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China Journal of Chinese Materia Medica ; (24): 5881-5887, 2023.
Article in Chinese | WPRIM | ID: wpr-1008786


This study aims to explore the effect and mechanism of Danggui Buxue Decoction(DBD)-containing serum in alleviating the H9c2 cell injury caused by the exposure to intermittent low oxygen. H9c2 cells were assigned into five groups: control(CON) group, intermittent low oxygen(IH) group, intermittent low oxygen plus DBD-containing serum(IH+DBD) group, intermittent low oxygen plus the autophagy enhancer rapamycin(IH+RAPA) group, and intermittent low oxygen plus DBD-containing serum and the autophagy inhibitor 3-methyladenine(IH+DBD+3-MA) group. Monodansylcadaverine(MDC) staining was employed to detect the changes of autophagosomes. Cell counting kit-8(CCK-8) assay was employed to determine the activity of myocardial cells, and lactate dehydrogenase(LDH) and creatine kinase(CK) kits were used to measure the LDH and CK levels in the cell culture, which would reflect the degree of cell damage. TdT-mediated dUTP nick-end labeling(TUNEL) staining was used to detect the apoptosis of myocardial cells, and JC-1 fluorescence probe to detect the changes in mitochondrial membrane potential. Western blot was employed to determine the expression levels of the autophagy-related proteins microtubule-associated proteins light chain 3Ⅱ(LC3Ⅱ), microtubule-associated proteins light chain 3Ⅰ(LC3Ⅰ), P62, Parkin and apoptosis related proteins pro caspase-3, caspase-3, B-cell lymphoma-2(Bcl-2), Bcl-2-associated X(Bax). The results showed that compared with the CON group, the IH group showed decreased fluorescence intensity of MDC staining, decreased LC3Ⅱ/LC3Ⅰ ratio, down-regulated Parkin expression, and up-regulated expression of P62. In addition, the IH group showed decreased cell survival rate, increased content of LDH and CK in the culture medium, increased number of TUNEL positive cells, and decreased pro caspase-3/caspase-3 and Bcl-2/Bax ratios and mitochondrial membrane potential. Compared with the IH group, the IH+DBD and IH+RAPA groups showed increased fluorescence intensity of MDC staining, increased LC3Ⅱ/LC3Ⅰ ratio, up-regulated Parkin expression, and down-regulated P62 expression. In addition, the two groups showed increased cell survival rate, reduced content of LDH and CK in the culture medium, decreased number of TUNEL positive cells, and increased pro caspase-3/caspase-3 and Bcl-2/Bax ratios and mitochondrial membrane potential. The IH+DBD+3-MA and IH groups showed no significant differences in the above indicators. Compared with the IH+DBD group, the IH+DBD+3-MA group showed decreased fluorescence intensity of MDC staining, decreased LC3Ⅱ/LC3Ⅰ ratio, down-regulated Parkin expression, and up-regulated P62 expression. In addition, the group had decreased cell survival rate, increased content of LDH and CK in the culture medium, increased number of TUNEL positive cells, decreased pro caspase-3/caspase-3 and Bcl-2/Bax ratios, and declined mitochon-drial membrane potential. To sum up, DBD could promote the mitophagy, inhibit the apoptosis, and alleviated the injury of H9c2 cells exposed to low oxygen.

Oxygen , bcl-2-Associated X Protein/metabolism , Caspase 3/genetics , Proto-Oncogene Proteins c-bcl-2/metabolism , Apoptosis , Autophagy , Ubiquitin-Protein Ligases , Microtubule-Associated Proteins
China Journal of Chinese Materia Medica ; (24): 2203-2211, 2023.
Article in Chinese | WPRIM | ID: wpr-981351


This study aims to investigate the therapeutic effect of alcohol extract of root and root bark of Toddalia asiatica(TAAE) on collagen-induced arthritis(CIA) in rats through phosphatidylinoinosidine-3 kinase/protein kinase B(PI3K/Akt) signaling pathway. To be specific, CIA was induced in rats, and then the rats were treated(oral, daily) with TAAE and Tripterygium Glycoside Tablets(TGT), respectively. The swelling degree of the hind leg joints was scored weekly. After 35 days of administration, the histopathological changes were observed based on hematoxylin and eosin(HE) staining. Enzyme-linked immunosorbent assay(ELISA) was employed to detect the levels of cytokines [tumor necrosis factor-α(TNF-α), interleukin(IL)-6)]. Terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL) staining was performed to detect the apoptosis of synoviocytes in rats. Western blot was used to detect the expression levels of apoptosis-related proteins B-cell lymphoma 2(Bcl-2)-associated X(Bax), Bcl-2, and caspase-3 and pathway-related proteins phosphoinositide 3-kinase(PI3K), phosphorylated(p)-PI3K, protein kinase B(Akt), and p-Akt. RT-qPCR was conducted to examine the mRNA levels of Bax, Bcl-2, caspase-3, TNF-α, IL-6, and IL-1β and pathway-related proteins PI3K, p-PI3K, Akt, and p-Akt. TAAE can alleviate the joint swelling in CIA rats, reduce serum levels of inflammatory cytokines, improve synovial histopathological changes, promote apoptosis of synoviocytes, and inhibit synovial inflammation. In addition, RT-qPCR and Western blot results showed that TAAE up-regulated the level of Bax, down-regulated the level of Bcl-2, and activated caspase-3 to promote apoptosis in synoviocytes. TAAE effectively down-regulated the protein levels of p-PI3K and p-Akt. In this study, TAAE shows therapeutic effect on CIA in rats and reduces the inflammation. The mechanism is that it suppresses PI3K/Akt signaling pathway and promotes synoviocyte apoptosis. Overall, this study provides a new clue for the research on the anti-inflammatory mechanism of TAAE and lays a theoretical basis for the better clinical application of TAAE in the treatment of inflammatory and autoimmune diseases.

Rats , Animals , Proto-Oncogene Proteins c-akt/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Caspase 3/genetics , Tumor Necrosis Factor-alpha/metabolism , bcl-2-Associated X Protein/metabolism , Plant Bark , Anti-Inflammatory Agents/therapeutic use , Arthritis, Experimental/chemically induced , Inflammation/drug therapy , Cytokines/metabolism , Proto-Oncogene Proteins c-bcl-2 , Apoptosis
Int. j. morphol ; 38(3): 523-529, June 2020. graf
Article in English | LILACS | ID: biblio-1098282


This study aimed to investigate the morphometric and the pattern of protein and gene expression related to the extrinsic apoptotic pathway in experimental focal cerebral ischemia and the hole of neuroprotection with hypothermia and ketoprofen. For this analysis, 120 rats were randomly divided into 3 groups (20 animals each): control - no surgery (20 animals); sham - simulation of surgery (20 animals); ischemic - focal ischemia for 1 hour, without reperfusion (80 animals) and divided into four subgroups with 20 animals each: ischemic + intraischemic hypothermia; ischemic + previous intravenous ketoprofen, and ischemic + hypothermia and ketoprofen. The infarct volume was measured using morphometric analysis of infarct areas defined by triphenyl tetrazolium chloride and the patterns of expression of the apoptosis genes (Fas, c-Flip, caspase-8 and caspase-3) and the apoptosis protein caspase-3 were evaluated by quantitative real-time PCR and immunohistochemistry, respectively. Hypo expression of genes of extrinsic pathway of apoptosis was observed: Fas receptor, c-Flip and caspase-8 in the ischemics areas. Increases in the gene and protein caspase-3 in the ischemic areas were also observed, and these increases were reduced by hypothermia and ketoprofen, also noted in the morphometric study. The caspases-3 increase suggests that this gene plays an important role in apoptosis, probably culminating in cell death and that the neuroprotective effect of hypothermia and ketoprofen is involved.

Este estudio tuvo como objetivo investigar la morfometría y el patrón de expresión de proteínas y genes relacionados con la vía apoptótica extrínseca en la isquemia cerebral focal experimental y el agujero de neuroprotección con hipotermia y ketoprofeno. Se dividieron aleatoriamente 120 ratas en 3 grupos (20 animales cada uno): control - sin cirugía (20 animales); simulación - simulación de cirugía (20 animales); isquemia isquemia focal durante 1 hora, sin reperfusión (80 animales) y dividida en cuatro subgrupos con 20 animales cada uno: isquemia + hipotermia intraisquémica; isquemia + ketoprofeno intravenoso previo, e isquemia + hipotermia y ketoprofeno. El volumen del infarto se midió utilizando un análisis morfométrico de áreas de infarto definidas por cloruro de trifenil tetrazolio y los patrones de expresión de los genes de apoptosis (Fas, c-Flip, caspase-8 y caspase-3) y la proteína de apoptosis caspase-3 fueron evaluados por PCR cuantitativa en tiempo real e inmunohistoquímica, respectivamente. Se observó hipoexpresión de genes de la vía extrínseca de la apoptosis: receptor Fas, c-Flip y caspasa-8 en las áreas isquémicas. También se observaron aumentos en el gen y la proteína caspasa-3 en las áreas isquémicas y estos aumentos se redujeron por hipotermia y ketoprofeno, también observado por estudio morfométrico. El aumento de caspasas-3 sugiere que este gen tiene un papel importante en la apoptosis, y probable causa de muerte celular, involucrando el efecto neuroprotector de la hipotermia y el ketoprofeno.

Animals , Rats , Brain Ischemia/genetics , Brain Ischemia/metabolism , Immunohistochemistry , Brain Ischemia/pathology , Brain Ischemia/therapy , Ketoprofen/pharmacology , Apoptosis/genetics , Neuroprotective Agents/pharmacology , Disease Models, Animal , Caspase 3/genetics , Caspase 8/genetics , Real-Time Polymerase Chain Reaction , Hypothermia, Induced
Rev. bras. anestesiol ; 66(1): 29-36, Jan.-Feb. 2016. graf
Article in Portuguese | LILACS | ID: lil-773481


BACKGROUND: Intra-articular injections of local anesthetics are commonly used to enhance post-operative analgesia following orthopedic surgery as arthroscopic surgeries. Nevertheless, recent reports of severe complications due to the use of intra-articular local anesthetic have raised concerns. OBJECTIVES: The study aims to assess use of vitamin C in reducing adverse effects of the most commonly employed anesthetics - ropivacaine, bupivacaine and lidocaine - on human chondrocytes. METHODS: The chondrocyte viability following exposure to 0.5% bupivacaine or 0.75% ropivacaine or 1.0% lidocaine and/or vitamin C at doses 125, 250 and 500 µM was determined by LIVE/DEAD assay and annexin V staining. Expression levels of caspases 3 and 9 were assessed using antibodies by Western blotting. Flow cytometry was performed to analyze the generation of reactive oxygen species. RESULTS: On exposure to the local anesthetics, chondrotoxicity was found in the order ropivacaine < bupivacaine < lidocaine. Vitamin C effectively improved the reduced chondrocyte viability and decreased the raised apoptosis levels following exposure to anesthesia. At higher doses, vitamin C was found efficient in reducing the generation of reactive oxygen species and as well down-regulate the expressions of caspases 3 and 9. CONCLUSIONS: Vitamin C was observed to effectively protect chondrocytes against the toxic insult of local anesthetics ropivacaine, bupivacaine and lidocaine.

JUSTIFICATIVA: Injeções de anestésicos locais por via intra-articular são comumente usadas para melhorar a analgesia no período pós-operatório de cirurgia ortopédica como artroscopia. No entanto, relatos recentes de complicações graves devido ao uso de anestésico local por via intra-articular causou preocupações. OBJETIVOS: O objetivo do estudo foi avaliar o uso de vitamina C para reduzir os efeitos adversos dos anestésicos mais comumente usados (ropivocaína, bupivacaína e lidocaína) sobre condrócitos humanos. MÉTODOS: A viabilidade dos condrócitos após a exposição à bupivacaína a 0,5% ou ropivacaína a 0,75% ou lidocaína a 1,0% e/ou vitamina C em doses de 125, 250 e 500 µM foi determinada pelo ensaio Vivo/Morto e coloração com anexina V. Os níveis de expressão das caspases 3 e 9 foram avaliados com o uso de anticorpos pela técnica Western blotting. Citometria de fluxo foi feita para analisar a geração de espécies reativas ao oxigênio. RESULTADOS: Na exposição aos anestésicos locais, condrotoxicidade foi encontrada na seguinte ordem: ropivacaína < bupivacaína < lidocaína. A vitamina C efetivamente melhorou a redução da viabilidade dos condrócitos e diminuiu os níveis elevados de apoptose após a exposição à anestesia. Em doses mais altas, a vitamina C foi eficiente para reduzir a geração de espécies reativas ao oxigênio e assim regular negativamente a expressão das caspases 3 e 9. CONCLUSÕES: Observamos que a vitamina C foi eficaz na proteção dos condrócitos contra a agressão tóxica dos anestésicos locais ropivacaína, bupivacaína e lidocaína.

Humans , Ascorbic Acid/pharmacology , Reactive Oxygen Species/metabolism , Chondrocytes/drug effects , Anesthetics, Local/toxicity , Ascorbic Acid/administration & dosage , Bupivacaine/toxicity , Down-Regulation/drug effects , Cells, Cultured , Apoptosis/drug effects , Chondrocytes/pathology , Dose-Response Relationship, Drug , Caspase 3/genetics , Caspase 9/genetics , Flow Cytometry , Ropivacaine , Amides/toxicity , Lidocaine/toxicity , Antioxidants/administration & dosage , Antioxidants/pharmacology
Journal of Veterinary Science ; : 475-483, 2014.
Article in English | WPRIM | ID: wpr-24553


Chronic enteritis can produce an excess of reactive oxygen species resulting in cellular damage. Stanniocalcin-1(STC-1) reportedly possesses anti-oxidative activity, the aim of this study was to define more clearly the direct contribution of STC-1 to anti-oxidative stress in cattle. In this study, primary intestinal epithelial cells (IECs) were exposed to hydrogen peroxide (H2O2) for different time intervals to mimic chronic enteritis-induced cellular damage. Prior to treatment with 200 microM H2O2, the cells were transfected with a recombinant plasmid for 48 h to over-express STC-1. Acridine orange/ethidium bromide (AO/EB) double staining and trypan blue exclusion assays were then performed to measure cell viability and apoptosis of the cells, respectively. The expression of STC-1 and apoptosis-related proteins in the cells was monitored by real-time PCR and Western blotting. The results indicated that both STC-1 mRNA and protein expression levels positively correlated with the duration of H2O2 treatment. H2O2 damaged the bovine IECs in a time-dependent manner, and this effect was attenuated by STC-1 over-expression. Furthermore, over-expression of STC-1 up-regulated Bcl-2 protein expression and slightly down-regulated caspase-3 production in the damaged cells. Findings from this study suggested that STC-1 plays a protective role in intestinal cells through an antioxidant mechanism.

Animals , Cattle , Male , Animals, Newborn , Blotting, Western/veterinary , Caspase 3/genetics , Cattle Diseases/etiology , Duodenum/metabolism , Enteritis/etiology , Epithelial Cells/metabolism , Gene Expression Regulation , Glycoproteins/genetics , Hydrogen Peroxide/pharmacology , Proto-Oncogene Proteins c-bcl-2/genetics , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction/veterinary
The Korean Journal of Parasitology ; : 459-469, 2014.
Article in English | WPRIM | ID: wpr-7401


Entamoeba histolytica is a tissue-invasive protozoan parasite causing dysentery in humans. During infection of colonic tissues, amoebic trophozoites are able to kill host cells via apoptosis or necrosis, both of which trigger IL-8-mediated acute inflammatory responses. However, the signaling pathways involved in host cell death induced by E. histolytica have not yet been fully defined. In this study, we examined whether calpain plays a role in the cleavage of pro-survival transcription factors during cell death of colonic epithelial cells, induced by live E. histolytica trophozoites. Incubation with amoebic trophozoites induced activation of m-calpain in a time- and dose-dependent manner. Moreover, incubation with amoebae resulted in marked degradation of STAT proteins (STAT3 and STAT5) and NF-kappaB (p65) in Caco-2 cells. However, IkappaB, an inhibitor of NF-kappaB, was not cleaved in Caco-2 cells following adherence of E. histolytica. Entamoeba-induced cleavage of STAT proteins and NF-kappaB was partially inhibited by pretreatment of cells with a cell-permeable calpain inhibitor, calpeptin. In contrast, E. histolytica did not induce cleavage of caspase-3 in Caco-2 cells. Furthermore, pretreatment of Caco-2 cells with a calpain inhibitor, calpeptin (but not the pan-caspase inhibitor, z-VAD-fmk) or m-calpain siRNA partially reduced Entamoeba-induced DNA fragmentation in Caco-2 cells. These results suggest that calpain plays an important role in E. histolytica-induced degradation of NF-kappaB and STATs in colonic epithelial cells, which ultimately accelerates cell death.

Humans , Caco-2 Cells , Calcium-Binding Proteins , Calpain/genetics , Caspase 3/genetics , Caspases , Cell Death , Colon/cytology , Entamoeba histolytica/physiology , Epithelial Cells/cytology , I-kappa B Proteins/metabolism , Intestinal Mucosa/cytology , NF-kappa B/genetics , RNA Interference , RNA, Small Interfering , STAT3 Transcription Factor/genetics , STAT5 Transcription Factor/genetics , Signal Transduction
Pakistan Journal of Pharmaceutical Sciences. 2013; 26 (3): 605-610
in English | IMEMR | ID: emr-142624


The Rho GTPases are the sub-group of Ras super family and identified in all eukaryotes. The Rho GTPases effect different cellular signaling pathways involved in a number of diseases such as cancer, neurological and cardiovascular disorders. Members of Rho GTPases including RhoA, RhoC and Rac1 play a major role in regulation of apoptosis in different kind of stress conditions. Here we investigated the Rho GTPase activating protein 15 [ArhGAP15] gene knock-down effect on apoptosis induced by ethanol in bovine fibroblast cells. The bovine Fibroblast cells were treated and transfected with two different concentrations [50 and 100 nM] of ArhGAP15 siRNA for 48 h respectively. Both concentrations of siRNA were effective and the results of RT-PCR revealed an efficient knock-down of ArhGAP15 mRNA in fibroblast cells. Further, the normal cells exposed to a 100 mM ethanol concentration showed a reduction in cell viability and induced the ratio of apoptosis related Bax/Bcl-2 proteins compared with ArhGAP15 siRNA transfected ethanol treated cells. Ethanol also increased caspase-3 expression in normal fibroblast cells compared with transfected cells. The ArhGAP15 knock-down cells treated with ethanol decreased Bax/Bcl-2 ratio and lower caspase-3 protein levels in ArhGAP15 knocked-down cells. Our results suggest that apoptosis induced by ethanol involves the activation of Rho GTPase activating protein 15 and silencing of the said gene protects apoptosis

Animals , RNA, Small Interfering/genetics , Transfection/methods , bcl-2-Associated X Protein/genetics , RNA, Messenger , Fibroblasts/drug effects , Ethanol/pharmacology , Cell Survival/genetics , Cells, Cultured , Apoptosis/genetics , Caspase 3/genetics , Cattle
Tehran University Medical Journal [TUMJ]. 2013; 71 (8): 536-540
in Persian | IMEMR | ID: emr-143043


Gastric cancer is the most prevalent cancer with poor survival in gastrointestinal tract. Caspase 3 and 9 play an important role in the development and progression of cancer. Polymorphisms in the genes for these enzymes can affect gene activity and thus may influence susceptibility to gastric cancer. In this study, caspase 3 and 9 genes polymorphisms in patients with gastric cancer were examined. In a case - control study, 100 patients with gastric cancer and 100 healthy individuals were evaluated in the region rs4647601: G> T for caspase-3 and -1263 A> G gene promoter for caspase 9. DNA extraction was performed from whole blood according to manufacture protocol. RFLP-PCR method was carrying out for detection of caspase 3 and 9 genes genotype in two groups. In this study, 143 men and 57 women were evaluated. All of them were selected from the same race and geographical area. The results indicated an increase of the mutant G allele in the control group, which leads to a decreasing in the incidence of gastric cancer [P<0.0001, OR: 0.096, [%0.95CL] =0.04-0.23]. It seems that screening of -1263 A> caspase 9 polymorphism could be a useful marker in personal sensitivity to gastric cancer and help to cancer treatment and prevention process. It is concluded that caspase gene variation may be a diagnostic factor in the gastric cancer.

Humans , Male , Female , Caspase 3/genetics , Caspase 9/genetics , Genetic Predisposition to Disease , Polymorphism, Single Nucleotide , Stomach Neoplasms/enzymology , Polymorphism, Restriction Fragment Length , Polymerase Chain Reaction
Journal of Korean Medical Science ; : 404-411, 2011.
Article in English | WPRIM | ID: wpr-52132


As one of trials on neuroprotection after spinal cord injury, we used pregabalin. After spinal cord injury (SCI) in rats using contusion model, we observed the effect of pregabalin compared to that of the control and the methylprednisolone treated rats. We observed locomotor improvement of paralyzed hindlimb and body weight changes for clinical evaluation and caspase-3, bcl-2, and p38 MAPK expressions using western blotting. On histopathological analysis, we also evaluated reactive proliferation of glial cells. We were able to observe pregabalin's effectiveness as a neuroprotector after SCI in terms of the clinical indicators and the laboratory findings. The caspase-3 and phosphorylated p38 MAPK expressions of the pregabalin group were lower than those of the control group (statistically significant with caspase-3). Bcl-2 showed no significant difference between the control group and the treated groups. On the histopathological analysis, pregabalin treatment demonstrated less proliferation of the microglia and astrocytes. With this animal study, we were able to demonstrate reproducible results of pregabalin's neuroprotection effect. Diminished production of caspase-3 and phosphorylated p38 MAPK and as well as decreased proliferation of astrocytes were seen with the administration of pregabalin. This influence on spinal cord injury might be a possible approach for achieving neuroprotection following central nervous system trauma including spinal cord injury.

Animals , Male , Rats , Apoptosis/drug effects , Astrocytes/drug effects , Blotting, Western , Body Weight/drug effects , Caspase 3/genetics , Cell Proliferation , Fluorescent Antibody Technique , Gene Expression , Hindlimb/drug effects , Inflammation , Methylprednisolone/therapeutic use , Microglia/drug effects , Motor Activity/drug effects , Neuroglia/drug effects , Neuroprotective Agents/therapeutic use , Paralysis/drug therapy , Proto-Oncogene Proteins c-bcl-2/genetics , Rats, Sprague-Dawley , Spinal Cord Injuries/drug therapy , gamma-Aminobutyric Acid/analogs & derivatives , p38 Mitogen-Activated Protein Kinases/genetics