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【Objective】 To explore the differential expression and functional analysis of circRNA from myocardial mitochondria in diabetes cardiomyopathy (DCM) mice. 【Methods】 The DCM mice model was established in 16-week-old db/db mice, and C57BL/KsJ mice were used as controls. RNA was extracted from the myocardium of two groups of mice, high-throughput sequencing was used to screen mitochondrial circRNA differentially expressed in the two groups, RT-qPCR was used to verify the sequencing results of the first 10 circRNAs with significant differential expression, and functional enrichment analysis was performed on the differentially expressed circRNA target genes, and miRNA target prediction software was used to analyze the circRNA-miRNA co-expression network. 【Results】 There were 147 mitochondrial circRNAs differentially expressed in the myocardium of DCM mice, including 89 highly expressed and 58 low expressed. The expression pattern of differentially expressed circRNAs in tissues was consistent with those of sequencing results. The enrichment analysis of GO and KEGG showed that the differentially expressed circRNA target genes were mainly enriched in cGMP/PKG, glucagon pathways, which were related to mitochondrial energy metabolism and cardiac hypertrophy. circRNA-miRNA co-expression analysis found that the most significantly up-regulated circRNA, chrM:1207-1536+, was associated with miR-491-3p, miR-99a-3p, and miR-99b-3p, and the most significantly down-regulated circRNA, chrM:1453-3205+, was associated with miR-181b-1-3p, miR-181b-2-3p, and miR-672-5p. 【Conclusion】 Compared to the control mice, there is differential expression of circRNAs in myocardial mitochondria of DCM mice. The differentially expressed circRNAs may interact with the corresponding miRNA to affect myocardial fibrosis and hypertrophy through regulation of energy metabolism, apoptosis and other pathways, thus participating in the pathogenesis of DCM.
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OBJECTIVE@#To evaluate the effect of rosmarinic acid (RA) on mitophagy and hypertrophy of cardiomyocytes exposed to high glucose (HG).@*METHODS@#Rat cardiomyocytes (H9c2) exposed to HG (25 mmol/L) were treated with 50 μmol/L RA or with both RA treatment and Parkin siRNA transfection, with the cells cultured in normal glucose (5.5 mmol/L) and HG as the controls. The expressions of PINK1, Parkin and LC3II/LC3I in the cells were detected by Western blotting. The formation of mitochondrial autophagosomes was observed by transmission electron microscope. Flow cytometry was employed to detect the level of reactive oxygen species (ROS) and apoptotic rate of the cells. The activities of respiratory chain complex enzymes were measured by spectrophotometry. Fluorescence enzyme labeling and @*RESULTS@#RA treatment significantly increased the expression levels of PINK1, Parkin and LC3-II/I (@*CONCLUSIONS@#RA can protect rat cardiomyocytes against oxidative stress injury and cardiomyocyte hypertrophy induced by HG by activating Parkin-mediated mitophagy.
Subject(s)
Animals , Rats , Cinnamates , Depsides , Glucose , Hypertrophy , Mitophagy , Myocytes, Cardiac , Protein Kinases , Reactive Oxygen Species , Ubiquitin-Protein Ligases/geneticsABSTRACT
AIM: To explore the influence of Huqi San on the Hedgehog signaling pathway in rats with prehe-patocarcinoma.METHODS: The model of prehepatocarcinoma in the rats was established by a modified solt-farber method.The rats were intragastric administrated with Huqi San solution for 3 d after subtotal hepatectomy.Four weeks after administration of the Huqi San solution, the hepatic damage was observed by histopathological analysis.The protein expression of glutathione S-transferase-π (GST-π), alpha-fetoprotein (AFP), OV6, albumin (ALB) and glioma-associated oncogene homolog 2 (Gli2) was detected by immunohistochemistry and immunofluorescence staining.The expression of Sonic hedgehog (Shh), Smoothened (Smo), Gli2, cyclin D and cyclin E at mRNA and protein levels in the rats was determined by RT-qPCR and Western blot, respectively.The serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and gamma-glutamyltransferase (GGT) were assayed using diagnostic kits.RESULTS: Compared with model group, Huqi San decreased the serum levels of ALT, AST and GGT, and alleviated the pathological changes in prehepatocarcinoma rats.Huqi San inhibited the protein expression of GST-π and AFP (P<0.05) in the prehepatocarcinoma rats.Huqi San also promoted the protein expression of OV6 and ALB (P<0.05).Furthermore, Huqi San activated Hedgehog signaling pathway and its downstream targeting molecules such as Shh, Smo, Gli2, cyclin D and cyclin E.In addition, the results in vitro showed that Huqi San may activate Hedgehog signaling pathway and promoted oval cell proliferation.CONCLUSION: Huqi San not only promotes hepatic progenitor cell proliferation, but also induces hepatic progenitor cell differentiation and inhibits prehepatocarcinoma in the rats probably via activating Hedgehog signaling pathway.
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Objective To reveal the role of serum ACE2/Ang (1-7)in the occurrence of atrial fibrillation (AF)and find new targets for the prevention and treatment of AF by analyzing the correlation between the serum concentration of ACE2/Ang (1-7 )in patients with rheumatic valvular heart disease and the occurrence of AF. Methods We collected the basic clinical information and peripheral venous blood of patients with rheumatic heart valve disease (totally 46 patients,including 24 with AF and 22 with SR).ELISA method was used to detect the serum concentration of ACE2,Ang (1-7)and AngⅡ in the serum samples.Then the differences and correlation between the two groups were analyzed.Results In the AF group ① the diameter of the left atrium was significantly greater than that in the SR group [(60.70±3.08 vs.48.15±2.16)mm,P<0.05];② the serum concentration of AngⅡ was significantly higher than that in the SR group [(45.88±2.87 vs.35.78±1.08)pg/mL, P<0.05],AngⅡ and left atrium diameter were positively correlated (Pearson test,P<0.05);③ the serum concentrations of ACE2 [(7.87±0.74 vs.11.65±0.57)U/L,P<0.05]and Ang (1-7)[(146.05±17.61 vs. 321.71±36.50)pg/mL,P<0.05]were significantly lower than those in the SR group,and negatively correlated with left atrium diameter (Pearson test,P<0.05);④ the serum concentration of Ang (1-7)was negatively correlated with AngⅡ concentration (Pearson test,P<0.05).Conclusion For patients with rheumatic valvular heart disease,ACE2/Ang (1-7 )may play a protective role in the occurrence of AF via antagonizing AngⅡ and inhibiting atrial remodeling.
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AIM:To study the effects of extracellular potassium on the protein expression of wild-type HERG and its mutant L539fs/47.METHODS:Wild-type HERG (WT) or its mutant HERG-L539fs/47 (MT) were transfected into HEK293 cells for 36 h.The cells were incubated in different media containing 0.8, 4.3 or 10 mmol/L potassium.Af-ter 6 h of incubation, the protein expression of HERG was detected by flow cytometry.After 12 h of incubation, the locali-zation and quantity of the proteins were detected by laser confocal imaging and Western blot.RESULTS: Different from the retention of mutant protein in cytoplasm, wild-type HERG protein was mainly distributed in the cell membrane.The 2 proteins both increased with the changes of extracellular potassium.Flow cytometry showed that the fluorescence in the 2 groups both increased with the changes of extracellular potassium ( P<0.01 ) .The fluorescence in WT group was signifi-cantly higher than that in MT group (P<0.01).Western blot showed that mutant HERG protein included only one 60 kD band, different from the 135 kD and 155 kD bands in wild-type HERG, which were affected by the changes of extracellular potassium (P<0.05).CONCLUSION:The retention of HERG mutant L539fs/47 protein in the cytoplasm is more than wild-type HERG.Chronic high extracellular potassium keeps the stability of wild-type and mutant HERG proteins on the cell membrane.Chronic low potassium reduces the expression of HERG channel proteins in a time-dependent manner.
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To investigate the cytotoxicity of the homemade peptide cationic liposome CDO14 and its efficacy of RNA interference (RNAi). MTT method was used to determine the cytotoxicity of the liposome to a human lung cancer cell line Luc-A549 that can express luciferase stably. Luciferase siRNA (Luc-siRNA) was transfected into Luc-A549 cells by CDO14. Contents of luciferase in the transfected cells were detected by luminous instrument and contents of total protein in these cells were detected by BCA method. Nude mice were inoculated with Luc-A549 cells in axilla to establish xenograft tumor model. Complexes of Luc-siRNA and the cationic liposomes were injected into the modeling mice via tail vein. Contents of luciferase in the transfected mice were detected by the whole body imaging system. The cytotoxicity of the homemade cationic liposome was similar to that of commercial liposome DOTAP, and lower than that of Lipo2000. The siRNA transfection efficacy mediated by CDO14 was higher than that mediated by DOTAP. The homemade peptide cationic liposome CDO14 is expected to serve as delivery vector in gene therapy because of its low cytotoxicity and high transfection efficiency.