[LIM kinases and their roles in the nervous system].

LIM kinase-1 (LIMK1) and LIM kinase-2 (LIMK2) are kinases that have serine/threonine and tyrosine dual specificity. Although they show significant structural similarity, LIMK1 and LIMK2 have different expression patterns, subcellular localization, and functions. Activation of LIM kinases regulates the downstream of Rho GTPases, and influences the architecture of the actin cytoskeleton by regulating the activity of cofilin. Recent studies have shown that LIM kinases play important roles in the nervous system. For example, development of the central nervous system is reliant upon the presence of LIMK1, and deletion of Limk1 gene is involved in the development of the human genetic disorder Williams syndrome. Therefore, it is of vital physiological significance to investigate the neuronal function of LIM kinases. In this review, we outline the structure, phosphorylation regulation and neuronal function of LIM kinases, so as to provide new ideas for the treatment of these neurological diseases.

Enhanced antitumor activity by the combination of dasatinib and combretastatin A-4 in vitro and in vivo.

The present study showed that the combination of dasatinib and combretastatin A-4 (CA-4) exhibited synergistic cytotoxicity in multiple types of cancer, including ovarian, hepatocellular, lung and prostate carcinoma. The enhanced apoptosis induced by dasatinib plus CA-4 was accompanied by a greater extent of mitochondrial depolarization, caspase-3 activation and PARP cleavage in HO-8910 cells. Furthermore, elevated expression of Mcl-1 led to a reduced apoptosis induced by dasatinib plus CA-4, highlighting that downregulated Mcl-1 was necessary for the potentiating effect of dasatinib to CA-4-triggered apoptosis. A clear increase in γ-H2AX expression was observed in the dasatinib+CA-4 group compared with the mono-treatment groups, indicating that dasatinib plus CA-4 may induce double-strand breaks (DSBs) in HO-8910 cells. Moreover, the increased anticancer efficacy of dasatinib combined with CA-4 was further validated in a human HO-8910 ovarian cancer xenograft model in nude mice. Our study is the first to show that the combination of dasatinib with CA-4 could be a novel and promising therapeutic approach for the treatment of cancer.

Wnt/ß-catenin signaling induces the aging of mesenchymal stem cells through promoting the ROS production.

Recent studies have demonstrated that the Wnt/ß-catenin signaling plays an important role in stem cell aging. However, the mechanisms of cell senescence induced by Wnt/ß-catenin signaling are still poorly understood. Our preliminary study has indicated that activated Wnt/ß-catenin signaling can induce MSC aging. In this study, we reported that the Wnt/ß-catenin signaling was a potent activator of reactive oxygen species (ROS) generation in MSCs. After scavenging ROS with N-acetylcysteine, Wnt/ß-catenin signaling-induced MSC aging was significantly attenuated and the DNA damage and the expression of p16(INK4A), p53, and p21 were reduced in MSCs. These results indicated that the Wnt/ß-catenin signaling could induce MSC aging through promoting the intracellular production of ROS, and ROS may be the main mediators of MSC aging induced by excessive activation of Wnt/ß-catenin signaling.

In vitro anti-cancer activity of chamaejasmenin B and neochamaejasmin C isolated from the root of Stellera chamaejasme L.

AIM: To examine the anti-cancer effects of chamaejasmenin B and neochamaejasmin C, two biflavonones isolated from the root of Stellera chamaejasme L (known as the traditional Chinese herb Rui Xiang Lang Du) in vitro. METHODS: Human liver carcinoma cell lines (HepG2 and SMMC-7721), a human non-small cell lung cancer cell line (A549), human osteosarcoma cell lines (MG63, U2OS, and KHOS), a human colon cancer cell line (HCT-116) and a human cervical cancer cell line (HeLa) were used. The anti-proliferative effects of the compounds were measured using SRB cytotoxicity assay. DNA damage was detected by immunofluorescence and Western blotting. Apoptosis and cell cycle distribution were assessed using flow cytometry analysis. The expression of the related proteins was examined with Western blotting analysis. RESULTS: Both chamaejasmenin B and neochamaejasmin C exerted potent anti-proliferative effects in the 8 human solid tumor cell lines. Chamaejasmenin B (the IC(50) values ranged from 1.08 to 10.8 µmol/L) was slightly more potent than neochamaejasmin C (the IC(50) values ranged from 3.07 to 15.97 µmol/L). In the most sensitive A549 and KHOS cells, the mechanisms underlying the anti-proliferative effects were characterized. The two compounds induced prominent expression of the DNA damage marker γ-H2AX as well as apoptosis. Furthermore, treatment of the cells with the two compounds caused prominent G(0)/G(1) phase arrest. CONCLUSION: Chamaejasmenin B and neochamaejasmin C are potential anti-proliferative agents in 8 human solid tumor cell lines in vitro via inducing cell cycle arrest, apoptosis and DNA damage.

[Expression of high mobility group box chromosomal protein 1 in mice with lupus nephritis].

OBJECTIVE: To determine the role of the novel proinflammatory cytokine high mobility group box chromosomal protein 1 (HMGB-1) in the pathogenesis of lupus nephritis. METHODS: Serum levels of anti-dsDNA antibodies were determined by enzyme linked immunosorbent assay (ELISA). Renal morphologic features were examined by light microscopy, electron microscopy, and immunohistologic analyses. The mRNA expression of HMGB-1 and monocyte chemoattractant protein-1 (MCP-1) was detected by RT-PCR. RESULT: MRL/lpr mice demonstrated characteristic alterations of serum immune parameters, with progressively increased anti-dsDNA antibodies with age, compared with age-matched C57BL/6J mice. MRL/lpr mice showed progressive development of renal damage, starting at 12 weeks of age and reached the peak at 20 weeks. The observed lesions included the presence of enlarged hypercellular glomeruli, with increased numbers of both resident cells and infiltrating leukocytes. Higher expression of HMGB-1 mRNA was found in MRL/lpr mice than what in C57BL/6J mice. Expression of HMGB-1 was positively correlated with that of MCP-1 mRNA. CONCLUSION: The results demonstrate that the higher expression of HMGB-1 may contribute to the pathogenesis of lupus nephritis.

[Expression of B7-H4 in prostate cancer and its clinical significance].

OBJECTIVE: To investigate the expression of B7-H4 in prostate cancer tissue and the relationship between the expression and the clinicopathological features. METHODS: Immunohistochemical staining was used to detect the expression of B7-H4 in prostate cancer tissue. And the relationship between the expressions and pathology was evaluated. RESULTS: The B7-H4 was diffusely expressed in cytoplasm and/or membrane of the prostate cancer tissue; the expression was much higher than that in normal prostate tissue (P<0.05). The expression of B7-H4 in the prostate cancer tissue was higher in patients with higher tumor grade. CONCLUSION: B7-H4 may be used as an new indicator for the diagnosis and prognosis of prostate cancer and a novel target for immunotherapy.

[Effects of Sema3A derived from tumor cells on functions of dendritic cells].

OBJECTIVE: To investigate the effects of tumor cell-derived Sema3A on the immunological functions of murine dendritic cells (DCs). METHODS: Lung adenocarcinoma A549 cells were transfected with small interference RNA, Si-Sema and Si-mut, and the interference efficiency was determined by real-time PCR and Western-blot. The concentrated supernatants from cultured tumor cells, Si-Sema and Si-mut-infected tumor cells were subjected to DCs respectively. The immunophenotypes of DCs were analyzed by flow cytometry, the production of IL-12P70 and the ability of DCs to stimulate DO11. 10 T cells secreting IFN-gamma and IL-2 were detected by enzyme linked immunosorbent assay (ELISA). RESULTS: Knockdown with Si-Sema3A significantly decreased the secretion of Sema3A by A549 cells in comparison with the Si-mut cells. DCs exposed to supernatants from Si-Sema cells showed elevated levels of MHC, CD40 and CD80, more production of IL-12P70, and enhanced capability of activating antigen-specific T cells, as evidenced by the remarkably increased levels of IFN-gamma and IL-2. CONCLUSION: A549 cells secrete Sema3A to inhibit the maturation and functions of DCs, which might be associated with the unidentified mechanism of immune evasion by tumor cells.

[Inhibitory effect of triptolide on interleukin-18 and its receptor in rheumatoid arthritis synovial fibroblasts].

AIM: To determine the effects of triptolide (TP) on the expression of interleukin-18 (IL-18) and its receptor in phorbol 12-myristate 13-acetate (PMA)-stimulated rheumatoid arthritis synovial fibroblasts (RASF). METHODS: RASF were pretreated with TP (0-100 microg/L) for 2 h before stimulation with PMA (50 microg/L). The bioactivity of IL-18 in the supernatant was detected based on IFN-gamma secretion from IL-18-responding human myelomonocytic KG-1 cells. IL-18 level was analyzed by ELISA. To estimate the protein and mRNA expression of IL-18 and IL-18Ralpha in RASF, Western blot and quantitative RT-PCR were performed. Nuclear factor-kappaB (NF-kappaB) activity in the whole-cell extract of treated RASF was also measured using an ELISA-based method. RESULTS: TP effectively inhibited the bioactivity of IL-18 in PMA-stimulated RASF. The expression of IL-18 and IL-18R at protein and gene levels was reduced by TP. NF-kappaB activity in PMA-stimulated RASF was profoundly suppressed by TP. These effects showed a high correlation with TP concentration (0-100 microg/L). CONCLUSION: TP effectively inhibited the expression of IL-18 and its receptor in PMA-stimulated RASF. These results suggest a mechanism of TP in RA therapy.

[Expression and identification of recombinant mouse gene B7-H4].

OBJECTIVE: To construct a eukaryotic expression vector encoding the gene of extracellular region of mouse B7-H4, to express it in yeast cell line and to determine its biological activity. METHODS: The extracellular region of the mouse B7-H4 gene was amplified with Xho I and EcoR I by PCR from a mouse B7-H4 chimeric plasmid. Digested with Xho I and EcoR I, the mB7-H4 gene was inserted into the yeast expression plasmid Ppic9. The DNA sequence was confirmed by double digestion and the Ppic9-mB7-H4 plasmid was transfected into the yeast cells. The expression of mB7-H4 was confirmed by PCR, Western Blot and ELISA analysis, and its biological function was determined. RESULT: Ppic9-mB7-H4 transfectants expressed mB7-H4 in yeast cells, and mB7-H4 effectively inhibited the proliferation of T lymphocytes with a fractional inhibition rate of 28.3 % and inhibited IL-2, IL-4, IL-10 and IFN-gamma production with fractional inhibition rates of 68.8%, 78.8%, 67.6% and 77.7%, respectively. CONCLUSION: The eukaryotic expression plasmid mouse B7-H4 has been successfully constructed and the expressed products of B7-H4 possess biological activity.

[Construction and identification of recombinant adenoviral vector encoding B7-H4 gene].

OBJECTIVE: To construct the recombinant adenovirus containing B7-H4 gene with AdEasy XL system and to identify its biological activities. METHODS: The full-length mouse B7-H4 gene was amplified by RT-PCR from C57 mouse lung and put into T vector, then verified by sequencing. Digested with Xhol I and EcoR V the B7-H4 gene was inserted into pshuttle-CMW(PSC). Pme I linearized shuttle plasmid was transformed into E.coli BJ5183-AD-1 to obtain the recombinant adenoviral plasmid pAd-mB7-H4 by efficient homologous recombination. Then the recombinant adenovirus-mB7-H4/Ad was obtained by packaging Pac I linearized in D-293 cells. The mRNA and protein expression of B7-H4 in mB7-H4/Ad infected AD-293 cells were detected by RT-PCR and Western blot, respectively. mB7-H4/Ad was used to infect L929 cells, the bioactivity of expressed B7-H4 in stimulation of T lymphocytes proliferation and cytokine production were tested. RESULTS: The full-length of mB7-H4 was cloned from mouse lung tissue cDNA and verified by sequencing. The recombinant plasmid pAd-m B7-H4 was successfully generated by homologous recombination, and the primary mB7-H4/Ad was obtained by packaging pAd-B7-H4 in AD-293 cells. Compared with the negative control, L929 cells infected with mB7-H4/Ad effectively inhibited the proliferation of T lymphocytes and cytokines production. CONCLUSION: The bioactive recombinant adenovirus mB7-H4/Ad has been successfully constructed.

[Construction and expression of recombinant adenovirus containing human thymic stromal lymphopoietin gene].

AIM: To construct and express recombinant adenovirus bearing human thymic stromal lymphopoietin (TSLP) gene. METHODS: TSLP gene amplified from human fetal lung cells was first cloned into eukaryotic expression vector pcDNA3.1, and then subcloned into shuttle vector pShuttle. The resultant plasmid was subsequently cotransformed into E. coli BJ5183 cells with adenoviral backbone plasmid pAdEasy-1. The recombinant adenovirus plasmid containing TSLP was digested with Pac I and transfected into HEK293 cells to package recombinant adenovirus particles. The TSLP gene of the recombinant virus was detected by PCR, and its expression was analyzed by Western blot. RESULTS: Recombinant adenovirus vector bearing human TSLP gene was constructed by homologous recombination in E.coli, and recombinant adenovirus was obtained by transfecting HEK293 cells with this infectious DNA. PCR test indicated that TSLP gene was successfully integrated into the adenoviral genome, and the titer of the recombinant Ad reached 1 x 10(11) pfu/L. Meanwhile, expression of TSLP in the infected Hela cells was confirmed by Western blot. CONCLUSION: The construction of recombinant adenovirus bearing human TSLP gene and its expression mediated by this adenovirus pave a foundation for the study on the biological function of this novel cytokine.

[Construction of the eukaryotic expression vector containing rat transforming growth factor beta type II receptor and interferon gamma fusion genes].

OBJECTIVE: To express a bioactive fusion protein comprising rat soluble transforming growth factor beta type II receptor and interferon gamma(rsTGFbetaR II-IFN gamma) in mammalian cells. METHODS: mRNA was extracted from rat liver and the sTGFbetaR II-IFN gamma genes amplified by RT-PCR, then the two gene segments were cloned into the same pSecTag2A expression vector, and pSecTag2A/rsTGFbetaR II-IFN gamma recombinant plasmid was obtained, which was later transfected into CHO cells using liposomes. The expression of pSecTag2A/rsTGFbetaR II-IFN gamma in the supernatant was detected by ELISA and Western blotting. The bioactivities of the fusion protein were tested by sTGF betaR II-IFN gamma and IFN gamma bioassays. RESULTS: pSecTag2A/rsTGFbetaR II-IFN gamma transfectants expressed rsTGF betaR II-IFN gamma fusion protein. The purified fusion protein exhibited anti-viral activity and antagonized the proliferation-inhibitive effect of TGFbeta1 on CCL-64 cells. It inhibits the HSC activation in vitro. CONCLUSION: The pSecTag2A/rsTGFbetaR II-IFN gamma recombinant plasmid constructed in this study can express bioactive rsTGFbetaR II-IFN gamma fusion protein.

[Construction and expression of the eukaryotic expression vector containing human soluble transforming growth factor beta receptor II].

OBJECTIVE: To construct a eukaryotic expression vector encoding the gene of extracellular region of type II transforming growth factor beta receptor (sTGFbetaR II), to express the protein in CHO cell line and to determine its biological activity. METHODS: The extracellular region (amino acids 1-159) of the human TGFbetaR II cDNA was amplified by PCR from a TGFbetaR II chimeric plasmid,and the eukaryotic expression plasmid pCDNA3.1/myc-his(-)B-sTGFbetaR II(pCDNA-sTGFbetaR II) was constructed by inserting the sTGFbetaR II cDNA into the EcoR I/Hind III-digested pCDNA. The DNA sequence was confirmed by double digestion and the pCDNA-sTGFbetaR II plasmid was transfected into the CHO cell line. The sTGFbetaR II protein was confirmed by Western blotting analysis and its biological function was determined. RESULTS: The specific protein was observed in western blotting, and the protein abrogated the growth-inhibitory effects of TGF-beta1 on mink lung epithelial cells (Mv1Lu). CONCLUSION: The eukaryotic expression plasmid pCDNA-sTGFbetaR II has been successfully constructed and the sTGFsTGFbetaR II protein with biological activity obtained.

[Effect of Triptolide on TNFalpha-induced activation of NF-kappaB and expression of COX-2 and iNOS in human rheumatoid arthritis synovial fibroblasts].

OBJECTIVE: To explore the effects of Triptolide (TP) on TNFalpha-induced cell proliferation and expressions of cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS) and their inducing products PGE2, NO in human rheumatoid arthritis synovial fibroblasts (RASF). METHODS: Fibroblasts (RASF) were obtained from synovial tissue of patients with RA and were cultured in vitro. RASF were stimulated with TNFalpha(20 microg/L) in the presence or absence of TP(0 - 100 microg/L) for 20 h. The RASF proliferation was determined by (3)H-TdR incorporation, and the productions of PGE2 and NO in culture supernatants of RASF were detected with competitive ELISA and enzyme reduction of nitrate. Expressions of COX-2 and iNOS mRNA in RASF were analyzed by semi-quantitative RT-PCR. Expressions of COX-2 and iNOS protein were estimated by Western-blot method and cellular enzyme immunoassay in synovial fibroblasts. NF-kappaB activity in whole-cell extract of RASF was also measured by an ELISA-based method. RESULTS: TP (>20 microg/L) down-regulated markedly TNFalpha-induced COX-2 and iNOS mRNA and protein expression, and their inducing products PGE2 and NO of synovial fibroblasts. This effect was positively correlated with TP concentrations. NF-kappaB activity in TNFalpha-stimulated synovial cells was suppressed profoundly by TP treatment (IC(50) approximately 35microg/L). The activity of NF-kappaB was correlated with the levels of COX-2 and iNOS expression in TNFalpha-stimulated RASF. No change was observed in proliferation of synovial cells after treatment of TP. CONCLUSION: TP could significantly down-regulate TNFalpha-induced COX-2, iNOS expression and production of PGE2, NO in human RASF, which is associated with the suppression of NF-kappaB activity.

[Enhancement by FK506 of triptolide-induced inhibition of expression of COX-2 and iNOS in human rheumatoid arthritis synovial fibroblasts].

To explore the effects of FK506 on the inhibition by triptolide (TP) of cell proliferation and expressions of cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS) and their inducing products PGE2, NO in human rheumatoid arthritis synovial fibroblasts (RASF), and to study the mechanisms of combination of FK506 and TP in RA therapy, RASF used in the experiments were obtained from synovial tissue of patients with RA and were cultured. RASF were pretreated with FK506(10-1000 nmol/L)for 2 h, then the cells were stimulated with TNF alpha(20 microg/L) in the presence or absence of TP (10 microg/L). The RASF proliferation was determined by [(3)H]-TdR incorporation, and the productions of PGE2 and NO in culture supernatants of RASF were detected with competitive ELISA and enzyme reduction of nitrate. Expression of COX-2 and iNOS mRNA in RASF were analyzed by semi quantitative RT-PCR. Expressions of COX-2 and iNOS protein were estimated by Western blot method and cellular enzyme immunoassay in synovial fibroblasts. NF-kappa B activity in whole-cell extract of RASF was also measured by an ELISA-based method. Results showed that neither FK506 nor TP at lower concentration (10 microg/L) alone affected TNF alpha-induced COX-2, iNOS expression and production of PGE2, NO in synovial cells. Combined treatment of FK506 and a lower concentration of TP (10 microg/L) down-regulated COX-2 and iNOS mRNA and protein expression, and their inducing products PGE2 and NO of synovial fibroblasts. This effect was positively correlated with FK506 concentrations (10-1000 nmol/L). NF-kappa B activity in TNF alpha-stimulated synovial cells was suppressed more profoundly by FK506 plus TP (10 microg/L) treatment than those with TP (10 microg/L) alone. No change was observed in inhibition of proliferation of synovial cells after combined treatment of FK506 and TP. In conclusion, FK506 enhanced TP-mediated down-regulation of COX-2, iNOS and their inducing products PGE2, NO in human RASF by suppressing the activity of NF-kappa B.

[Preliminary observation of CXCR3 expression on peripheral blood lymphocytes from patients with chronic hepatitis B infection].

AIM: To explore the role of chemokine receptor CXCR3 in pathogenesis of chronic hepatitis B. METHODS: The expression of CXCR3 on peripheral blood lymphocytes from chronic hepatitis B patients with various degrees of inflammation was detected, and the distributions of CXCR3 on CD4(+) and CD8(+) T lymphocytes were also evaluated by FACS. RESULTS: As compared with healthy control, the percentages of CXCR3(+) lymphocytes, monocytes and CD8(+) T cells were increased significantly in chronic hepatitis B patients. CONCLUSION: Above data suggest that chemokine receptor CXCR3 may play an important role in the recruitment of lymphocytes, especially CTLs recruited to inflammation sites.

[Serum levels of sFas, sICAM-1, IL-18 in patients with chronic hepatitis C and their clinical significance]

OBJECTIVE: To investigate the serum levels of soluble Fas antigen (sFas), soluble intercellular adhesion molecules-1 (sICAM-1), interleukin-18 (IL-18) in patients with chronic hepatitis C and to study their roles in pathogenesis of chronic hepatitis C. METHODS: Serum sFas, sICAM-1, IL-18 levels were measured in 30 cases of chronic hepatitis C before and after treatment of interferon-alpha by enzyme-linked immunosorbent assay (ELISA), serum titer of HCV-RNA was detected by quantitative PCR and serum ALT activity was also detected. RESULTS: Serum levels of sFas sICAM-1 IL-18 in chronic hepatitis C patients were significantly higher than those in normal controls (P<0.01), showing correlation with serum HCV-RNA titer (r=0.915, r=0.795, r=0.757, respectively, P<0.01), Serum levels of sICAM-1, IL-18 showed correlation with serum ALT level(gamma=0.952, gamma=0.969, respectively, P<0.01), but no relationship was observed between serum sFas and serum ALT level(P>0.05). Serum levels of sFsa sICAM-1 IL-18 markedly decreased in responsive patients while no change was observed in patients with no response after treatment. CONCLUSION: Soluble Fas, soluble ICAM-1, IL-18 may participate in the pathogenesis of chronic hepatitis C and show correlation with the severity of histological inflammation and viral titer.

[Effects of intrasplenic transplantation of IL-18 gene-modified fetal hepatocytes on mouse immune function]

OBJECTIVE: To investigate the effects of IL-18 gene-modified fetal hepatocytes (AdmIL-18/MNL.CL2) intrasplenic transplantation on mouse immune function. METHODS: Forty mice were evenly divided into 4 groups of 10. Each group received an intrasplenic transplantation one of the following: AdmIL-18/BNL.CL2, Ad-LacZ/BNL.CL2 (virus control), BNL.CL2 (cell control) and PBS (blank control). After two weeks, the mice were sacrificed. Serum cytokine levels, Mpsi and splenic cell culture supernatant and liver tissue extracts supernatants were measured using ELISA. Hepatic cytokines mRNA expression were determined by RT-PCR. THe cytotoxicity of peritoneal Mpsi and NK activity of spienocytes were detected by LDH release assay. The proliferation of splenic lymphocytes was determined by MTT assay. RESULTS: The IL-18, IL-2,IFN-gamma, TNF-alpha levels of serum, Mpsi and splenocyte culture supernatant, liver tissue extracts supernatants in mice transplanted with AdmIL-18/BNL.CL2 were higher and the IL-4, IL-10 levels were lower compared to their levels in other 3 groups. The highest IL-18, IL-2, IFN-gamma, TNF-alpha and the lowest IL-4, IL-10 mRNA expressions in the liver were observed in mice transplanted with AdmIL-18/BNL.CL2. The mice transplanted with AdmIL-18/BNL.Cl2 showed significantly increases cytotoxicity of Mpsi, NK activity and splenic cell proliferation compared with the other 3 groups. CONCLUSION: AdmIL-18 can be effectively transfected into mice fetal heptocytes which subsequently IL-18. Intransplenic transplantation of IL-18 gene-modified fetal hepatocytes may augment mouse immune function and provide an useful basis for targeted gene therapy of liver disease.