Application of RPN11 inhibitor Capzimin in treatment of type 2 diabetes mellitus

Abstract

The invention discloses an application of an RPN11 inhibitor Capzimin in treatment of type 2 diabetes mellitus. In an adult type 2 diabetes mouse model db / db, through intraperitoneal injection of Capzimin, it is found that type 2 diabetes related symptoms are obviously relieved, including reduction of intake blood glucose, reduction of fasting blood glucose, improvement of glucose tolerance and improvement of insulin sensitivity; therefore, the RPN11 inhibitor Capzimin can be used as a new intervention method and a treatment medicine for the type 2 diabetes mellitus, a new medicine is provided for effectively treating the type 2 diabetes mellitus clinically, and the RPN11 inhibitor Capzimin has good clinical application value and application prospects.

Description

Technical Field

[0001] The present invention relates to application of RPN11 inhibitor Capzimin in treating type 2 diabetes, and belongs to the technical field of biomedicine. Background Art

[0002] Type 2 diabetes mellitus (T2DM) has become a common disease worldwide, with a prevalence of more than 10% in my country. The main characteristics of type 2 diabetes include hyperglycemia, hyperinsulinemia, glucose intolerance, and insulin resistance. Identifying the pathogenic genes of type 2 diabetes and developing treatment strategies are both hot and difficult issues in type 2 diabetes.

[0003] RPN11 belongs to the deubiquitinase family, also known as POH1 or PSMD14, and works by removing ubiquitination modifications of target proteins and increasing the stability of target proteins. Previous studies have developed an RPN11-specific inhibitor, Capzimin, which was found to have an anti-tumor effect. However, there are no reports at home and abroad on whether Capzimin can be used as a therapeutic drug for type 2 diabetes. Summary of the invention

[0004] The purpose of the present invention is to provide an application of the RPN11 inhibitor Capzimin as a drug for treating type 2 diabetes.

[0005] In order to achieve the above object, the present invention provides the use of RPN11 inhibitor Capzimin in the preparation of a drug for treating type 2 diabetes. The CAS number of the RPN11 inhibitor Capzimin is: 2084868-04-0, and the chemical structure is as follows:

[0006]

[0007] Preferably, the medicament comprises a medically acceptable carrier and an effective amount of an active ingredient, and the active ingredient is Capzimin or a pharmaceutically acceptable salt thereof.

[0008] Preferably, the medicament improves type 2 diabetes by lowering blood glucose level and plasma insulin level, and improving glucose tolerance and insulin sensitivity.

[0009] The present invention also provides the use of RPN11 inhibitor Capzimin in the preparation of a drug for reducing blood sugar level and plasma insulin level. The CAS number of the RPN11 inhibitor Capzimin is: 2084868-04-0, and the chemical structure is as follows:

[0010]

[0011] Compared with the prior art, the present invention has the following beneficial effects:

[0012] The present invention found that the symptoms related to type 2 diabetes were significantly alleviated by intraperitoneal injection of Capzimin in an adult type 2 diabetes mouse model db / db, including reduced blood sugar during meals, reduced fasting blood sugar, improved glucose tolerance and increased insulin sensitivity; therefore, the RPN11 inhibitor Capzimin can provide a new intervention method and therapeutic drug for type 2 diabetes. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 :The mRNA expression of RPN11 in the liver of type 2 diabetic mice increased (A) and the protein expression increased (B);

[0014] Figure 2 :Capzimin reduces fed blood glucose (A) and fasting blood glucose (B) in type 2 diabetic mice;

[0015] Figure 3 :Capzimin improves glucose tolerance in type 2 diabetic mice;

[0016] Figure 4 :Capzimin improves insulin sensitivity in type 2 diabetic mice;

[0017] Figure 5 :Capzimin reduces the mRNA expression of gluconeogenic genes (G-6-pase and Pepck) in the liver of type 2 diabetic mice. DETAILED DESCRIPTION

[0018] In order to make the present invention more clearly understood, preferred embodiments are described in detail below with reference to the accompanying drawings.

[0019] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0020] Example

[0021] This example provides the use of the RPN11 inhibitor Capzimin as a drug for the treatment of type 2 diabetes

[0022] 1. Experimental materials and methods

[0023] 1.8-week-old db / db mice and littermate control normal mice were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. The mice were reared and experimented at the Department of Experimental Animal Science, School of Medicine, Shanghai Jiaotong University, and fed with a normal diet. The normal diet feed was purchased from Shanghai Slake Experimental Animal Co., Ltd., and the ingredients included: 10Kcal% fat, 70Kcal% carbohydrates, and 20Kcal% protein. All mice were euthanized after reaching the end point of the experiment. After anticoagulation and centrifugation of the whole blood, the supernatant was taken to obtain plasma and stored at -80°C. In subsequent experiments, it was used to detect plasma insulin content. Part of the fresh liver tissue was retained, quickly frozen in liquid nitrogen, and stored at -80°C. In subsequent experiments, RNA and protein were extracted, and real-time quantitative PCR experiments and protein immunoblotting experiments were carried out to detect the mRNA and protein expression abundance of the target gene.

[0024] 2. Glucose tolerance test, insulin sensitivity test and blood glucose concentration test: After fasting for 16 hours, mice were intraperitoneally injected with 20% D-glucose (2.0 mg / g body weight), and the blood glucose level at each time point was tested to carry out glucose tolerance test. After fasting for 6 hours, mice were intraperitoneally injected with 1.25 U / kg body weight of human insulin (purchased from Eli Lilly), and the blood glucose level at each time point was tested to carry out insulin sensitivity test. The tail of the mouse was cut open, 2-5 microliters of blood was squeezed out, and the blood glucose concentration was measured using an automatic blood glucose monitor (purchased from Johnson & Johnson).

[0025] 3. Detection of liver gene and protein expression: Real-time quantitative PCR was used to detect the mRNA expression of RPN11 gene and liver gluconeogenesis genes (PEPCK and G6Pase), and 36B4 gene was used as the internal reference of real-time quantitative PCR. Protein immunoblotting was used to detect RPN11 protein expression, and RPN11 antibody was purchased from Proteintech, catalog number 12059-1-AP; β-Actin protein was used as the internal reference of protein immunoblotting, and the antibody was purchased from Santa Cruz, catalog number sc-47778; α-Tubulin protein was used as the internal reference of protein immunoblotting, and the antibody was purchased from Proteintech, catalog number 11224-1-AP. The primers for real-time quantitative PCR experiments are as follows:

[0026] Mouse RPN11:

[0027] Forward: 5'-GGAGGTGGTGATAGCCGGTAT-3' (SEQ ID NO: 1);

[0028] Reverse:5'-TGGGTAATCCATAGAGCCCAG-3' (SEQ ID NO: 2);

[0029] Mouse 36B4:

[0030] Forward: 5'-AGATTCGGGATATGCTGTTGGC-3' (SEQ ID NO: 3);

[0031] Reverse:5'-TCGGGTCCTAGACCAGTGTTC-3' (SEQ ID NO: 4);

[0032] Mouse Pepck:

[0033] Forward: 5'-CTGCATAACGGTCTGGACTTC-3' (SEQ ID NO: 5);

[0034] Reverse:5'-CAGCAACTGCCCGTACTCC-3' (SEQ ID NO: 6);

[0035] Mouse G-6-pase:

[0036] Forward: 5'-CGACTCGCTATCTCCAAGTGA-3' (SEQ ID NO: 7);

[0037] Reverse:5'-GTTGAACCAGTCTCCGACCA-3' (SEQ ID NO: 8);

[0038] 4. The RPN11 inhibitor Capzimin is a conventional commercial reagent with a CAS number of 2084868-04-0 and a chemical structure as shown below:

[0039]

[0040] 5. Statistical analysis: In this study, all numerical variables were expressed as mean + standard error. Two-tailed Student-t test was used to compare the variables between the two groups. When P < 0.05, the two groups were considered to have statistically significant differences. * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001.

[0041] (II) Experimental results

[0042] 1. Increased expression of RPN11 in liver tissue of type 2 diabetic mouse model (db / db):

[0043] The db / db mouse is a leptin receptor gene-deficient mouse that exhibits hyperglycemia and insulin resistance. It is a common animal model for studying type 2 diabetes and is widely used in research and literature at home and abroad. We euthanized 8-week-old db / db male mice and normal control mice in the same litter, and took liver tissues from both groups to perform gene expression detection by quantitative real-time PCR and Western Blots. The results showed that compared with the normal control group mice, the messenger RNA (mRNA) and protein expression of RPN11 in the liver of db / db mice were significantly increased ( Figure 1 A and 1B). The results showed that RPN11 expression was increased in the liver tissue of the type 2 diabetic mouse model.

[0044] 2. RPN11 inhibitor Capzimin improves type 2 diabetes:

[0045] Eight-week-old db / db mice were randomly divided into two groups, with 8 mice in each group. They were given intraperitoneal injections of the RPN11 inhibitor Capzimin (2.5 mg / kg) or the control solvent (corn oil) once a day for 2 weeks. During this period, the mice's eating blood glucose and fasting blood glucose levels were tested, and glucose tolerance and insulin sensitivity tests were performed. After 2 weeks, the mice were euthanized, and liver tissues were taken to detect gluconeogenesis gene expression. The results showed that compared with the control group mice, the eating blood glucose and fasting blood glucose of the mice in the Capzimin treatment group decreased ( Figure 2 A and 2B), improved glucose tolerance ( Figure 3 ), improved insulin sensitivity ( Figure 4 ), the messenger RNA (mRNA) expression abundance of hepatic gluconeogenesis genes G-6-pase and Pepck was reduced ( Figure 5 The above results indicate that the RPN11 inhibitor Capzimin has the effect of improving and treating type 2 diabetes.

[0046] The above description is only a preferred embodiment of the present invention and is not any formal or substantial limitation of the present invention. It should be pointed out that a person skilled in the art can make several improvements and supplements without departing from the present invention, and these improvements and supplements should also be regarded as the protection scope of the present invention.

Claims

1. The use of RPN11 inhibitor Capzimin in the preparation of a drug for treating type 2 diabetes, characterized in that: The CAS number of the RPN11 inhibitor Capzimin is: 2084868-04-0, and the chemical structure is as follows:

2. The use according to claim 1, characterized in that The drug comprises a medically acceptable carrier and an effective amount of an active ingredient, wherein the active ingredient is Capzimin or a pharmaceutically acceptable salt thereof.

3. The use according to claim 1 or 2, characterized in that: The drug improves type 2 diabetes by lowering blood glucose levels and plasma insulin levels, and improving glucose tolerance and insulin sensitivity.

4. The use of RPN11 inhibitor Capzimin in the preparation of a drug for lowering blood sugar levels and plasma insulin levels, characterized in that: The CAS number of the RPN11 inhibitor Capzimin is: 2084868-04-0, and the chemical structure is as follows:

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