The cytoptrotection of small intestinal submucosa-derived gel in HL-1 cells during hypoxia/reoxygenation-induced injury.

Small intestinal submucosa (SIS)-derived gel injected into infarcted myocardium has been shown to promote repair and regeneration after myocardial infarction (MI); however, the specific impact of SIS gel on cardiomyocytes remained unknown. The aim of this study was to characterise SIS gel function in hypoxia-reoxygenation (H/R)-induced cardiomyocyte damage and its potential mechanism. HL-1 cardiomyocytes seeded on SIS matrix-coated plates, SIS gel, and uncoated plates were subjected to H/R, cell viability, apoptosis, expression of caspase-3, Bcl-2, and Bax were investigated. SIS gel and SIS matrix as coating substrates markedly improved cell viability, preventing cell apoptosis compared with uncoated plates, with SIS gel yielding the best cytoprotective effects. SIS gel down-regulated expression of pro-inflammatory cytokines (TNF-α, CCL2, and IL-6) by inhibiting the JNK-mitogen-activated protein kinase (MAPK)/NF-κB pathways. Furthermore, SIS gel protected cardiomyocytes from apoptosis by activating protein kinase B (AKT) and extracellular-signal-regulated kinase (ERK) pathways, and markedly up-regulated antiapoptotic Bcl-2 expression but inhibited that of proapoptotic Bax and c-caspase 3. Together, these findings show that SIS gel could decrease H/R-induced cell apoptosis through a mechanism potentially related to its ability to regulate expression of inflammatory cytokines and antiapoptosis signalling pathways to prevent cell apoptosis. Our findings thereby shed light on the mechanism related to SIS gel therapeutic efficacy for MI.

[Expression and clinical significance of long non-coding RNA LINC00520 in laryngeal squamous cell carcinoma].

Objective:To investigate the expression of LINC00520 in laryngeal squamous cell carcinoma(LSCC),and analyze its relevance and roles in carcinogenesis and development of LSCC.Method:The expression of LINC00520 in laryngeal squamous cell carcinoma tissue and paired adjacent normal tissue was determined by real-time PCR.The relationship between the expression of LINC00520 and the clinicopathological characteristics including clinical stage,pathological type,histological grade and lymph node metastasis of LSCC was analyzed.Result:(1)The LINC00520 expression level was significantly upregulated in LSCC tissues compared to that of paired adjacent normal tissues(P<0.000 1).(2)There were no statistical differences of the LINC00520 expression level among supraglottic,glottic and subglottic LSCCs(P>0.05).The LINC00520 expression level had no significant changes in poorly differentiated LSCC compared with that of well and moderately differentiated counterparts(P>0.05).Moreover,the expression of LINC00520 had no significant difference between T1+T2 stage and T3+T4 stage LSCC tissues(P>0.05).Interestingly,the LINC00520 level in LSCC with lymph node metastasis was significantly higher than that in patients without lymph node metastasis(P<0.01).Conclusion:Upregulation of LINC00520 in LSCC may contribute to its metastasis.

Integration of small RNAs, degradome and transcriptome sequencing in hyperaccumulator Sedum alfredii uncovers a complex regulatory network and provides insights into cadmium phytoremediation.

The hyperaccumulating ecotype of Sedum alfredii Hance is a cadmium (Cd)/zinc/lead co-hyperaccumulating species of Crassulaceae. It is a promising phytoremediation candidate accumulating substantial heavy metal ions without obvious signs of poisoning. However, few studies have focused on the regulatory roles of miRNAs and their targets in the hyperaccumulating ecotype of S. alfredii. Here, we combined analyses of the transcriptomics, sRNAs and the degradome to generate a comprehensive resource focused on identifying key regulatory miRNA-target circuits under Cd stress. A total of 87 721 unigenes and 356 miRNAs were identified by deep sequencing, and 79 miRNAs were differentially expressed under Cd stress. Furthermore, 754 target genes of 194 miRNAs were validated by degradome sequencing. A gene ontology (GO) enrichment analysis of differential miRNA targets revealed that auxin, redox-related secondary metabolism and metal transport pathways responded to Cd stress. An integrated analysis uncovered 39 pairs of miRNA targets that displayed negatively correlated expression profiles. Ten miRNA-target pairs also exhibited negative correlations according to a real-time quantitative PCR analysis. Moreover, a coexpression regulatory network was constructed based on profiles of differentially expressed genes. Two hub genes, ARF4 (auxin response factor 4) and AAP3 (amino acid permease 3), which might play central roles in the regulation of Cd-responsive genes, were uncovered. These results suggest that comprehensive analyses of the transcriptomics, sRNAs and the degradome provided a useful platform for investigating Cd hyperaccumulation in S. alfredii, and may provide new insights into the genetic engineering of phytoremediation.

Preparation and characterization of pro-angiogenic gel derived from small intestinal submucosa.

Gels derived from decellularized small intestinal submucosa (SIS) have been used to repair ischemic myocardium and deliver protein drug. However, their material properties and effects on cell behavior are not well understood, in part because of the difficulty of gelling in vitro. In this study, soluble SIS matrix, which was easily handled and could effectively gel, was successfully prepared using a modified method. Fourier transform infrared spectroscopy confirmed that the SIS gel contained not only collagen but also sulfated glycosaminoglycans (sGAGs). Interestingly, the sustained release of vascular endothelial growth factor and basic fibroblast growth factor within the SIS gel was detected, and no initial burst release was observed. The SIS gel was more capable of evoking neovascularization than collagen type I gel, as determined by tube formation experiments in human umbilical vein endothelial cells, the mouse aortic ring assay, and animal experiments. The upregulated expression of kinase insert domain receptor (KDR), Notch1, and Ang2, the key genes in angiogenesis that were evaluated in HUVECs seeded on the SIS gel, confirmed that angiogenesis bioactive factors contained in the SIS gel are indeed active and effective. The SIS gel significantly promoted neovascularization compared to the collagen type I gel in vivo. Histology revealed adequate host tissue response in engraftment both types of gels. Together, these data demonstrate that the SIS gel is a promising and attractive candidate for tissue engineering, especially in promoting vessel formation. STATEMENT OF SIGNIFICANCE: The material properties of small intestinal submucosa (SIS) gel and the effect of these properties upon cell behavior are not well understood, in part due to the difficulty of gelling in vitro. In this study, soluble SIS matrix, which was easily handled and gelled was prepared using modified method. The material properties and biocompatibility of SIS gel were explored. The sustained release of growth factors from this gel was observed along with its degradation in vitro. The results demonstrate that the SIS gel promote angiogenesis in vitro and in vivo. The SIS gel biological properties suggest that the constituent ECM molecules released from the gel remain activity. These findings suggested that the SIS gel was a promising candidate for tissue engineering, especially in promoting vessel formation.

Desensitization of G-protein-coupled receptors induces vascular hypocontractility in response to norepinephrine in the mesenteric arteries of cirrhotic patients and rats.

BACKGROUND: The increased beta-arrestin-2 and its combination with G-protein-coupled receptors (GPCRs) lead to GPCRs desensitization. The latter may be responsible for decreased contractile reactivity in the mesenteric arteries of cirrhotic patients and rats. The present study is to investigate the machinery changes of alpha-adrenergic receptors and G proteins and their roles in the contractility of mesenteric arteries of cirrhotic patients and animal models. METHODS: Patients with cirrhosis due to hepatitis B and cirrhotic rats induced by CCl4 were studied. Mesenteric artery contractility in response to norepinephrine was determined by a vessel perfusion system. The contractile effect of G protein-coupled receptor kinase-2 (GRK-2) inhibitor on the mesenteric artery was evaluated. The protein expression of the alpha1 adrenergic receptor, G proteins, beta-arrestin-2, GRK-2 as well as the activity of Rho associated coiled-coil forming protein kinase-1 (ROCK-1) were measured by Western blot. In addition, the interaction of alpha1 adrenergic receptor with beta-arrestin-2 was assessed by co-immunoprecipitation. RESULTS: The portal vein pressure of cirrhotic patients and rats was significantly higher than that of controls. The dose-response curve to norepinephrine in mesenteric arteriole was shifted to the right, and EC50 was significantly increased in cirrhotic patients and rats. There were no significant differences in the expressions of the alpha1 adrenergic receptor and G proteins in the cirrhotic group compared with the controls. However, the protein expressions of GRK-2 and beta-arrestin-2 were significantly elevated in cirrhotic patients and rats compared with those of the controls. The interaction of the alpha1 adrenergic receptor and beta-arrestin-2 was significantly aggravated. This interaction was significantly reversed by GRK-2 inhibitor. Both the protein expression and activity of ROCK-1 were significantly decreased in the mesenteric artery in patients with cirrhosis compared with those of the controls, and this phenomenon was not shown in the cirrhotic rats. Norepinephrine significantly increased the activity of ROCK-1 in normal rats but not in cirrhotic ones. Norepinephrine significantly increased ROCK-1 activity in cirrhotic rats when GRK-2 inhibitor was used. CONCLUSIONS: beta-arrestin-2 expression and its interaction with GPCRs are significantly upregulated in the mesenteric arteries in patients and rats with cirrhosis. These upregulations result in GPCR desensitization, G-protein dysfunction and ROCK inhibition. These may explain the decreased contractility of the mesenteric artery in response to vasoconstrictors.