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@#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.
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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.
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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.
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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.
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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.
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This study aimed at the molecular mechanism of Cordyceps sinensis polysaccharide-A(CPS-A) on angiotensin (Ang Ⅱ)-induced injury of L02 cells.The effect of AngⅡ and CPS-A on the proliferation of L02 cells was analyzed by MTT assay.PCR,Real-Time PCR and Western blot were also employed to determine the expression of IL-1β,AT1R,AT2R,NF-κB p65,TNFα and other inflammatory factors at mRNA and protein levels.The results showed that Ang Ⅱ and CPS-A could inhibit the proliferation of L02 cells by 1 × 10-5 mol/L and 200 μg/ mL,respectively.PCR,Real-Time PCR and Western blot showed that CPS-A could significantly down-regulate IL-1 β,TNF-α,NF-κB and AT1R.CPS-A has a good protective effect on AngⅡ-induced L02 cell injury.
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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.
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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.
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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.
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Aim To study the effect of emodin in Po-lygonum multiflorum on the expression of CYP450 isoenzymes in L02 hepatocytes and explore its mecha-nism of cytotoxicity. Methods L02 cells were treated with different concentrations of emodin. Cell viability was examined by MTS assay kit, and cell membrane injury was examined by detecting the release rate of lactate dehydrogenase( LDH) . The expression of cyto-chrome P450 mRNA was detected by real time PCR. Results The result of MTS assay showed that L02 cells viability was significantly reduced following expo-sure to emodin in a concentration and time dependent manner. The LDH release rate of L02 cells significant-ly increased after exposure to emodin for 48 h com-pared with the control group. On the mRNA level, compared with the control group,emodin had inductive effects on mRNA of each CYP450 enzyme, while had significant inductive effects on mRNA of CYP1 A1 and CYP1 B1 in a concentration and time dependent man-ner. Conclusion Emodin in Polygonum multiflorum may generate significant liver injury in L02 cells and has inductive effects on CYP450 enzyme activity.
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@#This research aimed to investigate the apoptotic effect of rhein on human primary liver cells(L-02)and the underlying mechanisms. MTT assay was used to detect the inhibitory activity of rhein on L-02 cells. Rhein-induced apoptosis in L-02 cells was evaluated by flow cytometry. Intracellular reactive oxygen species(ROS)was detected by DCFH-DA fluorescent probe. Endoplasmic reticulum stress-related gene and protein expression were detected by real-time quantitative PCR(RT-qPCR)and Western blot. The effect of antioxidant N-acetylcysteine(NAC)on these proteins, cysteinyl aspartate specific proteinase 4(caspase-4)and cysteinyl aspartate specific proteinase 3(caspase-3)activity was explored. The results showed that rhein inhibited the viability of L-02 cells in a dose-dependent and time-dependent manners. The apoptosis rate of L-02 cells in rhein-treated groups was increased significantly. Rhein induced generation of ROS which was blocked by NAC. The expressions of GRP 78, ATF-4, CHOP mRNA and the expressions of GRP 78, p-JNK, CHOP proteins were significantly increased in rhein-treated groups. However, NAC could not attenuate the expressions of GRP 78, p-JNK, CHOP proteins, caspase-4, caspase-3 activity induced by rhein significantly. In conclusion, rhein induces apoptosis in L-02 cells via a reactive oxygen species-independent endoplasmic reticulum stress pathway.
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This paper was aimed to investigate the protective effects of luteolin (Lut) against acetaminophen（APAP）-induced damage in L02 liver cells. CCK-8 was used to detect the cell activation of L02 cells treated by different Lut. The concentration and time of APAP induced L02 cell damage was screened. The effect of Lut on APAP induced apoptosis of L02 cells was detected by cell morphological observation, CCK-8 assay and flow cytometry. The contents of MDA, GSH and SOD activity in cell supernatant were detected by colorimetric assay. The expression of apoptosis-related genes Bax, Bcl-2 and caspase-3 was detected by RT-PCR. The results showed that Lut in 2.5-40 μmol•L⁻¹ range does not affect the activity of L02 cells; 12 mmol•L⁻¹ APAP incubated with L02 cell 12 h to establish damage model. Compared with the model group, the cell status of Lut group was significantly improved, the cell body was increased, the adherence ability was recovered, and the apoptosis rate was obviously decreased. MDA content decreased significantly (P<0.05, P<0.01), GSH and SOD activity significantly increased (P<0.05, P<0.01), at the same time, it could up-regulate expression of Bcl-2 mRNA and down-regulate the expression of Bax and caspase-3 mRNA. In conclusion,Lut has protective effect on APAP induced L02 cell injury, and its mechanism may be related to the reduction of oxidative stress and inhibition of apoptosis.
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AIM: To investigate the role of reactive oxygen species ( ROS)-mediated mitochondrial oxidative injury in isonicotinyl hydrazide ( INH)-induced DNA damage and the protective effect of quercetin on L-02 cells.ME-THODS:The injury model of hepatocyte L-02cells in vitro induced by INH was established .The cells were divided into control group, INH group, low-dose quercetin group and high-dose quercetin group.The DNA damage of L-02 cells was evaluated by the comet test .The mitochondrion was prepared , and the level of mitochondrial ROS and the value of mitochondrial membrane potential (ΔΨm ) were detected by fluorescent probes DCFH-DA and rhodamine 123.The content of MDA was measured by TBA method .The activity of SOD was assessed with the xanthine oxidase method .The protein expression of Bcl-2 and Bax was determined by Western blotting , and the value of Bax/Bcl-2 was calculated .RESULTS:INH induced obvious DNA damage , increased the level of mitochondrial ROS , the content of MDA and the value of Bax/Bcl-2, and markedly reduced the value of ΔΨm and the activity of SOD in the L-02 cells.Quercetin attenuated DNA dam-age, reduced the level of mitochondrial ROS , elevated the value of ΔΨm , declined the content of MDA , increased the ac-tivity of SOD and decreased the value of Bax/Bcl-2 in the L-02 cells.CONCLUSION:INH induces DNA damage in L-02 cells by generation of mitochondrial oxidative stress .Quercetin has a protective effect on L-02 cells to attenuate the INH-in-duced DNA damage by inhibiting ROS-mediated mitochondrial oxidative damage .
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Objective To study the toxicity and mechanism of fullerene(C_(60))on human embryo liver L-02 cells.Methods L-02 cells were exposed to C_(60)suspension of different concentrations(0.00,1.25,2.50,5.00,10.00,20.00 and 40.00?g/ml)for 24 h,then the content of GSH and the activity of LDH,SOD were determined,the viability of cells with/without NAC was also compared.Results Compared to the control group,the viability of cells exposed to 1.25,2.50,5.00,10.00,20.00 and 40.00?g/ml C_(60)suspension decreased in a dose-dependent manner,and the differences were significant(P