Application of iturin family lipopeptide in preparation of medicine for preventing and / or treating non-alcoholic fatty liver disease

Abstract

The invention discloses application of iturin family lipopeptide in preparation of a medicine for preventing and / or treating non-alcoholic fatty liver disease, and belongs to the field of biological medicine. Experiments prove that the iturin family lipopeptide can remarkably reduce weight gain and liver weight of an experimental model mouse, reduce the liver-weight ratio of the mouse, relieve HFD-induced hepatic distension and lipid droplet accumulation and improve hepatic lesion, and the effect of the iturin family lipopeptide is superior to that of a conventional liver protection drug silybin; in addition, according to verification of the invention, it is found that single iturin A or iturin B lipopeptide can also play a role in improving NAFLD of mice induced by high fat diet. The invention verifies the obvious effect and transformation potential of the iturin family lipopeptide in preventing and treating the non-alcoholic fatty liver disease, so that the iturin family lipopeptide can be applied to preparation of the medicine for preventing and treating the non-alcoholic fatty liver disease, and a new thought is provided for clinical treatment of preventing and treating the non-alcoholic fatty liver disease.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the use of iturin family lipopeptides in the preparation of drugs for preventing and / or treating non-alcoholic fatty liver disease. Background Art

[0002] Nonalcoholic fatty liver disease (NAFLD) is a common chronic liver disease worldwide and is becoming the most common indication for liver transplantation. NAFLD includes a series of histological features, ranging from simple fatty degeneration characterized by excessive lipid accumulation in hepatocytes to nonalcoholic fatty liver hepatitis associated with inflammation or fibrosis, and eventually develops into cirrhosis and liver malignancies. In addition, a large number of clinical studies have shown that NAFLD is not only closely related to type 2 diabetes, chronic kidney disease and cardiovascular disease, but is also a major risk factor for liver malignancies. It can be seen that NAFLD is serious and needs further attention. At present, some treatments for NAFLD have been proven to have good efficacy in clinical trials, such as pioglitazone, vitamin E, polyene phosphatidylcholine and other drugs commonly used in clinical treatment. However, the above drugs have some adverse reactions in clinical use. For example, taking pioglitazone can increase the patient's body weight by 3-5 kg, and also increase the risk of bladder cancer; vitamin E can increase the risk of prostate cancer and hemorrhagic stroke; vomiting, rash, diarrhea and other adverse reactions often occur during the use of polyene phosphatidylcholine, which seriously limits the promotion of the above drugs in clinical practice. Therefore, it is imperative to find safer and more effective drugs for the treatment of NAFLD.

[0003] Bioactive peptides are widely used active substances that can regulate the functions of animal bodies. They can protect the body from a variety of diseases and infections. The study of active peptides has been widely carried out in vitro (molecular and cellular levels) or in vivo (through animal experiments). So far, bioactive peptides have been used in drug research to solve clinical problems. Iturin lipopeptides are natural bioactive peptides that are both hydrophilic and lipophilic. They are mainly produced by Bacillus through the non-ribosomal peptide synthesis pathway (NRPS). Iturin family compounds were isolated from Bacillus subtilis by Delcambe and Devignat in 1957. So far, many lipopeptides belonging to the Iturins family have been discovered, such as Iturin A, B, C, D, E, bacillomycin D, F, L and mycosubtilin. Recent studies have found that other bacterial species that produce Iturins family lipopeptides include Bacillus, Bacillus amyloliquefaciens and Bacillus pumilus. The Iturins family are all cyclic heptapeptides connected to a β-amino fatty acid chain with a length of 14 to 17 carbons. The lipopeptides of the Iturins family have strong in vitro resistance to a variety of yeasts and fungi, but weak activity against bacteria and no antiviral activity. By comparing the different structures and different functions of various lipopeptides in the Iturins family, it was found that the position and length of the free carboxyl group have an effect on its biological activity, and as the number of carbon atoms in the β-amino fatty acid chain increases, the antibacterial activity of the active substance becomes stronger. There is no report in the prior art on the use of iturins family lipopeptides for the prevention and / or treatment of fatty liver or non-alcoholic fatty liver. Summary of the invention

[0004] The purpose of the present invention is to provide the use of iturin family lipopeptides in the preparation of drugs for preventing and / or treating non-alcoholic fatty liver disease, so as to solve the problems existing in the above-mentioned prior art. The present invention verifies the significant effect and transformation potential of iturin in preventing and treating non-alcoholic fatty liver disease, and provides a new idea for the clinical treatment of preventing and treating non-alcoholic fatty liver disease.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides the use of iturin family lipopeptides in the preparation of a drug for preventing and / or treating non-alcoholic fatty liver disease.

[0007] Furthermore, the iturin family lipopeptide comprises one or more of iturin A and iturin B.

[0008] The present invention also provides a medicine for preventing and / or treating non-alcoholic fatty liver disease, wherein the medicine comprises iturin family lipopeptides and pharmaceutically acceptable excipients.

[0009] Further, the iturin family lipopeptide comprises one or more of iturin A and iturin B. Further, the excipient comprises one or more of a diluent, a filler, an excipient, a binder, a wetting agent, a disintegrant, an absorption promoter, a surfactant, an adsorption carrier, a lubricant and a flavoring agent.

[0010] Furthermore, the dosage form of the drug includes injection, infusion, drops, powder, tablet, pill, granule, capsule, nasal drops or spray.

[0011] Furthermore, the drug is taken orally or parenterally.

[0012] The present invention discloses the following technical effects:

[0013] The present invention discloses a new application of iturin family lipopeptides, namely, application in the prevention or treatment of non-alcoholic fatty liver disease. It has been verified by a non-alcoholic fatty liver mouse model that iturin family lipopeptides can significantly reduce the weight gain and liver weight of experimental model mice, reduce their liver-to-weight ratio, alleviate HFD-induced liver swelling and lipid droplet accumulation, and improve liver lesions. The effect is better than the conventional liver protection drug silybin, and the present invention has been verified to find that a single iturin A or iturin B lipopeptide can also play a role in improving high-fat diet-induced NAFLD in mice. The present invention verifies the significant effect and conversion potential of iturin family lipopeptides in the prevention and treatment of non-alcoholic fatty liver disease. Therefore, iturin family lipopeptides can be used in the preparation of drugs for the prevention and treatment of non-alcoholic fatty liver disease, providing a new idea for the clinical treatment of the prevention and treatment of non-alcoholic fatty liver disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0015] Figure 1The figure is a validation diagram of the protective effect of iturin on the liver; A: grouping diagram; B: H&E staining diagram of the percentage of vacuolar areas in each group; C: Oil red O staining positive area statistics in each group; D: H&E and Oil red O staining; NCD: normal mouse diet control group; NCD+ITs: normal mouse diet treatment group; HFD: 60% high-fat diet control group; HFD+ITs: 60% high-fat diet treatment group; *: p < 0.05, **: p < 0.01, ***: p < 0.001;

[0016] Figure 2 Effects of iturin on HFD-induced non-alcoholic fatty liver mice; A: Schematic diagram of grouping; B: Statistical graph of weight gain of mice in each group; C: Statistical graph of weight of mice in each group; D: Statistical graph of liver weight of mice in each group; E: Statistical graph of the ratio of liver to body weight of mice in each group; F: Statistical graph of the proportion of vacuolar areas in H&E staining of each group; G: Statistical graph of Oil Red O positive areas in each group; H: H&E and Oil Red O staining; NCD: blank control group; HFD: control group; HFD+SI: positive control group; HFD+IT: iturin group; HFD+ITSI: iturin combined with silybin group; *: p<0.05, **: p<0.01, ***: p<0.001;

[0017] Figure 3 The effects of various components of iturin on HFD-induced non-alcoholic fatty liver mice; A: statistical graph of weight gain of mice in each group; B: statistical graph of vacuolar area ratio in H&E staining of each group; C: statistical graph of Oil Red O staining positive area in each group; NCD: blank control group; HFD: control group; HFD1-HFD12 are groups taking compounds 1-12 in Example 1 orally, respectively; **: p<0.01, ***: p<0.001, ****: p<0.0001. DETAILED DESCRIPTION

[0018] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0019] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0020] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only 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 associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0021] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0022] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0023] Example 1 Preparation of iturin

[0024] (1) Prepare 1.6 L of nutrient broth medium and dispense 800 mL into two 3 L shake flasks. Sterilize with high pressure steam and set aside.

[0025] The activated Bacillus Velez (deposit number: CGMCC No. 20130) was transferred to a shake flask containing NB medium at 32°C and 180 r / min. The culture period was set to 18 h. Then, it was inoculated into a 50 L fermenter containing 30 L NB medium at a 5% inoculum amount and cultured at 32°C and 120 r / min for 48 h.

[0026] (2) After the fermentation of the strain is completed, the fermentation liquid is collected and centrifuged at 10,000 r / min for 20 min. The fermentation supernatant is collected and the bacteria are removed. The filtrate is collected by filtering with a 0.22 μm filter membrane. The pH of the supernatant is adjusted to 2.0 with dilute hydrochloric acid and allowed to stand at 4°C overnight to allow the lipopeptide substances to be fully precipitated.

[0027] (3) After the lipopeptide substance was precipitated overnight, the mixture was centrifuged at 10,000 r / min for 20 min, the supernatant was removed, and the precipitate was dissolved with 300 mL of methanol. The mixture was centrifuged again at 10,000 r / min for 20 min. The methanol extraction and centrifugation operation were repeated three times. The extracts collected three times were combined, and the methanol in the extract was removed using a rotary evaporator at 30°C and 50 r / min. The water in the supernatant was removed using a freeze dryer. The obtained powder substance was the crude lipopeptide extract.

[0028] (4) The separation and purification of iturin was carried out by HPLC. The HPLC conditions were set as follows: mobile phase A: ultrapure water with 0.01% trifluoroacetic acid; mobile phase B: methanol mixture with 0.01% trifluoroacetic acid, and the sample volume was 3 mL. The above sample was loaded into a WondaSil C18 (4.6 mm × 150 mm) chromatographic column with a particle size of 5 μm, and eluted with a linear gradient of 10% to 90% acetonitrile, a flow rate of 3 mL / min, a detection wavelength of 214 nm, and a column temperature of 30°C. The components corresponding to 8-17 min were collected, and one portion was collected every 10 seconds. Mass spectrometry was used to confirm that the components of iturin obtained are shown in Table 1.

[0029] Table 1 Mass spectrometry results of purified products

[0030]

[0031] Example 2 Preventive effect of iturin on non-alcoholic fatty liver disease in mice

[0032] (1) 8-week-old C57BL / 6JGpt mice were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd. The mice were individually placed in ventilated cages, with 4 mice in each cage, provided with sufficient food and water, maintained at a temperature of 24.5±0.5℃, and followed a 12 / 12-hour light / dark cycle. After one week of adaptation, the mice were randomly divided into a normal mouse chow control group (NCD, fed with normal mouse chow and drinking normal water), a normal mouse chow treatment group (NCD-ITs, fed with normal mouse chow and drinking 50 mg / L iturin aqueous solution), a 60% high-fat diet control group (HFD, fed with 60% high-fat diet and drinking normal water) and a 60% high-fat diet treatment group (HFD+ITs, fed with 60% high-fat diet and drinking 50 mg / L iturin aqueous solution) (see Figure 1 Middle A), eight mice per group for 60 days.

[0033] (2) During the experiment, the clinical manifestations of mice in each group were closely observed. The coat color and mental state of the mice were observed and recorded daily. The weight of the mice was weighed and recorded daily. On the 60th day of the experiment, the mice were killed by dislocation of the neck and dissected to observe whether there was any damage or inflammation in the chest and abdominal cavities of the mice. The livers of the mice were removed and stored in 4% paraformaldehyde tissue fixative. Preparation of paraffin sections of liver tissue: Prepare the corresponding tissue sections according to the SOP of experiments such as pathological tissue sampling, fixation, embedding, paraffin sectioning, frozen sectioning, dehydration, and staining. Preparation of oil red sections of liver tissue: Prepare the corresponding tissue sections according to the SOP of experiments such as pathological tissue sampling, fixation, OCT embedding, frozen sectioning, and staining.

[0034] (3) Histological analysis by H&E and Oil Red O staining showed that mice receiving iturin solution showed significant liver protection, which was evident in both diet groups (NCD-ITs group and HFD+ITs group) ( Figure 1 BD). Compared with the HFD group, the number of vacuoles in liver tissue of mice in the HFD+ITs group was significantly reduced, the hepatocytes were arranged evenly, the amount of fat vacuoles was significantly reduced, and fat accumulation was significantly reduced. Compared with the NCD group, there was no significant change in the HE sections of the NCD+ITs group. The HE sections of the two groups showed that the hepatocytes were arranged evenly and regularly. Histological analysis of Oil Red O staining showed that the NCD+ITs group had less fat accumulation. Overall, these findings suggest that iturin has a hepatoprotective effect and can play a potential role in preventing and alleviating NAFLD.

[0035] Example 3 The therapeutic effect of iturin on non-alcoholic fatty liver disease in mice

[0036] 6-week-old C57BL / 6JGpt mice were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd. The mice were individually placed in ventilated cages, with 4 mice per cage, provided with sufficient food and water, maintained at a temperature of 24.5±0.5℃, and followed a 12 / 12-hour light / dark cycle. After one week of adaptation, they were treated with a 60% high-fat diet for 14 weeks to construct a NAFLD model.

[0037] After successful modeling, the mice were randomly divided into 5 groups (see Figure 2 Middle A): control group (HFD, NAFLD model mice orally administered with 0.9% normal saline), positive control group (HFD+SI, NAFLD model mice orally administered with 57 mg / kg silybin), iturin group (HFD+IT, NAFLD model mice orally administered with 14 mg / kg iturin), iturin combined with silybin group (HFD+ITSI, NAFLD model mice orally administered with 14 mg / kg iturin and 57 mg / kg silybin) and blank control group (NCD, normal mice orally administered with 0.9% normal saline). Each group included at least 8 mice for 4 weeks. Among them, silybin is the main active compound derived from milk thistle, which has anti-inflammatory, hepatoprotective, anti-fibrotic and antioxidant properties, and is used as a positive control drug.

[0038] In the HFD-fed mouse cohort, IT, SI, or ITSI all significantly reduced body weight gain compared with the control (HFD) mice treated with saline (SA) ( Figure 2 In addition, weight loss ( Figure 2 C) and liver weight ( Figure 2 D), and decreased liver-to-body weight ratio ( Figure 2Middle E). Histological analysis using H&E and Oil Red O staining showed that IT, SI, and ITSI attenuated HFD-induced liver swelling and lipid droplet accumulation, indicating that iturin has the potential to improve high-fat diet-induced NAFLD in mice ( Figure 2 FH), and to some extent was superior to the two treatments of silybin and iturin combined with silybin.

[0039] Example 4 Therapeutic effects of different components of iturin on non-alcoholic fatty liver disease in mice

[0040] 6-week-old C57BL / 6JGpt were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd. After C57 mice were adapted to the constant temperature animal room for one week, they were treated with a 60% high-fat diet for 14 weeks to construct NAFLD model mice.

[0041] After successful modeling, the mice were randomly divided into the following 14 groups:

[0042] NCD (blank control group): Normal mice were orally administered 0.9% saline,

[0043] HFD (control group): NAFLD model mice were orally administered 0.9% saline,

[0044] HFD1: NAFLD model mice were orally administered 14 mg / kg of compound 1 in Example 1.

[0045] HFD2: NAFLD model mice were orally administered 14 mg / kg of compound 2 in Example 1.

[0046] HFD3: NAFLD model mice were orally administered 14 mg / kg of compound 3 in Example 1.

[0047] HFD4: NAFLD model mice were orally administered 14 mg / kg of compound 4 in Example 1.

[0048] HFD5: NAFLD model mice were orally administered 14 mg / kg of compound 5 in Example 1.

[0049] HFD6: NAFLD model mice were orally administered 14 mg / kg of compound 6 in Example 1.

[0050] HFD7: NAFLD model mice were orally administered 14 mg / kg of compound 7 in Example 1.

[0051] HFD8: NAFLD model mice were orally administered 14 mg / kg of compound 8 in Example 1.

[0052] HFD9: NAFLD model mice were orally administered 14 mg / kg of compound 9 in Example 1.

[0053] HFD10: NAFLD model mice were orally administered 14 mg / kg of compound 10 in Example 1.

[0054] HFD11: NAFLD model mice were orally administered 14 mg / kg of compound 11 in Example 1.

[0055] HFD12: NAFLD model mice were orally administered 14 mg / kg of the compound 12 in Example 1.

[0056] The treatment lasted for 4 weeks. Each group included at least 8 mice.

[0057] Among the HFD-fed groups, HFD1, HFD2, HFD3, HFD4, HFD5, HFD6, HFD7, HFD8, HFD9, HFD10, HFD11, and HFD12 groups all significantly reduced body weight gain compared to saline-treated HFD mice ( Figure 3 Middle A). Histological analysis using H&E and Oil Red O staining showed that HFD1, HFD2, HFD3, HFD4, HFD5, HFD6, HFD7, HFD8, HFD9, HFD10, HFD11, and HFD12 groups all attenuated HFD-induced liver expansion and lipid droplet accumulation ( Figure 3 (B, C) showed that each component of iturin can improve NAFLD in mice induced by high-fat diet.

[0058] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Application of iturin family lipopeptides in the preparation of drugs for preventing and / or treating non-alcoholic fatty liver disease.

2. The use according to claim 1, characterized in that The iturin family lipopeptide comprises one or more of iturin A and iturin B.

3. A drug for preventing and / or treating non-alcoholic fatty liver disease, characterized in that: The medicine comprises iturin family lipopeptides and pharmaceutically acceptable excipients.

4. The drug according to claim 3, characterized in that The iturin family lipopeptide comprises one or more of iturin A and iturin B.

5. The drug according to claim 3, characterized in that The auxiliary materials include one or more of diluents, fillers, excipients, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers, lubricants and flavoring agents.

6. The drug according to claim 3, characterized in that The dosage form of the drug includes injection, infusion, drops, powder, tablet, pill, granule, capsule, nasal drops or spray.

7. The drug according to claim 3, characterized in that The drug can be taken orally or parenterally.

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