HUANG Xiao-feng,LIU Ze-gan,LEI Pan,MENG Zhong-ji,4,WANG Li-bo,DU Shi-ming,

(1.Department of Medicine,2.Taihe Affiliated Hospital of Hubei University of Medicine,3.Hubei Key Laboratory of Wudang Local Chinese Medicine Research,4.Institute of Biomedical Research,Hubei University of Medicine,Shiyan 442000,China)

Abstract:OBJECTlVE To investigate the effects of saikosaponin-d(SSd)on the proliferation of L02 cells induced by H2O2and the mechanism.METHODS①L02 cells were pretreated with SSd 0.5,1 and 2 μmol·L-1for 6 h,and administered with H2O2800 μmol·L-1for 24 h.Cell viability was analyzed by MTT assay,while the activities of glutamic-oxalacetic transaminase(GOT),glutamic-pyruvic transaminase(GPT),as well as the contents of malondialdehyde(MDA),and lactate dehydrogenase(LDH)in cell culture were detected by biochemical assay.② L02 cells were pretreated with SSd 2 μmol·L-1and tert-butylhydroquinone(tBHQ)50 μmol·L-1for 6 h,and administered with H2O2800 μmol·L-1for 24 h.The intracellular accumulation of reactive oxygen species(ROS)was analyzed by fluorescent probes,nuclear translocation of nuclear factor-erythroid 2-related factor 2(Nrf2)was determined by immunofluorescence assay,the protein expressions of Nrf2 and heme oxygenase-1(HO-1)were detected by Western blotting.RESULTS①Cell survival rate was decreased in model group significantly(vs control,P＜0.05),but increased in H2O2+SSd 1 and 2 μmol·L-1group significantly(vs H2O2,P＜0.05).The activities of GPT,GOT and the contents of MDA and LDH were significantly increased in model group(P＜0.05).Compared with model group,the activities of GPT,GOT and the contents of MDA and LDH were significantly decreased in H2O2+SSd 2 μmol·L-1(vs H2O2,P＜0.05). ② H2O2significantly increased ROS production(vs cell control,P＜0.05),in H2O2+SSd 2 μmol·L-1group,but the ROS levels were significantly reduced(vs H2O2,P＜0.05).Compared with the cell control group,the localization of Nrf2 in the nucleus was increased intuitively in SSd 2 μmol·L-1and tBHQ 50 μmol·L-1group.Compared with H2O2group,the protein expressions of Nrf2 and HO-1 in H2O2+SSd 2 μmol·L-1or H2O2+tBHQ 50 μmol·L-1 group were enhanced(all P＜0.05).SSd 2 μmol·L-1alone could significantly increase the expressions of Nrf2 and HO-1 in L02 cells(vs cell control and H2O2,P＜0.05).CONCLUSlON SSd can effectively protect against L02 cell injury induced by H2O2treatment,and the mechanism of which is related to the decrease of intracellular ROS,the up-regulation of Nrf2 and HO-1 protein expression.

Key words:saikosaponin-d;non-alcoholic fatty liver disease;nuclear factor-erythroid 2-related factor 2;heme oxygenase-1;oxidative stress

As a result of changes in the living standards and dietary habits of the population,the prevalence of non-alcoholic fatty liver disease(NAFLD)keeps increasing in China,and it is now the second major liver disease after viral hepatitis[1].Currently,the"two hit pathogenesis"theory of NAFLD is widely accepted[2-3].Oxidative stress and insulin resistance(IR)are important factors affecting the development of NAFLD.Oxidative stress refers to an imbalance between the process of oxidation and the levels of antioxidants in the body.When this balance is disturbed,the oxidation levels are affected.Studies show that the activation of the nuclear factor-erythroid 2-related factor 2(Nrf2)pathway can improve the ability of an individual to resist oxidative stress[4].Nrf2 is a gene transcription factor,which regulates and induces the expression of several antioxidant enzymes and phase Ⅱ drug metabolizing enzymes in vivo.During oxidative stress,the reactive cysteine residues of Keap1 are oxidized,resulting in the inactivation of Keap1,followed by the dissociation and translocation of Nrf2 into the nucleus,and then activate the expression of antioxidantand phase Ⅱ drug metabolizing enzymes,including heme oxygenase-1(HO-1)and quinone oxidoreductase 1(NOQ1),which can increase the ability of cells to resist oxidative stress.

In recent years,research on the efficacy and mechanism of traditional Chinese Medicine such as Crataegus pinnatififida,Bupleurum chinense,Salvia miltiorrhiza,Alisma orientalis,Cassia obtusifolia,and Rheum palmatum on NAFLD has become a hot topic[5-6].Bupleurum chinense has been used in the prevention and treatment of liver diseases for more than a thousand years in traditional Chinese medicine[7].Pharmacological studies show that saikosaponins are the principal active components of B.chinense,which exert anti-inflammatory,anti-cancer,and hepatoprotective effects.Among the saikosaponins,the pharmacological activity of saikosaponin-d(SSd)is found to be the most significant[8].Current reports have demonstrated the therapeutic effects of SSd against liver disease,but its mechanism has not been fully elucidated.

In this study,oxidative damage was induced in L02 cells using H2O2.The hepatoprotective effect of SSd was evaluated by determining the levels of lactic dehydrogenase(LDH),glutamicoxalacetic transaminase(GOT)and glutamicpyruvic transaminase(GPT),among other biochemical indices.The antioxidant effect of SSd was evaluated by determining the levels of malondialdehyde(MDA)and ROS.We also investigated the activation of Nrf2 by measuring the level of Nrf2/HO-1 protein in the nucleus and cytosol.

1 MATERlALS AND METHODS

1.1 Cell,drugs,reagents and instruments

L02 cells were a gift from the Institute of Biomedical Research,Taihe Hospital Hubei University of Medicine,and cultured in Dulbecco′s modified eagle medium(DMEM)supplemented with 10% fetal bovine serum(FBS).The cells were maintained at 37℃in a CO2incubator and the medium was renewed every 2 d.L02 cells were grown to 80% confluence and incubated in serum medium for 24 h before treatment.H2O2SSd and tBHQ were added to the culture medium.SSd and tBHQ were dissolved in dimethyl sulfoxide(DMSO)immediately before use(final concentration of DMSO＜0.1%).The materials used included DMSO(＜0.1%)was used as control.The materials used included LDH,MDA,GOT and GPT kits(A020-2-2,A003-4-1,C010-2-1,C009-2-1,Jiancheng Bioengineering Institute,China);3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide(MTT)(ST316 Sigma-Aldrich,Germany);ROS,RIPA lysis solution and BCA assay kit(S0033S,P0013B,P0010S,Beyotime Biotechnology,China);mouse anti-human Nrf2 monoclonal antibody(SC-365949,Santa Cruz Biotechnology,USA);rabbit anti-human HO-1 monoclonal antibody(PB9212,Boster Biological Technology,China);mouse anti-human β-actin monoclonal antibody,mouse anti-human histone monoclonal antibody,horseradish peroxidase(HRP)-labeled goat antirabbit IgG antibody and HRP-labeled goat antimouse IgG secondary antibody(58169s,3638s,7074s,7076s,Cell Signaling Technology,USA);Alexa Fluor 488 HRP-labeled goat anti-rabbit IgG antibody(ZF-0511,Zhongshanjinqiao,China);IVD microplate reader(Thermo Scientific,USA);IX71 fluorescence microscope and FV3000RS laser scanning confocal fluorescence microscopy(Olympus,Japan);ChemiDocTMTouch Imaging System(Bio-Rad,USA).

1.2 MTT assay for cell viability

The L02 cells were pretreated with SSd 0.5,1 and 2 μmol· L-1for 6 h before H2O2800 μmol· L-1was added to induce oxidative damage for 24 h.After that,10 μL of MTT solution(0.5 g·L-1)was added to each well and incubated for 4 h,and the absorbance at 570 nm(A570nm)in each well was measured with a microplate reader.Relative cell viability(%)=(A570nmof drug treatment group/A570nmof cell control group)×100%.

1.3 Detection of GPT,GOT,MDA and LDH

The cells were treated in the same way as in 1.2.Cell supernatant was collected to detect the contents of GPT,GOT,MDA and the activity of LDH by corresponding assay kits.

1.4 Fluorescent probe analysis for intracellular ROS detection

The cells were divided into 5 groups:the cell control group,SSd 2 μmol·L-1,H2O2800 μmol·L-1,SSd+H2O2and tBHQ 50 μmol·L-1+H2O2groups.The cells were pretreated with SSd 2 μmol· L-1and tBHQ 50 μmol· L-1for 6 h,and treated with H2O2800 μmol· L-1for 24 h.Then,the cells were treated with 2,7-dichlorodihydro fluoresce(DCFHDA in diacetate)10 mmol·L-1for 30 min and washed three times with PBS.DCFH-DA in a diacetate form did not fluoresce in the free state.When entering cells,it was deacetylated to DCFH and combined with ROS to form fluorescent DCF.DCF could not cross the cell membrane.Fluorescence intensity was observed using a fluorescence microscope,the cells were collected and detected with a fluorescence spectrophotometer within 30 min,and the fluorescence intensities which reflected the contents of ROS were analyzed using Image J software.

1.5 lmmunofluorescence for Nrf2 nuclear translocation detection

The cells were divided into three groups:the cell control groups，SSd 2 μmol· L-1and tBHQ 50 μmol· L-1group,covered with 200 μL of 4% formaldehyde for 10 min at room temperature and washed three times with PBS,treated in 0.1% Triton X-100 for 10 min and blocked with 5% goat serum for 30 min,and then labeled with Nrf2 monoantibody(1:200)overnight at 4℃,washed three times with PBS,and incubated for 2 h with goat anti-mouse antibody(Alexa Fluor 488,1∶200).Next,the cells were washed three times with PBS and stained with DAPI 0.1 mg·L-1at room temperature and washed three times.Images were acquired using laser scanning confocal fluorescence microscopy.

1.6 Western blotting analysis of Nrf2 and HO-1 protein expressions

3×105per well L02 cells were seeded into 6-well plates and cultured for 24 h.Grouping was the same as in 1.5.Proteins were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE)and transferred to polyvinylidene fluoride(PVDF)membranes that were incubated with a primary antibody of Nrf2(1∶1000),HO-1(1∶1000),histone 3(H3)(1∶2000),and β-actin(1∶1000)at 4℃ overnight.After being washed with Tris-buffered saline and Tween 20 four times for 10 min,the membranes were incubated with HRP-labeled goat anti-rabbit IgG and HRP-labeled goat anti-mouse IgG at room temperature.Images were acquired using a universal imaging system.The integrated absorbance(IA)of bands was obtained by ImageJ 1.36b imaging software.The relative protein expressions were expressed by the ratio of IA of target protein to IA of H3 or β-actin.

1.7 Statistics analysis

2 RESULTS

2.1 Effect of SSd on cell viability of L02 cells injured by H2O2

As shown in Fig.1.SSd 0.5,1 and 2 μmol·L-1treatment increased the viability of L02 cells induced with H2O2,suggesting that SSd could reverse the H2O2-induced damage in L02 cells.

Fig.1 Effect of saikosaponin-d(SSd)on viability of L02 cells injured by H2O2by MTT assay.L02 cells were pretreated with SSd 0.5,1 and 2 μmol·L-1for 6 h and exposure to H2O2 800 μmol·L-1for 24 h.x±s,n=6. ＊＊P＜0.01,compared with cell control group,#P＜0.05,compared with H2O2group.

2.2 Effect of SSd on activities of GPT and GOT and contents of LDH and MDH in supernatant of L02 cells injured by H2O2

As is shown in Tab.1,compared with cell control group,H2O2increased the levels of GPT,GOT,LDH and MDA to(11.70±0.31)U·L-1,(33.70±0.26)U·L-1,(571±10)U·L-1,and(3.97±0.08)μmol·g-1(allP＜0.05),respectively.Compare with H2O2group,SSd 2 μmol·L-1decreased the value of these indicators to(7.26±0.25)U·L-1,(19.04±0.72)U·L-1,(478±5)U·L-1,and(1.75±0.07)μmol·g-1protein,respectively(allP＜0.05).

Tab.1 Effect of SSd on activities of glutamic-pyruvic transaminase(GPT)and glutamic-oxalacetic transaminase(GOT)and contents of lactate dehydrogenase(LDH)and malondialdehyde(MDH)in supernatant of L02 cells injured by H2O2

2.3 Effects of SSd on ROS level in L02 cells injured by H2O2

As shown in Fig.2,fluorescence intensity in H2O2group was significantly increased(P＜0.05)compared to cell control group,which suggested that ROS levels in H2O2groups were significantly increased(P＜0.05).When the cells were pretreated with SSd 2 μmol· L-1,the fluorescence intensity was reduced significantly(P＜0.05).Compared to tBHQ(positive control)group,the fluorescence intensity was not statistically different between the two groups,indicating that oxidative damage induced by H2O2was substantially reversed by SSd.

Fig.2 Effect of SSd on reactive oxygen species(ROS)level in L02 cells injured by H2O2.L02 cells were pretreated with SSd 2 μmol·L-1or tert-butylhydroquinone(tBHQ)50 μmol·L-1(positive control)for 6 h,and then exposed to H2O2800 μmol·L-1for 24 h.FI:fluorescence intensity.x±s,n=3.＊P＜0.05,compared with cell control group;#P＜0.05,compared with H2O2group.

2.4 Effect of SSd on Nrf2 nuclear translocation in L02 cells

As is shown in Fig.3,in the cell control group,the Nrf2 was mainly localized in the cyto-plasm,and the localization of Nrf2 in the nucleus was increased intuitively after the cells were pretreated with SSd 2 μmol·L-1and tBHQ 50 μmol·L-1for 24 h.The results showed that SSd could promote the nuclear translocation of Nrf2.

Fig.3 Effect of SSd on nuclear factor-erythroid 2-related factor 2(Nrf2)nuclear translocation in L02 cells by immunofluorescence assay.The cells were pretreated with SSd 2 μmol·L-1and tBHQ 50 μmol·L-1for 24 h.Images were acquired using laser scanning confocal fluorescence microscopy.

2.5 Effect of SSd on protein expressions of Nrf2 and HO-1 in L02 cell injured by H2O2

As is shown in Fig.4,compared with cell control group,the protein expression of nuclear Nrf2 and HO-1 did not increase signifcantly,but the cyto-plasm Nrf2 was decreased(P＜0.05)in H2O2group.Compared with H2O2group,in SSd 2 μmol·L-1or tBHQ 50 μmol· L-1pretreatment and H2O2postprocessing group,the protein expression of nuclear Nrf2 was enhanced to 1.513±0.03 and 1.506±0.02(Fig.4B),and cytoplasm Nrf2 was enhanced to 0.287±0.01 and 0.362±0.01(Fig.4C),and HO-1 was enhanced to 0.898±0.04 and 1.218±0.05(Fig.4D)(all P＜0.05).SSd 2 μmol·L-1alone could significantly increase the expressions of nuclear Nrf2,cytoplasm Nrf2 and HO-1 in L02 cells(vs cell control and H2O2,P＜0.05),suggesting that nuclear translocation of Nrf2 was not upregulated after H2O2-treatment,and that SSd could promote the expression of Nrf2/HO-1.

Fig.4 Effect of SSd on protein expressions of Nrf2 in nucleus and cytoplasmia of L02 cell and heme oxygenase-1(HO-1)in L02 cells injured by H2O2detected by Western blotting.See Fig.2 for the cell treatment.B，C and D were the semiquantitative results of A.±s,n=3.＊P＜0.05,compared with cell control group;#P＜0.05,compared with H2O2group.

3 DlSCUSSlON

These results show that SSd can increase cell viability,decrease indicators of hepatocellular injury and oxidative stress and decrease ROS levels,which may be related to increase expressions of antioxidants protein Nrf2 and HO-1.

Oxidative stress is a major factor affecting NAFLD.Studies have shown that H2O2can induce oxidative damage of hepatocytes stimulate cells,produce excessive ROS,induce oxidative stress,and affect the Nrf2 signaling pathway[9-12].Nrf2 is a transcription factor that coordinates the basis and stress-induced activation of a large number of cellular protection genes,and can protect against oxidative stress[13].This study has confirmed that H2O2can promote the production of ROS,lipid peroxide MDA and other related factors in L02 cells.Additionally,the nuclear translocation of Nrf2 and HO-1 is upregulated slightly,but not sufficiently to resist the oxidative damage induced by H2O2.SSd is one of the pharmacological active components of Bupleurum,with antioxidant,antiinflammatory,anti-liver injury et al.In this study,SSd can reverse H2O2-induced oxidative stress and hepatocellular injury.The results show that SSd alone could significantly increase the expressions of Nrf2 and HO-1 in L02 cells,which indicates that SSd can not only promote the nuclear translocation level of Nrf2,but also increase the expression of Nrf2.After SSd pretreatment,the expression levels of Nrf2 in the nucleus and HO-1 are significantly increased,but there is no additive effect compared with SSd group and H2O2group,which may be caused by the competition between SSd and H2O2in the same pathway.

In this study,SSd ≤2 μmol·L-1has a protective effect on L02 cells,but exhibits cytotoxicity at ＞2 μmol·L-1.LIN et al[14]showed that SSd was toxic at doses higher than 2 μmol·L-1,whereas it showed protective effects against CCl4induced injury over a concentration range of 0.5-2 μmol·L-1.However,further research and in vivo studies are needed to better understand the pharmacological effects of SSd.The limitation of this study is that only the protein expressions of Nrf2 and HO-1 were measured,and the oxidation pathway will be extended out in the future.