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Progress in TcpC research
PAN Jian-Ping, XIN Xiao-Li
Journal of ZheJiang University(Medical Science), 2013, 42(5): 481-485.   https://doi.org/10.3785/j.issn.1008-9292.2013.05.001
Abstract( 658 )   HTML (   PDF(614KB)( 433 )

TcpC is a homolog of the Toll/interleukin-1 receptor (TIR) domain and is secreted by uropathogenic E. coli strain CFT073. TcpC can bind to MyD88,hereby exerting inhibitory effects on macrophages. TcpC represents an important virulence factor that promotes bacterial survival and pathogenicity. TcpC plays a critical role in urinary tract infection,particularly in the pathogenesis of pyelonephritis. In this review,the progress and prospects in TcpC research are discussed.

TcpC induces apoptosis of macrophages through promoting ROS production
ZHANG Da-Yong, LIN Yi-Qian, HE Fei, FANG Jie, ZHANG Chong, WANG Bao-Ming, PAN Jian-Ping
Journal of ZheJiang University(Medical Science), 2013, 42(5): 486-491.   https://doi.org/10.3785/j.issn.1008-9292.2013.05.002
Abstract( 928 )   HTML (   PDF(2580KB)( 401 )
Objective: To investigate the effects of Toll/interleukin 1 receptor domain-containing protein(TcpC)on macrophages and its mechanisms.
Methods: Murine macrophage J774A cells were co-cultured with TcpC producing wild type E. coli strain CFT073 (TcpCwt) or tcpc gene-deleted CFT073 mutant (TcpCmut) in Transwell system,respectively. Apoptosis of J774A cells co-cultured with TcpCwt or TcpCmut was analyzed by Annexin/PI double staining. The levels of reactive oxygen species (ROS) in J774A cells were determined by DCFH-DA staining after treatment with TcpCwt or TcpCmut at 6 h,12 h,24 h or 36 h. After the ROS was scavenged by N-acetylcysteine (NAC),the changes of J774A cell apoptosis were also examined. The expression of caspase-3 in J774A cells co-cultured with TcpCwt or TcpCmut in the presence or absence of 0.1 mmol NAC was detected by Western blot.
Results: J774A cells co-cultured with TcpCwt for 24 h or 36 h showed significantly increased apoptosis (27.39%±4.05% and 28.45%±4.55%,respectively) when compared to control group (7.96%±1.63% and 10.55%±1.44%,P<0.01) or TcpCmut group (11.45%±2.77% and 19.26%±2.89%,P<0.01). Levels of ROS in J774A cells treated with TcpCwt for 24 h (108.8±9.73) or 36 h (100.3±10.11) were significantly higher than those in control group (56.8±4.11 and 52.8±4.42,P<0.01) or TcpCmut (69.7±5.66 and 62.6±4.56,P<0.01). The pro-apoptotic effects of TcpCwt on J774A cells were reversed by 0.1 or 1 mMol NAC treatment. Expression of caspase-3 in J774A cells co-cultured with TcpCwt (0.43±0.04) decreased significantly when compared to control group (0.75±0.08,P<0.05) or TcpCmut group (0.80±0.12,P<0.05). However,total caspase-3 expression was restored in J774A cells co-cultured with TcpCwt in the presence of 0.1 mmol NAC (0.80±0.09).
Conclusion: TcpC can promote ROS production in macrophages,hereby inducing macrophage apoptosis.
TcpC induces apoptosis of human vascular endothelial cells and its mechanisms
ZHANG Chong, ZHOU Jia-Le, FANG Jie, ZHANG Da-Yong, WANG Bao-Ming, CHEN Rui-Ling, PAN Jian-Ping
Journal of ZheJiang University(Medical Science), 2013, 42(5): 492-497.   https://doi.org/10.3785/j.issn.1008-9292.2013.05.003
Abstract( 978 )   HTML (   PDF(1689KB)( 403 )

Objective: To investigate the effects of TcpC on human umbilical vascular endothelial cells (HUVECs) and its mechanisms.
Methods: HUVECs were co-cultured with TcpC secreting wild-type E. coli strain CFT073 (TcpCwt) or tcpc gene-deleted CFT073 mutant strain (TcpCmut) in transwell system,respectively. Apoptosis of HUVECs was analyzed by Annexin-V/PI double staining. Mitochondrial membrane depolarization was detected by JC-1 staining. Expression of apoptosis-related proteins in HUVECs was determined by Western blot.
Results: HUVECs showed morphological changes after co-cultured with TcpCwt for 24 h:the cells became detached and cell debris increased,and cell number was also decreased when compared to HUVECs co-cultured with TcpCmut. The apoptosis of HUVEC cells co-cultured with TcpCwt for 24 h significantly increased,compared to that of control group and TcpCmut group (60.1% 9.7% vs 9.0% 1.3% and 16.9% 0.4%,respectively, P<0.05); meanwhile the mitochondrial depolarization of HUVECs co-cultured with TcpCwt was significantly increased,compared to that in control and TcpCmut groups (64.5% 0.9% vs 14.5% 2.1% and 15.6% 3.3%,respectively,P<0.05). Cleavage of PARP and inhibition of Mcl-1 and XIAP expression were seen in HUVECs co-cultured with TcpCwt,but not in groups of control and TcpCmut.
Conclusion: TcpC secreted from CFT073 can induce apoptosis of HUVECs through mitochondrial pathway,in which PARP is cleaved and Mcl-1 and XIAP expressions are inhibited.

CCL21-CD40L fusion gene induce augmented antitumor activity in colon cancer
GONG Ting, LI Hong-Li, BA Yi
Journal of ZheJiang University(Medical Science), 2013, 42(5): 498-503.   https://doi.org/10.3785/j.issn.1008-9292.2013.05.004
Abstract( 583 )   HTML (   PDF(1005KB)( 330 )

Objective: Toinvestigate the anti-tumor activity of CCL21-exCD40Leukaryotic expression vector.
Methods: CCL21-exCD40L fusion gene were constructed by overlap PCR connecting CCL21 and exCD40L through a flexible linker (Gly3Ser)4 ,and then was cloned into expression vector pcDNA3.1+.pcDNA3.1+/CCL21 and pcDNA3.1+/exCD were constructed as negative control.Wsestern blot was used to identify the fusion protein.CHO cells was transfected with pcDNA3.1+/CCL21-exCD,pcDNA3.1+/CCL21 and pcDNA3.1+,respectively.The chemotatic function of the expressed product was detected by Transwell method and its anti-tumor activity was tested with vivo transfection.
Results: Gene sequencing and restrictive digestion proved the successful construction of pcDNA3.1+/CCL21-exCD40L,and its expression was conformed by western blot.The transfectant supernantes of pcDNA3.1+/CCL21-exCD40 group had a significant chmotactic function to DCs,of which the cell numbers passing through the film was 14.95 times of blank control every high power microscope visual field.After tumor orthotoic injection of plasmid carrying fusion gene in Balb/c mouse,the tumor mass reduced remarkablely,and all the mouse in fusion gene group survived after 4 weeks.
Conclusion: CCL21-exCD40L fusion protein had a remarkable function to DCs and it can inhibit tumor growth and prolong the mouse survival time,which is more effective than all controlgroup.

Pro-apoptotic effects of luteolin on hepatoma HepG2 cells
WANG Yuan-Peng, ZHOU Liang, GONG Xing-Guo
Journal of ZheJiang University(Medical Science), 2013, 42(5): 504-510.   https://doi.org/10.3785/j.issn.1008-9292.2013.05.005
Abstract( 564 )   HTML (   PDF(1379KB)( 428 )

Objective: To investigate the effect of luteolin on cell growth and apoptosis of HepG2 cells in vitro.
Methods: Cultured HepG2,HL60,A549 and LO2 cells were treated with luteolin for different doses (0 μg/ml,2.5 μg/ml,10 μg/ml and 20 μg/ml) and varied times (0 h,24 h,48 h and 72 h).Cell viability was measured with MTT assay and IC50 was calculated.The reactive oxygen species (ROS) levels in HepG2 cells treated with luteolin for 6 h and 12 h were measured with flow cytometry.Cell apoptosis of HepG2 cells treated with luteolin for 24h was examined with flow cytometry and Annexin V-FITC/PI.Expression levels of apoptosis pathway proteins (p53,ASPP2 and iASPP) in HepG2 cells were detected with western blot and the dose and time-effect was analyzed.
Results: Luteolin effectively inhibited tumor cell proliferation in a dose- and time-dependent manner,and the inhibition rates of 20μg/ml Luteolin for 72h were 39.34%,62.90%,57.57% and 62.90% to LO2,HepG2,HL60 and A549 cells,respectively.The intracellular ROS level was decreased in HepG2 cells by 13.88% and 41.11% after being treated with luteolin for 6 h and 12 h,respectively.The apoptosis rate of HepG2 cells treated with luteolin for 24 h was 14.43%,and western blot showed that luteolin reduced the expression level of iASPP by 77.07% and up-regulated the expression of p53 by 179.37% and ASPP2 by 725.02% in HepG2 cells treated with luteolin for 12 h.
Conclusion: Luteolin has anti-proliferative and pro-apoptotic activity on hepatoma HepG2 cells,which is associated with the altered expression of pro-apoptotic factors and decreased ROS level in HepG2 cells.

Preparation of anti-B7-H4 monoclonal antibody to investigate B7-H4 expression in pancreatic cancer
HONG Bo, QIAN Yun, YAO Hang-Ping
Journal of ZheJiang University(Medical Science), 2013, 42(5): 511-516.   https://doi.org/10.3785/j.issn.1008-9292.2013.05.006
Abstract( 517 )   HTML (   PDF(1585KB)( 409 )

Objective: To prepare a monoclonal antibody (mAb) against extracellular domain of B7-H4 and to investigate the expression of B7-H4 in pancreatic cancer tissue with the prepared mAb.
Methods: Balb/c mice were immunized with 3T3-B7-H4 cells and the splenic cells of the immunized mice were fused with Sp2/0 myeloma cells by conventional hybridoma techniques.An indirect ELISA method using 3T3-B7-H4 lysate as antigen was established to screen antibody-producing hybridoma cell lines.Western blott,immunoprecipitation (IP),and immunohistochemistry (IHC) were applied to characterize the mAb.Immunohistochemical staining was used to detect the expression of B7-H4 in human pancreatic cancer tissue.The correlation of B3-H4 expressions and pathological features of pancreatic cancer was analyzed.
Results: A hybridoma cell line secreting mAb against B7-H4 was obtained.The subclass of this mAb was IgM,and the light chain was Kappa.Western blot and IP showed that the mAb specifically recognized B7-H4.IHC staining revealed that the mAb stained in a predominantly diffuse plasmalemmal or cytoplasmic pattern when applied to certain tumor tissues.The B7-H4 was diffusely expressed in the cytoplasma and/ or membrane of pancreatic cancer tissue,which was much higher than that expressed in normal pancreatic tissue (4.00±1.44 vs 1.12±0.78,P<0.01).The expression of B7-H4 was higher in pancreatic cancer tissues with higher pathological grade or with lymph node metastasis as compared with that in pancreatic cancer tissues with lower grade or with no lymph mode metastasis (6.10±0.72 vs 3.55±1.12,P<0.01;6.14±0.66 vs 3.70±1.25,P<0.01).The expression level of B7-H4 was not related to patients′ age and gender.
Conclusion: Monoclonal antibody against B7-H4 with high activity and specificity has been prepared successfully.The expression of B7-H4 in pancreatic cancer tissue is up-regulated,which is closely related to the tumor grade and lymph node metastasis in pancreatic cancer.

Expression of PCNA, C-fos and Bax proteins in human embryonic tongue tissues
LIU Xue-Hong, ZHANG Yong
Journal of ZheJiang University(Medical Science), 2013, 42(5): 517-522.   https://doi.org/10.3785/j.issn.1008-9292.2013.05.007
Abstract( 399 )   HTML (   PDF(2323KB)( 302 )

Objective: To investigate of proliferating cell nuclear antigen (PCNA), C-fos and Bax proteins in human embryonic tongue tissue of different developmental stages.
Methods: Immunohistochemistry was used to detect the expressions of PCNA, C-fos and Bax proteins in embryonic tongue tissues of fetuses with 2, 3 and 4 month gestational age (n=16). One-way ANOVA and LSD-t test were employed to compare the number of positive expression cells in tongue tissues of fetuses with different gestational age.
Results: In the fetuses at 2, 3 and 4 months of gestation, the numbers of PCNA-positive cells in tongue epithelial tissues were 20.20±7.13, 39.10±13.44 and 26.00±9.02, respectively; those in tongue muscle and fiber tissues were 17.20±8.99, 22.30±6.57 and 32.40±14.72, respectively. In fetuses at 2 month of gestation, no C-fos-positive cells were found in tongue tissues; while at 3 and 4 months of gestation, the numbers of C-fos-positive cells in the tongue epithelial layers were 25.10±7.91, 17.40±2.80; those in tongue muscle and fiber tissues were 24.50±4.67 and 28.00±7.75, respectively. Only weak positive expression of Bax protein was observed in the third month of gestation in embryonic tongue tissues. A significant difference was noted in PCNA expression in tongue epithelial layers, the muscle and fiber tissues (P<0.01 and P<0.05) among 3 embryonic periods. A significant difference was found in C-fos expression in tongue epithelial layers (P<0.01), but not in tongue muscle and fiber tissues (P>0.05) among 3 periods.
Conclusion: Dynamic changes were seen in PCNA and C-fos expressions in embryonic tongue tissues in different gestational ages of fetus, indicating these two proteins may participate in regulation of the development and differentiation of tongue tissues in human embryos and fetuses.

Effects of sub-micro emulsion composition on cellular disposition of incorporated lipophilic drug
SUN Xiao-Yi, Xiang-Zhi-Qiang, WU Shuo, LV Yuan-Yuan, LIANG Wen-Quan
Journal of ZheJiang University(Medical Science), 2013, 42(5): 523-529.   https://doi.org/10.3785/j.issn.1008-9292.2013.05.008
Abstract( 415 )   HTML (   PDF(1260KB)( 351 )
Objective: To investigate the effects of sub-micro emulsion composition on cellular uptake and disposition of incorporated lipophilic drug.
Methods: Sub-micro emulsions containing 10% oil, 1.2% lecithin and 2.25% glycerol were prepared, and the fluorescent agent coumarin 6 was used as a model drug. The effects of oil types, co-surfactants and cationic lipid on uptake and elimination kinetics of 6-coumarin in HeLa cells were studied. The uptake mechanism of sub-micro emulsions was further investigated.
Results: Oil type and Tweens had no influence on the cellular uptake. Modifications of surfactants with Span series increased the cellular influx, among which Span 20 with hydrophilic-lipophilic balance (HLB) value of 8.6 was the best enhancer. The intracellular drug level reached up to (46.09±1.98)ng/μg protein which had significant difference with control group \[(38.54±0.34)ng/μg protein\]. The positively charged emulsions significantly increased the uptake rate constant and elimination rate constant which were 4 times and 1.5 times of those in anionic groups, respectively. The uptake enhancement was also observed in cationic emulsions, cellular concentrations at plateau were (42.73±0.84)ng/μg protein, which was about 3 times of that in anionic emulsions \[(15.71±0.74)ng/μg protein\], when extracellular drug concentration kept at 100 ng/ml. Cationic emulsions delivered the payload mainly by direct drug transfer to contacted cells, while the negative ones depended on both drug passive diffusion and clathrin-mediated endocytosis of drug containing oil droplets which accounted for 20% of the intracellular drug.
Conclusion: Interfacial characteristic of sub-micro emulsions such as co-surfactants HLB as well as zeta potentials can influence lipophilic drug both in cellular uptake and elimination.
Characteristics of microsomal phase Ⅱ metabolic enzymes in mouse embryonic stem cell-derived liver tissue
LI Tong, GUO Mei-Yuan, MA Kui-Fen, DU Yue, He-Liang-Yan, ZHU Dan-Yan, LOU Yi-Jia
Journal of ZheJiang University(Medical Science), 2013, 42(5): 530-537.   https://doi.org/10.3785/j.issn.1008-9292.2013.05.009
Abstract( 450 )   HTML (   PDF(2680KB)( 329 )

Objective: To investigate the characteristics of phaseⅡ metabolic enzymes in mouse embryonic stem (ES) cell-derived liver tissue.
Methods: Mature hepatocytes were differentiated from embryonic stem cells in cultured mouse embryoid bodies (EB) at d18.Western blot was used to detect the expression of uridine 5′-diphosphate glucronosyl transferase (UGT1a1,UGT1a6) and microsomal glutathione S-transferases 1(mGST1) during the differentiation course.The derived liver tissue was incubated with UDPGA and 7-HFC,the formation of 7-HFC glucuronide was detected by HPLC to examine the total activities of UGT1a1and UGT1a6.Furthermore,the microsomes were incubated with CDNB and GSH,and the mGST1 activity was measured by spectrometry.
Results: An increase tendency of UGT1a1 expression was noticed during the differentiation course.UGT1a6 and mGST1 were not detected in the earlier stage until d18 of differentiation.The metabolic activity of mGST1 in the derived hepatocytes was 7.65 nmol/min/mg on d18.
Conclusion: The ES cell-derived liver tissue possesses partial metabolic function of phase Ⅱenzymes on d18 of differentiation,which might be used as a model for in vitro research on hepatic pathophysiology and phase Ⅱ drug metabolism.

PP2 enhances intercellular communication of gap junction in breast cancer Hs578T cells
DONG Shu-Ying, ZHENG Chao, JIANG Guo-Jun, HAN Xi, TONG Xu-Hui
Journal of ZheJiang University(Medical Science), 2013, 42(5): 538-542.   https://doi.org/10.3785/j.issn.1008-9292.2013.05.010
Abstract( 506 )   HTML (   PDF(1135KB)( 342 )

Objective: To investigate the effect of Src kinase inhibitor PP2 on intercellular communication of gap junction in breast cancer cells.
Methods: Cultured breast cancer Hs578T cells were treated with various concentrations of pp2 (0,1,2,4,8,16,32 μmol/L) for 24h.Cell growth was determined by MTT assay; dye spread in Hs578T cells was measured by Parachute assay; and the expression of Src kinase in Hs578T cells was detected by Western blot.
Results: MTT assay showed that the survive rate of Hs578T cells treated with PP2 (1~8 μmol/L) was 98%±3%~94%±4%.Parachute assay showed that compared to control group the standard normalized dye spread rates of Hs578T cells treated with 1,2,4 and 8 μmol/L PP2 were 1.60±0.08,2.00±0.05,2.20±0.05 and 2.70±0.09,respectively (all P<0.01).Moreover,compared to control group at the same time points,the standard normalized dye spread of Hs578T cells treated with 8 μmol/L PP2 for 6,12 and 24 h were 1.4±0.05,1.7±0.06,and 2.2±0.07,respectively (all P<0.01).Western blot showed that the expression ratios of Src kinase/β-actin of Hs578T cells treated with 1,2,4 and 8 μmol/L PP2 for 24 h were 0.93±0.02,0.70±0.09,0.66±0.09 and 0.36±0.10,which were significantly inhibited compared with control group (P<0.05 or 0.01).And the expression ratio of Src kinase/β-actin of Hs578T cells treated with 8 μmol/L PP2 for 6,12 and 24h was 0.82±0.03,0.66±0.08 and 0.59±0.09,which were all inhibited significantly compared to control group (P<0.01).
Conclusion: PP2 enhances the gap junction function in breast cancer Hs578T cells,which is probably related to the inhibition of Src kinase.

Effect of Helicobacter Pylori lipopolysaccharide on expression of Gli and Ptch-1 proteins in sonic hedgehog signaling pathway of gastric mucosa GES-1 cells
ZHANG Jun, ZHANG Guo-Xin, CHEN Fei-Fei, HE Bang-Shun, YE Feng, PAN Xiao-Lin
Journal of ZheJiang University(Medical Science), 2013, 42(5): 543-549.   https://doi.org/10.3785/j.issn.1008-9292.2013.05.011
Abstract( 496 )   HTML (   PDF(1348KB)( 364 )
Objective: To investigate the effect of Helicobacter Pylori lipopolysaccharide (Hp-LPS) on expression of Gli and Ptch-1 proteins in sonic hedgehog (Shh) signaling pathway of gastric mucosa GES-1 cells.
Methods: The LPS was extracted from Hp by hot phenol water method, and then the concentration of LPS was detected by the kinetic turbidimetric assay. GES-1 cells were stimulated by different concentrations of Hp-LPS (0, 1, 10, 20, 30 and 40 μg/ml). The inhibition rates of cell growth were measured by MTT assay after treated with Hp-LPS for 24 h. The expression of Gli and Ptch-1 proteins were determined by Western Blot.
Results: MTT assay showed that the inhibition rates of GES-1 cell growth after treatment by different concentrations of Hp-LPS (1, 10, 20, 30 and 40μg/ml) were 25.8%±2.7%, 34.2%±3.1%, 46.3%±3.4%, 60.8%±2.1% and 82.9%±2.8% respectively (r=0.985, P<0.001). Western blot showed that the expressions of Gli and Ptch-1 proteins were decreased after Hp-LPS treatment (0, 1, 10, 20, 30 and 40 μg/ml): the relative expression values of Gli were 1.286±0.180, 0.963±0.067, 0.850±0.085, 0.566±0.058, 0.549±0.056 and 0.377±0.047, respectively (r=-0.945, P<0.001); those of Ptch-1 were 1.688±0.088, 1.466±0.061, 1.170±0.065, 1.042±0.064, 0.648±0.057 and 0.482±0.074, respectively (r=-0.985, P<0.001).
Conclusion: Hp-LPS can decrease the related protein expression of Shh signaling pathway, which indicates that Hp may interfere with the function of Shh signaling pathway in gastric mucosa via the effect of its LPS.
Effect of renal sympathetic denervation on left ventricular hypertrophy and inflammatory factors in spontaneously hypertensive rats
TAN Li-Hua, LI Xiao-Gang, GUO Yun-Zhong, TANG Xiao-Hong, YANG Kan, JIANG Wei-Hong
Journal of ZheJiang University(Medical Science), 2013, 42(5): 550-555.   https://doi.org/10.3785/j.issn.1008-9292.2013.05.012
Abstract( 320 )   HTML (   PDF(1315KB)( 394 )

Objective: To investigate the effect of renal sympathetic denervation on left ventricular hypertrophy and inflammatory factors in spontaneously hypertensive rats.
Methods: Thirty six spontaneously hypertensive rats (SHR) were divided into 3 groups with 12 animals in each group: SHR control group,operation group and sham operation group.Bilateral renal sympathectomy or sham operation were performed in operation and sham groups,respectively; another 12 WKY rats served as normal controls.The blood pressure and body weight were examined weekly.The animals were sacrificed at w1 and w6,rat hearts were collected and left ventricular mass index (LVMI) was calculated.The expression of TLR4,TNF-α and IL-6 in heart tissue were detected by immunohistochemistry and Western blot.
Results: The systolic blood pressure \[(201.67±11.09)mmHg vs (140.0±10.86)mmHg,P<0.05\],diastolic blood pressure \[(144.50±10.48)mmHg vs (78.50±7.32)mmHg,P<0.05\],LVMI (2.44±0.05 vs 1.93±0.05,P<0.05),the expression of TLR4 (0.298±0.004 vs 0.126±0.004,P<0.05),NF-κB (0.249±0.006 vs 0.195±0.005, P<0.05),TNF-α (0.323±0.004 vs 0.146±0.004,P <0.05),IL-6 (0.283±0.005 vs 0.207±0.006,P<0.05) in SHR control group were significantly higher than those in WKY group.Compared to sham operation group,the systolic blood pressure (157.30±9.35 vs 197.30±11.5,P<0.05), diastolic blood pressure (112.50±6.25 vs 146.80±7.6,P<0.05),LVMI (2.32±0.04 vs 2.57±0.09, P<0.05,TLR4 (0.198±0.006 vs 0.317±0.008,P<0.05),NF-κB (0.208±0.006 vs 0.332±0.007,P<0.05),TNF-α (0.27±0.009 vs 0.375±0.004,P<0.05),IL-6 (0.218±0.004 vs 0.376±0.009,P<0.05) in operation group were all decreased at w1 after sympathectomy.Six weeks after the operation,there were no significant differences in systolic blood pressure (197.50±12.13 vs 208.83±10.23,P>0.05) and diastolic blood pressure (150.33±7.74 vs 151.50±8.22,P>0.05) between denervated and sham-operated SHRs; however,the LVMI (2.46±0.07 vs 2.81±0.05,P<0.05) and the expression of TLR4(0.301±0.009 vs 0.567±0.006, P<0.05),NF-κB (0.251±0.004 vs 0.476±0.009,P<0.05),TNF-α (0.324±0.005 vs 0.535±0.006,P<0.05,IL-6 (0.285±0.009 vs 0.549±0.007,P<0.05) in operation group were still significantly lower than those in sham operation group.
Conclusion: Renal sympathetic denervation can significantly delay the progression of LVH in SHR,which may associated with lowering blood pressure and decreasing expression of TLR4,NF-κB,TNF-α,IL-6 in myocardial tissue.

Extract of Herba Siegesbeckiae on mouse rheumatoid arthritis induced by anthrogen-CIA monoclonal antibody
FU Xu-Chun, JIANG Fang-Ping, FAN Jia-Zhen, BAI Hai-Bo
Journal of ZheJiang University(Medical Science), 2013, 42(5): 556-560.   https://doi.org/10.3785/j.issn.1008-9292.2013.05.013
Abstract( 226 )   HTML (   PDF(1010KB)( 361 )

Objective: To investigate the anti-inflammatory effect of Herba Siegesbeckiae extracts on mouse rheumatoid arthritis induced by arthrogen-CIA monoclonal antibody.
Methods: The rheumatoid arthritis was induced by arthrogen-CIA arthritogenic monoclonal antibody in mice.The sandwich enzyme-linked immunosorbent assay was used to determine the concentration of IL-1β in mouse serum,and the content of IL-6,IL-17 and MMP-3 in supernatant of tissue homogenate of hind limb below the stifle of mice.One-way ANOVA was used for data analysis.
Results: The toe swelling was attenuated in Siegesbeckiae group than that in model group [(0.218±0.0307)cm3 vs (0.2545±0.0179)cm3,P<0.05].The serum IL-1β level in Siegesbeckiae group was lower than that in model group [(63.74±21.74)pg/ml vs (104.96±31.22)pg/ml,P<0.01].The contents of IL-6,IL-17 and MMP-3 in tissue supernatants of Siegesbeckiae group were all lower than those of model group [(171.10±48.35)pg/ml vs (249.64±75.08)pg/ml,P<0.05; (115.42±56.52)pg/ml vs (208.40±88.54)pg/ml,P<0.05; (3660.31±1680.99)pg/ml vs (5420.79±1201.43)pg/ml,P<0.05,respectively].
Conclusion: The extract of Herba Siegesbeckiae has anti-inflammatory effect on mouse rheumatoid arthritis induced by mixed arthrogen monoclonal antibody.

Effects of Corbrin Shugan capsule on dimethylnitrosamine-induced hepatic fibrosis in rats
QIAN Ying, FU Xu-Chun, HU Rong, SHEN Li-Mei, BAI Hai-Bo
Journal of ZheJiang University(Medical Science), 2013, 42(5): 561-566.   https://doi.org/10.3785/j.issn.1008-9292.2013.05.014
Abstract( 305 )   HTML (   PDF(2437KB)( 349 )
Objective: To investigate the effects of Corbrin Shugan capsule on dimethylnitrosamine (DMN)-induced hepatic fibrosis in rats.
Methods: Hepatic fibrosis was induced by DMN in AD rats.The serum concentrations of Ⅲ pro-collagen (Ⅲ PC),laminin (LN) and tissue inhibitor of metalloproteinase-1(TIMP-1) were determined with ELISA.The concentration of albumin (ALB) in sera and the content of hydroxyproline (Hyp) in liver tissues were determined with chemical colorimetric and HPLC,respectively.The fibrosis area was measured with Motic Med 6.0 digital medical image analysis system.
Results: Compared to model group the high-dose (450 mg·kg-1),mid-dose (270 mg·kg-1) and low-dose (90 mg·kg-1) groups of Corbrin Shugan capsule had significantly lower serum content of Ⅲ PC [(34.46±13.95),(36.15±9.46),and (40.58±7.72)ng·ml-1 vs (49.38±10.95)ng·ml-1,P<0.05 or P<0.01],TIMP-1 [(16.65±4.24),(16.66±4.34),and (18.99±6.05)ng·ml-1 vs (30.84±14.48)ng·ml-1,P<0.05 or P<0.01],LN [(12.94±4.29),(12.96±3.21),and (15.32±8.00)ng·ml-1 vs (30.22±17.00)ng·ml-1,P<0.05 or P<0.01] and smaller hepatic fibrosis area [(0.02240±0.01337),(0.02176±0.01460) and (0.02384±0.01405)μm2 vs (0.03929±0.01732)μm2,P<0.05 or P<0.01]; the high-dose and mid-dose groups of Corbrin Shugan capsule had significantly lower content of Hyp in liver tissues [(0.77±0.09) and (0.81±0.09)μg·mg-1 vs (1.06±0.33)μg·mg-1,P<0.05 or P<0.01]; and the high-dose group of Corbrin Shugan capsule significantly increased the content of ALB in sera [(34.02±4.17)g·L-1 vs (30.25±4.21)g·L-1,P<0.05].
Conclusion: Corbrin Shugan capsule is effective in treatment of DMN-induced hepatic fibrosis in rats.
MAPK p38 pathway may be involved in renal function improvement in chronic renal failure rats treated with Jianpi Qinghua Decoction
MA Xiao-Hong, ZOU Bin, ZHANG Yue, HE Li-Qun
Journal of ZheJiang University(Medical Science), 2013, 42(5): 567-572.   https://doi.org/10.3785/j.issn.1008-9292.2013.05.015
Abstract( 213 )   HTML (   PDF(1726KB)( 323 )

Objective: To investigate the involvement of MAPK p38 pathway in treatment of chronic renal failure with Jianpi Qinghua Decoction in rats.
Methods: Forty SPF SD rats were divided into sham group(n=10),model group(n=10),Jianpi Qinghua group(n=10) and losartan group(n=10). Rat chronic renal failure was induced by 5/6 nephrectomy (Platt method) in model,Jianpi Qinghua and losartan groups,and rats in sham group received sham operation. Jianpi Qinghua decoction (3.9 g·200 g-1) or losartan (3.3 g·200 g-1) daily were administrated by gavage in Jianpi Qinghua and losartan groups for 60 days,respectively. Rats in sham and model groups were orally administered with saline of the same volume. The serum levels of creatinine and urea nitrogen were measured by biochemical method,the expression of MAPK p38 was detected by Western Blot,and renal pathological changes were observed with hematoxylin-eosin staining.
Results: Compared to model group,serum creatinine levels after 60d in Jianpi Qinghua and losartan groups were decreased significantly (42.67±5.98 or 40.90±5.07 vs 60.90±9.54,both P<0.01), the expression of MAPK p38 was significantly down-regulated (0.555±0.004 or 0.587±0.045 vs 0.930±0.265,both P<0.01) and serum urea nitrogen was also decreased (8.56±0.75 or 7.97±0.86 vs 8.62±0.62,both P<0.05). The renal pathology in the model group presented glomerular mesangial proliferation,hyperplasia of glomenrulus mesangial cells and interstitial inflammation. Those pathological changes were attenuated significantly in Jianpi Qinghua and losartan groups.
Conclusion: Jianpi Qinghua Decoctions can improve the renal function and renal pathological changes in a rat with chronic renal failure,which may be associated with down-regulation of MAPK p38 immune inflammatory pathways.

Two-dimensional ultrasound speckle tracking imaging in detection of myocardial torsion for patients with chronic pulmonary heart disease
HUANG Jing-Yuan, Zheng-Zhe-Lan, Ye-Meng
Journal of ZheJiang University(Medical Science), 2013, 42(5): 573-577.   https://doi.org/10.3785/j.issn.1008-9292.2013.05.016
Abstract( 82 )   HTML (   PDF(865KB)( 318 )

Objective: To evaluate the application of two-dimensional ultrasound speckle tracking imaging (STI) in assessment of myocardial torsion and left ventricular function for patients with chronic pulmonary heart disease(CPHD).
Methods: Thirty six patients with CPHD and 38 normal subjects were enrolled in the study,and STI examinations were performed.The left ventricular short-axis views (mitral level,apical level) were observed,the rotation angles of the standardized time point were measured at each short-axis views and the corresponding left ventricular torsion angles were calculated.Simultaneously,the basal rotation peak,apical rotation peak,left ventricular twist peak,end-systolic basal rotation value,end-systolic apical rotation value and end-systolic left ventricular twist value were recorded.The correlations of left ventricular ejection fraction (LVEF) with left ventricular torsion peak,end-systolic left ventricular twist value in patients were analyzed.
Results: Compared to normal controls,the basal rotation peak,apical rotation peak,left ventricular twist peak,end-systolic basal rotation value,end-systolic apical rotation value and end-systolic left ventricular twist value were significantly lower (P<0.01) in CPHD patients.The LVEF was highly correlated with left ventricular twist peak and end-systolic left ventricular twist value in CPHD patients (r=0.967,0.952,P<0.001).
Conclusion: STI is sensitive to detect left ventricular myocardial torsion change; left ventricular torsion peak and end-systolic left ventricular twist value can be used to assess the left ventricular function in patients with CPHD.

Bone alkaline phosphatase and N-MID osteocalcin in monitoring of osteoporosis treatment with recombinant human parathyroid hormone 1-34
WENG Xiu-Mei, PAN Jian-Ping
Journal of ZheJiang University(Medical Science), 2013, 42(5): 578-582.   https://doi.org/10.3785/j.issn.1008-9292.2013.05.017
Abstract( 108 )   HTML (   PDF(517KB)( 354 )

Objective: To evaluate the application of bone turnover markers bone alkaline phosphatase (BALP) and N-MID osteocalcin (N-MID) in monitoring of osteoporosis treatment with recombined parathyroid hormone 1-34 (rhPTH1-34).
Methods: The bone mineral density (BMD) of the lumbar spine L2-L4 and the proximal femur were examined by dual energy X-ray absorptiometry (DXA) before and 6 and 12 months after rhPTH 1-34 treatment. Meanwhile, serum levels of BALP and N-MID were detected by electro-chemiluminescence assay.
Results: Six months after rhPTH 1-34 treatment, the BMD of proximal femur remained unchanged, and the BMD of the lumbar L2-L4 spine increased from 0.753 g/cm2 to 0.781 g/cm2(P<0.05); while serum levels of N-MID increased from 15.46 ng/ml to 27.07 ng/ml(P<0.01),BALP from 14.05 μg/ml to 24.31 μg/ml(P<0.01). Twelve months after drug administration, no significant changes were observed in BMD of proximal femur, and the BMD of the lumbar spine L2-L4 increased from 0.753 g/cm2 to 0.807 g/cm2(P<0.01) while serum levels of N-MID and BALP increased from 15.46 ng/ml and 14.05μg/ml to 49.38 ng/ml and 33.99 μg/ml, respectively (both P<0.01).
Conclusion: Serum levels of BALP and N-MID are more sensitive than BMD. Combination of two methods may provide better indicators for monitoring of osteoporosis treatment.

The roles of glutamate in sleep and wakefulness
SHI Yu-Feng, YU Yan-Qin
Journal of ZheJiang University(Medical Science), 2013, 42(5): 583-590.   https://doi.org/10.3785/j.issn.1008-9292.2013.05.018
Abstract( 151 )   HTML (   PDF(673KB)( 360 )

Glutamate as an excitatory neurotransmitter in the central nervous system,participate in initiation and maintaining of sleep and wakefulness.The paper presents an overview of the research progress of glutamate in the regulation of sleep and wakefulness,especially focuses on its role in the brainstem,lateral hypothalamus and basal forebrain.Glutamate in the brain stem regulates the brain activity and maintains muscle tone during the wakefulness,as well as adjusts the electroencephalograph (EEG) in rapid eye movement phase and leads to muscle weakness.Glutamate in the lateral hypothalamus participates in the lateral hypothalamic arousal system by activating orexins neurons.The basal forebrain glutamatergic neurons take part in EEG synchronization and cause the decrease of sleep.Finally,The glutamatergic neurons of the cerebral cortex is not just a target of the arousal system,but itself contribute to regulation of arousal.Meantime,the glutamatergic neurons can regulate sleep stages through interaction with other types of neurons,which forms a complex sleep-wake regulation network in the brain.These indicate that the switches between different phases of sleep and wakefulness have different neuronal circuits.So we also reviewed the neuronal circuits and mechanisms that glutamate may be involved in.This review will help us to get a better understanding of the roles of glutamate in sleep and wakefulness.

Crosstalk between canonical TGF-β/Smad and Wnt/β-catenin signaling pathway
RAO Cui, LIN Shan-Li, WEN Huan, DENG Hong
Journal of ZheJiang University(Medical Science), 2013, 42(5): 591-597.   https://doi.org/10.3785/j.issn.1008-9292.2013.05.019
Abstract( 224 )   HTML (   PDF(618KB)( 378 )

TGF-β signaling pathway plays a central role in the signaling networks that control the growth, differentiation of the cell, and the initiation of fibrosis and cancer. Wnt signaling pathway is critical for the embryonic development and the invasion and migration of cancer cells. TGF-β signaling and Wnt signaling, both of which play an important role in regulating embryonic development, fibrotic disease and tumor progression, have a close relationship. Researches find several typical cross points between these two signaling systems, such as Smad, Axin, Dvl and β-catenin. In this review, we focus on the crosstalk between TGF-β signaling and Wnt signaling through these typical factors, intending to better understand the process of fibrosis and tumor progression.

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