The surface antigen CD14 plays an important role in innate immunity, serving as a pattern recognition receptor for lipopolysaccharides (LPS). The aim of this study was to investigate the proliferation, NFκB activatio...The surface antigen CD14 plays an important role in innate immunity, serving as a pattern recognition receptor for lipopolysaccharides (LPS). The aim of this study was to investigate the proliferation, NFκB activation, and chemokine secretion of BEAS-2B cells, a human bronchial epithelial cell line, after LPS stimulation, and some details of inVolved signaling. The presence of CD14 was investigated by flow cytometry. Cell proliferation was measured with a [3H]-thymidine incorporation assay. sCD14, RANTES, and IL-8 concentrations in cell supernatants were measured by ELISA. BEAS-2B cells express CD14 on their surface and secrete soluble CD14 into the supernatant. Cells react on LPS with increased proliferation, activation of NFκB, and the secretion of the pro-inflammatory chemotactic cytokines IL-8 and RANTES, which proves the functionality of the CD14 receptor. Neither CD14 nor sCD14 are regulated by LPS. Specific inhibitors of various intracellular signaling pathways diminish the LPS-induced proliferation and IL-8 secretion: Thus MAP-Kinases p38 and JNK, tyrosine kinases, and PI3-kinase are involved in the signaling cascade from the LPS-CD14-complex on the cell surface to the increased cell proliferation and expression of IL-8;furthermore, ERK 1/2, IRAK 1/4, and the NFκB pathway are inVolved in the latter. The data show the existence and functionality of CD14 receptors on BEAS-2B cells and elucidate the signaling pathways inVolved. LPS is able to increase cell prolife-ration, various cytokines which are dependent on endogenous CD14. Three MAPK pathways, PI3 kinase and tyrosine kinase may be involved. Also CD14 is present/involved which was controversial.展开更多
目的:研究丁酸钠、曲古抑菌素A(trichostatin A,TSA)及腺病毒E1A相关的300 kD蛋白(adenoviral E1A binding protein of 300 kD,P300)介导的组蛋白乙酰化在人非小细胞肺癌A549细胞及人支气管上皮BEAS-2B细胞中对哮喘易感的解整合素金属...目的:研究丁酸钠、曲古抑菌素A(trichostatin A,TSA)及腺病毒E1A相关的300 kD蛋白(adenoviral E1A binding protein of 300 kD,P300)介导的组蛋白乙酰化在人非小细胞肺癌A549细胞及人支气管上皮BEAS-2B细胞中对哮喘易感的解整合素金属蛋白酶33(a disintegrin and metalloproteinase 33,ADAM33)基因表达的影响。方法:将A549细胞及BEAS-2B细胞分别分为丁酸钠对照组(双蒸水处理)和1、2.5、5 mmol/L丁酸钠组,TSA对照组(0.1%二甲基亚砜处理)和0.2、0.4、0.8μmol/L TSA组,按照组别分别予以相应处理;另将BEAS-2B细胞分为对照组(转染P300突变质粒)和P300组(转染P300表达质粒);采用双荧光素酶报告基因法分析ADAM33启动子活性的变化,实时荧光定量PCR(quantitative real time-PCR,qRT-PCR)检测ADAM33 mRNA表达,蛋白质印迹法检测ADAM33蛋白表达。结果:在人非小细胞肺癌A549细胞中,与对照组相比,1 mmol/L丁酸钠组及0.2μmol/L TSA组ADAM33基因启动子活性明显降低(P<0.01);在BEAS-2B细胞中,与对照组相比,1 mmol/L丁酸钠组及0.2μmol/L TSA组ADAM33基因启动子活性、mRNA及蛋白相对表达水平明显降低(P<0.05或P<0.01)。加大丁酸钠、TSA药物浓度,ADAM33表达无显著差异。在人支气管上皮BEAS-2B细胞中,与对照组相比,P300组ADAM33启动子活性、mRNA和蛋白相对表达水平明显降低(P<0.05)。结论:丁酸钠、TSA通过组蛋白乙酰化降低人非小细胞肺癌A549细胞和人支气管上皮BEAS-2B细胞中ADAM33表达,P300通过组蛋白乙酰化降低人支气管上皮BEAS-2B细胞中ADAM33表达。展开更多
Objective:To investigate the effects of stilbene glycoside(TSG)on okadaic acid-induced apoptosis in human neuroblastoma cells(SH-SY5Y)via the PI3K/AKT pathway.Methods:The optimal concentration of OA was screened by CC...Objective:To investigate the effects of stilbene glycoside(TSG)on okadaic acid-induced apoptosis in human neuroblastoma cells(SH-SY5Y)via the PI3K/AKT pathway.Methods:The optimal concentration of OA was screened by CCK-8 assay,and SH-SY5Y cells were divided into control group,model group,TSG group,LY294002 group and LY294002+TSG group.The proliferation and apoptosis in each group were detected by CCK-8 and TUNEL assays;Western blotting method and real-time fluorescence quantitative polymerase chain reaction was used to detect the expression of PI3K,P-PI3K(Y607),AKT,P-AKT(Ser473),Bcl-2 and Bax proteins.The relative protein expression was represented by P-PI3K(Y607)/PI3K,P-AKT(Ser473)/AKT and Bcl-2/Bax gray ratio.Results:CCK-8 screened the optimal concentration of OA as 40 nmol/L.Compared with the control group,the model group increased relative cell viability,decreased apoptosis rate,the pathway and apoptotic proteins expression levels of P-PI3K(Y607)/PI3K,P-AKT(Ser473)/AKT and Bcl-2/Bax were decreased,and the mRNA expression levels of PI3K,AKT and Bcl-2 were decreased.Bax mRNA expression level increased(P<0.05);Compared with model group,TSG group increased relative cell viability,decreased apoptosis rate,increased protein expression levels of P-PI3K(Y607)/PI3K,P-AKT(Ser473)/AKT,Bcl-2/Bax,and increased mRNA expression levels of PI3K,AKT,and Bcl-2.Bax mRNA expression decreased(P<0.05),LY294002 group decreased relative cell viability,increased apoptosis rate,P-PI3K(Y607)/PI3K protein expression levels were significantly decreased(P<0.05),P-AKT(Ser473)/AKT and Bcl-2/Bax protein expression levels were significantly decreased,but there was no statistical significance,PI3K,AKT and Bcl-2 mRNA expression levels were decreased,and Bax mRNA expression levels were increased(all P<0.05);Compared with LY294002 group,LY294002+TSG group increased relative cell viability,decreased apoptosis rate,and the protein expression levels of P-PI3K(Y607)/PI3K,P-AKT(Ser473)/AKT and Bcl-2/Bax were increased.The mRNA expression levels of PI3K,AKT,Bcl-2 were increased,Bax was decreased(all P<0.05).Conclusion:Stilbene glycoside may alleviate okadaic acid-induced apoptosis in SH-SY5Y cells by interfering with the PI3K/AKT signaling pathway,which in turn regulates the expression of apoptotic factors such as Bcl-2 and Bax.展开更多
Our previous studies showed that miR-23b was downregulated in patients with intracerebral hemorrhage(ICH). This indicates that miR-23b may be closely related to the patho-physiological mechanism of ICH, but this hypot...Our previous studies showed that miR-23b was downregulated in patients with intracerebral hemorrhage(ICH). This indicates that miR-23b may be closely related to the patho-physiological mechanism of ICH, but this hypothesis lacks direct evidence. In this study, we established rat models of ICH by injecting collagenase Ⅶ into the right basal ganglia and treating them with an injection of bone marrow mesenchymal stem cell(BMSC)-derived exosomal miR-23b via the tail vein. We found that edema in the rat brain was markedly reduced and rat behaviors were improved after BMSC exosomal miR-23b injection compared with those in the ICH groups. Additionally, exosomal miR-23b was transported to the microglia/macrophages, thereby reducing oxidative stress and pyroptosis after ICH. We also used hemin to mimic ICH conditions in vitro. We found that phosphatase and tensin homolog deleted on chromosome 10(PTEN) was the downstream target gene of miR-23b, and exosomal miR-23b exhibited antioxidant effects by regulating the PTEN/Nrf2 pathway. Moreover, miR-23b reduced PTEN binding to NOD-like receptor family pyrin domain containing 3(NLRP3) and NLRP3 inflammasome activation, thereby decreasing the NLRP3-dependent pyroptosis level. These findings suggest that BMSC-derived exosomal miR-23b exhibits antioxidant effects through inhibiting PTEN and alleviating NLRP3 inflammasome-mediated pyroptosis, thereby promoting neurologic function recovery in rats with ICH.展开更多
African dust storm events (ADE) travel across theAtlantic Ocean(ADEAO) and reach the Puerto Rican coast (ADEPRC), potentially impacting air quality and human health. To what extent seasonal variations in atmospheric p...African dust storm events (ADE) travel across theAtlantic Ocean(ADEAO) and reach the Puerto Rican coast (ADEPRC), potentially impacting air quality and human health. To what extent seasonal variations in atmospheric particulate matter (PM) size fractions, composition and sources trigger respiratory-adverse effects to Puerto Ricans is still unclear. In the present study, we investigated the pro-inflammatory and cytotoxic effects of PM samples harvested during ADEAO (PM10), ADEPRC (PM2.5 and PM10) and Non-ADE (Pre-and Post-ADEAO and Non-ADEPRC), using BEAS-2B cells. Endotoxins (ENX) in PM2.5 and PM10 extracts and traces of metals (TMET) in PM2.5 extracts were also examined. IL-6 and IL-8 secretion and cytotoxicity were used as endpoints. ADEAO and ADEPRC extracts were found to be more cytotoxic than Non-ADE and ADEAO were more toxic than ADEPRC extracts. PM10 extracts from ADEAO and Post-ADEAO caused significant secretion of IL-8. IL-6 and IL-8 secretion was higher following treatment with PM10 and PM2.5 ADEPRC than with Non-ADEPRC extracts. ENX levels were found to be higher in PM10 ADEAO than in the rest of the samples tested. TMET levels were higher in PM2.5 ADEPRC than in Non-ADEPRC extracts. Deferoxamine significantly reduced cytotoxicity and IL-6 and IL-8 secretion whereas Polymyxin B did not. TMET in PM2.5 fractions is a major determinant in ADEPRC-induced toxicity and work in conjunction with ENX to cause toxicity to lung cells in vitro. ENX and TMET may be responsible, in part, for triggering PM-respiratory adverse responses in susceptible and predisposed individuals.展开更多
文摘The surface antigen CD14 plays an important role in innate immunity, serving as a pattern recognition receptor for lipopolysaccharides (LPS). The aim of this study was to investigate the proliferation, NFκB activation, and chemokine secretion of BEAS-2B cells, a human bronchial epithelial cell line, after LPS stimulation, and some details of inVolved signaling. The presence of CD14 was investigated by flow cytometry. Cell proliferation was measured with a [3H]-thymidine incorporation assay. sCD14, RANTES, and IL-8 concentrations in cell supernatants were measured by ELISA. BEAS-2B cells express CD14 on their surface and secrete soluble CD14 into the supernatant. Cells react on LPS with increased proliferation, activation of NFκB, and the secretion of the pro-inflammatory chemotactic cytokines IL-8 and RANTES, which proves the functionality of the CD14 receptor. Neither CD14 nor sCD14 are regulated by LPS. Specific inhibitors of various intracellular signaling pathways diminish the LPS-induced proliferation and IL-8 secretion: Thus MAP-Kinases p38 and JNK, tyrosine kinases, and PI3-kinase are involved in the signaling cascade from the LPS-CD14-complex on the cell surface to the increased cell proliferation and expression of IL-8;furthermore, ERK 1/2, IRAK 1/4, and the NFκB pathway are inVolved in the latter. The data show the existence and functionality of CD14 receptors on BEAS-2B cells and elucidate the signaling pathways inVolved. LPS is able to increase cell prolife-ration, various cytokines which are dependent on endogenous CD14. Three MAPK pathways, PI3 kinase and tyrosine kinase may be involved. Also CD14 is present/involved which was controversial.
基金National Natural Science Foundation of China(No.81860709)Baise City Science and Technology Plan Project(No.Encyclopedia 20224139,Encyclopedia 20211807)2023 Youjiang Ethnic Medical College Graduate Innovation Program Project(No.YXCXJH2023013)。
文摘Objective:To investigate the effects of stilbene glycoside(TSG)on okadaic acid-induced apoptosis in human neuroblastoma cells(SH-SY5Y)via the PI3K/AKT pathway.Methods:The optimal concentration of OA was screened by CCK-8 assay,and SH-SY5Y cells were divided into control group,model group,TSG group,LY294002 group and LY294002+TSG group.The proliferation and apoptosis in each group were detected by CCK-8 and TUNEL assays;Western blotting method and real-time fluorescence quantitative polymerase chain reaction was used to detect the expression of PI3K,P-PI3K(Y607),AKT,P-AKT(Ser473),Bcl-2 and Bax proteins.The relative protein expression was represented by P-PI3K(Y607)/PI3K,P-AKT(Ser473)/AKT and Bcl-2/Bax gray ratio.Results:CCK-8 screened the optimal concentration of OA as 40 nmol/L.Compared with the control group,the model group increased relative cell viability,decreased apoptosis rate,the pathway and apoptotic proteins expression levels of P-PI3K(Y607)/PI3K,P-AKT(Ser473)/AKT and Bcl-2/Bax were decreased,and the mRNA expression levels of PI3K,AKT and Bcl-2 were decreased.Bax mRNA expression level increased(P<0.05);Compared with model group,TSG group increased relative cell viability,decreased apoptosis rate,increased protein expression levels of P-PI3K(Y607)/PI3K,P-AKT(Ser473)/AKT,Bcl-2/Bax,and increased mRNA expression levels of PI3K,AKT,and Bcl-2.Bax mRNA expression decreased(P<0.05),LY294002 group decreased relative cell viability,increased apoptosis rate,P-PI3K(Y607)/PI3K protein expression levels were significantly decreased(P<0.05),P-AKT(Ser473)/AKT and Bcl-2/Bax protein expression levels were significantly decreased,but there was no statistical significance,PI3K,AKT and Bcl-2 mRNA expression levels were decreased,and Bax mRNA expression levels were increased(all P<0.05);Compared with LY294002 group,LY294002+TSG group increased relative cell viability,decreased apoptosis rate,and the protein expression levels of P-PI3K(Y607)/PI3K,P-AKT(Ser473)/AKT and Bcl-2/Bax were increased.The mRNA expression levels of PI3K,AKT,Bcl-2 were increased,Bax was decreased(all P<0.05).Conclusion:Stilbene glycoside may alleviate okadaic acid-induced apoptosis in SH-SY5Y cells by interfering with the PI3K/AKT signaling pathway,which in turn regulates the expression of apoptotic factors such as Bcl-2 and Bax.
基金supported by the National Natural Science Foundation of China,No.81571120(to ZYH).
文摘Our previous studies showed that miR-23b was downregulated in patients with intracerebral hemorrhage(ICH). This indicates that miR-23b may be closely related to the patho-physiological mechanism of ICH, but this hypothesis lacks direct evidence. In this study, we established rat models of ICH by injecting collagenase Ⅶ into the right basal ganglia and treating them with an injection of bone marrow mesenchymal stem cell(BMSC)-derived exosomal miR-23b via the tail vein. We found that edema in the rat brain was markedly reduced and rat behaviors were improved after BMSC exosomal miR-23b injection compared with those in the ICH groups. Additionally, exosomal miR-23b was transported to the microglia/macrophages, thereby reducing oxidative stress and pyroptosis after ICH. We also used hemin to mimic ICH conditions in vitro. We found that phosphatase and tensin homolog deleted on chromosome 10(PTEN) was the downstream target gene of miR-23b, and exosomal miR-23b exhibited antioxidant effects by regulating the PTEN/Nrf2 pathway. Moreover, miR-23b reduced PTEN binding to NOD-like receptor family pyrin domain containing 3(NLRP3) and NLRP3 inflammasome activation, thereby decreasing the NLRP3-dependent pyroptosis level. These findings suggest that BMSC-derived exosomal miR-23b exhibits antioxidant effects through inhibiting PTEN and alleviating NLRP3 inflammasome-mediated pyroptosis, thereby promoting neurologic function recovery in rats with ICH.
文摘African dust storm events (ADE) travel across theAtlantic Ocean(ADEAO) and reach the Puerto Rican coast (ADEPRC), potentially impacting air quality and human health. To what extent seasonal variations in atmospheric particulate matter (PM) size fractions, composition and sources trigger respiratory-adverse effects to Puerto Ricans is still unclear. In the present study, we investigated the pro-inflammatory and cytotoxic effects of PM samples harvested during ADEAO (PM10), ADEPRC (PM2.5 and PM10) and Non-ADE (Pre-and Post-ADEAO and Non-ADEPRC), using BEAS-2B cells. Endotoxins (ENX) in PM2.5 and PM10 extracts and traces of metals (TMET) in PM2.5 extracts were also examined. IL-6 and IL-8 secretion and cytotoxicity were used as endpoints. ADEAO and ADEPRC extracts were found to be more cytotoxic than Non-ADE and ADEAO were more toxic than ADEPRC extracts. PM10 extracts from ADEAO and Post-ADEAO caused significant secretion of IL-8. IL-6 and IL-8 secretion was higher following treatment with PM10 and PM2.5 ADEPRC than with Non-ADEPRC extracts. ENX levels were found to be higher in PM10 ADEAO than in the rest of the samples tested. TMET levels were higher in PM2.5 ADEPRC than in Non-ADEPRC extracts. Deferoxamine significantly reduced cytotoxicity and IL-6 and IL-8 secretion whereas Polymyxin B did not. TMET in PM2.5 fractions is a major determinant in ADEPRC-induced toxicity and work in conjunction with ENX to cause toxicity to lung cells in vitro. ENX and TMET may be responsible, in part, for triggering PM-respiratory adverse responses in susceptible and predisposed individuals.
