The silent information regulator protein 2 (Sir2) and its homologues play an important role in the regulation of cellular physiological processes such as survival, apoptosis, and aging. SIRT1, the mammalian Sir 2 homologue, has been shown to deacetylate a wide range of non-histone substrates and histone substrates. It has been constantly reported that SIRT1 may be associated with the occurrence of metabolic syndrome, genomic homeostasis, tumors, and neurodegenerative diseases. Calorie restriction may mitigate many major diseases in rodent models by SIRT1-mediated deacetylase activity and prolong the life expectancies in these animals. Therefore, SIRT1 may be emphasized as a new therapy target for many different diseases.
Animals ; Caloric Restriction ; Humans ; Longevity ; Sirtuin 1 ; genetics ; physiology ; Substrate Specificity

OBJECTIVETo investigate the role of lysine-specific demethylase 1 (LSD1) in the process of THP-1 monocyte-to-macrophage differentiation.

METHODSQuantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blotting were performed to analyze the expression of LSD1 and interleukin-6 (IL-6) in THP-1 monocytes and THP-1-derived macrophages. Chromatin immunoprecipitation (ChIP) assay was applied to detect the occupancy of LSD1 and H3K4 methylation at IL-6 promoter during THP-1 monocyte-to-macrophage differentiation. IL-6 mRNA level and H3K4 methylation at IL-6 promoter were analyzed using qRT-PCR and ChIP assay in LSD1-knockdown THP-1 cells treated with 12-O-tetradecanoylphorbol-13-acetate (TPA) for 0, 4, 8, 12, and 24 hours. Fluorescence activated flow cytometry was performed to reveal the percentage of macrophages differentiated from THP-1 monocytes.

RESULTSThe expression of LSD1 reduced during THP-1 monocyte-to-macrophage differentiation (P<0.01). LSD1 occupancy decreased and H3K4 methylation increased at IL-6 promoter during the differentiation. With knockdown of LSD1, H3K4 methylation at IL-6 promoter was found increased after TPA treatment at different times points (all P<0.05, except 24 hours). The percentage of macrophages increased significantly in the THP-1 cells with LSD1 knockdown (P<0.05).

CONCLUSIONSLSD1 is repressed during the monocyte-to-macrophage differentiation of THP-1 cells. Suppression of LSD1-mediated H3K4 demethylation may be required for THP-1 monocyte-to-macrophage differentiation.

Cell Differentiation ; Cells, Cultured ; Dealkylation ; Histone Demethylases ; physiology ; Histones ; metabolism ; Humans ; Interleukin-6 ; genetics ; Macrophages ; cytology ; Monocytes ; cytology ; Promoter Regions, Genetic

OBJECTIVETo verify the regulation of acyl-coenzyme A:cholesterol acyltransferase 2 (ACAT 2), which is associated with cholesterol metabolism, by saturated fatty acids (SFAs).

METHODSPalmitic acid (PA), the most abundant saturated fatty acid in plasma, and oleic acid (OA), a widely distributed unsaturated fatty acid, were used to treat hepatic cells HepG2, HuH7, and mouse primary hepatocytes. In addition, PA at different concentrations and PA treatment at different durations were applied in HepG2 cells. In in vivo experiment, three-month male C57/BL6 mice were fed with control diet and SFA diet containing hydrogenated coconut oil rich of SFAs. The mRNA level of ACAT2 in those hepatic cells and the mouse livers was detected with real-time polymerase chain reaction (PCR).

RESULTSIn the three types of hepatic cells treated with PA, that SFA induced significant increase of ACAT2 expression (Pü0.01), whereas treatment with OA showed no significant effect. That effect of PA was noticed gradually rising along with the increase of PA concentration and the extension of PA treatment duration (both Pü0.05). SFA diet feeding in mice resulted in a short-term and transient increase of ACAT2 expression in vivo, with a peak level appearing in the mice fed with SFA diet for two days (Pü0.05).

CONCLUSIONSFA may regulate ACAT2 expression in human and mouse hepatic cells and in mouse livers.

Animals ; Base Sequence ; Cell Line, Tumor ; DNA Primers ; Dose-Response Relationship, Drug ; Fatty Acids ; pharmacology ; Humans ; Liver ; enzymology ; Male ; Mice ; Mice, Inbred C57BL ; Sterol O-Acyltransferase ; metabolism

OBJECTIVETo study the role of sirtuin 1 (SIRT1) in Fas ligand (FasL) expression regulation during vascular lesion formation and to elucidate the potential mechanisms.

METHODSSIRT1 and FasL protein levels were detected by Western blotting in either mouse arteries extract or the whole rat aortic vascular smooth muscle cell (VSMC) lysate. Smooth muscle cell (SMC)-specific human SIRT1 transgenic (Tg) C57BL/6 mice and their littermate wild-type (WT) controls underwent complete carotid artery ligation (ligation groups) or the ligation-excluded operation (sham groups). The carotid arteries were collected 1 day after operation. Reverse transcription-polymerase chain reaction was performed to detect the mRNA levels of SIRT1 and FasL. Luciferase reporter assays were performed to detect the effect of WT-SIRT1, a dominant-negative form of SIRT1 (SIRT1H363Y), and GATA-6 on the promoter activity of FasL. Flow cytometry assay was applied to measure the hypodiploid DNA content of VSMC so as to monitor cellular apoptosis.

RESULTSSIRT1 was expressed in both rat aortic VSMCs and mouse arteries. Forced SIRT1 expression increased FasL expression both in injured mouse carotid arteries 1 day after ligation (P<0.001) and VSMCs treated with serum (P<0.05 at the transcriptional level, P<0.001 at the protein level). No notable apoptosis was observed. Furthermore, transcription factor GATA-6 increased the promoter activity of FasL (P<0.001). The induction of FasL promoter activity by GATA-6 was enhanced by WT-SIRT1 (P<0.001), while SIRT1H363Y significantly relieved the enhancing effect of WT-SIRT1 on GATA-6 (P<0.001).

CONCLUSIONSOverexpression of SIRT1 up-regulates FasL expression in both flow-restricted mouse carotid arteries and serum-stimulated VSMCs. The transcription factor GATA-6 participates in the transcriptional regulation of FasL expression by SIRT1.

Animals ; Apoptosis ; Carotid Arteries ; physiology ; Fas Ligand Protein ; genetics ; GATA6 Transcription Factor ; physiology ; Male ; Muscle, Smooth, Vascular ; cytology ; metabolism ; Myocytes, Smooth Muscle ; metabolism ; RNA, Messenger ; analysis ; Rats ; Rats, Sprague-Dawley ; Sirtuin 1 ; physiology ; Up-Regulation

Objective To explore the role of cytidine/uridine monophosphate kinase 2( CMPK2) in the immune-mediated antitumor effect of IFNα in hepatocellular carcinoma. Methods RT-qPCR and Western blot were used to analyze the expression of CMPK2 in Huh7 after the treatment of IFNα. The CMPK2 overexpressing Huh7 cells were generated by stably infecting with lentivirus. The ATP level in the cells and the supernatant of CMPK2 overexpress-ing Huh7 cells were measured by CellTiter-Glo ATP fluorescence assay. RT-qPCR was applied to test the expression of inflammatory cytokines in macrophages under the treatment of the supernatant of CMPK2 overexpress-ing Huh7 cells. Results The transcription and protein level of CMPK2 were significantly enhanced after the treat-ment of IFNα for 6 hours ( P＜0.01) . CMPK2 increased the ATP level in the cells and supernatant of Huh7 cells ( P＜0.01) . The supernatant of CMPK2 overexpressing Huh7 cells activated the expression of IL1β, IL6 and CCL5 in macrophages( P＜0.01) . Conclusions IFNα increases the expression of CMPK2 in Huh7 cells to activate the expression of inflammatory cytokines in macrophages.

Our previous study has shown that p66^Shc plays an important role in the process of myocardial regeneration in newborn mice, and p66^Shc deficiency leads to weakened myocardial regeneration in newborn mice. This study aims to explore the role of p66^Shc protein in myocardial injury repair after myocardial infarction in adult mice, in order to provide a new target for the treatment of myocardial injury after myocardial infarction. Mouse myocardial infarction models of adult wild-type (WT) and p66^Shc knockout (KO) were constructed by anterior descending branch ligation. The survival rate and heart-to-body weight ratio of two models were compared and analyzed. Masson's staining was used to identify scar area of injured myocardial tissue, and myocyte area was determined by wheat germ agglutinin (WGA) staining. TUNEL staining was used to detect the cardiomyocyte apoptosis. The protein expression of brain natriuretic peptide (BNP), a common marker of myocardial hypertrophy, was detected by Western blotting. The results showed that there was no significant difference in survival rate, myocardial scar area, myocyte apoptosis, and heart weight to body weight ratio between the WT and p66^ShcKO mice after myocardial infarction surgery. Whereas the protein expression level of BNP in the p66^ShcKO mice was significantly down-regulated compared with that in the WT mice. These results suggest that, unlike in neonatal mice, the deletion of p66^Shc has no significant effect on myocardial injury repair after myocardial infarction in adult mice.
Animals ; Mice ; Body Weight ; Cicatrix/metabolism* ; Mice, Knockout ; Myocardial Infarction/genetics* ; Oxidative Stress ; Shc Signaling Adaptor Proteins/metabolism* ; Src Homology 2 Domain-Containing, Transforming Protein 1/metabolism*

Objective Angiotensin Ⅱ (Ang Ⅱ)-induced vascular damage is a major risk of hypertension. However, the underlying molecular mechanism of AngⅡ-induced vascular damage is still unclear. In this study, we explored the novel mechanism associated with Ang II-induced hypertension. Methods We treated 8- to 12-week-old C57BL/6J male mice with saline and Ang Ⅱ(0.72 mg/kg·d) for 28 days, respectively. Then the RNA of the media from the collected mice aortas was extracted for transcriptome sequencing. Principal component analysis was applied to show a clear separation of different samples and the distribution of differentially expressed genes was manifested by Volcano plot. Functional annotations including Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway were performed to reveal the molecular mechanism of Ang Ⅱ-induced hypertension. Finally, the differentially expressed genes were validated by using quantitative real-time PCR. Results The result revealed that a total of 773 genes, including 599 up-regulated genes and 174 down-regulated genes, were differentially expressed in the aorta of Ang Ⅱ-induced hypertension mice model. Functional analysis of differentially expressed genes manifested that various cellular processes may be involved in the Ang Ⅱ-induced hypertension, including some pathways associated with hypertension such as extracellular matrix, inflammation and immune response. Interestingly, we also found that the differentially expressed genes were enriched in vascular aging pathway, and further validated that the expression levels of insulin-like growth factor 1 and adiponectin were significantly increased (P<0.05). Conclusion We identify that vascular aging is involved in Ang Ⅱ-induced hypertension, and insulin-like growth factor 1 and adiponectin may be important candidate genes leading to vascular aging.
Aging ; Angiotensin II ; Animals ; Aorta/physiopathology* ; Blood Pressure/genetics* ; Gene Expression Profiling/methods* ; Gene Ontology ; Hypertension/genetics* ; Male ; Mice, Inbred C57BL ; Reverse Transcriptase Polymerase Chain Reaction