The Study Of The Treatment Of Yinxieling Tablets On The Mice Model Of Type 1 Diabetes

ObjectiveType 1 diabetes is a kind of acute and chronic disease mediated by T lymphocytes,which induced immune response to autologous islet beta cells,leading to islet damage and absolute deficit of insulin,that makes lifelong exogenous insulin replacement therapy for a necessary.Functional islet cells can secrete insulin,regulate blood glucose.With the complete destruction of T cells to islet cells and absolute lacking of insulin in the body,which can lead to overt hyperglycemia,often accompanied with“three more and one less”symptoms,such as polydypsia,polyphagia,polyuria,weight loss.Although,the incidence of type 1 diabetes in our country is low,but,a large population determines that the patients is not in minority,and today,a diagnosis of adolescents with type 1 diabetes patients also showed a trend of rising year by year,and the incidence also increased with the age,so looking for a kind of drug that can effectively treat and improve the disease has become an urgent task.Yinxieling tablets is an empirical formula used to treat psoriasis and owned by an old Chinese medicine professor xuan guowei of Traditional Chinese Medicine Hospital of Guangdong Province.As a hospital preparation,its bath number was Z20080123,issued by Guangdong Provincial Food and Drug Administration in 1998,and it has perfect effectiveness of treating psoriasis in clinical.Modern pharmacological studies have shown that Yinxieling tablets and its traditional Chinese medicine composition has many pharmacological activities,such as certain anti-inflammatory and immunosuppressive effects.At present,Yinxieling tablets as a drug for treating psoriasis in clinical application,yet there was no research report about the application of preventing and treating type 1 diabetes.Therefore,our study will explore the mechanism of Yinxieling tablets on type 1 diabetes from the level of animals.The study is helpful to recognize immunological mechanism of type 1 diabetes,and expand the treatment category of Yinxieling tablets.Methods1.To establish animal model of type 1 diabetes:when raised NOD(shi/Ltj)female mice for more than 4 months,we initially judged that the mice has got spontaneous type 1 diabetes or not by observing the degree of wetting,adhesion of the mice bedding and its water intake,then started to measure the blood glucose.If the blood glucose of mice reached 11.1 mmol/L,placed them separately with the mice not onset,and used them for making model.We successfully transplanted splenic lymphocytes from eleven NOD(shi/Ltj)mice which blood glucose reached 11.1 mmol/L into thirty-nine NOD-SCID recipient mice,about 0.5×107 splenic lymphocytes each recipient mice.2.Intragastric administration and blood glucose monitoring:we randomly divided the mice into two groups,nineteen female NOD-SCID mice of T1D model and twenty mice of that,as model group and treatment group,were respectively given ultrapure water and Yinxieling tablets' aqueous solution.The model group was used to be compared with treatment group.Eight NOD-SCID mice were subjected to no disposal,as normal control group,used to be compared with model group.Intragastric administration was started from day three after establishing mice models until executing mice and taking material.The gastric volume of model group was the same with treatment group,calculated according to 0.1mL/10g.The blood glucose(blood from mice tail vein)of all mice was detected and recorded on the fifteenth day,the seventeenth day,the twenty-first day,twenty-third day,twenty-fifth day,twenty-sixth day and until fortieth day after models established.3.Histopathological,immunohistochemical staining and gene expression:all groups of mice were executed by cervical dislocation on the fortieth day of making model.We took out the pancreas,preserved into three parts.One part was fixed in 10%neutral formalin for eighteen hours,then performed dehydration and paraffin embedding;one part was quickly put into liquid nitrogen with OCT-embedded for cooling,reserved at-80?;one part was placed in RNAlater(RNA Stabilization Solution,purchased in life),to extract RNA to detect the expression of genes in tissue samples.The paraffin samples of pancreatic tissue of was performed with HE staining and immunohistochemical staining of insulin antibody,and frozen samples of that was performed with immunohistochemical staining of CD3 antibody;Extraction of RNA samples was performed by reverse transcription and Q-PCR detection to detect the expression of disease-related genes,such as insulin,CD3,IL-17,IL-1?,IFN-?,IL-10,IL-4.Results1.The successfully establishing of animal models of type 1 diabetes:we judged that the mice was onset or not by blood glucose values of timing detection.The mice was onset approximately starting from the twenty-first day,and peaking on the thirtieth day.From the histopathological pictures,we can see that the pancreas was infiltrated with lots of T lymphocytes,and it illustrated that T lymphocytes was allotransplanted successfully to the pancreatic tissue and proliferated to attack islet cells in specificity,leading to an absolute lack of insulin,triggering type 1 diabetes.2.The treatment of Yinxieling tablets on the mice model of type 1 diabetes:The blood glucose of mice in all three groups was totally compared using Analysis of Variance of Repeated Measure,and the difference was extremely significant;the statistical P value of multiple compare between groups was less than 0.05,showing that the model was successfully constructed and the drug treatment was effective.We judged that the modelly NOD-SCID mice was onset or not based on the blood glucose which was more than 11.1 mmol/L.The morbidity of three groups' mice was compared by two groups.Because the total sample size of two groups was less than 40,we performed the compare using the Fisher' Exact test of Chi-square test.Compared treatment group with model group,after the 28th day,the difference was statistically significant,showing the effective drug treatment.The mice were defined as classical type 1 diabetes when the mice' blood glucose reached 16.7 mmol/L.We compared the days of mice onset between treatment group and model group through calculation,and found that the average onset days of mice in model group were one weeks more than that of treatment group,and the difference was extremely significant,illustrating the obvious effectiveness of Yinxieling tablets.3.The protection of Yinxieling tablets on islet cells in the pancreas of mice of type 1 diabetes:in presence of insulitis as the judgment standard,and the pancreatic pathology classification,the pancreatic epibiotic islet beta cells of treatment group was obviously more than model group,whats' more,the difference of the scope and degree of inflammatory infiltration was extremely significant compared with model group.Immunohistochemical staining of insulin antibodies showed that the difference of islet ? cells of the positive of insulin antibody in pancreas of treatment group was statistically significant compared with model group.The immunohistochemical staining of CD3 antibody showed that the difference of T cells in pancreas of treatment group was extremely significant compared with model group.Detection results of gene expression showed that the relative expression of CD3,IL-17,IL-1?,IFN-? of model group was significantly higher than that of treatment group,while gene of insulin,IL-10 and IL-4 was obviously lower than that of treatment group.Results showed that Yinxieling tablets can protect the function of pancreatic islet cells,and reduce lymphocyte infiltration in the islets.ConclusionMice of type 1 diabetes intervened and treated by Yinxieling tablets can reduce the infiltration of inflammation in the pancreas,protect islet cells,significantly increase survival rate of mice pancreatic islet cells of type 1 diabetes,delay onset,reduce the incidence degree,and extend the lifetime.The effect of Yinxieling tablets preventing and treating for type 1 diabetes is remarkable.