RESUMEN
Abstract Purpose: To investigate the protective effects of salvianolic acid A (SAA) on renal damage in rats with chronic renal failure (CRF). Methods: The five-sixth nephrectomy model of CRF was successfully established in group CRF (10 rats) and group CRF+SAA (10 rats). Ten rats were selected as sham-operated group (group S), in which only the capsules of both kidneys were removed. The rats in group CRF+SAA were intragastrically administrated with 10 mg/kg SAA for 8 weeks. The blood urine nitrogen (BUN), urine creatinine (Ucr), creatinine clearance rate (Ccr), and serum uperoxide dismutase (SOD) and malondialdehyde (MDA) were tested. The expressions of transforming growth factor-β1 (TGF-β1), bone morphogenetic protein 7 (BMP-7) and Smad6 protein in renal tissue were determined. Results: After treatment, compared with group CRF, in group CRF+SAA the BUN, Scr, serum MDA and kidney/body weight ratio were decreased, the Ccr and serum SOD were increased, the TGF-β1 protein expression level in renal tissue was decreased, and the BMP-7 and Smad6 protein levels were increased (all P < 0.05). Conclusion: SAA can alleviate the renal damage in CRF rats through anti-oxidant stress, down-regulation of TGF-β1 signaling pathway and up-regulation of BMP-7/Smad6 signaling pathway.
Asunto(s)
Animales , Masculino , Ratas , Ácidos Cafeicos/uso terapéutico , Proteína smad6/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Proteína Morfogenética Ósea 7/metabolismo , Fallo Renal Crónico/tratamiento farmacológico , Lactatos/uso terapéutico , Regulación hacia Abajo , Regulación hacia Arriba , Ratas Sprague-Dawley , Modelos Animales de Enfermedad , Fallo Renal Crónico/inducido químicamente , Fallo Renal Crónico/metabolismo , Pruebas de Función Renal , NefrectomíaRESUMEN
One of the major signaling pathways that determine the tumor aggression and patient outcome in pancreatic cancer is the transforming growth factor-beta (TGF-ß) pathway. It is inactivated at various levels in pancreatic cancer and plays a dual role in tumor initiation and progression. The Smad family of proteins transduce signals from the TGF-ß superfamily ligands that regulate cell proliferation, differentiation and death through activation of receptor serine/threonine kinases. This review discusses the structure, function and regulation of various participating Smad family members, and their individual roles in determining the progression and outcome of pancreatic cancer patients, with a special emphasis on Smad4.
Asunto(s)
Diferenciación Celular , Proliferación Celular , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Humanos , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Fosforilación , Receptores de Factores de Crecimiento Transformadores beta/genética , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Transducción de Señal , Proteína Smad4/química , Proteína Smad4/genética , Proteína Smad4/metabolismo , Proteína smad6/genética , Proteína smad6/metabolismo , Proteína smad7/genética , Proteína smad7/metabolismo , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Background: Smad4, Smad6 and Smad7 are important molecules in TGF-beta pathway, which plays an important role in pancreatic ductal adenocarcinoma (PDAC) biology. Aims : This study examined the expression profiles of Smad4, Smad6 and Smad7 mRNA in patient samples of PDAC and their relationship to Smad protein expression, SMAD4 gene mutations, clinicopathological parameters and patient survival. Settings and Design: Surgically resected, paired normal and tumor tissues of 25 patients of PDAC were studied. Materials and Methods: Protein and mRNA levels were assessed by immunohistochemistry and RT-PCR, respectively. Statistical Methods: Statistical analysis was done using Student's t-test, Pearson's chi-square test, Spearman's Rank Correlation, Pearson's Correlation test and Kaplan-Meier Logrank test. Results: While there was a highly significant difference in the protein levels of all three Smads in tumor as compared to normal samples, mRNA levels were significantly different only for Smad4. Protein levels did not correlate significantly with mRNA levels for any of the three Smads. The mRNA levels of Smad4 and Smad6, Smad4 and Smad7, and Smad6 and Smad7 in tumor samples showed a significant positive correlation. The relationship of Smad4 mRNA expression to SMAD4 gene status and Smad4 protein expression was discordant and there was no significant correlation between mRNA expression and clinicopathological parameters and patient survival. Conclusion : The absence of concordance between SMAD4 gene status, mRNA expression and Smad4 protein expression suggests the presence of other regulatory mechanisms in Smad4 transcription and translation in PDAC.
Asunto(s)
Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/secundario , Adulto , Anciano , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/secundario , Femenino , Humanos , Técnicas para Inmunoenzimas , Masculino , Persona de Mediana Edad , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Pronóstico , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína Smad4/genética , Proteína Smad4/metabolismo , Proteína smad6/genética , Proteína smad6/metabolismo , Proteína smad7/genética , Proteína smad7/metabolismo , Tasa de SupervivenciaRESUMEN
This experiment was designed to construct mouse Smad6 recombinant RNA interference vectors and determine their interference effects on bone marrow mesenchymal stem cells (BMSCs). Three recombinant Smad6 RNA interference vectors were constructed by molecular clone techniques with a lenti-virus vector expressing green fluorescent protein (GFP), and the correctness of recombinant vectors was verified by DNA sequencing. Mouse BMSCs were used for transfection experiments and BMP-2 was in use for osteogenic induction of MSCs. The transfection efficiency of recombinant vectors was examined by Laser confocal scanning microscope and the interference effect of recombinant vectors on Smad6 gene expression was determined by real-time RT-PCR and Western blot, respectively. Three Smad6 recombinant RNA interference vectors were successfully constructed and their correctness was proved by DNA sequencing. After transfection, GFPs were effectively expressed in MSCs and all of three recombinant vectors gained high transfection efficiency (> 95%). Both real-time PCR and Western blot examination indicated that among three recombinant vectors, No. 2 Svector had the best interference effect and the interference effect was nearly 91% at protein level. In conclusion, Mouse recombinant Smad6 RNA interference (RNAi) vector was successfully constructed and it provided an effective tool for further studies on BMP signal pathways.
Asunto(s)
Animales , Ratones , Proteínas Morfogenéticas Óseas , Genética , Metabolismo , Vectores Genéticos , Genética , Proteínas Fluorescentes Verdes , Genética , Lentivirus , Genética , Metabolismo , Células Madre Mesenquimatosas , Metabolismo , Interferencia de ARN , Proteínas Recombinantes , Genética , Proteína smad6 , Genética , TransfecciónRESUMEN
The inhibitory Smad6 and Smad7 are responsible for cross-talk between TGF-beta/bone morphogenic protein (BMP) signaling and other cellular signaling pathways, as well as negative feedback on their own signaling functions. Although inhibitory Smads are induced by various stimuli, little is known about the stimuli that increase Smad6 transcription, in contrast to Smad7. Here we demonstrate that etoposide, which induces double strand breaks during DNA replication, significantly up-regulates the transcription of the Smad6 gene in CMT-93 mouse intestinal cells by increasing specific DNA binding proteins. In addition, endogenous inhibition of the Smad6 gene by RNAi interference led to transient accumulation of G1 phase cells and reduction in incorporation of bromodeoxyuridine (BrdU). These findings strongly suggest that Smad6 plays a distinct role in the signaling of etoposide-induced DNA damage.
Asunto(s)
Animales , Ratones , Secuencia de Bases , Línea Celular , Proteínas de Unión al ADN/metabolismo , Enterocitos/citología , Etopósido/farmacología , Fase G1/efectos de los fármacos , Datos de Secuencia Molecular , Regiones Promotoras Genéticas/genética , ARN Interferente Pequeño/metabolismo , Fase S/efectos de los fármacos , Proteína smad6/genética , Activación Transcripcional/efectos de los fármacosRESUMEN
The present study was designed to observe the expressions of bone morphogenetic protein-7 (BMP-7) and inhibitory Smads in kidney of rats with diabetic nephropathy (DN), and explore the possible mechanism of DN. Male Wistar rats weighing 180-220 g were single injected with streptozocin (STZ, 55 mg/kg body weight) for 2, 4, 8 and 16 weeks to induce DN. Blood glucose, kidney weight/body weight and 24-hour urine protein in the control and DN rats were examined; the expressions of BMP-7, Smad6 and Smad7 were detected by using immunohistochemical techniques, Western blot and real-time PCR. The results showed that blood glucose and 24-hour urine protein in DN rats were higher than that in the control rats and kidney weight/body weight was also elevated in DN rats, especially in 16-week STZ-induced rats. The expressions of BMP-7 and Smad6 proteins in DN rats were elevated, while BMP-7 mRNA expression was increased 2 weeks after STZ injection and decreased 16 weeks after STZ injection. The expressions of Smad7 protein and mRNA were elevated in DN rats 2 weeks after STZ injection and decreased 16 weeks after STZ injection. In addition, the expressions of transforming growth factor-beta1 (TGF-beta1) and collagen type I (COL-I) mRNA were increased in DN rats. These results suggest in the early stage of DN, increase in BMP-7 and inhibitory Smad expression may play a role in the feedback regulation and restrain the development of DN.