The Study On Chymase Pathway In Renin-Angiotensin System Of Syrian Golden Hamster Islets

BackgroundDiabetes is one of the major diseases that the whole humankind are facing in the 21st century, which will lead to population health status decline and various vascular diseases. Patients with diabetes and its complications will have their life quality decreased, and the whole society will also take enormous economic burden. Studies have shown that, during the development of type 2 diabetes, the most important mechanism is insulin resistance and pancreatic P-cell dysfunction. Insulin resistance can influence type 2 diabetes in each session, while the isletsβ-cell dysfunction is the core of the pathogenesis of type 2 diabetes. A hot point that scholars at home and abroad focus on is to explore the causes and specific mechanism of type 2 diabetes andβ-cell dysfunction.The study on Renin-angiotensin-aldosterone system (RAS), which has been recognized as an important body systematic mechanism, has a history of 100 years. A lot of evidences have proved that it plays an important part in controlling the blood pressure and its volume, liquid, and the electrolyte balance, etc. In the classic cycle of RAS, the rennin produced by kidneys acts on circulating angiotensinogen (AGT), and convert into a lower activity substance--angiotensinⅠ(AngⅠ) through the enzyme exscind. Angiotensin-converting enzyme (ACE), existing in endothelial cells of the lung and other organs, cut Ang I terminal amino acid and generate angiotensin II (Ang II),which is the most important factor in the entire cycle RAS.Aside from the function of causing hypertension by stimulating blood vessel contraction, Ang II can also regulate tissue differentiation and mediate storage of water. What's more, the relationship between the downstream aldosterone generation and insulin resistance (IR) has also become one of the domestic and foreign research hot spot recently. However, as an active peptide, the specific mechanisms of AngⅡfor insulin resistance has not been fully clarified. Studies found that AngⅡcan also cause the insulin resistance of peripheral target tissues, such as fat, muscle, liver and other organs, by the mechanism that inducing inflammation, oxidative stress and damaging insulin signaling pathway. Blocking RAS can significantly reduce the peripheral tissue inflammation, oxidative shock response and increase insulin sensitivity. Therefore, to explore the generation of AngⅡand its mechanism in type 2 diabetes will become one of the most important research direction.In last few years, studies have shown that RAS carry out its functions not only through the classical pathway, but the generation of angiotensinⅡcould also give the regulating effect in local autocrine or paracrine level. In fact, as early as 1970s, there are scholars who have found the serine protease existed in the hamster cheek pouch blood vessel, which could convert Ang I to AngⅡ.The study also found that in many part of the organs, in addition to the classic ACE-mediated AngⅡgeneration way, there is another important local pathway that define as chymase channels. Local RAS has all the components that can be tested in classical RAS.In normal state, it could keep the balance with the regulation system. However, under some pathological states, local RAS may lead to metabolic abnormalities and break the balance of the whole RAS.The increasing in local secretion of Ang II usually play a negative role, and its over active may finally lead to some certain diseases. The activated local RAS has been found in a variety of tissues and organs presently and the expression of local pancreatic RAS has aroused great and widespread attention of the whole world. Studies found that the local pancreatic RAS has many functions: including the stimulation and inhibition of cell proliferation, inducing apoptosis, resulting in reactive oxygen species family, and regulating hormone secretion, as well as constriction and extend of blood vessels and so on. Research of animal and human islets also pointed out that the local RAS has great relevance to the generation and development of type 2 diabetes, and the mechanism is thought to be related to Ang II-mediated oxidative stress, pancreatic hormone starch,β-cell apoptosis and Islet fibrosis and so on, but the exact mechanism has not been found and need further research.In recent years, such as the HOPE, LIFE and other large-scale clinical studies have shown that, using classical drugs-angiotensin converting enzyme inhibitors(ACEI) or angiotensin II receptor blockers (ARB) to block the RAS, the incidence of diabetes in high-risk groups can significantly reduced, which aroused many people re-thinking of the relationship between RAS and type 2 diabetes and that between islet's P-cell function and the RAS. The DREAM study also found that the application of a kind of ACEI-ramipril makes the proportion of patient in the intervention group increased obviously, whose blood glucose returns to normal level, comparing with the placebo group. It's proved that, by improving RAS,glucose metabolism can also be changed, so patients with diabetes could obtain great benefits. But this study also pointed out that there is no significant reduction of people in the intervention group progressed to diabetes and death. The latest NAVIGATOR study involved more than 9,000 patients, making it one of the largest and longest global trials to date in pre-diabetic patients and it gives us the results that valsartan based regimen reduced risk of developing new-onset diabetes by 14%, but did not reduce risk of cardiovascular events. However, nateglinide based regimen did not reduce incidence of new-onset diabetes or of cardiovascular eventsAll of the above results give us a new perspective of the relationship between type 2 diabetes and RAS.In addition, there are a number of studies have showed that, after the intervention and blocking the RAS,the blood glucose levels will decreased significantly for patients with type 2 diabetes. A series of RAS research has already revealed that it plays an important role in cardiovascular and endocrine-related. metabolism diseases, but the specific mechanism is still needed to be clarified. At the same time, the cause of type 2 diabetes and its complications mechanisms are still not clear, which is a reason for unvisible control of type 2 diabetes at present.In summary, the activation of circulating and local RAS in islets may be the important factors for damage ofβ-cell, and it may thereby mediate the development of type 2 diabetes.Evidence-based studies have shown that blocking the RAS can reduce the incidence of diabetes-risk populations and improve glucose metabolism disorders in patients with type 2 diabetes, which is thought to be a new target for prevention and treatment of metabolism syndrome.Based on the above viewpoints and conclusions, we choose the pancreatic islets of Syrian golden hamster as a research model and hope to find out the mechanism of generation of Ang II.What's more, to observe its change in generation in local RAS, especially in the disease state. We also want to find out the change in islets of Syrian golden hamster after we intervene chymase pathway and use inhibitors to shut off it. Through the above methods to identify the pathogens of type 2 diabetes and to provide a basis theory for treatment of type 2 diabetes. Purpose1.Select male Syrian golden hamster, after the anesthesia, perfuse collagenase V in situ, using water bath to digestion and Ficoll density gradient to purification, then using DTZ to staining. On the purpose of obtaining high survival rate of the Syrian hamster islets and identify its function after collection. To observe the hyperlipidemia with fructose-drinking and high-fat diet feeding of Syrian golden hamster, analysis the rational application of different hyperlipidemia animal models.2.Adopt different inhibitors:the chymostatin, captopril, a-antitrypsin, aprotinin to block the renin-angiotensin system in Syrian golden hamster pancreatic islets and observe the changes in AngⅡproduction. Analyse the Ang II generation pathway in islets' local RAS system of Syrian golden hamster and explore its formation mechanism. Use different inhibitors:the chymostatin, captopril, a-antitrypsin, aprotinin to block the renin-angiotensin system in hyperlipidemia model of Syrian golden hamster pancreatic islets and observe the changes in AngⅡproduction.Methods1.Select healthy male Syrian golden hamster, fasting 24h and anesthetize with the intraperitoneal injection of chloral hydrate (3ml/kg). Laparotomy and find out the main pancreatic duct from the duodenum. Ligate the choledoch and its branches from the intestinal side. Puncture bile duct and intubation. Inject collagenase V to make the pancreas full of expansion. Then cut the pancreas immediately and put into a bottle which is placed in 37℃water bath to digest. After the digestion is completed, adding serum-containing culture fluid to terminate the digestion. Collect the precipitation liquid and use discontinuous density gradient to purify islets. Add DTZ to the purified precipitation and stained in dark place at room temperature.Observe the final production under inverted microscope, combined with image analysis software to counts the number of islets. Observe islets' livability rates by using trypan blue staining.2.Use 45 Syrian golden hamsters and randomized divided into 3 groups:control group, high-fat group, fructose-drinking high-fat group. Feed with free access to food and water for 4 weeks. Body weight were measured every month, and measure plasma Glu, TG, TC and Cr level in Oth and 4th week. Analysis the characters and application of different feedstuff to raise dyslipidemia model of Syrian golden hamster.3.After separation and purification, selected the islet cells from healthy Syrian golden hamster and the hyperlipidemia model,add the islets into 24-well plates, with inverted microscope. Add fetal calf serum and pre-incubated for 30min. Mark each group and divide into:①control group.②captopril group.③chymostatin group.④aprotinin group.⑤α-antitrypsin group.⑥captopril+chymostatin group. Each group added angiotensin I (0.02μmol/l) and then added PBS,captopril (final concentration:1mmol/L), chymostatin (final concentration:1mmol/L), aprotinin (final concentration lmmol/L), a-antitrypsin (final concentration:50μg/ml), captopril and chymostatin (both final concentration:lmmol/L) to interfere islets. After constant temperature incubate for 120min, collect the liquid in 24-well plates, gather supernatant liquid after centrifugate. Adopt competition ELISA to test concentrations of Ang II in the supernatant liquid. Mensurate of Ang II concentration in the following stock solution:captopril, chymostatin, aprotinin, a-antitrypsin and PBS to exclude false positives that may interfere the whole test.Results1.After DTZ staining, islets of Syrian golden hamster become red or scarlet under microscopy. Islets are morphological integrity and cell membrane is clearly visible, with good refraction and no damage. Islets are basically without exocrine organization after purified. Each hamster pancreas can gain islets about 400 IEQ, islets purified rate>90%, islets live rate>90%. After cultured for 1 week, purified islets grow in good condition in vitro. All of the high-fat diet groups of Syrian hamsters show hyperlipidemia compares to the control group, fructous-high-fat group has the dramatically high plasma TC and TG level.2.By comparing captopril, chymostatin, aprotinin, a-antitrypsin and the PBS stock solution(negative control) added in this experiment we found that, in addition to the concentration of AngⅡin the control group is 82.49 pg/ml, the concentration of AngⅡin the rest reagent stock solutions is less than 2.00 pg/ml (P<0.01).The solution of various reagents and inhibitors don't contain any Ang II basically.3.Through the competitive ELISA method and analyse the its result of local islets'AngⅡsecretion after intervention, we found that:Comparing with the control group, the concentration of AngⅡin captopril group, chymostatin group, aprotinin group, a-antitrypsin group and captopril+chymostatin group is reduced,which in Captopril group and chymostatin group were respectively reduced 42.50% and 50.94%(P<0.01)compared with the control group, but the difference between the two groups doesn't have statistically significance. The inhibition effect ofα-antitrypsin group and the aprotinin group is not the same as former two groups and compared with the control group, the concentration still respectively reduced 20.04% and 18.67%(P<0.05). Contrasting to the control group, the generation of AngⅡin captopril+chymostatin group shrinks to 81.82%(P<0.01).4. Analysing the ELISA result of local islets' of hyperlipidemia hamsters' AngⅡsecretion after intervention, we found that:Comparing with the control group, the concentration of AngⅡin captopril group, chymostatin group, aprotinin group, a-antitrypsin group and captopril+chymostatin group is reduced, which in Captopril group and chymostatin group were respectively reduced 39.49% and 56.70%(P<0.01) compared with the control group, but the difference between the two groups doesn't have statistically significance. The inhibition effect of a-antitrypsin group and the aprotinin group is not the same as former two groups and compared with the control group, the concentration still respectively reduced 22.02% and 24.88%(P<0.05). Contrasting to the control group, the generation of AngⅡin captopril+ chymostatin group shrinks to 78.34%(P<0.01).Conclusion1. In situ collagenase perfusion combined with bile duct intubation digestion may obtain high-purity, high-living rate of the Syrian hamster islets.2.All the high-fat diet group Syrian hamster appear hyperlipidemia, but among them there is not significant defference between high-fat group and fucose-drinking high-fat group.3.In addition to angiotensin-converting enzyme, chymase is another pathway of angiotensinⅡgeneration in Syrian golden hamster's islets.4. The generation of AngⅡof healthy Syrian golden hamster's islets don't have significant difference between the angiotensin-converting enzyme pathway and chymase pathway.5.In hyperlipidemia Syrian golden hamster's islets, with addition to angiotensin-converting enzyme, chymase is still another pathway of angiotensinⅡgeneration. Not as what has been prove in the early test, the generation of AngⅡin hyperlipidemia Syrian golden hamster's islets have significant difference between the angiotensin-converting enzyme pathway and chymase pathway.