Experimental Research In The Mechanisms Of Liuwei Dihuang Pills On Prevention Of Diabetes Mellitus In OLETF Rats

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^-1d^-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 ratsOBJECTIVE: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 ratsOBJECTIVE: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.