Targeting of miR-96-5p by catalpol ameliorates oxidative stress and hepatic steatosis in LDLr-/- mice via p66shc/cytochrome C cascade

Catalpol ameliorated hepatic steatosis

At first, whether catalpol was able to protect against HFD-induced NAFLD in LDLr-/- mice was investigated by liver morphologic and histological examination. As shown in Figure 1A and 1B, ND-treated mice exhibited no apparent abnormalities, whereas long-term HFD feeding significantly increased the liver size, changed the color of the liver and induced a large amount of lipid deposition in hepatocytes, as well as massive nuclear pleomorphism and inflammatory cell infiltration. However, catalpol (200mg/kg) treatment markedly alleviated the liver injury and attenuated the hepatic lipid accumulation caused by the HFD. As shown in Figure 1C, in agreement, the results of transmission electron microscopy demonstrated a pronounced increase of hepatic lipid droplets in HFD-treated mice. However, hepatic lipid droplets accumulation was significantly decreased in catalpol treatment group compared with HFD-fed group. In addition, catalpol produced inhibitory effect on hepatic steatosis also confirmed in oxLDL or PA-treated hepG2 cells (Supplementary Figure 2A, 2B). Taken together, these results indicated that catalpol confered protection against HFD-induced NAFLD.

Figure 1. Catalpol ameliorated hepatic steatosis in LDLr-/- mice. The mice were fed with normal or western diet for 16-week, and then sacrificed for morphological and pathological observation. (A) Representative morphological images of liver sections. (B) H&E- stained sections of liver sections. H&E-stained sections were photographed at 200× magnification. (C) Transmission electron microscopy of liver sections. The liver sections were photographed at 10 000× magnification. The lipid droplets were indicated by arrows. ND: normal diet; WD: western diet.

Catalpol attenuated HFD-induced liver injury and lipid accumulation in HFD-treated LDLr-/- mice

We fortnightly measured body weights of mice, which were shown in Figure 2A. The body weight of mice in HFD-fed group didn’t change much compared with the body weight in ND-fed mice. However, catalpol decreased the body weight in ND-fed mice, without affecting food intake in mice (Supplementary Figure 3). Typical biochemical markers were employed to further elucidate the protective effects of catalpol against NAFLD. As shown in Figure 2B, the liver/body weight ratios in HFD-fed mice were markedly greater than those in the control group, and catalpol treatment obviously reversed this trend in a dose-dependent manner. Serum ALT and AST levels were also measured to elucidate the protective effect of catalpol. As shown in Figure 2C, compared with the control group, HFD-fed mice developed liver injury, as demonstrated by increased serum levels of ALT and AST. However, these changes were partly reversed by catalpol treatment. Plasma TG and TC were synthesized into the blood by the liver, thus plasma levels of TG and TC were detected to reflect liver function. As expected, after 16 weeks of HFD feeding, the serum levels of TG and TC were dramatically higher than those in the control group, whereas catalpol treatment partly abrogated these increases (Figure 2D). Above results suggested a protective effect of catalpol on HFD-induced hepatic injury.

Figure 2. Catalpol ameliorated liver index, hepatic injury in LDLr-/- mice. (A) Body weights variation during 16 weeks period. (B) Liver index. (C) Serum levels of ALT and AST. (D) Serum levels of TC, TG. The results are the mean ± SD (n=10), ^**P< 0.05 vs. Control group, ^##P < 0.05 vs. WD group. ND: normal diet; WD: western diet.

Catalpol ameliorated HFD-induced cell oxidative stress and cell apoptosis in HFD-treated LDLr-/- mice

Lipid accumulation is closely link to oxidative stress and cellular apoptosis which may form a feedback loop that significantly aggravates NAFLD induced hepatic injury. We therefore evaluated the state of oxidative stress and cellular apoptosis. As shown in Figure 3A, compared with the control group, the HFD-fed group exhibited a significantly decreased SOD level. However, catalpol treatment effectively increased the SOD level. Additionally, catalpol could also decrease the MDA level increased by HFD-fed in LDLr-/- mice (Figure 3B). Moreover, compared with the control group, CAT level was significantly decreased in HFD-fed mice. In contrast, catalpol treatment markedly reversed the decreased CAT level (Figure 3C). Decreased ATP generation was also considered to be a result of oxidative stress and early progress to induce cell apoptosis. As shown in Figure 3D and 3E, ATP content and ATPase activity were significantly decreased in NAFLD mice compared with the control group (p<0.05). DNA oxidative damage level can be evaluated by 8-OHdG level. As shown in Figure 3F, 8-OHdG level was significantly increased in HFD-fed mice, and catalpol-treatment effectively reversed the increase induced by HFD. Catalpol-treatment resulted in increased ATP content and ATPase activity respectively. Further, hepatic cell apoptosis was detected by TUNEL staining. As shown in Figure 3G, compared with the control group, TUNEL positive cells which were stained in green were notably increased in HFD-fed mice. However, catalpol treatment significantly reduced the number of TUNEL positive cells. Additionally, effect of catalpol on inhibiting apoptosis was also detected in PA-treated hepG2 cells (Supplementary Figure 2C). Together, these results indicated that catalpol attenuated oxidative stress and apoptosis in NAFLD.

Figure 3. Catalpol inhibited oxidative stress, promoted ATP production and inhibited hepatic apoptosis. (A) SOD level. (B) MDA level. (C) CAT level. (D) ATP content. (E) ATPase activity. (F) 8-OHdG level. The results are the mean± SD (n=10), ^**P< 0.05 vs. Control group, ^##P < 0.05 vs. WD group. (G) Tunel staining for hepatic apoptosis. Tunel stained sections were photographed at 200× magnification. ND: normal diet; WD: western diet.

Potential role of P66Shc in NAFLD

P66Shc expression was investigated in the high-fat diet and normal mice. Raw data were retrieved using the search terms ‘GSE94754’ in the GEO dataset. A total of128 different genes with p<0.05 and log F≥1.0 were identified. R_4.4 was used to screen the differentially expressed genes. The results indicated that 92 genes were significantly upregulated and 36 genes were remarkably decreased (Figure 4A). The analysis also indicated that p66shc expression in high-fat diet mice was significantly higher compared with the control group (Figure 4B). Together, the results suggested that p66shc might play an important role in the process of NAFLD.

Figure 4. Potential role of P66Shc in NAFLD. (A) Differentially expressed genes retrieved from ‘GSE94754’ in the GEO dataset. (B) Heat map of differentially expressed genes retrieved from ‘GSE94754’ in the GEO dataset.

Protective effects of catalpol against NAFLD involved down-regulation of p66shc/cytochrome C pathway

In order to ensure whether p66shc/cytochrome C pathway was involved in NAFLD, p66shc and cytochrome C protein level was measured. As shown in Figure 5A, 5B, HFD-fed significantly increased p66shc and cytochrome C protein expression, however, catalpol reversed this increase in a dose-dependent manner. As expected, in PA or oxLDL-treated hepG2 cells, high expression of p66shc and cytochrome C level and the decrease after catalpol treatment were also observed (Figure 5C, 5D and supplementary Figure 4A, 4B). The results suggested a potential role of p66shc in NAFLD. Thus, p66shc expression was blocked by its specific siRNA for further investigation. In Figure 5E, p66shc protein expression was knocked down effectively. Then, the role of p66shc in ROS production, steatosis and apoptosis was measured. P66shc siRNA transfection significantly increased mitochondrial membrane potential (Figure 5F), inhibited ROS overproduction (Figure 5G), hepatic steatosis (Figure 5H) and cellular apoptosis (Figure 5I). These results indicated that decreased p66shc protein expression benefited to NAFLD through inhibiting oxidative stress, hepatic steatosis and these induced hepatic cell apoptosis.

Figure 5. P66shc was involved in catalpol-mediated protective effects. (A) P66shc protein expression in NAFLD mice. (B) Cytochrome C (Cyto C) protein expression in NAFLD mice. (C) P66shc protein expression in PA- treated hepG2 cells. (D) Cyto C protein expression in PA-treated hepG2 cells. (E) P66shc protein expression was down-regulated by its specific siRNA. (F) JC-1 staining. (G) ROS level. (H) Nile red staining. (I) Tunel staining (Tunel positive cells were stained in green; DAPI was stained to show nuclei). The results are the mean± SD (n=10), ^**P< 0.05 vs. Control group, ^##P < 0.05 vs. WD or PA group.

miR-96-5p was decreased among the miRNAs targeting p66shc in NAFLD

miRNAs are potent gene regulators that have been implicated in a wide range of diseases. Because NAFLD induced p66shc overexpression might be mediated by miRNAs, we investigated potential p66shc-regulating miRNAs. At First, the miRNA target prediction programs miRanda, TargetScan, and PicTar. were used to identify miRNAs that target the SIRT1 3′ untranslated region (3′UTR). The differential expression in the liver of the putative p66shc-targeting miRNAs was determined by real-time PCR with or without HFD As shown in Table 1, the levels of miR-9-5p and miR-124-3p in NAFLD mice were significantly increased to 2.69-fold and 3.04-fold compared with that in control mice. However, the level of miR-96-5p in NAFLD mice was decreased to 0.23-fold compared with that in control mice. In the current view, miRNAs can negatively regulate gene expression by binding with the 3′-UTR of a specific mRNA, causing its degradation or translational repression. Based on this mechanism, we hypothesized that the down-regulated miR-96-5p might play important roles in the regulation of NAFLD, possibly through inhibiting p66shc.

Increased miR-96-5p inhibits p66shc expression

To evaluate the potential roles of miR-96-5p in NAFLD, agomir targeting miR-96-5p was employed in the in vitro NAFLD model. MiRNA function requiring them to be uploaded in to the RISC, where targeting of miRNAs to mRNA occurs. Argonaute-2 was eluted from mice liver or PA-treated and miRNA was extracted. Levels of Ago-2-bound miR-96-5p were significantly decreased in NAFLD models both in vivo and in vitro compared with controls (Figure 6A, 6B). Agomir-96-5p treatment significantly increased miR-96-5p levels in the control and PA-treated cells compared with each agomir-NC (Figure 6C), p66shc protein expression was significantly down regulated following agomir-96-5p transfection as well as cytochrome C protein expression (Figure 6D, 6E). However, no apparent change in p66shc mRNA was observed in either group (Figure 6F). These data suggested that miR-96-5p had the potential to regulate hepatic p66shc expression, and this effect might involve repressing translation rather than affecting mRNA degradation.

Figure 6. miR-96-5p regulated p66shc expression in hepG2 cells. HepG2 cells were transfected with ago-96-5p to upregulate miR-96-5p expression. Ago-NC was used as a normal control. (A, B) Argonaute-2 (Ago2) – immunoprecipitated miR-96-5p expression in NAFLD mice and PA-treated hepG2 cells. (C) miR-96-5p expression. (D, E) P66shc and cyto C protein expressions. (F) P66shc mRNA expression. (G) Schematic of the wild-type p66shc 3′UTR (3′UTR-wt) and mutated p66shc 3′UTR (3′UTR-mut) luciferase constructs. (H) HepG2 cells were transfected with 3′UTR-wt or 3UTR-mut and with ago-96-5p or ago-NC, as indicated. ^**P< 0.05. Error bars depict the standard deviation. BS: binding site; NC: negative control.

To further validate whether p66shc was a direct target of miR-96-5p in the liver, luciferase fusion construct containing either the wild type or mutated p66shc 3′- UTR was transfected into hepG2 cells (Figure 6G). Then, these cells were con-transfected with agomir-96-5p and the corresponding control. As shown in Figure 6H, agomir-96-5p clearly suppressed the luciferase activity of the wild-type reporter. However, this repression was not observed for the mutated p66shc 3′-UTR. Collectively, these data indicated that miR-96-5p could indeed regulate p66shc expression in the liver.

Catalpol alleviated ROS, apoptosis and hepatic steatosis might through upregulating miR-96-5p/p66shc pathway

To better understand the role of miR-96-5p in ROS production and hepatic steatosis, agomir-96-5p was transfected into hepG2 cells, also, the effect of catalpol was evaluated comparing with agomir-96-5p. Agomir-96-5p and catalpol significantly increased miR-96-5p levels compared with PA-treated hepG2 cells (Figure 7A). To further explore the potential mechanisms, we examined the expression of p66shc. In PA-treated group, p66shc expression was markedly increased; however, agomir-96-5p or catalpol treatment enhanced miR-96-5p expression, and suppressed p66shc and cytochrome C protein expression effectively (Figure 7B, 7C). As expected, PA-induced ROS overproduction and hepatic steatosis were also attenuated by agomir-96-5p or catalpol (Figure 7D, 7E). Furthermore, agomir-95-5p or catalpol preserved mitochondrial membrane potential and reduced cell apoptosis (Figure 7F, 7G). Moreover, catalpol treatment significantly reversed the decreased miR-96-5p expression induced by PA. On the other hand, Antagomir-96-5p aggregated PA-induced hepatic steatosis and oxidative stress in hepG2 cells, moreover antagomir-96-5p blocked the inhibitory effects of catalpol on p66shc protein expression, hepatic steatosis and oxidative stress (Supplementary Figure 5). Taken together, the results indicated that catalpol performed such protective effects might through upregulate miR-96-5p expression level.

Figure 7. Catalpol ameliorated oxidative stress, hepatic steatosis and apoptosis through upregulating miR-96-5p. (A) miR-96-5p level. (B) P66Shc protein expression. (C) Cyto C protein expression. (D) ROS level. (E) Nile red staining. (F) JC-1 level. (G) Tunel staining. The results are the mean ± SD (n=8), ^**P< 0.05 vs. Control group, ^##P < 0.05 vs. WD group.

Gene expression profile data

Data of GEO series GSE94754 was obtained from Gene Expression Omnibus database. The mice used for GSE94754 were exposed to control or high-fat diet for 12 weeks. Samples for microarrays analysis were gained from the liver tissue, with 8 samples of high fat diet and 8 samples of normal diet as control group.

Raw data preprocessing and screening and integration of differentially expressed genes

R_4.4 was used to screen the differentially expressed genes between two different diet groups. Genes integration of differentially expressed genes identified from gene chips was performed using RoubustRank Aggreg. Genes with statistic significance (p<0.05, fold change>2) were filtered. Heat maps were generated by ‘pheatmap’ package using data with expression.

Reagents

Catalpol (98%) was obtained from Nanjing jingzhu Biotech Ltd. Co (Nanjing, China). 1640 medium was bought from Gibco-BRL Company (Gaithersburg, MD, USA). DCFH-DA fluorescent probe and ECL Plus were obtained from Beyotime (Jiangsu, China). Antibodies specific for P66Shc, cytochrome C was obtained from Proteintech Group (Wuhan, China). Antibody specific for β-actin was purchased from Beyotime (Jiangsu, China).

Ethics statement

All experiments were approved by the Animal Care and Use Committee of Dalian Medical University, and the experimental procedures were performed in strict accordance with Legislation Regarding the Use and Care of Laboratory Animals of China. Before the experiments, the animals were allowed to suit the new environment for 7 days, and housed in a room under 12 h light/dark cycle, a controlled temperature at 22± 3 °Cand a relative humidity at 60± 10%.

Animals and diets

Male eight-week-old LDL receptor knockout (LDLr-/-) mice with a C57BL/6 background were purchased from Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). To detect the effect of catalpol on atherosclerosis, sixty LDLr-/- mice were divided into five groups: chow diet (n=12), catalpol (100mg/kg/D, n=12), western diet (HFD, n=12), western diet with catalpol (100mg/kg/D, n=12) and western diet with catalpol (200mg/kg/D, n=12) for 16 weeks. The diet was a commercially prepared mouse food (MD12017) supplemented with 20.0% (wt/wt) cocofat,1.25% (wt/wt) cholesterol, and 22.5% (wt/wt) protein and 45.0% carbohydrate (Jiangsu Medicine Ltd., Jiangsu,, China). At week-16, mice were anesthetized with 2% isoflurane (Forene®, Abbott), one-milliliter of blood was collected by abdominal aorta, and tissues were collected for further analysis.

Biochemical analyses

Blood samples were centrifuged (3000 rpm, 10 min, 4 C) and serum was separated for analysis. Total cholesterol (TC), triglyceride (TG), alanine aminotransferase (ALT), aspartate aminotransferase (AST), superoxide dismutase (SOD), malondialdehyde (MDA), catalase (CAT), ATP content and ATPase activity levels of serum were measured using commercial kits purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). All kits were used according to the corresponding manufacturers' instructions.

Liver histological analysis

For pathological analysis, the isolated left lateral segment of the liver lobes was fixed into 4% paraformaldehyde solution. Then the fixed-livers were embedded in paraffin and sliced (5 μM sections). Finally, the sections were stained with hematoxylin and eosin for histologic analysis.

Electron microscopy

For ultrastructural analysis by electron microscopy, the liver samples of control, high fat diet (HFD) and high-dose catalpol treatment group were fixed in 2.5% glutaraldehyde solution and embedded according to standard protocols. Briefly, the samples were rinsed in PBS and then postfixed in 1% osmium tetroxide. After rinsing in PBS for 45 min, the samples were dehydrated in a graded series of ethanol (70–100%) and then routinely embedded in epon. Then, embedded specimens were cut into ultrathin sections of 0.5 μM. Finally, ultrathin sections were stained with uranyl acetate and lead citrate.

Preparation of ox-LDL

Fresh whole blood from normal human was added with 0.5% EDTA-2Na for anticoagulation. Plasma was isolated by spinning the blood at 4°C, 7000r/min for 15 min. Native LDL (density: 1.019~1.063 g/mL) was separated from the fresh nomolipidemic human serum by discontinuous density-gradient ultracentrifugation using a Beckman coulter optima L-100 XP Ultracentrifuge and then oxidatively modified [43]. LDL was oxidized with 50 μM CuSO₄ at 37°C for 24 h, and then transferred into EDTA (200 mmol/L) in PBS for 24 h at 4°C. Subsequently, oxidation was stopped by extensive dialysis against PBS with 0.01% EDTA and sterilized by filtration. LDL oxidation was confirmed by thiobarbituric acid reaction substances with malondialdehyde as the standard [44]. LDL and oxLDL protein concentrations were determined with a bicinchoninic acid (BCA) protein assay kit (Beyotime) which used bovine serum albumin as the standard and was expressed as micrograms per milliliter of solution. LDL prepared every 2 weeks.

Cell culture and treatment

Human hepatocellular carcinoma cell line HepG2 cells were purchased from ScienCell company (CA, USA) and cultured in Dulbecco's Modied Eagle's Medium (DMEM) with 10% (v/v) fetal bovine serum (Gibco, CA, USA), 20 mg/mL penicillin, and 20 mg/mL streptomycin and maintained at 37°C in a humidified atmosphere of 5% CO₂. One day before treatment, the culture medium was changed to DMEM medium without FBS. Palmitate (PA) was dissolved in PBS at 70°C and then mixed with 10% BSA at 55°C for 10 min to achieve a final PA concentration of 500 μM. The experiments were divided into six groups: control; induction with 20 μM catalpol; hepatic steatosis induction with 500 μM PA or ox-LDL (200μg/mL); concomitant induction with 5, 20, 80μM catalpol and 500 μM PA or ox-LDL (200μg/mL). Briefly, HepG2 cells were seeded on 6-well plates at a density of 1×10⁶ per well and allowed to grow to desired confluence. Then the cells were treated with indicated concentrations of PA or ox-LDL and catalpol for 24 hours and the cells or culture media were collected to analysis.

Transfection of agomirs/antagomirs and siRNA

HepG2 cells were transiently transfected using Lipofectamine 2000 (Life Technologies-Invitrogen, Carlsbad, CA, USA) in Opti MEM according to the manufacturer’s protocol, as described [45]. Agomirs/antagomirs, siRNA targeting p66shc (siRNA) and the corresponding NC were obtained from GenePharma. The transfection procedures were performed according to the manufacturer’s protocols.

Measurement of intracellular ROS

2’, 7’-dichlorodihydrofluorescein diacetate (H₂DCFDA) probe was employed to measure ROS level as previously described [46]. HepG2 cells were incubated with PA and different concentrations of catalpol for 24h at 37°C and then collected and incubated with 20 μM H₂DCFHDA for 30min at 37°C. The fluorescence intensity was immediately measured using Fluorescent Activated Cell Sorting (FACS) Calibur Flow Cytometer (Becton Dickinson Immunocytometry Systems, CA, USA) equipped with an argon ion laser (488nm excitation) and 20 000 cells per sample were measured.

Mitochondria membrane potential assay

The mitochondrial membrane potential was assessed in the parasite using JC-1 (5, 5’, 6, 6’-tetrachloro-1, 1′, 3, 3′- tetraethylbenzimidazolylcarbocyanine iodide), that remains in monomeric form in the cytoplasm and has a green fluorescence (525 nm). However, the membrane potential of functional mitochondria establishes a negative charge that allows the lipophilic dye to accumulate and form aggregates in the mitochondria, which have red fluorescence (590 nm). HepG2 were collected from parasite cultures in control and experimental sets at different time points and incubated with JC-1 dye (at a final concentration of 10 μM) for 30 min at 37°C. Cells were washed with PBS and analyzed by flow cytometry using FACSCalibur flow cytometer and CellQuestPro software (Becton Dickinson, San Jose, CA, USA).

Nile red staining

Nile red was prepared as previously described [47]. The lipid-bound Nile Red fluorescence was observed with a fluorescence microscope.

Argonaute-2 (Ago-2) immunoprecipitation

Pools of three individual liver for each group were homogenized in immunoprecipitation buffer (300 mM NaCl, 5 mM MgCl2, 0.1% NP-40, 50 mM Tris–HCl, pH7.5), centrifuged, and 400 μL lysate was incubated overnight at 4°C with 5 μg of antibodies to Argonaute-2 (Cell Signaling Technology, Beverly, MA, USA). 20 μL of 50% Protein-A/G-agarose beads (Beyotime Institute of Biotechnology) was then added for 1h at 4°C and the bead complexes were centrifuged at 16,000 ×g for 15 min at 4°C. The pellet was washed and processed for miRNA extraction, stem-loop RT, and RT–PCR (Applied Biosystems).

Western blot analysis

Cells and tissues were harvested and protein extracts prepared according to established methods [48]. Extracts were separated in sodium dodecyl sulfate–polyacrylamide electrophoresis gels (8~15%) and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA, USA). The membranes were blocked with 5% milk, and then incubated with indicated primary antibodies at 4°C overnight. After washing, the membranes were then incubated with the appropriate secondary antibodies. The membranes were exposed to enhance chemiluminescence-plus reagents (Beyotime Institute of Biotechnology, Hangzhou, China). The emitted light was captured by a Bio-rad imageing system with a Chemi HR camera 410 and analyzed with a Gel- Pro Analyzer Version 4.0 (Media Cybernetics, MD, USA).

miRNA expression

Total RNA was extracted using the miRNeasy kit (Qiagen Shanghai, China) and 250 ng was reverse transcribed using stem-loop Multiplex primer pools (Applied Biosystems, Foster City, CA, U.S.A.). Reverse transcription (RT)–specific primers for rat miRNAs miR-9-5p, miR-96-5p, miR-124-3p (life technology) were used for all miRNA RT. Quantitative polymerase chain reaction(qPCR) was carried out using a 7500HT Fast Realtime System (Applied Biosystems) and TaqMan microRNA assays (Applied Biosystems). Endogenous RNA U6 small nuclear 2 (RNU6B) was used for normalization. The relative fold change in expression of the target gene transcript was determined using the comparative cycle threshold method (2^-ΔΔCT).

TUNEL staining

Apoptosis was quantified in paraffin-embedded liver using a terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labelling (TUNEL) Assay kit (TUNEL Apo-Green Detection Kit, Biotool, Houston, USA)) according to manufacturer instructions. Green fluorescence staining indicated positive TUNEL staining.

Luciferase activity assay

Plasmids containing the SHC1 3′-UTR response element (3’UTR-wt) and the corresponding mutant (3’UTR-mut) were obtained from Obio Technology Corp., Ltd (Shanghai, China). Plasmid DNA and the agomir or the NC was conducted 36 h after transfection. The luciferase activity was determined with a Dual-Luciferase Reporter Assay Kit (TransGen) using a Dual-Light Chemiluminescent Reporter Gene Assay System (Berthold) and was normalized to the Renilla luciferase activity.

Measurement of 8OH-dG contents

DNA was isolated according to the protocol’s instruction, and the RNA-free DNA obtained was used to determine 8-OHdG levels using the 8-OHdG ELISA kit (Huamei, Wuhan, China) according to the manufacturer’s instructions.

Statistical analysis

All results are expressed as the mean± standard deviation (SD) from more than three independent experiments, and data analyses were performed with the SPSS software package, version 19.0. Comparison of quantitative variables was performed by either Student’s t test or ANOVA followed by the student-newman-keuls (SNK) test. p values <0.05 (two-tailed) were considered statistically significant.