The protective effect of trigonelline on H / 中国药理学通报

Aim To study the protective effect of trigonelline on H

Study on spectrum-toxicity relationship of in vitro hepatotoxicity of aqueous extract from Euodia rutaecarpa / 中国药房

OBJECTIVE To study the spectru m-toxicity relationship of in vitro hepatotoxicity of aqueous extract from Euodia rutaecarpa. METHODS The aqueous extract from 16 batches of E. rutaecarpa from different habitats were prepared. The fingerprints of aqueous extract from E. rutaecarpa were established by ultra high performance liquid chromatography （UPLC） method and Similarity Evaluation System of TCM Fingerprint （2012A edition ），and common peaks were identified and the similarity was evaluated. Using normal human hepatocytes L 02 as subject ，inhibitory effect of aqueous extract from 16 batches of E. rutaecarpa to them were investigated. The spectrum-toxicity relationship of UPLC fingerprint of aqueous extract from E. rutaecarpa with the hepatotoxicity of hepatocytes L 02 was analyzed by grey relational analysis （GRA）and partial least squares regression analysis （PLSR）. The corresponding compound of the chromatographic peak with the greatest correlation with the in vitro hepatotoxicity of E. rutaecarpa were isolated ，prepared and identified. RESULTS There were 27 common peaks in UPLC fingerprints of aqueous extract from 16 batches of E. rutaecarpa ，with similarity of 0.375-0.995. Totally 9 peaks were confirmed ，i.e. neochlorogenic acid （peak 5），chlorogenic acid （peak 9），cryptochlorogenic acid （peak 10），caffeic acid （peak 12），rutin （peak 16），hyperin（peak 17），dehydroevotarine（peak 19），evotarine（peak 24），rutecarpine（peak 25）. The aqueous extract from 16 batches of E. rutaecarpa showed significant inhibitory effect on the growth of L 02 cells（P＜0.05 or P＜0.01），and the inhibitory rate ranged from 6.68％ to 67.95％. GRA showed that there were 18 common peaks with correlation degree greater than 0.8，which were peak 8＞peak 3＞peak 23＞peak 7＞peak 4＞peak 9＞peak 12＞peak 2＞peak 19＞peak 6＞ 4928381。E-mail：799247687@qq.com peak 15＞peak 5＞peak 1＞peak 17＞peak 21＞peak 26＞ peak 20＞peak 14 in descending order of correlation degree. PLSR showed that there were 14 peaks with regression coefficient＞0 and variable importance projection value ＞1，and the order of regression coefficient was peak 8＞peak 3＞peak 23＞ peak 2＞peak 7＞peak 4＞peak 12＞peak 9＞peak 19＞peak 5＞peak 17＞peak 26＞peak 10＞peak 15. Peak 8 had the greatest correlation with in vitro hepatotoxicity，and the corresponding compound of this peak was identified as 6-O-trans caffeoyl gluconic acid. CONCLUSIONS The in vitro hepatotoxicity of aqueous extract from E. rutaecarpa is the result of multiple component interaction，among which 6-O-trans caffeoyl gluconic acid shows closest relation with in vitro hepatotoxicity.

Preparation and characterization of fenugreek leaf flavonoids and their protective effects against oxidative damage to hepatocytes / 中国中药杂志

The present study investigated the main components of fenugreek(Trigonella foenum-graecum L.) leaf flavonoids(FLFs) and their antioxidant activity. FLFs were prepared and enriched by solvent extraction, and the flavonoids were characterized by high-performance liquid chromatography-quadrupole-time-of-flight tandem mass spectrometry(HPLC-Q-TOF-MS/MS). The protective effect of FLFs against H_2O_2-induced stress damage to L02 hepatocytes was also investigated. Firstly, the cell viability was measured by MTT assay. The oxidative stress injury model was induced by H_2O_2 in L02 cells. The release of lactate dehydrogenase(LDH), the content of reduced glutathione(GSH) and malondialdehyde(MDA), and the activities of superoxide dismutase(SOD) and catalase(CAT) were measured by assay kits. Hoechst fluorescence staining was performed to observe the cell apoptosis. The expression levels of c-Jun N-terminal kinase(JNK), extracellular signal-regulated kinase 1/2(ERK1/2), nuclear factor erythroid-2 related factor 2(Nrf2), heme oxygenase 1(HO-1), and their phosphorylated proteins were detected by Western blot. Based on the MS fragment ion information and data in databases, FLFs contained eight flavonoids with quercetin and kaempferol as the main aglycons. The cell viabi-lity assay revealed that as compared with the conditions in the H_2O_2 treatment group, 3.125-25 μg·mL~(-1) FLFs could increase the viability of L02 cells, reduce LDH release and MDA content in a dose-dependent manner, potentiate the activities of SOD, CAT, and GSH, decrease the phosphorylation of JNK and ERK1/2 proteins, and up-regulate the expression of Nrf2 and HO-1. The results of fluorescence staining showed that the nucleus of the H_2O_2 treatment group showed concentrated and dense strong blue fluorescence, while the blue fluorescence intensity of the FLFs group decreased significantly. FLFs showed a protective effect against H_2O_2-induced oxidative damage in L02 cells, and the underlying mechanism is associated with the enhancement of cell capability in clearing oxygen free radicals and the inhibition of apoptosis by the activation of the MAPKs/Nrf2/HO-1 signaling pathway. The antioxidant effect of fenugreek leaf is related to its rich flavonoids.

Role of NRF2 signaling pathway in trichloromethane-induced oxidative stress in L02 cells / 中国职业医学

@#Objective - （ ） To investigate the effects of nuclear factor erythroid 2 related factor 2 NRF2 on the oxidative stress （ ） Methods ） ，， induced by trichloromethane TCM in human normal hepatocyte L02 cells. i L02 cells were stimulated with 1 2 ， ， ， （ ）， 4 8 12 16 and 20 mmol/L TCM solution dissolved in dimethyl sulfoxide and the control group and blank group were set ， - ， up. After culturing for 24 hours the cell viability was detected by CCK 8 colorimetric method and the concentration of TCM ） -， - stimulation was screened. ii L02 cells in logarithmic growth phase were randomly divided into control group and low medium - ， ， ， and high dose groups. After 24 hours of exposure to 0 4 8 and 12 mmol/L TCM the cells were collected. The activity of （ ）， （ ）， （ - ） （ ） superoxide dismutase SOD catalase CAT glutathione peroxidase GSH Px and the level of malondialdehyde MDA NRF2， - （HO-1）， were detected by colorimetric analysis. The mRNA expression levels of heme oxygenase 1 glutamate cysteine （GCLC） （） （NQO1） - ligase catalytic subunit and NAD P H quinone dehydrogenase 1 were detected by real time fluorescence ， - ， polymerase chain reaction. The protein levels of NRF2 HO 1 GCLC and NQO1 were detected by Western blotting.Results ） ， ， ， ， i When the concentration of TCM was 4 8 12 16 and 20 mmol/L the survival rate of L02 cells decreased （ P ） ， ， significantly compared with the control group all <0.05 . The concentration of 0 4 8 and 12 mmol/L were selected as the ） ， - stimulation doses for subsequent experiments. ii Compared with the control group the activities of SOD and GSH Px in L02 （ P ） （ P ）， - cells in the three doses groups decreased all <0.05 and the levels of MAD increased all <0.05 with a dose effect - （P ）， relationship. The CAT activity of L02 cells in the medium dose group was lower than that in the control group <0.05 and the - （ P ） CAT activity of L02 cells in the high dose group was lower than that in the others three groups all <0.05 . Compared with the ， NRF2 - （P ），NRF2 control group the relative expression levels of mRNA in L02 cells in the low dose group decreased <0.05 - （P ）， NRF2 mRNA in L02 cells in the medium dose group increased <0.05 mRNA and NRF2 protein expression in L02 cells in （ P ） HO-1，GCLC， NQO1 ， the highdose group increased both <0.05 . The relative expression level of mRNA and GCLC NQO1 （ P ） protein expression in L02 cells in the three doses groups increased compared with the control group all <0.05 . The relative NRF2 - - - expression level of mRNA in L02 cells in the high dose group was higher than that in the low and medium dose groups （ P ）， - （P ）， both <0.05 and the relative expression of NRF2 protein was higher than that in the low dose group <0.05 but the HO-1 GCLC - - （ relative expression levels of and mRNA and HO 1 protein level were lower than those in the medium dose group all P ）Conclusion - <0.05 . TCM exposure can inhibit the proliferation of L02 cells by inducing oxidative stress with a dose effect ， relationship. In this process the antioxidant mechanism mediated by NRF2 was activated. The expression of antioxidant defense ， - ， and detoxification related target genes downstream of NRF2 signaling pathway was activated and the expression of HO 1 - GCLC and NQO1 was up regulated to alleviate the oxidative damage caused by TCM.

Phillygenin Mitigates LPS/ATP-induced L02 Cell Inflammation by Regulating P2X7R/NF-κB/NLRP3 Inflammasome Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo explore the effects of phillygenin （PHI） on the inflammation in L02 cells induced by lipopolysaccharide （LPS） and adenosine triphosphate （ATP） and the expression of purinergic 2X7 receptor （P2X7R）， NOD-like receptor family pyrin domain containing 3 （NLRP3）， and nuclear factor kappa B （NF-κB） expression. MethodIn this study， the inflammation model was induced in L02 cells by 100 μg·L-1 LPS treatment for 24 h and 5 mmoL·L-1 ATP treatment for 5 h. The cells in the PHI groups were cultured with PHI （100， 50， 25 mg·L-1） for 6 h in the LPS treatment period， followed by LPS treatment for another 18 h. After ATP treatment for 5 h， the mRNA and protein expression of interleukin-1β （IL-1β）， interleukin-18 （IL-18）， P2X7R， NLRP3， Caspase-1 precursor （pro-Caspase-1）， cleaved Caspase-1， NF-κB， and NF-κB inhibitor protein α （IκBα） in L02 cells was detected by real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） and Western blot， respectively. Molecular docking was used to predict whether P2X7R could bind to PHI， and DCFH-DA was employed to detect the accumulation of reactive oxygen species （ROS） in cells. P2X7R was silenced by small interfering ribonucleic acid （siRNA）， and then the mRNA expression of IL-1β， IL-18， P2X7R， NLRP3， Caspase-1， NF-κB， and IκBα was detected by Real-time PCR. ResultReal-time PCR and Western blot showed that compared with the normal group， the model group showed increased expression of IL-1β and IL-18 （P<0.05）， and compared with the model group， the PHI groups showed down-regulated IL-1β， IL-18 mRNA and protein expression （P<0.05）. Molecular docking suggested a good binding effect of PHI to P2X7R. Real-time PCR and Western blot analysis showed that the expression of P2X7R in the model group was significantly up-regulated compared with that in the normal group （P<0.05）， and compared with the model group， the PHI groups showed down-regulated mRNA and protein expression of P2X7R （P<0.05）. DCFH-DA results showed that compared with the normal group， the model group showed increased content of ROS （P<0.05）， and compared with the model group， the PHI groups decreased the accumulation of ROS （P<0.05）. As demonstrated by Real-time PCR and Western blot， compared with the normal group， the model group showed increased expression of NLRP3 inflammasome and NF-κB （P<0.05）， and compared with the model group， the PHI groups significantly decreased the mRNA and protein expression of NLRP3 and cleaved Caspase-1， and up-regulated the mRNA and protein expression of NF-κB and IκBα （P<0.05）. Real-time PCR analysis showed that compared with the results in the model group， after silencing P2X7R by siRNA， the mRNA expression of IL-1β， IL-18， P2X7R， NLRP3， Caspase-1， NF-κB， and IκBα was decreased （P<0.05）. PHI exerted the same effect， and the mRNA expression was further reduced after the combination of them. ConclusionPHI is presumed to suppress the expression of the NLRP3/NF-κB signaling pathway by down-regulating upstream P2X7R to alleviate the LPS/ATP-induced inflammation in L02 cells， suggesting that P2X7R may be the target of PHI against inflammation.

Lipid Accumulation and IL-6 Production in L02 Hepatocytes Induced by Sodium Oleate: Dose and Time Dependence / 生物医学与环境科学（英文）

To explore interleukin-6 (IL-6) production and characterize lipid accumulation in L02 hepatocytes induced by sodium oleate. L02 hepatocytes were incubated with 0, 37.5, 75, 150, 300, 600, or 1,200 μmol/L sodium oleate for 24 h, and the supernatant was collected to detect the concentration of IL-6. L02 hepatocytes were incubated with 300, 150, 75, or 0 μmol/L sodium oleate for 0-24 h. The supernatant was collected for detection of IL-6 and free fatty acids. L02 hepatocytes treated with 300 μmol/L sodium oleate for 0-24 h were stained with Oil Red O. With extended sodium oleate incubation time, IL-6 levels increased, and free fatty acids decreased. After 24 h incubation, IL-6 levels increased as sodium oleate increased from 37.5 to 300 μmol/L (

Safety study and compositional analysis of polyoxyethylene 35 castor oil by UPCC-Q-TOF-MS / 药学学报

A UPCC-Q-TOF-MS method was established to analyze the components of polyoxyethylene 35 castor oil. The separation was performed at 50 ℃ on a Waters Acquity UPCC system by an Torus Diol column (3.0 mm × 100 mm, 1.7 μm) with gradient elution of CO2 and methanol - acetonitrile (50∶50); the flow rate was 1.0 mL·min-1, the back pressure was 2 000 psi, and methanol containing 2.5 mmol·L-1 ammonium formate was used as ionization reagent, whose flow rate was 0.2 mL·min-1. Positive ion electrospray ionization (ESI) mode and MSE technology were used. The qualitative analyses were carried out by using precise mass information of the parent and product ions and a PCA model was established by UPCC-Q-TOF-MS. L-02 cells and RBL-2H3 cells were used to study the cytotoxicity and histamine release of CrEL samples in vitro. A total of 13 kinds of CrEL components were obtained and their structures were identified by UPCC-Q-TOF-MS, with 255 compounds in total. The percentage content of 13 types of components was calculated by the normalization method. The content of polyoxyethylene glycerol tri-ricinoleate (PGTri-ricinoleate) in all samples was 0.36% - 2.80% and the main components were polyethylene glycol, polyethylene glycerol and polyoxyethylene glycerol mono-ricinoleate. All samples have different degrees of cytotoxicity and histamine release, which is negatively correlated with the content of PGTri-ricinoleate and positively correlated with the content of polyoxyethylene glycol fatty acid esters. The UPCC-Q-TOF-MS method is simple and rapid, has strong separation ability and high accuracy. It is suitable for the analysis of CrEL components. It is suggested that the fatty acid composition should be included in the monograph of CrEL for injection to increase the content of PGTri-ricinoleate and decrease the content of polyoxyethylene glycol fatty acid esters, so as to improve the product safety.

Effect and Mechanism of Celastrol on Endoplasmic Reticulum Stress in Hepatic L02 Cells of Nonalcoholic Fatty Liver Disease / 中国实验方剂学杂志

Objective:To explore the effect of celastrol in inhibiting the lipid metabolism disorder in hepatic L02 cells and its possible mechanism on endoplasmic reticulum stress (ERS) of non-alcoholic fatty liver cells by intervening non-alcoholic fatty liver disease(NAFLD) cell model with celastrol. Method:Hepatic L02 cells were divided into control group, model group, low-dose celastrol treatment group (Cel 0.5 mg·L-1), high-dose celastrol treatment group (Cel 1 mg·L-1) and simvastatin group (SIM 6 mg·L-1) for cultivation. The contents of total cholesterol (TC) and total triglyceride (TG) in hepatic L02 cells were detected, and the oil red staining was used to detected the lipid accumulation in hepatic L02 cells. Reverse transcription polymerase chain reaction (RT-PCR) and Western blot were used to detect the mRNA and protein expression levels of endoplasmic reticulum stress (ERS)-related signal molecules activating transcription factor 6 (ATF6), glucose regulated protein 78 (GRP78), inositol-requiring enzyme 1 (IRE1), sterol regulatory element-binding protein cleavage-activating protein (SCAP), sterol regulatory element-binding protein-1c (SREBP-1c) and sterol regulatory element-binding protein-2 (SREBP-2) in hepatic L02 cell model respectively. Result:The contents of TC and TG in hepatic L02 cells of NAFLD group were significantly higher than those in control group (P-1 group, Cel 1 mg·L-1 group and SIM 6 mg·L-1 group were significantly lower than those in NAFLD group (P-1 group, the Cel 1 mg·L-1 group, and the SIM 6 mg·L-1 group were lower than the NAFLD group to different degrees. According to the results of RT-PCR and Western blot, the mRNA transcription and protein expression levels of ERS-related signaling molecules ATF6, GRP78, IRE1, SCAP, SREBP-1c and SREBP-2 in hepatic L02 cells of NAFLD group were higher than those of control group (P-1 group, Cel 1 mg·L-1 group and SIM 6 mg·L-1 group were lower than those of NAFLD group (P-1 group and the SIM 6 mg·L-1 group. Conclusion:Celastrol can reduce the lipid metabolism disorder in hepatic L02 cells by down-regulating the expressions of ERS-related signaling molecules ATF6, GRP78, IRE1, SCAP, SREBP-1c and SREBP-2 in hepatic L02 cells, so as to improve NAFLD.

Trichloroethylene-induced abnormal methylation on promoter region of SET in hepatic L-02 cells / 中华劳动卫生职业病杂志

Objective@#To explore the trichloroethylene-induced alteration of methylation on the promoter region of SET and related mechanisms in hepatic L-02 cells.@*Methods@#L-02 cells were treated with different concentrations of TCE(0 mmol/L, 1 mmol/L, 2 mmol/L, 4 mmol/L, 8 mmol/L) for 24 h. The genomic DNA were then extracted and modified by bisulfite sodium. The DNA methylation was then analyzed using bisulfite sequencing PCR (BSP).@*Results@#The overall methylation on promoter region of SET was decreased along with the increased concentrations of TCE in hepatic L-02 cells. Moreover, 73 CpG islands were found abnormally altered, among which 9 were predicted in transcriptional factor binding regions.@*Conclusion@#The decreased levels of CpG islands in the transcriptional factor binding region may contribute to the elevation of SET in TCE-induced hepatotoxicity.

Establishment and optimization of hepatocyte steatosis model / 中华肝脏病杂志

Objective@#To establish a new model of hepatic steatosis cells by optimizing the original ethanol or high fat, the present study proposed an in vitro hepatocyte steatosis model for the study of fatty liver.@*Methods@#Oil red O staining was used to observe the effects of fetal bovine serum, oleic acid and ethanol on lipid accumulation in human liver cell line L02 in a concentration- and time-dependent manner. RT-PCR was used to detect the mRNA expression levels of PPAR-γ and AP-2, and the suitable conditions for the establishment of hepatocyte steatosis model were screened out. A t-test was used for comparison between the two groups, and one-way Analysis of Variance (ANOVA) was used in more than three groups.@*Results@#Oil red O staining showed the number of reddish-orange lipid droplets in L02 cells gradually increased with the increase of fetal bovine serum, oleic acid and ethanol in a concentration - and time-dependent manner. Compared with 0.00% oleic acid and 2% ethanol, the count value of red particle was 100.00% ± 17.63% at the beginning and after 24 h, 0.003% oleic acid and 2% ethanol jointly acted in L02 cells. After incubation for 48 hours with 2% ethanol and serum-free DMEM medium, the accumulation of lipid droplets was the highest with a count value of 802.38%+71.06%(t = 42.36, P < 0.001). RT-PCR analysis showed the lipid accumulation induced by this method was positively correlated with the mRNA expression of PPAR-γ and AP-2.@*Conclusion@#L02 cells were successfully exposed to high fat and ethanol, and the hepatocyte steatosis model was established and optimized, suggesting that the occurrence of hepatic cell steatosis was related to the up-regulation of PPAR-γ and AP-2.

The regulation effect of emodin on human embryonic hepatocyte L02 cell strain Farnesoid X receptor / 中华实用儿科临床杂志

Objective To investigate the regulation effect of emodin on human embryonic liver L02 cells strain farnesoid X receptor (FXR) pathways.Methods By using Guggulsterones,FXR genes were intervened with in L02 cells as model group,in three different concentrations of emodin (50.0 μ mol/L,25.0 μmol/L,12.5 μmol/L) of emodin in the model group cells,FXR,small heterodimer parter (SHP),UDP-glucuronosyltransferase 2B4 (UGT2 B4),bile salt export pump(BSEP) mRNA and protein expressions were detected by real-time fluorescent quantitative PCR and Western blot test.Results (1) The relative expressions of FXR mRNA and protein in the model group (0.240 ± 0.021,0.385 ±0.119) decreased significantly than those of control group (1.000 ± 0.088,1.000 ± 0.223),the differences were statistically significant (t =14.62,4.21,all P ＜ 0.01).Compared with the model group,the relative expressions of FXR mRNA in the high,the middle and the low-dose emodin groups (0.755 ±0.083,0.817 ±0.097,0.547 ± 0.080) were significantly higher (t =10.42,10.03,6.39,all P ＜ 0.01).The relative expressions of FXR protein in the medium-dose group (0.865 ± 0.203) increased significantly (t =3.53,P ＜ 0.01).The relative expressions of FXR mRNA in the high and the medium-dose groups (0.755 ± 0.083,0.817 ± 0.097) were higher than those in the low-dose group (0.547 ± 0.080),the differences were statistically significant (t =3.11,3.70,all P ＜ 0.01).(2)The relative expressions of SHP,UGT2B4,BSEP mRNA and protein in the model group (0.148 ±0.025,0.205 ± 0.039,0.184 ± 0.020;0.458 ± 0.130,0.255 ± 0.170,0.303 ± 0.100) were significantly lower than those in the control group (1.000 ±.0.099,1.000 ±0.104,1.000 ±0.125;1.000 ±0.129,1.000 ±0.157,1.000 ±0.162),the differences were statistically significant (t =14.50,12.44,11.19,5.13,5.57,6.33,all P ＜ 0.01).The relative expressions of SHP,UGT2B4 and BSEP mRNA in the high,the middle and the low-dose groups (0.610 ± 0.058,0.514 ± 0.041,0.707 ± 0.062;0.755 ± 0.108,0.800 ± 0.086,0.727 ± 0.076;0.470 ± 0.070,0.582 ± 0.050,0.500±0.108) were significantly lower than those in the model group (0.148 ± 0.025,0.205 ± 0.039,0.184 ± 0.020),the differences were statistically significant (t =12.75,9.38,13.94,9.46,10.90,11.96,7.53,10.31,5.00,all P ＜0.01).The relative expressions of SHP,UGT2B4 and BSEP mRNA in the high and the middle-dose emodin group (0.658 ±0.091,0.624 ±0.113,0.607 ±0.097;0.868 ±0.194,0.883 ±0.099,0.913 ±0.131) were significantly higher than those in the low-dose group (0.458 ±0.130,0.255 ±0.170,0.303 ±0.100),the differences were statistically significant (t =2.18,3.13,3.78,3.05,5.53,6.41,all P ＜ 0.01).The relative expression of SHP and BSEP protein in the low-dose group (0.645 ±0.135,0.572 ±0.076) increased,the differences were statistically significant (t =1.73,P ＜ 0.05,t =3.72,P ＜ 0.01).The relative expression of BSEP protein in the high and the medium-dose groups (0.607 ±0.097,0.913 ± 0.131) was significantly higher than those in the low-dose group (0.572 ± 0.076),the differences were statistically significant (t =1.99,3.90,all P ＜ 0.01).The relative expressions of SHP and UGT2B4 mRNA in the high and the low-dose group (0.610 ±0.058,0.470 ±0.070;0.514 ± 0.041,0.582 ± 0.050) were significantly lower than those in the medium-dose group (0.800 ± 0.086),the differences were statistically significant (t =3.75,6.47,3.83,3.42,all P ＜ 0.01).The expression levels of UGT2B4 and BSEP in the high and the low-dose groups (0.624 ± 0.113,0.644 ± 0.097;0.607 ± 0.097,0.572 ± 0.076) were significantly lower than those in the medium-dose group (0.883 ± 0.099,0.913 ± 0.131),the differences were statistically significant (t =4.27,2.98,6.30,3.90,all P ＜ 0.01).Conclusions Guggulsterones can inhibit FXR and downstream genes SHP,UGT2B4,BSEP expressions in L02,and emodin can enhance FXR gene expression,promote SHP,UGT2B4,BSEP gene expression,inhibit cholestasis pathway,protection of liver cell,which shows a dosage discreapancy.

Effects of Glycyrrhiza uralensis Extract on the Expression of UGT1A and MRP2 in Human Liver L-02 Cells Damaged by Triptolide / 中国药房

OBJECTIVE:To investigate the effects of Glycyrrhiza uralensis extract (GE) on the expression of uridine diphosphate glucuronyltransferase 1A (UGT1A) and multidrug resistance associated protein 2 (MRP2) in human liver L-02 cells damaged by triptolide (TP),and to study attenuated mechanism of G.uralensison for TP.METHODS:The survival rates of L-02 cells were determined by MTT assay after cultured with 0 (blank control),40,80,160 nmol/L TP for 12,18,24 h.L-02 cells were divided into blank control group (blank culture medium),model control group (80 nmol/L TP) and GE pretreatment group (adding 80 nmol/ L TP after pretreated with 30,60,90 mg/L GE for 24 h);after cultured for 18 h,survival rates of L-02 cells were determined by MTT assay.Rifampin (RIF) group (positive control,adding 80 nmoi/L TP after pretreated with 10 μmol/L RIF for 24 h) was added on the basis of the above grouping (GE concentration of 60 mg/L in GE pretreatment group).After cultured for 24 h,the protein expressions of UGT1A and MRP2 were detected.RESULTS:The inhibition effect of TP on cell proliferation was positively correlated with the concentration and the time.Compared with blank control group,cell survival rate of model control group was decreased significantly (P＜0.05),and the protein expression of MRP2 was decreased significantly (P＜0.01).Compared with model control group,cell survival rates of 30,60,90 mg/L GE pretreatment groups were all increased significantly (P＜0.01).The protein expressions of UGT1A and MRP2 were increased significantly in 60 mg/L GE pretreatment group (P＜0.01).CONCLUSIONS:GE pretreatment can relieve TP-induced human liver L-02 cell damage,and its attenuated mechanism may be associated with the increase the expression of UGT1A and MRP2.

Preventive effect and underlying mechanism of dihydrocumin on NAFLD in oleic acid-treated HepG2 cells / 中草药

Objective: To evaluate the preventive effect of dihydrocumin (DHC) on an in vitro model of nonalcoholic fatty liver disease (NAFLD) and investigate the signal transduction pathways underlying DHC treatment. Methods: Oleic acid (OA, 0.5 mmol/L) induced hepatic steatosis was established in HepG2 cells as in vitro model of NAFLD. After cells were co-treated by OA and DHC (0, 5, 10, and 20 μmol/L) for 24 h, the cellular contents of triglyceride (TG), reactive oxygen species (ROS) and NO were determined by cellular biochemical assays. Signaling pathways involved in glucolipid metabolism and oxidative stress including SREBP-1C, PNPLA3, PPARα, PI3K, the phosphorylation of AKT (pAKT), AKT, and Nrf2 on the mRNA and protein levels were determined by RT-qPCR and Western blotting. The glucose uptake was determined by fluorospectrophotometry using 2-NBDG as a fluorescence probe. Results: Compared with the control group, the content of TG, ROS and NO was significantly increased, the uptake of 2-NBDG was decreased. and the expression of SREBP-1C and PNPLA3on the mRNA and protein levels were up-regulated and the protein expression levels for PPARα, PI3K and Nrf2, as well as the ratio of pAKT to AKT were down-regulated in OA-induced cells. Compared with OA treated group, DHC decreased the levels of cellular TG and NO, as well as the mRNA and protein expression levels of SREBP-1C and PNPLA3, and increased the uptake of 2-NBDG, while at the same time increasing the cellular glucose uptake and the protein expression levels of PPARα, PI3K, pAKT/AKT, and Nrf2 in OA-induced HepG2 cells. Conclusion: DHC protected OA-induced hepatic steatosis by inhibiting lipid accumulation and oxidative/nitrative stress and increasing hepatic insulin sensitivity. Furthermore, the effect of DHC is likely associated with its role in the regulation of SREBP-1C, PNPLA3, PPARα, Nrf2, PI3K and AKT signaling pathways. DHC may have the effects on relieving the resistance of insulin and promoting the uptake of glucose in the hepatocytes by upregulating PI3K expression and pAKT/AKT level. Through increasing the expression of Nrf2 and reducing the content of NO in the cells, DHC could alleviate the inflammatory reaction and oxidative damage of liver cells.

Effects of sodium arsenite on Cyclin D1 and Cyclin dependent kinase 4 of human normal liver cells / 中华地方病学杂志

Objective To investigate the effects of sodium arsenite (NaAsO2) on the expressions of Cyclin D1 and Cyclin dependent kinase 4 (CDK4) in human normal liver cells (L-02).Methods L-02 cells were exposed to different doses of NaAsO2 (0,50,100,150 μmol/L) for 24 h,flow cytometry was used to detect the cell cycle,real time quantitative PCR and Western blotting were used to detect the Cyclin D1,CDK4 mRNA and protein expression.Results Cell cycle detection:compared with the control group G0-G1 phase [(60.33 ± 0.40)％],except 50 μmol/L NaAsO2 group [(54.58 ± 0.40)％],the numbers of cells in 100 and 150 μmol/L NaAsO2 groups [(64.60 ± 0.62)％,(83.13 ± 0.25)％] were increased,the differences were statistically significant (all P ＜ 0.05);cell proportion of S phase in the control group,50,100 and 150 μmol/L NaAsO2 groups [(34.35 ± 0.30)％,(29.89 ± 0.32)％,(29.50 ± 0.44)％,(11.93 ± 0.12)％] were decreased,the differences were statistically significant (all P ＜ 0.05);cell proportion of G2-M phase was compared between the control group,50,100 and 150 μmol/L NaAsO2 groups [(5.32 ± 0.11)％,(15.54 ± 0.14)％,(5.90 ± 0.20)％,(4.93 ± 0.15)％],the difference was statistically significant (F =908.359,P ＜ 0.05).Cyclin D1 and CDK4 detection:the expressions of Cyclin D1 mRNA (0.48 ± 0.17,1.00 ± 0.31,1.00 ± 0.21,2.06 ± 0.53) and protein (0.65 ± 0.02,0.64 ± 0.05,0.71 ± 0.10,0.84 ± 0.05) were compared betwee the control group,50,100 and 150 μmol/L NaAsO2 groups,the differences were statistically significant (F =167.886,46.575,all P ＜ 0.05);the expressions of CDK4 mRNA (1.04 ± 0.19,1.00 ± 0.21,1.29 ± 0.22,2.31 ± 0.31) and protein (0.67 ± 0.04,0.74 ± 0.03,0.83 ± 0.07,0.94 ± 0.04) were compared betwee the control group,50,100 and 150 μ mol/L NaAsO2 groups,the differences were statistically significant (F =95.171,145.848,all P ＜ 0.05).Conclusions Treatment of L-02 cells with NaAsO2 has changed the expressions of Cyclin D1,CDK4 mRNA and protein,which leads to L-02 cell cycle arrested at G0-G1 phase,ultimately leads to cell damage.

The level of autophagy induced by oxygen glucose deprivation/reperfusion injury in L02 cell / 安徽医科大学学报

Objective To explore the level of autophagy induced by oxygen glucose deprivation/reperfusion(OGD/R) injury in L02 cell.Methods L02 cells were cultured to establish the model of OGD/R injury and simulate clinical hepatic ischemia-reperfusion injury.The L02 cells were randomly divided into 5 groups : normal control group, oxygen-glucose deprivation 6 h/reperfusion 1,3,6,12 h group (OGD 6 h/R 1,3,6,12 h).Then observe the form changes of the L02 cells by optical microscope.The appreciation of the company's relative L02 cells was detected by MTT.The expression of autophagy related proteins such as Beclin-1, LC3 and p62 were evaluated by Western blot.Results Compared with the normal control group, the form damaged and the cells proliferation activity of L02 cells in the OGD/R group were gradually increased in a time-dependent manner.Compared with the normal control group, autophagy related proteins LC3 , Beclin-1 were increased at OGD 6 h/R 1 h.The expression of LC3 was gradually increased as the time went on and was increased gradually at OGD 6 h/R 6 h, reached a peak at OGD 6 h/R 12 h(P<0.01).The expression of Beclin-1 was gradually increased as the time went on and was increased gradually at OGD 6 h/R 6 h and OGD 6 h/R 12 h (P<0.01).The expression of p62 had no obvious change at OGD 6 h/R 1 h and OGD 6 h/R 3 h, began to increase sharply at OGD 6 h/R 6 h and reached a peak at OGD 6 h/R 12 h(P<0.01).Conclusion Our data suggests that oxygen-glucose deprivation/reperfusion may increase the level of autophagy and lead to autophagic cell death in L02 cell.

Effects of sodium arsenite on the reactive oxygen species levels and cell apoptosis of human normal liver cells / 中华地方病学杂志

Objective To investigate the effects of sodium arsenite (NaAsO2) on cell survival circumstance,reactive oxygen species (ROS) and cell apoptosis in human normal hepatic cells (L-02).Methods L-02 cells were exposed to different doses of NaAsO2 (0,50,100,150 μmol/L) for 24 h.MTT assay was used to detect the survival of L-02 cells,and flow cytometry (FCM) was used to detect the ROS levels and the early (Q4),late (Q2) apoptosis of L-02 cells.Results Cell survival rate:cell survival rate was compared between groups,the difference was statistically significant (F =350.51,P ＜ 0.05),the cell survival rates of 50,100 and 150 μmol/L NaAsO2 groups [(87.30 ± 3.74)％,(49.03 ± 4.72)％,(13.44 ± 4.01)％] were significantly lower than that of the control group [(100.00 ± 0.00)％,all P ＜ 0.05];compared with 50 μmol/L NaAsO2 group,the cell survival rates of 100 and 150 μmol/L NaAsO2 groups were significantly decreased (all P ＜ 0.05);compared with 100 μmol/L NaAsO2 group,the cell survival rate of 150 μmol/L NaAsO2 group was significantly decreased (P ＜ 0.05).The ROS levels:ROS levels were compared between groups,the difference was statistically significant (F =407.78,P ＜ 0.05),the ROS levels of 100 and 150 μ mol/L NaAsO2 groups (3 212.00 ± 221.93,5 521.33 ± 179.63) were significantly higher than that of the control group (1 691.67 ± 73.98,all P＜ 0.05);compared with 50 μmol/L NaAsO2 group (1 927.67 ± 62.45),the ROS levels of 100 and 150 μmol/L NaAsO2 groups were significantly increased (all P ＜ 0.05);compared with 100 μmol/L NaAsO2 group,the ROS level of 150 μ mol/L NaAsO2 group was significantly increased (P ＜ 0.05).Cell apoptosis:cell apoptosis rates of Q2,Q4 and Q2 + Q4 were compared between groups,the differences were statistically significant (F =256.84,26.53,63.89,all P ＜ 0.05);excecpt the cell apoptosis rate of Q4 in 50 μ mol/L NaAsO2 group [(5.43 ± 0.57) ％],the cell apoptosis rates of Q2 [(5.67 ± 0.21)％] and Q2 + Q4 [(11.10 ± 0.40) ％] in 50 μ mol/L NaAsO2 group,the cell apoptosis rates of Q2 [(13.60 ± 0.79) ％],Q4 [(7.37 ± 2.01) ％] and Q2 + Q4 [(20.97 ± 2.38) ％] in 100 μmol/L NaAsO2 group,the cell apoptosis rate of Q2 [(13.47 ± 0.78) ％],Q4 [(16.97 ± 3.45) ％] and Q2 + Q4 [(30.43 ± 3.84) ％] in 150 μmol/L NaAsO2 group were significantly higher than those of the control group [Q2:(3.47 ± 0.12) ％,Q4:(2.90 ± 0.90) ％,Q2 + Q4:(6.37 ± 1.00) ％,all P ＜ 0.05];compared with 50 μmol/L NaAsO2 group,the cell apoptosis rates of Q2,Q4 and Q2 + Q4 in 100 and 150 μmol/L NaAsO2 groups were increased,except the cell apoptosis rate of Q4 in 100 μ mol/L NaAsO2 group,the differences were statistically significant (all P＜0.05);the cell apoptosis rates of Q4 and Q2 + Q4 in 150 μmol/L NaAsO2 group compared with 100 μmol/L NaAsO2 group were significantly increased (all P ＜ 0.05).Conclusions NaAsO2 can induce L-02 cells to increase ROS levels,and inhibit L-02 cell proliferation.In addition,NaAsO2 can induce early apoptosis and late apoptosis in L-02 cells.

Mild hypothermia pretreatment attenuates liver L02 cells hypoxic-ischemia reperfusion injury through inhibiting JNK activation / 中华器官移植杂志

Objective To investigate the protective effects and the possible mechanisms of mild hypothermia against liver L02 cells hypoxic-ischernia reperfusion injury by using mild hypothermia pretreatment.Methods L02 cells were randomly divided into three groups:normal control group (N group),hypoxic-ischemia reperfusion group (control group) and hypoxic-ischemia reperfusion with mild hypothermia pretreatment group (experimental group).Before hypoxic-ischemia reperfusion,cells in experimental group were pretreated with mild hypothermia for 6 h,while the other groups were given the normal culture.Thereafter,the hypoxic-ischemia reperfusion models of L02 cells were performed by a tri-gas incubator to hypoxic-ischemia culture for 12 h,followed by reperfusion with normal conditions for 4 hours.Cells in N group were cultured in normal conditions.The temperature of experimental groups was set to 32 C.The samples were collected,and the cell injury,the cell vitality,the cell apoptosis and the expression of JNK in different groups were detected.Results Compared to N group,the levels of alanine aminotransferase (ALT),aspartate transaminase (AST) and lactic dehydrogenase (LDH) were significantly increased,the cell vitality was significantly decreased,the cell apoptosis and the expression of p-JNK were significantly increased in control and experimental groups (P ＜ 0.05).Compared to control group,all these changes were significantly ameliorated in experimental group.The levels of ALT,AST and LDH in control group were (30.0 ± 4.6),(26.3 ± 3.8) and (1129.0 ± 134.3) U/L,and those in the experimental group were (21.0 ± 2.7),(18.7 ± 2.1)and (898.3 ± 79.2),respectively.The cell vitality in control group and experimental group was (64.33 ± 2.32)％ and (78.17± 3.01)％ respectively.The cell apoptosis in control group and experimental group was (32.4 ± 2.3) ％ and (18.8 ± 1.4) ％ respectively.The expression of p-JNK in experimental group was significantly decreased.All these differences were statistically significant (P＜0.05).Conclusion Mild hypothermia pretreatment could significantly attenuate liver L02 cells hypoxicischemia reperfusion injury probably by inhibiting JNK activation.

Mild hypothermia pretreatment attenuates liver L02 cells hypoxic-ischemia reperfusion injury through inhibiting JNK activation / 中华器官移植杂志

Objective To investigate the protective effects and the possible mechanisms of mild hypothermia against liver L02 cells hypoxic-ischernia reperfusion injury by using mild hypothermia pretreatment.Methods L02 cells were randomly divided into three groups:normal control group (N group),hypoxic-ischemia reperfusion group (control group) and hypoxic-ischemia reperfusion with mild hypothermia pretreatment group (experimental group).Before hypoxic-ischemia reperfusion,cells in experimental group were pretreated with mild hypothermia for 6 h,while the other groups were given the normal culture.Thereafter,the hypoxic-ischemia reperfusion models of L02 cells were performed by a tri-gas incubator to hypoxic-ischemia culture for 12 h,followed by reperfusion with normal conditions for 4 hours.Cells in N group were cultured in normal conditions.The temperature of experimental groups was set to 32 C.The samples were collected,and the cell injury,the cell vitality,the cell apoptosis and the expression of JNK in different groups were detected.Results Compared to N group,the levels of alanine aminotransferase (ALT),aspartate transaminase (AST) and lactic dehydrogenase (LDH) were significantly increased,the cell vitality was significantly decreased,the cell apoptosis and the expression of p-JNK were significantly increased in control and experimental groups (P ＜ 0.05).Compared to control group,all these changes were significantly ameliorated in experimental group.The levels of ALT,AST and LDH in control group were (30.0 ± 4.6),(26.3 ± 3.8) and (1129.0 ± 134.3) U/L,and those in the experimental group were (21.0 ± 2.7),(18.7 ± 2.1)and (898.3 ± 79.2),respectively.The cell vitality in control group and experimental group was (64.33 ± 2.32)％ and (78.17± 3.01)％ respectively.The cell apoptosis in control group and experimental group was (32.4 ± 2.3) ％ and (18.8 ± 1.4) ％ respectively.The expression of p-JNK in experimental group was significantly decreased.All these differences were statistically significant (P＜0.05).Conclusion Mild hypothermia pretreatment could significantly attenuate liver L02 cells hypoxicischemia reperfusion injury probably by inhibiting JNK activation.

Effects of magnesium isoglycyrrhizinate on the expressions of UGT1A, MRP2 protein and mRNA in L-02 cells damaged by triptolide / 天津医药

Objective To observe the effect of magnesium isoglycyrrhizinate on the expressions of UGT1A, MRP2 protein and mRNA of L-02 cells damaged by triptolide, and to investigate hepatoprotective mechanism of magnesium isoglycyrrhizinate in terms of drug metabolism. Methods L-02 cells were divided into 4 groups:normal group, triptolide group, magnesium isoglycyrrhizinate group and rifampicin group. Magnesium isoglycyrrhizinate group and rifampicin group were pretreated by magnesium isoglycyrrhizinate and rifampicin for 24 h and the remaining two groups added medium. Triptolide were added for 18 h except normal group. Cell survival rate was tested by MTT. The expression levels of UGT1A, MRP2 protein and mRNA were detected by Western blot assay and RT-PCR. Results Compared with triptolide group, cell survival rate was significantly higher in magnesium isoglycyrrhizinate group (P<0.05). Meanwhile, the expression levels of UGT1A, MRP2 protein and mRNA were significantly lower in triptolide group compared with those of control group (P<0.05). The expression levels of UGT1A, MRP2 protein and mRNA were significantly up-regulated in magnesium isoglycyrrhizinate pretreatment group than those of triptolide group (P<0.05). The UGT1A protein and mRNA expressions were significantly decreased in rifampicin pretreatment group than those of magnesium isoglycyrrhizinate group ( P<0.05), but there were no significant differences in MRP2 protein and mRNA expressions between the two groups. Conclusion Magnesium isoglycyrrhizinate shows protective effects on triptolide induced L-02 cell injury, which may be involved with the activation of UGT1A and MRP2.

Effect of chlorogenic acid at high concentration on lipid accumulation and oxidative stress injury in L02 cells / 中草药

Objective To investigate the effects of chlorogenic acid (CGA) at high concentration ( > 100 μmol/L) on the lipid accumulation and oxidative stress in L02 cells under normal and non alcoholic liver disease (NAFLD) status induced by oleic acid (OA) and palmic acid (PA) treatment. Methods L02 cells were treatment by OA (666 μmol/L)-PA (333 μmol/L) for 24 h to induce intracellular steatosis. After normal and OA-PA treated cells were treated by CGA (0, 0.5, 1, and 2 mmol/L) for 24 h, the intracellular contents of lipid droplets and reactive oxygen species (ROS) were determined by Oid red staining and fluorospectrophotometry, respectively. And the mRNA and protein expression levels of SREBP-1C, PNPLA3, and CYP2E1 were detected by Real-time PCR and Western blotting, respectively. Results CGA treatment dose-dependently increased the levels of intracellular lipid droplets and ROS, as well as the mRNA and protein expression levels of SREBP-1C, PNPLA3, and CYP2E1 in normal and OA-PA treated L02 cells. Conclusion CGA treatment at high concentration can accelerate the lipid accumulation and oxidative stress injury in L02 cells under normal and NAFLD status.