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Xuezhikang Attenuated the Functional and Morphological Impairment of Pancreatic Islets in Diabetic Mice Via the Inhibition of Oxidative Stress

Author: WangJun
Tutor: JiangShiSen
School: Nanjing University
Course: Internal Medicine
Keywords: Xuezhikang db/db mice Type2diabetes mellitus Glucosemetablism Pancreatic islet First-Phase Insulin Secretion microenvironment Oxidative stress Nicotinamide adenine dinucleotide phosphate oxidase
CLC: R587.1
Type: PhD thesis
Year: 2013
Downloads: 32
Quote: 0
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BACKGROUND:Type2Diabetes Mellitus (T2DM) and its complications severely influence the human’s healthy status and life quality as well as increase the social burden. One of the final manifestations in T2DM is the progressive secretory dysfunction of pancreatic islet and β-cell, which play the crucial role in the pathogenesis and development of T2DM. Meanwhile, the hyperglycemia-induced oxidative stress is one of the mechanisms involved in the injury of islet function. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) is the major source of reactive oxygen species. So the inhibition of oxidative stress is essential for the conservation of islet function. Recently, the concept of "Islet microenvironment" was proposed by some researchers, who regarded that the local regulation of islet microenvironment was the central point of the T2DM pathogenesis. The improvement of islet microenvironment would be another promising therapeutical target of diabetes. The increasing incidence of diabetes associated with statin has raised concerns about the effect of statins on glucose metabolism. To date, it remains questionable whether statin treatment has a detrimental or favorable effect on diabetes. Xuezhikang, purified from red yeast rice, is a traditional Chinese medicine with the major potency, lipid-lowering. Described as the "natural statin", xuezhikang was proved to have potential benefits on glucose metabolism by the clinical trials. So far, there has not been basic research involved in the effect of xuezhikang on glucose metabolism. The db/db mouse is a genetically obese mouse model of T2DM, and has been adopted as the ideal T2DM model for experimental study. In our study, the db/db mice were subjected to the xuezhikang intake for eight weeks. In vitro, the islet perifusion system was used to evaluate the first-phase insulin secretory function of β cells after xuezhikang treatment, which has not been investigated yet. In vivo, we measured the metabolic parameters and observed the change of glucose metabolism, islet microenviroment, untrastructure and so on. In addition, the glucose-sensing apparatus was analyzed for the attempt to investigate whether xuezhikang could modulate the glucose-sensing signal pathway of β-cell in db/db mice. We moved a forward step to explore the possible target and underlying mechanism of xuezhikang in the oxidative stress reaction.OBJECTIVES:Through the db/db T2DM mouse model, the present study aims to:1. To assess the influence of xuezhikang on metabolism profile including TC, LDL-C, TG, FBG, and FSI in serum.2. To observe the impact of xuezhikang on the glucose tolerance and islet endocrine function.3. To investigate the protection of xuezhikang against the injury in the context of pancreatic β-cell content, microenvironment and ultrastructure.4. To evaluate the role of xuezhikang in regulating the expression of glucose-sensing apparatus in mRNA and protein level.5. To explore the bioactivity of xuezhikang in the inhibition of oxidative stress.METHODS:A total of40male genetically diabetic C57BL/KsJ-db/db mice, weighing 34-42g, were randomly assigned into two groups (n=20per group).These animals were treated with xuezhikang at300mg/kg/d (xuezhikang group) or placebo (placebo group) for8weeks. In addition, another20aged-matched lean non-diabetic C57BL/KsJ-db/m littermates served as a wild-type control (non-diabetic control, n=20).During8-week treatment, the physiological and metabolic parameters (BW, TC, LDL-C, TG, FBG and FSI) were measured every week. After8-week treatment,10mice were randomly selected from each group and received intraperitoneal glucose tolerance test (IPGTT). The concentration of glucose and insulin were determined in the blood samples collected at different time points:0,30,60,120min. At the end of experiment, the in vitro insulin release kinetics was studied with the perifusion system.Meanwhile, animals were anesthetized with sodium phenobarbital prior to pancreatectomy. We applied the semi-quantitative analysis to assess the content of β-cell with Image-Pro Plus5.0.1after immunohistochemistry for insulin. The similar method was used for the detection of CD31expression surrounding the β-cell. The pancreas tissues were cut into small pieces with razor blades, which were examined on a electron microscope in order to observe the ultrastructure of organelles in the β-cell. In addition, the expression level of glucose-sensing apparatus, GLUT-2, GCK was measured in mRNA level by qRT-PCR and in protein level by western blot.On the other hand, with respect to immunohistochemistrical analysis, ABC method was employed for the detection of insulin, gp91phox,8-hydroxy-20-deoxyguanosine (8-OHdG),4-hydroxynonenal modified protein (4-HNE).All values were expressed as mean±standard deviation. Means among groups were compared with one way analysis of variance, and those between two groups were compared with LSD test. Data before and after experiment were compared with paired test. RESULTS:1. At baseline, there were no marked differences in the BW, FBG, FSI, TC, LDL-C and TG between xuezhikang group and placebo group. However, all of these values were lower in db/m group than those of two db/db groups. In particular, the db/db mice aged8weeks had the FBG of>15mmol/L. After treatment with xuezhikang, the development of hyperglycemia was alleviated, and FBG and FSI at the terminal stage of treatment in the db/db mice was markedly lower than that in the placebo-treated db/db mice (P<0.05), although there is no difference between before and after xuezhikang treatment. Likewise, TC, LDL-C and TG in the xuezhikang group were markedly reduced as compared to the db/db control (P<0.05). Besides, the8-week of xuezhikang intake blunted weight gain in db/db mice (P<0.05).2. During the120-minute glucose tolerance test, the blood glucose level after glucose loading in the xuezhikang group was lower than that of placebo group (P<0.05), which was standardized to that of non-diabetic group. Moreover, insulin secretion stimulated by glucose loading in the xuezhikang group was higher than that of db/db control. The AUCINS0-30in the xuezhikang-treated mice at30minutes after the test was higher than that of the placebo-treated db/db mice (P<0.05). The experiments involving the islets perifusion in vitro revealed that in contrast to the db/db control, the insulin secretion derived from isolated islets was not noticeably raised in xuezhikang group pretreated with a low-concentration glucose solution (2.8mM). However, it was moderately increased at1minute after perfusion with the high-concentration glucose solution (16.7mM)(P<0.05).3. The less intense dark brown staining, which implied the reduced amount of P-cells, could be observed in islets from db/db control mice compared to db/m mice. Xuezhikang therapy seemed to decline the trend of β-cell reduction (P<0.05). Similarly, the favorable effect of xuezhikang treatment was presented again by the restored β-cell mass (P<0.05). As an endothelial cell marker, the CD31expression was depleted in db/db diabetic animals. Interestingly, the staining intensity of CD31was higher in xuezhikang group than in placebo group. On the other hand, the alleviated mitochondria swelling and the increased proportion of intact mitochondria were achieved in xuezhikang-treated mice vs. the placebo-treated counterpart.4. We measured the mRNA and protein levels of GLUT-2and GCK in an attempt to determine a possible impact of xuezhikang on glucose sensing. The result of qRT-PCR showed the reduced mRNA level of GLUT-2and GCK in db/db mice compared with db/m mice. Western blotting revealed decreased levels of both GLUT-2and GCK in db/db mice compared with db/m mice. The final analysis further validated that the expression of GLUT-2and GCK ascended relatively, both in mRNA and protein level, in xuezhikang group as opposed to placebo group (P<0.01).5. The percentage of β-cells expressing8-OHdG and4-HNE was higher in db/db mice than in db/m mice (P<0.05). Xuezhikang significantly reduced the expression levels of these two markers to the normal level. Furthermore, we studied the expression levels of gp91phox, which is one of the major components of NADPH oxidase. Semiquantitative analysis revealed that the uptake of xuezhikang cut down the expression of gp91phox almost to the extent of wild-type control, lower than that of db/db control (P<0.01).CONCLUSIONS:1. Xuezhikang not only reduced the blood lipids, but also alleviated blood high glucose, improved the glucose tolerance and elevated the serum insulin concentration of db/db mice.2. Xuezhikang could alleviate blood high glucose and protect the function of β cells by improving first phase insulin secretion.3. Xuezhikang-treatment alleviated blood high glucose and protected the function of β cells db/db mice by the conservation of islet microenvironment and the increment of β cell content.4. Xuezhikang increased the expressions of glucose-sensing apparatus, GLUT-2&GCK, in both mRNA and protein level. It implied that xuezhikang would possibly serve as the mediator in the pathway of insulin resistance.5. Xuezhikang reduced the expression of reactive oxygen species and inhibited one of the active subunits of nicotinamide adenine dinucleotide phosphate oxidase. Xuezhikang could exert the protection against oxidative stress.

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CLC: > Medicine, health > Internal Medicine > Endocrine diseases and metabolic diseases > Islet disease > Diabetes
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