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

We reported in this manuscript that TGF-beta1 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-beta1-induced apoptosis and PAI-1 promoter activity. Treatment of cells with TGF-beta1 activates p38. Furthermore, over-expression of dominant negative mutant p38 also reduced the TGF-beta1-induced apoptosis. The data indicate that the activation of p38 is involved in TGF-beta1-mediated gene expression and apoptosis.

Correlation between expression of two transforming growth factor-beta 1 receptors and microvascular density in a rat model of cerebral ischemia and reperfusion injury

The effects of transforming growth factor-β1 （TGF-β1） are currently controversial. Whether TGF-β1 promotes or inhibits revascularization under different conditions remains poorly understood. Based on previous studies, the current experiment established rat models of cerebral ischemia and reperfusion injury （IRI）, and demonstrated that pathological and functional damage was also increased after IRI. The most serious damage was observed at 3 days after reperfusion, at which time microvascular density fell to its lowest level. Soon afterwards, microvascular density increased, new collateral circulation was gradually established at 4 to 7 days after reperfusion, and pathological damage and neurological deficits were improved. TGF-β1, activin receptor-like kinase 5 （ALK5） mRNA and protein expression levels increased gradually over time. In contrast, ALK1 mRNA and protein expression decreased over the same period. A significant negative correlation was detected between microvascular density and expression of the ALK5 gene transcript. There was no correlation between microvascular density and ALK1 gene transcriptional expression following cerebral IRI in a rat model. These findings suggest that ALK5, rather than ALK1, is the critical receptor in the TGF-β1 signal pathways after cerebral IRI.

Metformin attenuates angiotensin II induced cardiac fibrosis and transforming growth factor-β1 production through the inhibition of hepatocyte nuclear factor4

Aim In diabetic patients, metformin appears to provide cardiovascular protection that cannot be attribu- ted only to its antihyperglycemic effects. Metformin is also known as the AMP-activated protein kinase （AMPK） ac- tivator. Our previous study suggested that metformin inhibits transforming growth factor-β1 （TGF-β1） production in a mouse heart failure model of pressure overload. TGF-β1 is a key factor in cardiac fibrosis and is usually induced by Angiotensin Ⅱ （Ang Ⅱ ） in the pressure overload mouse models. This study investigated the effect of metformin on cardiac fibrosis and TGF-β production induced by AngII and the underlying mechanisms. Methods C57/BL6 wild-type and AMPKα2 knockout mice were used. AngII （3 mg · kg-1 · d-1） was infused subcutaneously into mice for 7 days. Adult mouse cardiac fibroblasts were isolated and treated with AngII （ 1 μmol · L-1） and/or met- formin （1 mmol · L-l）. Results In C57/BL6 mice, metformin inhibits AngII-induced cardiac fibrosis. In cardi-ac fibroblasts, metformin inhibits TGF-β1 expression and production induced by AngII. AMPK inhibitor, com- pound C, reversed the effects of metformin. In vivo, AMPKα2 deficiency further increases AngII-induced TGF-β1 production. In cardiac fibroblasts, metformin inhibited AngII induced hepatocyte nuclear factor4 （HNF4ot protein level increase and HNF4α binding with TGF-β1 promoter using chromatin immunoprecipitation assay. In vivo, AMPKα2 deficiency further increased AngII-induced HNF4α protein level. Using HNF4α adenovirus, overexpress- ing HNF4α led to a 1.5-fold increase in TGF-β1 mRNA expression. HNF4a siRNA blocked AngII induced TGF- β1 production. Luciferase reporter with deleted HNF4a binding sites showed decreased TGFbl transcriptional activ- ity induced by AngII. In AMPK or2-/- heart, the inhibition of metformin on HNF4a protein was attenuated. Con- clusion Metformin inhibits AngII induced cardiac fibrosis and TGF-β1 production through AMPK activation. The underlying mechanism is that AMPK activation inhibits AngII induced HNF4α and then decreases TGF-β1 expres- sion.

Effects of retinoic acid on proliferation,phenotype and expression of cyclin-dependent kinase inhibitors in TGF-β1-stimulated rat hepatic stellate cells

AIM To study the molecular mechanisms ofretinoic acid(RA)on proliferation andexpression of cyclin-dependent kinase inhibitors(CKI),i.e.p16,p21 and p27 in cultured rathepatic stellate cells(HSC)stimulated withtransforming growth factor beta 1(TGF-β1).METHODS HSC were isolated from healthy ratlivers and cultured.After stimulated with1 mg/L TGF-β1,subcultured HSC were treatedwith or without 1 nmol/L RA.MTT assay,immunocytochemistry(ICC)for p16,p21,p27and α-smooth muscle actin(α-SMA)protein,insitu hybridization(ISH)for retinoic acidreceptor beta 2(RAR-β2)and p16,p21 and p27mRNA and quantitative image analysis(partially)were performed.RESULTS RA inhibited HSC proliferation(41.50%,P【0.05),decreased the protein levelof α-SMA(55.09%,P【0.05),and induced HSCto express RAR-β2 mRNA.In addition,RAincreased the protein level of p16(218.75%,P【0.05)and induced p21 protein expression;meanwhile,p27 was undetectable by ICC in bothcontrol and RA-treated HSC.However,RA hadno influence on the mRNA levels of p16,p21 orp27 as determined by ISH.CONCLISION Up-regulation of p16 and p21 on post-transcriptional level may contribule, in part to RA inhibition of TGF-β1-initiated rat HSC activation in vitro.

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.

Role of activin receptor-like kinase 1 in vascular development and cerebrovascular diseases

Activin receptor-like kinase 1(ALK1)is a transmembrane serine/threonine receptor kinase of the transforming growth factor beta(TGFβ)receptor superfamily.ALK1 is specifically expressed in vascular endothelial cells,and its dynamic changes are closely related to the proliferation of endothelial cells,the recruitment of pericytes to blood vessels,and functional differentiation during embryonic vascular development.The pathophysiology of many cerebrovascular diseases is today understood as a disorder of endothelial cell function and an imbalance in the proportion of vascular cells.Indeed,mutations in ALK1 and its co-receptor endoglin are major genetic risk factors for vascular arteriovenous malformation.Many studies have shown that ALK1 is closely related to the development of cerebral aneurysms,arteriovenous malformations,and cerebral atherosclerosis.In this review,we describe the various roles of ALK1 in the regulation of angiogenesis and in the maintenance of cerebral vascular homeostasis,and we discuss its relationship to functional dysregulation in cerebrovascular diseases.This review should provide new perspectives for basic research on cerebrovascular diseases and offer more effective targets and strategies for clinical diagnosis,treatment,and prevention.

基于Traf6/TAK1通路探讨维生素D对甲状腺功能减退肾损伤幼鼠肾小管上皮细胞间充质转化的影响

目的探讨维生素D(VD)对甲状腺功能减退(HT)肾损伤幼鼠肾小管上皮细胞间充质转化(EMT)的影响,以及其对肿瘤坏死因子受体相关因子6(Traf6)/转化生长因子-β活化激酶1(TAK1)通路的调控机制。方法通过丙基硫尿嘧啶(PTU)灌胃构建幼鼠HT模型,以过表达TAK1(pc DNA3.1-TAK1)作功能挽救实验;50只SPF级雄性SD大鼠分为正常组、HT组、VD低剂量(HT+VD-L)组、VD高剂量(HT+VD-H)组、HT+VD-H+pc DNA3.1-TAK1(HT+VD-H+pc)组,每组10只。全自动生化仪检测各组大鼠血清血肌酐(Scr)和血尿素氮(BUN)的含量;脱氧核糖核苷酸末端转移酶介导的缺口末端标记法试剂盒(TUNEL)检测肾组织中的细胞凋亡;免疫组化检测肾组织中转化生长因子β1(TGF-β1)、α-平滑肌肌动蛋白(α-SMA)和上皮钙黏蛋白(E-cadherin)的表达;Western blot法检测肾组织中B淋巴细胞瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)、Traf6、TAK1和磷酸化TAK1(p-TAK1)的表达。结果VD能明显降低HT幼鼠血清中Scr和BUN的含量,下调肾组织中的细胞凋亡率,降低肾组织中TGF-β1和α-SMA的表达,上调E-cadherin的表达;抑制肾组织中Traf6、p-TAK1和Bax的表达,升高肾组织中Bcl-2的表达,差异均具有统计学意义(均P<0.05)。结论维生素D能抑制HT幼鼠肾小管上皮细胞的EMT,降低肾组织中的细胞凋亡率,减轻肾组织的病理损伤,改善其肾功能,这与抑制Traf6/TAK1信号的激活有关。

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.

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.

Electroacupuncture attenuates chronic salpingitis via transforming growth factor-β1/p38 mitogen-activated protein kinase signaling pathway

OBJECTIVE:To explore the effect of electroacupuncture(EA)on rats with chronic fallopian tube inflammation and its potential mechanisms.METHODS:Thirty-six female Sprague-Dawley rats were divided into Control,Model and EA groups.The pathological morphology of the fallopian tubes was observed by hematoxylin-eosin(HE)and Masson staining.The results of transforming growth factor-β1(TGF-β1),P38 mitogen-activated protein kinase(MAPK),phosphorylation(p)-p38MAPK in rat oviduct tissues were detected by immunohistochemistry.Results of P38MAPK,p-P38MAPK and TGF-β1 in rat oviduct tissues were detected by immunofluorescence.The expression level of p38MAPK,p-P38MAPK,TGF-β1 protein in rats was detected by Western blot.Quantitative real-time polymerase chain reaction(RT-q PCR)was used to detect m RNA expression levels of TGF-β1.RESULTS:It found that collagen fibers counts decreased significantly in EA group compared to Model group.The phosphorylation of P38MAPK in EA group was significantly reduced compared to Model group.The serum TGF-β1 expressions in EA group increased decreased significantly.CONCLUSION:Electroacupuncture was able to attenuate chronic salpingitis through down-regulating TGF-β1/MAPK signaling pathway.

Huangqi decoction(黄芪汤) attenuates renal interstitial fibrosis via transforming growth factor-β1/mitogen-activated protein kinase signaling pathways in 5/6 nephrectomy mice

OBJECTIVE: To investigate the effect of Huangqi decoction( 黄芪汤) on renal interstitial fibrosis and its association with the transforming growth factor-β1(TGF-β1)/mitogen-activated protein kinase(MAPK) signaling pathway. METHODS: 120 C57/BL mice were randomly divided into six groups: sham group, Enalapril(20 mg/kg) group, 5/6 nephrectomy model group, and 5/6 nephrectomy model plus Huangqicoction(0.12, 0.36 and 1.08 g/kg respectively) groups. Detecting 24hours urinary protein, blood pressure, serum creatinine, urea nitrogen content changes. Periodic Acid-Schiff stain(PAS) and Masson’s trichrome staining was used to observe the renal tissue pathological changes. Protein expression of TGF-β1, Phosphorylated P38 mitogen activated protein kinases(P-P38), Phosphorylated c-jun N-terminal kinase(P-JNK), Phosphorylated extracellular regulated proteinhnase(PERK), Fibroblast-specific protein-1(FSP-1), Alpha smooth muscle actin(α-SMA), Type Ⅲ collagen(Collagen Ⅲ), Connective tissue growth factor(CTGF),Bcl-2 Assaciated X protein(Bax) and B cell lymphoma 2(Bcl-2) were measured with western blot and immunohistochemical. RESULTS: Both Huangqi decoction and Enalapril improved the kidney function, 24 h urinary protein and the fibrosis in 5/6 nephrectomy mice, Huangqi decoction downregulated the expressions of TGF-β1, FSP-1, α-SMA, Collagen Ⅲ and CTGF in a dose-dependent manner, and it has a significant difference(P < 0.01) compared with model group.Huangqi decoction downregulated the expressions of P-P38, P-JNK, P-ERK and Bcl-2 in a dose-dependent manner, while upregulated the expression of Bax. CONCLUSIONS: The protective effect of Huangqi decoction for renal interstitial fibrosis in 5/6 nephrectomized mice via the inhibition of EpithelialMesenchymal Transitions and downregulating the TGF-β1/MAPK signaling pathway.

胰岛素样生长因子1对人RPE细胞分泌TGF-β2、MMP-2的影响及机制研究

目的研究胰岛素样生长因子1(IGF-1)对人视网膜色素上皮细胞(ARPE-19)表达转化生长因子β2(TGF-β2)、基质金属蛋白酶2(MMP-2)的影响,并探索其作用机制。方法ARPE-19细胞分别按不同浓度IGF-1和不同浓度LY294002培养6 h、12 h、24 h、48 h,采用CCK-8法检测细胞活力,确定IGF-1、LY294002的最佳作用浓度与时间。细胞划痕法检测细胞迁移活性。ELISA法检测细胞培养上清液中TGF-β2浓度。将ARPE-19细胞分为对照组、IGF-1组(80μg·L^(-1) IGF-1)、IGF-1+LY294002组(80μg·L^(-1) IGF-1+30 mmol·L^(-1) LY294002)、LY294002组(30 mmol·L^(-1) LY294002),使用无血清DMEM/F12培养基培养,对照组不做任何处理,分别采用RT-PCR、Western blot检测细胞中TGF-β2、MMP-2、磷脂酰肌醇-3-激酶(PI3K)、蛋白激酶B(AKT)的mRNA和蛋白表达量。结果与0μg·L^(-1) IGF-1比较,80μg·L^(-1) IGF-1的细胞活力24 h变化显著(P<0.05),故确定其为IGF-1最佳作用浓度和时间。与0 mmol·L^(-1) LY294002比较,24 h的30 mmol·L^(-1) LY294002接近半数抑制浓度,故确定其为LY294002最佳作用时间和浓度。细胞划痕法检测结果显示,0μg·L^(-1) IGF-1组、40μg·L^(-1) IGF-1组、80μg·L^(-1) IGF-1组细胞迁移率整体比较及两两比较差异均有统计学意义(均为P<0.05)。ELISA检测结果显示,0μg·L^(-1) IGF-1组、40μg·L^(-1) IGF-1组、80μg·L^(-1) IGF-1组细胞上清液中TGF-β2浓度整体比较及两两比较差异均有统计学意义(均为P<0.05)。RT-PCR、Western blot检测结果显示,IGF-1、LY294002培养24 h,与对照组比较,IGF-1组细胞中TGF-β2、MMP-2、PI3K、AKT的mRNA与蛋白表达水平均升高,而LY294002组细胞中TGF-β2、MMP-2、PI3K、AKT的mRNA与蛋白表达水平均下降(均为P<0.05);与IGF-1组比较,IGF-1+LY294002组细胞中TGF-β2、MMP-2、PI3K、AKT的mRNA与蛋白表达水平均下降(均为P<0.05)。结论IGF-1能促进ARPE-19细胞增殖、迁移;IGF-1可能通过PI3K/AKT信号通路上调ARPE-19细胞中TGF-β2、MMP-2的表达,参与近视的发生与发展。

RNF99通过TAK1/NF-κB信号通路参与泛素化与脓毒症性休克的潜在联系

目的 探讨环指蛋白99(RNF99)介导的转化生长因子激酶1(TAK1)/核因子-κB(NF-κB)信号通路参与泛素化与脓毒症性急性呼吸窘迫综合征(ARDS)的潜在联系。方法 进行质粒和siRNA转染以过表达或敲低小鼠肺泡上皮细胞(MLE12)中RNF99,分析磷酸p65和p65蛋白表达。免疫沉淀分析RNF99与TRAF6和TAK1的蛋白相互作用关系。将40只小鼠随机分成WT+PBS、WT+LPS、RNF99特异性表达(TG)+PBS和TG+LPS组,每组10只。通过腹膜内注射30 mg/kg LPS诱导脓毒症。结果 与Vector组相比,RNF99组MLE12细胞中TRAF6和TAK1的蛋白表达水平显著降低(P<0.05)。泛素化TRAF6蛋白在RNF99敲低的MLE12细胞中增加。与LPS+Vector组相比,在LPS+RNF99组MLE12细胞中p65的磷酸化水平明显降低(P <0.05)。与si-NC组相比,si-RNF99组MLE12细胞中RNF99、IκBα的蛋白表达水平显著降低(P <0.05)。与LPS+si-NC组相比,在LPS+si-RNF99组MLE12细胞中p65的磷酸化水平明显增加(P <0.05)。TG+LPS组小鼠肺组织中CD68巨噬细胞染色百分比较WT+LPS组显著降低(P <0.05)。TG+LPS组小鼠肺组织中p65的磷酸化水平显著低于WT+LPS组小鼠(P <0.05)。结论 RNF99通过与NF-κB信号通路的关键调节因子(TRAF6/TAK1)相互作用来调节NF-κB信号通路,并改善小鼠腹腔注射LPS后肺损伤。

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。

宫颈上皮内瘤变患者血清中DCLK1、LTBP2的表达及与临床病理特征、术后复发的关系

目的:探讨宫颈上皮内瘤变(cervical intraepithelial neoplasia,CIN)患者血清双皮质素样激酶1(bicorticoid like kinase 1,DCLK1)、潜在转化生长因子结合蛋白2(latent transforming growth factor binding protein 2,LTBP2)水平与临床病理特征和术后复发的关系。方法:收集整理2019年03月至2022年09月期间于本院确诊的342例CIN患者的相关临床资料并作为CIN组,依据术后复发情况分为未复发组293例和复发组49例;收集351例健康体检者为对照组。采用酶联免疫吸附法检测血清DCLK1、LTBP2水平;采用受试者工作特征(ROC)曲线分析血清DCLK1、LTBP2水平对CIN患者术后复发的预测价值;采用Cox比例风险回归模型筛选CIN复发的影响因素。结果:CIN组血清DCLK1、LTBP2水平高于对照组(P<0.05)。CIN分级Ⅲ级、有高危人乳头瘤病毒(HPV)感染的CIN患者血清DCLK1、LTBP2水平高于CIN分级Ⅰ-Ⅱ级、无高危HPV感染患者(P<0.05)。复发组血清DCLK1、LTBP2水平及CIN分级Ⅲ级、有高危HPV感染的CIN患者比例高于未复发组(P<0.05)。DCLK1、LTBP2单独及联合预测CIN患者术后复发的AUC分别为0.803、0.710、0.870。DCLK1、LTBP2、CIN分级是CIN患者术后复发的独立危险因素(P<0.05)。结论:CIN患者血清DCLK1、LTBP2高表达与CIN分级增加和高危HPV感染有关,且二者联合可有效预测术后复发。

血清DCLK1、LTBP2联合经阴道实时剪切波弹性成像对宫颈癌的早期诊断价值

目的探讨血清双皮质素样激酶1(DCLK1)、潜在转化生长因子结合蛋白2(LTBP2)联合经阴道实时剪切波弹性成像(SWE)对宫颈癌的早期诊断价值。方法选取2021年8月至2022年12月于本院就诊的宫颈病变患者155例为研究对象,经手术病理确诊宫颈癌患者75例(宫颈癌组)和宫颈上皮内瘤变(CIN)患者80例(CIN组),另取同期于本院体检的无宫颈相关疾病志愿者80例作为对照组。各组受试者均进行血清DCLK1、LTBP2水平检测及经阴道SWE检查。受试者工作特征(ROC)曲线分析血清DCLK1、LTBP2对宫颈癌的诊断价值;采用四格表法分析血清DCLK1、LTBP2联合经阴道SWE对宫颈癌的诊断价值。结果宫颈癌组患者血清DCLK1、LTBP2水平显著高于CIN组及对照组(P<0.05)。与术前相比,宫颈癌患者术后1、3、6个月的血清DCLK1、LTBP2水平逐渐下降,各时间点两两比较差异均有统计学意义(P<0.05)。血清DCLK1、LTBP2诊断宫颈癌的曲线下面积分别为0.868、0.754,敏感度为86.67%、78.67%,特异度为81.25%、60.00%,最佳截断值为1.49 ng/mL、19.02μg/mL。宫颈癌组患者病灶组织的最大弹性模量、平均弹性模量均显著高于CIN组(P<0.05)。血清DCLK1、血清LTBP2、经阴道SWE诊断宫颈癌的阳性率分别为86.67%、78.67%、82.67%,与金标准均具有一致性(Kappa=0.624、0.501、0.673,P<0.001),三项联合诊断宫颈癌的阳性率为97.33%,与金标准的一致性较高(Kappa=0.846,P=0.001);三项联合诊断的敏感度、准确度均显著高于血清DCLK1、血清LTBP2、经阴道SWE单一指标诊断,三项联合诊断的特异度显著高于血清DCLK1、LTBP2单一指标诊断,三项联合的误诊率均显著低于血清DCLK1、LTBP2单一指标诊断,差异均有统计学意义(P<0.05)。结论血清DCLK1、LTBP2联合经阴道SWE在宫颈癌诊断中应用价值较高,可提升诊断的敏感度、准确度,降低误诊率。

增生性瘢痕形态学观察及血管内皮生长因子和转化生长因子β激活性激酶1表达的检测与意义

目的研究血管内皮生长因子(VEGF)和转化生长因子β激活性激酶1(TAK1)在正常皮肤及增生性瘢痕组织中的表达,并结合增生性瘢痕的形态学观察,探讨其在病理性瘢痕发病机制中的作用,为病理性瘢痕的临床治疗提供一定的依据与参考。方法采用常规HE、Masson染色、免疫荧光方法和荧光定量PCR法对10例来自体正常皮肤及15例增生性瘢痕进行VEGF和TAK1在组织中的定位与表达量的检测、病理形态学观察。结果形态学观察显示增生性瘢痕的表皮形态异常,增生性瘢痕组织中的真皮成纤维细胞较正常皮肤排列紊乱、致密。免疫荧光结果显示增生性瘢痕组织中VEGF和TAK1的表达强度明显高于正常皮肤组织。荧光定量PCR结果显示VEGF和TAK1在增生性瘢痕组织中表达均增强(P<0.01,P<0.05)。结论增生性瘢痕组织胶原纤维化程度明显高于正常皮肤组织,且瘢痕组织中VEGF与TAK1表达程度均高于正常组织,对增生性瘢痕的形成可能发挥促进作用。VEGF与TAK1可作为增生性瘢痕诊断与鉴别诊断的参考指标,为防治病理性瘢痕形成提供新的治疗靶点。

TGF-β1及Ⅰ型受体ALK1在人脑胶质瘤细胞的表达及意义

目的观察转化生长因子β1(TGF-β1)及活化素受体样激酶1(ALK1)在人脑胶质瘤细胞中的表达及其在胶质细胞瘤病理分级中的意义。方法采用半定量RT-PCR、Western blot及免疫组织化学染色等方法检测32例人脑胶质瘤及3例正常脑组织中TGF-β1及ALK1mRNA及蛋白的表达水平。结果人脑胶质瘤细胞中存在TGF-β1及ALK1mRNA和蛋白表达。与正常组比较,高级别胶质瘤组TGF-β1及ALK1mRNA和蛋白表达均显著升高(P<0.05),其中ALK1的高表达与胶质瘤病理级别呈明显正相关(r=0.297,P<0.05)。结论人脑胶质细胞瘤中,ALK1可能参与了胶质瘤的恶变过程。

新型过氧化物酶Peroxiredoxin-1对大鼠肺成纤维细胞增殖和JNK通路的影响

目的探讨新型过氧化物酶Peroxiredoxin-1(Prx-1)对大鼠肺成纤维细胞(FB)增殖和JNK通路的影响。方法体外培养FB,随机分为:对照组、TGF-β1组、空转染组、Prx-1转染组。对照组:以0.4%血清浓度MEM作为基础培养液;TGF-β1组:0.4%血清培养条件下,给予TGF-β1(5μg/L)孵育细胞;空转染组:利用脂质体Lipo2000将空载体转染到FB 36 h,给予同步化处理12 h后,在0.4%血清培养条件下,给予TGF-β1(5μg/L)孵育细胞;Prx-1转染组:Prx-1真核表达质粒转染到FB 36 h,给予同步化处理12 h后,在0.4%血清培养条件下,给予TGF-β1(5μg/L)孵育细胞。质粒转染采用脂质体转染法;免疫荧光检测质粒转染和8-OhdG(DNA氧化产物)的水平;MTT法检测FB增殖;Western blot检测JNK和Prx-1的表达情况。结果质粒成功转染入FB,转染Prx-1真核表达质粒使Prx-1在FB内蛋白表达水平增高。与对照组比较,TGF-β1组的FB增殖、8-OhdG及磷酸化的JNK表达均明显增加。与TGF-β1组比较,空转染组中的上述观察指标无明显变化,但在Prx-1转染组,这些指标均明显下降。结论 TGF-β1能够诱导FB生成反应性氧物质(ROS),并由此促进JNK的激活和FB增殖,而Prx-1可通过抑制ROS来抑制TGF-β1诱导的FB增殖。

TGFβ1及I型受体ALK1/ALK5mRNA在脑动静脉畸形中的表达及意义

目的探讨转化生长因子β1(TGFβ1)及其I型受体ALK1、ALK5在脑动静脉畸形中的表达,并探讨其在脑动静脉畸形发生、发展中的作用。方法应用半定量RT-PCR方法检测脑动静脉畸形中TGFβ1、ALK1及ALK5mRNA的表达。结果TGFβ1及其受体ALK5mRNA在脑动静脉畸形中的表达均显著性升高,其相对表达量分别为0.777±0.047和0.585±0.074;受体ALK1mRNA表达则显著性降低,相对表达量为0.173±0.044,与正常对照组的0.720±0.098比较差异有显著性(P<0.01)。结论TGFβ1及其受体ALK1、ALK5mRNA的表达平衡在脑动静脉畸形发生、发展形成中具有重要作用。