Chrysin inhibited epithelial-mesenchymal transition of type Ⅱ alveolar epithelial cell by regulating NF-κB/Twist 1 signaling pathway / 中国中药杂志

This study aimed to assess whether chrysin(ChR) can inhibit epithelial-mesenchymal transition(EMT) of type Ⅱ alveolar epithelial cell and produce anti-pulmonary fibrosis effect by regulating the NF-κB/Twist 1 signaling pathway. Sixty rats were randomly divided into the control group, the bleomycin(BLC) group, BLC+ChR(50 mg·kg~(-1)) group and BLC+ChR(100 mg·kg~(-1)) group, with 15 rats in each group. The pulmonary fibrosis model was induced by intratracheal injection of BLC(7 500 U·kg~(-1)). Rats were orally administered with different doses of ChR after BLC injection for 28 days. The cells were divided into control group, TGF-β1 group(5 ng·mL~(-1)), and TGF-β1+ChR(1, 10, 100 μmol·L~(-1)) groups. The type Ⅱ alveolar epithelial cells were treated with TGF-β1 for 24 h, and then treated with TGF-β1 for 48 h in the presence or absence of different doses of ChR(1, 10 and 100 μmol·L~(-1)). The morphological changes and collagen deposition in lung tissues were analyzed by HE staining, Masson staining and immunohistochemistry. The mRNA and protein expression levels of collagen Ⅰ, E-cadherin, zonula occludens-1(ZO-1), vimentin, alpha smooth muscle actin(α-SMA), inhibitor of nuclear factor kappa B alpha(IκBα), nuclear factor-kappa B p65(NF-κB p65), phospho-NF-κB p65(p-p65) and Twist 1 in lung tissues and cells were detected by qPCR and Western blot, respectively. The animal experiment results showed that as compared with the BLC group, after administration of ChR for 28 days, bleomycin-induced pulmonary fibrosis in rats was significantly relieved, collagen Ⅰ expression in lung tissues was significantly reduced(P<0.05 or P<0.01), and EMT of alveolar epithelial cells was obviously inhibited [the expression levels of E-cadherin and ZO-1 were increased and the expression levels of vimentin and α-SMA were decreased(P<0.05 or P<0.01)], concomitantly with significantly reduced IκBα and p65 phosphorylation level in cytoplasm and decreased NF-κB p65 and Twist 1 expression in nucleus(P<0.05 or P<0.01). The cell experiment results showed that different doses of ChR(1, 10 and 100 μmol·L~(-1)) significantly reduced TGF-β1-induced collagen Ⅰ expression(P<0.05 or P<0.01), significantly inhibited EMT of type Ⅱ alveolar epithelial cells[the expression levels of E-cadherin and ZO-1 were increased and the expression levels of vimentin and α-SMA were decreased(P<0.05 or P<0.01)], and inhibited IκBα and p65 phosphorylation in cytoplasm and down-regulated NF-κB p65 and Twist 1 expression in nucleus induced by TGF-β1(P<0.05 or P<0.01). The results suggest that ChR can reverse EMT of type Ⅱ alveolar epithelial cell and alleviate pulmonary fibrosis in rats, and its mechanism may be associated with reducing IκBα phosphorylation and inhibiting NF-κB p65 phosphorylation and nuclear transfer, thus down-regulating Twist 1 expression.

Effect of small dose capsaicin for treatment of pulmonary fibrosis in mice and its mechanism / 中国应用生理学杂志

To observe whether the mechanism of small dose capsaicin (Cap) against pulmonary fibrosis in mouse is mediated by agitating transient receptor potential vanilloid 1 (TRPV1). Methods: A total of 60 BALB/c mice were randomly divided into control (CON) group, bleomycin (BLM)group, Cap (0.5, 1,2 mg/kg) groups and Cap (2 mg/kg) plus SB-452533 (2.5 mg/kg) group. C57BL/6 mice were intratracheally injected with 3.5 mg/kg BLM to induce pulmonary fibrosis model. Animals for drugs treatment received daily drug via subcutaneous injection for 21 days. The morphological changes and collagen deposition in lung tissues were analysed by HE staining, Masson staining and immunohistochemistry. The concentration of calcitonin gene-related peptide (CGRP) in plasma was determined by ELISA. The mRNA and (or) proteins levels of α-CGRP, β-CGRP, collagen I, collagen III, E-Cadherin, zonula occludens-1 (ZO-1), vimentin, alpha smooth muscle actin (α-SMA), TRPV1, p-ERK1/2 and eukaryotic initiation factor 3a (eIF3a) were detected by qPCR and (or) Western blot. Compared with the BLM group, small dose Cap significantly reduced bleomycin-induced pulmonary fibrosis in mice and obviously reversed alveolar epithelial cells epithelial-mesenchymal transition (EMT) (the expression of E-cadherin and ZO-1 were increased(P＜0.05 or P＜0.01)and the expression of α-SMA and Vimentin were decreased (P＜0.05 or P＜0.01) after drugs treatment for 21 day, concomitantly with the increase the expressions of TRPV1 and CGRP (P＜0.05 or P＜0.01), and inhibiting ERK1/2 phosphorylation and eIF3a expression (P＜0.05 or P＜0.01). These effects of small dose Cap were abolished in the presence of TRPV1 receptor antagonist SB-452533. The results suggest that small dose Cap can reverse alveolar epithelial cells EMT and alleviate bleomycin-induced pulmonary fibrosis in mice by inhibiting ERK1/2/eIF3asignaling pathway, which is related to agitating TRPV1 receptor and releasing of CGRP.

Effects of EGCG on Proliferation, Cell Cycle and DAPK1 Gene Methylation of Acute Promyelocytic Leukemia NB4 Cell Line / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the effects of epigallocatechin-3-gallate (EGCG) on proliferation and cell cycle of acute promyelocytic leukemia NB4 cell line and to clarify the molecular mechanism.</p><p><b>METHODS</b>NB4 cells were treated with 0,50,75,100 and 125µmol/L of EGCG for 24, 48, 72 and 96 h, respectively. The proliferation level of NB4 cells was measured by CCK-8 assay. The cell cycle progression of NB4 cells was assayed by flow cytometry. The mRNA expression levels of DNMT1, DNMT3a and DAPK1 were detected by RT-PCR. The methylation status of gene was tested by methylation specific PCR, and the expression level of DAPK1 protein was detected by Western blot.</p><p><b>RESULTS</b>The proliferation and cell cycle progression of NB4 cells treated with EGCG were inhibited and showed the characteristic of time-dependent and dose-dependent manner. The expression level of DAPK1 and DNMT3a decreased in NB4 cells treated with EGCG. The expression level of DAPK increased in NB4 cells treated with EGCG, while the methylation of DAPK1 gene decreased.</p><p><b>CONCLUSION</b>EGCG inhibits the proliferation and cell cycle progression of NB4 cells by inhibiting the expression of DNMT1 and DNMT3a and down-regulating the methylation status of DAPK1 gene.</p>

Pharmacokinetics and brain distribution of NMD/TMP-nanoparticles / 中国中药杂志

This study aims to prepare nimodipine/tetramethylpyrazine-loaded poly(D, L-lactide-co-glycolide) dual-drug nanoparticles (NMD/TMP-NPs) and investigate pharmacokinetics and brain distribution to evaluate the possibility of enhancing the drug effect of dual-drug nanoparticles. NMD/TMP-NPs were prepared via W/O/W emulsion solvent evaporation. Entrapment efficiency and drug loading of NMD/TMP-NPs were investigated by ultracentrifugation, and drug release behavior in vitro was studied by dialysis method. The pharmacokinetic and brain distribution were studied in SD mice administered intravenously with NMD/TMP-NPs in comparison with NMD-suspension, NMD/TMP-suspension and NMD-NPs, (NMD-NPs+TMP)-suspension. According to the results, the entrapment efficiency and drug loading of NMD were (79.71±0.73)%, (1.74±0.02)%, those of TMP were (40.26±1.51)% and (4.38±0.16)%. The nanoparticles showed the property of sustained release. On the basis of the major parameters for in vivo pharmacokinetic and brain distribution, t1/2β of NMD-suspension, NMD/TMP-suspension and NMD-NPs, (NMD-NPs+TMP)-suspension, NMD/TMP-NPs were (1.097±0.146), (1.055±0.06), (1.950±0.140), (1.860±0.096), (2.497±0.475） h, CL were (0.778±0.098), (1.133±0.111), (0.247±0.023), (0.497±0.040), (0.297±0.024) h•L-1, AUC0-t in rat plasma were (514.218±60.383), (352.916±33.691), (1 618.429±240.198), (804.110±75.804), (1 349.058±215.497) μg•h•L⁻¹, respectively, and AUC0-t in brain were 0.301 9, 0.624 8, 1.068 6, 1.313 0, 1.046 5 mg•h•L⁻¹, respectively. According to the in vivo study, the pharmacokinetic behavior of NMD were markedly prolonged by adding TMP or prepared dual-drug nanoparticles.

Effect of As₂O₃on Hedgehog Pathway in Chronic Myeloid Leukemia Cells / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To explore the effect of As₂O₃on Hedgehog pathway in chronic myeloid leukemia (CML) cells.</p><p><b>METHODS</b>The apoptosis of K562 cells was detected by MTT method and flow cytometry; the expressions of PTCH and SMO protein and mRNA in Hedgehog pathway were determined by Western blot and real-time PCR, respectively. Retults: The As₂O₃ could induce the apoptosis of K562 cells with optimal concentration 2 µmol/L and optimal time 24 hours. The expressions of PTCH and SMO protein and mRNA in Hedgehog pathway of K562 cells treated with As₂O₃at optimal concentration and optimal time were down-regulated.</p><p><b>CONCLUSION</b>The As₂O₃can down-regulate the expression of PTCH and SMO in Hedgehog pathway.</p>

Vimentin significantly promoted gallbladder carcinoma metastasis / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>The precise molecular mechanisms underlying the gallbladder carcinoma (GBC) metastasis has not been fully elucidated.</p><p><b>METHODS</b>In the present study, metastasis-associated proteins were identified by comparative proteomic analysis. The functional study of the candidate protein vimentin was further investigated. First, a pair of higher and lower metastatic sublines (termed GBC-SD/M3 and GBC-SD, respectively), originated from the same parental cell line, was screened by spontaneous tumorigenicity and metastasis in vivo in animal study and further characterized by metastatic phenotypes analysis in vitro. Subsequently, a proteomic approach comprised two-dimensional gel electrophoresis analysis and mass spectroscopy was used to identify and compare the protein expression patterns between higher metastatic GBC-SD/M3 and lower metastatic GBC-SD cell lines. Then twenty-six proteins were identified.</p><p><b>RESULTS</b>Among the 26 proteins identified, fourteen proteins were up-regulated and 12 proteins were down-regulated in GBC-SD/M3. Vimentin was identified and found to be overexpressed in GBC-SD/M3 as compared with GBC-SD. This result was further confirmed by quantitative PCR and Western blotting analysis. Furthermore, the cell migration and invasion potency of GBC-SD/M3 in vitro was remarkably suppressed after small interference RNA-mediated knockdown of vimentin. Moreover, immunoblot and immunohistochemical analysis on 12 human GBC specimens showed consistently increased vimentin expression in metastases compared with primary tumors.</p><p><b>CONCLUSION</b>Tumor vimentin level may reflect the pathological progression in some GBC and may be a useful marker for predicting tumor metastasis and a therapeutic target for the treatment of GBC patients with metastases.</p>

Molecular and functional comparisons of the vacuolar Na+/H+ exchangers originated from glycophytic and halophytic species / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

A novel vacuolar Na+/H+ exchanger, CgNHX1, was cloned from a halophytic species Chenopodium glaucum by using reverse transcriptase-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) technique. Sequence alignment and phylogenetic analysis of 22 NHX genes from GenBank as well as the new CgNHX1 gene indicate that NHX genes shared a great degree of similarity, regardless of their glycophytic or halophytic origin. Expression of the CgNHX1 gene was induced by NaCl and peaked at 400 mmol/L NaCl. Overexpression of NHX1 genes in rice enhanced their tolerance to salt stress. However, there is no significant difference in salt tolerance among the transgenic rice plants overexpressing the NHX1 genes from either glycophytic or halophytic species. The Na+ content of both the wild type (WT) and transgenic plants increased when exposed to 50 and 100 mmol/L NaCl, and the Na+ concentration in transgenic plants was marginally higher than that of WT. Our data demonstrate that the overexpression of the NHX1 gene from either glycophytic or halophytic species resulted in the enhanced tolerance to salt stress at a similar level, suggesting that NHX gene per se might not be the reason accounting for the difference in salt tolerance between glycophytes and halophytes.

Expression of recombination-activating genes and T cell receptor gene recombination in the human T cell leukemia cell line / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Recent studies have suggested that mature T cells can change their specificity through reexpression of recombination-activating genes (RAG) and RAG-mediated V(D)J recombination. This process is named receptor revision and has been observed in mature peripheral T cells from transgenic mice and human donors. However, whether thebreceptor revision in mature T cells is a random or orientated process remains poorly understood. Here we used the Jurkathuman T cell line, which represents a mature stage of T cell development, as a model to investigate the regulation of Tcell receptor (TCR) gene recombination.</p><p><b>METHODS</b>TCR Dbeta-Jbeta signal joint T cell receptor excision DNA circles (sjTRECs) were determined by nested and seminested PCR. Double-strand DNA breaks at recombination signal sequences (RSSs) in the TCRVbeta chain locus were detected by ligation-mediated-PCR. Further analysis of the complementarity-determining region 3 (CDR3) size of the TCRVbeta chain was examined by the TCR GeneScan technique.</p><p><b>RESULTS</b>RAG1, RAG2, and three crucial components of the nonhomologous DNA end-joining (NHEJ) pathway were readily detected in Jurkat. Characteristics of junctional diversity of Dbeta2-Jbeta2 signal joints and ds RSS breaks associated with the Dbeta2 5' and Dbeta 2 3' sites were detected in DNA from Jurkat cells. CDR3 size and the gene sequences of the TCRVbeta chain did not change during cell proliferation.</p><p><b>CONCLUSIONS</b>RAG1 and RAG2 and ongoing TCR gene recombination are coexpressed in Jurkat cells, but the ongoing recombination process may not play a role in modification of the TCR repertoire.However, the results suggest that Jurkat could be used as a model for studying the regulation of RAGs and V(D)J recombination and as a "special" model of the coexistence of TCR gene rearrangements and "negative" receptor revision.</p>

Research Progress on Rcombinant Hepatitis B e Antigen / 中国生物工程杂志

HBeAg is an ungranular secretory protein,which encoded by C gene of HBV DNA and it increases with the replication of HBV. So it is one of the markers of active replication of HBV in clinical diagnosis. HBeAg is important biologic raw materials which is widely used in preparation of related diagnostic articles with HBV infection serological detection. The technology of expression and purification of recombinant HBeAg is quite mature,which had successfully expressed the target protein in various expression systems. The key factors on HBeAg expression include important site mutation in precore region ,the choice of vectors,effects of RNA interference(RNAi)and so on. Therefore,in order to meet requirements of related diagnostic products,it need to improve expression level and purity of recombinant HBeAg and avoid cross-reaction with HBcAg. In a word,it showed that acquisition of high quality recombinant HBeAg could lay substantial foundation for improving diagnostic products,provide a reliable evidence for exploiting new type of therapeutic and preventive HBV vaccine and offer possibility of HBeAb detection methodological optimization.