The involvement of p38 MAPK in transforming growth factor β1-induced apoptosis in murine hepatocytes

We reported in this manuscript that TGF-β1 induces apoptosis in AML12 murine hepatocytes, which is associated with the activation of p38 MAPK signaling pathway. SB202190, a specific inhibitor of p38 MAPK, strongly inhibited the TGF-β1-induced apoptosis and PAI-1 promoter activity. Treatment of cells with TGF-β1 activates p38. Furthermore, over-expression of dominant negative mutant p38 also reduced the TGF-β1-induced apoptosis. The data indicate that the activation of p38 is involved in TGF-β1-mediated gene expression and apoptosis.

Expression of Connective Tissue Growth Factor in Renal Tubulointerstitial Fibrosis in Rats and Its Pathogenic Role

Summary： In order to explore the role of connective tissue growth factor （CTGF） in the pathogenesis of renal tubulointerstitial fibrosis, 48 Wistar rats were randomly divided into sham-operated and unilateral ureteral obstruction （UUO） group. On the postoperative day 1, 3, 7 and 14, the rats were killed and the kidneys were removed. The renal tubulointerstitial injury index was evaluated according to the MASSON staining. The mRNA levels of CTGF, transforming growth factor β1 （TGF-β1）. collagen [ （col I ）, and plasminogen activator inhibitor-1 （PAI 1） were detected using rexerse transcriptional-polymerase chain reaction （RT PCR）. Immunohistochemistry was performed to evaluale the protein expression of the above factors, and the relations among them were analyzed. Quantitative expression of CTGF protein in the kidneys was also assessed using Western blot. The results showed that TGF-β1 mRNA level was increased at first day after UUO, followed by a marked elevation of CTGF mRNA level, which began to increase 3 days after UUO （P〈0.01）. With the progression of the disease, the mRNA expression of CTGF, col I and PAI-1 was increased progressively. Immunohistochemistry revealed that the CTGF protein expression was significantly increased in fibrotic areas and tubular epithelial cells 3 days after UUO. On the post-UUO day 7, the protein level of CTGF was positively related to the renal tubulointerstitial injury index （r =0.62, P〈0.01）, the expression of TGF-β1 （r=0.85, P〈0.01）, colI （r=0.78, P〈0.01）, and PAI-1（r=0.76, P〈0.01）. Upon Western blot analysis, CTGF protein expression began to increase 3 days after UUO, and appeared progressively throughout the time course （P〈0.01, as compared with sham-operated group）. It is concluded that CTGF can be induced by TGF-β and mediate various profibrotic actions of this cytokine, such as increasing extracellular matrix （ECM） synthesis and decreasing ECM degradation. The increased expression of CTGF may play a crucial role in the development and progression of tubulointerstitial fibrosis.

Flagellin of Pseudomonas aeruginosa induces transforming growth factor beta 1 expression in normal bronchial epithelial cells through mitogen activated protein kinase cascades

Background Acute lung infection due to Pseudomonas aeruginosa （P. Aeruginosa） is a serious problem, especially in patients with structural lung conditions or immune compromised hosts, leading to an overwhelming threat with a high risk of morbidity and mortality. As an outcome of infection, fibrosis can be linked with chronic lung diseases. But some fibrotic manifestations, such as an irreversible decrease of lung function and fibrous bands seen on chest imaging, have been found after an acute infection with P. Aeruginosa. Fibrogenesis/remodeling resulting from acute lung infection by P.aeruginosa is rarely reported. This study was designed to explore the relation between fibrogenesis/remodeling and acute infection by P. Aeruginosa in vitro. We used flagellin protein from P. Aeruginosa, a key initiator of acute P.aeruginosa lung infection, to elucidate mechanisms by which acute lung infection with P. Aeruginosa can cause fibrogenesis/remodeling.Methods We studied the effect of flagellin from P. Aeruginosa （flagellin for short） on the transforming growth factor beta 1 （TGF-β1） and interleukin-8 （IL-8） expression, and the possible involvement of the signaling pathway, tumor necrosis factor receptor-associated factor 6 （TRAF6）/mitogen activated protein kinase （MAPK） pathway. Flagellin was purified from the P. Aeruginosa standard strain, PAO1. Normal bronchial epithelial cells BEAS-2B were challenged with different concentrations of flagellin, and cell viability assessment was performed by cell counting kit-8. BEAS-2B cells were incubated with flagellin with the specific MAPK inhibitors or TRAF6 siRNA. Cell lysates and the cultured supernatant were collected. The level of TGF-β1 and IL-8 were detected by enzyme-linked immunosorbant assay （ELISA）. Western blotting was used to detect the protein levels of MAPK signal proteins p38, c-Jun NH2-terminal kinase （JNK） and extracellular regulated kinase （ERK）.Results Expression of TGF-β1 in BEAS-2B cells was elevated by flagellin vs. Control groups （（104.3±20.8） vs.（44.6±4.4） pg/ml （P 〈0.01）） and was ablated by either p38 or JNK inhibitors compared with flagellin treatment （（45.1±18.8）vs. （104.3±20.8） pg/ml and （48.1±20.8） vs. （104.3±20.8） pg/ml, respectively （P 〈0.05））. Flagellin also elevated the expression of IL-8 in BEAS-2B cells vs. The control groups （（554.9±57.7） vs. （51.4±2.2.9） pg/ml （P 〈0.01））, and p38 MAPK inhibitors weaken the expression by flagellin （（301.1 ±155.1） vs. （554.9±57.7） pg/ml （P 〈0.05））. Western blotting revealed that all three MAPK proteins, p38, JNK and ERK were activated by flagellin challenge in an early phase, respectively in 15 minutes （P 〈0.01）, 30 minutes （P 〈0.01） and 15 minutes （P 〈0.01）. TRAF6 siRNA which decreased expression of TRAF6, altered the activation of JNK, p38, and ERK following flagellin treatment, but its influence on the expression of TGF-β1 and IL-8 has no statistical significance.Conclusions Flagellin from P. Aeruginosa PAO1 induces TGF-β1 expression in normal bronchial epithelial cells,BEAS-2B, through the MAPK signal cascade in vitro. It suggests that the fibrogenesis/remodeling process may be initiated from an early stage of acute lung infection due to P. Aeruginosa.

Inhibition of proliferation and transforming growth factor β3 protein expression by peroxisome proliferators-activated receptor γ ligands in human uterine leiomyoma cells

Background Rosiglitazone is known as the most potent and specific peroxisome proliferators-activated receptor γ （PPAR-γ） ligand. It has potentially far-reaching effects on pathophysiological processes, from cancer to atherosclerosis and diabetes. However, it is not clear whether rosiglitazone affects the protein expression of transforming growth factor β3 （TGF-β3） and the cell proliferation in human uterine leiomyoma cells in vitro.Methods Human uterine leiomyoma tissues were dissected and cultured. Cells were divided into 5 groups： one control group and other four groups with different concentrations of rosiglitazone （10^-7, 10^-8, 10^-9 and 10^-10 mol/L）. Cells were cultured for 72 hours in serum-free Dulbecco＇s modified Eagle＇s medium. MTT reduction assay was used to detect the cell proliferation. Reverse transcription polymerase chain reaction （RT-PCR） was used to detect the mRNA expression of PPAR-γ and TGF-β3. Immunofluorescence staining was used to detect the expressions of PPAR-γ and TGF-β3 proteins. Results MTT reduction assay indicated that the treatment with rosiglitazone （from 10^-7 to 10^-9 mol/L） resulted in an inhibition of the cell growths after 72 hours （P〈0.01）. RT-PCR analysis revealed that 10^-7 mol/L rosiglitazone significantly affected the gene expression at 72-hour： PPAR-γ mRNA expression was up-regulated and TGF-β3 mRNA was down-regulated and rosiglitazone at the concentration of 10-7 mol/L affected these most effectively （P〈0.01）. Immunofluorescence staining demonstrated that treatment with 10^-7 mol/L rosiglitazone resulted in the significant changes of PPAR-γ and TGF-β3 protein expressions compared with the other treatment groups and the control group at 72-hour （P〈0.01）. All the effects of rosiglitazone on uterine leiomyoma cells were dose- and time-dependent in vitro. Conclusions The present study demonstrates that the PPAR-γ activator, rosiglitazone, inhibits the cell proliferation partly through the regulations of PPAR-γ and TGF-β3 expressions. The cross-talk between the signal pathways of PPAR-γ and TGF-β3 may be involved in the process.

Follistatin-Like 1 Promotes Bleomycin-lnduced Pulmonary Fibrosis through the Transforming Growth Factor Beta 1/Mitogen-Activated Protein Kinase Signaling Pathway

Background： Follistatin-like I （FSTL 1） is a novel profibrogenic factor that induces pulmonary fibrosis （PF） through the transforming growth factor-beta 1 （TGF-[B 1 ）/Smad signaling. Little is known about its effects on PF through the non-Smad signaling, like the mitogen-activated protein kinase （MAPK） pathway. Therefore, this study aimed to investigate the role ofFSTL 1 in PF through the MAPK signaling pathway and its mechanisms in lung fibrogenesis. Methods： PF was induced in Fstll~ and wild-type （WT） C57BL/6 mice with bleomycin. After 14 days, the mice were sacrificed, and lung tissues were stained with hematoxylin and eosin; the hydroxyproline content was measured to confirm PF. The mRNA and protein level of FSTLI and the change of MAPK phosphorylation were measured by quantitative polymerase chain reaction and Western blotting. The effect of Fst11 deficiency on fibroblasts differentiation was measured by Western blotting and cell immunofluorescence. MAPK signaling activation was measured by Western blotting in Fst11＋/ and WT fibroblasts treated with recombinant human FSTLI protein. We pretreated mouse lung fibroblast cells with inhibitors of the extracellular signal-regulated kinase （ERK）, p38, and Jun N-terminal kinase （JNK） signaling and analyzed their differentiation, proliferation, migration, and invasion by Western blotting, 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide analysis, and transwell assays. The Student＇s t-test was used to compare the differences between two groups. Results： Fstll deficiency attenuated phosphorylation of the ERK, p38, and JNK signaling in bleomycin-induced fibrotic lung tissue 14 days after injury （0.67 ± 0.05 vs. 1.22 ± 0.03, t = 14.92, P = 0.0001; 0.41 ± 0.01 vs. 1.15 ± 0.07; t = 11.19; P = 0.0004; and 0.41 ± 0.01 vs. 1.07± 0.07, t = 8.92, P = 0.0009; respectively）, compared with WT lungs at the same time and in primary lung fibroblasts （0.82 ± 0.01 vs. 1.01 ±0.04, t = 4.06, P = 0.0150; 1.04 ±0.03 vs. 1.24 ± 0.03, t= 4.44, P = 0.0100： and 0.76 ±0.05 vs. 0.99± 0.05, t = 4.48, P = 0.0100; respectively）, compared with TGF-β1-stimulated WT group. Recombinant human FSTLI protein in lung fibroblasts enhanced TG F-β1 -mediated phosphorylation of the ERK （ 1.19± 0.08 vs, 0.55 ± 0.04, t = 6.99, P = 0.0020）, p38 （ 1.18 ±0.04 vs. 0.66 ± 0.03, t = 11.20, P = 0.0020）, and .INK （ 1,11± 0.01 vs. 0.84 ± 0.04, t = 6.53, P = 0.0030）, compared with the TGF-β1-stimulated WT group. Fstll-deficient fibroblasts showed reduced alpha-smooth muscle actin （α-SMA） expression （0.70 ± 0.06 vs. 1.28 ±0.11, t = 4.65, P = 0.0035, compared with the untreated WT group; 1.40 ± 0.05 vs. 1.76± 0.02, t = 6.31, P = 0.0007; compared with the TGF-β1-treated WT group）. Compared with the corresponding condition in the control group, the TGF-β1/FSTL 1-mediated α-SMA expression was significantly suppressed by pretreatment with an inhibitor of p38 （0.73± 0.01 vs. 1.13 ± 0.10, t = 3.92, P = 0.0078） and JNK （0.78 ± 0.03 vs. 1.08 ± 0.06, t = 4.40,P = 0.0046） signaling. The proliferation of mouse lung fibroblast cells （MLgs） significantly decreased after treatment of an inhibitor of p38 （0.30 ±0.01 vs. 0.46 ±0.03, t = 4.64, P = 0.0009）, JNK （0.30 ± 0.01 vs. 0.49 ± 0.01, t = 12.84, P = 0.0001）, and Smad2/3 （0.18 ± 0.02 vs. 0.46 ±0.02, t = 12.69, P = 0.0001） signaling compared with the dimethylsulibxide group. The migration and invasion cells of MLgs significantly decreased in medium pretreated with an inhibitor of p38 （70.17 ±3.28 vs. 116.30 ± 7.11, t = 5.89, P = 0.0042 for the migratory cells; 19.87 ± 0.84 vs. 32.70 i 0.95, t =10.14, P = 0.0005 for the invasive cells）, JNK （72.30 ±3.85 vs. 116.30 ± 7.11, t = 5.44, P = 0.0056 for the migratory cells; 18.03 ± 0.94 vs. 32.70 ± 0.95, t = 11.00, P = 0.0004 for the invasive cells）, and Smad2/3 （64.76 ± 1.41 vs. 116.30 ± 7.11, t = 7.11, P = 0.0021 for the migratory cells; 18.03 ± 0.94 vs. 32.70 ±0.95, t = 13.29, P = 0.0002 for the invasive cells） signaling compared with the corresponding condition in the dimethylsulfoxide group. Conclusion： FSTL1 affects lung fibroblast differentiation, proliferation, migration, and invasion through p38 and JNK signaling, and in this way, it might influence the development of PF.

Influence of baicalin on the expression of receptor activator of nuclear factor-κB ligand and osteoprotegerin in human periodontal ligament cells

Objective To study the effect of baicalin on the expression of receptor activator of nuclear factor-κB ligand(RANKL)and osteoprotegerin(OPG)in cultured human periodontal ligament(HPDL)cells.Methods Small interfering RNA(siRNA)eukaryotic expression vector targeted transforming growth factor βⅡ receptor(TGF-β RⅡ)was constructed and transfected into T cells.HPDL cells with T cells transfected with siRNA or not were placed in the culture medium that had been added with lipopolysaccharide(LPS)and baicalin.The obtained solution was divided into six groups according to the components(group Ⅰ:HPDL cells+LPS+T cells transfected with siRNA1+baicalin;group Ⅱ:HPDL cells+LPS+T cells transfected with siRNA1;group Ⅲ:HPDL cells+LPS+T cells+baicalin;group Ⅳ:HPDL cells+LPS+T cells;group Ⅴ:HPDL cells+baicalin;group Ⅵ:HPDL cells)and was cultured for 48 hours.RT-PCR was used to observe the effect of baicalin on the expression of OPG-RANKL in HPDL cells.Results The ratio of RANKL/OPG in group Ⅰ was lower than that in group Ⅱ(P<0.01)and higher than that in group Ⅲ(P<0.01);The ratio of RANKL/OPG in group Ⅲ was lower than that in group Ⅳ(P<0.01);the ratio of RANKL/OPG in group Ⅳ was higher than that in group Ⅵ(P<0.01);the ratio of RANKL/OPG in group Ⅴ was lower than that in group Ⅵ(P<0.05).Conclusion ① Baicalin could decrease the ratio of RANKL/OPG in HPDL cells.② The TGF-β signaling transduction plays an important role in the effect of baicalin on the RANKL/OPG ratio in HPDL cells.③ Baicalin acts not only through TGF-β to regulate RANKL/OPG in HPDL cells,but also through other pathways.

Mechanism of ELL-associated factor 2 and vasohibin 1 regulating invasion,migration,and angiogenesis in colorectal cancer

BACKGROUND As a novel endogenous anti-angiogenic molecule, vasohibin 1(VASH1) is not only expressed in tumor stroma, but also in tumor tissue. Moreover, studies have shown that VASH1 may be a prognostic marker in colorectal cancer(CRC). Knockdown of VASH1 enhanced transforming growth factor-β1(TGF-β1)/Smad3 pathway activity and type Ⅰ/Ⅲ collagen production. Our previous findings suggest that ELL-associated factor 2(EAF2) may play a tumor suppressor and protective role in the development and progression of CRC by regulating signal transducer and activator of transcription 3(STAT3)/TGF-β1 signaling pathway. However, the functional role and mechanism of VASH1-mediated TGF-β1 related pathway in CRC has not been elucidated.AIM To investigate the expression of VASH1 in CRC and its correlation with the expression of EAF2. Furthermore, we studied the functional role and mechanism of VASH1 involved in the regulation and protection of EAF2 in CRC cells in vitro.METHODS We collected colorectal adenocarcinoma and corresponding adjacent tissues to investigate the clinical expression of EAF2 protein and VASH1 protein in patients with advanced CRC. Following, we investigated the effect and mechanism of EAF2 and VASH1 on the invasion, migration and angiogenesis of CRC cells in vitro using plasmid transfection.RESULTS Our findings indicated that EAF2 was down-regulated and VASH1 was upregulated in advanced CRC tissue compared to normal colorectal tissue. KaplanMeier survival analysis showed that the higher EAF2 Level group and the lower VASH1 Level group had a higher survival rate. Overexpression of EAF2 might inhibit the activity of STAT3/TGF-β1 pathway by up-regulating the expression of VASH1, and then weaken the invasion, migration and angiogenesis of CRC cells.CONCLUSION This study suggests that EAF2 and VASH1 may serve as new diagnostic and prognostic markers for CRC, and provide a clinical basis for exploring new biomarkers for CRC. This study complements the mechanism of EAF2 in CRC cells, enriches the role and mechanism of CRC cellderived VASH1, and provides a new possible subtype of CRC as a therapeutic target of STAT3/TGF-β1 pathway.

Differential Mechanisms of the Effect of Peroxisome Proliferator-Activated Receptor Gamma Agonists on Bleomycin-Induced Lung Fibrosis

Background and Objectives: Peroxisome proliferator-activated receptor-g (PPAR-g) is a nuclear receptor whose activation regulates inflammation and fibrosis in various organs. We aimed to investigate the effect of two PPAR-g ligands, telmisartan and rosiglitazone, on lung injury and fibrosis induced by intratracheal bleomycin (BLM). Methods: Lung injury and fibrosis was induced in female C57Bl/6 mice by intratracheal instillation of 1.0 mg/kg of BLM. Some of the animals received rosiglitazone intraperitoneally or telmisartan in drinking water. Bronchoalveolar lavage (BAL) was performed 2, 7, 14 or 21 days after BLM instillation for cell counting and measurement of mediators in the lung. In a separate series, the lungs were sampled for collagen assay and histopathological evaluation. Results: Treatment with rosiglitazone or telmisartan significantly attenuated the BLM-induced increases in lung collagen content, pathological score, and inflammatory cells in BAL fluid. Rosiglitazone significantly suppressed BLM-induced elevation of TGF-b1, MCP-1, and IL-6 levels in the lung. In contrast, telmisartan made no changes in these cytokines, whereas it mitigated the BLM-induced increase in prostaglandin F2a in the lung. Higher concentrations of rosiglitazone and telmisartan attenuated proliferation of lung fibroblasts in vitro. Conclusions: Two PPAR-g ligands, rosiglitazone and telmisartan, exert protective effects on BLM-induced lung fibrosis through the suppression of different profibrotic mediators.

Idiopathic pulmonary fibrosis in relation to gene polymorphisms of transforming growth factor-β1 and plasminogen activator inhibitor 1

Background Idiopathic pulmonary fibrosis （IPF） is a progressive and lethal fibrotic lung disease of unknown etiology. Host susceptibility or genetic factors may be important for the predisposition to it. Transforming growth factor-β1 （TGF-β1 a potent profibrotic cytokine） and plasminogen activator inhibitor 1 （PAl-1） play important roles in the development of pulmonary fibrosis. The objective of the study was to investigate the association between the gene polymorphisms of TGF-β1 869 T〉C and PAl-1 4G/5G and the susceptibility to IPF in Han ethnicity. Methods Polymerase chain reaction （PCR） and restriction fragment length polymorphism were performed to analyse the gene polymorphisms of TGF-β1 in 869T〉C and PAl-1 4G/5G in 85 IPF patients and 85 healthy controls matched in age, gender, race and smoker status. Results There was a significant difference in 869T〉C genotype distribution of TGF-β1 between IPF cases and controls, a significant negative association between TC genotype and the development of IPF （OR=0.508, 95% CI： 0.275-0.941） and a positive association between CC genotype and the development of IPF （OR=1.967, 95% CI： 1.063-3.641）. There was a significant positive association between PAl-1 5G/5G genotype and the development of IPF （OR=0.418, 95% CI： 0.193-0.904）. Conclusions Gene polymorphisms of TGF-β1 in 869T〉Cand PAl-1 4G/5G may affect the susceptibility to IPF in Han ethnicity. Further investigations are needed to confirm these findings and assess their biological significance in the development of the disease in this ethnic population.

草鱼TAB2与TAK1蛋白互作鉴定及其对两种抗菌肽基因表达的影响

为了探究草鱼TAB2(Ci TAB2)与Ci TAK1能否互作及其对2种草鱼抗菌肽基因(Cihepcidin与Ciβ-defensin1)表达的影响,实验首先采用实时荧光定量PCR(qPCR)方法分析拟态弧菌感染后Citab2和Citak1在草鱼免疫相关组织中的时空表达模式。然后利用荧光共定位、免疫共沉淀及Western blot技术鉴定Ci TAB2与Ci TAK1在细胞内共定位及相互作用情况。最后将过表达质粒pEGFP-N1-Citak1与pEGFP-N1-Citab2共同转染草鱼肾细胞(CIK细胞),检测Cihepcidin与Ciβ-defensin1的相对mRNA表达水平。结果显示,拟态弧菌感染能够显著改变Citab2和Citak1的相对表达水平,前者于感染后不同时间在各检测组织中表现出不同的时空表达模式,而后者均呈现先上调后下调的表达模式;荧光显微镜下观察到Ci TAB2与Ci TAK1共定位于转染后的HEK293T和CIK细胞的胞质中,且在HEK293T细胞内能够形成Ci TAB2-Ci TAK1蛋白复合物;共同过表达Ci TAB2与Ci TAK1后,CIK细胞内Cihepcidin与Ciβ-defensin1的相对mRNA表达水平在各检测时间点均显著上调。结果表明,Ci TAB2与Ci TAK1存在互作关系且二者互作能够促进上述两种抗菌肽的转录表达。本研究从蛋白互作调控抗菌肽表达的角度为防治鱼类弧菌病提供了新策略。

3-甲基腺嘌呤对转化生长因子-β诱导大鼠肝脏星形细胞株HSC-T6活化及自噬的影响观察

目的观察3-甲基腺嘌呤(3-MA)对转化生长因子-β(TGF-β)诱导的大鼠肝星形细胞株HSC-T6活化及自噬的影响。方法取对数生长期的HSC-T6细胞分为空白组、TGF-β+PBS组、TGF-β+3-MA组。TGF-β+3-MA组细胞加入浓度为2 ng/mL TGF-β培养72 h后加入3-MA(0.5 mg/mL)处理24 h,TGF-β+PBS组细胞加入浓度为2 ng/mL TGF-β培养72 h后加入等量PBS处理24 h,空白组加入等量PBS处理,采用实时荧光定量PCR法检测各组细胞活化标志物α-SMA、TGF-βmRNA和自噬标志物LC3、Beclin-1、Atg5 mRNA,采用Western blotting法检测各组细胞活化标志物α-SMA、TGF-β蛋白和自噬标志物LC3、Beclin-1、Atg5蛋白。结果TGF-β+PBS组、TGF-β+3-MA组细胞活化标志物α-SMA、TGF-βmRNA和蛋白均高于空白组,且TGF-β+3-MA组均低于TGF-β+PBS组(P均<0.05)。TGF-β+PBS组、TGF-β+3-MA组细胞自噬标志物LC3、Beclin-1、Atg5 mRNA和蛋白均高于空白组,且TGF-β+3-MA组均低于TGF-β+PBS组(P均<0.05)。结论3-MA可抑制TGF-β诱导的大鼠肝星形细胞株HSC-T6活化及自噬。

支气管哮喘病儿外周血单核细胞中LncRNA MEG3的表达及其与肺功能、免疫功能的相关性研究

目的探讨支气管哮喘病儿外周血单核细胞中长链非编码RNA母系表达基因3(LncRNA MEG3)的表达及其与肺功能和免疫功能的相关性。方法选取2017年10月至2018年9月安阳市妇幼保健院收治的支气管哮喘病儿60例为观察组,根据疾病严重程度分为轻度组(20例),中度组(20例),重度组(20例),同时选取同期体检的40例健康儿童作为对照组。均行肺功能检测,采用qRT-PCR检测各组外周血单核细胞中LncRNA MEG3表达量;血细胞分析仪检测嗜酸性粒细胞计数水平,ELISA法检测免疫球蛋白E(IgE)水平血清白细胞介素-4(IL-4)、白细胞介素-17A(IL-17A)、γ干扰素(IFN-γ)、转化生长因子-β(TGF-β)水平,比较各组肺功能、免疫功能指标、血清炎性介质及LncRNA MEG3水平差异,Pearson相关检验分析支气管哮喘病儿外周血单核细胞中LncRNA MEG3表达量与肺功能指标、免疫功能指标及炎性介质的相关性。多元线性回归分析LncRNA MEG3表达量与病情严重程度、肺功能、免疫功能的关联影响关系。结果重度组,中度组及轻度组LncRNA MEG3表达水平高于对照组[(0.30±0.07)、(0.42±0.06)、(0.67±0.10)比(1.01±0.13)](P<0.05),重度组LncRNA MEG3表达水平高于中度组及轻度组(P<0.05),中度组LncRNA MEG3表达水平高于轻度组(P<0.05);重度组,中度组及轻度组FEV1[(52.19±9.86)、(59.73±8.02)、(73.18±12.90)比(93.06±10.43)],FEV1/FVC[(65.23±9.65)、(72.05±5.28)、(80.12±8.12)比(93.25±10.90)]及PEF[(42.27±5.98)%、(59.94±5.67)%、(70.22±6.59)%比(86.52±3.57)%]低于对照组(P<0.05),重度组FEV1,FEV1/FVC及PEF低于中度组及轻度组(P<0.05),中度组FEV1,FEV1/FVC及PEF低于轻度组(P<0.05),重度组,中度组及低度组IgE[(110.25±13.32)U/mL、(84.92±12.98)U/mL、(56.61±11.21)U/mL比(23.21±5.81)U/mL]、嗜酸性粒细胞计数水平[(0.45±0.04)×10~9/L、(0.36±0.03)×10~9/L、(0.20±0.02)×10~9/L比(0.13±0.03)×10~9/L]、IL-4[(20.24±4.07)ng/L、(16.71±3.17)ng/L、(12.23±1.59)ng/L比(8.03±2.25)ng/L]、IL-17A[(119.51±8.52)ng/L、(100.89±6.28)ng/L、(85.74±9.05)ng/L比(52.67±13.24)ng/L]水平高于对照组(P<0.05),重度组IgE、嗜酸性粒细胞计数水平、IL-4、IL-17A水平高于中度组及轻度组(P<0.05),中度组IgE、嗜酸性粒细胞计数水平、IL-4、IL-17A水平高于轻度组(P<0.05),重度组、中度组及轻度组IFN-γ[(40.13±5.39)ng/L、(50.01±6.45)ng/L、(60.30±5.42)ng/L比(69.87±6.51)ng/L]、TGF-β[(709.74±36.53)ng/L、(836.77±42.64)ng/L、(1,002.35±53.34)ng/L比(1,940.88±224.39)ng/L]低于对照组(P<0.05),重度组IFN-γ、TGF-β低于中度组及轻度组(P<0.05),中度组IFN-γ、TGF-β低于轻度组(P<0.05),相关分析显示,支气管哮喘病儿LncRNA MEG3表达水平与IgE、IL-4、IL-17A呈负相关(P<0.05),与IFN-γ、TGF-β呈正相关(P<0.05),与FEV1,FEV1/FVC及PEF呈正相关(P<0.05)。多元线性回归显示,病情严重程度、肺功能、免疫功能与LncRNA MEG3表达量有密切的关联影响关系(P<0.05)。结论支气管哮喘病儿外周血单核细胞中LncRNA MEG3的表达量降低,并且与病情相关,其可能通过对免疫功能及炎症介质的调节参与支气管哮喘的发病及进展过程。

LncRNA NAMA对胶质瘤细胞U87侵袭能力的影响

目的:探讨lnc RNA NAMA对胶质瘤细胞U87侵袭能力的影响及机制。方法:收集2018年6月~2020年1月在我院接受手术治疗的胶质瘤患者组织样本共30例。逆转录PCR(RT-PCR)检测肿瘤组织及瘤旁组织中lnc RNA NAMA的表达。在U87胶质瘤细胞中,分别转染lnc RNA NAMA过表达质粒(pc DNA-NAMA,pc DNA-VC为对照)或干扰质粒(si-NAMA,si-NC为对照),transwell检测细胞侵袭能力,RT-PCR及Western blot检测细胞内转化生长因子β受体1(TGFBR1)表达量。结果:USCS数据库提示,TGFBR1为lnc RNA NAMA临近基因。lnc RNA NAMA在胶质瘤组织中表达量高于瘤旁组织,且其在Ⅲ+Ⅳ级组织中含量高于Ⅰ+Ⅱ级组织样本(均P<0.05)。Transwell结果显示,pc DNA-NAMA组透过细胞数多于pc DNA-VC组,si-NAMA组少于si-NC组。RT-PCR及Western blot检测显示,pc DNA-NAMA组TGFBR1表达较pc DNA-VC组升高,si-NAMA组TGFBR1表达较si-NC组降低(均P<0.05)。si-TGFBR1+pc DNA-VC组与si-TGFBR1+pc DNA-NAMA组相比,transwell小室透过细胞数无统计学差异(P>0.05)。结论:lnc RNA NAMA在胶质瘤组织中高表达,可增强胶质瘤细胞U87的侵袭能力,其作用机制可能与上调TGFBR1有关。

黄芩苷通过调控LncRNA-MALAT1/miR-361-3p/TGF-β轴治疗低氧性肺动脉高压

目的:探讨黄芩苷(Baicalin)对低氧性肺动脉高压的治疗作用及其潜在机制。方法:选取18只C57BL/6雄性小鼠随机分为常氧组、低氧组及低氧+黄芩苷组,每组各6只。21 d后采用导管法检测小鼠右心室收缩压(RVSP)及平均颈动脉压力(mCAP),行苏木素-伊红(HE)染色观察肺动脉血管重塑程度,采用免疫组化染色检测TGF-β通路蛋白表达。常氧/低氧条件下培养小鼠肺动脉平滑肌细胞(MPASMCs)48 h,采用CCK-8法测MPASMCs细胞活力,Transwell和Wound healing检测MPASMCs细胞迁移能力,蛋白质印迹(Western blot)法检测TGF-β通路蛋白表达差异,生物信息学分析和双荧光素酶报告基因检测验证LncRNA-MALAT1/miR-361-3p/TGF-β轴的调控关系。结果:与低氧组比,黄芩苷干预组MPASMCs的增殖和迁移能力显著下降(P<0.05),小鼠的RVSP明显下降(P<0.05),肺血管重塑改善。与常氧组比,低氧组中TGF-β信号通路相关蛋白TGF-β、p-smad2、p-smad3表达显著升高,而黄芩苷干预后TGF-β信号通路显著抑制(P<0.05)。结论:黄芩苷可能通过调控LncRNA-MALAT1/miR-361-3p轴调控TGF-β表达,改善低氧MPASMCs异常增殖、低氧性肺动脉高压小鼠肺血管结构重塑,进而降低肺动脉压力。

All-trans Retinoic Acid Diminishes Collagen Production in a Hepatic Stellate Cell Line via Suppression of Active Protein-1 and c-Jun N-terminal Kinase Signal

Following acute and chronic liver injury,hepatic stellate cells (HSCs) become activated to undergo a phenotypic transformation into myofibroblast-like cells and lose their retinol content,but the mechanisms of retinoid loss and its potential roles in HSCs activation and liver fibrosis are not understood.The influence of retinoids on HSCs and hepatic fibrosis remains controversial.The purpose of this study was to evaluate the effects of all-trans retinoid acid (ATRA) on cell proliferation,mRNA expression of collagen genes [procollagen α1 (Ⅰ),procollagen α1 (Ⅲ)],profibrogenic genes (TGF-β 1,CTGF,MMP-2,TIMP-1,TIMP-2,PAI-1),fibrolytic genes (MMP-3,MMP-13) and the upstream element (JNK and AP-1) in the rat hepatic stellate cell line (CFSC-2G).Cell proliferation was evaluated by measuring BrdU incorporation.The mRNA expression levels of collagen genes [procollagen α1 (Ⅰ),procollagen α1 (Ⅲ)],profibrogenic genes (TGF-β 1,CTGF,MMP-2,TIMP-1,TIMP-2,PAI-1),and fibrolytic genes (MMP-3,MMP-13) were quantitatively detected by using real-time PCR.The mRNA expression of JNK and AP-1 was quantified by RT-PCR.The results showed that ATRA inhibited HSCs proliferation and diminished the mRNA expression of collagen genes [procollagen α1 (Ⅰ),procollagen α1 (Ⅲ)] and profibrogenic genes (TGF-β 1,CTGF,MMP-2,TIMP-1,TIMP-2,PAI-1),and significantly stimulated the mRNA expression of MMP-3 and MMP-13 in HSCs by suppressing the mRNA expression of JNK and AP-1.These findings suggested that ATRA could inhibit proliferation and collagen production of HSCs via the suppression of active protein-1 and c-Jun N-terminal kinase signal,then decrease the mRNAs expression of profibrogenic genes (TGF-β 1,CTGF,MMP-2,TIMP-1,TIMP-2,PAI-1),and significantly induce the mRNA expression of MMP-3 and MMP-13.

Smad4 and ERK2 stimulated by transforming growth factor betal in rhabdomyosarcoma

Background Transforming growth factor beta （TGF-beta） plays an essential role in the regulation of normal physiologic processes of cells. TGF-beta has been shown to regulate several mitogen-activated protein kinases （MAPK） pathways in several epithelial cells. However, the effects of TGF-beta on soft tissue sarcoma are seldom reported. Our previous studies suggested that there should be some other signal transduction pathways besides Smads, which are important to regulate the growth of human embryonal rhabdomyosarcoma （RMS） cells. In the present study, we examined the expression and functional relations of extracellular signal-regulated kinase 2 （ERK2） and Smad4 in human RMS tissue and a RMS cell line, RD. Methods RD cells and normal human primary skeletal myoblasts （Mb） were treated with TGF-betal to establish the expression profile of ERK2 at the mRNA and protein levels detected by RT-PCR and immunofluorescence. Immunohistochemistry was used to detect the expression of ERK2 and Smad4 in 50 tissue specimens of human RMS and 23 specimens of normal skeletal muscles. Follow-up of specimens was performed 6 months to 70 months later. Results RD cells and human RMS tissues showed the higher expression of ERK2 and Smad4 than the normal control, either the protein level or the mRNA level. And, exogenous TGF-betal stimulation can lead to higher expression of ERK2 and its nuclear translocation, so TGF-betal can also activatedMAPK （ERK2） pathway, resulting in a sustained activation of ERK2 for at least 2 hours. Immunohistochemistry analysis, however, showed that there was no correlation between ERK2 and Smad4 protein. The overexpression of ERK2 and Smad4 had no indicative effects on histological subtypes, histological grading, gender, age, and prognosis. Conclusions In RMS, signaling of TGF-betal from cell surface to nucleus can also be directed through the MAPK （ERK2） pathway besides the TGF-betal/Smads pathway. The activation of ERK2 by TGF-betal may be Smad4 independent. Moreover, there may be some other tanglesome relationships between the TGF-betal/Smads pathway and the MAPK pathway which takes part in the development, invasion and metastasis of tumor cells.

Overexpression of ELL-associated factor 2 suppresses invasion,migration,and angiogenesis in colorectal cancer

BACKGROUND The androgen responsive gene,ELL-associated factor 2(EAF2),expressed in benign prostate tissues,has been shown to play an important role in tumor suppression in a variety of malignant tumors.In addition,some scholars found that EAF2 frameshift mutations are associated with intratumor heterogeneity in colorectal cancer(CRC)and inactivation of EAF2 in microsatellite instability-high CRC.However,the molecular mechanism by which EAF2 is involved in CRC invasion and metastasis remains unclear.AIM To determine the clinical value of expression of EAF2 protein in CRC,and to study the effects of EAF2 on the invasion,migration,and angiogenesis of CRC cells in vitro.METHODS In this study,we collected colorectal adenocarcinoma and corresponding adjacent tissues to investigate the clinical expression of EAF2 protein in patients with advanced CRC.Subsequently,we investigated the effect of EAF2 on the invasion,migration,and angiogenesis of CRC cells in vitro using plasmid transfection.RESULTS EAF2 protein was lowly expressed in cancer tissues of patients with advanced CRC.Kaplan-Meier survival analysis showed that the survival rate of the high EAF2 level group was higher than that of the low EAF2 level group.CONCLUSION Our results demonstrated that EAF2,as a tumor suppressor,may inhibit the invasion,metastasis,and angiogenesis of CRC cells by regulating the signal transducer and activator of transcription 3/transforming growth factor-β1 crosstalk pathway,and play a cancer suppressive and protective role in the occurrence and development of CRC.Our findings are of great significance to provide a new idea and theoretical basis for the targeted diagnosis and treatment of CRC.

EFFECTS OF TGF-β_1 ON THE EXPRESSION OF PLASMINOGEN ACTIVATOR INHIBITOR TYPE 1 IN CULTURED HUMAN RENAL INTERSTITIAL FIBROBLASTS

Objective To investigate the effects of transforming growth factor-β1 (TGF-β1 ) on the expression of plasminogen activator inhibitor type 1 (PAI-1 ) mRNA in renal interstitial fibrosis in vitro. Methods Human renal interstitial fibroblasts were isolated and cultured in vitro. The cells wers stimulated by TGF-β1 with different concentration (0 to 10ng/ml ) at different time (0 to 48h). The expression of PAI-1 mRNA was assayed by RT-PCR. Results TGF-β1, had dose-dependent and time-dependent effects on the expression of PAI-1 mRNA in renal interstitial fibroblasts. Conclusion TGF-β1 may partic- ipate in renal fibrosis with inducing the expression of PAI-1 mRNA in renal fibroblasts and affecting the synthesis and degradation of extracellular matrix (ECM).

NLRP6诱导胶质瘤细胞生物活性及对TGF-β1/Smad蛋白的作用

目的探讨核苷酸结合寡聚化结构域样受体(NLR)P6诱导胶质瘤细胞生物活性及对转化生长因子(TGF)-β1/Smad蛋白的作用。方法将胶质瘤细胞U251分为正常组(U251细胞)、NC组(U251细胞+NC-空转)和干预组(U251细胞+NLRP6-shRNA),四甲基偶氮唑蓝(MTT)法检测24、48和72 h U251细胞增殖,Transwell小室法、流式细胞法、Western印迹和聚合酶链反应(PCR)法分别检测24 h后U251细胞迁移数目、凋亡率、TGF-β1/Smad蛋白和mRNA表达。结果与正常组比较,NC组及干预组细胞侵袭数目、TGF-β1 mRNA及蛋白表达水平显著降低;细胞凋亡率、Smad4 mRNA及蛋白表达水平显著升高(均P<0.05)。与NC组比较,干预组细胞侵袭数目、TGF-β1 mRNA及蛋白表达水平显著降低;细胞凋亡率、Smad4 mRNA及蛋白水平显著升高(均P<0.05)。结论沉默NLRP6能够降低胶质瘤细胞U251细胞活性,其作用机制可能与抑制TGF-β1水平及激活Smad4水平相关。

TGF-β_(1)联合aPTT对急性ST段抬高型心肌梗死患者高血栓负荷的预测价值

目的分析转化生长因子-β_(1)(TGF-β_(1))联合活化部分凝血活酶时间(aPTT)对急性ST段抬高型心肌梗死(STEMI)患者高血栓负荷(HTB)的预测价值。方法选取140例STEMI患者,视血栓负荷情况分为HTB组66例和低血栓负荷(LTB)组74例,采用酶联免疫吸附法与凝固法检测血清TGF-β_(1)水平与aPTT。通过多因素Logistic回归分析STEMI患者HTB的影响因素,受试者工作特征(ROC)曲线分析血清TGF-β_(1)水平和aPTT对STEMI患者HTB的预测价值。结果HTB组男性、吸烟比例和年龄、白细胞计数、TGF-β_(1)水平高于LTB组,aPTT短于LTB组(P均<0.05)。多因素Logistic回归分析显示,年龄增加(OR=1.033,95%CI:1.005~1.063)、吸烟(OR=1.880,95%CI:1.102~3.208)、TGF-β_(1)升高(OR=1.924,95%CI:1.047~3.536)为STEMI患者HTB的独立危险因素,aPTT延长(OR=0.893,95%CI:0.820~0.972)为独立保护因素(P均<0.05)。ROC曲线分析显示,血清TGF-β_(1)水平和aPTT单独与联合预测STEMI患者HTB的ROC曲线下面积(AUC)分别为0.786、0.711、0.840,灵敏度分别为64.89%、59.54%、83.21%,特异度分别为81.88%、71.81%、70.47%。血清TGF-β_(1)水平和aPTT联合预测STEMI患者HTB的AUC大于二者单独预测(P均<0.05)。结论血清TGF-β_(1)水平升高和aPTT缩短与STEMI患者HTB密切相关,可作为STEMI患者HTB的辅助预测指标,二者联合对STEMI患者HTB的辅助预测价值较高。