Circular RNA circRSF1 binds to HuR to promote radiation-induced inflammatory phenotype in hepatic stellate cells / 南方医科大学学报

OBJECTIVE@#To investigate whether circular RNA circRSF1 regulates radiation-induced inflammatory phenotype of hepatic stellate cells (HSCs) by binding to HuR protein and repressing its function.@*METHODS@#Human HSC cell line LX2 with HuR overexpression or knockdown was exposed to 8 Gy X-ray irradiation, and the changes in the expression of inflammatory factors (IL-1β, IL-6 and TNF-α) were detected by qRT-PCR. The expressions of IκBα and phosphorylation of NF-κB were detected with Western blotting. The binding of circRSF1 to HuR was verified by RNA pull-down assay and RNA-binding protein immunoprecipitation (RIP). The expressions of inflammatory factors, IκBα and the phosphorylation of NF-κB were detected after modifying the interaction between circRSF1 and HuR.@*RESULTS@#Knockdown of HuR significantly up- regulated the expressions of IL-1β, IL-6 and TNF-α, decreased IκBα expression and promoted NF-κB phosphorylation in irradiated LX2 cells, whereas overexpression of HuR produced the opposite changes (P < 0.05). Overexpression or knockdown of circRSF1 did not significantly affect the expression of HuR. RNA pull-down and RIP experiments confirmed the binding between circRSF1 and HuR. Overexpression of circRSF1 significantly reduced the binding of HuR to IκBα and down-regulated the expression of IκBα (P < 0.05). Overexpression of circRSF1 combined with HuR overexpression partially reversed the up-regulation of the inflammatory factors, down-regulated IκBα expression and increased phosphorylation of NFκB in LX2 cells, while the opposite effects were observed in cells with knockdown of both circRSF1 and HuR (P < 0.05).@*CONCLUSION@#circRSF1 reduces IκBα expression by binding to HuR to promote the activation of NF-κB pathway, thereby enhancing radiation- induced inflammatory phenotype of HSCs.

Evaluation of anti-tuberculosis effect of Mycobacterium tuberculosis polyphosphate kinase 2 aptamer / 上海交通大学学报（医学版）

Objective·To investigate the bacteriostasis effect of Mycobacterium tuberculosis (MTB) polyphosphate kinase 2 (PPK2) aptamer on MTB in vitro.Methods·The bioinformatics method was used to analyze the homology of MTB PPK2 and common pathogens of respiratory tract,and the PPK2 phylogenetic tree was constructed.The binding affinity of the PPK2 aptamer to H37Rv,BCG,Mycobacterium smegmatis,Pseudomonas aeruginosa and Acinetobacter baumannii was analyzed by enzyme-linked oligonucleotide assay (ELONA).The PPK2 aptamer was incubated for 24 h in serum and its biological stability in serum was analyzed by agarose gel electrophoresis.The minimum inhibitory concentration (MIC) of the PPK2 aptamer to H37Rv was determined by micro-azure method.H37Rv was inoculated with 1 μmol/L PPK2 aptamer or random sequence on Roche culture medium for 10 d and colony growth status was observed.H37Rv was co-cultured with different concentrations of PPK2 aptamer for 10 d,absorbance at 600 nm was measured by microplate reader.The effect of PPK2 aptamer on the growth of H37Rv was observed.Results·PPK2 phylogenetic tree constructed by bioinformatics analysis showed that PPK2 protein of H37Rv was not closely related to the common pathogens of respiratory tract,and it was relatively close to Pseudomonas aeruginosa.The ELONA assay results showed that the PPK2 aptamer binded selectively to H37Rv.Agarose gel electrophoresis analysis showed PPK2 aptamer in serum was at least stable for 8 h.The MIC of the PPK2 aptamer to H37Rv was 50 nmol/L.The colony growth of Roche culture showed that PPK2 aptamer had an inhibitory effect on H37Rv growth.Growth inhibition test showed that the absorbance at 600 nm of H37Rv showed a decreasing trend with the increase of PPK2 aptamer concentration,which indicated that PPK2 aptamer had an inhibitory effect on H37Rv growth.Conclusion·PPK2 aptamer has good antibacterial activity against H37Rv in vitro.

Effect of SAHA on Maturation of Dendritic Cells and Its Mechanism / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the effect of SAHA on the maturation of human dendritic cells (DC) and to explore its underlying mechanism.</p><p><b>METHODS</b>Peripheral blood mononuclear cells (PBMNC) were isolated from human peripheral blood and cultured in RPMI 1640 medium with 100 ng/ml rhGM-CSF and 500 U/ml rhIL-4. In the LPS induced maturation process, dendritic cells treated with or without SAHA were used as test group, and dendritic cells treated without LPS or SAHA were used as control group. DC was observed under inverted microscope. Flow cytometer was used to detect the surface antigen molecules expressed by DC. The mixed lymphocyte culture (MLC) was used to observe the allogeneic lymphocyte stimulation. The NF-κB signaling pathway was detected by electrophoretic mobility shift assay (EMSA).</p><p><b>RESULTS</b>The SAHA could effectively suppress the maturation of DC induced by LPS, the DC treated with SAHA+LPS had immature morphological characteristics; the expression of CD80, CD83 and HLA-DR in SAHA+LPS group and control group were significantly down-regulated as compared with single LPS group (P<0.01); the ability of DC to stimulate the proliferation of allogeneic T lymphocytes in SAHA+LPS group and control group was significantly weaker than that in single LPS group (P<0.01); EMSA results showed that NF-κB activity decreased after SAHA and LPS treatment and was significantly lower than that of single LPS group.</p><p><b>CONCLUSION</b>SAHA can effectively suppress DC maturation induced by LPS and also weaken the ability to stimulate allogeneic T lymphocyte. NF-κB signaling pathway is involved in regulating DC maturation.</p>

Interaction between hydrogen sulfide and nitric oxide on cardiac protection in rats with metabolic syndrome.

OBJECTIVE: To investigate the interaction between hydrogen sulfide (H2S)/cystathionine gamma-lyase (CSE) system and nitric oxide (NO)/nitric oxide synthase (NOS) system on cardiac protection in metabolic syndrome (MS) rats. METHODS: Forty one male Sprague-Dawley rats were randomly divided into 6 groups: control group, MS group, H2S donor group, CSE inhibitor group, NOS inhibitor group, and NO donor group. The MS rat model was established by a high-fat diet of 16 weeks. Rats in control and MS groups were subjected to normal saline and the other four groups were respectively subjected to sodium hydrosulfide (NaHS, 56 µmol/kg), D,L-propargylglycine (PPG, 37.5 mg/kg), Nψ-nitro-L-arginine methyl ester (L-NAME, 18 mg/kg), L-Arginine (500 mg/kg) every day. Four weeks later, the obesity indices, blood sugar of oral glucose tolerance test in each time point (0,30,60, and 120 minutes) and blood lipids (cholesterol, triglyceride, high density lipoprotein, low density lipoprotein) were measured. The computer-based electrophysiological recorder system was used to measure the changes of the left ventricular systolic pressure (LVSP), the left ventricular end diastolic pressure (LVEDP), the maximal rate of pressure increase in the contraction phase (+dP/dtmax), and the maximal rate of pressure decrease in the diastole phase (-dP/dtmax). H2S and NO concentration in plasma and myocardium, as well as CSE, constitutive NOS (cNOS), and inducible NOS (iNOS) activities in myocardium were measured with colorimetric method. Reverse transcription-polymerase chain reaction was used to assess the gene expression of CSE and endothelial NOS (eNOS) mRNAs. RESULTS: Compared with control group, the obesity indices, blood sugar at each time point, and blood lipids significantly increased in MS group (P<0.05). H2S and NO concentration in plasma and myocardium, CSE and cNOS activities in myocardium, the expressions of CSE mRNA and eNOS mRNA, and the myocardial function significantly decreased in MS group (P<0.05). Compared with MS group, NO concentration in plasma and myocardium, cNOS and iNOS activities in myocardium, and the expression of eNOS mRNA significantly increased in CSE inhibitor group (P<0.05). However, activities of cNOS and iNOS in myocardium and the expression of eNOS mRNA were significantly decreased in H2S donor group (P<0.01), while the myocardial function significantly increased (P<0.05). H2S concentration in plasma and myocardium, and the expression of CSE mRNA significantly increased in NOS inhibitor group (P<0.05). However, in NO donor group, the CSE activity in myocardium and the expression of CSE mRNA significantly decreased (P<0.05). And the myocardial function was improved significantly (P<0.05). CONCLUSIONS: Both the H2S/CSE and NO/NOS systems appear to have a mutual down-regulation effect on myocardium in MS rats. Meanwhile, exogenous H2S and NO supplement is cardioprotective in rat model of MS.