六味地黄丸预防OLETF鼠糖尿病发病机制的实验研究
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摘要
【背景】
随着社会经济的发展和人们生活方式的改变,糖尿病已经成为继心血管疾病和肿瘤之后的第三大严重危害人类健康的慢性非传染性疾病,其患病率在全球范围内呈逐年增高的趋势,给个人和社会带来巨大的经济负担。目前,临床上仍然缺乏有效的手段来控制糖尿病及其并发症的发生与发展,预防糖尿病的发生,并从根本上消除糖尿病带来的危害,成为最受人们关注的问题之一。美国糖尿病预防试验(DPP)和STOP-NIDDM等循证医学研究表明:占全部糖尿病患者90%以上的2型糖尿病是可以预防的,采取干预措施可以使2型糖尿病发病危险降低31%～58%。然而,目前的预防手段都不同程度面临着干预药物毒副反应大、药物价格昂贵、患者依从性差等明显问题,因此,寻找更为有效、经济、不良反应小的预防糖尿病药物成为目前药物预防高危人群糖尿病发病研究的热点。
2型糖尿病的发病机制尚未完全阐明,目前认为胰岛素抵抗和胰岛β细胞功能受损是2型糖尿病最重要的两个发病因素,其中胰岛素抵抗在2型糖尿病的发病过程中有可能起始动作用并贯穿该病的全过程。因此,改善胰岛素抵抗对于预防和治疗2型糖尿病至关重要。
胰岛素抵抗是许多临床疾病(如高血压、动脉粥样硬化等),尤其是常见的内分泌代谢性疾病的共同危险因素,已经成为许多学科共同感兴趣的研究热点。胰岛素抵抗是亚细胞、细胞、组织或机体的一种病理生理状态,当机体出现胰岛素抵抗时,胰岛素刺激的葡萄糖利用减少,同时不能有效抑制肝糖原分解输出葡萄糖,从而导致血糖升高并最终导致糖尿病的出现。随着对胰岛素抵抗研究的深入,人们发现许多激素和细胞因子,尤其是脂肪细胞分泌的多个细胞因子,在胰岛素抵抗的发病中起着重要作用。脂联素是脂肪组织分泌的细胞因子之一,与能量代谢、胰岛素抵抗的关系非常密切。研究发现,血浆脂联素的浓度与身体脂肪构成比、腰臀比例、空腹胰岛素浓度以及餐后2h葡萄糖浓度呈负相关,与胰岛素敏感性呈正相关,血浆脂联素水平的降低在肥胖早期已经存在,在发生2型糖尿病后继续降低,提示血浆脂联素水平的降低与胰岛素抵抗有着密切的联系。脂联素有望成为一种胰岛素敏感性的指标,甚至可能成为一种治疗胰岛素抵抗和糖尿病的有效药物。
炎症及炎症介质在2型糖尿病发病机制中的作用也日益引起学术界的关注。其中,核因子-κB(Nuclear.factor-κB,NF-κB)作为炎症启动、调节的关键核因子,是研究的热点。但是,NF-κB如何调控炎症继而导致2型糖尿病的发生的具体机制,以及炎症与胰岛素抵抗在2型糖尿病发病中的相互关系目前尚未明确。为更好地阐明2型糖尿病的发病机制,我们有必要探讨炎症与胰岛素抵抗的关系。目前,国内外已有文献报道,NF-κB抑制物激酶((IκB kinase,IKK)是联系炎症与胰岛素抵抗的重要纽带。生理情况下,NF-κB与其抑制物IκB(Inhibitor of NF-κB)结合,以无活性的形式存在于细胞浆内,一旦IκB激酶IKK被肿瘤坏死因子(Tumor necrosis factor-α,TNF-α)等炎症因子激活后,使IκB磷酸化并与NF-κB解离,解除抑制的NF-κB进入细胞核内,调节一系列炎症反应。IKK既是调节炎症的重要因素,同时IKK又是胰岛素受体和胰岛素受体底物(Insulin receptor substrate,IRS)的丝氨酸磷酸化激酶,它可使IRS 307位的丝氨酸磷酸化,导致正常的酪氨酸磷酸化受抑制,并减弱胰岛素受体与IRS的结合以及终止胰岛素信号向磷脂酰肌醇-3激酶(Phosphatidylinositol-3 kinase,PI3-K)传递;另外,IRS的丝氨酸磷酸化还可以增加IRS的降解,并使IRS成为胰岛素受体激酶的抑制物。综上所述,IKK是将炎症和IR联系起来的重要枢纽。因此,在对糖尿病干预的研究中,探讨干预药物对IKK/NF-κB通路的影响,不仅对阐明胰岛素抵抗和炎症在2型糖尿病的发病机制中的作用有着重要意义,而且对于干预药物的作用机制的阐明也非常必要。
近年来,随着中西医结合治疗糖尿病研究的不断进展,中药复方制剂及其活性成分在治疗糖尿病方面的作用得到越来越多的重视。我们在前期研究中发现,六味地黄丸能够延缓糖尿病动物模型OLETF(Otsuka Long-Evans TokushimaFatty)鼠的餐后高血糖的出现,降低糖尿病发病率,对糖尿病的预防具有重要的作用。国内外研究显示,六味地黄丸具有调节糖代谢、增加肝糖原含量、抗炎和抗氧化作用。因此我们推测,六味地黄丸可能通过影响炎症的有关通路,改善胰岛素抵抗,从而预防2型糖尿病的发病。故本实验以IKK/NF-κB通路为切入点,探讨六味地黄丸对糖尿病大鼠胰岛素敏感器官中IKK的干预作用及其对2型糖尿病的预防作用机制。
第1部分六味地黄丸对OLETF鼠胰岛素抵抗的影响
【目的】
探讨六味地黄丸能否改善OLETF鼠的胰岛素抵抗,观察六味地黄丸对OLETF鼠血浆脂联素水平、脂肪组织中脂联素mRNA表达量以及胰岛素敏感性的影响。
【方法】
1.以自发性2型糖尿病大鼠模型OLETF鼠和其同系非糖尿病对照鼠LETO鼠为实验对象。大鼠皆为雄性,共分三组。OLETF鼠干预组(干预组)和OLETF鼠对照组(对照组),各20只;LETO鼠10只作为正常对照组(LETO组)。从8周龄开始,干预组予六味地黄丸溶液灌胃(2.4g·kg~(-1)d~(-1)),其余两组以等量蒸馏水灌胃。分别于8周龄、32周龄和40周龄时随机处死大鼠,采集血标本,分离大鼠胰腺及肝脏组织,置于液氮中保存备用;
2.检测空腹血清葡萄糖、游离脂肪酸、甘油三酯、胆固醇、血浆胰岛素和脂联素水平;
3.采用RT-PCR,检测脂肪组织中脂联素mRNA的表达;
4.计算胰岛素敏感指数(ISI);
5.分析血浆脂联素水平与各相关指标的相关性。
【结果】
1.与LETO组相比,对照组除空腹血糖升高不显著外,空腹血浆胰岛素、血清甘油三酯、胆固醇均显著升高,胰岛素敏感指数显著降低;
2.对照组血浆脂联素水平和脂肪组织脂联素mRNA表达量与LETO组相比显著降低;
3.干预组空腹血糖与对照组相比无明显变化,但空腹胰岛素和血清甘油三酯、胆固醇均显著降低,胰岛素敏感指数显著增高;
4.与对照组相比,干预组的血浆脂联素水平和脂肪组织脂联素mRNA表达量均明显增加;
5.对照组血浆脂联素与胰岛素敏感指数呈正相关,而与空腹胰岛素、甘油三酯、游离脂肪酸、胆固醇呈负相关。
【结论】
1.OLETF鼠脂联素水平与胰岛素敏感性呈正相关;
2.OLETF鼠脂联素水平与游离脂肪酸、甘油三酯水平呈负相关;
3.六味地黄丸能明显增加OLETF鼠脂肪组织脂联素mRNA表达水平,升高血浆脂联素水平;
4.六味地黄丸能有效调节血脂异常和改善胰岛素抵抗。
第2部分六味地黄丸对OLETF鼠肝脏及胰腺的形态学影响
【目的】
通过观察六味地黄丸对OLETF鼠肝脏及胰腺的形态学影响,探讨六味地黄丸对OLETF鼠肝脏和胰腺组织是否具有保护作用。
【方法】
1.实验动物、分组和干预措施同第1部分;
2.从液氮中取出已分离的大鼠胰腺及肝脏组织后,置于10%多聚甲醛中固定24小时,包埋蜡块;
3.石蜡切片机间隔4um连续切片,相邻切片分别用标准HE染色观察大鼠肝脏及胰腺组织学改变;
4.特殊染色:根据HE结果,对对照组及干预组大鼠肝脏及胰腺以冰冻切片进行MASSON染色及PAS染色,以进一步明确病变性质。
【结果】
1.8周龄时,各组大鼠胰岛结构无明显差别。32周龄,干预组和对照组胰岛明显比LETO组肥大,并有纤维组织长入,对照组大鼠胰岛纤维化更加明显,胰岛结构排列紊乱。40周龄,对照组大鼠胰岛形状更不规则,纤维组织大量增生,多数胰岛失去正常结构,胰岛明显萎缩。干预组大鼠胰岛基本保持正常结构,无明显肥大或萎缩;
2.8周龄时,各组大鼠肝脏结构亦无明显差别。32周龄,对照组及干预组大鼠肝细胞均出现明显空泡样变性,而LETO组大鼠肝小叶结构依然整齐,肝细胞以中央静脉为中心排列成索状。40周龄,对照组大鼠肝细胞空泡样变性更加明显,干预组较32周时改善,可见轻度肝索排列紊乱,部分肝细胞空泡化;
3.8周龄时,各组大鼠肝脏PAS染色无明显差别,肝细胞中未见明确的红色颗粒;
32周龄,PAS染色对照组及干预组肝细胞的胞质及胞核均可见到的红色颗粒;
40周龄,对照组肝细胞中红色颗粒更加丰富,而干预组肝细胞中红色颗粒明显减少;
4.各组各周龄大鼠肝脏未见到纤维组织增生。
【结论】
1.六味地黄丸有助于保持大鼠肝脏及胰腺的正常结构;
2.随着糖尿病病程进展,OLETE大鼠肝脏的脂肪浸润、糖原累积及胰腺纤维变性呈渐进性加重;
3.六味地黄丸能够通过保护OLETF大鼠胰岛及肝脏的正常形态结构,达到维持胰岛素敏感器官胰腺和肝脏的生理功能作用。
第3部分六味地黄丸对OLETF鼠IKK/NF-κB通路的影响
【目的】
观察六味地黄丸对OLETF鼠IKK/NF-κB通路的影响,探讨六味地黄丸是否可以通过抑制IKK、NF-κB的表达而起到改善OLETF鼠胰岛素抵抗的作用。
【方法】
1.实验动物、分组和干预措施同第1部分;
2.免疫荧光染色检测IKKβ在各组大鼠胰腺组织中的表达情况;
3.免疫荧光染色检测IKKβ在各组大鼠肝脏组织中的表达情况;
4.免疫荧光染色法检测各组大鼠胰腺组织中P-IRS-1的表达情况;
5.免疫荧光染色法检测各组大鼠肝脏组织中P-IRS-1的表达情况;
6.免疫组化法检测NF-κB在各组大鼠肝脏及胰腺的表达情况,用图像分析系统分析结果;
7.分析IKKβ、NF-κB及P-IRS-1的相关性;
8.实验数据计量资料以均数±标准差((?)±s)。采用SPSS10.0软件进行统计分析,两样本间计量资料的比较采用t检验。多组间资料比较采用单因素方差分析(ANOVA)检验。各因子之间的双变量相关性采用等级相关系数(Spearman秩相关系数)的非参数方法。
【结果】
1.六味地黄丸对肝脏中IKKβ表达的影响
各组IKKβ的表达都随周龄延长而增加,且这种差异有统计学意义。8周龄时,对照组IKKβ表达已高于LETO组(P＜0.001)。32周龄时,对照组IKKβ表达显著高于LETO组和干预组(P＜0.001);与LETO组相比,干预组IKKβ表达情况无显著差别(P＞0.05)。40周龄时,IKKβ表达仍然是在对照组最高,干预组其次,LETO组最低,差异有统计学意义(P＜0.001)。
2.六味地黄丸对胰腺中IKKβ表达的影响
各组IKKβ的表达都随周龄延长而逐渐增加,但在LETO组和对照组32周龄与40周龄时的差异无统计学意义(P分别为0.414和0.390)。8周龄时,对照组IKKβ蛋白表达高于LETO组,差异有统计学意义(P＜0.05)。32周龄时,IKKβ蛋白的表达在对照组最高,干预组其次,LETO组最低,差异有统计学意义(P＜0.05)。40周龄时,各组IKKβ的表达都有不同程度升高,IKKβ表达依然是对照组最高,干预组其次,LETO组最低,且各组间的差异均有统计学意义(P＜0.001)。
3.六味地黄丸对大鼠肝脏NF-κβ表达的影响
同周龄各组间比较,8周龄时,对照组NF-κβ的表达显著高于LETO组(P＜0.05)。32周龄时,与LETO组相比,对照组NF-κβ的表达水平显著升高,干预组次之,各组间差别有统计学意义(P＜0.001)。40周龄时,对照组NF-κB的表达进一步升高,显著高于干预组和LETO组,干预组仍然介于对照组和LETO组之间,各组间差异有统计学意义(P＜0.001)。
4.六味地黄丸对大鼠胰腺NF-κβ表达的影响
同周龄各组间比较,8周龄时,对照组胰腺中NF-κB的表达就开始显著高于LETO组(P＜0.05):32周龄时,与LETO组大鼠相比,对照组和干预组NF-κB的表达量均显著增高(P＜0.05);干预组低于对照组,但差异无统计学意义(P=0.082)。40周龄时,对照组和干预组胰腺中NF-κB表达量仍然都显著低于LETO组(P＜0.05),干预组胰腺中NF-κB表达量仍低于对照组,但差异无统计学意义(P=0.060)。
5.六味地黄丸对大鼠肝脏P-IRS-1表达的影响
同周龄各组间比较,8周龄时,LETO组P-IRS-1的表达即显著高于对照组(P＜0.05)。32周龄时,P-IRS-1在肝脏的表达在LETO组最高,干预组次之,对照组最低,差异显著(P＜0.001)。40周龄时,LETO组P-IRS-1的表达进一步升高,显著高于干预组和对照组,对照组则呈下降趋势,仍在三组中表达最低,各组间差异有统计学意义(P＜0.001)。
6.六味地黄丸对大鼠胰腺P-IRS-1表达的影响
8周龄时,LETO组P-IRS-1的在胰腺中的表达显著高于对照组(P＜0.001)。32周龄时,对照组显著低于LETO组和干预组,干预组低于LETO组,各组间差异具有统计学意义(P＜0.001)。40周龄时,P-IRS-1在LETO组最高,干预组次之,对照组最低,差异有统计学意义(P＜0.001)。
7.肝脏中IKKβ、NF-κB与P-IRS-1的相关性分析
相同周龄时,肝脏中IKKβ、NF-κB与P-IRS-1的相关分析结果:8周龄,IKKβ与NF-κB的表达呈正相关,相关性显著(r_s=0.909,P＜0.001);IKKβ、NF-κB均与P-IRS-1呈负相关,相关性不显著(P=0.208)。32周龄时,IKKβ与NF-κB的表达呈显著正相关(r_s=0.884,P＜0.001),IKKβ与P-IRS-1呈负相关(r_s=-0.75,P=0.02),NF-κB与P-IRS-1呈显著负相关(r_s=-0.867,P=0.002)。40周龄时,IKKβ与NF-κB的仍呈显著正相关(r_s=0.913,P＜0.001),且均与P-IRS-1显著负相关(P＜0.001)。
8.胰腺中IKKβ、NF-κB与P-IRS-1的相关性分析
相同周龄时,胰腺中IKKβ、NF-κB与P-IRS-1的相关分析结果:8周龄,IKKβ与NF-κB的表达呈显著正相关(r_s=0.951,P＜0.001),均与P-IRS-1呈负相关,相关性不显著(P=0.198)。32周龄时,IKKβ与NF-κB的呈正相关,相关性显著(r_s=0.903,P＜0.001),IKKβ与P-IRS-1呈显著负相关(r_s=-0.828,P＜0.05),NF-κB与P-IRS-1呈负相关(r_s=-0.75,P＜0.05)。40周龄时,IKKβ与NF-κB的仍呈显著正相关(r_s=0.901,P＜0.001),且均与P-IRS-1显著负相关(P＜0.001)。
【结论】
1.六味地黄丸能降低OLETF鼠IKKβ、NF-κB的表达,减轻炎症反应对肝脏和胰腺的损害;
2.六昧地黄丸能增加OLETF鼠肝脏及胰腺组织中P-IRS-1的表达水平,改善胰岛素抵抗;
3.OLETF鼠肝脏中IKKβ与NF-κB的表达在各周龄时均呈高度正相关(P＜0.001);与IRS-1表达呈负相关,其中32周龄和40周龄时相关性有统计学意义(P＜0.02)。在胰腺组织中有同样的趋势。提示了IKK在炎症与胰岛素抵抗中具有桥梁作用;
4.六味地黄丸通过降低IKKβ的表达,达到改善胰岛素抵抗和抗炎作用,可能是其预防2型糖尿病发病的基本机制之一。
BACKGROUD:
With the development of economy and alteration of life style,the incidence rate of diabetes mellitus roars up year by year.Diabetes mellitus has become the third disease following the cardiovascular diseases and tumor which damage people's health seriously.It brings enormous economic burden for individual and society.With the knowledge of insulin,the development and application of drugs stimulating secretion of insulin and insulin-sensitizing drugs etc,more methods are available in the treatment of diabetes.However,although progress has been made in the treatment of diabetes,we still can't control the development of diabetes and its complications. So preventing diabetes becomes one of the most important issues in order to solve the problem radically.Whether diabetes can be prevented and what is the most economical,safe,effective way to prevent diabetes becoming a must to tackle this problem.The evidence-based research of DPP and stop-NIDDM showed T2DM which accounts for 90%of all patients with DM can be prevented and the risk of development of diabetes can be decreased by 31%-58%through pharmacological interventions.However,researches above uncover some problems to extent,such as the compliance of patients,the side-effects and high price of the drugs,the high cost of diabetes prevention and so on.Finding more economical,little side-effect and more effective drugs to prevent diabetes from occurring in susceptible populations or individuals become indispensable in pharmacological interventions.
The mechanism of type 2 diabetes mellitus has not been acknowledged yet. Insulin resistance(IR) andβ-cell dysfunction are the most two important reasons for the onset of type 2 diabetes mellitus.IR plays a key role in the onset of T2DM and run through the whole process of diabetes mellitus.So it is important to improve IR for prevention and therapy of T2DM.
IR is a pathophysiologic condition that the reactivity of organ to insulin is decreased.It is the congenerous risk of many clinical diseases(such as hypertension and atherosclerosis),especially in endocrine and metabolism diseases.IR is manifested by decreased insulin-stimulated glucose transport and metabolism in adipocytes and skeletal muscle and by impaired suppression of hepatic glucose output.
Nowadays,inflammation plays an important part in the mechanism of T2DM more than ever before.As the initial factor of inflammation,NF-κB is the hotspot in recent studies.We want to know if there is relationship between inflammation and IR in the pathogenesis of T2DM.IKKβwas thought to be the bridge factor between inflammation and Iron one hand,IKKβis crucially important for the activation of NF-κB which is the key factor in the startup of inflammation;on the other hand,as serine-tyrosine kinases,IKKβcan cause the serine phosphorylation of insulin receptor and insulin receptor substrate,which can block the normal insulin signal,as a result, IR will happen.Threrfore,it is necessary for us to investigate the IKK/NF-κB pathway in the mechanism of T2DM.Furthmore,we can find the new target for the prevention and therapy of T2DM.
With the development of combination of TCM with western medicine about treatment of diabetes,Chinese herbs and their active component have come into spotlight.It is feasible to find effective drug to prevent diabetes.In recent years, many scholars have carried out researches in prevention of diabetes with Chinese herbs and found Liuwei Dihuang Pills can regulate glucose metabolism and immune function,anti-damage,stabilize membrance,etc.As well as increase glycogen, decrease the activity of G-6-PDH,decrease the blood glycose,TG,FFA and anti-oxidation.These researches show that Liuwei Dihuang Pills can decrease blood glucose,ameliorate lipid metabolism,improve hyperinsulinsm and insulin resistance.In recent years,Liuwei Dihuang Pills could inhibit the expression of inflammatory mediators independent with reducing glucose.We think Liuwei Dihuang Pills may have some effects on prevention of T2DM.,which is achieved by intervening the IKK/NF-κB pathway.
OLETF(Otsuka Long- Evans Tokushima Fatty) rats,a spontaneously diabetic rat with polyuria,polydispia,and mild obesity,were used in this study to observe the effects of Liuwei Dihuang Pills on incidence rate of diabetes,and explore the probable mechanism,such as the mutual effect of inflammation and insulin resistance in order to provide theoretical and experimental proofs.See the following three parts:
Part 1 Effects of Liuwei Dihuang Pills on insulin resistance in OLETF rats OBJECTIVE:
To study the effects of Liuwei Dihuang Pills on insulin resistance in OLETF rats, and observe the influence on plasma adiponectin level and the expression level of adiponectin mRNA in adipose tissue of OLETF rats.Other indexes related to insulin sensitivity were measured to identify the effects of Liuwei Dihuang Pills and to provide beneficial evidence of Liuwei Dihuang Pills on prevention of diabetes mellitus.
METHODS:
1.OLETF rats,spontaneous NIDDM model rats,with their lean nondiabetic counterparts,Long-Evans Tokushima Otsuka(LETO) rats,were supplied at 4 week of age.Forty male OLETF rats were randomly divided into two groups,Liuwei Dihuang Pills treated group(Group Liuwei ) and the control group(Group Control). Ten male LETO rats were in normal control group(Group LETO).Rats of Group Liuwei were given Liuwei Dihuang Pills(2.4mg·kg~(-1)d~(-1)) by intragastric administration from 8 week of age on.Except for Group Liuwei,all rats were given distilled water by intragastric administration.Fasting blood glucose,serum triglyceride,serum cholesterol,serum FFA,plasma insulin and plasma adiponectin concentration were determined.
2.The adiponectin mRNA expression level in adipose tissue was tested by RT-PCR.
3.Insulin sensitivity index(ISI) was calculated according to the fasting blood glucose and fasting plasma insulin concentration.
4.The correlation between plasma adiponectin level and other related indexes were analyzed.
RESULTS:
1.Compared with Group LETO,rats of Group Control had significantly higher fasting plasma insulin concentration,serum lipid and lower ISI,but the FBG had not obvious change.
2.Rats of Group Control had significantly lower serum adiponectin and the expression level of adiponectin mRNA in adipose tissue decreased compared with rats of Group LETO.
3.Compared with Group LETO,Group Liuwei had lower plasma insulin concentration,serum lipid and higher ISI,but the FBG had not obvious change.
4.The plasma adiponectin concentration and the expression level of adiponectin mRNA in adipose tissue in Group Liuwei was obvious higher than those of Group Control.
5.There was a positive correlation between plasma adiponectin concentration and ISI,while there was a negative correlation between plasma adiponectin level and fasting plasma insulin,serum TG,FFA,CHOL.
CONCLUSIONS:
1.Plasma adiponectin level is positively correlated to insulin sensitivity in OLETF rats.
2.The level of adiponectin is negatively correlated to fasting plasma insulin,serum FFA and triglyceride.
3.Liuwei Dihuang Pills can increase the plasma level of adiponectin and the expression level of adiponectin mRNA in adipose tissue.
4.Liuwei Dihuang Pills can decrease hyperlipemia and improve insulin resistance.
Part 2 Effects of Linwei Dihuang Pills on the pathology of livers and pancreas in OLETF rats
OBJECTIVE:
To study the effects of Liuwei Dihuang Pills on the pathology of livers and pancreas in OLETF(Otsuka Long-Evans Tokushima Fatty) rats and explore the probability of Liuwei Dihuang Pills in preventing diabetes in laboratory animals.
METHODS:
1.The laboratory animal,drug and method are same as Part 1.
2.Rats were sacrificed at the age of 8,32 and 40 week.Pancreas and livers were removed after rats were sacrificed and stored in formaldehydum polymerisatum.
3.The pancreatic and hepatic tissues were stained with hemotoxylin and erosin,PAS and Masson to observe the morphological change.
RESULTS:
1.The structure of islets in age 8 week is not different from each other.Hemotoxylin and erosin staining disclosed severe fibrosis in pancreatic islet of control group in 32 week of age and it is more severer in 40 week of age which featured abnormal round appearance and disorganised islets with atrophy islet observed.There were relatively normal round islet in Liuewi group and there were not serious atrophy islets.
2.The structure of hepatic cells in age 8 week is not different from each other. Granules were found in the control group and Liuwei group of the 32 and 40 week-old age.
3.Through PAS staining:glycogen degeneration was found in the control group and Liuwei group of the 32 and 40 week-old age.The former ones were severer than the latter ones.
4.Through Masson staining:there is no fibrosis found in livers of the LETO group, control group and Liuwei group of the 40 week-old age.
CONCLUSIONS:
1.With the development of Diabetes mellitus,there are more and more fatty degeneration,and glycogen degeneration in livers in OLETF rats,and severe fibrosis in pancreatic islet.
2.Liuwei Dihuang Pills can prevent the pancreatic and hepatic tissues from degeneration in OLETF rats.
Part 3 Primary research of IKK/NF-κB pathway in the mechanisms of Liuwei Dihuang Pills on prevention of diabetes mellitus in OLETF rats
OBJECTIVE:
To study the effects of Liuwei Dihuang Pills on IKK/NF-κB pathway in OLETF rats,and observe the influence on IKKβand NF-κB level in hepatic and pancreatic tissues of OLETF rats.Meanwhile,measure P-IRS-1 values as an index of insulin resistance.All the indexes were measured to identify the effects of Liuwei Dihuang Pills and to provide beneficial evidence of Liuwei Dihuang Pills on prevention of T2DM.
METHODS:
1.The laboratory animal,drug and method are same as Part 1.
2.Livers and pancreas were removed from the liquid nitrogen.The expression of IKKβand P-IRS-1 in livers and pancreas were determined by fluoroimmunoassay, then use subsequent image analysis to present the IKKβand P-IRS-1 values with Positive Unit.
3.The hepatic and pancreatic paraffin section were stained with the method of immunohistochemistry to observe the change of NF-κB,then use subsequent image analysis to present the NF-κB values with Positive Unit.
4.The correlation between IKKβlevel、NF-κB level and P-IRS-1 value were analyzed.
RESULTS:
1.In the livers,IKKβlevels in each group increased with week age,and significant difference were found(P＜0.001).IKKβlevels in Control group were significant higher than in LETO group from 8 week-old(P＜0.001).The values of IKKβin Liuwei group were between the two groups in the 32 week-old and 40 week-old,and significant difference were found in 40 week-old(P＜0.001).
2.In the pancreas,IKKβlevels in each group increased with week age,and no significant difference were found in LETO group and Liuwei group between the 32 and 40 week-old(P=0.414 and 0.390).IKKβlevels in Control group were significant higher than in LETO group from 8 week-old(P=0.002).
3.In the livers,NF-κB levels in LETO group and Liuwei group increased with week age(P＜0.05).NF-κB levels in Control group were obvious higher than in LETO group from 8 week-old(P=0.002).The values of NF-κB in Liuwei group were between the two groups in the 32 week-old and 40 week-old(P＜0.001).The values of NF-κB among groups has significant difference(P＜0.001).
4.In the pancreas,NF-κB levels in LETO group and control group increased with week age(P＜0.01),while in Liuwei group had the same trend without significant difference.NF-κB levels in Control group were obvious higher than in LETO group from 8 week-old(P=0.005).The values of NF-κB in Liuwei group were between the two groups in the 32 week-old and 40 week-old(P＜0.008),but no significant difference were found between the control group and Liuwei group(P＞0.060).
5.In the livers,P-IRS-1 levels in LETO group increased with week age(P=0.590), while decreased in control(P=0.001) and Liuwei group(P=0.748).P-IRS-1 levels in LETO group were obvious higher than in control group from 8 week-old(P=0.002). The values of P-IRS-1 in Liuwei group were between the two groups in the 32 week-old and 40 week-old(P＜0.01).The values of P-IRS-1 among groups has significant difference(P＜0.01).
6.In the pancreas,P-IRS-1 levels in LETO group in age 32-week old was the highest age(P＜0.001),while in the other two groups no significant difference were found with week age(P＞0.05).P-IRS-1 levels in LETO group were obvious higher than in control group from 8 week-old(P＜0.001).The values of P-IRS-1 in Liuwei group were between the two groups in the 32 week-old and 40 week-old(P＜0.001).The values of P-IRS-1 among groups has significant difference(P＜0.001 ).
7.In the livers,there was a highly positive correlation between IKKβlevel and NF-κB level,while there was a negative correlation between IKKβlevel and P-IRS-1 value、NF-κB level and P- IRS-1 value.
8.In the pancreas,there was a highly positive correlation between IKKβlevel and NF-κB level,while there was a negative correlation between IKKβlevel and P-IRS-1 value、NF-κB level and P-IRS-1 value.
CONCLUSIONS:
1.Liuwei Dihuang Pills can decrease the IKKβlevel、NF-κB level to lighten the damage in the livers and pancreas which are important insulin resistant organs in OLETF rats.
2.Liuwei Dihuang Pills can increase the P-IRS-1 level in the livers and pancreas which means to improve IR.
3.There was a highly positive correlation between IKKβlevel and NF-κB level, while there was a negative correlation between IKKβlevel and P-IRS-1 value,which can prove the bridge role IKKβin the inflammation and IR.
4.Decreasing IKKβmay be one of the mechanisms of Liuwei Dihuang Pills' preventing onset of T2DM.
随着社会经济的发展和人们生活方式的改变,糖尿病已经成为继心血管疾病和肿瘤之后的第三大严重危害人类健康的慢性非传染性疾病,其患病率在全球范围内呈逐年增高的趋势,给个人和社会带来巨大的经济负担。目前,临床上仍然缺乏有效的手段来控制糖尿病及其并发症的发生与发展,预防糖尿病的发生,并从根本上消除糖尿病带来的危害,成为最受人们关注的问题之一。美国糖尿病预防试验(DPP)和STOP-NIDDM等循证医学研究表明:占全部糖尿病患者90%以上的2型糖尿病是可以预防的,采取干预措施可以使2型糖尿病发病危险降低31%～58%。然而,目前的预防手段都不同程度面临着干预药物毒副反应大、药物价格昂贵、患者依从性差等明显问题,因此,寻找更为有效、经济、不良反应小的预防糖尿病药物成为目前药物预防高危人群糖尿病发病研究的热点。
2型糖尿病的发病机制尚未完全阐明,目前认为胰岛素抵抗和胰岛β细胞功能受损是2型糖尿病最重要的两个发病因素,其中胰岛素抵抗在2型糖尿病的发病过程中有可能起始动作用并贯穿该病的全过程。因此,改善胰岛素抵抗对于预防和治疗2型糖尿病至关重要。
胰岛素抵抗是许多临床疾病(如高血压、动脉粥样硬化等),尤其是常见的内分泌代谢性疾病的共同危险因素,已经成为许多学科共同感兴趣的研究热点。胰岛素抵抗是亚细胞、细胞、组织或机体的一种病理生理状态,当机体出现胰岛素抵抗时,胰岛素刺激的葡萄糖利用减少,同时不能有效抑制肝糖原分解输出葡萄糖,从而导致血糖升高并最终导致糖尿病的出现。随着对胰岛素抵抗研究的深入,人们发现许多激素和细胞因子,尤其是脂肪细胞分泌的多个细胞因子,在胰岛素抵抗的发病中起着重要作用。脂联素是脂肪组织分泌的细胞因子之一,与能量代谢、胰岛素抵抗的关系非常密切。研究发现,血浆脂联素的浓度与身体脂肪构成比、腰臀比例、空腹胰岛素浓度以及餐后2h葡萄糖浓度呈负相关,与胰岛素敏感性呈正相关,血浆脂联素水平的降低在肥胖早期已经存在,在发生2型糖尿病后继续降低,提示血浆脂联素水平的降低与胰岛素抵抗有着密切的联系。脂联素有望成为一种胰岛素敏感性的指标,甚至可能成为一种治疗胰岛素抵抗和糖尿病的有效药物。
炎症及炎症介质在2型糖尿病发病机制中的作用也日益引起学术界的关注。其中,核因子-κB(Nuclear.factor-κB,NF-κB)作为炎症启动、调节的关键核因子,是研究的热点。但是,NF-κB如何调控炎症继而导致2型糖尿病的发生的具体机制,以及炎症与胰岛素抵抗在2型糖尿病发病中的相互关系目前尚未明确。为更好地阐明2型糖尿病的发病机制,我们有必要探讨炎症与胰岛素抵抗的关系。目前,国内外已有文献报道,NF-κB抑制物激酶((IκB kinase,IKK)是联系炎症与胰岛素抵抗的重要纽带。生理情况下,NF-κB与其抑制物IκB(Inhibitor of NF-κB)结合,以无活性的形式存在于细胞浆内,一旦IκB激酶IKK被肿瘤坏死因子(Tumor necrosis factor-α,TNF-α)等炎症因子激活后,使IκB磷酸化并与NF-κB解离,解除抑制的NF-κB进入细胞核内,调节一系列炎症反应。IKK既是调节炎症的重要因素,同时IKK又是胰岛素受体和胰岛素受体底物(Insulin receptor substrate,IRS)的丝氨酸磷酸化激酶,它可使IRS 307位的丝氨酸磷酸化,导致正常的酪氨酸磷酸化受抑制,并减弱胰岛素受体与IRS的结合以及终止胰岛素信号向磷脂酰肌醇-3激酶(Phosphatidylinositol-3 kinase,PI3-K)传递;另外,IRS的丝氨酸磷酸化还可以增加IRS的降解,并使IRS成为胰岛素受体激酶的抑制物。综上所述,IKK是将炎症和IR联系起来的重要枢纽。因此,在对糖尿病干预的研究中,探讨干预药物对IKK/NF-κB通路的影响,不仅对阐明胰岛素抵抗和炎症在2型糖尿病的发病机制中的作用有着重要意义,而且对于干预药物的作用机制的阐明也非常必要。
近年来,随着中西医结合治疗糖尿病研究的不断进展,中药复方制剂及其活性成分在治疗糖尿病方面的作用得到越来越多的重视。我们在前期研究中发现,六味地黄丸能够延缓糖尿病动物模型OLETF(Otsuka Long-Evans TokushimaFatty)鼠的餐后高血糖的出现,降低糖尿病发病率,对糖尿病的预防具有重要的作用。国内外研究显示,六味地黄丸具有调节糖代谢、增加肝糖原含量、抗炎和抗氧化作用。因此我们推测,六味地黄丸可能通过影响炎症的有关通路,改善胰岛素抵抗,从而预防2型糖尿病的发病。故本实验以IKK/NF-κB通路为切入点,探讨六味地黄丸对糖尿病大鼠胰岛素敏感器官中IKK的干预作用及其对2型糖尿病的预防作用机制。
第1部分六味地黄丸对OLETF鼠胰岛素抵抗的影响
【目的】
探讨六味地黄丸能否改善OLETF鼠的胰岛素抵抗,观察六味地黄丸对OLETF鼠血浆脂联素水平、脂肪组织中脂联素mRNA表达量以及胰岛素敏感性的影响。
【方法】
1.以自发性2型糖尿病大鼠模型OLETF鼠和其同系非糖尿病对照鼠LETO鼠为实验对象。大鼠皆为雄性,共分三组。OLETF鼠干预组(干预组)和OLETF鼠对照组(对照组),各20只;LETO鼠10只作为正常对照组(LETO组)。从8周龄开始,干预组予六味地黄丸溶液灌胃(2.4g·kg~(-1)d~(-1)),其余两组以等量蒸馏水灌胃。分别于8周龄、32周龄和40周龄时随机处死大鼠,采集血标本,分离大鼠胰腺及肝脏组织,置于液氮中保存备用;
2.检测空腹血清葡萄糖、游离脂肪酸、甘油三酯、胆固醇、血浆胰岛素和脂联素水平;
3.采用RT-PCR,检测脂肪组织中脂联素mRNA的表达;
4.计算胰岛素敏感指数(ISI);
5.分析血浆脂联素水平与各相关指标的相关性。
【结果】
1.与LETO组相比,对照组除空腹血糖升高不显著外,空腹血浆胰岛素、血清甘油三酯、胆固醇均显著升高,胰岛素敏感指数显著降低;
2.对照组血浆脂联素水平和脂肪组织脂联素mRNA表达量与LETO组相比显著降低;
3.干预组空腹血糖与对照组相比无明显变化,但空腹胰岛素和血清甘油三酯、胆固醇均显著降低,胰岛素敏感指数显著增高;
4.与对照组相比,干预组的血浆脂联素水平和脂肪组织脂联素mRNA表达量均明显增加;
5.对照组血浆脂联素与胰岛素敏感指数呈正相关,而与空腹胰岛素、甘油三酯、游离脂肪酸、胆固醇呈负相关。
【结论】
1.OLETF鼠脂联素水平与胰岛素敏感性呈正相关;
2.OLETF鼠脂联素水平与游离脂肪酸、甘油三酯水平呈负相关;
3.六味地黄丸能明显增加OLETF鼠脂肪组织脂联素mRNA表达水平,升高血浆脂联素水平;
4.六味地黄丸能有效调节血脂异常和改善胰岛素抵抗。
第2部分六味地黄丸对OLETF鼠肝脏及胰腺的形态学影响
【目的】
通过观察六味地黄丸对OLETF鼠肝脏及胰腺的形态学影响,探讨六味地黄丸对OLETF鼠肝脏和胰腺组织是否具有保护作用。
【方法】
1.实验动物、分组和干预措施同第1部分;
2.从液氮中取出已分离的大鼠胰腺及肝脏组织后,置于10%多聚甲醛中固定24小时,包埋蜡块;
3.石蜡切片机间隔4um连续切片,相邻切片分别用标准HE染色观察大鼠肝脏及胰腺组织学改变;
4.特殊染色:根据HE结果,对对照组及干预组大鼠肝脏及胰腺以冰冻切片进行MASSON染色及PAS染色,以进一步明确病变性质。
【结果】
1.8周龄时,各组大鼠胰岛结构无明显差别。32周龄,干预组和对照组胰岛明显比LETO组肥大,并有纤维组织长入,对照组大鼠胰岛纤维化更加明显,胰岛结构排列紊乱。40周龄,对照组大鼠胰岛形状更不规则,纤维组织大量增生,多数胰岛失去正常结构,胰岛明显萎缩。干预组大鼠胰岛基本保持正常结构,无明显肥大或萎缩;
2.8周龄时,各组大鼠肝脏结构亦无明显差别。32周龄,对照组及干预组大鼠肝细胞均出现明显空泡样变性,而LETO组大鼠肝小叶结构依然整齐,肝细胞以中央静脉为中心排列成索状。40周龄,对照组大鼠肝细胞空泡样变性更加明显,干预组较32周时改善,可见轻度肝索排列紊乱,部分肝细胞空泡化;
3.8周龄时,各组大鼠肝脏PAS染色无明显差别,肝细胞中未见明确的红色颗粒;
32周龄,PAS染色对照组及干预组肝细胞的胞质及胞核均可见到的红色颗粒;
40周龄,对照组肝细胞中红色颗粒更加丰富,而干预组肝细胞中红色颗粒明显减少;
4.各组各周龄大鼠肝脏未见到纤维组织增生。
【结论】
1.六味地黄丸有助于保持大鼠肝脏及胰腺的正常结构;
2.随着糖尿病病程进展,OLETE大鼠肝脏的脂肪浸润、糖原累积及胰腺纤维变性呈渐进性加重;
3.六味地黄丸能够通过保护OLETF大鼠胰岛及肝脏的正常形态结构,达到维持胰岛素敏感器官胰腺和肝脏的生理功能作用。
第3部分六味地黄丸对OLETF鼠IKK/NF-κB通路的影响
【目的】
观察六味地黄丸对OLETF鼠IKK/NF-κB通路的影响,探讨六味地黄丸是否可以通过抑制IKK、NF-κB的表达而起到改善OLETF鼠胰岛素抵抗的作用。
【方法】
1.实验动物、分组和干预措施同第1部分;
2.免疫荧光染色检测IKKβ在各组大鼠胰腺组织中的表达情况;
3.免疫荧光染色检测IKKβ在各组大鼠肝脏组织中的表达情况;
4.免疫荧光染色法检测各组大鼠胰腺组织中P-IRS-1的表达情况;
5.免疫荧光染色法检测各组大鼠肝脏组织中P-IRS-1的表达情况;
6.免疫组化法检测NF-κB在各组大鼠肝脏及胰腺的表达情况,用图像分析系统分析结果;
7.分析IKKβ、NF-κB及P-IRS-1的相关性;
8.实验数据计量资料以均数±标准差((?)±s)。采用SPSS10.0软件进行统计分析,两样本间计量资料的比较采用t检验。多组间资料比较采用单因素方差分析(ANOVA)检验。各因子之间的双变量相关性采用等级相关系数(Spearman秩相关系数)的非参数方法。
【结果】
1.六味地黄丸对肝脏中IKKβ表达的影响
各组IKKβ的表达都随周龄延长而增加,且这种差异有统计学意义。8周龄时,对照组IKKβ表达已高于LETO组(P＜0.001)。32周龄时,对照组IKKβ表达显著高于LETO组和干预组(P＜0.001);与LETO组相比,干预组IKKβ表达情况无显著差别(P＞0.05)。40周龄时,IKKβ表达仍然是在对照组最高,干预组其次,LETO组最低,差异有统计学意义(P＜0.001)。
2.六味地黄丸对胰腺中IKKβ表达的影响
各组IKKβ的表达都随周龄延长而逐渐增加,但在LETO组和对照组32周龄与40周龄时的差异无统计学意义(P分别为0.414和0.390)。8周龄时,对照组IKKβ蛋白表达高于LETO组,差异有统计学意义(P＜0.05)。32周龄时,IKKβ蛋白的表达在对照组最高,干预组其次,LETO组最低,差异有统计学意义(P＜0.05)。40周龄时,各组IKKβ的表达都有不同程度升高,IKKβ表达依然是对照组最高,干预组其次,LETO组最低,且各组间的差异均有统计学意义(P＜0.001)。
3.六味地黄丸对大鼠肝脏NF-κβ表达的影响
同周龄各组间比较,8周龄时,对照组NF-κβ的表达显著高于LETO组(P＜0.05)。32周龄时,与LETO组相比,对照组NF-κβ的表达水平显著升高,干预组次之,各组间差别有统计学意义(P＜0.001)。40周龄时,对照组NF-κB的表达进一步升高,显著高于干预组和LETO组,干预组仍然介于对照组和LETO组之间,各组间差异有统计学意义(P＜0.001)。
4.六味地黄丸对大鼠胰腺NF-κβ表达的影响
同周龄各组间比较,8周龄时,对照组胰腺中NF-κB的表达就开始显著高于LETO组(P＜0.05):32周龄时,与LETO组大鼠相比,对照组和干预组NF-κB的表达量均显著增高(P＜0.05);干预组低于对照组,但差异无统计学意义(P=0.082)。40周龄时,对照组和干预组胰腺中NF-κB表达量仍然都显著低于LETO组(P＜0.05),干预组胰腺中NF-κB表达量仍低于对照组,但差异无统计学意义(P=0.060)。
5.六味地黄丸对大鼠肝脏P-IRS-1表达的影响
同周龄各组间比较,8周龄时,LETO组P-IRS-1的表达即显著高于对照组(P＜0.05)。32周龄时,P-IRS-1在肝脏的表达在LETO组最高,干预组次之,对照组最低,差异显著(P＜0.001)。40周龄时,LETO组P-IRS-1的表达进一步升高,显著高于干预组和对照组,对照组则呈下降趋势,仍在三组中表达最低,各组间差异有统计学意义(P＜0.001)。
6.六味地黄丸对大鼠胰腺P-IRS-1表达的影响
8周龄时,LETO组P-IRS-1的在胰腺中的表达显著高于对照组(P＜0.001)。32周龄时,对照组显著低于LETO组和干预组,干预组低于LETO组,各组间差异具有统计学意义(P＜0.001)。40周龄时,P-IRS-1在LETO组最高,干预组次之,对照组最低,差异有统计学意义(P＜0.001)。
7.肝脏中IKKβ、NF-κB与P-IRS-1的相关性分析
相同周龄时,肝脏中IKKβ、NF-κB与P-IRS-1的相关分析结果:8周龄,IKKβ与NF-κB的表达呈正相关,相关性显著(r_s=0.909,P＜0.001);IKKβ、NF-κB均与P-IRS-1呈负相关,相关性不显著(P=0.208)。32周龄时,IKKβ与NF-κB的表达呈显著正相关(r_s=0.884,P＜0.001),IKKβ与P-IRS-1呈负相关(r_s=-0.75,P=0.02),NF-κB与P-IRS-1呈显著负相关(r_s=-0.867,P=0.002)。40周龄时,IKKβ与NF-κB的仍呈显著正相关(r_s=0.913,P＜0.001),且均与P-IRS-1显著负相关(P＜0.001)。
8.胰腺中IKKβ、NF-κB与P-IRS-1的相关性分析
相同周龄时,胰腺中IKKβ、NF-κB与P-IRS-1的相关分析结果:8周龄,IKKβ与NF-κB的表达呈显著正相关(r_s=0.951,P＜0.001),均与P-IRS-1呈负相关,相关性不显著(P=0.198)。32周龄时,IKKβ与NF-κB的呈正相关,相关性显著(r_s=0.903,P＜0.001),IKKβ与P-IRS-1呈显著负相关(r_s=-0.828,P＜0.05),NF-κB与P-IRS-1呈负相关(r_s=-0.75,P＜0.05)。40周龄时,IKKβ与NF-κB的仍呈显著正相关(r_s=0.901,P＜0.001),且均与P-IRS-1显著负相关(P＜0.001)。
【结论】
1.六味地黄丸能降低OLETF鼠IKKβ、NF-κB的表达,减轻炎症反应对肝脏和胰腺的损害;
2.六昧地黄丸能增加OLETF鼠肝脏及胰腺组织中P-IRS-1的表达水平,改善胰岛素抵抗;
3.OLETF鼠肝脏中IKKβ与NF-κB的表达在各周龄时均呈高度正相关(P＜0.001);与IRS-1表达呈负相关,其中32周龄和40周龄时相关性有统计学意义(P＜0.02)。在胰腺组织中有同样的趋势。提示了IKK在炎症与胰岛素抵抗中具有桥梁作用;
4.六味地黄丸通过降低IKKβ的表达,达到改善胰岛素抵抗和抗炎作用,可能是其预防2型糖尿病发病的基本机制之一。
BACKGROUD:
With the development of economy and alteration of life style,the incidence rate of diabetes mellitus roars up year by year.Diabetes mellitus has become the third disease following the cardiovascular diseases and tumor which damage people's health seriously.It brings enormous economic burden for individual and society.With the knowledge of insulin,the development and application of drugs stimulating secretion of insulin and insulin-sensitizing drugs etc,more methods are available in the treatment of diabetes.However,although progress has been made in the treatment of diabetes,we still can't control the development of diabetes and its complications. So preventing diabetes becomes one of the most important issues in order to solve the problem radically.Whether diabetes can be prevented and what is the most economical,safe,effective way to prevent diabetes becoming a must to tackle this problem.The evidence-based research of DPP and stop-NIDDM showed T2DM which accounts for 90%of all patients with DM can be prevented and the risk of development of diabetes can be decreased by 31%-58%through pharmacological interventions.However,researches above uncover some problems to extent,such as the compliance of patients,the side-effects and high price of the drugs,the high cost of diabetes prevention and so on.Finding more economical,little side-effect and more effective drugs to prevent diabetes from occurring in susceptible populations or individuals become indispensable in pharmacological interventions.
The mechanism of type 2 diabetes mellitus has not been acknowledged yet. Insulin resistance(IR) andβ-cell dysfunction are the most two important reasons for the onset of type 2 diabetes mellitus.IR plays a key role in the onset of T2DM and run through the whole process of diabetes mellitus.So it is important to improve IR for prevention and therapy of T2DM.
IR is a pathophysiologic condition that the reactivity of organ to insulin is decreased.It is the congenerous risk of many clinical diseases(such as hypertension and atherosclerosis),especially in endocrine and metabolism diseases.IR is manifested by decreased insulin-stimulated glucose transport and metabolism in adipocytes and skeletal muscle and by impaired suppression of hepatic glucose output.
Nowadays,inflammation plays an important part in the mechanism of T2DM more than ever before.As the initial factor of inflammation,NF-κB is the hotspot in recent studies.We want to know if there is relationship between inflammation and IR in the pathogenesis of T2DM.IKKβwas thought to be the bridge factor between inflammation and Iron one hand,IKKβis crucially important for the activation of NF-κB which is the key factor in the startup of inflammation;on the other hand,as serine-tyrosine kinases,IKKβcan cause the serine phosphorylation of insulin receptor and insulin receptor substrate,which can block the normal insulin signal,as a result, IR will happen.Threrfore,it is necessary for us to investigate the IKK/NF-κB pathway in the mechanism of T2DM.Furthmore,we can find the new target for the prevention and therapy of T2DM.
With the development of combination of TCM with western medicine about treatment of diabetes,Chinese herbs and their active component have come into spotlight.It is feasible to find effective drug to prevent diabetes.In recent years, many scholars have carried out researches in prevention of diabetes with Chinese herbs and found Liuwei Dihuang Pills can regulate glucose metabolism and immune function,anti-damage,stabilize membrance,etc.As well as increase glycogen, decrease the activity of G-6-PDH,decrease the blood glycose,TG,FFA and anti-oxidation.These researches show that Liuwei Dihuang Pills can decrease blood glucose,ameliorate lipid metabolism,improve hyperinsulinsm and insulin resistance.In recent years,Liuwei Dihuang Pills could inhibit the expression of inflammatory mediators independent with reducing glucose.We think Liuwei Dihuang Pills may have some effects on prevention of T2DM.,which is achieved by intervening the IKK/NF-κB pathway.
OLETF(Otsuka Long- Evans Tokushima Fatty) rats,a spontaneously diabetic rat with polyuria,polydispia,and mild obesity,were used in this study to observe the effects of Liuwei Dihuang Pills on incidence rate of diabetes,and explore the probable mechanism,such as the mutual effect of inflammation and insulin resistance in order to provide theoretical and experimental proofs.See the following three parts:
Part 1 Effects of Liuwei Dihuang Pills on insulin resistance in OLETF rats OBJECTIVE:
To study the effects of Liuwei Dihuang Pills on insulin resistance in OLETF rats, and observe the influence on plasma adiponectin level and the expression level of adiponectin mRNA in adipose tissue of OLETF rats.Other indexes related to insulin sensitivity were measured to identify the effects of Liuwei Dihuang Pills and to provide beneficial evidence of Liuwei Dihuang Pills on prevention of diabetes mellitus.
METHODS:
1.OLETF rats,spontaneous NIDDM model rats,with their lean nondiabetic counterparts,Long-Evans Tokushima Otsuka(LETO) rats,were supplied at 4 week of age.Forty male OLETF rats were randomly divided into two groups,Liuwei Dihuang Pills treated group(Group Liuwei ) and the control group(Group Control). Ten male LETO rats were in normal control group(Group LETO).Rats of Group Liuwei were given Liuwei Dihuang Pills(2.4mg·kg~(-1)d~(-1)) by intragastric administration from 8 week of age on.Except for Group Liuwei,all rats were given distilled water by intragastric administration.Fasting blood glucose,serum triglyceride,serum cholesterol,serum FFA,plasma insulin and plasma adiponectin concentration were determined.
2.The adiponectin mRNA expression level in adipose tissue was tested by RT-PCR.
3.Insulin sensitivity index(ISI) was calculated according to the fasting blood glucose and fasting plasma insulin concentration.
4.The correlation between plasma adiponectin level and other related indexes were analyzed.
RESULTS:
1.Compared with Group LETO,rats of Group Control had significantly higher fasting plasma insulin concentration,serum lipid and lower ISI,but the FBG had not obvious change.
2.Rats of Group Control had significantly lower serum adiponectin and the expression level of adiponectin mRNA in adipose tissue decreased compared with rats of Group LETO.
3.Compared with Group LETO,Group Liuwei had lower plasma insulin concentration,serum lipid and higher ISI,but the FBG had not obvious change.
4.The plasma adiponectin concentration and the expression level of adiponectin mRNA in adipose tissue in Group Liuwei was obvious higher than those of Group Control.
5.There was a positive correlation between plasma adiponectin concentration and ISI,while there was a negative correlation between plasma adiponectin level and fasting plasma insulin,serum TG,FFA,CHOL.
CONCLUSIONS:
1.Plasma adiponectin level is positively correlated to insulin sensitivity in OLETF rats.
2.The level of adiponectin is negatively correlated to fasting plasma insulin,serum FFA and triglyceride.
3.Liuwei Dihuang Pills can increase the plasma level of adiponectin and the expression level of adiponectin mRNA in adipose tissue.
4.Liuwei Dihuang Pills can decrease hyperlipemia and improve insulin resistance.
Part 2 Effects of Linwei Dihuang Pills on the pathology of livers and pancreas in OLETF rats
OBJECTIVE:
To study the effects of Liuwei Dihuang Pills on the pathology of livers and pancreas in OLETF(Otsuka Long-Evans Tokushima Fatty) rats and explore the probability of Liuwei Dihuang Pills in preventing diabetes in laboratory animals.
METHODS:
1.The laboratory animal,drug and method are same as Part 1.
2.Rats were sacrificed at the age of 8,32 and 40 week.Pancreas and livers were removed after rats were sacrificed and stored in formaldehydum polymerisatum.
3.The pancreatic and hepatic tissues were stained with hemotoxylin and erosin,PAS and Masson to observe the morphological change.
RESULTS:
1.The structure of islets in age 8 week is not different from each other.Hemotoxylin and erosin staining disclosed severe fibrosis in pancreatic islet of control group in 32 week of age and it is more severer in 40 week of age which featured abnormal round appearance and disorganised islets with atrophy islet observed.There were relatively normal round islet in Liuewi group and there were not serious atrophy islets.
2.The structure of hepatic cells in age 8 week is not different from each other. Granules were found in the control group and Liuwei group of the 32 and 40 week-old age.
3.Through PAS staining:glycogen degeneration was found in the control group and Liuwei group of the 32 and 40 week-old age.The former ones were severer than the latter ones.
4.Through Masson staining:there is no fibrosis found in livers of the LETO group, control group and Liuwei group of the 40 week-old age.
CONCLUSIONS:
1.With the development of Diabetes mellitus,there are more and more fatty degeneration,and glycogen degeneration in livers in OLETF rats,and severe fibrosis in pancreatic islet.
2.Liuwei Dihuang Pills can prevent the pancreatic and hepatic tissues from degeneration in OLETF rats.
Part 3 Primary research of IKK/NF-κB pathway in the mechanisms of Liuwei Dihuang Pills on prevention of diabetes mellitus in OLETF rats
OBJECTIVE:
To study the effects of Liuwei Dihuang Pills on IKK/NF-κB pathway in OLETF rats,and observe the influence on IKKβand NF-κB level in hepatic and pancreatic tissues of OLETF rats.Meanwhile,measure P-IRS-1 values as an index of insulin resistance.All the indexes were measured to identify the effects of Liuwei Dihuang Pills and to provide beneficial evidence of Liuwei Dihuang Pills on prevention of T2DM.
METHODS:
1.The laboratory animal,drug and method are same as Part 1.
2.Livers and pancreas were removed from the liquid nitrogen.The expression of IKKβand P-IRS-1 in livers and pancreas were determined by fluoroimmunoassay, then use subsequent image analysis to present the IKKβand P-IRS-1 values with Positive Unit.
3.The hepatic and pancreatic paraffin section were stained with the method of immunohistochemistry to observe the change of NF-κB,then use subsequent image analysis to present the NF-κB values with Positive Unit.
4.The correlation between IKKβlevel、NF-κB level and P-IRS-1 value were analyzed.
RESULTS:
1.In the livers,IKKβlevels in each group increased with week age,and significant difference were found(P＜0.001).IKKβlevels in Control group were significant higher than in LETO group from 8 week-old(P＜0.001).The values of IKKβin Liuwei group were between the two groups in the 32 week-old and 40 week-old,and significant difference were found in 40 week-old(P＜0.001).
2.In the pancreas,IKKβlevels in each group increased with week age,and no significant difference were found in LETO group and Liuwei group between the 32 and 40 week-old(P=0.414 and 0.390).IKKβlevels in Control group were significant higher than in LETO group from 8 week-old(P=0.002).
3.In the livers,NF-κB levels in LETO group and Liuwei group increased with week age(P＜0.05).NF-κB levels in Control group were obvious higher than in LETO group from 8 week-old(P=0.002).The values of NF-κB in Liuwei group were between the two groups in the 32 week-old and 40 week-old(P＜0.001).The values of NF-κB among groups has significant difference(P＜0.001).
4.In the pancreas,NF-κB levels in LETO group and control group increased with week age(P＜0.01),while in Liuwei group had the same trend without significant difference.NF-κB levels in Control group were obvious higher than in LETO group from 8 week-old(P=0.005).The values of NF-κB in Liuwei group were between the two groups in the 32 week-old and 40 week-old(P＜0.008),but no significant difference were found between the control group and Liuwei group(P＞0.060).
5.In the livers,P-IRS-1 levels in LETO group increased with week age(P=0.590), while decreased in control(P=0.001) and Liuwei group(P=0.748).P-IRS-1 levels in LETO group were obvious higher than in control group from 8 week-old(P=0.002). The values of P-IRS-1 in Liuwei group were between the two groups in the 32 week-old and 40 week-old(P＜0.01).The values of P-IRS-1 among groups has significant difference(P＜0.01).
6.In the pancreas,P-IRS-1 levels in LETO group in age 32-week old was the highest age(P＜0.001),while in the other two groups no significant difference were found with week age(P＞0.05).P-IRS-1 levels in LETO group were obvious higher than in control group from 8 week-old(P＜0.001).The values of P-IRS-1 in Liuwei group were between the two groups in the 32 week-old and 40 week-old(P＜0.001).The values of P-IRS-1 among groups has significant difference(P＜0.001 ).
7.In the livers,there was a highly positive correlation between IKKβlevel and NF-κB level,while there was a negative correlation between IKKβlevel and P-IRS-1 value、NF-κB level and P- IRS-1 value.
8.In the pancreas,there was a highly positive correlation between IKKβlevel and NF-κB level,while there was a negative correlation between IKKβlevel and P-IRS-1 value、NF-κB level and P-IRS-1 value.
CONCLUSIONS:
1.Liuwei Dihuang Pills can decrease the IKKβlevel、NF-κB level to lighten the damage in the livers and pancreas which are important insulin resistant organs in OLETF rats.
2.Liuwei Dihuang Pills can increase the P-IRS-1 level in the livers and pancreas which means to improve IR.
3.There was a highly positive correlation between IKKβlevel and NF-κB level, while there was a negative correlation between IKKβlevel and P-IRS-1 value,which can prove the bridge role IKKβin the inflammation and IR.
4.Decreasing IKKβmay be one of the mechanisms of Liuwei Dihuang Pills' preventing onset of T2DM.
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11.张汝学,周金黄,张永祥,等.去胸腺对大鼠糖代谢的影响及地黄寡糖对其的调节作用.中国药理学通报,2002,18(2):194-197
12.王敏.六味地黄丸合用消渴丸对糖尿病大鼠血糖及血脂代谢的试验研究.中华实用中西医杂志,2003,3(16):810-811
13.胡合营,郑军萍.糖尿病患者血中SOD含量变化与维生素E、六味地黄丸关系的临床观察,放射免疫学杂志,2001,14(3):140-141
14.Reaven GM.Role of insulin resistance in human disease.[J]Diabetes,1988,37(12):1595-1607
15.McGarry JD.Banting Lecture 2001:Dysregulation of fattyacidmetabolism in the etiology of type 2 diabetes.Diabe-tes,2002;51(1):7-18
16.Damaris Christensen.Inflammatory ideas:new thoughts about causes of diabetes.Science New,2002,162(9):136
17.Barzilay J,Freedland E.Inflammation and its association with glucose disorders and cardiovascular disease.Treaat Endocrinol,2003,2(2):85-94
18.Syed MA,Barinas-Mitchell,Pietropaolo SL,et al.Is type 2 diabetes a chronic inflammatory/autoimmune disease? Diabetes Nutr Metab,2002,15(2):68-83
19.Tak P P,Firestein GS.NF- κ B:a key role in inflammatory diseases[J].J Clin Invest,2001,107:7-11
20.Li Z W,Chu W M,Hu YL,et al.The IKK β subunit of I κ B kinase(IKK) is essential for nuclear factor κ B activation and prevention of apoptosis[J].J Exp Med,1999,189:1839-45
21.DixitV,Mak TW.NF-kappaB signaling:Many roads lead to Madrid[J].Cell,2002,111(5):615-9
22.Vermeulen L,De Wilde G,Notebaert S,et al.Regulation of the transcriptional activity of the nuclear factor-kappaB p65 subunit[J].B iochem Pharm acol,2002,64(526):963-70
23.Jijon H,Allard B,Jobin C.NF-kappaB inducing kinase activates NF-kappaB transcriptional activity independently of IkappaB kinase gamma through a p38MAPK2 dependent RelA phosphorylation path-way[J].Cell Signal,2004,16(9): 1023-32
24.Ducut Sigala JL,Bottero V,Young DB,et al.Activation of transcription factor NF-kappaB requires ELKS,an IkappaB kinase regulatory subunit[J].Science,2004,304(5679):1963-7
25.Rahman I.Regulation of nuclear factor-kappa B,activator protein21,and glutathione levels by tumor necrosis factor-alpha and dexamethasone in alveolar epithelial cells[J].Biochem Pharmacol,2000,60:1041-9
26.Romashkova J A,Makarov S S.NF-kappaB is a target of AKT in anti-apoptotic PDGF signalling[J].Nature,1999,284:316
27.Yuan M Y,Konstantopoulos N,Lee J S,et al.Reversal of obesity and diet induced insulin resistance with salicylates or targeted disruption of IKK β[J].Science,2001,293:1673-7
28.Kulkarni RN,Winnay JN,Kahn CR.Tissue-specific knockout of insulin receptor in pancreaticβ3cells creates an insulin secretory defect similar to that in type 2diabetes.Cell,1999,96:329-39
29.Leibiger IB,Leibiger B,Berggren PO.Insulin feedback action on pancreaticβ2cell function.FEBS Letters,2002,532:1-6
30.潘长玉,尹士男.胰岛素抵抗-2型糖尿病发病机制的重要因素.中华内分泌代谢杂志.2000,16(1):56-57
31.李光伟.胰岛素抵抗评估及其临床应用.中华老年多器官疾病杂志2004,3(1):11-12
32.Scherer PE,Willinas S,Fogliano M,et al.A movel serum protein similar to Clq,produced exclusively in dipocytes.BiolChem,1995,270:26746-26749
33.Hu E,Liang P,Splegelman BM.AdipoQ is a novel adipose-specific gene dysregulated in obesity.J Biol Chem 1996,271:10697-10703
34.Berg AH,Combs TP,Scherer PE.Acrp30/adiponectin:an adipocytokine regulating glucose and lipid metabolism.Trends and Clinical Metabolism,2002,13:84-99
35.Yamauchi T,Kamon J,Waki H,et al:The fat derived hormone adiponectin reverses insulin resistance associated with both lioatrophy and obesity. Nat Med, 2001,7:941-946
36. Hotta K, Funahashi T, Bodkin NL, et al. Circulating concentrations of the adipocyte protein adiponectin are decreased in parallel with reduced insulin sensitivity during the progression to type 2 diabetes in rhesus monkeys .Diabetes, 2001, 50(5): 1126-1130.
37. Kubota N, Terauchi Y, Yamauchi T, et al. Disruption of adiponectin causes insulin resistance and neointimal formation. J Biol Chem, 2002, 277: 25863-25866.
38. Bogan JS, Lodish HF: Two ompartments for insulin-stimulated exocytosis in 3T3-1 adipocytes defined by endogenous ACRP30 and GLUT4. J Cell Biol , 1999,146:609-620.
39. Smith U, Axelsen M, Carvalho E, et al. Insulin signaling and action in fat cells:associations with insulin resistance and type 2 diabetes. Ann N Y Acad Sci, 1999,892:119-126.
40. Yu JG, Javorschi S, Hevener AL, et al. The effect of thiazolidinediones on plasma adiponectin levels in normal,obese and type 2 diabetic subjects. Diabetes,2002, 51:2968-2974.
41. Shimabukuro M, Zhou YT, Levi M, et al. Fatty acid-induced beta cell apoptosis: a link between obesity and diabetes. Proc Natl Acad Sci U S A. 1998, 95(5):2498-502.
42. Perseghin G, Ghosh S, Gerow K. Metabolic defects in lean nondiabetic offspring of NIDDM parents:a cross-sectional study. Diabetes, 1997, 46: 1001-1009
43. Weyer C, Bogardus C, Mott DM, et al.The natural history of insulin secretory dysfunction and insulin resistance in the pathogenesis of type 2 diabetes mellitus [J]. J Clin Invest, 1999, 104(6):787-94
44. Kawano K, Hirashima T, Mori S, et al.OLETF rat:a new NIDDM rat strain. Diabetes Rea Clin Pract, 1994:24(supp l):S317-322
45. Curtis J, Wilson C. Preventing type 2 diabetes mellitus [J]. J Am Board Fam Pract, 2005; 18(1):37-43
46. Kawano K, Hirashima T, Mori S, et al. Spontaneous long-term hyperglycemic rat with diabetic complications. Otsuka Long-Evans Tokushima Fatty (OLETF) strain [J]. Diabetes, 1992,41(11):1422-1428
47. Sato T. Insulin resistance in skeletal muscle of the male Otsuka Long-Evans Tokushima Fatty ,a new model of NIDDM.Diabetologia,1995,38:1033-1036
48. DiBisceglia AM.What every hepatologist should know about endocrinology: obesity, diabetes, and liver disease. Gastroenterology. 2004 Feb; 126(2):604-6.
49. Nepomnyashchikh GI, Pavlenko OA, Aidagulova SV, et al. Ultrastrutural analysis of liver biopsy specimens in diabetes mellitus associated with chronic Opisthorchaiasis.Bull Exp Biol Med,2001,132:1190-4
50. Scheen AJ, Luyckx FH. Nonalcoholic steatohepatitis and insulin resistance: interface between gastroenterologists and endocrinologists. Acta Clin Belg. 2003 Mar-Apr; 58(2):81-91
51. Lee Y, Yu X, Gonzales F, et al. PPAR alpha is necessary for the lipopenic action of hyperleptinemia on white adipose and liver tissue. Proc Natl Acad Sci USA , Med Sci,2002, 19 : 11848-53
52. Sung CK, She H, Xiong S, et al. Tumor necrosis factor2alpha inhibits peroxisome proliferators-actived receptor gamma activity at a posttranslational level in hepatic stellate cells. Am J Physiol Gastrointest Liver Physiol, 2004 , 286 : 722-9
53. Neuschwander-Tetri BA, Brunt EM, Wehmeier KR, et al. Improved nonalcoholic steatohepatitis after 48 week of treatment with the PPAR- gamma ligand rosiglitazone. Hepatology. 2003 ,10 : 1008-17
54. Eun Hee Koh, Min-Seon Kim, Joong-Yeol Park, et al. Peroxisome proliferator-activated receptor (PPAR)-alpha Activation Prevents Diabetes in OLETF Rats.Diabetes,2003,52(9),2331-7
55. Castoldi G, Galimberti S, Riva C ,et al. Association between serum values of C-reactive protein and cytokine production in whole blood of type 2 diabetic patients. Clin Sci (Lond). 2007 Mar 16; [Epub ahead of print]
56. Ye J. Role of insulin in the pathogenesis of free fatty acid-induced insulin resistance in skeletal muscle. Endocr Metab Immune Disord Drug Targets. 2007 Mar; 7(1):65-74.
57. Balagopal P, Graham TE, Kahn BB, et al. Reduction of Elevated Serum Retinol Binding Protein (RBP4) in Obese Children by Lifestyle Intervention: Association with Sub-clinical Inflammation. J Clin Endocrinol Metab. 2007 Mar 6; [Epub ahead of print]
58. Alexandraki K, Piperi C, Kalofoutis C, et al. Inflammatory process in type 2 diabetes: The role of cytokines[J ]. Ann N Y Acad Sci. 2006 Nov; 1084:89-117.
59. Mita T, Watada H, Uchino H .Association of C-reactive protein with early-stage carotid atherosclerosis in Japanese patients with early-state type 2 diabetes mellitus[J ]. Endocr J. 2006 Oct; 53(5):693-8.
60. Li J , Peet GW, Pullen S S , et al. Recombinant I κ B kinase α and β are direct kinase of I κ B α [J ]. J Biol Chem, 1998, 273:30736-41
61.Kiyoshi T, Osamu T, Tohru T, et al. Limb and Skin Abnormalities in Mice Lacking IKK α [J]. Science, 1999,284:313-6
62. Yinling H,Veronipue B, Mireille D, et al.Abnormal Morphogenesis But Intact IKK Activation in Mice Lacking the IKK α Subunit of I κ B Kinase[J ]. Science, 1999,284:316-20
63. ChristopherJ.MacKenzie, Elwyn Ritchie, Andrew Paul, et al. IKKα and IKKβ function in TNFa- stimulated adhesion molecule expression in human aortic smooth muscle cells[J ]. Cell Signal, 2007, 19: 75-80
64. SE Shoelson, J Lee, M Yuan. Inflammation and the IKK β /I κ B/NF- κ B axis in obesity-and diet-induced insulin resistance[J ]. Int J of Obe, 2003, 27: S49-S52
65. Chietti G, Gbaguidi FG, Grigli S, et al.CLA-1/SR-BI is expressed in atherosclerotic lesion macrophages and regulated by activators of peroxisome proliferators-activated receptors[J ].Circulation.2000, 101(20):2411-7
66. KimJ K, Kim YJ , Fillnore J J ,et al. Prevention of fat-induced insulin resistance by salicylate [ J ]. J Clin Invest, 2001, 108:437-46
67. Hundal R S, Petersen K F, Mayerson A B, et al. Mechanism by which high2dose aspirin improves glucose metabolism in type 2 diabetes [J]. J Clin Invest, 2002, 109: 1321-6
68. Ruan H , Hacohen N , Golub T R ,et al. Tumor necrosis factor-alpha suppresses adipocyte-specific genes and activates expression of preadipocyte genes in 3T3-L1 adipocytes: nuclear factor-KappaB activation by TNF-alpha is obligatory [J]. Diabetes, 2002, 51:1319-36
69. Rossi A, Kapali P, Natoli G, et al. Anti-inflammatory cyclopentenone prostaglandins are direct inhibitors of I κ B kinase[J ].Nature,2000,403:103-8
2.《中国糖尿病防治指南》总论,2006
3.胡予,陈世耀,王吉耀.糖尿病预防治疗的费用效益分析.临床流行病学,2004,25(5):431-434
4.Swinburn BA,Metcalf PA,Ley SJ.Long-term(5-year) effects of a reduced-fat diet intervention in individuals with glucose intolerance.Diabetes Care,2001,24:619-24
5.Tuomilehto J,Lindstr(o|¨)m J,Eriksson JG,et al.Prevention of type 2 diabetes mellitus by changes in lifestyle among subjects with impaired glucose tolerance.N Engl J Med 2001;344:1343-50
6.Diabetes Prevention Program Research Group.Reduction in the incidence of type 2 diabetes with lifestyle intervention or metformin[J].N Engl J Med,2002,346:393-403
7.Lindstrom J,Peltonen M,Tuomilehto J.Lifestyle strategies for weight control:experience from the Finnish Diabetes Prevention study.Proc Nutr Soc.2005,64(1):81-8
8.Laaksonen DE,Lindstrom J,Lakka TA,et al.Physical activity in the prevention of type 2 diabetes:the Finnish Diabetes Prevention study.Diabetes,2005,54(1):158-65
9.Chiasson JL,Josse RG,Gomis R,et al.Acarbose for prevention of type 2diabetes mellitus:the STOP -NIDDM randomised trial[J].Lancet,2002,359:2072-7
10.Li G,Hu Y,Yang W,Jiang Y,et al.Effects of insulin resistance and insulin secretion on the efficacy of interventions to retard development of type 2 diabetes mellitus:the DA Qing IGT and Diabetes Study.Diabetes Res Clin Pract,2002,58:193-200
11.张汝学,周金黄,张永祥,等.去胸腺对大鼠糖代谢的影响及地黄寡糖对其的调节作用.中国药理学通报,2002,18(2):194-197
12.王敏.六味地黄丸合用消渴丸对糖尿病大鼠血糖及血脂代谢的试验研究.中华实用中西医杂志,2003,3(16):810-811
13.胡合营,郑军萍.糖尿病患者血中SOD含量变化与维生素E、六味地黄丸关系的临床观察,放射免疫学杂志,2001,14(3):140-141
14.Reaven GM.Role of insulin resistance in human disease.[J]Diabetes,1988,37(12):1595-1607
15.McGarry JD.Banting Lecture 2001:Dysregulation of fattyacidmetabolism in the etiology of type 2 diabetes.Diabe-tes,2002;51(1):7-18
16.Damaris Christensen.Inflammatory ideas:new thoughts about causes of diabetes.Science New,2002,162(9):136
17.Barzilay J,Freedland E.Inflammation and its association with glucose disorders and cardiovascular disease.Treaat Endocrinol,2003,2(2):85-94
18.Syed MA,Barinas-Mitchell,Pietropaolo SL,et al.Is type 2 diabetes a chronic inflammatory/autoimmune disease? Diabetes Nutr Metab,2002,15(2):68-83
19.Tak P P,Firestein GS.NF- κ B:a key role in inflammatory diseases[J].J Clin Invest,2001,107:7-11
20.Li Z W,Chu W M,Hu YL,et al.The IKK β subunit of I κ B kinase(IKK) is essential for nuclear factor κ B activation and prevention of apoptosis[J].J Exp Med,1999,189:1839-45
21.DixitV,Mak TW.NF-kappaB signaling:Many roads lead to Madrid[J].Cell,2002,111(5):615-9
22.Vermeulen L,De Wilde G,Notebaert S,et al.Regulation of the transcriptional activity of the nuclear factor-kappaB p65 subunit[J].B iochem Pharm acol,2002,64(526):963-70
23.Jijon H,Allard B,Jobin C.NF-kappaB inducing kinase activates NF-kappaB transcriptional activity independently of IkappaB kinase gamma through a p38MAPK2 dependent RelA phosphorylation path-way[J].Cell Signal,2004,16(9): 1023-32
24.Ducut Sigala JL,Bottero V,Young DB,et al.Activation of transcription factor NF-kappaB requires ELKS,an IkappaB kinase regulatory subunit[J].Science,2004,304(5679):1963-7
25.Rahman I.Regulation of nuclear factor-kappa B,activator protein21,and glutathione levels by tumor necrosis factor-alpha and dexamethasone in alveolar epithelial cells[J].Biochem Pharmacol,2000,60:1041-9
26.Romashkova J A,Makarov S S.NF-kappaB is a target of AKT in anti-apoptotic PDGF signalling[J].Nature,1999,284:316
27.Yuan M Y,Konstantopoulos N,Lee J S,et al.Reversal of obesity and diet induced insulin resistance with salicylates or targeted disruption of IKK β[J].Science,2001,293:1673-7
28.Kulkarni RN,Winnay JN,Kahn CR.Tissue-specific knockout of insulin receptor in pancreaticβ3cells creates an insulin secretory defect similar to that in type 2diabetes.Cell,1999,96:329-39
29.Leibiger IB,Leibiger B,Berggren PO.Insulin feedback action on pancreaticβ2cell function.FEBS Letters,2002,532:1-6
30.潘长玉,尹士男.胰岛素抵抗-2型糖尿病发病机制的重要因素.中华内分泌代谢杂志.2000,16(1):56-57
31.李光伟.胰岛素抵抗评估及其临床应用.中华老年多器官疾病杂志2004,3(1):11-12
32.Scherer PE,Willinas S,Fogliano M,et al.A movel serum protein similar to Clq,produced exclusively in dipocytes.BiolChem,1995,270:26746-26749
33.Hu E,Liang P,Splegelman BM.AdipoQ is a novel adipose-specific gene dysregulated in obesity.J Biol Chem 1996,271:10697-10703
34.Berg AH,Combs TP,Scherer PE.Acrp30/adiponectin:an adipocytokine regulating glucose and lipid metabolism.Trends and Clinical Metabolism,2002,13:84-99
35.Yamauchi T,Kamon J,Waki H,et al:The fat derived hormone adiponectin reverses insulin resistance associated with both lioatrophy and obesity. Nat Med, 2001,7:941-946
36. Hotta K, Funahashi T, Bodkin NL, et al. Circulating concentrations of the adipocyte protein adiponectin are decreased in parallel with reduced insulin sensitivity during the progression to type 2 diabetes in rhesus monkeys .Diabetes, 2001, 50(5): 1126-1130.
37. Kubota N, Terauchi Y, Yamauchi T, et al. Disruption of adiponectin causes insulin resistance and neointimal formation. J Biol Chem, 2002, 277: 25863-25866.
38. Bogan JS, Lodish HF: Two ompartments for insulin-stimulated exocytosis in 3T3-1 adipocytes defined by endogenous ACRP30 and GLUT4. J Cell Biol , 1999,146:609-620.
39. Smith U, Axelsen M, Carvalho E, et al. Insulin signaling and action in fat cells:associations with insulin resistance and type 2 diabetes. Ann N Y Acad Sci, 1999,892:119-126.
40. Yu JG, Javorschi S, Hevener AL, et al. The effect of thiazolidinediones on plasma adiponectin levels in normal,obese and type 2 diabetic subjects. Diabetes,2002, 51:2968-2974.
41. Shimabukuro M, Zhou YT, Levi M, et al. Fatty acid-induced beta cell apoptosis: a link between obesity and diabetes. Proc Natl Acad Sci U S A. 1998, 95(5):2498-502.
42. Perseghin G, Ghosh S, Gerow K. Metabolic defects in lean nondiabetic offspring of NIDDM parents:a cross-sectional study. Diabetes, 1997, 46: 1001-1009
43. Weyer C, Bogardus C, Mott DM, et al.The natural history of insulin secretory dysfunction and insulin resistance in the pathogenesis of type 2 diabetes mellitus [J]. J Clin Invest, 1999, 104(6):787-94
44. Kawano K, Hirashima T, Mori S, et al.OLETF rat:a new NIDDM rat strain. Diabetes Rea Clin Pract, 1994:24(supp l):S317-322
45. Curtis J, Wilson C. Preventing type 2 diabetes mellitus [J]. J Am Board Fam Pract, 2005; 18(1):37-43
46. Kawano K, Hirashima T, Mori S, et al. Spontaneous long-term hyperglycemic rat with diabetic complications. Otsuka Long-Evans Tokushima Fatty (OLETF) strain [J]. Diabetes, 1992,41(11):1422-1428
47. Sato T. Insulin resistance in skeletal muscle of the male Otsuka Long-Evans Tokushima Fatty ,a new model of NIDDM.Diabetologia,1995,38:1033-1036
48. DiBisceglia AM.What every hepatologist should know about endocrinology: obesity, diabetes, and liver disease. Gastroenterology. 2004 Feb; 126(2):604-6.
49. Nepomnyashchikh GI, Pavlenko OA, Aidagulova SV, et al. Ultrastrutural analysis of liver biopsy specimens in diabetes mellitus associated with chronic Opisthorchaiasis.Bull Exp Biol Med,2001,132:1190-4
50. Scheen AJ, Luyckx FH. Nonalcoholic steatohepatitis and insulin resistance: interface between gastroenterologists and endocrinologists. Acta Clin Belg. 2003 Mar-Apr; 58(2):81-91
51. Lee Y, Yu X, Gonzales F, et al. PPAR alpha is necessary for the lipopenic action of hyperleptinemia on white adipose and liver tissue. Proc Natl Acad Sci USA , Med Sci,2002, 19 : 11848-53
52. Sung CK, She H, Xiong S, et al. Tumor necrosis factor2alpha inhibits peroxisome proliferators-actived receptor gamma activity at a posttranslational level in hepatic stellate cells. Am J Physiol Gastrointest Liver Physiol, 2004 , 286 : 722-9
53. Neuschwander-Tetri BA, Brunt EM, Wehmeier KR, et al. Improved nonalcoholic steatohepatitis after 48 week of treatment with the PPAR- gamma ligand rosiglitazone. Hepatology. 2003 ,10 : 1008-17
54. Eun Hee Koh, Min-Seon Kim, Joong-Yeol Park, et al. Peroxisome proliferator-activated receptor (PPAR)-alpha Activation Prevents Diabetes in OLETF Rats.Diabetes,2003,52(9),2331-7
55. Castoldi G, Galimberti S, Riva C ,et al. Association between serum values of C-reactive protein and cytokine production in whole blood of type 2 diabetic patients. Clin Sci (Lond). 2007 Mar 16; [Epub ahead of print]
56. Ye J. Role of insulin in the pathogenesis of free fatty acid-induced insulin resistance in skeletal muscle. Endocr Metab Immune Disord Drug Targets. 2007 Mar; 7(1):65-74.
57. Balagopal P, Graham TE, Kahn BB, et al. Reduction of Elevated Serum Retinol Binding Protein (RBP4) in Obese Children by Lifestyle Intervention: Association with Sub-clinical Inflammation. J Clin Endocrinol Metab. 2007 Mar 6; [Epub ahead of print]
58. Alexandraki K, Piperi C, Kalofoutis C, et al. Inflammatory process in type 2 diabetes: The role of cytokines[J ]. Ann N Y Acad Sci. 2006 Nov; 1084:89-117.
59. Mita T, Watada H, Uchino H .Association of C-reactive protein with early-stage carotid atherosclerosis in Japanese patients with early-state type 2 diabetes mellitus[J ]. Endocr J. 2006 Oct; 53(5):693-8.
60. Li J , Peet GW, Pullen S S , et al. Recombinant I κ B kinase α and β are direct kinase of I κ B α [J ]. J Biol Chem, 1998, 273:30736-41
61.Kiyoshi T, Osamu T, Tohru T, et al. Limb and Skin Abnormalities in Mice Lacking IKK α [J]. Science, 1999,284:313-6
62. Yinling H,Veronipue B, Mireille D, et al.Abnormal Morphogenesis But Intact IKK Activation in Mice Lacking the IKK α Subunit of I κ B Kinase[J ]. Science, 1999,284:316-20
63. ChristopherJ.MacKenzie, Elwyn Ritchie, Andrew Paul, et al. IKKα and IKKβ function in TNFa- stimulated adhesion molecule expression in human aortic smooth muscle cells[J ]. Cell Signal, 2007, 19: 75-80
64. SE Shoelson, J Lee, M Yuan. Inflammation and the IKK β /I κ B/NF- κ B axis in obesity-and diet-induced insulin resistance[J ]. Int J of Obe, 2003, 27: S49-S52
65. Chietti G, Gbaguidi FG, Grigli S, et al.CLA-1/SR-BI is expressed in atherosclerotic lesion macrophages and regulated by activators of peroxisome proliferators-activated receptors[J ].Circulation.2000, 101(20):2411-7
66. KimJ K, Kim YJ , Fillnore J J ,et al. Prevention of fat-induced insulin resistance by salicylate [ J ]. J Clin Invest, 2001, 108:437-46
67. Hundal R S, Petersen K F, Mayerson A B, et al. Mechanism by which high2dose aspirin improves glucose metabolism in type 2 diabetes [J]. J Clin Invest, 2002, 109: 1321-6
68. Ruan H , Hacohen N , Golub T R ,et al. Tumor necrosis factor-alpha suppresses adipocyte-specific genes and activates expression of preadipocyte genes in 3T3-L1 adipocytes: nuclear factor-KappaB activation by TNF-alpha is obligatory [J]. Diabetes, 2002, 51:1319-36
69. Rossi A, Kapali P, Natoli G, et al. Anti-inflammatory cyclopentenone prostaglandins are direct inhibitors of I κ B kinase[J ].Nature,2000,403:103-8