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Effect of tankyrase 1 on autophagy in the corpus cavernosum smooth muscle cells from ageing rats with erectile dysfunction and its potential mechanism.

ABSTRACT: This study compared tankyrase 1 expression and autophagy quantity between erectile dysfunction (ED) and non-ED rats' corpus cavernosum smooth muscle cells (CSMCs). This study aslo explored the effect and possible mechanism of tankyrase 1 on autophagy and cell proliferation in ageing ED rats' CSMCs. The intracavernous pressure and mean systemic arterial pressure were measured to investigate erectile function so that eight 24-month-old ED and eight 8-month-old male Wistar rats were chosen respectively. The rat CSMCs were isolated and cultured by enzyme digestion, in which tankyrase 1 expression and autophagy quantity were compared. Tankyrase 1 overexpression was induced with plasmid transfection by Lipofectamine. The effect of tankyrase 1 overexpression on proliferation, autophagy and mTOR pathway in 24-month-old ED rats' CSMCs was measured by the cell growth curve in MTT assay, cell cycle analysis in flow cytometry (FCM), key protein expression in Western blot, autophagy quantity in transmission electron microscopy, monodansylcadaverine staining and GFP-LC3 fluorescence. The primary CSMCs were confirmed by immunofluorescence, and the purity was 99.1% in FCM. Compared with that of 8-month-old rats, tankyrase 1 expression and autophagy quantity significantly decreased in 24-month-old ED rats' primary CSMCs (P < 0.01). Tankyrase 1 overexpression significantly increased the growth rate (P < 0.05) and increased the S phase of cell cycle (P < 0.01). The autophagosome quantity was remarkably increased (P < 0.01), LC3-I/II and Beclin 1 were upregulated (P < 0.01 and P < 0.05), and p-p70S6K (Thr(389)) was downregulated in 24-month-old ED rat CSMCs (P < 0.05). In conclusion, Tankyrase 1 and autophagy decrease in the CSMCs from aging rats with ED, and tankyrase 1 may have a positive effect on proliferation by enhancing autophagy and regulating the mTOR signalling pathway.

SUBMITTER: Zhang J

PROVIDER: S-EPMC3739314 | biostudies-other | 2010 Sep

REPOSITORIES: biostudies-other

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Project description:ObjectiveTo construct a corpus cavemosum smooth muscle cell (CCSMCs) line with TEAD1 knockout from diabetic rats with erectile dysfunction (ED) using CRISPR/Cas9 technology and explore the role of TEAD1 in phenotypic modulation of CCSMCs in diabetic rats with ED.ObjectiveModels of diabetic ED were established in male Sprague-Dawley rats by intraperitoneal injection of streptozotocin. CCSMCs from the rat models were primarily cultured and identified with immunofluorescence assay. Three sgRNAs (sgRNA-1, sgRNA-2 and sgRNA-3) were transfected via lentiviral vectors into 293T cells to prepare the sgRNA-Cas9 lentivirus. CCSMCs from diabetic rats with ED were infected by the lentivirus, and the cellular expression of TEAD1 protein was detected using Western blotting. In CCSMCs infected with the sgRNA-Cas9 lentivirus (CCSMCs-sgRNA-2), or the empty lentiviral vector (CCSMCs-sgRNA-NC) and the blank control cells (CCSMCs-CK), the expressions of cellular phenotypic markers SMMHC, calponin and PCNA at the mRNA and protein levels were detected using real-time fluorescence quantitative RT-PCR (qRT-PCR) and Western blotting, respectively.ObjectiveThe primarily cultured CCSMCs from diabetic rats with ED showed a high α-SMA-positive rate of over 95%. The recombinant lentivirus of TEAD1-sgRNA was successfully packaged, and stable TEAD1-deficient CCSMC lines derived from diabetic rat with ED were obtained. Western blotting confirmed that the protein expression of TEAD1 in TEAD1-sgRNA-2 group was the lowest (P < 0.05), and this cell line was used in subsequent experiment. The results of qRT-PCR and Western blotting showed significantly up-regulated expressions of SMMHC and calponin (all P < 0.05) and down-regulated expression of PCNA (all P < 0.05) at both the mRNA and protein levels in TEAD1-deficient CCSMCs from diabetic rats with ED.ObjectiveWe successfully constructed a stable CCSMCs line with CRISPR/Cas9-mediated TEAD1 knockout from diabetic rats with ED. TEAD1 gene knockout can induce phenotype transformation of the CCSMCs from diabetic rats with ED from the synthetic to the contractile type.

Project description:IntroductionStem cell therapy is a novel method for the treatment of diabetic erectile dysfunction (ED). Many relative animal studies have been done to evaluate the efficacy of this therapy in rats.AimsThis meta-analysis was performed to compare the efficacy of different stem cell therapies, to evaluate the influential factors and to determine the optimal stem cell therapeutic strategy for diabetic ED.MethodsWe searched the studies analyzing the efficacy of stem cell therapy for diabetic ED in rats published before September 30, 2015 in PubMed, Web of Science and EBSCO. A random effects meta-analysis was conducted to assess the outcomes of stem cell therapy. Subgroup analysis was also performed by separating these studies based on their different characteristics. Changes in the ratio of intracavernous pressure (ICP) to mean arterial pressure (MAP) and in the structure of the cavernous body were compared.Results10 studies with 302 rats were enrolled in this meta-analysis. Pooled analysis of these studies showed a beneficial effect of stem cell therapy in improving erectile function of diabetic rats (SMD 4.03, 95% CI = 3.22 to 4.84, P< 0.001). In the stem cell therapy group, both the smooth muscle and endothelium content were much more than those in control group. There was also significant increase in the expression of endothelial nitric oxide synthase (eNOS) and neuronal nitric oxide synthase (nNOS), the ratio of smooth muscle to collagen, as well as the secretion of vascular endothelial growth factor (VEGF). Besides, apoptotic cells were reduced by stem cell treatment. The subgroup analysis indicated that modified stem cells were more effective than those without modification.ConclusionsOur results confirmed that stem cell therapy could apparently improve the erectile function of diabetic rats. Some specific modification, especially the gene modification with growth factors, could improve the efficacy of stem cell therapy. Stem cell therapy has potential to be an effective therapeutic strategy for diabetic ED.

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Effect of tankyrase 1 on autophagy in the corpus cavernosum smooth muscle cells from ageing rats with erectile dysfunction and its potential mechanism.

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