| Objective: To develop the biphasic insulin aspart 40 injection and study on the effect of the biphasic insulin aspart 40 injection on T1 DM rats blood glucose and its immune regulation.Methods:(1) The biphasic insulin aspart 40 injection was developed by means of prescription study and process exploitation, and then we study its quality of the preparation.(2) Type I diabetes model rats received injection of insulin aspart 40, once a day, and the injections last for 6 weeks. General conditions of rats in each group were observed during the experiment. including monitoring the weight, quantity of drinking water, urination amount, food intake amount and fasting blood glucose.(3) Flow cytometry was used to detect the changes of CD4+CD25+Treg cells in peripheral blood of rats before and after medication for 3 weeks and 6 weeks. The levels of serum INF-γ, IL-10 and TGF-β in rat serum were detected by ELISA method. After 6 weeks medication, RT-PCR method was adopted to detect the Foxp3 m RNA level in peripheral blood lymphocytes of rats, and HE staining method was used to observe the pathological changes of rats’ pancreatic tissue in each group. The experiment was set up with normal control group, diabetic model group and positive drug control group.Results:(1) According to the Chinese Pharmacopoeia and enterprise standard indicators,the developed insulin aspart 40 injection were in line with the requirements of various indexs and its preparation stability was good;(2) General situation of rats in the experimental group: rats in the experimental group were generally in good condition, typical symptoms improve significantly, and the weight shows a rising trend. The weight of rats at 6 weeks(258.4g) was significantly higher than that of rats in model group(1.543g)(P<0.05). The HE staining pancreatic tissue show that after 6 weeks’ medication, the islet tissue of rats in the experimental group was relatively good, and worse than normal pancreatic tissue, but better than model group. Compared with the drug control group, it had no significant difference. After the drug intervention, the blood glucose of the experimental group(7.75mmol/L) was significantly lower than model group(24.93mmol/L) at the 7 days, and the blood glucose of the experimental group was lower than model group at the 21 days and 42 days(P<0.05).(3) Flow cytometry results of CD4+CD25+ Treg cells: after drug intervention for 6 weeks, Treg cells in the experimental group(1.84%) were higher than model group(1.46%), showing significant differences(P<0.05), and lower than the normal group(2.23%), showing significant differences(P<0.05).(4) Relative quantitative statistical results of Foxp3 m RNA: after 6 weeks’ medication, the experimental group(3.2368) was significantly higher than the normal group(P<0.05), and significantly lower than the model group(6.7928)(P<0.05).(5) Detection of results cytokine: 1after 6 weeks’ medication, INF-γ content(111.87pg/ml) in the experimental group was lower than that before the medication(140.04pg/ml), and obviously lower than that in model group(185.90pg/ml)(P<0.05). 2after 6 weeks’ medication, IL-10 content(1.45ng/ml) in the experimental group was higher than that before the medication(1.15ng/ml), and also was higher than that in model group(0.93ng/ml)(P<0.05). 3after 6 weeks’ medication, TGF-β content(114.21pg/ml) in the experimental group was higher than that before the medication(97.19pg/ml), and also was higher than that in model group(79.13pg/ml)(P<0.05).Conclusion:(1) The biphasic insulin aspart 40 injection developed by this method, has stable and uniform quality.(2) Biphasic insulin aspart 40 injection could reduce blood glucose amount in type I diabetic rats, lessen its typical symptoms, and could alleviate injury on islet tissue of type I diabetic rats.(3) Adopting the biphasic insulin aspart 40 injection to treat diabetic rats can increase the number of CD4+CD25+Treg cells, up-regulate the expression of Foxp3 m RNA, while reduce the IFN-γ of diabetic rats and up-regulate the IL-10 and TGF-β at the same time. The treatment effect is achieved through the cytokine and Treg cell immunity adjustment. |
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