Dihydromyricetin mediates epithelial mesenchymal transformation and regulates the proliferation and apoptosis of esophageal squamous cell carcinoma cells / 中华肿瘤杂志

Objective: To study the effects of dihydromyricetin (DMY) on the proliferation, apoptosis and epithelial mesenchymal transition (EMT) of esophageal squamous cell carcinoma (ESCC) cell KYSE150 and KYSE410. Methods: KYSE150 and KYSE410 cells were treated with different concentrations of DMY (0, 25, 50, 100, 150, 200 μmol/L) for 24 hours. The median inhibition concentration (IC50) values of KYSE150 and KYSE410 were detected by cell counting kit-8 (CCK-8) method. Then 0.5‰ dimethyl sulfoxide (DMSO) was used as control group, dihydromyricetin (DMY), dihydromyricetin and transforming growth factor-β1 (DMY+ TGF-β1), transforming growth factor-β1 (TGF-β1) were used as experimental group. Cell proliferation and apoptosis rates were measured by clonal formation and flow cytometry. Transwell invasion and wound healing assay were used to detect cell invasion and migration. The protein expression levels of Caspase-3, Caspase-9, Bcl-2, Bax, Smad2/3, phosphorylation-Smad2/3 (p-Smad2/3) and Vimentin were detected by western blot. Results: The IC50 values of DMY on KYSE410 and KYSE150 cells were 100.51 and 101.27 μmol/L. The clone formation numbers of KYSE150 and KYSE410 in DMY group [(0.53±0.03) and (0.31±0.03)] were lower than those in DMSO group [(1.00±0.10) and (1.00±0.05), P<0.05]. The apoptosis rates of KYSE150 and KYSE410 cells in DMY group [(1.84±0.22)% and (2.80±0.07)%] were higher than those in DMSO group [(1.00±0.18)% and (1.00±0.07)%, P<0.05]. The invasion numbers of KYSE150 and KYSE410 cells in DMY group [(0.42±0.03) and (0.29±0.05)] were lower than those in DMSO group [(1.00±0.08) and (1.00±0.05), P<0.05]. The migration rates of KYSE150 and KYSE410 cells in DMY group [(0.65±0.14)% and (0.40±0.17)%] were lower than those in DMSO group [(1.00±0.10)% and (1.00±0.08)%, P<0.05]. The clone formation numbers of KYSE150 and KYSE410 in TGF-β1 group [(1.01±0.08) and (0.99±0.25)] were higher than those in DMY+ TGF-β1 group [(0.73±0.10) and (0.58±0.05), P<0.05]. The apoptosis rates of KYSE150 and KYSE410 cells in TGF-β1 group [(0.81±0.14)% and (1.18±0.10)%] were lower than those in DMY+ TGF-β1 group [(1.38±0.22)% and (1.85±0.04)%, P<0.05]. The invasion numbers of KYSE150 and KYSE410 cells in TGF-β1 group [(1.19±0.11) and (1.39±0.11)] were higher than those in DMY+ TGF-β1 group [(0.93±0.09) and (0.93±0.05), P<0.05]. The migration rates of KYSE150 and KYSE410 cells in TGF-β1 group [(1.87±0.19)% and (1.32±0.04)%] were higher than those in DMY+ TGF-β1 group [(0.86±0.16)% and (0.77±0.12)%, P<0.05]. The protein expression levels of Bax, Caspase-3 and Caspase-9 in KYSE150 and KYSE410 cells in DMY group were higher than those in DMSO group, while the protein expression level of Bcl-2 was lower than that in DMSO group (P<0.05). The protein expression levels of p-Smad2/3, Smad2/3 and Vimentin in KYSE150 and KYSE410 cells in DMY group were lower than those in DMSO group (P<0.05). The protein expression levels of Bax, Caspase-3 and Caspase-9 in KYSE150 and KYSE410 cells in TGF-β1 group were lower than those in DMY+ TGF-β1 group, and the protein expression level of Bcl-2 was higher than that in DMY+ TGF-β1 group (P<0.05). The protein expression levels of Bax, Caspase-3 and Caspase-9 in KYSE150 and KYSE410 cells in DMY+ TGF-β1 group were lower than those in DMY group, and the protein expression level of Bcl-2 was higher than that in DMY group (P<0.05). The protein expression levels of p-Smad2/3, Smad2/3 and Vimentin in KYSE150 and KYSE410 cells in TGF-β1 group were higher than those in DMY+ TGF-β1 group (P<0.05). Conclusion: DMY can inhibit the proliferation and EMT of ESCC mediated by TGF-β1 and promote cell apoptosis.

Effects and molecular mechanism of exogenous L-carnitine on excessive endoplasmic reticulum stress-mediated hepatic pyroptosis in severely scald rats / 中华烧伤杂志

Objective: To investigate the effects and molecular mechanism of exogenous L-carnitine on hepatic pyroptosis mediated by excessive endoplasmic reticulum stress in severely scald rats. Methods: The experimental research method was adopted. According to the random number table (the same group method below), fifteen female Sprague Dawley rats aged 6-8 weeks were divided into sham-injury group, scald alone group, and scald+carnitine group (with 5 rats in each group), and full-thickness scald of 30% total body surface area were made on the back of rats in scald alone group and scald+carnitine group, and rats in scald+carnitine group were additionally given intraperitoneal injection of L-carnitine. At post injury hour (PIH) 72, The levels of aspartate aminotransferase (AST) and alanine dehydrogenase (ALT) of biochemical indicators of liver injury were detected by automatic biochemical analyzer with the sample number of 5. At PIH 72, liver tissue damage was detected by hematoxylin-eosin staining. At PIH 72, The mRNA levels of nucleotide-binding oligomerization domain-containing protein-like receptor family pyrin domain containing 3 (NLRP3), cysteine aspartic acid specific protease 1 (caspase-1), gasderminD (GSDMD), and interleukin 1β(IL-1β) in liver tissue as pyroptosis-related markers and glucose regulatory protein 78 (GRP78) and CCAAT/enhancer binding protein homologous protein (CHOP) in liver tissue as endoplasmic reticulum stress-related markers were detected by real-time fluorescence quantitative reverse transcription polymerase chain reaction (RT-qPCR). Protein expression levels of GRP78, CHOP, NLRP3, caspase-1, caspase-1/p20, GSDMD-N, and cleaved IL-1β in liver tissue were detected by Western blotting, and the sample numbers were all 5. HepG2 cells as human liver cancer cells were divided into dimethyl sulfoxide (DMSO) group, 0.1 μmol/L tunicamycin (TM) group, 0.2 μmol/L TM group, 0.4 μmol/L TM group, and 0.8 μmol/L TM group and were treated accordingly. After 24 h of culture, cell viability was detected by cell counting kit 8, and the intervention concentration of TM was screened, and the sample number was 5. HepG2 cells were divided into DMSO group, TM alone group, and TM+carnitine group, and treated accordingly. After 24 h of culture, the protein expression levels of GRP78, CHOP, NLRP3, caspase-1, caspase-1/p20, GSDMD-N, and cleaved IL-1β in cells were detected by Western blotting, and the sample numbers were all 3. Data were statistically analyzed with one-way analysis of variance and least significant difference-t test. Results: At PIH 72, the AST and ALT levels of serum in scald alone group were (640±22) and (157±8) U/L, which were significantly higher than (106±13) and (42±6) U/L in sham-injury group, respectively, with t values of -46.78 and -25.98, respectively, P<0.01. The AST and ALT levels of serum in scald+carnitine group were (519±50) and (121±10) U/L, which were significantly lower than those in scald alone group, respectively, with t values of 4.93 and 6.06, respectively, P<0.01. At PIH 72, the morphology of liver tissue of rats in sham-injury group were basically normal with no obvious inflammatory cell infiltration; compared with those in sham-injury group, the liver tissue of rats in scald alone group showed a large number of inflammatory cell infiltration and disturbed cell arrangement; compared with that in scald alone group, the liver tissue of rats in scald+carnitine group showed a small amount of inflammatory cell infiltration. At PIH 72, the mRNA expression on levels of NLRP3, caspase-1, GSDMD, and IL-1β in liver tissue of rats in scald alone group were significantly higher than those in sham-injury group (with t values of 34.42, 41.93, 30.17, and 15.68, respectively, P<0.01); the mRNA levels of NLRP3, caspase-1, GSDMD, and IL-1β in liver tissue of rats in scald+carnitine group were significantly lower than those in scald alone group (with t values of 34.40, 37.20, 19.95, and 7.88, respectively, P<0.01). At PIH 72, the protein expression levels of NLRP3, caspase-1, caspase-1/p20, GSDMD-N, and cleaved IL-1β in liver tissue of rats in scald alone group were significantly higher than those in sham-injury group (with t values of 12.28, 26.92, 5.20, 10.02, and 24.78, respectively, P<0.01); compared with those in scald alone group, the protein expression levels of NLRP3, caspase-1, caspase-1/p20, GSDMD-N, and cleaved IL-1β in liver tissue of rats in scald+carnitine group were significantly decreased (with t values of 10.99, 27.96, 12.69, 8.96, and 12.27, respectively, P<0.01). At PIH 72, the mRNA levels of GRP78 and CHOP in liver tissue of rats in scald alone group were significantly higher than those in sham-injury group (with t values of 21.00 and 16.52, respectively, P<0.01), and the mRNA levels of GRP78 and CHOP in liver tissue of rats in scald+carnitine group were significantly lower than those in scald alone group (with t values of 8.92 and 8.21, respectively, P<0.01); the protein expression levels of GRP78 and CHOP in liver tissue of rats in scald alone group were significantly higher than those in sham-injury group (with t values of 22.50 and 14.29, respectively, P<0.01), and the protein expression levels of GRP78 and CHOP in liver tissue of rats in scald+carnitine group were significantly lower than those in scald alone group (with t values of 14.29 and 5.33 respectively, P<0.01). After 24 h of culture, the cell survival rates of 0.1 μmol/L TM group, 0.2 μmol/L TM group, 0.4 μmol/L TM group, and 0.8 μmol/L TM group were significantly decreased than that in DMSO group (with t values of 4.90, 9.35, 18.64, and 25.09, respectively, P<0.01). Then 0.8 μmol/L was selected as the intervention concentration of TM. After 24 h of culture, compared with that in DMSO group, the protein expression levels of GRP78 and CHOP in cells in TM alone group were significantly increased (with t values of 10.48 and 17.67, respectively, P<0.01), and the protein expression levels of GRP78 and CHOP in TM+carnitine group were significantly lower than those in TM alone group (with t values of 8.08 and 13.23, respectively, P<0.05 or P<0.01). After 24 h of culture, compared with those in DMSO group, the protein expression levels of NLRP3 and GSDMD-N in cells in TM alone group were significantly increased (with t values of 13.44 and 27.51, respectively, P<0.01), but the protein expression levels of caspase-1, caspase-1/p20, and cleaved IL-1β in cells were not significantly changed (P>0.05); compared with that in TM alone group, the protein expression levels of NLRP3 and GSDMD-N in cells in TM+carnitine group were significantly decreased (with t values of 20.49 and 21.95, respectively, P<0.01), but the protein expression levels of caspase-1, caspase-1/p20, and cleaved IL-1β in cells were not significantly changed (P>0.05). Conclusions: In severely scald rats, exogenous L-carnitine may play a protective role against liver injury by inhibiting the pathways related to excessive endoplasmic reticulum stress-mediated pyroptosis.

Shexiang Baoxin Pill Regulates Intimal Hyperplasia, Migration, and Apoptosis after Platelet-Derived Growth Factor-BB-Stimulation of Vascular Smooth Muscle Cells via miR-451 / 中国结合医学杂志

OBJECTIVE@#To investigate the regulatory roles of Shexiang Baoxin Pill (SXBXW) in neointimal formation and vascular smooth muscle cells (VSMCs) invasion and apoptosis as well as the potential molecular mechanisms using cultured VSMCs model of vascular injury (platelet-derived growth factor (PDGF)-BB-stimulated) in vitro.@*METHODS@#VSMCs were randomly assigned to 5 groups: blank, PDGF-BB (20 ng/mL+ 0.1% DMSO), SXBXW-L (PDGF-BB 20 ng/mL + SXBXW low dose 0.625 g/L), SXBXW-M (PDGF-BB 20 ng/mL + SXBXW medium dose 1.25 g/L) and SXBXW-H (PDGF-BB 20 ng/mL+ SXBXW high dose 2.5 g/L) group. Cell proliferation was assessed using cell counting kit-8 (CCK-8) assay and bromodeoxyuridine (BrdU) incorporation assay, the migration effects were detected by Transwell assay, cell apoptosis rate was measured by the Annexin V/propidium iodide (PI) apoptosis kit. The markers of contractile phenotype of VSMCs were detected with immunofluorescent staining. To validate the effects of miR-451 in regulating proliferation, migration and apoptosis treated with SXBXW, miR-451 overexpression experiments were performed, the VSMCs were exposed to PDGF-BB 20 ng/mL + 0.1% DMSO and later divided into 4 groups: mimic-NC (multiplicity of infection, MOI=50), SXBXW (1.25 g/L) + mimic-NC, mimic-miR451 (MOI=50), and SXBXW (1.25 g/L) + mimic-miR451, and alterations of proteins related to the miR-451 pathway were analyzed using Western blot.@*RESULTS@#PDGF-BB induced VSMCs injury causes acceleration of proliferation and migration. SXBXW inhibited phenotypic switching, proliferation and migration and promoted cell apoptosis in PDGF-BB-induced VSMCs. In addition, miR-451 was shown to be down-regulated in the VSMCs following PDGF-BB stimulation. SXBXW treatment enhanced the expression of miR-451 in PDGF-BB-induced VSMCs (P<0.05). Compared with SXBXW + mimic-NC and mimic-miR451 groups, the expression of tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta (Ywhaz) and p53 was further reduced in SXBXW + mimic-miR451 group, while activating transcription factor 2 (ATF2) was increased in VSMCs (P<0.05).@*CONCLUSION@#SXBXW regulated proliferation, migration and apoptosis via activation of miR-451 through ATF2, p53 and Ywhaz in PDGF-BB-stimulated VSMCs.

Effects of dimethylsulfoxide and pentoxifylline in the vitality of cutaneous flaps in rats / Efeitos do dimetilsulfóxido e da pentoxifilina na vitalidade de retalhos cutâneos em ratos

ABSTRACT Objectives: to verify the influence of dimethylsulfoxide and pentoxifylline on the vitality of cutaneous flaps in rats and the tissue repair process. Methods: were studied 30 Wistar rats, submitting them to a 2cm wide by 8cm long dorsal cutaneous flap, of caudal base. We distributed the animals in three groups: Control Group (n=10) with application gauze moistened with 0.9% Saline in the flap bed for 30 seconds; Dimethylsulfoxide group (n=10), with administration of 1ml of 5% dimethylsulfoxide divided into five injections of 0.2ml in the transition of the flap segments; Pentoxifylline group (n=10), with administration of pentoxifylline 20mg/kg, diluted to 1ml and divided into five injections of 0.2ml in the transition of the flap segments. Drugs were administered intraoperatively, in a single dose and subcutaneously. We observed the skin flaps for changes in color and texture. On the 10th postoperative day, we checked the dimensions of viable and necrotic tissues, followed by excision of the specimen for histological analysis. Results: the measurements of length of the viable and necrotic tissues between groups showed no differences. Histological analysis showed that the Dimethylsulfoxide group presented neovascularization, inflammatory infiltrate with leukocytes and more structured conjunctival stroma. The Pentoxifylline group showed neovascularization and inflammatory infiltrate, with moderate to intense granulation. The control group evolved with a higher rate of necrosis in the distal segment. Conclusion: dimethylsulfoxide and pentoxifylline influenced the vitality of the flap and the tissue repair process. However, they did not prevent necrosis macroscopically.

RESUMO Objetivos: verificar a influência do dimetilsulfóxido e da pentoxifilina na vitalidade e no processo de reparo tecidual de retalhos cutâneos em ratos. Método: foram estudados 30 ratos Wistar, nos quais foi confeccionado retalho cutâneo dorsal de 2cm de largura por 8cm de comprimento, de base caudal, e distribuídos em três grupos: Grupo Controle (n=10) com aplicação de gaze umedecida com solução salina a 0,9%, no leito do retalho, por 30 segundos; Grupo dimetilsulfóxido (n=10) com injeção de 1ml de dimetilsulfóxido a 5% divididos em cinco injeções de 0,2ml na transição dos segmentos do retalho; Grupo pentoxifilina (n=10) com injeção de 1ml pentoxifilina 20mg/kg, divididos em cinco injeções de 0,2ml na transição dos segmentos do retalho. Os fármacos foram administrados no transoperatório, em dose única e por via subcutânea. Os retalhos cutâneos foram observados quanto às alterações de cor e textura. No décimo dia de pós-operatório aferiu-se a dimensão do tecido viável e de necrose, seguido da exérese da peça para análise histológica. Resultados: a medida da dimensão de tecido viável e de necrose dos grupos não apresentou diferenças. A análise histológica mostrou que o grupo dimetilsulfóxido apresentou neovascularização, infiltrado inflamatório com leucócitos e estroma conjuntivo mais estruturado. O grupo pentoxifilina, mostrou neovascularização e infiltrado inflamatório com granulação moderada e intensa. O grupo controle evoluiu com maior índice de necrose no segmento distal. Conclusão: dimetilsulfóxido e pentoxifilina influenciaram na vitalidade do retalho e no processo de reparo tecidual. Entretanto, não evitaram a necrose macroscopicamente.

Criopreservación de células estromales derivadas del tejido adiposo: comparación de diferentes medios de criopreservación / Cryopreservation of adipose derived stromal cells: comparison of different cryopreservation media

Objetivo Obtener células estromales derivadas del tejido adiposo, medir y comparar las tasas de viabilidad antes e inmediatamente después un ciclo de criopreservación con diferentes combinaciones de criopreservantes de manera de obtener el mejor medio de criopreservación. Material y método Medición de la tasa de viabilidad poscriopreservación de células estromales derivadas del tejido adiposo obtenidas de 5 pacientes utilizando medios definidos (DMEM/Ham F12) libres de suero bovino y suplementados con una de los siguientes combinaciones de compuestos: dimetilsulfóxido (DMSO) 10%; DMSO 10% + trehalosa 7,6%; DMSO 10% + albúmina humana 10% y DMSO 10% + trehalosa 7,6% + albúmina humana 10%, mediante citometría de flujo con ioduro de propidio. Resultados No existen diferencias estadísticamente significativas en las tasas de viabilidad de las células estromales posterior a un ciclo de criopreservación. Sin embargo, se observa una tendencia a mejorar la tasa de recuperación de células vitales al agregar albúmina humana. Conclusiones No se observaron diferencias significativas entre las condiciones estudiadas, sugiriendo que ninguna es superior a las demás en cuanto a rendimiento. Es así como podemos afirmar que la criopreservación de las células estromales derivadas del tejido adiposo en un medio que combine DMEM/F12 con DMSO 10% + trehalosa 7,6% + albúmina humana 10% no logra una tasa de recuperación de células vitales significativamente mayor que las congeladas solo con DMSO 10%.

Aim To obtain stromal cells derived from adipose tissue, to measure and compare viability rates before and immediately after cryopreservation cycle, using different combinations of cryoprotective agents in order to identify the best cryopreservation medium. Material and method Viability rate after cryopreservation of stromal cells derived from adipose tissue were assessed by flow cytometry with propidium iodide. Samples of stromal cells obtained from 5 patients were kept defined, bovine serum-free media (DMEM/Ham-F12), supplemented with one of the following combinations of compounds: 10% dymethylsulfoxide (DMSO); Trehalose 10% DMSO + 7.6%; 10% DMSO + 10% human albumin and 10% DMSO + 7.6% Trehalose + 10% human albumin. Results No statistically significant differences were observed in the viability rates of stromal cells derived from adipose tissue after a cryopreservation cycle. However, we observed a tendency towards improvement of recovery rate when human albumin was added to the medium. Conclusions None of the studied conditions proved superior to others in terms of cell vitality after a cryopreservation cycle. Hence, we conclude that the cryopreservation of stromal cells derived from adipose tissue in an environment that combines DMEM/F12 with 10% DMSO + 7.6% Trehalose + human albumin 10% does not achieve a significantly higher recovery rate than only frozen solely with DMSO 10%.

Pharmacological study of the mechanisms involved in the vasodilator effect produced by the acute application of triiodothyronine to rat aortic rings

A relationship between thyroid hormones and the cardiovascular system has been well established in the literature. The present in vitro study aimed to investigate the mechanisms involved in the vasodilator effect produced by the acute application of 10-8–10-4 M triiodothyronine (T3) to isolated rat aortic rings. Thoracic aortic rings from 80 adult male Wistar rats were isolated and mounted in tissue chambers filled with Krebs-Henseleit bicarbonate buffer in order to analyze the influence of endothelial tissue, inhibitors and blockers on the vascular effect produced by T3. T3 induced a vasorelaxant response in phenylephrine-precontracted rat aortic rings at higher concentrations (10-4.5–10-4.0 M). This outcome was unaffected by 3.1×10-7 M glibenclamide, 10-3 M 4-aminopyridine (4-AP), 10-5 M indomethacin, or 10-5 M cycloheximide. Contrarily, vasorelaxant responses to T3 were significantly (P<0.05) attenuated by endothelium removal or the application of 10-6 M atropine, 10-5 M L-NG-nitroarginine methyl ester (L-NAME), 10-7 M 1H-(1,2,4)oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), 10-6 M (9S,10R,12R)-2,3,9,10,11,12-Hexahydro-10-methoxy-2,9-dimethyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3′,2′,1′-kl]pyrrolo[3,4-i](1,6)benzodiazocine-10-carboxylic acid, methyl ester KT 5823, 10-2 M tetraethylammonium (TEA), or 10-7 M apamin plus 10-7 M charybdotoxin. The results suggest the involvement of endothelial mechanisms in the vasodilator effect produced by the acute in vitro application of T3 to rat aortic rings. Possible mechanisms include the stimulation of muscarinic receptors, activation of the NO-cGMP-PKG pathway, and opening of Ca2+-activated K+ channels.

Re-emergence of Chorioptes bovis (Acari: Psoroptidae) in cattle in the state of Rio Grande do Sul, Brazil / Reemergência de Chorioptes bovis (Acari: Psoroptidae) em bovinos no Estado do Rio Grande do Sul, Brasil

Here we describe an outbreak of chorioptic mange in cattle, 56 years after its first identification in Brazil. Between the months of June and July 2011, dermatitis characterized by alopecia and crusted and thickened skin at the insertion of the tail and in the ischiorectal fossa was recognized in 40 (35.7%) out of 112 Holstein cows on a farm in the northeastern mesoregion of the state of Rio Grande do Sul, Brazil. After diagnosing mange caused by Chorioptes bovis, the cows were weighed and treated with 0.5% ivermectin, as a pour-on single dose, and were separated into two groups: cows in early lactation and those in late lactation. The survival rate of C. bovis and the healing rate in the two groups of infested cows were monitored every seven days through skin scrapings. After 28 days of evaluation, the cure rate through treatment was greater among cows in early lactation (p <0.0001). The survival rate of C. bovis was higher in cows in late lactation.

O objetivo deste estudo foi descrever um surto de sarna corióptica em bovinos, 56 anos após a sua primeira identificação no Brasil. Entre os meses de junho a julho de 2011, a dermatite caracterizada por alopecia, com crosta e espessamento da pele na inserção da cauda e na fossa isquiorretal, foi observada em 40 (35,7%) de 112 vacas holandesas de uma propriedade rural pertencente à Mesorregião do Nordeste do Estado do Rio Grande do Sul, Brasil. Após o diagnóstico da sarna causada por Chorioptes bovis, as vacas foram pesadas, tratadas com 0,5% de ivermectina pour on em dose única e separadas em dois grupos: vacas no início da lactação e no final da lactação. A taxa de sobrevivência de C. bovis e a taxa de cura dos dois grupos de vacas infestadas foram monitoradas a cada sete dias por meio de raspas de pele. Após 28 dias do estudo, a taxa de cura com o tratamento foi maior em vacas no início da lactação (p <0,0001). A taxa de sobrevivência de C. bovis foi maior em vacas no final da lactação.

[Inductive effect of deprenyl and dimethyl sulfoxide on proliferation and survival of the mesenchymal stem cells]

Research have been focused on the applying the chemical inducer for transdifferentiation the adult BMSCs into neural cell. So that, at the first should investigate the toxcity effect of the chemical inducer on the induced cells. Plasticity and easy accessibility of bone marrow mesenchymal stem cells is a unique charactristic for treatment of neural disorderies. This study was desgined to determine the inductive effect of Deprenyl and Dimethyl sulfoxide on proliferation and survival of the mesenchymal stem cells. In this experimental study, BMSCs isolated from the adult rat bone marrow and cultured in alpha MEM containing 10% FBS. Cell identity for surface antigens was performed in third passage by immunocytochemistry and multipotancy capacity of BMSCs was done by BMSC differentiation into adipocytes and osteocytes. The cells were exposed to chemical agents [a: the alpha MEM medium supplemented with 2% DMSO, b: the alpha MEM medium supplemented with 10[-8]M Deprenyl] for 24 hours and then transferred to alpha MEM containing 10% FBS cell survival and proliferation was evaluated after the 24, 48, 72 and 96 houres by MTT [3-[4-5-Dimethylthiazolyl-2-y1]-2,5-diphenyltetrazolium romid] test. Data were analyzed using SPSS-16, One-Way ANOVA and Tukey tests. In addition to expression the surface antigens and adipogenic and osteogenic differentiation by BMSCs, MTT test results showed that proliferation and survival of induced-deprenyl and DMSO cells within 48, 72 and 96 hours after the induction was increased significantly than negative control group. Deprenyl increases survival and cell proliferation compared to Dimethyl Sulfoxide. It can be used as cell inducer

Avaliação da trealose como crioprotetor de células-tronco hematopoiéticas de sangue de cordão umbilical e placentário / Evaluation of trehalose as a cryoprotectant natural of hematopoietic stem cells from umbilical cord blood and placental

O sangue do cordão umbilical e placentário (SCUP) tem sido usado como fonte de células-tronco hematopoiéticas (CTH) para reconstituir a função medular (hematopoiese). A maioria das vezes, esta modalidade de transplante requer a criopreservação das CTH, que permanecem congeladas até uma possível utilização futura. Na criopreservação de CTH, o reagente químico dimetilsulfóxido (DMSO) tem sido utilizado como um crioprotetor. No entanto, tem sido provado que DMSO tem efeitos tóxicos para o corpo humano. Muitos organismos na natureza possuem uma capacidade de sobreviver ao congelamento e à desidratação acumulando dissacarídeos, como a trealose e sacarose, por isso a trealose, tem sido investigada como um crioprotetor alternativo para diversos tipos celulares. Outro dano muito comum durante o congelamento é a formação de espécie reativas de oxigênio (ERO) que diminui a viabilidade celular, por isso a adição de bioantioxidantes na solução de criopreservação das células é passo muito importante. Este estudo foi dividido em duas fases na primeira foram avaliados os resultados obtidos com a adição de antioxidantes na solução de criopreservação das células de SCUP e na segunda fase avaliou-se a hipótese que a solução de criopreservação contendo trealose intracelular e extracelular melhora a recuperação e a viabilidade das células-tronco do SCUP, após a criopreservação. SCUP foi processado e submetido à criopreservação em soluções contendo na primeira fase: soluções com diferentes concentrações de DMSO (10%, 5% e 2,5%), assim como as combinações de DMSO (5%, 2,5%) com um dos dissacarídeos (60mmol/L) e ácido ascórbico e/ou catalase (10mg/mL); e na segunda fase: soluções contendo diferentes concentrações de DMSO (10% e 2,5%), assim como as combinações de DMSO (2,5%) com trealose intra (a trealose foi introduzida na célula por meio de lipossomas) e extracelular e soluções contendo trealose intra e extracelular sem DMSO, armazenados por duas semanas em N2L, e descongeladas...

The umbilical cord blood (UCB) has been used as a source of primitive hematopoietic stem cells (HSC) to reconstitute the hematopoiesis. Most often, it is required the cryopreservation of HSC, which remain frozen in banks for possible future use. For cryopreservation of HSC, the chemical reagent dimethylsulfoxide (DMSO) has been used as a cryoprotectant. Many organisms in nature have a capacity of survive freezing and dehydration by accumulating disaccharides, so the trehalose, has been actively investigated as an alternative cryoprotector, other damage which is very common during freezing is oxygen free radicals formation which decreases the cellular viability after thawing, so the addition of bioantioxidants in the solution of cryopreservation of cells is very important. This study was divided into two phases: first, we evaluated the results obtained with the addition of antioxidants in the solution for cryopreservation of cord blood cells and the second phase: evaluate the hypothesis that the cryopreservation solution containing intracellular and extracellular trehalose improves recovery and viability of cord blood stem cells after cryopreservation. UBC was processed and subjected to cryopreservation solutions containing for the first phase: solutions with different concentrations of DMSO (10%, 5% and 2.5%), as well as combinations of DMSO (5%, 2.5 %) with a disaccharide (60 mmol/L), ascorbic acid and/or catalase (10mg/mL), and for the second phase: solutions containing different concentrations of DMSO (10% and 2.5%), as well as combinations of DMSO (2.5%) with intracellular trehalose (trehalose was introduced into the cell by means of liposomes) and solutions containing extra and intracellular trehalose without DMSO, stored for two weeks in N2L, and thawed. The thawed cells were assessed by flow cytometry, MTT and colony forming units (CFU) assays. In the first phase of the study our analysis showed catalase improved the preservation CD34+ and CD123+...

Testis-mediated gene transfer in mice: comparison of transfection reagents regarding transgene transmission and testicular damage

Testis-mediated gene transfer (TMGT) has been used as in vivo gene transfer technology to introduce foreign DNA directly into testes, allowing mass gene transfer to offspring via mating. In this study, we used plasmid DNA (pEGFP-N1) mixed with dimethylsulfoxide (DMSO), N,N-dimethylacetamide (DMA) or liposome (Lipofectin) in an attempt to improve TMGT. Males receiving consecutive DNA complex injections were mated to normal females to obtain F0 progeny. In vivo evaluation of EGFP expression, RT-PCR and PCR were used to detect the expression and the presence of exogenous DNA in the progeny. We also evaluated possible testicular damage by histological procedures. PC R and RT-PCR analyses revealed that liposome and DMSO increased the rate of TMGT. Histological analyses demonstrated that repeated (4 times) injections of DNA complexes can affect spermatogenesis. DMSO was the most deleterious among the reagents tested. In this study, we detected the presence of transgene in the progeny, and its expression in blood cells. Consecutive injections of DNA complexes were associated with impaired spermatogenesis, suggesting requirement of optimal conditions for DNA delivery through TMGT.

Influence of 2-methoxyestradiol on MCF-7 cells: an improved differential interference contrasting technique and Bcl-2 and Bax protein expression levels

Proteins of the B-cell lymphoma 2 family are crucial for the regulation of apoptosis. B-cell lymphoma 2-associated X is a pro-apoptotic protein, while B-cell lymphoma 2 protein opposes apoptosis. The influence of 1 microM 2-methoxyestradiol was investigated on the expression levels of these two proteins in MCF-7 cells. 2-Methoxyestradiol exposure did not influence B-cell lymphoma 2 protein expression levels after 24 h of exposure. In contrast, B-cell lymphoma 2-associated X protein levels were significantly reduced. An improved differential interference contrasting technique revealed compromised cell density and the presence of a mitotic block in exposed cells. The study proposes that the influence of 2-methoxyestradiol on the expression of these proteins may be time- and cell type dependent and thus not evident d uring the mitotic block observed. Investigation of the regulation of the B-cell lymphoma 2 family will allow researchers to consider signaling pathways for diseases where apoptosis can potentially be controlled.

Trypanocidal constituents in plants: 7. Mammea-type coumarins

Calophyllum brasiliense and Mammea americana (Clusiaceae) are two trees from the tropical rain forests of the American continent. A previous screening showed high trypanocidal activity in the extracts of these species. Several mammea-type coumarins, triterpenoids and biflavonoids were isolated from the leaves of C. brasiliense. Mammea A/AA was obtained from the fruit peels of M. americana. These compounds were tested in vitro against epimastigotes and trypomastigotes of Trypanosoma cruzi, the etiologic agent of Chagas disease. The most potent compounds were mammea A/BA, A/BB, A/AA, A/BD and B/BA, with MC100 values in the range of 15 to 90 g/ml. Coumarins with a cyclized ,-dimethylallyl substituent on C-6, such as mammea B/BA, cyclo F + B/BB cyclo F, and isomammeigin, showed MC100 values > 200 g/ml. Several active coumarins were also tested against normal human lymphocytes in vitro, which showed that mammea A/AA and A/BA were not toxic. Other compounds from C. brasiliense, such as the triterpenoids, friedelin, canophyllol, the biflavonoid amentoflavone, and protocatechuic and shikimic acids, were inactive against the epimastigotes. The isopropylidenedioxy derivative of shikimic acid was inactive, and its structure was confirmed by X-ray diffraction. Our results suggest that mammea-type coumarins could be a valuable source of trypanocidal compounds.

Urinary glycosaminoglycans excretion and the effect of dimethyl sulfoxide in an experimental model of non-bacterial cystitis

PURPOSE: We reproduced a non-bacterial experimental model to assess bladder inflammation and urinary glycosaminoglycans (GAG) excretion and examined the effect of dimethyl sulfoxide (DMSO). MATERIALS AND METHODS: Female rats were instilled with either protamine sulfate (PS groups) or sterile saline (control groups). At different days after the procedure, 24 h urine and bladder samples were obtained. Urinary levels of hyaluronic acid (HA) and sulfated glycosaminoglycans (S-GAG) were determined. Also to evaluate the effect of DMSO animals were instilled with either 50 percent DMSO or saline 6 hours after PS instillation. To evaluate the effect of DMSO in healthy bladders, rats were instilled with 50 percent DMSO and controls with saline. RESULTS: In the PS groups, bladder inflammation was observed, with polymorphonuclear cells during the first days and lymphomononuclear in the last days. HA and S-GAG had 2 peaks of urinary excretion, at the 1st and 7th day after PS injection. DMSO significantly reduced bladder inflammation. In contrast, in healthy bladders, DMSO produced mild inflammation and an increase in urinary HA levels after 1 and 7 days and an increase of S-GAG level in 7 days. Animals instilled with PS and treated with DMSO had significantly reduced levels of urinary HA only at the 1st day. Urinary S-GAG/Cr levels were similar in all groups. CONCLUSIONS: Increased urinary levels of GAG were associated with bladder inflammation in a PS-induced cystitis model. DMSO significantly reduced the inflammatory process after urothelial injury. Conversely, this drug provoked mild inflammation in normal mucosa. DMSO treatment was shown to influence urinary HA excretion.

Análise ultraestrutural e de fatores de crescimento de diferentes métodos de preservação da membrana amniótica utilizada em cirurgia ocular / Ultrastructural and growth factor analysis of amniotic membrane preserved by different methods for ocular surger

OBJETIVO: Comparar, por microscopia eletrônica, a integridade anatômica e a presença de fatores de crescimento e citocinas da membrana amniótica preservada com glicerol/MEM (1:1) e dimetilsulfóxido puro. MÉTODOS: As membranas amnióticas preservadas em glicerol/MEM (1:1) ou dimetilsulfóxido puro foram processadas para microscopia eletrônica de transmissão e varredura. Como controle, membrana amniótica fresca foi imediatamente fixada após coleta e processada para microscopia eletrônica. As citocinas e os fatores de crescimento avaliados foram: TGF-beta- fator transformador de crescimento beta; TGF-beta ativ- fator transformador de crescimento beta ativado; EGF- fator recombinante de crescimento epitelial humano; FGF-4- fator de crescimento fibroblástico 4; FGF-beta- fator de crescimento fibroblástico básico; IL-4- interleucina 4; PGE2- prostaglandina E2; IL-10- interleucina 10; KGF- fator de crescimento de queratinócito; HGF- fator de crescimento de hepatócito. RESULTADOS: As membranas amnióticas do grupo controle apresentavam epitélio íntegro, com microvilos na superfície e complexos juncionais entre as células e a membrana basal. As membranas amnióticas preservadas em glicerol/MEM tinham aspecto semelhante às do controle, com maior altura das células epiteliais. Já as membranas amnióticas preservadas em dimetilsulfóxido mostraram redução das junções intercelulares e destacamento do epitélio da membrana basal. As citocinas e fatores de crescimento não apresentaram diferenças entre os grupos, exceto FGF-4, FGF-beta, PGE2 e KGF. CONCLUSÕES: A membrana amniótica preservada em meio glicerol/MEM apresentou melhor integridade tecidual, com menor desprendimento do epitélio da membrana basal, em comparação com a preservada no dimetilsulfóxido puro. Os fatores de crescimento e citocinas estavam, em sua maior parte, preservados com as duas técnicas de preservação.

PURPOSE: To compare the anatomical structure and the presence of growth factors and cytokines of amniotic membrane preserved in glycerol/MEM (1:1) or undiluted dimethyl sulfoxide through electron microscopy. METHODS: Amniotic membrane preserved in glycerol/MEM (1:1) or undiluted dimethyl sulfoxide were processed for transmission and scaning electron microscopy. As control, freshly collected amniotic membrane was fixed and processed for electron microscopy. The cytokines and growth factors assessed were: TGF-beta (transforming growth factor beta); TGF-b activ (activated transforming growth factor beta); EGF (epidermal growth factor); FGF-4 (fibroblast growth factor 4); bFGF (basic fibroblast growth factor); IL-4 (interleukin 4); PGE2 (prostaglandin E2); IL-10 (interleukin 10); KGF (keratinocyte growth factor); HGF (hepatocyte growth factor). RESULTS: Amniotic membrane from the control group showed intact epithelium, with surface microvilli and junctional complexes between the cells and the basal membrane. Glycerol/MEM preserved amniotic membrane had similar aspect to the control, with higher epithelial cells. Those amniotic membranes preserved in dimethyl sulfoxide disclosed less intercellular junction and detachment of the epithelium from the basal membrane. The cytokines and growth factors did not disclose significant differences, except for FGF-4, bFGF, PGE2 and KGF. CONCLUSIONS: Amniotic membrane preserved in glycerol/MEM showed a better tissue structure, with less detachment of the epithelium from the basal membrane, in comparison to undiluted dimethyl sulfoxide. The majority of the growth factors and cytokines were kept with both techniques of preservation.

Effect of sphingosine and phorbol-12-myristate-13-acetate on the growth and dimethylsulfoxide-induced differentiation in the insect trypanosomatid Herpetomonas samuelpessoai

We investigated the effect of two modulators of protein kinase C, sphingosine and phorbol-12-myristate-13-acetate (PMA), on the growth and dimethylsulfoxide (DMSO)-induced differentiation in Herpetomonas samuelpessoai. Sphingosine did not stimulate the transformation of undifferentiated-promastigotes in differentiated-paramastigotes. PMA alone or in association with DMSO increased the number of paramastigotes in comparison to control cells. DMSO inhibited the parasite growth (35 percent) and several unusual morphological features resembling aberrant cell division were observed. Sphingosine did not significantly reduce the growth in contrast to PMA. Collectively, our results demonstrated that the reduction of the proliferation translates in an increase of the differentiation rate in the insect trypanosomatid H. samuelpessoai.

Perspectives of contraceptive choices for men.

Apart from condoms and vasectomy, which have several limitations of their own, no other methods of contraception are available to men. Various chemical, hormonal, vas based and herbal contraceptives have been examined and few of them have reached the stage of clinical testing. Promising leads have been obtained from testosterone buciclate/undecanoate, alone or in combination with levonorgestrel butanoate or cyproterone acetate, RISUG, an injectable intravasal contraceptive and a few herbal products, particularly the seed products of Carica papaya. It is feasible that an ideal male contraceptive, that meets out all the essential criteria will be made available to the community in the near future.

Standardization of serum free media for cryopreservation of RK13 cells.

Foetal calf serum present in the media used for cryopreservation was replaced by various synthetic polymer such as gelatin, glycerol, carboxymethyl cellulose and dimethyl sulphoxide at various concentration. Growth pattern of cells, % survival and karyological studies have been done in the present study. It was found that optimum concentration of carboxymethyl cellulose was 0.1% in combination with 10% glycerol and 10% DMSO. At this concentration percentage survival of cells was found maximum and karyotype was found normal without any abnormality in the chromosomes. It was concluded from the study that serum free media can be employed for the cryopreservation of these cells which are further used for production of tissue culture vaccines without causing any adverse affects.

Cord blood from collection to expansion: feasibility in a regional blood bank.

This article reviews the various aspect of the experimental phase preceding the establishment of an umbilical cord blood (UCB) bank within a regular blood bank, a situation totally different from that of de novo establishing a cord blood bank having human and financial resources. An ethically approved two-year study has been conducted to determine the technical feasibility, and the practical problems that might be encountered such as public compliance, the additional workload, introduction of new activities ranging from collection and processing to progenitor expansion, infectious disease testing, development of a quality control system, record keeping and documentation, development of specific procedures and definitions of requirements. The cost benefit aspect, which will ultimately depend on the frequency of units release, was not considered in this study.

Relaxant effect of 6-deoxyclitoriacetal on smooth muscle preparations.

The pharmacological effect of 6-deoxyclitoriacetal (6-DA), a rotenoid compound isolated from the roots of Clitoria macrophylla Wall. (Papilionaceae), was examined on different smooth muscle preparations. 6-Deoxyclitoriacetal 0.2 mg/ml produced a significant decrease in the spontaneous contraction of isolated rat uterus. It also suppressed the contraction induced by acetylcholine 5x10(-6) M and oxytocin 5x10(-3) IU/ml. The cumulative contractile responses of rat aortic strips caused by serotonin 10(-8)-10(-4) M and norepinephrine 10(-11)-10(-7) M were reduced by 6-DA 0.4 mg/ml. In calcium free Kreb's solution, 6-DA inhibited the aortic contraction produced by a cumulative dose of calcium chloride (0.1-30 mM). In guinea-pig ileum, 6-DA 0.15 mg/ml exerted the spasmolytic activity by inhibition of the contractile response evoked by various contractile agents e.g. acetylcholine 10(-9)-10(-5) M, serotonin 10(-9)-10(-5) M and histamine 10(-9)-10(-5) M. All of the results indicated that 6-DA could induce a smooth muscle relaxant effect by interference with intracellular calcium metabolism.

Effect of dimethylsulfoxide on sphingomyelinase activity and cholesterol metabolism in Niemann-Pick type C fibroblasts

Niemann-Pick type C (NPC) fibroblasts present a large concentration of cholesterol in their cytoplasm due to a still unidentified deficiency in cholesterol metabolism. The influence of dimethylsulfoxide (DMSO) on the amount of intracellular cholesterol was measured in 8 cultures of normal fibroblasts and in 7 fibroblast cultures from NPC patients. DMSO was added to the fibroblast cultures at three different concentrations (1, 2 and 4 percent, v/v) and the cultures were incubated for 24 h. Sphingomyelinase activity was significantly increased in both groups of cells only when incubated with 2 percent DMSO (59.4 9.1 and 77.0 9.1 nmol h-1 mg protein-1, controls without and with 2 percent DMSO, respectively; 47.7 5.2 and 55.8 4.1 nmol h-1 mg protein-1, NPC without and with 2 percent DMSO, respectively). However, none of the DMSO concentrations used altered the amount of cholesterol in the cytoplasm of NPC cells (0.704 0.049, 0.659 0.041, 0.688 0.063 and 0.733 0.088 mg/mg protein, without DMSO, 1 percent DMSO, 2 percent DMSO and 4 percent DMSO, respectively). This finding suggests that sphingomyelinase deficiency is a secondary defect in NPC and shows that DMSO failed to remove the stored cholesterol. These data do not support the use of DMSO in the treatment of NPC patients