ABSTRACT
BACKGROUND@#Sarcoplasmic reticulum calcium ATPase 2a (SERCA2a) is a key protein that maintains myocardial Ca 2+ homeostasis. The present study aimed to investigate the mechanism underlying the SERCA2a-SUMOylation (small ubiquitin-like modifier) process after ischemia/reperfusion injury (I/RI) in vitro and in vivo .@*METHODS@#Calcium transient and systolic/diastolic function of cardiomyocytes isolated from Serca2a knockout (KO) and wild-type mice with I/RI were compared. SUMO-relevant protein expression and localization were detected by quantitative real-time PCR (RT-qPCR), Western blotting, and immunofluorescence in vitro and in vivo . Serca2a-SUMOylation, infarct size, and cardiac function of Senp1 or Senp2 overexpressed/suppressed adenovirus infected cardiomyocytes, were detected by immunoprecipitation, triphenyltetrazolium chloride (TTC)-Evans blue staining, and echocardiography respectively.@*RESULTS@#The results showed that the changes of Fura-2 fluorescence intensity and contraction amplitude of cardiomyocytes decreased in the I/RI groups and were further reduced in the Serca2a KO + I/RI groups. Senp1 and Senp2 messenger ribose nucleic acid (mRNA) and protein expression levels in vivo and in cardiomyocytes were highest at 6 h and declined at 12 h after I/RI. However, the highest levels in HL-1 cells were recorded at 12 h. Senp2 expression increased in the cytoplasm, unlike that of Senp1. Inhibition of Senp2 protein reversed the I/RI-induced Serca2a-SUMOylation decline, reduced the infarction area, and improved cardiac function, while inhibition of Senp1 protein could not restore the above indicators.@*CONCLUSION@#I/RI activated Senp1 and Senp2 protein expression, which promoted Serca2a-deSUMOylation, while inhibition of Senp2 expression reversed Serca2a-SUMOylation and improved cardiac function.
Subject(s)
Animals , Mice , Calcium/metabolism , Cysteine Endopeptidases/metabolism , Myocardial Reperfusion Injury/metabolism , Myocardium/metabolism , Myocytes, Cardiac/metabolism , Proteins/metabolism , Sarcoplasmic Reticulum Calcium-Transporting ATPases/geneticsABSTRACT
Objective:To investigate the effects of histone deacetylase 3 (HDAC3) on the pyroptosis of breast cancer (BC) cells via regulating miR-625/anti-silencing function 1B (ASF1B) and its mechanism.Methods:The expression level of HDAC3, miR-625 and ASF1B in BC tissue, adjacent normal tissue, BC cell lines (T47D, MCF7 and MDA-MB-231) and human normal breast epithelial cell MCF-10A was detected by qRT-PCR. The expression level of cell pyroptosis related protein NLRP3, Caspase-1 and GSDMD was detected by Western blot. The expression level of IL-18 and IL-1βwere detected by ELISA. ChIP experiment was used to determine the interaction between HDAC3 and miR-625. The dual luciferase reporter assay was used to verifiy the targeted regulation between miR-625 and ASF1B.Results:Compared with adjacent normal tissue and MCF-10A cells, the expression of HDAC3 and ASF1B was increased and the expression of miR-625 was decreased in BC tissue and cells (all P<0.05) . Compared with si-NC group, the protein expression level of NLRP3, Caspase-1 and GSDMD in si-HDAC3 group was increased, and the concentration of IL-18 and IL-1β in cell culture supernatant was increased (all P<0.05) . HDAC3 inhibited the expression of miR-625 by binding to the promoter region of miR-625 ( P<0.05) . Compared with si-HDAC3+miR-NC group, The expression of NLRP3, Caspase-1, GSDMD, IL-18 and IL-1β in si-HDAC3+miR-625 inhibitor group was decreased (all P<0.05) . ASF1B was confirmed as a target gene of miR-625, the level of pyroptosis related factors in si-HDAC3+pcDNA3.1-ASF1B group was significantly lower than that in si-HDAC3 + pcDNA3.1-NC group. Conclusion:HDAC3 up regulates the expression of ASF1B by inhibiting miR-625, and then inhibits BC cell pyroptosis.
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Objective To investigate the effect of low-dose arsenious acid solution (As(III)) combined with total particulate matter (TPM) from cigarette smoke on cellular oxidative stress in human lung cancer cell line A549 cells. Methods A549 cells were divided into four groups: negative control group (0.75% DMSO), low dose As(III) group (0.88% μg/mL, 75% DMSO), cigarette smoke TPM group (75 μg/mL), and combined exposure group (75 μg/mL TPM, 0.88 μg/mL As (III)). After 24 hours' exposure, the superoxide dismutase (SOD) level in cell culture medium and intracellular 8-hydroxy-2-deoxyguanosine (8-OHdG) content were detected by ELISA, and intracellular reactive oxygen species (ROS) level was detected by fluorescent probe DCFH-DA. 2×2 factorial design was used to evaluate the interaction. Results Compared with the control group, the level of SOD in the combined exposure group was significantly increased (P<0.05). In addition, the ROS content in the combined exposure group and TPM alone group was significantly increased (P<0.05). The levels of 8-OHdG in the combined exposure group and low-dose As(III) treated group were significantly higher than those in the control group(P<0.05). The results of the factorial analysis showed that low-dose As(III) and TPM had interaction on SOD levels, ROS and 8-OHdG contents in A549 cells. The effects on SOD and ROS were synergistic, while the effect on 8-OHdG was antagonistic. Conclusion Low-dose arsenious acid solution As(III) and TPM in cigarette smoke have interaction on oxidative stress in A549 cells.
ABSTRACT
In order to understand the current development of cell transformation assay (CTA) and its application in the evaluation of cigarette smoke carcinogenesis, the relevant literatures were analyzed and combed from these two aspects. CTA can evaluate the carcinogenicity of various genotoxic and non-genotoxic carcinogens in a short period of time, and has a strong consistency with the results of animal carcinogenic test. After malignant transformation, the cells show changes in cell morphology, immortalization of cells, disappearance of cell-cell contact inhibition, and the ability to form tumors when injected into animals. The identification methods of transformed cells include transformed cell focus count, agglutination test, soft agar culture and inoculation of nude mice, etc. At present, BALB/c 3T3 cells, Bhas 42 cells and SHE cells are the most widely used cells for CTA. Cigarette smoke is a complex aerosol containing a variety of non-genetic carcinogenic chemicals. Cell transformation tests are often used as an in vitro alternative method to evaluate the carcinogenic effects of cigarette smoke, which is different from the short-term genetic toxicity test. It simulates the long-term state of human smoking induced malignant transformation of cells, through the long-term exposure of cells for several decades, which is closer to the occurrence of cancer caused by human smoking. Therefore, CTA can evaluate the carcinogenicity of cigarette smoke and other tobacco products.
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Objective Establishing a fingerprint method to identify the characteristic chemicals in the roots of Gentiana macrophylla and evaluate their quality.Methods RP-HPLC was developed for fingerprint analysis and determination of four ingredients in G macrophylla roots from different sources.LC-ESI-TOF-MS was employed to identify the chromatographic peaks of the fingerprint.Results Five common peaks were identified by comparing their retention time with reference secoiridoid glucosides.Eight major peaks in chromatographic fingerprint were analyzed by on-line LC-ESI-TOF-MS.Four secoiridoid glucosides were identified based on their MS data.Conclusion The method is specific and could be served for the quality identification and comprehensive evaluation of G macrophylla.