Endothelial progenitor cells (EPCs) participate in the neovascularization processes in the development of hepatocellular carcinoma (HCC). We investigated whether interactions between EPCs and HCC cells affect chemotactic and pro-inflammatory activities of EPCs. Two distinct phenotypes of circulating EPCs, i.e., myeloid-derived EPCs (colony forming unit-endothelial cells, CFU-ECs) and outgrowth EPCs (endothelial-colony forming cells, ECFCs), were co-cultured with Huh7 and Hep3B cells by using transwell chamber and IBIDITM Culture-Inserts and μ-slide plates. Transwell and horizontal migration/invasion assays and time-lapse microscopy were used to monitor and analyze the migration and invasion of EPCs induced by these HCC cells. A human cytokine antibody array was used to compare protein expression profiles in EPCs and HCC cells. Flow cytometry and electromobility shift analysis were used to detect nuclear factor-κB (NF-κB)–DNA binding activity and pro-inflammatory adhesion molecule expression in EPCs. Ectopic full-length CC chemokine receptor 6 (CCR6) plasmid was used to transfect into ECFCs to investigate the role of CCR6 in HCC-induced EPC migration and invasion. The results show that co-culture with Huh7 and Hep3B cells induces the expression of endothelial cell (EC) markers KDR, Flt1, CD31 and VE-cadherin in CFU-ECs, but down-regulates the expressions of CD31 and VE-cadherin in ECFCs. These HCC cells induce migration and invasion of CFU-ECs, but not ECFCs, and do not affect the cell cycle distribution in these EPCs. Cytokine protein array identifies macrophage inflammatory protein-3α (MIP-3α) produced by HCC cells as a critical factor responsible for the HCC-induced chemotaxis of CFU-ECs, which highly express the specific MIP-3α counterreceptor CCR6. Overexpressing CCR6 in ECFCs significantly increases their chemotaxis in response to HCC cells. Co-culturing EPCs with HCC cells results in decreases in NF-κB binding activity and hence intracellular adhesion molecule-1, vascular cell adhesion molecule-1, and E-selectin expressions in EPCs. Our results indicate that HCC cells exert differential effects on CFU-ECs and ECFCs, with increased chemotaxis for CFU-ECs, but not ECFCs. This HCC-induced chemotaxis of CFU-ECs is mediated by MIP-3α produced by HCC cells, which targets to CCR6 on CFU-ECs. Tumors may provide a humoral microenvironment to attenuate the pro-inflammatory activity of EPCs, which might be associated with the tumor escape mechanism.

Adlay has long been used in traditional Chinese medicine and as a nourishing food. The acetone extract of adlay hull had previously been demonstrated to possess potent antimutagenic activity. The aims of this study were to identify the antimutagenic constituents from adlay hull by using Ames antimutagenic activity-guide isolation procedures and to investigate their chemopreventive efficacies in cultured cells. The results demonstrated that six compounds showing great antimutagenic activity were identified by spectroscopic methods and by comparison with authentic samples to be p-hydroxybenzaldehyde, vanillin, syringaldehyde, trans-coniferylaldehyde, sinapaldehyde, and coixol. Two of them, trans-coniferylaldehyde and sinapaldehyde, exhibit relatively potent scavenging of DPPH radicals, inhibit TPA stimulated superoxide anion generation in neutrophil-like leukocytes, and induce Nrf2/ARE-driven luciferase activity in HSC-3 cells. Moreover, trans-coniferylaldehyde possesses cytoprotective efficacy against tert-butyl hydroperoxide-induced DNA double-strand breaks in cultured cells, and the chemopreventive potency induced by trans-coniferylaldehyde may be through the activation of kinase signals, including p38, ERK1/2, JNK, MEK1/2, and MSK1/2. In summary, we first identified six antimutagenic constituents from adlay hull. Among them, trans-coniferylaldehyde would be a highly promising agent for cancer chemoprevention and merits further investigation.

OBJECTIVE To study the effects of transforming growth factor-beta1/integrin-linked kinase (TGF-beta1/ILK) signal way in interleukin-1beta (IL-1beta)-induced rat tubular epithelial-myofibroblast transdifferentiation (TEMT), and to investigate whether emodin inhibits IL-1beta-induced TEMT through the TGF-beta1/ILK signal way-dependent mechanism. METHODS Normal rat kidney epithelial cell line (NRK52E) was used in this study. NRK52E cells were divided into blank control group, emodin control group, IL-1beta-induced group, emodin-inhibited group, SB431542 (TGF-beta 1 type I receptor blocker)-inhibited group, emodin plus SB431542-inhibited group, emodin-pretreated group and emodin-reversed group. After 48-hour culture, morphological changes of the NRK52E cells were observed by an inverted phase contrast microscope. The expressions of alpha-smooth muscle actin (alpha-SMA) and E-cadherin were detected by two-color immunohistochemical staining, while the expressions of TGF-beta1 and ILK were detected by one-color immunohistochemical staining. We also performed the imaging analysis to quantitatively analyze the result of the immunohistochemical staining. The secretion of fibronectin (FN) was analyzed by enzyme-linked immunosorbent assay. RESULTS Compared with the blank control group, IL-1beta might induce TEMT, which was showed in increasing expression of alpha-SMA, increasing secreting of FN and decreasing expression of E-cadherin, and at the same time the expressions of TGF-beta1 and ILK were enhanced (P<0.05). Emodin might inhibit all of those changes induced by IL-1beta (P<0.05). When TGF-beta1 signal way was intercepted, IL-1beta induced-TEMT was suppressed and the expression of ILK was decreased, however, there was no significant difference in expression of TGF-beta1 between the SB431542 group and the IL-1beta-induced group. Compared with emodin-inhibited group, emodin-pretreatment could not prevent IL-1beta induced-TEMT in a certain extent, but emodin could not revert IL-1beta-induced TEMT. Spearman correlation analysis showed that TGF-beta1 expression had positive correlation with expressions of alpha-SMA, FN, ILK and negative correlation with E-cadherin expression, and the expression of ILK was positively correlated with the expressions of alpha-SMA and FN and negatively correlated with E-cadherin expression. CONCLUSION IL-1beta induces TEMT partly depending on TGF-beta1/ILK signal way, partly via which emodin inhibits the TEMT induced by IL-1beta.

Objective It is to explore the role of p38 mitogenactivated protein kinase(p38MAPK) on the IL-1β-induced tubular epithelial-myofibroblast transdifferentiation(TEMT) and to explore intervention of Emodin(EMD).Methods The cultured NRK52E cells in vitro were divided into control group,IL-1β-induced group,IL-1β+SB203580 group and IL-1β+EMD group.After the cells had been cultured for 48 hours,the morphology of cells was observed under the inverted phase-contrast microscope and the expressions of α-SMA,CK,p38MAPK and p-p38MAPK were measured by immuno-cytochemistry method.Results IL-1β could induce some NRK52E became fibroblast-like-shaped and the expressions of CK decreased significantly,but α-SMA p38MAPK and p-p38MAPK increased respectively.After p38MAPK been blocked by SB 203580,the changes of morphology induced by IL-1β were inhibited,and the expressions of α-SMA and p-p38MAPK decreased respectively,CK increased significantly.EMD could significantly inhibit the morphologic changes and the expressions of α-SMA p38MAPK and p-p38MAPK.The inhibitory effects of EMD were similar to that of SB 203580.Conclusion p38MAPK takes a part in the process of TMET induced by IL-1β.EMD can inhibit the process of TMET through interventing the p38MAPK signaling pathway.

AIMTo observe the change of ILK expression in interleukin-1beta(IL-1beta)-induced tubular epithelial-myofibroblast transdifferentiation, and to investigate whether emodin inhibit IL-1beta-induced tubular epithelial-myofibroblast transdifferentiation through an intergern linked kinase-dependent mechanism.METHODSNormal rat kidney epithelial cell line (NRK52E) was cultured and then divided into blank group, emodin control group, IL-1beta-induced group and emodin-inhibited group. When the cells were cultured for 48 h, their morphological changes were observed by an inverted phase contrast microscope. The expression of a-smooth muscle actin (a-SMA) and E-cadherin were detected using a two-color immunohistochemistry staining technique, while the expression of integrin-linked kinase (ILK) was detected using a one-color immunohistochemistry staining technique. The secretion of fibronectin (FN) was analyzed by ELISA.RESULTSNRK52E cells cultured with IL-1 became fibroblast-like in appearance. The expression of a-SMA was enhanced (65h5+/-1h7 vs 140h4+/-3h0, P<0h05), the expression of E-cadherin was decreased (82h5+/-1h0 vs 36h0+/-2h8, P<0h05), the expression of ILK was enhanced (36h1+/-3h1 vs 82h4+/-1h2, P<0h05), and the secretion of FN was increased (54h6+/-3h1 vs 124h8+/-3h2 mg/L, P<0h05). Emodin markedly inhibited all of those changes induced by IL-1beta.CONCLUSIONThe expression of ILK is up-regulated in IL-1beta-induced tubular epithelial-myofibroblast transdifferentiation. Emodin might inhibit TEMT by a down-regulation the expression of ILK.

Objective To observe the effect of Emodin(EMD)on the tubular epithelial-myofibroblast transdifferentiation(TEMT)induced by IL-1β in vitro.Methods The cultured NRK52E cells were induced by IL-1β,and at the same time co-incubated with different concentrations EMS.After cells had been cultured for 12、24、48 and 72 hours respectively,the morphology of cells was observed under the inverted phase-contrast microscope;the expressions of a-SMA,CK were measured by immuno-cytochemistry method and semiquantified by mean intergrated opitical density(IOD).Results Compared with IL-1β group,the expressions of a-SMA and CK of NRK52E cells treated only with different concentrations EMD have not significant difference.EMD can reduce the IL-1β-induced NRK52E cells' morphology changes,and up-regulate the expression of CK but down-rugulate a-SMA's significantly.Conclusion EMD can inhibit partly the process of TMET induced by IL-1β.

Aim: To observe the influence of interleukin (IL)-1β on tubular epithelial-myofibroblast transdifferentiation (TEMT) and the secretion of fibronectin (FN) in cells. Methods: The experiment was carried out in the Staff Room of Infection Immunity, Affiliated Hospital of Luzhou Medical College from June 2005 to January 2006. The normal rat kidney epithelial cell line (NRK52E) was cultured In Vitro and then co-incubated with IL-1β (10 μg/L), When the cells were cultured for 12, 24, 48, 72 hours, their morphological changes were observed through inverted phase contrast microscope; The expressions of α-smooth muscle actin (α-SMA) and E-cadherin were detected by a two-color immunohistochemistry staining technique; Supernatant liquid of every well was analyzed for secretion of FN by ELISA. Results: NRK52E cells cultured with IL-1β for 48 hours became fibroblast-like in appearance; when the cells were cultured With IL-1β for 12 and 72 hours, the expression of α-SMA were enhanced (64.80±2.19, 83.23±2.71, 170.53±3.94, P < 0.05), the expression of E-cadherin was decreased (81.77±1.27, 64.50±1.31, 26.20±2.32, P < 0.05). These changes were time-dependent. The secretion of FN was increased with the time prolonged (P < 0.05) and doubled at 72 hours of culture. Conclusion: IL-1β can induce TEMT and enhance the secretion of FN in cultured NRK52E cells.

AIM:Emodin could significantly inhibit the differentiation of NRK52F cell induced by interleukin-1β(IL-1β).In this study,we investigate the effects of transforming growth factor β1(TGF-β1) on the differentiation of tubular epithelial-myofibroblast transdifferentiation(TEMT) induced by IL-1β and on the inhibition of emodin.METHODS:The experiment was carried out in Immunity Laboratory of Affiliated Hospital of Luzhou Medical College from October 2006 to May 2007.The cultured NRK52E cells of rats were divided into ①control group,in which the cells were cultured in high glucose DMEM medium added by 0.05 volume fraction calf serum,②IL-1β induction group,in which the high glucose DMEM medium containing 10 μg/L IL-1β was used,③SB431542 blocking group,in which the cells were cultured in high glucose DMEM medium containing 10 μg/L IL-1β and 10 μmol/LSB431542,and ④IL-1β+emodin group,in which the cells were cultured in high glucose DMEM medium containing 10 μg/L IL-1β and 25 mg/L emodin.After the cells were treated for 48 hours,the morphology of NRK52E cells was observed under the inverted phase-contrast microscope and the expressions of creatine kinase(CK),α-smooth muscle actin(α-SMA) and TGF-β1 were measured by immunocytochemistry method.RESULTS:①Some cells became fibroblast-like-shaped after induced by IL-1β,and the expressions of CK decreased significantly(P < 0.01),but α-SMA and TGF-β1 increased(P < 0.01).②In SB431542 blocking group,the percentage of elongated cells was less than that of IL-1β group and the expression of α-SMA was decreased(P < 0.01),but CK increased significantly(P < 0.01).No obvious change was found in the expression of TGF-β1.③The inhibitory effect of emodin on the morphous of IL-1β induced cells and the expressions of CK and α-SMA was significantly and similar to that of SB431542.Meanwhile,emodin remarkably inhibited the expression of TGF-β1 induced by IL-1β.CONCLUSION:TGF-β1 may mediate the process of TMET induced by IL-1β,and the inhibitory effect of emodin on the process of TMET induced by IL-1β.

整合素连接激酶(Integrin-linked kinase,ILK)是一种近期发现的Ser/Thr蛋白激酶.ILK能够通过与整合素β1亚单位的结合介导细胞与胞外基质的连接,以依赖于PI3K的方式激活,并通过磷酸化下游底物PKB/AKT、GSK3等使胞外信号得以向下游传递,参与多种信号传导通路,与肾间质纤维化的发生相关.