| Diabetes Mellitus(DM)is a syndrome mainly characterized by the elevated plasma glucose and impaired glucose tolerance with metabolism disorders of glucose,fat and protein.Type 2 Diabetes Mellitus(T2DM),which accounts for more than 90%of diabetic patients,is mainly caused by insulin resistance and insulin secretion disorder.Western medicine has a markedly hypoglycemic effect but has several defects as well,such as hypoglycemia reaction,liver and kidney toxicity,secondary failure.Therefore,it is still a difficult scientific problem for the whole world to research for a new anti-diabetic drug with high efficiency and low toxicity.Traditional Chinese medicine(TCM)has advantages of curative effect and few side effects on treating diabetic patients.LM Granules(LMG),a kind of TCM prescription created by Professor Wu Bingcai,has shown a curative effect on treating T2DM patients in clinical observations,which is an empirical prescription in differential treatment of diabetes from the Liver.In order to make LMG more widely used in clinical,experiments for verifying its hypoglycemic effect and exploring the related mechanisms to provide scientific data support are so imperative.ObjectiveDevelop Type 2 diabetes mellitus(T2DM)GK rat model to investigate the pharmacological effects of LM granules(LMG)on the diabetic GK rats.The observed indictors included general symptoms,glucose metabolisms,islet function,pancreatic pathological changes,inflammatory cytokines,oxidative stress,and JNK signal pathway.To study the hypoglycemic effect and the related mechanisms of LMG,so that to provide experimental evidences for the clinical application of LMG in T2DM area.Methods1.Developing animal model:GK rats were induced to be T2DM models by being feeded with high-fat diet for two weeks consecutively.The rats with fasting blood glucose not less than 11.1 mmol/L were considered as successful models.2.Grouping and administering:Eight SD rats were adopted as normal control group(NC).The successful model rats were randomly divided into six groups with eight GK rats in each group:untreated diabetic model group(DM);groups treated by LMG with a low,midium,and high dose,respectively(LML,LMM,LMH);group treated by Sheng Qi Hypoglycemic granules(SQ)and group treated by Gliclazide Tablets(GL).After grouping,the drugs were administered correspondingly via intragastric administration once a day for nine weeks;the NC group and DM group were administered with equal volume of purified water via the same way.3.Sample collections and index determinations:During administration of drugs,general symptoms were observed every day,body weights were recorded weekly,and fasting blood glucose(FBG)levels were measured every three weeks in rats.After the last administration,rats were fasting for 12 hours,then oral glucose tolerance test(OGTT)was conducted and the area under the curve(AUC)was calculated in each group.Afterwards,the rats were anesthetized to collect blood samples via abdominal aorta.And the pancreas of rats were rapidly removed by dissecting at last.Enzyme-linked immunosorbent assay(ELISA)was applied to detect glycated hemoglobin(HbAlc)and insulin(INS)levels in the serum,and then insulin resistance index(IRI)and insulin sensitivity index(ISI)were calculated;levels of inflammatory cytokines tumor necrosis factor(TNF)-? and interleukin(IL)-1 ? were determined by ELISA as well.Biochemical assay was used to test the serum superoxide dismutase(SOD)and malondialdehyde(MDA)levels.Hematoxylin and eosin(HE)staining and aldehyde fuchsin staining were adopted to observe the pathological changes of islets and ratio of islet ? cells,respectively.And the expression levels of proteins JNK,p-JNK(Thr183/Tyrl85),Akt,p-Akt(Ser473)were examined by western blot(WB).4.Statistical analysis:All data were analysed by SPSS 17.0 software and presented by mean ± standard deviation(x ± s).Differences among groups were compared with one-way analysis of variance(ANOVA)and P<0.05 was considered statistically significant.Results1.General symptoms:Compared with the normal rats,the model rats were lean,unresponsive and lethargy with coarse and dirty hair and significantly decreased body weight from the second week after administration(P<0.01).There showed some improvement of rats in the treatment groups compared with the model group,while the body weights were not changed significantly(P>0.05).2.Indicators of glucose metabolism:After the 9-week-administration,the FBG level of the model rats was much higher than the normal rats(P<0,01);compared with the DM group,the FBG levels of rats in the treatment groups were all diminished significantly(P<0.05 or P<0.01);meanwhile,contrasted to pre-administration,the FBG levels of rats were decreased in all the drugged groups except for the LML group(P<0.05 or P<0.01);however,compared with the SQ and GL group,the FBG levels of rats in LMM and LMH group were not changed significantly(P>0.05).In terms of the HbAlc level of rats,it was escalated significantly in the DM group than the NC group(P<0.01);and it was diminished in each of the treatment groups except for the LMM group compared with the DM group(P<0.05 or P<0.01);and in contrast to the SQ and GL group,no significant differences of the HbAlc levels of rats were found in the three LMG groups(P>0.05).3.Islet function:Compared with the NC group,the AUC and IRI volumes were increased,while the INS level and ISI volume were decreased markedly in the DM group rats(P<0.01).Compared with the DM group,the AUC volumes of rats were diminished in all the treatment groups,significantly(P<0.05 or P<0.01);the levels of INS were not significantly boosted in each drugged group(P>0.05),but the mean difference of INS between the LMH and DM group was more than 2 ? IU/mL,as well as the GL group.In addition,rats treated by the high-dose LMG,SQ and GL were induced to lower IRI and higher ISI volumes than the untreated diabetic rats,and the differences were statistically significant(P<0.05 or P<0.01).4.The pathological changes of pancreas:Compared with the normal rats,the islet tissues of model rats had very irregular shapes and edges,and much smaller volumes,consisting of significantly reduced and unevenly distributed 3 cells,and the cell nucleus of some islets appeared aggregation.Compared with the DM group,the shapes of islets of rats had been improved in all the treatment groups,and the cell nucleus aggregation were not found in the LMH,SQ and GL group,but the ratio of ? cells were not obviously increased contrasted to the NC group.5.Inflammatory cytokines:Contrasted to the NC group,the levels of TNF-a and IL-1? were both arised markedly in the DM group(P<0.01).Compared with the DM group,different dosed LMG decreased the TNF-a and IL-1? levels significantly(P<0.05 or P<0.01);SQ and GL also cut down them a lot,the differences were statistically significant(P<0.01).6.Indicators of oxidative stress:The serum SOD level of the DM rats was lower than the normal rats,while the MDA level was higher(P<0.01).Compared with the DM group,LMG markedly enhanced the SOD level of rats with different doses,and cut down the MDA level with the midium and high dose(P<0.01);meanwhile,the drug of SQ showed a significant effect of accelerating the SOD and diminishing the MDA levels of rats,as well as GL(P<0.01).7.Influence on JNK signal pathway:In terms of the model rats,the value of p-JNK/JNK was larger,and the value of p-Akt/Akt was smaller than the normal rats(P<0.01),suggesting that the phosphorylation level of JNK protein was higher and that of Akt protein was lower in the DM group.The values of p-JNK/JNK were much larger in all the treatment groups(P<0.01),and the values of p-Akt/Akt were much smaller in the LMM,LMH,SQ and GL group,than that in the DM group(P<0.01),hinting that LMG could inhibit the JNK phosphorylation and promoted the Akt phosphorylation,as well as the drug of SQ and GL.Conclusion1.LMG could cut down the levels of fasting blood glucose and glycated hemoglobin in T2DM model GK rats,indicating a role of regulating glucose metabolism and maintaining glucose homeostasis.2.LMG could heighten the glucose tolerance,alleviate the insulin resistance and add to the insulin sensitivity of T2DM model GK rats,hinting that the islet function of the diabetic rats had been protected to a certain degree,that could be used to explain for its hypoglycemic effect.3.LMG was abled to improve the morphologies and structures of islet tissues in T2DM model GK rats,pointing a mitigation of the islet pathological damage,which might be a reason for the improved islet function.4.The levels of inflammatory factor TNF-a and IL-1? could be reduced by LMG in T2DM model GK rats,manifesting some anti-inflammatory properties of LMG,which was probablely one of the reasons for the improvement of insulin resistance,and protection of islet structure and function.5.LMG might enhance the activity of the antioxidant enzyme SOD,and reduce the content of the peroxidation product MDA in T2DM model GK rats,demenstrating an ability of inhibiting oxidative stress,which could be another reason for alleviating insulin resistance,and protecting the structure and function of islets by LMG.6.LMG suppressed the phosphorylation of JNK protein and promoted the phosphorylation of Akt protein in pancreatic tissue of T2DM model GK rats,preliminarily clarifying the effect link of protecting the islet structure and function due to LMG.In summary,our results suggested that LMG could reduce the fasting blood glucose and glycated hemoglobin levels,improve the glucose tolerance and insulin resistance,protect the islet cells from progressive loss of mass and function for a better durability of glucose control in the T2DM model of GK rats.The potential mechanisms might be related with the abilities of anti-inflammatory and anti-oxidative stress,and the inhibition of the JNK signal pathway in the diabetic rats. |
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