© Borgis - New Medicine 1/2012, s. 21-24
*Ji-An Li1, Shu-Ying Han1, Chun-Yu Tian1, Jin-Xiu Chu1, Xiang-Bo Gou1, Ting Han1, Jing Bai1, Yan-Ping Zhang1, Peng Cui1, Bo-Nan Zhang1, Hai-mei Bo1, Chen Zhen2, Júlia Bartos3
The Effects of DAIPING (Diabalance) granule on the Glycolipid Metabolism and PTP1B Expression in Liver of type 2 Diabetes Rats1)
1Hebei United University
2Central-European Traditional Chinese Medicine Association, Budapest, Hungary
President: Dr. Chen Zhen
3Semmelweis University, Budapest, Hungary doctorate student
Aim of the study. The aim of the study is to investigate the effects of the DAIPING (Diabalance) granule on triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C) and the free fatty acids (FFA) in type 2 diabetes rats. At the same time the PTP1B expression in liver of type 2 diabetes rats was evaluated with the use of western blotting and real-time quantitative PCR.
Results. The Daiping particle can decrease the TG, TC, LDL-C and FFA, and can also significantly inhibit the PTP1B expression in liver of type 2 diabetes rats.
Conclusions. The DAIPING (Diabalance) granule can improve the type 2 diabetes.
During the therapy of type 2 diabetes, traditional Chinese drug becomes very important because of its multicomponent characteristics and little side effect. Thus the investigation on the inhibitor of PTP1B in traditional Chinese drug aspect has significant purpose. We discovered that Daiping had many effects, including antioxidation, decreasing the blood glucose and blood fat and inhibiting the formation of advanced glycation end products (AGEs). Subsequently, we sought to identify the effect of DAIPING (Diabalance) granule on PTP1B protein expression at the molecular level and investigated the mechanism of DAIPING’s (Diabalance) improvement on type 2 diabetes.
Materials and Methods
Sixty male Sprague-Dawley rats weighting 200±15 g were purchased from Beijing Medicinal Institute as our experimental animals (Certificated SCXK-2009-0004). The high-fat alimentation was provided by Beijing AoKe Company Limited (Certificated SCXK-2007-00013). All rats were fed normal alimentation and tap water was provided ad libitum.
The DAIPING (Diabalance) granule is composed of Tremella, Mulberry Leaf, Radix Polygoni Multiflori Preparata etc., and it is manufactures by Traditional Chinese Medical College Laboratory of Hebei United University.
Modeling and the evaluation of the diabetes model
Eight rats forming the control group (N) were fed with normal diet for 4 weeks. The other rats were fed with high-fat diet at the same time, then they received streptozotocin (STZ) (Sigma, 25 mg·kg-1) by single tail intravenous injection to establish the type 2 diabetes models and the N group received the citric acid buffer solution accordingly. After five days the type 2 diabetes model was successfully established as the type 2 diabetic rats with fasting blood glucose (FBG) (72 hour abrosia) ≥ 11.1 mmol·L-1.
Subgroup and administration
The type 2 diabetes rats were randomly divided into four groups, ten rats per group. The Metformin rats group (Met) were administered Metformin (0.14 g·kg-1·d-1) orally once a day for four weeks, and the Daiping rats groups were administered Daiping orally at 0.345 g·kg-1·d-1 (DL) and 1.38 g·kg-1·d-1 (DH) respectively. At the same time, the other type 2 diabetes rats group (M) was administered orally with the same dose.
Euglycemic hyperinsulinemic clamp technique
After 12 hours of fasting, blood was collected from the tail vein of rats, and FBG was measured using a glucometer. In vivo, insulin resistance was measured with the euglycemic hyperinsulinemic clamp technique with an infusion of insulin (1 mU·ml-1·min-1 body weight). Blood samples were collected at 10-minute intervals for the immediate measurement of plasma glucose concentrations and 20% glucose was infused at variable rates to maintain glucose level at the steady state. The glucose injection rate (GIR) was measured at 60 minutes of steady state. The general mean of six blood glucose measurements at the steady state is the steady-state blood glucose, and the general mean of the six GIR is the steady state GIR. At the end of the clamps, rats were euthanized, and tissues were obtained for molecular analysis.
The blood serum index
After the last administration, rats were anesthetized and blood samples were obtained from abdominal aorta. The triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol (HDL-C) plasma levels were measured with ACA.
The liver was extracted, frozen in liquid nitrogen and kept at -80°C until required. Subsequently, tissues were homogenized in ice-cold lysis buffer 1% Nonident P-40, 1% sodium deoxycholate, 150 mmol NaCl, 1 mmol EDTA, 1 mmol PMSF, 1 mmol NaF, 1 mmol sodium vanadate, 50 mmol Tris-HC1 (pH 7.4), 10 μg·ml-1 Aprotinin, 10 μg·ml-1 Leupeptin. For Western blotting test, equal amounts (either 60 or 90 μg depending on the protein of interest) of protein extract were subjected to SDS-PAGE (10%), and transferred to polyvinylidene difluoride membranes (Millipore) at a constant current (depending on the size of membrane) for 2 h. The membranes were blocked in 10% milk for 1 h. Membranes were probed with the indicated first antibody (BD Biosciences) against specific PTP1B overnight at 4°C, followed by 2-hour incubation with horseradish peroxidase-labeled goat anti-mouse IgG (Beyotime). Immunoreactive proteins were detected using enhanced chemiluminescence (ECL) according to the manufacturer’s manual and quantified by densitometry.
Quantification of PTP1B mRNA expression by real-time quantitative PCR
Total RNA was extracted from the liver of rats using Trizol reagent according to the manufacturer’s instructions. The quality of the extracted RNA was confirmed by a value of 2 for the 28S ribosomal RNA/18S ribosomal RNA ratio after agarose gel electrophoresis. Levels of mRNA for a housekeeping gene (β-actin) and PTP1B gene were measured by real-time quantitative PCR. Total RNA (1 μg) was reverse transcribed into first-strand cDNA using SYBR®primeScript® RT-PCR kit. PCR was performed using 2 μl cDNA, 10 μl SYBR® Primix Ex TaqTM , and the 1.0 μl of primer in a total volume of 20 μl. The PCR primer sequences used were as follows: PTP-1B forward, 5’-CGAGCAGATCGACAAGTCCGG-3’, PTP-1B reverse, 5’-AACTCCTTTGGGCTTCTTCCA-3’, β-actin forward, 5’-ACGGTCAGGTCATCACTATC-3’, β-actin reverse, 5’-TGCCACAGGATTCCATACC-3’. PCR amplification was performed in the following conditions: initial activation of the hot start DNA polymerase for 10 seconds at 95°C followed by 40 cycles of 2 steps (95°C for 15 seconds and 55°C for 20 seconds)
Data were expressed as mean ± SEM. The significance of differences between the groups was analyzed using Student’s test.
Insulin resistance of the euglycemic hyperinsulinemic clamp technique
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