utilizando-se histomorfometria em lâminas coradas em HE e submetidas à reação de TUNEL. Além disso, avaliou-se a expressão de Bcl-2 e das caspases 8 e 3, pela reação de polimerase em cadeia em tempo real, em placentas saudáveis em diferentes estádios de gestação. Amostras de placentônios de vacas com quatro, seis e nove meses de gestação foram colhidas e processadas. O índice apoptótico aumentou progressivamente com o avanço da gestação. Tanto o Bcl-2 quanto as caspases 3 e 8 foram expressas nos três períodos estudados, sendo a expressão de Bcl-2 menor que a de caspase 8, que é menor que a de caspase 3. Estes resultados indicam que essas moléculas estão envolvidas na via apoptótica ativada na maturação placentária, exibindo um padrão de expressão ao longo da gestação e contribuindo para o equilíbrio fisiológico da celularidade e renovação celular na placenta bovina.


Apoptosis and its regulating mechanisms are crucial physiological events for the maintenance of the placental homostasis; and disequilibrium of these processes may compromise placental function and the success of the pregnancy. In this study, apoptosis was investigated by histomorphometry using slides stained with HE and TUNEL reaction. Besides that, Bcl-2 and caspases 8 and 3 expression were evaluated by real time polymerase chain reaction in healthy placentas under different gestacional ages. Samples of placentones of cows at 4th, 6th, and 9th months of gestation were harvested and processed. The apoptotic index gradually increased with the advance of the gestation. Bcl-2 and caspases 3 and 8 were expressed in all the studied periods, being the expression of Bcl-2 lower than that of caspase 8, which was lower than caspase 3. These results indicate that these molecules are involved in the activated apoptotic way in the placental maturation, showing a standard expression throughout the gestation and contributing for the physiological balance of the cellularity and cellular turn over in bovine placenta.


Assuntos
Animais , Bovinos , Caspases/análise , Caspases/efeitos adversos , Caspases/isolamento & purificação , Fator de Indução de Apoptose/análise , Fator de Indução de Apoptose/deficiência , Placenta/anatomia & histologia , Placenta/embriologia , Bovinos/anatomia & histologia , Bovinos/anormalidades , Bovinos/cirurgia , Homeostase , Marcação In Situ das Extremidades Cortadas/métodos , Marcação In Situ das Extremidades Cortadas/veterinária , Prenhez
10.
Arq. bras. med. vet. zootec ; 57(4): 457-464, ago. 2005.
Artigo em Português | LILACS | ID: lil-415186

RESUMO

Determinou-se a expressão gênica das caspases 3 e 8 mediante transcrição reversa de mRNA total e reação em cadeia da polimerase (RT-PCR) para avaliar a apoptose em timo e baço de ratas imunossuprimidas por glicocorticóides. Utilizou-se dexametasona para indução da apoptose e atrofia linfóide. Quarenta e cinco fêmeas Wistar recém-desmamadas foram separadas em três grupos: as ratas de A (n=18) e B (n=18) foram tratadas com 250 e 500mg de glicocorticóide, via intramuscular, respectivamente, e as do C (n=9) não foram tratadas. Após 24, 48 e 72 horas, seis animais de cada grupo tratado e três do controle foram anestesiados, pesados e sacrificados. O baço e o timo foram coletados e pesados. Fragmentos dos órgãos foram fixados em formol tamponado a 10 por cento e processados segundo técnica para inclusão em parafina. Os blocos foram seccionados em 5æm, e os cortes corados em hematoxilina e eosina. A análise histopatológica aliada ao peso dos órgãos nas diferentes doses e tempos demonstrou que a dexametasona induziu hipotrofia linfóide, que ocorreu com maior intensidade no tempo de 72 horas em animais do grupo B. Fragmentos de timo e de baço foram imediatamente congelados em nitrogênio líquido para extração de mRNA e DNA. Para a padronização da técnica de RT-PCR, utilizaram-se pool de amostras de mRNA dos animais-controle e pool de mRNA de animais tratados em cada tempo de experimento. A técnica de RT-PCR foi sensível o suficiente para a detecção dos mRNAs que codificam as caspases 3 e 8, e ambas participaram do processo de apoptose induzido por dexametasona.


Assuntos
Animais , Apoptose , Baço/fisiologia , Caspases/efeitos adversos , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Timo/fisiologia , Dexametasona/administração & dosagem
11.
Chem Res Toxicol ; 18(3): 420-7, 2005 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-15777082

RESUMO

The role of mitochondria and apical caspases in apoptosis induced by the benzene metabolite hydroquinone (HQ) remains to be elucidated. Here, we investigated the involvement of mitochondria and activation of the apical caspases-8 and -9 in HQ induced apoptosis in myeloperoxidase (MPO)-rich HL-60 and MPO-deficient Jurkat T cells. Treatment of HL-60 and Jurkat cells with HQ resulted in apoptosis as assessed by phosphatidyl serine (PS) exposure, loss of mitochondrial transmembrane potential (MTP), release of cytochrome c, and processing of apical caspases-8 and -9 and executioner caspase-3. In HQ-treated HL-60 cells, pretreatment with the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (ZVAD), which did not inhibit PS exposure, also failed to abrogate the loss of MTP and release of cytochrome c. However, complete processing of caspase-9 was inhibited in the presence of ZVAD. In marked contrast, in HQ-treated Jurkat cells, ZVAD significantly abrogated PS exposure, loss of MTP, and caspase-9 processing but not release of cytochrome c. Although ZVAD-sensitive caspase-8 processing occurred in both cell types, pretreatment with either fas-receptor blocking ZB4 or fas-ligand NOK1 neutralizing antibodies did not inhibit HQ-induced apoptosis. In conclusion, our results demonstrate that HQ induced apoptosis in Jurkat cells occurs via a ZVAD-inhibitable, caspase-dependent process, while in HL-60 cells, apoptosis occurs predominantly via caspase-independent mechanisms. Our results emphasize that both caspase-dependent and independent mechanisms should be considered in the intrinsic apoptotic pathway induced by HQ.


Assuntos
Apoptose/efeitos dos fármacos , Caspases/biossíntese , Células HL-60/efeitos dos fármacos , Hidroquinonas/toxicidade , Células Jurkat/efeitos dos fármacos , Clorometilcetonas de Aminoácidos/farmacologia , Caspases/efeitos adversos , Combinação de Medicamentos , Inibidores Enzimáticos/farmacologia , Citometria de Fluxo , Células HL-60/enzimologia , Células HL-60/patologia , Humanos , Células Jurkat/enzimologia , Células Jurkat/patologia , Potenciais da Membrana/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/enzimologia , Fosfatidilserinas/metabolismo
12.
J Pharmacol Sci ; 97(1): 138-45, 2005 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-15655286

RESUMO

Gagam-whanglyun-haedoktang (GWH) is a newly designed herbal drug formula based on the traditional oriental pharmacological knowledge for the purpose of treating tumorous diseases. Apoptosis is an evolutionarily conserved suicide program residing in cells. In the present study, apoptosis inducing activities of the decocted water extract of GWH were studied. Results of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay showed that GWH had a strong cytotoxic effect on HL-60 cells. The number of live cells was less than 20% after exposure to 1 mg/ml GWH for 48 h. GWH increased cytotoxicity of HL-60 cells in a dose- and time-dependent manner. The percentage of apoptotic cells by flow cytometric analysis of the DNA-stained cells increased to 28%, 31%, and 37% at 24 h and to 37%, 44%, and 81% at 48 h after treatment with 0.01, 0.1, and 1 mg/ml GWH, respectively. DNA fragmentation also occurred in apoptosis and was characterized by a ladder pattern on agarose gel. In addition, GWH increased the secretion of tumor necrosis factor-alpha. GWH-induced apoptosis was accompanied by activation of caspase-3. These results suggest that GWH induces activation of caspase-3 and eventually leads to apoptosis.


Assuntos
Caspases/metabolismo , Fragmentação do DNA , Ativação Enzimática/efeitos dos fármacos , Medicina Tradicional do Leste Asiático , Preparações de Plantas/efeitos adversos , Caspase 3 , Caspases/efeitos adversos , Ciclo Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Relação Dose-Resposta a Droga , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo/métodos , Células HL-60 , Humanos , Fatores de Tempo , Fator de Necrose Tumoral alfa/metabolismo
13.
J Control Release ; 99(2): 301-14, 2004 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-15380639

RESUMO

N-(2-Hydroxypropyl)methacrylamide (HPMA) copolymer carrier containing the anticancer drug doxorubicin bound either by a proteolytically degradable bond (non-targeted PK1 or targeted with alpha-CD71 mAb) or by a hydrolytically degradable bond were synthesised and tested in vivo for various biological properties. Mouse 38C13 B-cell lympoma was used as a well established and defined cell line for this study. 38C13 cells are sensitive to free doxorubicin and IC50 was very low, about 0.014 microM. PK1 showed a strongly decreased cytostatic effect, IC50 being 12.6 microM. alpha-CD71 targeted conjugate, which can be considered as an antibody-targeted form of PK1, had IC50 0.358 microM. HPMA copolymer with doxorubicin bound via a hydrolytically sensitive bond (HYD conjugate) showed a high cytostatic effect with IC50 about 0.052 microM. We demonstrated that HYD conjugate inhibited DNA synthesis and induced p21(Waf1/Cip1) protein expression (p21(Waf1/Cip1) is cyclin-dependent kinase inhibitor which blocks cell cycle progression) as quickly as free doxorubicin, whereas PK1 acted much more slowly. Similarly, apoptosis induction measured by Annexin V binding and Caspase 3 activity was detected later after incubation of cells with PK1 or alpha-CD71 targeted conjugate. Apoptosis was manifested by elevation of bax and bad mRNA levels, which was much more rapid and intense in the case of free doxorubicin and HYD conjugate. Expression of antiapoptotic genes as well as cyclin-dependent kinases was surprisingly not affected.


Assuntos
Acrilamidas/síntese química , Acrilamidas/farmacologia , Doxorrubicina/síntese química , Doxorrubicina/farmacologia , Ligantes , Acrilamidas/metabolismo , Animais , Apoptose/efeitos dos fármacos , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Caspase 3 , Caspases/efeitos adversos , Caspases/efeitos dos fármacos , Caspases/metabolismo , Ciclo Celular/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Membrana Celular/patologia , Proliferação de Células/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p21 , DNA/antagonistas & inibidores , DNA/genética , DNA/metabolismo , Doxorrubicina/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/genética , Genes myc/efeitos dos fármacos , Genes myc/genética , Hidrazonas/síntese química , Hidrazonas/metabolismo , Hidrazonas/farmacologia , Hidrólise , Concentração Inibidora 50 , Camundongos , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Mensageiro/genética , Receptores da Transferrina/efeitos dos fármacos , Receptores da Transferrina/genética , Timidina/metabolismo , Trítio , Proteína X Associada a bcl-2 , Proteína de Morte Celular Associada a bcl
14.
Toxicol Sci ; 79(1): 137-46, 2004 May.
Artigo em Inglês | MEDLINE | ID: mdl-14976342

RESUMO

Rotenone is a naturally derived pesticide that has recently been shown to evoke the behavioral and pathological symptoms of Parkinson's disease in animal models. Though rotenone is known to be an inhibitor of the mitochondrial complex I electron transport chain, little is known about downstream pathways leading to its toxicity. We used human dopaminergic SH-SY5Y cells to study mechanisms of rotenone-induced neuronal cell death. Our results suggest that rotenone, at nanomolar concentrations, induces apoptosis in SH-SY5Y cells that is caspase-dependent. Furthermore, rotenone treatment induces phosphorylation of c-Jun, the c-Jun N-terminal protein kinase (JNK), and the p38 mitogen activated protein (MAP) kinase, indicative of activation of the p38 and JNK pathways. Importantly, expression of dominant interfering constructs of the JNK or p38 pathways attenuated rotenone-induced apoptosis. These data suggest that rotenone induces apoptosis in the dopaminergic SH-SY5Y cells that requires activation of the JNK and p38 MAP kinases and caspases. These studies provide insights concerning the molecular mechanisms of rotenone-induced apoptosis in neuronal cells.


Assuntos
Apoptose/efeitos dos fármacos , Receptores Dopaminérgicos/efeitos dos fármacos , Rotenona/toxicidade , Proteínas Quinases p38 Ativadas por Mitógeno/efeitos adversos , Caspases/efeitos adversos , Caspases/efeitos dos fármacos , Caspases/metabolismo , Linhagem Celular Tumoral , Clorpirifos/toxicidade , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/efeitos adversos , Proteínas Quinases JNK Ativadas por Mitógeno/efeitos dos fármacos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , MAP Quinase Quinase 4 , Quinases de Proteína Quinase Ativadas por Mitógeno/efeitos adversos , Quinases de Proteína Quinase Ativadas por Mitógeno/efeitos dos fármacos , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Receptores Dopaminérgicos/genética , Receptores Dopaminérgicos/metabolismo , Rotenona/química , Transdução de Sinais/efeitos dos fármacos , Transfecção/métodos , Proteínas Quinases p38 Ativadas por Mitógeno/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
15.
Arch Pharm Res ; 27(1): 68-76, 2004 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-14969342

RESUMO

Mistletoe lectin has been reported to induce apoptosis in different cancer cell lines in vitro and to show antitumor activity against a variety of tumors in animal models. We previously demonstrated the Korean mistletoe lectin (Viscum album var. coloratum, VCA)-induced apoptosis by down-regulation of Bcl-2 and telomerase activity and by up-regulation of Bax through p53- and p21-independent pathway in hepatoma cells. In the present study, we observed the induction of apoptotic cell death through activation of caspase-3 and the inhibition of telomerase activity through transcriptional down-regulation of hTERT in the VCA-treated A253 cells. We also observed the inhibition of telomerase activity and induction of apoptosis resulted from dephosphorylation of Akt in the survival signaling pathways. In addition, combining VCA with the inhibitors of phosphatidylinositol 3-kinase (PI3-kinase) upstream of Akt, wortmannin and LY294002 showed an additive inhibitory effect of telomerase activity. In contrast, the inhibitor of protein phosphatase 2A (PP2A), okadaic acid inhibited VCA-induced dephosphorylation of Akt and inhibition of telomerase activity. Taken together, VCA induces apoptotic cell death through Akt signaling pathway in correlated with the inhibition of telomerase activity and the activation of caspase-3. From these results, together with our previous studies, we suggest that VCA triggers molecular changes that resulting in the inhibition of cell growth and the induction of apoptotic cell death of cancer cells, which suggest that VCA may be useful as chemotherapeutic agent for cancer cells.


Assuntos
Apoptose , Linhagem Celular Tumoral , Lectinas/efeitos adversos , Lectinas/química , Erva-de-Passarinho/química , Fosfatidilinositol 3-Quinases/metabolismo , Telomerase/antagonistas & inibidores , Caspase 3 , Caspases/efeitos adversos , Caspases/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Cromonas/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Sinergismo Farmacológico , Humanos , Coreia (Geográfico) , Lectinas/antagonistas & inibidores , Lectinas/isolamento & purificação , Morfolinas/farmacologia , Fosfatidilinositol 3-Quinases/farmacologia , Inibidores de Fosfoinositídeo-3 Quinase , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Monoéster Fosfórico Hidrolases/farmacologia , Fosforilação/efeitos dos fármacos , Extratos Vegetais/química , Preparações de Plantas/efeitos adversos , Preparações de Plantas/antagonistas & inibidores , Preparações de Plantas/química , Proteínas de Plantas/efeitos adversos , Proteínas de Plantas/antagonistas & inibidores , Proteínas de Plantas/química , Proteínas Inativadoras de Ribossomos Tipo 2 , Telomerase/genética , Toxinas Biológicas/efeitos adversos , Toxinas Biológicas/química , Regulação para Cima
16.
Trends Neurosci ; 23(1): 20-6, 2000 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-10631785

RESUMO

Both necrotic and apoptotic neuronal death are observed in various neurological and neurodegenerative disorders. Calpain is activated in various necrotic and apoptotic conditions, while caspase 3 is only activated in neuronal apoptosis. Despite the difference in cleavage-site specificity, an increasing number of cellular proteins are found to be dually susceptible to these cysteine proteases. These include alpha- and beta-fodrin, calmodulin-dependent protein kinases, ADP-ribosyltransferase (ADPRT/PARP) and tau. Intriguingly, calpastatin is susceptible to caspase-mediated fragmentation. Neurotoxic challenges such as hypoxia-hypoglycemia, excitotoxin treatment or metabolic inhibition of cultured neurons result in activation of both proteases. Calpain inhibitors can protect against necrotic neuronal death and, to a lesser extent, apoptotic death. Caspase inhibitors strongly suppress apoptotic neuronal death. Thus, both protease families might contribute to structural derangement and functional loss in neurons under degenerative conditions.


Assuntos
Apoptose/efeitos dos fármacos , Encefalopatias/metabolismo , Calpaína/metabolismo , Caspases/metabolismo , Animais , Encefalopatias/tratamento farmacológico , Calpaína/efeitos adversos , Caspases/efeitos adversos , Inibidores de Cisteína Proteinase/metabolismo , Humanos , Necrose , Fármacos Neuroprotetores/metabolismo
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