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Effects of D-pinitol on insulin resistance through PI3K/ Akt signaling pathway in type 2 diabetes mellitus rats. Yunfeng Gao, Mengna Zhang, Tianchen Wu, Mengying Xu, Haonan Cai, and Zesheng Zhang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b01238 • Publication Date (Web): 11 Jun 2015 Downloaded from http://pubs.acs.org on June 18, 2015

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Journal of Agricultural and Food Chemistry

Effects of D-pinitol on insulin resistance through PI3K/Akt signaling pathway in type 2 diabetes mellitus rats

Yunfeng Gao, Mengna Zhang, Tianchen Wu, Mengying Xu, Haonan Cai Zesheng Zhang*

Key Laboratory of Food Nutrition and Safety, Ministry of Education, College of Food Engineering and Biotechnology, Tianjin University of Science & Technolo gy, Tianjin 300457, China

Corresponding Author Tel: (+86) 022 60912431 Fax: (+86) 022 60912431 Email: [email protected] ; [email protected]

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Abstract: D-pinitol, a compound isolated from pinaceae and leguminosae

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plants, has been reported to possess insulin-like properties. Although

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hypoglycemic activity of D-pinitol was recognized in recent years, the molecule

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mechanism of D-pinitol in the treatment of diabetes mellitus remains unclear.

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In this investigation, a model of type 2 diabetes mellitus (T2DM) with insulin

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resistance was established by feeding a high-fat diet (HFD) and injecting

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streptozocin (STZ) to the Sprague Dawley (SD) rats, targeting to explore more

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details of mechanism in the therapy of T2DM. D-pinitol was administrated to

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the diabetic rats as two doses [30, 60 mg/ (kgbodyweightd)]. The level of

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fasting blood glucose (FBG) was decreased 12.63% in high-dosage group.

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And the ability of oral glucose tolerance was improved in D-pinitol treated

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groups. The biochemical indexes revealed that D-pinitol had a positive effect

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on hypoglycemic activity. Western boltting suggested that D-pinitol could

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promote the expression of the phosphatidylinositol-3-kinase (PI3K) p85,

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PI3Kp110 as well as the downstream target protein kinase B/Akt (at Ser473).

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Besides, D-pinitol inhibited the expression of glycogen synthesis kinase-3β

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(GSK-3β) protein and regulated the expression of glycogen synthesis (GS)

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protein and then accelerated the glycogen synthesis. From above all, D-pinitol

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played a positive role in regulating insulin mediated glucose uptake in liver

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through translocation and activation of PI3K/Akt signaling pathway in T2DM

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rats.

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Key words: D-pinitol; Diabetes mellitus; PI3K/Akt signaling pathway 2


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Introduction

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Diabetes mellitus is a complex disease which is called “The Silent Killer” due

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to the amount of patients and lots of chronic complications. Around 90% of the

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diabetes cases are T2DM, which is known as non-insulin-dependent diabetes

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mellitus.1 Patients of T2DM mostly are characterized by insulin resistance and

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impaired insulin secretion because of the dysfunction of β-cell caused by the

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high level of blood glucose.2 Irregularly increasing adipose of body tissues

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leads to obesity, raising the risk of insulin resistance and then impairing

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insulin-stimulated glucose uptake in the peripheral tissues.3, 4

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D-pinitol, 3-O-methyl form of D-chiro-inositol, is one of the naturally

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occurring inositol derivatives.5 It is rich in pinaceae plants and has already

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been found in leguminosae plants.6 D-pinitol is regarded as a bioactive

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compound for it can be used for treatments connected with diabetes mellitus

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and its chronic complications. For the most part, many reports have shown the

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favorable effect of D-pinitol on the treatment of disorders associated with

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insulin because of its insulin-like function.7 It is useful in treating diabetes by

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reducing the level of blood glucose in patients with T2DM.8,9,10 And it also

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showed that D-pinitol could stimulate translocation of glucose transporter 4 in

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skeletal muscles of C57BL/5 mice in order to improve insulin sensitivity of the

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diabetic mice.11 However, the molecule mechanism of D-pinitol in the treatment

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of diabetes mellitus, especially on liver, still remains unclear. Liver is the

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predominant visceral organ to regulate the glucose metabolism by means of 3



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plenty of signaling pathways. PI3K/Akt is one of the most primary signaling

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pathways which is believed to be a major mechanism involved in the

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development of insulin resistance.12,

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evaluate the effect of D-pinitol on insulin resistance through PI3K/Akt signaling

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pathway in T2DM rats.

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The aim of this investigation is to

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Materials and Methods

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Materials and chemicals

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D-pinitol (95%) used in this study was purchased from Yipin Biological

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Technology Co., Ltd (Xi’an, China). STZ was purchased from Sigma Chemical

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Co. (St. Louis, MO, USA). Blood glucose meter and blood glucose test strips

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were obtained from Johnson & Johnson, Co. (New Buren Zwick, NJ, USA).

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Rat insulin elisa kit was purchased from Dingguo Changsheng Biotechnology

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Co. Ltd (Beijing, China). Insulin was obtained from Gen-view Scientific INC

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(Tallahassee, FL, USA). Trizol was purchased from TransGen Biotech Co., Ltd.

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(Beijing, China). RIPA, nitrocellulose membrane and SABC (rabbit IgG) – POD

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Kit were brought from Solarbio science & technology Co., Ltd (Beijing, China).

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Antibodies PI3Kp85, PI3Kp110, Akt, phospho-Akt, GSK-3β and GS were

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obtained from Cell Signaling Technology lnc. (Danvers, MA, USA). Secondary

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antibodies were brought from ZSGB-BIO technology Co., Ltd (Beijing, China).

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The other laboratory chemicals were of the analytical grade.

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Animals

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SD rats (200±20 g), purchased from the animal house of Beijing University

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Science Center, were chosen for our experiment. All rats were kept in internal

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flawless animal rooms which were under a controlled environment at 23±2 °C

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with humidity of 55±10%, 12h light/dark cycle and unrestricted food and water.

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The animals were approved by the Animal Care and Use Committee, all the

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related facilities and experimental procedures were executed according to the

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Technical Standards for Testing & Assessment of Health Food (2003).

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Induction of T2DM in rats and medicinal dosage

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All the experimental animals had been acclimatized to the laboratory

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environment for 7 days. Then the animals were randomly divided into normal

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control group (NC) and diabetic control group (DC). NC group, consisting of 20

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rats, was fed with a normal chow diet during the whole study, while the other

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group was fed with HFD. After a month, the HFD rats were injected

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intraperitoneally with STZ solution at a dose of 30 mg / (kgb.w.). The STZ was

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dissolved in citrate buffer at the pH of 4.2-4.5. The rats in NC group were

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injected with the citrate buffer vehicle. 72h after injection, the rats in DC group

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with blood glucose level ≥ 11.1mmol/L were considered as T2DM rats and

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would be used in future experiments. These T2DM rats were fed with HFD

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throughout the whole study. Then the T2DM rats were randomly divided into

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three groups with 10 animals each. The first group (low-dosage group, LD)

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received D-pinitol at 30 mg / (kgb.w.) per day, the second group (high-dosage

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group, HD) received D-pinitol at 60 mg / (kgb.w.) per day and the last one

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received distilled water only (diabetes control group, DC). 10 rats were chosen

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from the normal control group to receive D-pinitol at 60 mg / (kgb.w.) per day

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as the normal high-dosage control group (NH).

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Oral glucose tolerance test (OGTT) and insulin tolerance test (ITT)

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OGTT was performed in overnight fasted rats from every group. Vehicle

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(distilled water) was orally administered to NC and DC groups. 30 mg/kg, 60

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mg/kg and 60 mg/kg D-pinitol were orally administered to LD, HD and NH

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groups. 20 min later, all the rats were orally administrated glucose with a dose

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of 2g/kg. Blood was withdrawn from the tip of the tail at 0, 30, 60 and 120 min

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from all 5 groups to measure the blood glucose level.

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ITT were performed in overnight fasted rats from all groups 3 days after the

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OGTT. 20 min after administered the vehicle (distilled water) and D-pinitol, the

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rats were injected insulin intraperitoneally with a dose of 0.15 U/kg. The same

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as OGTT had shown that the blood samples were withdrawn at 0, 30, 60 and

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120 min after injected insulin, and the blood glucose level was measured.

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Biochemical analysis

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FBG level was determined by the blood glucose meter and test strips each

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week. The fasting serum insulin (FINS) was determined by ELISA kit. The

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insulin sensitivity index (ISI) was calculated following the formula ISI = Ln

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(1/FINS*FBG). And the Homeostasis model assessment-β (HOMA-β) were

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calculated following the formula HOMA-β = (20*FINS) / (FBG-3.5). All the

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parameters were performed according to the manufactures’ instructions. The

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blood samples and viscera organs were stored at -80°C in the refrigerator for

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future research.

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Real-time polymerase chain reaction (RT-PCR) analysis

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RT-PCR was operated to determine the expression levels of the genes.

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Total RNA was extracted from liver using Trizol reagent, and the concentration

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of the total RNA was determined by the ultraviolet spectrophotometer. It was

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converted to DNA by RT-PCR kit and the reaction was performed according to

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the manufacturer’s instructions. The specific primers which include both sense

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and anti-sense were used for the amplification of DNA (Table1).

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Western blot analysis

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The expression of protein including PI3Kp85 (85kDa), PI3Kp110 (110 kDa),

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Akt (60kDa), p-Akt (60kDa), GSK-3β (46kDa) and GS (81-85kDa) were

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analyzed by western blot. In brief, 100mg liver was homogenized in RIPA

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buffer for 10 min, followed by centrifugation at 10,000 rpm for 5 min at 4°C. The

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supernatant was transferred to a new clean centrifuge tube, and Bradford

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colorimetric method was used to determine the concentration of the total

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protein. The tissue protein (50-70 µg) was subjected to 10% SDS-PAGE for

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0.5h at 80V and 2.5h at 100V, in order to separate the aim protein from the

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others. Proteins were transferred to a nitrocellulose membrane for 3h at 300

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mA by wet transfer equipment. The membrane was incubated in blocking

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solution containing 5% non-fat dried milk for 2h at room temperature.

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Subsequently, the membrane was exposed to desire the primary antibodies,

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PBS containing PI3Kp85 antibody (1:1000), PI3Kp110 antibody (1:1000), Akt

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antibody (1:1000), p-Akt (Ser473) antibody (1:2000), GSK-3β （1:1000）and

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GS (1:1000) were incubated to the membrane at 4°C overnight and β-actin (43 8


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kDa) was used as control protein. After incubated with the secondary antibody

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for 2h at room temperature, the membrane was exposed to chemiluminescent

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reagent (ECL) for about 5-10 min. The expressions of the proteins on the

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membranes with fluorescence were exposed to the X-ray photographic films in

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the darkroom and the band densities were quantified.

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Immunohistochemistry

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Liver tissues embedded in paraffin were performed on 5-µm paraffin

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sections for immunohistochemical staining. The paraffin sections were

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dewaxed and rehydrated before dipped into 0.01M citrate buffer (pH 6.0) with

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a boiling water reaction in order to retrieve antigen. The paraffin sections were

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incubated with 3% H2O2 in order to quench the activity of endogenous

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peroxidase. And then the sections were incubated in the solution containing 3%

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BSA to block non-specific binding sites, while endogenous avidin-binding

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activity was inhibited by continuously treated with avidin-biotin. And the

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following steps of the immunodetection experiment were operated according to

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the manufactures’ instruction of SABC (rabbit IgG) – POD Kit. For each

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antibody, a negative control was included in which the primary antibody was

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replaced with phosphate buffer saline (PBS).

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Statistical analysis

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All the data were presented as means ± S.D. T-test was used to exhibit the

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significance of differences between samples. These differences were

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considered significant when p

Akt

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