Saikosaponin-d regulates angiogenesis in idiopathic pulmonary fibrosis through angiopoietin/Tie-2 pathway.

OBJECTIVE: Idiopathic pulmonary fibrosis (IPF) is considered as a chronic interstitial lung disease with underlying mechanism of IPF remaining unclear, while there are no definitive treatment options. In recent years, scientists have gradually paid attention to the influence of angiogenesis on IPF. Because IPF is a progressive with microvascular remodeling disorder, scientists have postulated that angiogenesis may also be one of the initiating and contributing factors of the disease. Bupleurum is a common natural Chinese herbal medicine with antibacterial, anti-inflammatory, anti-tumor and other pharmacological effects. As the most important active monomer of Bupleurum, Saikosaponin-d (SSd) is a new discovery with anti-pulmonary fibrosis (PF) activity. This study attempts to investigate the role of SSd in the interference of PF through regulation of angiogenesis in IPF through Angiopoietin (Angpt) /Tie receptor 2 (Tie2) pathway. METHODS: Randomly, we allocated C57BL/6 mice into four groups (n = 20 in each group). Afterwards, establishment of IPF model was accomplished via intratracheal administration of bleomycin (BLM, 5 mg/kg), while corresponding drug intervention was given accordingly. On 3rd, 7th, 14th and 28th days after modeling, we performed histopathological examination through staining. Meanwhile, immunohistochemistry (IHC) of PF and the expression of related factors were observed, while Ang/Tie2 pathway was assessed by ELISA with the effect of SSd on angiogenesis related proteins in IPF being explored with IHC and Western Blot technique. RESULTS: Our results showed that SSd could reduce inflammation and PF levels in lung tissue of experimental mice, while levels of angiogenesis-related factors, namely Tie-2, Ang-1 and ANGPT2 (Ang-2), fibrosis- associated factors like Alpha-smooth muscle actin (α-SMA), collagen-I and hydroxyproline in SSd and dexamethasone (DXM) mice were significantly reduced at each time point compared to BLM (p < 0.01). Additionally, we discovered substantial decreased expressions of Ang-1, Ang-2, Tie-2, α-SMA and collagen-I at protein level in SSd and DXM mice at each time point compared to BLM (p < 0.05). Besides, insignificant differences were observed between SSd and DXM groups (p > 0.05). CONCLUSION: This study has demonstrated that SSd could down-regulate the expression of ANG-1, Ang-2 and Tie2 in the Ang/Tie2 pathway, and may reduce lung inflammation and PF in BLM-induced mice via inhibition of angiogenesis.

SIgA in various pulmonary diseases.

Secretory immunoglobulin A (SIgA) is one of the most abundant immunoglobulin subtypes among mucosa, which plays an indispensable role in the first-line protection against invading pathogens and antigens. Therefore, the role of respiratory SIgA in respiratory mucosal immune diseases has attracted more and more attention. Although the role of SIgA in intestinal mucosal immunity has been widely studied, the cell types responsible for SIgA and the interactions between cells are still unclear. Here, we conducted a wide search of relevant studies and sorted out the relationship between SIgA and some pulmonary diseases (COPD, asthma, tuberculosis, idiopathic pulmonary fibrosis, COVID-19, lung cancer), which found SIgA is involved in the pathogenesis and progression of various lung diseases, intending to provide new ideas for the prevention, diagnosis, and treatment of related lung diseases.

[Saikosaponin D inhibits proliferation and collagen production of human embryonic lung fibroblasts by regulating TGF-ß1/Smads signaling pathway].

Objective To investigate the effect of saikosaponin D (SSD) on the proliferation and transformation of human embryonic lung fibroblasts (HELFs) induced by transforming growth factor-beta 1 (TGF-ß1) and the regulation of signal pathway of TGF-ß1/Smads family. Methods HELFs were cultured in vitro and divided into 5 groups: a control group, 1 ng/mL TGF-ß1-induced group, 1 ng/mL TGF-ß1 combined with 0.5 µmol/L SSD treatment group, 1 ng/mL TGF-ß1 combined with 1 µmol/L SSD treatment group, and 1 ng/mL TGF-ß1 combined with 2 µmol/L SSD treatment group. Cell viability of HELFs was detected by CCK-8 assay. The expression of Smad2, Smad3 and Smad7 mRNA were detected by real-time fluorescence quantitative PCR. The protein levels of α-smooth muscle actin (α-SMA), type 1 collagen (Col1), Smad2, Smad3, phosphorylated Smad2 (p-smad2), p-smad3 and Smad7 were assessed by Western blot analysis. Results Compared with the control group, TGF-ß1-induced group showed the apparently increased proliferation ability, the increased protein levels of Col1 and α-SMA, the significantly increased mRNA and protein phosphorylation levels of Smad2 and Smad3, and the significantly decreased mRNA and protein expression of Smad7. Compared with the TGF-ß1-induced group, the cell proliferation of HELFs in different concentrations of SSD treatment groups was reduced, which could reverse the changes of the above indicators in a dose-dependent manner. Conclusion SSD plays an important role in anti-pulmonary fibrosis by regulating TGF-ß1/Smads signaling pathway.

IL-33 Can Promote the Process of Pulmonary Fibrosis by Inducing the Imbalance Between MMP-9 and TIMP-1.

IL-33 played an important role in inflammatory diseases as evidenced by their high levels of expression in diseased tissues. Previous studies showed that IL-33/ST2L signal transduction pathway participated in epithelial-mesenchymal transition (EMT) of A549 cells. Cytokine IL-1ß can increase the expression of MMPs by activating NF-kB. The excessive or inappropriate expression of MMP-9 may randomly and non-selectively destroy the extracellular matrix. TIMP-1 (tissue inhibitor of MMP-9) effects on ebb and flow of ECM by inhibiting activation of MMP-9. Therefore, IL-33 may take part in the process of pulmonary fibrosis by regulating expressions of MMP-9 and TIMP-1. To explore the acting mechanism of IL-33 in pulmonary fibrosis, proliferation of the human embryonic lung fibroblasts and expressions of related signal molecules was analyzed in vitro. We cultured HELF cells and stimulated HELF with rhIL-33 at different time points (24, 48, 72 h) and different concentrations respectively. The expression of the receptor ST2L was analyzed by RT-PCR and the proliferative rate of HELF was tested by MTT. The expressions of collagen IV, MMP-9, TIMP-1, and critical signal transducer TRAF-6 and NF-kappaB were tested by Western blotting. The rhIL-33 can promote proliferation of HELF and the concentration of 10 ng/ml was most significant at 72 h (P < 0.05). Hence, this experiment chose 10 ng/ml as stimulated concentration at following experiments. The expressions of collagen IV, MMP-9, TIMP-1, TRAF-6, and NF-kappaB increased and then reduced in protein levels at different time points (0, 6, 12, 24, 48, 72 h) (P < 0.05). IL-33 participates in the production of profibrotic cytokines and formation of mesenchymal substances in early inflammatory responses of pulmonary fibrosis. IL-33 can regulate deposition of ECM and promote the process of pulmonary fibrosis by inducing the imbalance between MMP-9 and TIMP-1.

NF-κB dependent up-regulation of TRPC6 by Aß in BV-2 microglia cells increases COX-2 expression and contributes to hippocampus neuron damage.

The deposition of amyloid ß-protein (Aß) has been involved in neurodegeneration of Alzheimer's disease (AD). Besides Aß plaques and neuronal loss, microglia activation is also common in AD patient brains, suggesting its important role in the pathogenesis of AD. Although activation of microglia by Aß plaques has been demonstrated, the mechanism underlying it is still largely unclear. Here, we found that TRPC6 has a crucial role in microglia activation by Aß. Aß up-regulates the level of TRPC6 via NF-κB in BV-2 microglia and increases the expression of pro-inflammatory factors and oxidative enzyme, COX-2. Knock-down of TRPC6 reduces the Aß-induced expression of pro-inflammatory factors and COX-2 and the damage of hippocampus neurons. Furthermore, inhibition of COX-2 also protects hippocampus neurons from Aß-induced inflammatory damage. Collectively, our studies suggest that Aß increase the expression of TRPC6 via NF-κB in BV-2 microglia and promotes the production of COX-2, which induces hippocampus neuron damage.

Anti-Inflammatory Effects of Schisandrin B on LPS-Stimulated BV2 Microglia via Activating PPAR-γ.

Schisandrin B (Sch B), a dibenzocyclooctadiene lignan isolated from Schisandra chinensis (Turcz.) Baill, has been shown to have anti-inflammatory effect. The purpose of this study was to evaluate the effect of Sch B on LPS-induced inflammation in microglia and to investigate the molecular targets of Sch B. BV2 cells were stimulated by LPS in the presence or absence of Sch B. The results showed that the levels of TNF-α, IL-6, IL-1ß, and PGE2 upregulated by LPS were significantly suppressed by Sch B. LPS-induced NF-κB activation was also inhibited by Sch B. Furthermore, Sch B was found to upregulate the expression of PPAR-γ in a concentration-dependent manner. In addition, the inhibition of Sch B on TNF-α, IL-6, IL-1ß, and PGE2 production were reversed by PPAR-γ antagonist GW9662. In conclusion, these results suggested that Sch B inhibited LPS-induced inflammatory response by activating PPAR-γ.

[Downregulation of miR-18a or miR-328 inhibits the invasion and migration of lung adenocarcinoma A549 cells].

Objective To investigate the expressions of miR-18a and miR-328 in lung adenocarcinoma A549 cells, and explore the effect of miR-18a or miR-328 on invasion and migration of A549 cells. Methods The expressions of miR-18a and miR-328 in A549 cells were detected by real-time quantitative PCR. Then the specific miR-18a or miR-328 inhibitor sequences were transfected into A549 cells to downregulate the expression of miR-18a or miR-328. The invasion and migration abilities of A549 cells were evaluated by Transwell(TM) assay. Results The miR-18a and miR-328 were overexpressed in A549 cells. And with the corresponding inhibitors being transfected, the expressions of miR-18a and miR-328 in A549 cells were downregulated. In addition, TranswellTM assay showed that decreased expression of miR-18a or miR-328 significantly inhibited the invasion and migration of A549 cells. Conclusion Downregulation of miR-18a or miR-328 can inhibit the invasion and migration abilities of A549 cells effectively.

IL­33/ST2 pathway in a bleomycin­induced pulmonary fibrosis model.

The present study aimed to investigate the interleukin (IL)­33/ST2 pathway in a model of acute pulmonary fibrosis, and to examine the pathogenesis of pulmonary fibrosis. The pulmonary fibrosis model was established by a single exposure to bleomycin (BLM group) endotracheally to represent idiopathic pulmonary fibrosis, and a control (Cont) group was treated with the same volume of saline. The degrees of acute injury, inflammation and fibrosis were detected using hematoxylin and eosin and Masson's staining. The IL­33, ST2, myeloid differentiation primary response 88 (MyD88) and tumor necrosis factor receptor­associated factor 6 (TRAF6) proteins were detected using Western blotting. The serum levels of IL­4 and IL­13 were detected using an enzyme­linked immunosorbent assay. The results indicated that, compared with the Cont group, there were significant differences in the alveolitis scores in the BLM group on days 3, 7, 14 and 28 (P<0.01). The grades of fibrosis were also significantly different on days 7, 14 and 28 (P<0.01). On examining the dynamic protein expression levels of IL­33, ST2, MyD88 and TRAF6, the expression of IL­33 in the BLM group increased initially, and then decreased gradually following a peak on day 7. The significant differences between the BLM and Cont groups were observed on days 3 and 7 (P<0.05). Compared with the Cont group, the protein levels of ST2, MyD88 and TRAF6 in the BLM group exhibited an increasing trend from day 3, with significant differences, compared with the Cont group, on days 3, 7, 14 and 28 (P<0.05). On examination of the serum levels of IL­4 and IL­13 in each group, the levels of IL­4 and IL­13 in BLM group remained higher from day 7, with peaks on day 28, and were significantly different, compared with the Cont group, on days 7, 14 and 28 (P<0.05). In conclusion, the IL­33/ST2 signaling pathway was found to be involved in the rodent model of pulmonary fibrosis induced by bleomycin.

The ratio of Th17/Treg cells as a risk indicator in early acute respiratory distress syndrome.

INTRODUCTION: Recent studies have revealed that lung inflammation mediated by CD4+ T cells may contribute to the pathogenesis of acute respiratory distress syndrome (ARDS). The imbalance between CD4 + CD25 + Foxp3 + regulatory T (Treg) cells and T helper (Th)17 cells has been found in a number of different inflammation and autoimmune diseases, while the role of the Th17/Treg balance in ARDS remains largely unknown. The aim of this study was to investigate the Th17/Treg pattern and its impact on disease severity and outcomes in patients with ARDS. METHODS: This prospective, observational study enrolled 79 patients who fulfilled the Berlin definition of ARDS and 26 age- and sex-matched healthy controls. Circulation Th17 and Treg cell frequencies were analyzed by flow cytometry, and the expressions of Th17- and Treg-related cytokines in serum were measured by enzyme-linked immunosorbent assay (ELISA). Acute Physiologic and Chronic Health Evaluation (APACHE) II score, Sequential Organ Failure Assessment (SOFA) score, and the Lung Injury Score were also calculated at enrollment. RESULTS: Within 24 hours after the onset of ARDS, the changes of peripheral circulating Th17 and Treg cell frequencies gradually increased from mild to severe ARDS. Th17/Treg ratio was positively correlated with APACHE II score, SOFA score, and Lung Injury Score, while negatively correlated with PaO2/FiO2. The areas under the receiver operating characteristic (AUC) curves of Th17/Treg ratio for predicting 28-day mortality in ARDS patients was higher than that of APACHE II score, SOFA score, Lung injury score, as well as PaO2/FiO2. Using a Th17/Treg ratio cutoff value of >0.79 to determine 28-day mortality, the sensitivity was 87.5% with 68.1% specificity. Multivariate logistic regression showed Th17/Treg ratio >0.79 (odds ratio = 8.68, P = 0.002) was the independent predictor for 28-day mortality in patients with ARDS. Finally, cumulative survival rates at 28-day follow-up also differed significantly between patients with Th17/Treg ratio >0.79 and ≤0.79 (P <0.001). CONCLUSIONS: The Th17/Treg imbalance favoring a Th17 shift represents a potential therapeutic target to alleviate lung injury and a novel risk indicator in patients with early ARDS.

ß-catenin induces A549 alveolar epithelial cell mesenchymal transition during pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease characterized by expansion of the fibroblast and myofibroblast population and extracellular matrix deposition. Although the pathogenic mechanisms of IPF remain to be fully elucidated, there is emerging evidence that fibroblasts and myofibroblasts may be derived partially from alveolar epithelial cells by epithelial­mesenchymal transition (EMT). In the present study, A549 cells were treated with different concentrations of Wnt1 and the results indicated that the mRNA and protein expression levels of vimentin, α­smooth muscle actin (α­SMA) and collagen Ⅰ gradully increased and those of E­cadherin gradully decreased in a concentration­dependent manner. Furthermore, the A549 cells were transfected with ß­catenin plasmid cells, revealing phenotypic changes in the cells from a pebble to a fusiform shape. The mRNA and protein expression levels of of vimentin, α­SMA and collagen Ⅰ increased significantly, whereas those of E­cadherin decreased significantly. The present study examined the roles of alveolar epithelial cell injury and profibrogenic cytokine release in EMT and their association with the Wnt/ß­catenin signaling pathway in a mouse model of bleomycin­induced pulmonary fibrosis. Bronchoalveolar fluid was obtained 7 days after treatment with bleomycin and the A549 cells were incubated for 48 h. An increase in the expression levels of the mesenchymal markers, α­SMA, vimentin and collagen Ⅰ, and a concomitant decrease in the expression of the epithelial marker, E­cadherin were observed. This corresponded with an increased expression of ß­catenin. When the A549 cells were infected with a lentivirus expressing ß­catenin shRNA, no significant increase was observed in the expression of the mesenchymal cell markers and the expression of E­cadherin did not decrease. These findings demonstrated that activation of the Wnt signaling pathway was capable of inducing an EMT program in the lung epithelial cells through ß­catenin and that injured alveolar epithelium activated the Wnt/ß­catenin signaling pathway, thereby inducing the expansion of the fibroblast/myofibroblast population through EMT. These results suggested that ß­catenin was involved in the formation of lung fibrosis and may provide a theoretical basis for the treatment of IPF.

Dexamethasone attenuates bleomycin-induced lung fibrosis in mice through TGF-ß, Smad3 and JAK-STAT pathway.

In order to find the possible mechanism of Dexamethasone (Dex) during curing fibrosis, the bleomycin (BLM)-induced mice model was used. After fibrosis were induced by BLM, histopathological evaluation and RT-PCR were employed to detect the expression of TGF-ß1, Smad3 and STAT1. It was found that BLM promoted the development of inflammation, leading to severe pulmonary fibrosis with the increasing of TGF-ß1, Smad3 and STAT1. After Dex treatment, the expression of TGF-ß1, Smad3 and STAT1 showed a little higher with alleviation of the fibrosis. Thus it is concluded that there is a possible pathway of mouse pulmonary fibrosis model through TGF-ß, Smad3 and JAK-STAT pathway.

Effect of the Wnt1/ß-catenin signalling pathway on human embryonic pulmonary fibroblasts.

Idiopathic pulmonary fibrosis (IPF) is a fibrotic lung disease associated with a high rate of mortality, characterised by an accumulation of fibroblasts/myofibroblasts in the fibroblastic foci (FF) and by an excessive deposition of extracellular matrix (ECM) in the lung parenchyma. The pathogenesis of this fatal disorder remains unclear. Previous evidence suggests that myofibroblasts are key effectors of the deposition of ECM. In the present study, human embryonic pulmonary fibroblast (HEPF) cells were incubated with different concentrations of Wnt1. The present study revealed that cell proliferation improved following stimulation using different concentrations of Wnt1 in a concentration-dependent manner. When the concentration exceeded 20 µg/l, cell proliferation was significant (P<0.05) and the cell expression of α-SMA, vimentin and collagen I mRNA, as well as protein expression, significantly increased (P<0.05). Bronchoalveolar lavage fluid (BALF) was then obtained from bleomycin (BLM)-induced models of pulmonary fibrosis. HEPF cells were cultured with Dulbecco's modified Eagle's medium plus BALF. The mRNA and protein expression of α-SMA, vimentin and collagen I significantly increased and these increases were associated with ß-catenin. Furthermore, following being infected with the lentivirus expressing ß-catenin shRNA, HEPF cells were cultured with BALF. However, the mRNA and protein expression of α-SMA, vimentin and collagen I did not increase significantly. The present study suggested that the Wnt1/ß-catenin signalling pathway can promote HEPF cell proliferation and induced HEPF cells can change into myofibroblasts and promote ECM deposition. These findings may provide a theoretical basis for the treatment of IPF.

Pathogenesis pathways of idiopathic pulmonary fibrosis in bleomycin-induced lung injury model in mice.

Our objective was to investigate the pathogenesis pathways of idiopathic pulmonary fibrosis (IPF). Bleomycin (BLM) induced animal models of experimental lung fibrosis were used. CHIP assay was executed to find the link between Smad3 and IL-31, and the expressions of TGF-ß1, Smad3, IL-31 and STAT1 were detected to find whether they were similar with each other. We found that in the early injury or inflammation of the animal model, BLM promoted the development of inflammation, leading to severe pulmonary fibrosis. Then the expression of TGF-ß1 and Smad3 increased. Activated Smad3 bound to the IL-31 promoter region, followed by the activation of JAK-STAT pathways. The inhibitor of TGF-ß1 receptor decreased the IL-31 expression and knocking-down of IL-31 also decreased the STAT1 expression. We conclude that there is a pathway of pathogenesis in BLM-induced mouse model that involves the TGF-ß, IL-31 and JAKs/STATs pathway.

Inhibition of epithelial-mesenchymal transition in A549 cell by transfected Napsin A.

BACKGROUND: Epithelial-mesenchymal transition is a cellular process characterized by the loss of cell adhesion, inhibition of E-cadherin expression, and increased cell mobility. Cells without Napsin A are susceptible to transition. Further studies are required to investigate whether this transition can be reversed by restoration of Napsin A. METHODS: A Napsin A expression vector PLJM1-Napsin A plasmid was constructed and then transfected into the epithelial cell line A549 by lentivirus transfection to obtain A549-PLJM1-Napsin A cell line. Cell proliferation was assayed by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide and cell cycle was measured by flow cytometry. The E-cadherin, type I collagen, and focal adhesion kinase mRNA level was detected by reverse transcription-polymerase chain reaction. The Napsin A, E-cadherin, type I collagen, and focal adhesion kinase protein level in A549 cells was detected by Western blotting. RESULTS: Transforming growth factor-b1 induced epithelial-mesenchymal transition in A549 cells, as demonstrated by significant reduction of E-cadherin mRNA and protein levels (P < 0.01) as well as up-regulation of type I collagen (P < 0.01). Transfection of Napsin A in A549 cells can partially block the transforming growth factor-b1-regulated expression of E-cadherin and type I collagen (P < 0.01). In addition, transforming growth factor-b1-induced cell proliferation was inhibited by Napsin A (P < 0.01). Further study demonstrated that Napsin A caused G(0)/G(1) arrest and inhibited the expression of focal adhesion kinase (P < 0.01), a key protein in the integrin signaling pathway, in the in vitro epithelial-mesenchymal transition model. CONCLUSIONS: Sustained Napsin A expression in A549 cells can inhibit the transforming growth factor-b1-induced epithelial-mesenchymal transition. This may be due to the Napsin A-mediated inhibition of focal adhesion kinase expression and integrin signaling pathway.

[Therapeutic effects and mechanism of saikosaponin-d in mice with bleomycin-induced pulmonary fibrosis].

OBJECTIVE: To study the therapeutic effects and mechanism of saikosaponin-d (SSd) in mice with bleomycin (BLM)-induced pulmonary fibrosis. METHODS: According to the random number table, 180 mice were randomly divided into 5 groups. Four groups were pulmonary fibrosis models. Fibrosis model mice were established by intratracheal injection of bleomycin (5 mgxkg(-1)). They were BLM, DXM, SSd and SSd + DXM groups (n = 40 each). At 1 hour post-modeling, DXM, SSd and SSd + DXM groups were injected ip with dexamethasone (DXM, 5 mgxkg(-1)xd(-1), 0.1 ml), SSd (1.8 mgxkg(-1)xd(-1), 0.18 ml), DXM + SSd (0.28 ml) respectively qd until Day 28. BLM group was similarly dosed with normal saline. In addition, a normal control group (NC group, n = 20) treated likewise. The mice were anesthetized and sacrificed at Days 3, 7, 14, 28 for harvests of serum and lung tissue samples. The conventional histopathological changes of lung tissue were observed. Except for NC group, modeling groups of mice were used to observe the natural survival rate. Such indices as superoxide dismutase (SOD) and malonaldehyde (MDA) were examined both in lung tissue and serum samples. And hydroxyproline (HYP) was tested only in lung tissue. RESULTS: SSd could markedly increase the survival rate (80.0% in SSd and SSd + DXM groups vs 50.0% in BLM group, P < 0.05) and reduce alveolitis and fibrosis in mice. In comparison with BLM group, the levels of HYP of three treatment groups (DXM, SSd and SSd + DXM) in lung tissue was significantly lower (P < 0.05) at Days 14 and 28. The levels of MDA both in serum and lung tissue were significantly lower at Days 3, 7 and 14 (P < 0.05). The serum level of SOD was significantly higher at Days 3, 7 and 14 while the level of SOD in lung tissue was significantly higher at Days 3 and 7 (P < 0.05, P < 0.01). CONCLUSIONS: SSd has marked therapeutic effects upon bleomycin-induced pulmonary fibrosis in mice. And the mechanism may be associated with its anti-lipid peroxidation effect.

[Effect of Napsin A transfection into type II alveolar epithelial cells on pulmonary fibrosis].

OBJECTIVE: To study the in vitro effect and mechanism of Napsin A gene transfection into type II alveolar epithelial cells on pulmonary fibrosis. METHODS: A recombinant lentiviral plasmid PLJM1-Napsin A was constructed and transfected into human type II alveolar epithelial cell line A549. The model of pulmonary fibrosis was established by the in vitro stimulation of A549 cells by transforming growth factor beta-1 (TGF-ß1). The morphological changes were observed continuously under inverted microscopy. The proliferation of transgenic and non-transgenic cells was detected by MTT. To observe the degree of epithelial-mesenchymal transition (EMT) by TGF-ß1 intervening A549 cells, the expressions of E-cadherin and fibronectin were determined by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. Lastly the protein expression of focal adhesion kinase (FAK) was detected by Western blot to investigate the mechanism. RESULTS: The result of sequencing the recombinant lentiviral plasmid PLJM1-Napsin A was the same as the design sequence. Napsin A mRNA and protein were expressed in transgenic A549 cells (P<0.01). The model of pulmonary fibrosis was established successfully based on the morphology of transformed interstitial cell. As compared with the control group, the proliferation rate of transgenic cells decreased significantly (P<0.05). The mRNA and protein expression of E-cadherin significantly decreased in the model of pulmonary fibrosis (P<0.01), while the expression of fibronectin markedly increased (P<0.01). But the change rate of transgenic cells decreased (P<0.01, P<0.05). The expression of FAK was significantly elevated after the stimulation of TGF-ß1 (P<0.01). But the upward trend of the transgenic cells was smaller as compared with the control group (P<0.01). CONCLUSION: Pulmonary fibrosis may be suppressed by the transfection of Napsin A gene into type II alveolar epithelial cells. And the mechanism may be through the inhibition of integrin signal transduction.

[Human lamada-interferon expressed in BHK-21 cell line and its bioactivity].

AIM: To construct the eukaryotic expressing vector PCAGG -HuIFN-lambda1 and PCAGG-HuIFN-lambda2 and to study the biological activity of HuIFN-lambda1and HuIFN-lambda2. METHODS: The cDNA fragment encodding HuIFN-lambda1 and HuIFN-lambda2 was amplified from the total RNA extracted from virus-induced HeLa cells by RT-PCR. Then it was cloned into the eukaryotic expressing vector PCAGG-EGFP. The recombinant was transfected into BHK-21 cells. VSV*GFP-A549 system was used to measure the anti-virus activity.The constructed cell line MDBK-Mxp-Luc was used to study the characteristics of MxA protein induced by the products of PCAGG-HuIFN-lambda1 and PCAGG-HuIFN-lambda2. RESULTS: The recombinant vector HuIFN-lambda1-PMD18-T Vector was enzymed by Sac I and Xho I while HuIFN-lambda2-PMD18-T Vector was enzymed by Sac I and Sal I. The fragments were both 610 bp and they were consistent with nucleotide sequences reported in GenBank. The anti-virus activity of protein expressed by PCAGG-HuIFN-lambda1 and PCAGG-HuIFN-lambda2 was 10(4) IU/mL and 10(2) IU/mL, respectively. The protein expressed by PCAGG-HuIFN-lambda1 and PCAGG-HuIFN-lambda2 induced the expression of the anti-virus protein MxA. The expression of protein MxA induced by PCAGG-HuIFN-lambda1 increased with the passage of time, reaching the peak during 9 to 12 hours and disappearing in 24 hours. CONCLUSION: The eukaryotic expressing vector of PCAGG-HuIFN-lambda1 and PCAGG-HuIFN-lambda2 has been successfully constructed and transiently expressed in BHK-21 cells. The anti-virus activity of the products is closely correlated with inducing the expression of anti-virus protein MxA.

Effect of NaCl and Helicobacter pylori vacuolating cytotoxin on cytokine expression and viability.

AIM: To determine whether Helicobacter pylori (H pylori) vacuolating cytotoxin (VacA) regulates release of pro-inflammatory cytokines (IL-1beta, IL-8, TNF-alpha, and IL-6) or alters gastric epithelial cell viability and to determine whether NaCl affects these VacA-induced changes. METHODS: Vacuolating activity was determined by measuring the uptake of neutral red into vacuoles of VacA-treated human gastric epithelial (AGS) cells. AGS cell viability was assessed by direct cell counting. Specific enzyme-linked immunosorbent assays (ELISA) and reverse transcriptase-polymerase chain reaction(RT-PCR) were performed to examine the effects of H pylori VacA and NaCl on cell pro-inflammatory cytokine production in AGS cells. Immunohistochemical staining of gastric tissue from Mongolian gerbils was used to confirm VacA-induced pro-inflammatory cytokine production and the effects of NaCl on this VacA-induced response. RESULTS: Addition of VacA alone reduced AGS cell viability (P < 0.05), and this reduction was enhanced by high doses of NaCl (P < 0.05). VacA alone induced expression of TNF-alpha, IL-8 and IL-1beta, while NaCl alone induced expression of TNF-alpha and IL-1beta. Changes in mRNA levels in the presence of both VacA and NaCl were more complicated. For the case of TNF-alpha, expression was dose-dependent on NaCl. IL-6 mRNA was not detected. However, low levels of IL-6 were detected by ELISA. Positive immunohistochemical staining of IL-1, IL-6, and TNF-alpha was found in gastric tissue of H pylori-infected gerbils fed with either a normal diet or a high salt diet. However, the staining of these three cytokines was stronger in H pylori-infected animals fed with a 5 g/kg NaCl diet. CONCLUSION: VacA decreases the viability of AGS cells, and this effect can be enhanced by NaCl. NaCl also affects the production of pro-inflammatory cytokines induced by VacA, suggesting that NaCl plays an important role in H pylori-induced gastric epithelial cell cytotoxicity.

[Detection of telomerase activity in bronchoscopic brush-off samples in patients with lung cancer].

BACKGROUND AND OBJECTIVE: Recent studies have shown that activation of telomerase plays an important role in carcinogenesis. However, there was few report on the level of telomerase activity in small samples from the patients with lung cancer. This study was designed to investigate the diagnostic significance of the detection of telomerase activity in bronchoscopic brush-off cells from the patients with lung cancer. METHODS: Telomeric repeat amplification protocol(TRAP)-based telomerase polymerase chain reaction(PCR)-enzyme-linked immunosorbent assay (ELISA) TRAP-PCR-silver staining were employed to detect telomerase activity in 56 samples of brushing cells from the patients with lung cancer and 10 samples with inflammation. RESULTS: The positive rate of telomerase activity in 56 biopsy samples of lung cancer group was significantly higher than that in inflammation group (P < 0.001). The sensitivity, specificity, and accuracy of detection of telomerase activity was 87.5%, 83.3%, and 86.3%, respectively. There was no significant difference in the positive rate of telomerase activity between central lung cancer and peripheral lung cancer. Positive rate of detection of telomerase activity in bronchoscopic brush-off cells was 46.4%. The positive rate of telomerase activity detected in TRAP-PCR-ELISA was higher than that detected in TRAP-silver staining, but the significant difference was not found. It was found that samples with low absorbing value detected in the quantified way would show weak positive with less ladder bands or vague ladder bands if detected in the latter way. CONCLUSION: The telomerase activity may be a good marker for diagnosis of lung cancer. Combined with cytologic measure, it is possible to raise the early diagnostic rate of lung cancer.