Objective: To investigate the inhibitory effect of Rh2 on HepG2 cells and explore the underlying mechanism.Methods: We used lentivirus carrying β-catenin to infect HepG2 cell, and detected expression of β-catenin using fluorescence microscopy.The effect of Rh2 on proliferation of HepG2-β-catenin and HepG2 cells was measured by CKK-8 assay,and flow cytometry was used to detect cell cycle and apoptosis.The activity of Gsk-3βwas checked by ELISA kit.The expression of Gsk-3β,β-catenin,Bax,Bcl2,CyclinD1,MMP3 genes were measured by qRT-PCR.In order to checked the relationship between β-catenin and TCF4,CHIP assay kit was used,the expression of Bax,Bcl2,CyclinD1,MMP3 genes were measured by PCR.The expressions of Gsk-3β,β-catenin,Bax,Bcl2,CyclinD1,MMP3 proteins were examined by Western blot.Results:HepG2 cells were successfully infected by pLOV-EF1a-MCS-3FLAG-β-catenin lentivirus,named HepG2-β-catenin.CCK-8 showed that ginsenoside Rh2 could effectively inhibit the proliferation of HepG2 and HepG2-β-catenin cells in vitro,which exhibits a dose-dependent manner at range of 10-160 μmol/L Rh2.The IC50 of Rh2 exposure on HepG2 cell for 48,72 h were 100 μmol/L and 58.12 μmol/L,but the IC50 of Rh2 exposure on HepG2-β-catenin for 48,72 h were 129.2 μmol/L,83.33 μmol/L,respectively.The IC50 of Rh2 exposure on HepG2-β-catenin cell was higher than HepG2 cell, compared with HepG2 group the differences was statistically significant ( P<0.01 ).Flow cytometry indicated that Rh2 could arrest HepG2 and HepG2-β-catenin cells in G0/G1 phase;the cell population in G0/G1 phase of HepG2+Rh2 group was(64.57±0.65)%,HepG2-β-catenin+Rh2 group was(58.61±2.01)%.Flow cytometry indicated that Rh2 could induced early apoptosis in HepG2 and HepG2-β-catenin cells.The apoptosis rate of HepG2 +Rh2 group was (17.27 ±2.77)%,HepG2-β-catenin +Rh2 group(9.02 ±1.76)%.The ELISA results indicated that HepG2 cells was induced by Rh2 for 12,24,48,72 h,the activity of Gsk-3βgradually increased,peak in 48 h,then decreased.Compared with control group,Rh2 induced HepG2 and HepG2-β-catenin cells for 48 hours, Gsk-3βactivity were increased, and their activity reduced after adding Bio, there were no significant differences between HepG2+Rh2 and HepG2-β-catenin+Rh2 groups.The PCR,CHIP and WB results showed that the expression of Gsk-3β,Bax gene and proteins increased,while theβ-catenin,CyclinD1,Bcl2,MMP3 gene and proteins down-regulation in HepG2 and HepG2-β-catenin cell induced by Rh2.Compared with HepG2-β-catenin +Rh2 group, the expression of other gene and proteins changed significantly,however,Gsk-3βwas no significant difference.Conclusion:Over-expression of β-catenin may weaken the phar-macological effects of ginsenoside Rh2 on HepG2 cells.The activity of Gsk-3βwas increased by ginsenoside Rh2 to degradeβ-catenin, affecting the expression of downstream genes,promoting apoptosis of liver cancer cells and inhibiting metastasis.

Objective:To study the mechanism of ginsenoside Rh2 inhibiting HepG2 cells migration.Methods:HepG2 cells in logarithmic growth phase were cultured in 96-well plates,which were induced by different concentration Rh2,respectively for 24,48,72 hours.The cell inhibition was detected by Cell Counting Kit.Transwell chambers was used to checked HepG2 cell migration ability;luciferase was tested by Luciferase Reporter Assay system reagent;The expressions of P-ERK,ERK,P-P38,P-38,P-JUK,JUK,MMP3 proteins were detect by Western blot;the expression of AP1,MMP3 gene were detected by Quantitative PCR;The expression of AP1, MMP3 fluorescence protein were observed by fluorescence microscopy.Results:Administrated with different concentration of Rh2 after 24 ,48 ,72 h,the proliferation of HepG2 cells were inhibited ( P<0.05) ,and in dose-and time-dependent manner.Transwell assay showed Rh2 could significantly inhibited migration of HepG2 cells.The expressions of P-ERK , MMP3 proteins were significantly decreased,the expressions of P-JUK, P-P38 proteins were significantly increased, expression levels of ERK, P-38, JUK were no significant difference.Expression of AP1,MMP3 gene were significantly decreased,the expressions of AP1,MMP3 fluorescence proteins were significantly decreased.Conclusion:Ginsenoside Rh2 can activate MAPK pathway to inhibit the migration of HepG2 cells.

Objective To investigate the effects of functional magnetic resonance imaging (fMRI)with acute ischemic stroke (AIS) patients,and to evaluate the relationship between brain reorganization and motor recovery.Methods Nine AIS patients and 9 healthy volunteers were assessed by fMR1 during passive finger clenching at a pace of 1 Hz.The fMRI results were analyzed using SPM2 software.Lateral indices (LIs) and activated regions were calculated,and the relationship between LI and muscle strength was examined.Results In the control group,activation was observed in the contralateral sensorimotor cortex (SMC) and the bilateral supplementary area (SMA) during the passive movement.In the AIS group,similar results were recorded dur- ing unaffected hand movement,but the ipsilateral activation areas were greater than those on the eontralateral side during movement of the affected hand.LI results confirmed that movement of the affected hand mainly elici- ted activation in the ipsilateral hemisphere.Conclusion The different fMRI manifestations of patients and nor- mal subjects reflect brain compensation,and fMRI is valuable for studying the correlation between motor function and brain reorganization.

Objective To investigate the clinical effect of sitagliptin therapy in patients with type 2 diabetes and its effect on glucose trans-porter-4 ( GLUT4).Methods Sixty four patients with type 2 diabetes were randomly divided into two groups , and patients in the observation group (n=32) were given sitagliptin 100 mg qd orally, and those in the control group ( n=32 ) were given acarbose 50 mg tid within meals.The treatment lasted for 12 weeks.The data of the clinical efficacy of the two groups were compared before and after treatment.While 30 healthy controls were selected , only given health education.Data of the expre-ssion of GLUT4 in serum of three groups were detected.Results In ob-servation group , the data of fasting blood glucose ( FBG) , 2 h postprandi-al blood glucose(2 h PG)and glycosylated hemoglobin(HbA1c) were ob-viously better than those in the control group [(6.32 ±0.44 ),(8.76 ± 0.93),(6.85 ±0.37)mmol· L-1 vs(6.44 ±0.46),(9.15 ±0.94), (6.98 ±0.28)mmol· L-1],(P<0.05).The islet beta cell function index in observation group was improved significantly compared with the control group [(2.59 ±0.22),(66 ±18) vs(2.78 ±0.39),(62 ±13)], (P<0.05).The data of serum levels of GLUT4 in the observation group was significantly higher than that in control group [(6.07 ±0.59)vs(2.58 ±0.45)μg· L-1](P <0.05).Conclusion Sitagliptin can effectively control the level of blood sugar , improve islet beta cell function , and the effect may be related to the up -regulation of GLUT4 ex-pression.

OBJECTIVETo study the effects of deltamethrin (DM) on cell survival rate and intracellular Ca(2+) ([Ca(2+)]i) concentration in primary cultured astrocytes of rat.

METHODSThe cell survival rate was measured by Typan Blue assay; the intracellular [Ca(2+)]i concentration was determined by the fluorescent Ca(2+) indicator Fura-2/AM.

RESULTSThe survival rate of astrocytes was decreased to 91.9% after astrocytes were incubated with 1 x 10(-5) mol/L DM for 72 h (P < 0.05). The cell survival rates were 89.0%, 84.8%, 81.2% and 79.2% respectively when astrocytes were administered with 1 x 10(-4) mol/L DM for 4, 12, 24 and 72 h, which were remarkably lower than control groups (P < 0.01). Comparing with controls and before DM treatment, sharp increases in [Ca(2+)]i concentration [(451.4 +/- 42.3), (536.9 +/- 47.5) and (870.9 +/- 100.5) nmol/L respectively] were observed when astrocytes were incubated with 1 x 10(-7), 1 x 10(-6) and 1 x 10(-5) mol/L DM for 5 minutes (P < 0.01). After astrocytes were treated with 1 x 10(-8), 1 x 10(-7), 1 x 10(-6), 1 x 10(-5) mol/L DM for 15 minutes, the [Ca(2+)]i concentrations were decreased to (124.3 +/- 6.0), (131.3 +/- 19.1), (118.9 +/- 1.4), (136.6 +/- 3.8) nmol/L respectively, which were significantly different from those of controls and before treatment. And this situation was almost keeping stable to 30 min.

CONCLUSIONThe cell survival rate was decreased and the [Ca(2+)]i concentration was temporarily increased when astrocytes were treated with DM.

Animals ; Astrocytes ; cytology ; drug effects ; metabolism ; Calcium ; metabolism ; Cell Survival ; drug effects ; Cells, Cultured ; Insecticides ; toxicity ; Nitriles ; Pyrethrins ; toxicity ; Rats

OBJECTIVETo investigate the effects of deltamethrin on the filial brain nitric oxide synthase (NOS) activity and neurobehavioral development of the exposed lactational rats.

METHODSPregnant rats were randomizedly divided into the treated group and the control group. The treated group was administered orally with 3.35, 6.70 mg/kg deltamethrin every other day from postnatal day (PND) 1 to PND 19 while the control group was administered with the corn oil of same amount in the same period. The activity of NOS of filial brain and neurobehavioral functions of the filial rats were observed.

RESULTSThe lactational survival rate (81.80%:78.60%) in both treated groups was decreased significantly (P < 0.01) compared with that in the control group. The body weight of filial rats on PND 10, 21 in 6.70 mg/kg DM treated group [(16.62 +/- 2.2 8), (31.34 +/- 6.94) g] was less than those in the control group (P < 0.05). The delayed time in the filial rats in 6.70 mg/kg group was (3.05 +/- 1.20) s and the positive rates of passive escaping response in 3.35 and 6.70 mg/kg DM treated group were 22.5% and 21.5% respectively. There was the trend of the developmental increase of the activity of filial brain NOS between PND 5 and PND 21 and the NOS activity of rat brain on PND 5 in 6.70 mg/kg group [(0.60 +/- 0.07) U.mg pro(-1).h(-1)] was lower than that in the control group (P < 0.05).

CONCLUSIONExposure to high dose of deltamethrin in lactational female rats will decrease the activity of NOS of brain and retard the neurobehavioral development of their filial rats.

Animals ; Animals, Newborn ; Brain ; drug effects ; enzymology ; Female ; Learning ; drug effects ; Male ; Memory ; drug effects ; Nitric Oxide Synthase ; metabolism ; Nitriles ; toxicity ; Pregnancy ; Prenatal Exposure Delayed Effects ; Pyrethrins ; toxicity ; Rats ; Rats, Wistar

OBJECTIVETo investigate the inhibitory effect of trichostatin A (TSA) on the proliferation of HepG2 cells and explore the underlying mechanism.

METHODSHepG2 cells exposed to different concentrations of TSA for 24, 48, or 72 h were examined for cell growth inhibition using a cell counting kit, changes in cell cycle distribution with flow cytometry, cell apoptosis with annexin V-FTIC/PI double staining, and cell morphology changes under inverted microscope. The expressions of beta-catenin, HDAC1, HDAC3, H3K9, cyclinD1 and Bax proteins in the exposed cells were detected by Western blotting, and the expressions of HDAC1 and HDAC3 mRNAs by quantitative fluorescent PCR.

RESULTSExposure to TSA caused significant dose- and time-dependent inhibition of HepG2 cell proliferation (P<0.05) and resulted in increased cell percentage in G0/G1 and G2/M phases and decreased cell percentage in S phase. The apoptotic index in the control group was (6.22 ± 0.25)%, which increased to (7.17 ± 0.20)% and (18.14 ± 0.42)% after exposure to 250 and 500 nmol/L TSA, respectively. Exposure to 250 and 500 nmol/L TSA also caused cell morphology changes with numerous floating cells. The expressions of beta-catenin, H3K9 and Bax proteins were significantly increased and CyclinD1, HDAC1, and HDAC3 protein expressions decreased in TSA-treated cells, but the expressions of HDAC1 and HDAC3 mRNAs showed no significant changes.

CONCLUSIONSTSA can inhibit the proliferation of HepG2 cells and induce cell cycle arrest and apoptosis by inhibiting HDAC activity, promoting histone acetylation, and activating Wnt/beta-catenin signaling pathway.

Acetylation ; Apoptosis ; Cell Cycle Checkpoints ; Cell Proliferation ; drug effects ; Cyclin D1 ; metabolism ; Hep G2 Cells ; drug effects ; Histone Deacetylase 1 ; metabolism ; Histone Deacetylases ; metabolism ; Histones ; metabolism ; Humans ; Hydroxamic Acids ; pharmacology ; Wnt Signaling Pathway ; bcl-2-Associated X Protein ; metabolism ; beta Catenin ; metabolism

OBJECTIVETo elucidate the mechanism of damage on central nervous system (CNS) caused by deltamethrin (DM).

METHODSThe mRNA and protein expressions of brain-derived neurotrophic factor (BDNF) in the cerebral cortex and hippocampus of the rats exposed to DM were measured by retro-transcription-polymerase chain reaction (RT-PCR), dot blot, flow cytometry analysis and immunohistochemistry.

RESULTSAfter exposure to DM at high-dose (DM1, 25.0 mg x kg(-1) x d(-1), i.p.) once and low-dose (DM2, 12.5 mg x kg(-1) x d(-1), i.p.) for 5 days, the level of BDNF mRNA and protein expression in the cerebral cortex and hippocampus of the rats increased significantly. The levels of BDNF mRNA and protein expression in the cerebral cortex and hippocampus measured by of RT-PCR in the rats with DM1 and DM2 were higher than those in the controls by 48% and 56%, and 59% and 54%, respectively. And, those measured by dot blot in the rats with MD1 and MD2 were 186% and 161%, and 148% and 158% of those in the controls, respectively, basically similar to those measured by RT-PCR. Flow cytometric analysis showed that the levels of BDNF mRNA and protein expression in the cerebral cortex and hippocampus in the rats with DM1 and DM2 were higher than those in the controls by 53% and 89%, and 45% and 46%, respectively. Immunohistochemical analysis showed that protein expression in the cerebral cortex of the rats with DM1 and DM2 were 129% and 147% of those in the controls, same as the flow cytometric analysis, but those were significantly higher in the hippocampus mainly in the CA1 and DG areas of the rats with MD1 and the CA3 and DG areas of the rats with DM2.

CONCLUSIONSDM could induce BDNF mRNA and protein expression in the cerebral cortex and hippocampus of the rats, which could play an important role in repairing of nerve damage.

Animals ; Brain-Derived Neurotrophic Factor ; biosynthesis ; genetics ; Cerebral Cortex ; drug effects ; metabolism ; Hippocampus ; drug effects ; metabolism ; Insecticides ; toxicity ; Male ; Nitriles ; Pyrethrins ; toxicity ; RNA, Messenger ; biosynthesis ; drug effects ; Rats ; Rats, Wistar ; Reverse Transcriptase Polymerase Chain Reaction

This report aims to investigate the dynamical changes of HBcAg18-27 epitope specific cytotoxic T lymphocytes(CTL), alanine aminotransferase (ALT), HBV DNA and HBsAg in peripheral blood of acute hepatitis B patients, and to explore the roles of HBcAg18-27-specific CTLs in virus clearance and liver injury. Acute hepatitis B (AHB) and chronic hepatitis B (CHB) patients were divided into two groups according to results of HLA-A0201. Patients with positive HLA-A0201 were classified into HBcAg-specific CTL group and those with negative HLA-A0201 were referred as control group. The specific CTLs were stained with HLA-A0201 limited HBcAg18-27 epitope MHC-Pentamer and the frequencies of CTLs, T, B, NK and NKT cells were detected by flow cytometry (FCM). The serum ALT, HBV DNA and HBsAg were examined using speed analysis, quantitative PCR and abbott chemiluminescent technology. The frequencies of HBcAg18-27-specific CTLs in AHB patients were higher in the early three weeks as compared to the late three weeks. The apex time of HBV-specific CTL frequencies lagged behind those of HBV DNA, HBsAg and ALT. The loss of HBsAg in patients with high frequencies of HBV-specific CTL was earlier than that in patients with low frequencies (t = 2.018, P value is less than 0.05). In the second week the peak frequencies of CD3+CD8+ cells overlapped with that of HBcAg18-27-specific CTLs and with a positive correlation between (r = 0.420, P value is less than 0.05). During the early stages of AHB, the frequencies of NK and NKT cells were found significantly lower than that of control group and CHB group and the levels were back to normal after recovery. Moreover, a negative correlation existed between the frequencies of NK cells and the dynamic changes of HBcAg18-27-specific CTLs (r = -0.435, P value is less than 0.01) in AHB group. The frequencies of HBcAg18-27-specific CTLs were significantly higher as compared to CHB group in the first three weeks (z = -3.258, -4.04, and -3.259, P value is less than 0.01). The early loss of HBsAg was closely related to the high frequencies of HBcAg18-27 specific CTLs in AHB patients. HBcAg-specific CTL frequencies in peripheral blood could be used to predict clinical outcome after HBV infection. The frequencies of CD8+ T cells can reflect the changes of frequencies of HBcAg-specific CTL during acute HBV infection.
Adolescent ; Adult ; Case-Control Studies ; Female ; HLA-A2 Antigen ; immunology ; Hepatitis B ; immunology ; Hepatitis B Core Antigens ; blood ; immunology ; Humans ; Killer Cells, Natural ; immunology ; Lymphocyte Count ; Male ; Middle Aged ; T-Lymphocytes, Cytotoxic ; cytology ; immunology ; Young Adult

Ketamine (KTM), a N-methyl-D-aspartate (NMDA) receptor antagonist, was found to has an anti-inflammatory effect, but some patients suffered from exacerbated pro-inflammatory reactions after anesthesia with KTM. The present study was aimed to examine the underlying mechanism of pro-inflammatory effects of KTM. In this study, RAW264.7 cells were exposed to KTM and NMDA alone or combined for 30 min before lipopolysaccharide (LPS) stimulation. The expression levels of IL-6 and TNF-α were detected by RT-PCR and ELISA, and those of NMDA receptors by RT-PCR in RAW264.7 cells. Additionally, the TLR4 expression was determined by RT-PCR and flow cytometry, respectively. The results showed that in RAW264.7 cells, KTM alone promoted the TLR4 expression, but did not increase the expression of IL-6 or TNF-α. In the presence of LPS, KTM caused a significantly higher expression of IL-6 and TNF-α than LPS alone. NMDA could neither alter the IL-6 and TNF-α mRNA expression, nor reverse the enhanced expression of IL-6 and TNF-α mRNA by KTM in LPS-challenged cells. After TLR4-siRNA transfection, RAW264.7 cells pretreated with KTM no longer promoted the IL-6 and TNF-α expression in the presence of LPS. In conclusion, KTM accelerated LPS-induced inflammation in RAW264.7 cells by promoting TLR4 expression, independent of NMDA receptor.
Anesthetics, Dissociative ; pharmacology ; Animals ; Cell Survival ; drug effects ; Gene Expression Regulation ; Inflammation Mediators ; pharmacology ; Interleukin-6 ; genetics ; Ketamine ; pharmacology ; Lipopolysaccharides ; pharmacology ; Macrophages ; drug effects ; metabolism ; Male ; Mice ; N-Methylaspartate ; pharmacology ; RAW 264.7 Cells ; Signal Transduction ; drug effects ; Toll-Like Receptor 4 ; genetics ; metabolism ; Tumor Necrosis Factor-alpha ; genetics