Givinostat inhibition of hepatic stellate cell proliferation and protein acetylation

AIM: To explore the effect of the histone deacetylase inhibitor givinostat on proteins related to regulation of hepatic stellate cell proliferation.METHODS: The cell counting kit-8 assay and flow cytometry were used to observe changes in proliferation, apoptosis, and cell cycle in hepatic stellate cells treated with givinostat. Western blot was used to observe expression changes in p21, p57, CDK4, CDK6, cyclin D1, caspase-3, and caspase-9 in hepatic stellate cells exposed to givinostat. The scratch assay was used to analyze the effect of givinostat on cell migration. Effects of givinostat on the reactive oxygen species profile, mitochondrial membrane potential, and mitochondrial permeability transition pore opening in JS-1 cells were observed by laser confocal microscopy.RESULTS: Givinostat significantly inhibited JS-1 cell proliferation and promoted cell apoptosis, leading to cell cycle arrest in G0/G1 phases. Treatment with givinostat downregulated protein expression of CDK4, CDK6, and cyclin D1, whereas expression of p21 and p57 was significantly increased. The givinostat-induced apoptosis of hepatic stellate cells was mainly mediatedthrough p38 and extracellular signal-regulated kinase 1/2. Givinostat treatment increased intracellular reactive oxygen species production, decreased mitochondrial membrane potential, and promoted mitochondrial permeability transition pore opening. Acetylation of superoxide dismutase(acetyl K68) and nuclear factor-κB p65(acetyl K310) was upregulated, while there was no change in protein expression. Moreover, the notable beneficial effect of givinostat on liver fibrosis was also confirmed in the mouse models.CONCLUSION: Givinostat has antifibrotic activities via regulating the acetylation of nuclear factor-κB and superoxide dismutase 2, thus inhibiting hepatic stellate cell proliferation and inducing apoptosis.

Effects of IκBα and its mutants on NF-κB and p53 signaling pathways

AIM： To study the effects of IκBα and its mutants （IκBαM, IκBα3N, IκBαM44C） on NF-κB, p53 and their downstream target genes. The relationship of NF-κB, p53, and IκBα was further discussed. METHODS： pECFP-IκBα, pECFP-IκBαM （amino acides 1-317, Ser32, 36A）, pECFP-IκBα243N （amino acides 1-243）, pECFP-IκBα244C （amino acides 24±317）, pEYFP-p65 and pp53-DsRed were constructed and transfected to ASTC-α-1 cells. Cells were transfected with pECFP-Cl as a control. 30 h after the transfection, location patterns of NF-κB, p53 and IκBα（IκBαM, IκBα243N, IκBα224C） were observed by a laser scanning microscope （LSM510/ConfoCor2, Zeiss）. RNA extraction and reverse transcription were performed in cells transfected or co-transfected with different plasmids. Effects of IκBα and its mutants on the transprition level of NF-κB, NF-κB downstream target gene TNF-α, p53 and p53 downstream target gene Bax were observed by real time QT-PCR. In all experiments β-actin was reference. Results are expressed as the target/reference ratio of the sample divided by the target/reference ratio of the control. Different transfected cells were incubated with CCK-8 for 2 h in the incubator. Then the absorbance at 450 nm was measured by using a microplate reader. RESULTS： Cells that were transfected with p53- DsRed revealed a predominant nuclear localization. YFP-p65 mainly existed in the cytoplasm. Cells were transfected with CFP-IκBα, CFP-IκBαM, and CFP-IκBα243N respectively and revealed a predominant cytosolic localization. However, cells transfected of CFP-IκBα244C revealed a predominant nuclear localization. The rnRNA levels of p65, TNF-α, p53 and Bax in CFP-IκBα transfected cells did not change significantly, while in YFP-p65/CFP-IκBα co-transfected cells, IκBα decreased the transcription of p65 downstream gene TNF-α （2.24 ± 0.503） compared with the YFP-p65/ CFP-C1 co-transfected cells （5.08 ± 0.891） （P 〈 0.05）. Phosphorylation defective IκBα （IκBαM） decreased the transcription levels of all the four genes compared with the control （P 〈 0.05）. The N terminus of IκBα（IκBα243N） increased the transcription of NF-κB （1.84 ± 0.176） and TNF-α （1.51 ± 0.203） a little bit. However, the C terminus of IκBα（IκBα244C） increased the transcription of NF-κB, TNF-α, p53 and Bax significantly （8.29 ± 1.662, 14.16 ± 2.121, 10.2 ± 0.621, 3.72 ± 0.346） （P 〈 0.05）. The CCK-8 experiment also showed that IκBα244C and p53 synergistically mediate apoptosis. CONCLUSIONS： IκBα and its mutants （IκBαM, IκBα243N, IκBαM244C） have different effects on NF- KB and p53 signaling pathways, according to their different structures. IκBαbounds with NF-KB and p53 in cytoplasm steadily, and inhibits both of the two signaling pathways, p53 and IκBα244C may be co-factor in inducing apoptosis. The C terminal of IκBαnhanced cell death, which suggests that it may be a pro-apoptotic protein existed in cells.

(5R)-5-hydroxytriptolide inhibits the inflammatory cascade reaction in astrocytes

Many studies have shown that(5R)-5-hydroxytriptolide is the optimal modified analogue of triptolide, possessing comparable immunosuppressive activity but much lower cytotoxicity than triptolide. Whether(5R)-5-hydroxytriptolide has preventive effects on neuroinflammation is unclear. This study was designed to pretreat primary astrocytes from the brains of neonatal Sprague-Dawley rats with 20, 100 and 500 nM(5R)-5-hydroxytriptolide for 1 hour before establishing an in vitro neuroinflammation model with 1.0 μg/mL lipopolysaccharide for 24 hours. The generation of nitric oxide was detected by Griess reagents. Astrocyte marker glial fibrillary acidic protein was measured by immunohistochemical staining. The levels of tumor necrosis factor-α and interleukin-1β in the culture supernatant were assayed by enzyme linked immunosorbent assay. Nuclear factor-κB/p65 expression was examined by immunofluorescence staining. The phosphorylation of inhibitor of nuclear factor IκB-α and the location of nuclear factor-κB/P65 were determined using western blot assay. Our data revealed that(5R)-5-hydroxytriptolide inhibited the generation of nitric oxide, tumor necrosis factor-α and interleukin-1β from primary astrocytes activated by lipopolysaccharide, decreased the positive reaction intensity of glial fibrillary acidic protein, reduced the expression of tumor necrosis factor alpha and interleukin-1β in culture supernatant, inhibited the phosphorylation of IκB-α and the translocation of nuclear factor-κB/P65 to the nucleus. These results have confirmed that(5R)-5-hydroxytriptolide inhibits lipopolysaccharide-induced glial inflammatory response and provides cytological experimental data for(5R)-5-hydroxytriptolide in the treatment of neurodegenerative diseases.

孕激素在子痫前期患者TLR4-MyD88依赖的信号通路中的作用

目的研究孕激素在子痫前期(preeclampsia,PE)孕妇Toll样受体4(toll-like receptor4,TLR4)-髓样细胞分化蛋白88(myeloid differentiation factor 88,My D88)依赖的信号通路中的作用。方法原代培养子痫前期孕妇外周血单个核细胞(peripheral blood mononuclear cell,PBMC),以不同浓度孕激素(0 mol/L、10-8mol/L、10-6mol/L、10-4mol/L)处理,实时定量聚合酶联反应(Real-time polymerase Chain reaction,real-time PCR)检测TLR4 mRNA、My D88 mRNA、核转录因子-κB(nuclear factorκB,NF-κB)mRNA的表达;免疫印迹法Western blot检测IκB-α蛋白的表达,用凝胶图像处理系统分析各条目标带灰度值酶联免疫吸附试验(Enzyme-linked immunosorbent assay,ELISA)检测细胞上清液中肿瘤坏死因子-α(tumor necrosis factors,TNF-α)及白介素-6(interleukin-6,IL-6)的表达。结果随着孕酮浓度的增加,各组TLR4 mRNA、My D88mRNA及NF-κB mRNA的相对表达量2-△△Ct均逐渐降低,IκB-α蛋白的表达逐渐增加,差异均有统计学意义(均P<0.05);同时,细胞上清液中细胞因子TNF-α及IL-6的表达均逐渐减少,差异均有统计学意义(P<0.05)。结论孕激素可显著抑制TLR4-My D88依赖的信号传导通路,对子痫前期患者具有保护作用。