Application of anagloside C in preparation of medicine for treating diabetes mellitus

Abstract

The invention discloses application of anagloside C in preparation of a medicine for treating diabetes, and relates to the technical field of biological medicines. The research finds that after ANC administration treatment is carried out on a T2DM animal model db / db mouse, the blood glucose level is reduced in a dose-dependent manner, the insulin sensitivity is obviously increased, the lipid metabolism disorder is improved, and the ballooning and fatty degeneration accumulation conditions in the liver tissue are relieved; a Western blot detection result shows that the ANC has the effects of activating a db / db mouse liver tissue AMPK signal, inhibiting a TXNIP / NLRP3 inflammasome signal and protecting an insulin signal. The results show that ANC has a therapeutic effect on T2DM. On the basis, the ANC can inhibit TXNIP / NLRP3 inflammasome signals through AMPK dependence to improve liver insulin resistance, and therefore the effect of treating diabetes mellitus is achieved.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the application of angoroside C in the preparation of drugs for treating diabetes. Background Art

[0002] Diabetes is a group of diseases characterized by high blood sugar and related to impaired insulin secretion or its physiological effects. Among them, type 2 diabetes mellitus (T2DM) is the most common, accounting for about 90-95%. China has the largest number of diabetic patients in the world, and the prevalence rates of diabetes and prediabetes have reached 12.8% and 35.2% respectively. The prevalence of diabetes has greatly increased the burden on individuals and society and has become a major public health problem. The country is increasingly attaching importance to the role of traditional Chinese medicine in "inheriting the essence" and "innovating while maintaining integrity" in the construction of a healthy China. Therefore, it is of great significance to explore drugs for effectively preventing and treating diabetes from traditional Chinese medicine resources.

[0003] Insulin resistance (IR) refers to the pathological process in which insulin target organs and cells have reduced sensitivity to insulin, resulting in impaired insulin regulation of glucose and lipid metabolism. It includes the entire process from the initial stage of the body compensatorily secreting excessive insulin and gradually developing hyperinsulinemia to the stage of pancreatic islet dysfunction decompensation and the appearance of T2DM symptoms. Therefore, effectively improving IR is the key to treating T2DM. The occurrence and development of IR are related to metabolic inflammatory responses. A large number of studies have shown that the NLRP3 inflammasome is an important mediator of this inflammatory response. The NLRP3 inflammasome is a protein complex composed of NLRP3, ASC, and caspase-1. After the activation of the NLRP3 inflammasome, caspase-1 generates enzymatic catalytic activity through self-cleavage and cleaves pro-IL-1β to produce a large amount of active IL-1β; IL-1β acts on the cell itself or other cells, causing an inflammatory cascade amplification reaction, interfering with insulin signal transduction, and leading to the occurrence and development of IR.

[0004] The liver is one of the main target organs of insulin. Under the action of insulin, the liver maintains the homeostasis of glucose and lipid metabolism in the body by regulating activities such as hepatic glycogen synthesis and hepatic glucose output. However, in the state of IR, the regulation of liver insulin signaling is impaired, the ability of hepatocytes to take up and utilize glucose decreases, the level of gluconeogenesis increases, and lipid deposition and hepatic glucose output increase. Among them, the increase in hepatic glucose output is the main reason for the relatively high blood sugar (especially fasting blood sugar) levels in T2DM patients. Therefore, effectively improving liver IR is the key to treating T2DM.

[0005] Scrophularia ningpoensis Hemsl. is the root of the plant Scrophularia ningpoensis Hemsl. of the genus Scrophularia, which is mainly produced in Hubei, Sichuan and other places. According to traditional Chinese medicine theory, Scrophularia ningpoensis Hemsl. "nourishes yin and clears heat, and can treat consumptive thirst", and can be used alone or in combination with other traditional Chinese medicines for the treatment of diabetes. Modern research shows that the water decoction of Scrophularia ningpoensis Hemsl., polysaccharide components, etc. have therapeutic effects on experimental diabetic animals, but the mechanism of action and the material basis of pharmacodynamics are still unclear at present.

[0006] Angoroside C (ANC) is a phenylpropanoid glycoside compound contained in Scrophularia ningpoensis Hemsl., which has the effects of antioxidant, inhibiting the release of macrophage inflammatory cytokines and improving ventricular remodeling. It is one of the active ingredients of Scrophularia ningpoensis Hemsl., and its chemical structural formula is as follows:

[0007]

[0008] At present, there is no report on the application of Angoroside C in the treatment of diabetes. Summary of the Invention

[0009] The purpose of the present invention is to provide the application of Angoroside C in the preparation of drugs for the treatment of diabetes, so as to solve the problems existing in the above-mentioned prior art. The present invention finds that Angoroside C can improve hepatic insulin resistance by AMPK-dependent inhibition of TXNIP / NLRP3 inflammasome signaling, thereby playing a role in the treatment of diabetes.

[0010] To achieve the above purpose, the present invention provides the following solutions:

[0011] The present invention provides the application of Angoroside C in the preparation of drugs for the treatment of diabetes

[0012] Further, the diabetes is type 2 diabetes.

[0013] Further, the Angoroside C is the only active ingredient of the drug.

[0014] The present invention also provides a drug for the treatment of diabetes, and the active ingredient includes Angoroside C.

[0015] Further, the diabetes is type 2 diabetes.

[0016] Further, the Angoroside C is the only active ingredient of the drug.

[0017] Further, the drug also includes pharmaceutically acceptable excipients.

[0018] Further, the dosage form of the drug includes granules, tablets, powders, pills, capsules or oral liquids.

[0019] The present invention discloses the following technical effects:

[0020] The present invention's research found that after treatment with ANC in the db / db mouse, an animal model of T2DM, the blood glucose level decreased in a dose-dependent manner, insulin sensitivity increased significantly, lipid metabolism disorders were improved, and the accumulation of ballooning degeneration and steatosis in liver tissues was alleviated; the Western blot detection results showed that ANC had the effects of activating the AMPK signal, inhibiting the TXNIP / NLRP3 inflammasome signal, and protecting the insulin signal in the liver tissues of db / db mice. The above results indicate that ANC has a therapeutic effect on T2DM. Based on this, the present invention believes that ANC can improve hepatic insulin resistance by AMPK-dependent inhibition of the TXNIP / NLRP3 inflammasome signal, thereby playing a role in treating diabetes. Description of the Drawings

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0022] Figure 1 Detection results of blood glucose (A) and body weight (B) of mice in each experimental group;

[0023] Figure 2 Detection results of glucose tolerance (A) and insulin sensitivity (B) of mice in each experimental group;

[0024] Figure 3 Detection results of serum insulin level (A), HOMA-IR (B), glycogen content level in liver tissues (C), serum TC level (D), serum TG level (E), serum HDL-C level (F), serum LDL-C level (G), serum NEFA level (H), serum IL-1β level (I), and serum TNF-α level (J) of mice in each experimental group;

[0025] Figure 4 Detection results of TC and TG content levels (A), HE staining of liver tissues (B), steatosis score of liver tissues (C), lobular inflammation score of liver tissues (D), ballooning degeneration score of hepatocytes in liver tissues (E), and NAFLD activity score of liver tissues (F) of mice in each experimental group; The scale bar in B is 400 μm;

[0026] Figure 5The detection results of the phosphorylation levels of AMPK and ACC in the liver tissues of mice in each experimental group (A), the TXNIP / NLRP3 inflammasome signal in the liver tissues (B), and the insulin signal in the liver tissues (C). Detailed implementation manners

[0027] The various exemplary implementation manners of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0028] It should be understood that the terms described in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0029] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0030] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific implementation manners of the present invention specification, which are obvious to those skilled in the art. Other implementation manners obtained from the present invention specification are obvious to those skilled in the art. The present invention specification and examples are only exemplary.

[0031] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.

[0032] Example 1

[0033] 1. Experimental materials

[0034] ANC, normal saline, blood glucose test strips, insulin detection kits, metformin hydrochloride (Metformin, Met) tablets; kits for detecting the contents of insulin, liver glycogen, TC, TG, HDL-C, LDL-C, NEFA, IL-1β, and TNF-α; antibodies against AMPK, p-AMPK, ACC, p-ACC, TXNIP, NLRP3, Caspase-1, IL-1β, Akt, GSK3β, and β-actin.

[0035] Spontaneously diabetic C57BLKS / Leprdb (db / db) mice and wild-type control C57BLKS (WT) mice were purchased from Jiangsu Jicui Yakang Biotechnology Co., Ltd. All experimental procedures were approved by the Institutional Animal Ethics Review Board of our unit. The experiment began after the animals were adaptively fed for 1 week.

[0036] 2. Methods

[0037] 2.1 Experimental grouping and drug administration

[0038] Twelve C57BLKS mice were used as the wild-type (WT) control group, and 60 db / db mice were randomly divided into 5 groups:

[0039] Group 1 - WT group: Twelve C57BL / 6BSK mice were given an equal volume of normal saline by gavage as a control for 8 consecutive weeks.

[0040] Group 2 - db / db group: Twelve db / db mice were given an equal volume of normal saline by gavage as a control for 8 consecutive weeks.

[0041] Group 3 - db / db + Met group: Twelve db / db mice were given an equal volume of 300 mg / kg Met by gavage for 8 consecutive weeks.

[0042] Group 4 - db / db + low-dose ANC group: Twelve db / db mice were given an equal volume of 20 mg / kg ANC by gavage for 8 consecutive weeks.

[0043] Group 5 - db / db + medium-dose ANC group: Twelve db / db mice were given an equal volume of 40 mg / kg ANC by gavage for 8 consecutive weeks.

[0044] Group 6 - db / db + high-dose ANC group: Twelve db / db mice were given an equal volume of 80 mg / kg ANC by gavage for 8 consecutive weeks.

[0045] Among them, ANC was administered after being dissolved in normal saline.

[0046] During the administration period, the body weight and blood glucose of the animals were monitored weekly, and intraperitoneal glucose tolerance test (IPGTT) and insulin tolerance (IPITT) experiments were conducted. After 8 weeks, the mice were anesthetized and sacrificed under fasting conditions, blood samples were collected and serum was separated and stored at -80°C, and liver tissues were snap-frozen in liquid nitrogen and then stored at -80°C.

[0047] For the above serum samples, after thawing on ice, the levels of insulin, hepatic glycogen, blood lipids (TC, TG, HDL-C, LDL-C and NAFA) and the contents of serum inflammatory cytokines (IL-1β and TNF-α) were detected according to the methods described in the kit instructions. And the insulin resistance index (HOMA-IR) was calculated based on the insulin and fasting blood glucose values.

[0048] For the above frozen liver tissues, after homogenizing on ice, the contents of glycogen, TC and TG in the tissues were detected according to the methods described in the kit instructions, and the results were normalized by protein concentration. For the above frozen liver tissues, they were fixed with 10% formalin solution and then paraffin-embedded. Sections with a thickness of 5 μm were prepared and HE staining was performed. After observing and photographing under a microscope, the scores of hepatic steatosis, intra-lobular inflammation, ballooning degeneration of hepatocytes and NAFLD activity score items were calculated respectively. Another part of the above liver tissues was thawed on ice and proteins were extracted with RIPA lysis buffer, and then protein samples were prepared and the expression or activation levels of proteins such as AMPK, ACC, TXNIP, NLRP3, Caspase-1, IL-1β, Akt, GSK3β, etc. were detected by Western blot method, using β-actin as an internal reference.

[0049] 2.2 Data analysis

[0050] The experimental results were expressed as mean ± standard error (Mean ± SEM). One-way ANOVA was used for the means among multiple groups, and the q-test was performed by the SNK method for pairwise comparison. Statistical significance was indicated by P < 0.05, ** P < 0.01 vs. WT; # P < 0.05, ## P < 0.01 vs. db / db.

[0051] 3. Results

[0052] 3.1 Effects of ANC administration on blood glucose and body weight of db / db mice, an animal model of T2DM

[0053] As Figure 1 shown, ANC administration intervention could dose-dependently reduce the blood glucose of db / db mice, an animal model of T2DM, and had no obvious effect on body weight.

[0054] 3.2 Effects of ANC administration on glucose tolerance and insulin sensitivity of db / db mice, an animal model of T2DM

[0055] As Figure 2 shown, ANC administration intervention can dose-dependently improve glucose tolerance and insulin sensitivity in db / db mice, an animal model of T2DM.

[0056] 3.3 Effects of ANC administration on hyperinsulinemia, insulin resistance index, hepatic glycogen synthesis, blood lipid levels (TC, TG, HDL-C, LDL-C, and NAFA), and serum inflammatory cytokine levels (IL-1β and TNF-α) in db / db mice, an animal model of T2DM.

[0057] As Figure 3 shown, ANC intervention can alleviate hyperinsulinemia, reduce the insulin resistance index, increase hepatic glycogen synthesis, correct blood lipid level disorders (TC, TG, HDL-C, LDL-C, and NAFA), and reduce serum inflammatory cytokine levels (IL-1β and TNF-α) in db / db mice, an animal model of T2DM.

[0058] 3.4 Effects of ANC administration on lipid accumulation, hepatic steatosis, intra-lobular inflammation, ballooning degeneration of hepatocytes, and NAFLD activity score in the liver tissue of db / db mice, an animal model of T2DM

[0059] As Figure 4 shown, ANC intervention can reduce lipid accumulation in the liver tissue of db / db mice, an animal model of T2DM, and improve the scores of hepatic steatosis, intra-lobular inflammation, ballooning degeneration of hepatocytes, and NAFLD activity score.

[0060] 3.5 Effects of ANC administration on AMPK activity, TXNIP / NLRP3 inflammasome, and insulin signaling in the liver tissue of db / db mice, an animal model of T2DM

[0061] As Figure 5 shown, ANC has an improving effect on abnormal AMPK activity, TXNIP / NLRP3 inflammasome, and insulin signaling in the liver tissue of db / db mice, an animal model of T2DM.

[0062] The embodiments described above are only descriptions of the preferred modes of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention should fall within the protection scope determined by the claims of the present invention.

Claims

1. Use of angoroside C in the preparation of a medicament for treating diabetes.

2. The application according to claim 1, wherein The diabetes is type 2 diabetes.

3. The application according to claim 1, wherein The angoroside C is the only active ingredient of the medicament.

4. A drug for treating diabetes, characterized in that, The active ingredient includes angoroside C.

5. The drug according to claim 4, characterized in that, The diabetes is type 2 diabetes.

6. The drug according to claim 4, characterized in that, The angoroside C is the only active ingredient of the medicament.

7. The drug according to claim 4, wherein The medicament further comprises a pharmaceutically acceptable excipient.

8. The drug according to claim 7, characterized in that, The dosage form of the medicament includes granule, tablet, powder, pill, capsule or oral liquid.

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