Application of adiporon combined with steviol glycoside in preparation of drugs related to sepsis

Abstract

The application relates to the field of biological medicine, in particular to application of AdipoRon combined with steviol glycoside in preparation of a drug related to sepsis. The application provides application of AdipoRon combined with steviol glycoside in preparation of a drug related to sepsis, can solve the problem of sepsis skeletal muscle atrophy which is common in clinic but has no solution; and provides a drug related to sepsis containing AdipoRon and steviol glycoside, which is good in treatment effect, high in safety and easy to prepare.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of biological medicine, in particular to application of AdipoRon combined with steviol glycoside in preparation of a drug for sepsis. BACKGROUND

[0002] Sepsis is a common complication of trauma with extremely high mortality, affecting millions of people every year. Sepsis is defined as a life-threatening organ dysfunction syndrome caused by the host's immune response to infection, and is the leading cause of death in hospitalized patients. Sepsis can cause various organ damage, including brain damage, heart dysfunction, skeletal muscle atrophy, kidney damage, liver damage and lung damage. Among them, skeletal muscle atrophy accounts for a high proportion in patients with severe sepsis, not only increases the mortality of sepsis patients, but also worsens the prognosis of surviving patients. Skeletal muscle, as the largest tissue in the human body, provides structural support, movement, breathing and expression functions, so it is crucial to maintain skeletal muscle mass and integrity.

[0003] At present, there is no drug in the medical field to cure sepsis muscle atrophy, and prevention is mainly used in clinical treatment. SUMMARY

[0004] The purpose of the present application is to provide application of AdipoRon combined with steviol glycoside in preparation of a drug for sepsis, so as to solve at least one technical problem in the prior art.

[0005] The first aspect of the present application provides application of AdipoRon combined with steviol glycoside in preparation of a drug for sepsis.

[0006] AdipoRon is a kind of adiponectin receptor agonist, and steviol glycoside is a kind of glycoside existing in stevia rebaudiana leaves; when AdipoRon is combined with steviol glycoside, muscle atrophy caused by sepsis can be well treated.

[0007] Further, the drug for sepsis is a drug for treating skeletal muscle atrophy caused by sepsis. Preferably, the drug for sepsis is a drug for treating tibialis anterior muscle atrophy caused by sepsis or a drug for treating gastrocnemius muscle atrophy caused by sepsis.

[0008] The second aspect of the present application provides a drug for sepsis, which comprises AdipoRon and steviol glycoside.

[0009] Steviol glycoside is a kind of natural glycoside, which has been approved by the Food and Agriculture Organization of the United Nations and the World Organization Expert Committee since 2006 as a food sweetener additive. Adiponectin receptor agonist AdipoRon has the advantages of being orally taken, having remarkable effect and high serum stability. Therefore, the drug for sepsis has high safety, is easy to prepare, and has a broad application prospect.

[0010] Further, the mass ratio of AdipoRon to steviol glycosides is 10-60:10-60. Preferably, the mass ratio of AdipoRon to steviol glycosides is 20-40:20-40. Most preferably, the mass ratio of AdipoRon to steviol glycosides is 40:40.

[0011] Furthermore, the sepsis-related drugs also include pharmaceutically acceptable excipients.

[0012] Further, the pharmaceutically acceptable excipients are one or more of starch, lactose, microcrystalline cellulose, β-cyclodextrin, sodium carboxymethyl starch, water, physiological saline, DMSO, PEG300, and Tween80.

[0013] Furthermore, the sepsis-related drug is in the form of an oral preparation. For example, the sepsis-related drug may be in the form of an oral tablet, oral pill, oral solution, oral suspension, or oral powder.

[0014] The technical solutions of the embodiments of the present invention have the following beneficial effects:

[0015] This invention provides the application of AdipoRon in combination with steviol glycosides in the preparation of sepsis-related drugs, which can solve the common but intractable problem of skeletal muscle atrophy in sepsis; and provides sepsis-related drugs containing AdipoRon and steviol glycosides, which have good therapeutic effects, high safety and are easy to prepare. Attached Figure Description

[0016] Figure 1 This is a diagram showing the HE staining results of the tibialis anterior muscle in mice in the sham-operated group of this invention.

[0017] Figure 2 This is a diagram showing the HE staining results of the tibialis anterior muscle in mice in the cecal ligation and puncture group in this embodiment of the invention.

[0018] Figure 3 This is a diagram showing the HE staining results of the tibialis anterior muscle in mice treated with the combined drugs in this embodiment of the invention.

[0019] Figure 4 This is a diagram showing the HE staining results of the tibialis anterior muscle in mice treated with AdipoRon in this embodiment of the invention.

[0020] Figure 5 This is a diagram showing the HE staining results of the tibialis anterior muscle in mice treated with stevioside in this embodiment of the invention.

[0021] Figure 6 This is a statistical diagram of the cross-sectional area of ​​the tibialis anterior muscle fibers in each group of mice in the embodiments of the present invention.

[0022] Figure 7 This is a diagram showing the HE staining results of the gastrocnemius muscle of mice in the sham-operated group in this embodiment of the invention.

[0023] Figure 8 This is a diagram showing the HE staining results of the gastrocnemius muscle of mice in the cecal ligation and puncture group in this embodiment of the invention.

[0024] Figure 9 This is a diagram showing the HE staining results of the gastrocnemius muscle of mice in the combined drug treatment group in this embodiment of the invention.

[0025] Figure 10 This is a diagram showing the HE staining results of the gastrocnemius muscle of mice in the AdipoRon treatment group in this embodiment of the invention.

[0026] Figure 11 This is a diagram showing the HE staining results of the gastrocnemius muscle of mice in the stevioside treatment group in this embodiment of the invention.

[0027] Figure 12 This is a statistical diagram of the cross-sectional area of ​​the gastrocnemius muscle fibers in each group of mice in the embodiments of the present invention.

[0028] Figure 13 This is a diagram showing the results of detecting mouse tibialis anterior muscle tissue using Western blotting in an embodiment of the present invention.

[0029] Figure 14 This is a statistical chart showing the results of detecting mouse tibialis anterior muscle tissue using Western blotting in an embodiment of the present invention. Detailed Implementation

[0030] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0031] Adiponectin (ApN) is a hormone secreted in large quantities by adipose tissue and circulating in the blood. It promotes insulin sensitization, accelerates fat burning, and has anti-atherosclerotic effects, effectively combating various metabolic disorders. However, as a large protein molecule, the structure and function of adiponectin are not fully understood, making it difficult to develop into a drug. Furthermore, the poor water solubility of its C-terminal domain and larger peptide segments necessitates injection administration, hindering the implementation of clinical treatment strategies based on intact adiponectin. To overcome these limitations, researchers have identified several small-molecule peptides that can target the adiponectin receptor and mimic its function—adiponectin receptor agonists. Among them, the orally bioactive synthetic small molecule AdipoRon is the most promising, as it can mimic the main functions of adiponectin and is an ideal therapeutic alternative with broad application prospects.

[0032] Stevia is a low-calorie sweet plant and a natural source of biological sweeteners. Its leaves contain zero-calorie steviol glycosides (STE), which also have anti-inflammatory properties. At the same time, the high economic value of steviol glycosides makes stevia an ideal crop for large-scale cultivation.

[0033] In view of the current situation where there is no cure for skeletal muscle atrophy caused by sepsis, this invention provides the application of AdipoRon in combination with steviol glycosides in the preparation of sepsis-related drugs, based on the functions of AdipoRon and steviol glycosides.

[0034] The following experiments verified the effect of AdipoRon combined with steviol glycosides. AdipoRon was purchased from Selleckchem, and steviol glycosides were purchased from MedChemExpress. The mice used were male C57BL / 6 mice, purchased from Nanchang Kangxuhe Biotechnology Co., Ltd.

[0035] Mice were randomly divided into the following groups: sham operation group (SC), cecal ligation and puncture group (CLP), solvent control group (SC-solvent), AdipoRon treatment group (CLP-AdipoRon), steviol glycoside treatment group (CLP-STE), and combination drug treatment group (CLP-combination), and were treated as follows:

[0036] Sham surgery was performed on mice in the SC group;

[0037] CLP group mice underwent cecal ligation and puncture.

[0038] Mice in the SC-solvent group were administered a mixed solvent (10% DMSO, 40% PEG300, 5% Tween 80, and 45% saline) by gavage for 7 consecutive days (once a day) and underwent sham surgery.

[0039] CLP-AdipoRon mice were administered AdipoRon by gavage for 7 consecutive days (once a day) and underwent cecal ligation and puncture. The administration method of AdipoRon was as follows: it was dissolved in a mixed solvent (10% DMSO, 40% PEG300, 5% Tween 80, 45% physiological saline) and administered by gavage for 7 consecutive days (once a day) before cecal ligation and puncture, at a dose of 40 mg / kg.

[0040] CLP-STE mice were administered steviol glycosides by gavage for 7 consecutive days (once a day) and underwent cecal ligation and puncture. The administration method of steviol glycosides was as follows: it was dissolved in a mixed solvent (10% DMSO, 40% PEG300, 5% Tween 80, 45% physiological saline) and administered by gavage for 7 consecutive days (once a day) before cecal ligation and puncture, at a dose of 40 mg / kg.

[0041] Mice in the CLP-combination group were administered the combination drug (AdipoRon, steviol glycosides, and a solvent) by gavage for 7 consecutive days (once daily), followed by cecal ligation and puncture. AdipoRon was administered by dissolving it in a mixed solvent (10% DMSO, 40% PEG300, 5% Tween 80, and 45% physiological saline) and then administered by gavage for 7 consecutive days (once daily) before cecal ligation and puncture. Steviol glycosides were administered by dissolving them in a mixed solvent (10% DMSO, 40% PEG300, 5% Tween 80, and 45% physiological saline) and then administered by gavage for 7 consecutive days (once daily) before cecal ligation and puncture. The CLP-combination group was further divided into multiple dosage groups, as shown in Table 1.

[0042] Table 1. Dosage of the combined drug treatment group in mice

[0043]

[0044] A mouse model of septic muscular atrophy was established by cecal ligation and puncture: After fasting overnight, the mice were anesthetized with sodium pentobarbital. A 1.5 cm incision was made in the midline of the abdomen to expose the cecum. The same location was selected for ligation, and the cecal wall was punctured with a 2 mL syringe needle to allow a small amount of contents to spill out. The cecum was then returned to the abdominal cavity and sutured. The wound was treated with povidone-iodine postoperatively, and the mice were allowed to drink water. They were allowed to eat again after 12 hours.

[0045] The sham surgery method was as follows: After fasting overnight, the mice were anesthetized with sodium pentobarbital, and a 1.5 cm incision was made in the midline of the abdomen. The cecum was located and carefully separated, and then placed back into the abdominal cavity and sutured. After the operation, the wound was treated with povidone-iodine, water was restored, and the mice were fed again after 12 hours.

[0046] Mice were sacrificed 48 hours after surgery. The tibialis anterior and gastrocnemius muscles were separated, fixed in 4% paraformaldehyde solution for 24 hours, rinsed with water, and then dehydrated until transparent. They were then embedded in paraffin and cut into paraffin sections about 4 µm thick. After dewaxing, they were stained with HE reagent, dehydrated, sealed with glue, and placed under a microscope to observe pathological changes and take images.

[0047] Three sets of HE staining results were collected for each group. The cross-sectional area of ​​100 muscle fibers in each set was counted and the average value was taken. Then, a bar chart was made based on these data.

[0048] from Figures 1-5 It can be seen that the tibialis anterior muscle fibers of mice after the CLP group model atrophied and the cross-sectional area of ​​the muscle fibers decreased, indicating that the sepsis model was successful. The effects of different drug combinations were different, with the C group mice having the largest cross-sectional area of ​​muscle fibers and the best treatment effect.

[0049] Table 2 shows the statistical data of the cross-sectional area of ​​the tibialis anterior muscle fibers in each group of mice. Figure 6 The chart shows the statistical cross-sectional area of ​​tibialis anterior muscle fibers in each group of mice. A p-value < 0.05 was considered statistically significant; * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, and ns indicates p > 0.05 (no statistically significant difference). It can be seen that the cross-sectional area of ​​tibialis anterior muscle fibers in mice in groups B (20 mg / kg AdipoRon + 40 mg / kg STE), C (40 mg / kg AdipoRon + 40 mg / kg STE), and D (40 mg / kg AdipoRon + 20 mg / kg STE) was significantly higher than that in the cecal ligation and puncture group (CLP), with group C having the largest cross-sectional area. Furthermore, the cross-sectional area of ​​tibialis anterior muscle fibers in group C was significantly higher than that in the AdipoRon treatment group (CLP-AdipoRon) and the steviol glycoside treatment group (CLP-STE).

[0050] Table 2. Statistical data on the cross-sectional area of ​​the tibialis anterior muscle fibers in each group of mice.

[0051]

[0052] from Figures 7-11 It can be seen that the gastrocnemius muscle fibers of mice in the CLP group atrophied and the cross-sectional area of ​​the muscle fibers decreased after the modeling, indicating that the sepsis model was successful. The effects of different drug combinations were different, with the C group mice having the largest cross-sectional area of ​​muscle fibers and the best treatment effect.

[0053] Table 3 shows the statistical data of the cross-sectional area of ​​the gastrocnemius muscle fibers in each group of mice. Figure 12The chart shows the statistical distribution of gastrocnemius muscle fiber cross-sectional area in each group of mice. It can be seen that the gastrocnemius muscle fiber cross-sectional area in group B (treated with 20 mg / kg AdipoRon + 40 mg / kg STE), group C (treated with 40 mg / kg AdipoRon + 40 mg / kg STE), and group D (treated with 40 mg / kg AdipoRon + 20 mg / kg STE) was significantly higher than that in the cecal ligation and puncture group (CLP), with group C exhibiting the largest gastrocnemius muscle fiber cross-sectional area. Furthermore, the gastrocnemius muscle fiber cross-sectional area in group C was significantly higher than that in the AdipoRon treatment group (CLP-AdipoRon) and the steviol glycoside treatment group (CLP-STE).

[0054] Table 3 Statistical data on the cross-sectional area of ​​gastrocnemius muscle fibers in each group of mice

[0055]

[0056] After analyzing the grayscale values ​​of the three sets of Western blot results using ImageJ, the results were normalized using the study gene / internal reference gene to obtain the relative expression level of p-Akt protein for each set of proteins, and then statistically analyzed or plotted.

[0057] Table 4 shows the relative expression levels of p-Akt protein in each group of mice. Figure 13 This image shows the results of Western blot analysis of mouse tibialis anterior muscle tissue. Figure 14 This is a statistical graph showing the results of Western blot analysis of mouse tibialis anterior muscle tissue. It can be seen that the combination of AdipoRon and steviol glycosides may improve septic skeletal muscle atrophy by activating the PI3K / Akt pathway.

[0058] Table 4. Relative expression levels of p-Akt protein in mice of each group

[0059]

[0060] In summary, combining the HE staining results of the tibialis anterior and gastrocnemius muscles and the statistical results of the muscle fiber cross-sectional area, it can be concluded that AdipoRon and steviol glycosides have a synergistic effect. The combination of 40 mg / kg AdipoRon + 40 mg / kg STE can effectively improve skeletal muscle atrophy in septic mice. In addition, the expression of p-Akt protein in skeletal muscle samples from mice in the sham-operated group, solvent control group, cecal ligation and puncture group, and combination drug treatment group was examined to investigate the mechanism of action of adiponectin. The results indicate that the combination of AdipoRon and steviol glycosides may improve sepsis-induced skeletal muscle atrophy by activating the PI3K / Akt pathway.

[0061] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any form or substance. It should be noted that those skilled in the art can make various improvements and additions without departing from the method of the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention. Any modifications, alterations, and equivalent changes made by those skilled in the art based on the above-disclosed technical content without departing from the spirit and scope of the present invention are equivalent embodiments of the present invention. Furthermore, any modifications, alterations, and evolutions made to the above embodiments based on the essential technology of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. The application of AdipoRon in combination with steviol glycosides in the preparation of sepsis-related drugs, characterized in that, The sepsis-related drug is a drug for treating skeletal muscle atrophy caused by sepsis, and the mass ratio of AdipoRon to steviol glycoside is 40:

40.

2. The application according to claim 1, characterized in that, The sepsis-related drugs are drugs for treating tibial muscle atrophy caused by sepsis or drugs for treating gastrocnemius muscle atrophy caused by sepsis.

3. A sepsis-related drug, characterized in that, The drug includes AdipoRon and steviol glycosides, and the sepsis-related drug is a drug for treating skeletal muscle atrophy caused by sepsis, wherein the mass ratio of AdipoRon to steviol glycosides is 40:

40.

4. The sepsis-related drug according to claim 3, characterized in that, The sepsis-related drugs also include pharmaceutically acceptable excipients.

5. The sepsis-related drug according to claim 4, characterized in that, The pharmaceutically acceptable excipients are one or more of starch, lactose, microcrystalline cellulose, β-cyclodextrin, sodium carboxymethyl starch, water, physiological saline, DMSO, PEG300, and Tween80.

6. The sepsis-related drug according to claim 3, characterized in that, The sepsis-related drugs are in the form of oral preparations.

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