Roles of Smad3 and Smad7 in rat pancreatic stellate cells activated by transforming growth factor-beta 1

BACKGROUND: Pancreatic stellate cells (PSCs) play a major role in promoting pancreatic fibrosis. Transforming growth factor beta 1 (TGF-beta 1) is a critical mediator of this process. This study aimed to determine the expression of the Smad3 and Smad7 genes in the process of PSC activation, and explore the mechanisms of chronic pancreatitis. METHODS: The expressions of Smad3 and Smad7 in PSCs before and after TGF-beta 1 treatment were detected by reverse transcription-polymerase chain reaction and Western blotting analysis. Smad3 expression was detected in PSCs after treatment with 5 ng/ml of TGF-beta 1 for 24 hours. RESULTS: Smad7 expression was decreased in TGF-beta 1 -activated PSCs (P<0.05) in a dose-dependent manner. When TGF-beta 1 concentration reached 10 ng/ml, the expression of p-Smad3, Smad3, and Smad7 was inhibited (P<0.05). CONCLUSIONS: TGF-beta 1 promotes the expression of Smad3 and inhibits the expression of Smad7 during the activation of PSCs. In contrast, high-dose TGF-beta 1 downregulates the expression of Smad3 in completely activated PSCs.

Interference of Y-27632 on the signal transduction of transforming growth factor beta type 1 in ocular Tenon capsule fibroblasts

AIM: To investigate the interfering effect of Y-27632, a ROCK-I selective inhibitor, on the signal transduction pathway of transforming growth factor-beta 1 (TGF-beta 1) in ocular Tenon capsule fibroblasts (OTFS) in vitro. METHODS: After OTFS from passages 4 to 6 47 vitro were induced by TGF-beta 1 and then treated by Y-27632, the changes of the OTFS cell cycles were analyzed via flow cytometry, and the proteins expression of the alpha -smooth muscular actin (alpha -SMA), connective tissue growth factor (CTGF), collagen I were calculated by Western blot. After OTFS treated by the different concentrations of Y-27632, the expression levels of the alpha -SMA, CTGF and collagen I mRNA were assayed by RT-PCR. RESULTS: Y-27632 had no markedly effect on the OTFS cell cycles. After treated by TGF-beta 1, OTFS in G1 period significantly increased. The cell cycles distribution by both TGF-beta 1 and Y-27632 had no remarkable difference from that in control group. Y-27632 significantly inhibited the proteins expressions of both alpha -SMA and CTGF, while to some extent inhibited that of collagen I. TGF-beta 1 significantly promoted the proteins expressions of alpha -SMA, CTGF and collagen I. After OTFS treated by both TGF-beta 1 and Y-27632, of alpha -SMA, the protein expression was similar with that in control group (P=0.066>0.05), but the protein expression of CTGF or collagen I, respectively, was significantly different from that in control group (P=0.000<0.01). The differences of expressions of the alpha -SMA, CTGF and collagen I mRNA in 30, 150, 750 mu mol/L Y-27632 group were statistically significant, compared with those in control group, respectively (alpha -SMA, P=0.002, 0.000, 0.000; CTGF, P=0.014, 0.002, 0.001; collagen I,P=0.003, 0.002, 0.000). CONCLUSION: Blocking the Rho/ROCK signaling pathway by using of Y-27632 could inhibit the cellular proliferation and the expression of both CTGF and alpha -SMA whatever OTFS induced by TGF-beta 1 or not. Y-27632 suppressed the expression of collagen I mRNA without induction.

Effects of RNA interference targeting transforming growth factor-beta 1 on immune hepatic fibrosis induced by Concanavalin A in mice

BACKGROUND: Previous studies have shown that transforming growth factor-beta 1 (TGF-beta 1) is the most potent means of stimulating liver fibrogenesis by myofibroblast-like cells derived from hepatic stellate cells. Thus, TGF-beta 1 could be a target for treating hepatic fibrosis. This study aimed to investigate the inhibitory effects of specific TGF-beta 1 small interference RNA (siRNA) on immune hepatic fibrosis induced by Concanavalin A (Con A) in mice. METHODS: Three short hairpin RNAs targeting different positions of TGF-beta 1 were designed and cloned to the plasmid pGenesil-1 to obtain three recombinant expression vectors (pGenesil-TGF-beta 1-ml, pGenesil-TGF-beta 1-m2 and pGenesil-TGF-beta 1-m3). Thirty male Kunming mice were randomly divided into 6 groups: normal, model, control, and three treatment groups. The immune hepatic fibrosis models were constructed by injecting Con A via the tail vein at 8 mg/kg per week for 6 weeks. At weeks 2, 4 and 6, pGenesil-TGF-beta 1-ml, pGenesil-TGF-beta 1-m2 or pGenesi1-TGF-beta 1-m3 was injected by a hydrodynamics-based transfection method via the tail vein at 0.8 ml/10 g within 24 hours after injection of Con A in each of the three treatment groups. The mice in the control group were injected with control plasmid pGenesil-HK at the same dose. All mice were sacrificed at week 7. The levels of hydroxyproline in liver tissue were determined by biochemistry. Liver histopathology was assessed by Van Gieson staining. The expression levels and localization of TGF-beta 1, Smad3, and Smad7 in liver tissue were detected by immunohistochemistry. The expression of TGF-beta 1, Smad3, Smad7 and alpha-smooth muscle actin (alpha-SMA) mRNAs in the liver were assessed by semi-quantitative RT-PCR. RESULTS: The levels of hydroxyproline in the liver tissue of the treatment groups were lower than those of the model group (P<0.01). Histopathologic assay showed that liver fibrogenesis was clearly improved in the treatment groups compared with the model group. The expression levels of TGF-beta 1 and Smad3 of liver tissue were also markedly lower in the treatment groups than in the model group (P<0.01), while the levels of Smad7 were higher in the treatment groups than in the model group (P<0.01). RT-PCR further showed that the expression of TGF-beta 1, Smad3 and alpha-SMA mRNA was significantly inhibited in the treatment groups compared with the model group, while the levels of Smad7 were increased. There was no difference in the above parameters among the three treatment groups or between the control and model groups (P>0.05), but the inhibitory effect of pGenesil-TGF-beta 1-ml was the highest among the treatment groups. CONCLUSIONS: Specific siRNA targeting of TGF-beta 1 markedly inhibited the fibrogenesis of immune hepatic fibrosis induced by Con A in mice. The anti-fibrosis mechanisms of siRNAs may be associated with the down-regulation of TGF-beta 1, Smad3 and alpha-SMA expression and up-regulation of Smad7 expression in liver tissue, which resulted in suppressing the activation of hepatic stellate cells. (Hepatobiliary Pancreat Dis Int 2009; 8: 300-308)

Pre-ischemia electro-acupuncture potentiates the expression of Bcl-2 and transforming growth factor-beta 1 in rat brains

The expression of the anti-apoptotic molecules Bcl-2 and transforming growth factor-beta 1 is known to confer protective effects on the cerebral ischemia-reperfusion injury.The current study investigated the expression levels of Bcl-2 and transforming growth factor-beta 1 in response to multiple pre-ischemia electro-acupuncture at acupoints Zusanli（ST36）and Fengchi（GB20） stimulation.Rats were divided into five groups：uninjured,control,non-acupoint,GB20 and ST36. Rats in the non-acupoint,GB20 and ST36 groups received 30 minutes（3 times or 18 times）of electro-acupuncture stimulation before experimental cerebral ischemia was induced.Bcl-2 and transforming growth factor-beta 1 were found to be significantly increased in the ST36 groups with either 3 or 18 electro-acupuncture treatments（P〈0.05）.The production was higher with 18 electro-acupuncture treatments in the ST36 groups（P〈0.05）.In the GB20 groups,significant increase was only observed in transforming growth factor-beta 1 with 18 electro-acupuncture treatments（P〈0.05）.No significant elevation of the level of transforming growth factor-beta 1 was observed in the non-acupoint groups.However,the production of Bcl-2 increased with 18 treatments in the non-acupoint groups（P〈0.05）.The data suggest that multiple pre-ischemia electro-acupuncture at ST36 was effective in conferring neuroprotective effect on the brain by means of upregulation of Bcl-2 and transforming growth factor-beta 1 and the effect was increase with the number of treatment.

Plasma Levels of Transforming Growth Factor-Beta 1 in Women with Pelvic Organ Prolapse

Objective: In women with pelvic organ prolapse (POP), decreased expression of transforming growth factor-beta 1 (TGF-β1) has been shown in POP tissues. However, no studies have evaluated plasma TGF-β1 levels in patients with POP, so it is unknown whether they are also changed or not. Therefore, we compared plasma TGF-β1 levels in women with and without POP. Methods: Participants were 49 women with POP and 23 healthy control women. All participants were postmenopausal. We measured plasma TGF-β1 and compared data between patients with POP and controls, and between patients with uterine prolapse (UP, n = 19) and those with a cystocele (CC, n = 30). In addition, in patients, we assessed the POP quantification system (POP-Q) stage. Results: Plasma TGF-β1 levels were significantly lower in patients than in healthy controls. POP-Q stage was not significantly different between the UP and CC subgroups, but POP-Q stage IV was diagnosed in 63% of patients with UP and 7% of those with CC. Plasma TGF-β1 levels were significantly lower in the CC subgroup than in the UP subgroup. Conclusion: Plasma TGF-β1 is decreased in POP. It remains unclear whether the lower levels indicate a reduction in systemic TGF-β1 activity, but they can be assumed to reflect reduced TGF-β1 expression in POP tissues.

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.

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.

Transforming growth factor-β and toll-like receptor-4 polymorphisms are not associated with fibrosis in haemochromatosis

AIM:To investigate the role of genetic polymorphisms in the progression of hepatic fibrosis in hereditary haemochromatosis.METHODS:A cohort of 245 well-characterised C282Y homozygous patients with haemochromatosis was studied,with all subjects having liver biopsy data and DNA available for testing.This study assessed the association of eight single nucleotide polymorphisms(SNPs)in a total of six genes including toll-like receptor 4(TLR4),transforming growth factor-beta(TGF-β),oxoguanine DNA glycosylase,monocyte chemoattractant protein 1,chemokine C-C motif receptor 2 and interleukin-10 with liver disease severity.Genotyping was performed using high resolution melt analysis and sequencing.The results were analysed in relation to the stage of hepatic fibrosis in multivariate analysis incorporating other cofactors including alcohol consumption and hepatic iron concentration.RESULTS:There were significant associations between the cofactors of male gender(P=0.0001),increasing age(P=0.006),alcohol consumption(P=0.0001),steatosis(P=0.03),hepatic iron concentration(P<0.0001)and the presence of hepatic fibrosis.Of the candidate gene polymorphisms studied,none showed a significant association with hepatic fibrosis in univariate or multivariate analysis incorporating cofactors.We also specifically studied patients with hepatic iron loading above threshold levels for cirrhosis and compared the genetic polymorphisms between those with no fibrosis vs cirrhosis however there was no significant effect from any of the candidate genes studied.Importantly,in this large,well characterised cohort of patients there was no association between SNPs for TGF-βor TLR4and the presence of fibrosis,cirrhosis or increasing fibrosis stage in multivariate analysis.CONCLUSION:In our large,well characterised group of haemochromatosis subjects we did not demonstrate any relationship between candidate gene polymorphisms and hepatic fibrosis or cirrhosis.

Saponin Ⅰ from Shuitianqi(Rhizoma Schizocapasae Plantagineae) inhibits metastasis by negatively regulating the transforming growth factor-β1/Smad7 network and epithelial-mesenchymal transition in the intrahepatic metastasis Bagg's Albino/c mouse model

OBJECTIVE:To examine the influence of SaponinⅠfrom Shuitianqi(Rhizoma Schizocapasae Plantagineae)(SSPHⅠ)on hepatocellular carcinoma(HCC)metastasis,and to elucidate the underlying mechanism.METHODS:The intrahepatic metastasis Bagg's Albino/c(BALB/c)mouse model was established with human hepatocellular carcinomas(HepG2)cells,then treated with normal saline(once per day),cisplatin(2 mg/kg,once every 2 d),and SSPHⅠ(25,50,and 75 mg/kg,once per day).Then,we assessed alterations in the hepatic pathology and target protein expressions in the intrahepatic metastasis BALB/c mouse model using a series of molecular biology techniques.RESULTS:Based on our analysis,SSPHⅠsignificantly alleviated hepatocyte necrosis and tumor cells infiltration.Moreover,SSPHⅠsuppressed extracellular matrix(ECM)degradation and angiogenesis via a decrease in matrix etalloproteinase-2(MMP-2),MMP-9,CD31,CD34,and vascular endothelial growth factor(VEGF)levels.Furthermore,SSPHⅠrepressed invasion and metastasis by suppressing the transforming growth factor-β1(TGF-β1)/Smad7 axis and epithelial-mesenchymal transition(EMT),as evidenced by the scarce TGF-β1,Ncadherin,and Vimentin expressions,and elevated Smad7 and E-cadherin expressions.CONCLUSION:The SSPHⅠ-mediated negative regulation of the TGF-β1/Smad7 axis and EMT are critical for the inhibition of HCC invasion and metastasis.

儿童慢性粒细胞白血病慢性期红细胞参数及血清bFGF、TGF-β1、VEGF表达变化分析

目的探究儿童慢性粒细胞白血病(CML)慢性期红细胞参数及血清碱性成纤维细胞生长因子(bFGF)、转化生长因子β1(TGF-β1)及血管内皮生长因子(VEGF)表达变化。方法前瞻性选取2020年1月至2023年1月在首都医科大学附属北京儿童医院进行治疗的54例CML慢性期患儿为研究组,另随机抽取46名同期在本院进行体检的健康儿童为健康对照组。研究组给予酪氨酸激酶抑制剂治疗。比较两组间红细胞参数及血清bFGF、TGF-β1、VEGF表达变化,并比较研究组治疗前后红细胞参数及血清bFGF、TGF-β1、VEGF表达水平。结果研究组的RBC、血红蛋白、红细胞压积(HCT)及平均红细胞血红蛋白浓度(MCHC)水平分别为(3.45±0.04)×10^(12)/L、(102.33±1.15)g/L、(32.03±0.61)%、322.15±2.58,均显著低于对照组[(4.98±0.03)×10^(12)/L、(149.78±1.88)g/L、(44.33±0.31)%、334.12±0.77],平均红细胞体积(MCV)、平均红细胞血红蛋白含量(MCH)及红细胞体积分布宽度(RDW)水平分别为(91.44±0.77)fL、(33.15±2.55)pg、(17.55±0.12)%,均显著高于对照组[(89.88±0.34)fL、(30.24±0.16)pg、(12.66±0.11)%],差异均有统计学意义(P<0.05)。研究组的血清bFGF、VEGF水平分别为(30.66±9.66)、(128.68±30.58)pg/mL,均显著高于对照组[(5.26±1.54)、(70.66±11.26)pg/mL],TGF-β1水平为(38.22±8.06)μg/L,显著低于对照组[(78.66±8.13)μg/L],差异均有统计学意义(P<0.05)。治疗后,研究组患儿的RBC、血红蛋白、HCT、MCV及MCH水平均较治疗前显著降低,MCHC及RDW水平均较治疗前显著升高,差异均有统计学意义(P<0.05)。研究组治疗后的血清bFGF、VEGF水平均较治疗前显著降低,TGF-β1水平较治疗前显著升高,差异均有统计学意义(P<0.05)。结论在儿童CML慢性期患儿中可见血细胞参数明显异常,血清bFGF、VEGF水平显著升高,TGF-β1水平显著降低。酪氨酸激酶抑制剂治疗CML慢性期能有效改善患儿红细胞形态及功能,抑制肿瘤细胞生长,临床疗效显著,值得临床推广使用。

心房颤动患者血清脂蛋白a、Apelin-13、转化生长因子β1水平与左心房内径的关系

目的 探讨心房颤动(AF)患者血清脂蛋白a[Lp(a)]、Apelin-13、转化生长因子β1(TGF-β1)及左心房内径(LAD)间的关系。方法 2022年12月至2024年1月,选择在滁州市第一人民医院心内科住院患者100例,按照是否发生AF分为2组,AF组50例,非AF(NAF)组50例。检测2组患者血清Lp(a)、Apelin-13、TGF-β1水平,采用经胸超声心动图检查左心房内径并进行相关性分析。结果 AF组Lp(a)、TGF-β1水平及LAD升高,Apelin-13水平降低,与NAF组比较差异均有统计学意义(P<0.05)。多因素logistic回归分析显示,Lp(a)、TGF-β1、舒张压及LAD为AF的危险因素,Apelin-13为AF的保护因素(P<0.05),且Lp(a)、TGF-β1、LAD及Apelin-13对AF具有预测价值(P<0.05)。线性回归结果显示,Lp(a)、TGF-β1、Apelin-13对LAD的回归系数分别为0.01(P>0.05)、-0.04(P>0.05)、-1.22(P<0.05)。结论 Lp(a)、TGF-β1及LAD为AF的独立危险因素;但Lp(a)、TGF-β1与LAD无明确相关性;Apelin-13作为AF独立保护因素,与LAD呈负相关。

鉴定LTBP1作为胃癌预后的生物标志物及其与肿瘤免疫微环境的相关性

目的探索潜在转化生长因子β结合蛋白1(latent transforming growth factor beta binding protein 1,LTBP1)在胃癌发生发展及免疫微环境中的生物学功能。方法在TCGA数据库中分析LTBP1在泛癌中的表达,然后通过胃癌组织和癌旁组织进行验证。通过回归比例分析法分析LTBP1表达与临床病理变量之间的相关性。Cox回归分析和Kaplan-Meier图用于评估LTBP1在胃癌中的预后价值。通过TIMER分析LTBP1的表达水平与胃癌中免疫细胞浸润之间的相关性。免疫组织化学染色检测LTBP1蛋白在胃癌组织及癌旁组织中的表达水平。结果与正常胃组织相比,胃癌组织中LTBP1表达显著上调。其表达与病理TNM分期显著相关。LTBP1高表达的胃癌患者的总体生存率(overall survival,OS)缩短。免疫组化结果显示,与癌旁组织相比,LTBP1在胃癌组织中显著高表达。TIMER检测发现LTBP1的表达与3种免疫细胞浸润呈正相关。结论LTBP1可能是胃癌预后的一个潜在生物标志物,并影响癌症的肿瘤免疫微环境。

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.

Effects of Qingguang'an(青光安)containing serum on the expression levels of autophagy-related genes in human Tenon's fibroblasts induced by transforming growth factor beta 1

OBJECTIVE:To explore the effects of Qingguang'an(青光安)containing serum on the expression levels of autophagy related genes in the transforming growth factor beta 1(TGF-β1)-activated human Tenon's fibroblasts(HTFs).METHODS:(a)Primary HTFs were stimulated by TGF-β1 and underwent immunohistochemistry,which established a cell model after Glaucoma filtration surgery(GFS).(b)The cell models were divided into 4 group:normal group(normal cells),model group(+TGF-β1),treatment group(+TGF-β1+medicated serum),and positive control group(TGF-β1+rapamycin).Then,Qingguang'an medicated serum with optimum concentration was added to the corresponding group.The autophagy positive cells were identified by the Cyto-ID autophagy detection kits under fluorescent microscope and Cytation 5 multifunctional instrument for cell imaging.And the mean fluorescence intensity of autophagy positive cells was determined by flow cytometry.The expression levels of autophagy related genes—Beclin-1,autophagy related gene 5(ATG-5),and microtubule-associated protein 1 light chain 3(LC-3Ⅱ)were detected by quantitative reverse transcription-polymerase chain reaction and Western blot analysis.RESULTS:Compared with the normal group and the model group,the relative mRNA expression levels of autophagy-related genes(Beclin-1,ATG-5 and LC-3Ⅱ)in the experimental group were notably increased(P<0.05,P<0.01),and with the extension of treatment time,it had an increasing trend(48 h was more obvious),which showed a certain time dependency;the protein expression levels of autophagy-related genes(Beclin-1,ATG-5,and LC-3Ⅱ)were significantly increased in the experimental group(P<0.05,P<0.01).With the prolongation of treatment time,there was an increasing trend(48 h was relatively obvious),and it revealed a certain time dependency CONCLUSION:The Qingguang'an medicated serum could up-regulate autophagy related genes(Beclin1,ATG5,and LC3Ⅱ)in the TGF-β1-activated HTFs.

Expression of ectonucleotide pyrophosphatase-1 in end-plate chondrocytes with transforming growth factor beta 1 siRNA interference by cyclic mechanical tension

Background Ectonucleotide pyrophosphatase/phosphodiesterase （ENPP）-I is a membrane-bound protein that catalyzes the hydrolysis of extracellular nucleoside triphosphates to monophosphate and extracellular inorganic pyrophosphate （ePPi）. Mechanical stimulation regulates ENPP-1 expression. This study sought to investigate the changes in ENPP-1 expression after stimulation using cyclic mechanical tension （CMT）.

牛磺熊去氧胆酸通过调节内质网应激抑制转化生长因子β1所诱导的心肌成纤维细胞纤维化

目的探讨牛磺熊去氧胆酸(TUDCA)对转化生长因子β1(TGF-β1)所诱导的心肌成纤维细胞(CFs)活化的作用及相关机制,为TUDCA治疗糖尿病心肌纤维化提供新的治疗目标。方法提取原代SD乳鼠CFs和心肌细胞,通过免疫荧光法鉴定CFs的纯度。分别将高糖培养下的CFs用不同时间的TGF-β1干预,用Western blot检测内质网应激(ERS)通路相关蛋白和纤维化指标的表达,探讨TGF-β1对CFs活化及ERS相关通路的影响。用Western blot检测CFs和心肌细胞内同型半胱氨酸内质网应激泛素样结构域1(Herpud1)的表达情况。通过沉默Herpud1的表达及用Transwell和Western blot检测沉默Herpud1后CFs纤维化指标的表达,探讨Herpud1在TGF-β1诱导的CFs活化中的作用。用CCK-8检测不同浓度的TUDCA处理下CFs的活力。用免疫荧光和Western blot检测TUDCA对TGF-β1诱导的CFs中Herpud1、葡萄糖调节蛋白78(GRP78)、转录激活因子6(ATF6)、α-平滑肌肌动蛋白(α-SMA)、波形蛋白(Vimentin)和Ⅰ型胶原蛋白(CollagenⅠ)表达的影响。为进一步明确Herpud1在TUDCA抑制CFs活化中的作用,用Western blot检测过表达Herpud1后相关蛋白的表达情况。结果在成功构建CFs纤维化模型的基础上,TGF-β1能够诱导CFs活化及ERS通路相关蛋白的表达;Herpud1在CFs中的表达高于心肌细胞;敲除Herpud1可抑制TGF-β1所诱导的CFs活化;TUDCA能显著降低TGF-β1诱导的CFs中GRP78、ATF6、α-SMA、Vimentin和CollagenⅠ的表达水平;此外,过表达Herpud1还可逆转TUDCA对TGF-β1诱导的CFs活化的抑制。结论下调Herpud1基因的表达是TUDCA抑制TGF-β1诱导的CFs纤维化的机制之一。

Moxibustion enables protective effects on rheumatoid arthritis-induced myocardial injury via transforming growth factor beta1 signaling and metabolic reprogramming

OBJECTIVE:To examine the effects of moxibustion on myocardial injury and myocardial metabolomics in rats with rheumatoid arthritis(RA)based on the transforming growth factor beta1(TGF-β1)/Smads signaling pathway.METHODS:One hundred rats were treated with saline[normal control(NC)group]or complete Freund’s adjuvant(CFA)by right plantar injection for the RA model group,and the latter were randomly divided into 4 groups.Tripterygium wilfordii polyglycoside tablets(雷公藤多苷片,TPT)have anti-inflammatory and are widely used in the clinical treatment of RA,therefore serving as a positive control group.Three days post injection rats were given TPT tablet(TPT group),acupuncture therapy(APT group),and moxibustion treatment(MOX group)for 15 consecutive days,while NC group and model group were equally grasped and fixed and received normal saline.Rat joint swelling scores and arthritis index(AI)were evaluated in each group before the CFA challenge,therapy and after receiving therapy.Myocardial ultrastructure was observed by electron microscope.Enzyme-linked immunosorbent assay was used to detect cardiac troponin I(cTnI)levels in rat myocardial tissue.Quantitative reverse transcription polymerase chain reaction and Western blotting analysis were used to measure the mRNA and protein levels of TGF-βsignaling molecules including TGF-β1,Smad2,Smad3,Smad4,and Smad7.Myocardial metabolomics was analyzed using gas chromatography-mass spectrometer.RESULTS:Compared with model group,RA model rats receiving TPT,acupuncture,or moxibustion therapy all showed reduced joint swelling scores and AI(all P<0.01)and improved myocardial damage,whereas rats treated with moxibustion were found to be more marked.Consistently,the expressions of cTnI,TGF-β1,Smad2,Smad3,and Smad4 were found to be elevated in model rat group in contrast to NC rats and were significantly downregulated in TPT,APT and MOX group when compared with model group,while the levels of Smad7 showed the opposite result(all P<0.01).Moreover,the dissection of metabolomics suggested a novel metabolite biomarker panel including D-Xylulose 5-phosphate,dihydroxyacetone phosphate,arachidonic acid,etc was defined and implicated in amino acid,glucose,and fatty acid metabolic processes as revealed by principal component analysis and partial least squares discriminant analysis.CONCLUSION:Moxibustion prevents RA-induced inflammatory response and offers potent therapeutic effects on myocardial dysfunctions.The protective effects might be associated with its role in TGF-β1 inactivation and metabolic reprogramming.

除风益损汤对兔角膜损伤后瘢痕形成及TGF-β1、Smad3、α-SMA蛋白表达的影响

目的探讨除风益损汤对兔角膜损伤后瘢痕形成及转化生长因子-β1(ransforming growth factor-beta 1,TGF-β1)、果蝇母本抗生存因子蛋白3(small mothers against decapentaplegic 3,Smad3)、α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)蛋白表达的影响。方法取健康月龄新西兰兔48只,按随机数字法将其分成4组,每组12只,设为空白组、模型组、除风益损汤组(以下简称中药组)、重组牛碱成纤维细胞生长因子滴眼组(以下简称贝复舒组);中药组予除风益损汤灌胃,1次/d,空白组、模型组、贝复舒组予等体积生理盐水灌胃,1次/d,贝复舒组予重组牛碱成纤维细胞生长因子滴眼液滴眼,3次/d;建模后各组于7、14、28 d在高倍显微镜下观察角膜损伤修复情况,共聚焦激光断层扫描观察角膜细胞,Masson染色观察角膜组织病理改变,Western blot检测角膜组织中TGF-β1、Smad3、α-SMA蛋白表达。结果眼前节拍照:可见兔角膜7、14、28 d时中药组、贝复舒组角膜损面积伤较模型组角膜损伤面积明显减小。共聚焦激光断层扫描角膜浅层细胞:在造模后28 d,模型组呈现出大量瘢痕组织,同时存在肌成纤维细胞;中药组和贝复舒组可见成纤维细胞及角膜基质细胞,以及极少量瘢痕组织。角膜混浊度分级:与空白组比较,模型组在造模后7、14、28 d的角膜混浊分级均明显升高(P<0.01),呈时间相关性,但28 d时模型组的角膜混浊分级出现回落,与模型组相比,中药组和贝复舒组在造模后7、14、28 d的角膜混浊分级均明显降低(P<0.01),且呈时间相关性(P<0.01)。Masson切片染色:在造模后28 d,空白组可见角膜上皮整齐连续排列,基质层纤维排列整齐,染色致密,厚度均一;模型组伤口处角膜上皮细胞不连续分布,且角膜前部基质浅蓝色胶原纤维排列紊乱,呈不连续状态,厚度不一,角膜后部基质浅蓝色胶原纤维排列较混乱,呈紊乱纤维束;中药组可见角膜前部基质浅蓝色胶原纤维较模型组排列有序,稍疏松,呈连续状态,角膜后部基质浅蓝色胶原纤维排列有序;贝复舒组见角膜前部基质浅蓝色胶原纤维较模型组排列有序,稍疏松,呈连续状态,仍存在纤维束,角膜后部基质浅蓝色胶原纤维排列有序。Western blot检测:与模型组比较,中药组、贝复舒组的角膜组织中TGF-β1、Smad3、α-SMA蛋白表达在7、14、28 d时均显著降低(P<0.05)。结论除风益损汤能促进角膜损伤修复,抑制角膜瘢痕化,并对TGF-β1、Smad3、α-SMA蛋白产生影响。

老年患者NLR、TGF-β1、Cav-1水平与颅内大动脉急性闭塞机械取栓术后出血风险的关系

目的探讨老年患者中性粒细胞/淋巴细胞比值(NLR)、转化生长因子-β1(TGF-β1)、陷窝蛋白1(Cav-1)与颅内大动脉急性闭塞机械取栓术后出血风险的关系。方法选取2017年9月—2021年12月河北中石油中心医院行颅内大动脉急性闭塞机械取栓术的老年颅内大动脉急性闭塞患者120例。根据术后是否出血分为出血组和非出血组,比较2组患者临床资料和NLR、TGF-β1、Cav-1水平差异。采用多因素Logistic回归分析评价术后出血的影响因素;采用受试者工作特征(ROC)曲线评价NLR、TGF-β1、Cav-1预测术后出血的价值。结果出血组美国国立卫生研究院卒中量表(NIHSS)评分为(19.91±2.28)分,NLR和Cav-1分别为(7.20±1.12)和(19.29±5.53)ng·mL^(-1),均高于非出血组(P<0.05);Alberta卒中项目早期电子计算机断层扫描评分(ASPECTS)评分为(5.40±0.77)分,TGF-β1为(20.20±8.28)pg·mL^(-1),均低于非出血组(P<0.05)。多因素Logistic回归分析结果显示,NIHSS评分、NLR是患者术后出血的危险因素(P<0.05),ASPECTS评分是患者术后出血的保护因素(P<0.05)。ROC曲线分析结果显示,基于ASPECTS评分、NIHSS评分、NLR建立的预测模型预测术后出血的曲线下面积为0.851,敏感性和特异性分别为64.00%和84.00%。结论NLR、Cav-1、TGF-β1均与老年患者颅内大动脉急性闭塞机械取栓术后出血有关。基于ASPECTS评分、NIHSS评分、NLR建立的预测模型在预测患者术后出血中有一定的应用价值。