淫羊藿苷對H2O2誘導的PC12細胞內質網應激的保護作用

張洋洋，李祖高，劉義偉，陳 霞，李 菲

(遵義醫科大學 藥理學教研室暨基礎藥理教育部重點實驗室暨特色民族藥教育部國際合作聯合實驗室，貴州 遵義 563099)

Eukaryotic cells execute various functions in subcellular compartments or organelles.Endoplasmic reticulum (ER) plays a major role in proteostasis by the correct folding,modification and quality control of approximately one third of all secretory and membrane proteins[1].Reactive oxygen species (ROS) has been shown to modulate ER function.ROS,ER stress,and protein aggregation are associated with the etiology of diseases such as type 2 diabetes,cancer,and Alzheimer’s disease (AD).AD,a central neurodegenerative disease and no remission in its progression,now afflicts more than 50 million people worldwide[2].The discovery of disease-modifying drugs is now of the neuropharmacological research priorities.

Icariin (ICA),a Chinese traditional herbal medicine extracted from Epimedium,exhibits a wide range of pharmacological functions including anti-inflammation,antioxidant actions[3].It also has a protection neurons against oxygen-glucose deprivation and oxidative stress[4].ICA is widely distributed in the body and can pass through the blood-brain barrier[5].We have carried out that ICA can protect neurons against ER stress by inducing Hrd1 expression[6].However,the effect of ICA on ER stress is not clearly,therefore,this present study is aimed to evaluate the effect of ICA on ER stress in PC12 cells induced by H2O2.

1 Materials and methods

1.1 Materials Icariin (purity≥98% by HPLC) was purchased from Nanjing Zelang Medical Technology Corporation Ltd (Nanjing,China),and dissolved in dimethyl sulfoxide (DMSO) and filtered.Other reagents were reagent-graded and commercially available.

1.2 Cell culture and treatment Highly differentiated rat pheochromocytoma PC12 cells (Rockville,MD,USA) were cultured in DMEM supplemented with 100 U/ml penicillin,100 U/ml streptomycin,and 10% FBS at 37℃ in a humidified atmosphere of 95% air and 5% CO2.The medium was changed every other day.First,we tested the effect of ICA alone on PC12 cells by treating cells with various concentrations of ICA (3.125,6.25,12.5,25,50 and 100 μM) for 48 h.Subsequently,the cells injury induced by H2O2(100,200,400,800 μM) was evaluated.To investigate the protective effect of ICA on H2O2-induced PC12 cells injury,cells were pretreated with ICA at various concentrations (6.25,12.5 and 25 μM) for 2 h,and then co-incubated with 400 μM H2O2for another 48 h.

1.3 Cell viability assay Cell viability was measured by the MTT assay (Beijing Solarbio Scienc-e & Technology,China).Cells were seeded onto 96 wells plate at 1×105cells/well density in 100 μl culture medium for the indicated time points Another while cells were incubated with MTT for 4 h at 37oC till the end of experiment.The dark blue crystals in cells were solubilized in DMSO.The optical density (OD) of each well was measured at 570 nm with a Microplate Reader.Cell viability was calculated as percentage of cell viability (OD treated/OD control) ×100.

1.4 LDH leakage assay The PC12 cells (2×104cells per well) were pre-treated with or without ICA as described above and the supernatant was analyzed by a LDH detection kit (Nanjing jiancheng Bioengineering Institute,China),according to the manufacturer’s protocol.The leakage of LDH was determined at 490 nm wavelength using a Microplate Reader.

1.5 Real-time quantitative PCR Total RNA from PC12 cells was isolated with Trizol reagent.Then 1 μg mRNA was reverse transcribed to cDNA with the qScript cDNA synthesis kit.After 30 s pre-denature at 95℃,40 cycles will be performed:denature at 95℃ for 15 s,annealing and extension at 60℃ for 30 s.Dissociation curve was performed after finishing 40 cycles to verify the quality of primers and amplification.Quantitative real-time PCR (qPCR) was performed with SYBR?Green Supermix (BioRad).Relative expression was normalized to β-actin.Specific primers used in this study were list in table 1.

Table1SepicificqPCRprimersusedinthisstudy

1.6 Western Blot Western blot analysis was used to determine the level of PERK,eIF2α,ATF4,CHOP,phospho-PERK (Thr982),phospho-eIF2α (Thr51),β-actin and Tubulin (Affinity,USA).Cells were homogenized with RIPA lysis buffer,which contained the protease inhibitor and phosphatase inhibitor cocktails.Total proteins were separated on 10% SDS-PAGE gels and transferred to PVDF membranes,which were blocked with non-fat milk for 2 h at room temperature,and then incubated with first-antibodies over night at 4oC with rotation.After washing,membranes were incubated with appropriate HRP conjugated second-antibodies (Beyotime,China) for 1 h at room temperature; the blots were visualized by BeyoECL (Thermo,Waltham,MA,USA).The image was scanned,and the band intensity was quantified using Quantity One softwarev 4.52 (BioRad).

1.7 Statistical Analysis Statistical analysis was performed using SPSS 18.0.All data were expressed as mean ± SD and were analyzed for statistical significance using a one-way analysis of variance (ANOVA),followed by Bonferroni multiple comparisons test.P<0.05 was considered statistically significant.

2 Results

2.1 ICA inhibits H2O2-induced PC12 cells injury The cell viability was measured using MTT assay in this study after various treatments.As shown in Fig 1A,less than 50 μM ICA did not affect cell viability and caused no cell toxicity.The results indicated that H2O2reduced cell injury in a concentration-dependent manner,and the concentration at 400 μM for 48 h time point was confirmed to be an appropriate ER stress modelinvitrowith approximately 50% cell viability inhibition rate [Fig 1B].As shown in Fig 1C,H2O2decreased cell viability,which was partly reversed by ICA (12.5 and 25μM).In parallel,400 μM H2O2significantly increased LDH release.However,pretreatment with ICA reduced the amount of LDH release [Fig 1D].This protective effect of ICA was also evidenced by morphologic observations of PC12 cells.As shown in Fig 2,H2O2induced cells shrinkage and floatation,pretreated with ICA reduced the injury.

Cell viability was measured by MTT assay followed treatment with indicated concentrations of ICA (A) and H2O2 (B) for 48 h.Cells were pre-treated with indicated concentrations of ICA for 2 h followed co-incubation with 400 μM H2O2 for 48 h,cell viability was measured by MTT assay (C) and LDH release assay (D). independent experiments,every experiment contained 5 samples/group) *：P<0.05,**：P<0.01 vs control,#：P<0.05,##：P<0.01vs H2O2.Fig 1 Effect of ICA on H2O2-induced PC12 cells injury

Fig 2 The effect of ICA on H2O2-induced morphological alternations in PC12 cells(Arrows:injured cells)

2.2 ICA represses H2O2induced-ER stress in PC12 cells In order to observe the effect of ICA on ER stress,we tested the level of marker of ER stress:glucose-regulated protein 78 (GRP78),and the results showed that H2O2increased high expression of GRP78 protein,interestingly,pretreated with ICA significantly decreased GRP78 level [Fig 3A and D].We further explored whether the protein kinase RNA-like ER kinase (PERK) pathway is involved in the effect of ICA on H2O2-induced-ER stress.The phosphorylation level of PERK and α-subunit of eukaryotic translation initiation factor-2 (eIF2α),as well as their total proteins levels were tested by Western blot.The results showed in Fig 3 that PERK and eIF2α phosphorylation levels were induced higher by H2O2than control group,however,pretreated with ICA reduced their phosphorylation levels.Next,we explored the downstream factors of PERK-eIF2α signaling pathway:activating transcription factor-4 (ATF4) and C/EBP homologous protein (CHOP),and found that their protein levels reduced in ICA group compared with alone H2O2group.

A：Representative bands of GRP78 and p-PERK;B:p-eIF2α and eIF2α;C:ATF4 and CHOP;D-H:respectively,the relative value normalized with that of tubulin,β-actin,or total independent experiments) *:P<0.05,**:P<0.01 vs control,#:P<0.05,##:P<0.01 vs H2O2.Fig 3 Effect of ICA on ER stress-induced by H2O2 in PC12 cells

This study also tested the mRNA level of markers of ER stress,as shown in Fig 4,H2O2dramatically increased the mRNA level of GRP78,PERK,ATF4 and CHOP in PC12 cell.Pretreated with ICA could decrease the mRNA level of GRP78,ATF4 and CHOP,but not affect PERK mRNA level.

The mRNA expression of abovementioned genes were normalized to independent experiments) *：P<0.05,**：P<0.01 vs control,#：P<0.05,##：P<0.01 vs H2O2.Fig 4 Effect of ICA on the mRNA level of GRP78,ATF4,and CHOP in H2O2-induced PC12 cell

3 Discussion

As an important organelle in eukaryotic cell,ER is closely related to the synthesis of proteins,the production of lipids and the storage of calcium ions[7-8].It has been reported that a variety of causes,such as ischemia,low oxygen,drug or poison,oxidative stress and other pathological physiology stimuli could induce ER stress further to induce the cell self-adjustment and even induce apoptosis[9].It also has been reported that ER stress widely presents in the brain of AD patients,deteriorates neurons loss and neurological function[10],and could be one of the main causes of AD[11-12].We have been demonstrated that ICA could ameliorate the cognitive function of AD and neuron injury in multiple models[13-14].This study further supported the conclusion that ICA protected neuron with evidences of the increased cell viability and decreased LDH release using H2O2-induced cell injuries modelinvitro.

There are three main sensors,PERK,activating transcription factor-6 (ATF6) andinositol-requiring protein-1(IRE1) located in ER membrane,normally combined with GRP78 which closes their signals.Under ER stress condition,GRP78 protein dissociates from the sensors and binds to non-folded and folded proteins,which is an important marker to induce and reflect the stress level of ER[15].Then the sensor proteins:PERK,ATF6 and IRE1 will be activated and trigger their downstream signaling pathway,respectively.ER stress reaction usually includes three mechanisms:the instantaneous adaptive response courses in the early phases with reducing protein synthesis thereby the number of proteins transfer into the ER has been decreased; the second phase cells trigger the long-term adaptive response,regulate the corresponding gene transcription to enhance the ability of ER treated with non-folding protein; if the first two mechanisms do not reestablish cell homeostasis,then the apoptosis reaction will be triggered to protect organisms from damaged cells[16-17].H2O2at the concentration of 400 μM incubated PC12 cells for 48 h induced cells loss and severe ER stress evidenced by the increased of level GRP78,PERK and its downstream eIF2α,as well as alleviated the phosphorylation of PERK and eIF2α.Pretreated with ICA for 2 h reduced the expression of GRP78 protein and mRNA in H2O2-induced PC12 cells; suppressed the activity of PERK and eIF2α through decreasing their phosphorylation level,but not altered the mRNA level of PERK and the total protein of eIF2α.This evidence maybe indicates that ICA regulates the function of PERK and eIF2α via inhibiting their activity,not their expression.

Activated PERK phosphorylates and activates its downstream eIF2α at serine 51site.p-eIF2α suppresses the translation initiation complex eIF2-GTP-Met-tRNAiMet,and then reduces the mostly proteins synthesis load in the ER[18],but increases ATF4 protein synthesis.ATF4 encodes a cAMP reaction element that can promote cell survival through inducing genes expression in amino acid metabolism,redox reaction,stress response and protein secretion reaction,or promote apoptosis through inducing CHOP expression[19-20].The level of ATF4 and CHOP has been increased in PC12 cells after incubation with H2O2.ICA pretreatment for 2 h could repress the protein and mRNA level of ATF4 and CHOP,therefore improved the cells survival.

In conclusion,this study supported the hypothesis that ICA possesses the protection against ER stress,especially suppressing the activation of PERK/eIF2α signaling pathway.