| Diabetes Mellitus is a common and frequently-occurring disease in many countries around the world at present, with its mortality rate has been ranked in the tumor, cardiovascular after third. The number of diabetes mellitus patients in China ranks first in the word, and about95%are type2diabetes mellitus patients. Therefore, the tasks brook of treatment of diabetes cannot delay.Pancreatic islet beta call dysfunction and the insulin resistance of peripheral tissue are two key factors of the pathogeny of type2diabetes. C-Jun NH2-terminal kinase (JNK) is a key number of the super family of mitogen activated protein kinase (MAPK), the family also includes ERK and p38. JNK signaling pathway is an important pathway in the pathogeny of type2diabetes. In pancreatic cells in type2diabetes mellitus, oxidative stress can lead to the activation of JNK and insulin signaling pathway of nucleus damaged, therefore the insulin gene expression level decreased, cause destruction of insulin synthesis. In addition phosphorylation of IRS-lser307induced by the activation of JNK, leading to insulin resistance. Mulberry leaf and its effective components have been reported to have good effect of reducing blood glucose, but its hypoglycemic mechanism is rarely reported. JNK signaling pathway is an important pathway involved in pathogeny of diabetes mellitus. Is there exists close correlation between the therapeutic effect of mulberry leaf on diabetes and JNK signaling pathway? That is a question we must answer in this paper.1. Literature studyThis part is a comprehensive literature review on the pathogenesis associated with type2diabetes mellitus and currents status of TCM in the treatment of type2diabetes researches. Based on this, the theoretical foundation of this research was offered. We mainly reviewed the following respects:1.Current situation of diabetes epidemiology and the progress in research on the pathogenesis of diabetes mellitus;2. Current situation of TCM and its effective components in the treatment of type2diabetes;3. Research progress on hypoglycemic effects and mechanism of the effective component of mulberry leaves;4. Current research on the important relationship between JNK signaling pathway and T2DM;5. Research progress on microRNAs associated with T2DM.2. Experimental study2.1Effect of mulberry leaves effective extracts on glucose and lipid metabolism in T2DM rats.ObjectiveEstablish rat model of type2diabetes mellitus (T2DM), study the effect of total flavones, total polysaccharide and water extracts of mulberry leaves on glucose and lipid metabolism in T2DM rats.MethodsSD rats were randomly divided into one normal control group (group NC, n=8)and one diabetes mellitus group(DM), the T2DM rats model were induced by feeding with high-sugar and high-lipid fodder for8weeks and a single intraperitoneal injection of streptozotocin (STZ), the successful models were divided into5groups at random:diabetes mellitus model group(DM), diabetes with Mulberry leaves flavones extract (30mg·kg-1) treatment group (MTF), diabetes with mulberry leaves polysaccharide extract (100mg·kg-1) treatment group (MTP), diabetes with mulberry leaves aqueous extract (6g·kg-1) treatment group(MWE), diabetes with rosiglitazone (4mg·kg-1)treatment group (RSG),10rats per group. The drug was administered by intragastric administration for6weeks, and the rats were measured the fasting plasma glucose level weekly. Afterwards, the blood plasma was collected by the abdominal aortic method in these anesthetized rats and measure NEFA, CHO, TC levels. The function of islets and fat levels in the blood were evaluated.Results(1) Compare with NC group rats, the DM group rats show polydipsia, excretion, hyperglycemia, hyperlipemia symptoms, and later weight loss, consistent with the clinical features of T2DM.(2) After6weeks of treatment, the body weight of the NC group rats increased, and compared with NC group the DM group rat decreased with statistical significance (P<0.01);Compared with DM group, the body weight of MWE、MTP、MTF group rats were heavier(P <0. OlorO.05).(3) The FBG level of MTF and MTP group in the4th week and MWE group in the5th week began to decrease; the data were significantly different compared with the moment without drug treatment (P<0.05).(4) The measurements of NEFA、TG、CHO of DM group had significant difference with the NC group after the treatment for6weeks (P<0.05), meanwhile, in the MWE group, the TG, CHO levels decreased compared with the DM group (P<0.05), there was no significant difference in the NEFA levels (P>0.05); The levels of TG、 CHO reduced (P<0.05), there was no significant difference in NEFA level (P>0.05);The levels of TG declined in the MTP group (P<0.05), there was no significant difference in the NEFA and CHO group (P>0.05);In the RSG group, there was no significant difference between them (P>0.05)ConclusionThe T2DM rat model we established can meet the experimental requirements. Each mulberry leaves effective extracts could reduce fasting blood glucose and improve the living condition; meanwhile, they had the effects of decreasing blood glucose, reducing the blood lipid at the same time. Rosiglitazone has good effect on lowering the blood glucose but could not reduce the blood lipid. Furthermore, each effective parts of mulberry leaf had different performances in the aspects of decreasing the blood glucose and the blood lipid.2.2Effect of mulberry leaves effective extracts on protecting pancreata and its function in T2DM rats.ObjectiveTo estimate the effect of different parts of mulberry leaves effective extracts on islet tissue and islet cell function in T2DM rats.MethodsUsed the same T2DM rat model and treatments as the above we established. The glucose tolerance factor (OGTT assay) was evaluated in the6th week, afterwards, the blood plasma were collected by the abdominal aortic method in these anesthetized rats and measure INS, SOD and MDA levels, the function of islets in the blood and anti-oxidative ability were evaluated accordingly, finally, took islet tissue to observe the changes in morphology and pathology under a light microscope and a transmission electron microscope (TEM). Results(1)The islet HE staining showed that islet beta cells seldom exist and were almost vacuolar, the islet cells atrophied and cell nuclear were condensed, meanwhile, the pancreas were severely damaged, however, the MTF、MTP、MWE、 RSG levels of pancreas cells in the medicated group improved significantly compared the DM group.(2)The islet TEM told us that β cells were in the center of islet for NC group rats, there was a large number of different size of secretory granules stockpiling in the cytoplasm. In these granules we saw a lot of dense core in all shapes and size. The electron density of granule was not on the same height, and limiting membrane wrapped outside the granules. There was a clear and wide gap between the dense core and limiting membrane. Mitochondria scattered in cytoplasm, round or elongated. The cell nucleus of other groups showed different degrees of pykonsis and cell damage.(3) The blood sugar levels of the MWE, MTF, MTP and RSG groups were lower than those of model group in OGTT2h and had statistical significance (P<0.05).(4) The measurements of INS、SOD、MDA of DM group had significant difference with the NC group after the treatment for6weeks (P<0.05),meanwhile, in the. MWE group, the INS and SOD level increased compared with the DM group (P<0.05) there was no significant difference in the MDA levels (P>0.05);The INS and SOD level of MTF group elevated and the levels of MDA reduced (P<0.05); The INS、SOD level increased while the levels of MDA declined in the MTP group (P<0.05), so did RSG group.ConclusionEach effective parts of mulberry leaves can increase secretion of insulin and had repair effect on islet function in diabetic rats. There was no significant difference between these three effective extracts. But from the OGTT, the serum insulin level and pathological examination data, effect of mulberry leaves total flavonoids on islet function in T2DM rats was more obvious.2.3Effect of mulberry leaves effective extracts on the expression of JNK、 AKT、PDX-1、IRS-1mRNA in T2DM rats.ObjectiveTo investage the mechanism of mulberry leaves effective extracts hypoglycemic, protect the islet function and its relationship with JNK signaling pathway, determine the effect on JNK、AKT、PDX-1、IRS-1mRNA expression of mulberry leaves effective extracts in T2DM rats.MethodsUsed the same T2DM rat model and treatments as the above we established. Took islet tissue to extract total RNA and then determine its purity, meanwhile measure the expression of islet tissue of rat with JNK、AKT、PDX-1、IRS-1mRNA by Q-pcr.Results(1) Compared with NC group, the expression level of JNK mRNA in DM group rat islet significantly increased (P<0.01), and AKT、PDX-1、IRS-1mRNA significantly decreased (P<0.01).(2)Compared with DM group, mulberry leaves total polysaccharide down regulated the expression of JNK mRNA (P<0.05), up-regulated the expression of AKT、IRS-1mRNA (P<0.01), and with no significant effect on PDX-1mRNA expression; mulberry leaves total flavonoids could significantly up-regulate the expression of AKT and PDX-1mRNA(P<0.05), had a certain effect on IRS-1mRNA and JNK mRNA, though there was no significant differences exit; mulberry leaves water extraction down regulated the expression of AKT、PDX-1and IRS-1mRNA (P<0.01), and with no significant effect on JNK mRNA expression;(4) Rosiglitazone had a significant effect on down regulating JNK mRNA expression (P<0.01) and up-regulating AKT、PDX-1and IRS-1mRNA expression (P<0.01)ConclusionJNK, AKT、PDX-1and IRS-1target of JNK signaling pathway were affected by mulberry leaves total polysaccharide, total flavonoids and water extraction to varying degrees. We believe their molecular mechanism of hypoglycemic activity, protection of islet tissue and repair effect of islet function has business with JNK signaling pathway.2.4Effect of mulberry leaves effective extracts on the expression of JNK、 AKT、PDX-1、IRS-1protein expression and phosphorylation in T2DM rats.ObjectiveTo explore the mechanism of mulberry leaves effective extracts hypoglycemic, protect the islet function and its relationship with JNK signaling pathway, determine the effect on JNK、AKT、PDX-1、IRS-1protein expression and phosphorylation of mulberry leaves effective extracts in T2DM rats.MethodsUsed the same T2DM rat model and treatments as the above we established. Took islet tissue, cut a small piece then fixed by formalin and embedded by paraffin, afterwards sliced, detected the expression of JNK, p-JNK by immunohistochemistry (IHC); The rest to extract total protein and then determine its purity, meanwhile examined the expression of JNK, p-JNK (Thr183/Tyr185), AKT, p-AKT ser473, IRS-1, p-IRS-lser307, PDX-1in rat islet tissue by western blot.ResultsIHC showed that JNK, p-JNK has different degrees of expression in each group in the pancreas. Western blot showed that:compared with NC group, phosphorylation of JNK and IRS-lser307in DM group increased significantly(P <0.01), phosphorylation of Aktser473decreased (P<0.01) and the expression of PDX-1decreased (P<0.01). Compared with DM group:(1) mulberry leaves total polysaccharide, total flavonoids and water extraction could significantly decrease level of the phosphorylation of JNK (Thrl83/Tyrl85). IRS-1ser307(P <0.01), increase the expression of PDX-1(P<0.01), have no notable effect on the phosphorylation of Aktser473;(2) Rosiglitazone could significantly reduce the level of JNK (Thr183/Tyrl85)、IRS-lser307phosphorylation (P<0.01), increase the level of PDX-1expression (P<0.01) and Aktser473phosphorylation(P<0.05).ConclusionMulberry leaves total polysaccharide, total flavonoids and water extraction could reduce the level of JNK phosphorylation, protect the pancreatic tissue from JNK damage. The hypoglycemic activity, protecting islet function of mulberry leaves related to JNK signaling pathway. |
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