Application of anoectochilus glycoside combined with aspirin in the preparation of drugs for treating MAFLD

Abstract

The present invention discloses the application of anoectin combined with aspirin to prepare a drug for treating MAFLD, belongs to the field of biomedicine technology, and provides the application of anoectin combined with aspirin in preparing a drug for preventing and / or treating metabolic-associated fatty liver disease (MAFLD), and the application of anoectin combined with aspirin in preparing a drug for preventing and / or treating metabolic-associated fatty liver disease (MASH). The present invention uses anoectin and aspirin in combination to treat metabolic-associated fatty liver disease, which greatly alleviates liver lesions and liver damage, significantly reduces lipid droplet accumulation, reduces the degree of inflammatory cell infiltration, alleviates inflammation, and almost no collagen fiber hyperplasia is observed, compared with the use of anoectin alone or aspirin alone.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to the application of anoectochilus glycoside combined with aspirin in preparing a drug for treating MAFLD. Background Art

[0002] Metabolic-associated steatohepatitis (MASH) is a form of metabolic-associated fatty liver disease (MAFLD), which is mainly manifested by fat deposition in the liver, accompanied by inflammation and liver cell damage. The occurrence of MAFLD is closely related to metabolic syndromes such as obesity, type 2 diabetes, dyslipidemia, and hypertension. These metabolic abnormalities lead to the accumulation of fat in the liver, which in turn causes liver inflammation and liver cell damage. If not effectively intervened, it may further develop into liver fibrosis or even cirrhosis.

[0003] The specific pathogenesis of MASH has not been fully elucidated, but it is known that it involves multiple aspects, including insulin resistance, abnormal lipid metabolism, oxidative stress, and endoplasmic reticulum stress. These factors work together to lead to liver fat deposition, aggravated inflammatory response, and liver cell damage. At present, clinical treatment drugs for MASH are mainly focused on therapeutic targets targeting metabolism, inflammation, or fibrosis. Although these drugs can relieve some symptoms, such as reducing the degree of fibrosis, they cannot completely cure MASH. At the same time, drug treatment is often accompanied by strong adverse reactions, which limits its long-term effectiveness and safety. Therefore, there is an urgent need to find new treatment strategies that can more effectively solve the fundamental problems of MASH.

[0004] In the process of MAFLD developing into MASH, the main features are the pathological changes in the liver and the gradual aggravation of the accompanying metabolic and inflammatory responses. As the disease progresses, fatty degeneration in the liver is further aggravated, and the fat droplets in the hepatocytes increase and their size increases, resulting in severe damage to the structure and function of the hepatocytes. In the MASH stage, in addition to fatty degeneration, hepatocytes will also experience ballooning, accompanied by lobular inflammation. Inflammatory cells (such as neutrophils and lymphocytes) infiltrate in the hepatic lobules, forming local inflammatory foci, further aggravating the damage and necrosis of hepatocytes. These pathological changes make the treatment of MASH more complex and challenging, so there is an urgent need to find effective treatment strategies targeting the pathological mechanisms.

[0005] Anoectin is a natural chemical component extracted from the plant of Anoectinus roxburghii and belongs to the class of alkaloid compounds. Anoectin is a traditional Chinese medicinal material with extremely high medicinal value. Anoectin is the most important component of Anoectin. It has been proven to have multiple biological activities such as anti-inflammatory, antioxidant, and immunomodulatory. Therefore, it has attracted much attention in traditional medicine and modern medical research. Studies have shown that anoectin can regulate multiple physiological functions in the body and may have a positive effect on improving immunity, delaying aging, and promoting metabolism. Therefore, anoectin can be used to develop health products and potential therapeutic drugs. Since the plants from which anoectin is derived are rare and there are few existing studies, our research is of great significance and can fill the gap in this field and provide a scientific basis for the development of its medicinal potential.

[0006] Aspirin is currently widely used to prevent cardiovascular disease, especially to reduce the risk of heart attack and stroke, because it can inhibit platelet aggregation and reduce the probability of thrombosis. Its relationship with MASH has attracted the attention of researchers in recent years. Aspirin inhibits cyclooxygenase (COX) activity, reduces the production of inflammatory factors, reduces platelet aggregation, and improves lipid metabolism. These effects not only help prevent and treat cardiovascular disease, but may also have a potential positive effect on the treatment of fatty liver by reducing liver inflammation, improving metabolic disorders and slowing down liver fat accumulation. Summary of the invention

[0007] In order to solve the above technical problems, the present invention proposes the use of roxburghii glycoside and aspirin in combination to prepare a drug for treating MAFLD. The combined use of roxburghii glycoside and aspirin can significantly reduce liver fat accumulation, can more efficiently exert the therapeutic effect, and significantly reduce liver lesions and inflammation.

[0008] To achieve the above objectives, the present invention provides the use of anoectochiloside in combination with aspirin in the preparation of a drug for preventing and / or treating metabolism-related fatty liver disease.

[0009] Preferably, the mass ratio of acanthoside to aspirin in the drug is 8:1.

[0010] The present invention also provides a medicine for preventing and / or treating metabolism-related fatty liver disease, wherein the effective ingredients of the medicine include anoectochilus glycoside and aspirin.

[0011] Preferably, the mass ratio of acanthoside to aspirin in the drug is 8:1.

[0012] Preferably, the drug further comprises a pharmaceutically acceptable excipient.

[0013] The present invention also provides the use of anoectochilus glycoside combined with aspirin in the preparation of a drug for preventing and / or treating metabolism-related fatty hepatitis.

[0014] Preferably, the mass ratio of acanthoside to aspirin in the drug is 8:1.

[0015] The present invention also provides a medicine for preventing and / or treating metabolism-related fatty hepatitis, wherein the effective ingredients of the medicine include anoectochilus glycoside and aspirin.

[0016] Preferably, the mass ratio of acanthoside to aspirin in the drug is 8:1.

[0017] Preferably, the drug further comprises a pharmaceutically acceptable excipient.

[0018] Compared with the prior art, the present invention has the following advantages and technical effects:

[0019] The present invention uses anoectochilus glycoside and aspirin in combination to treat metabolism-related fatty hepatitis. Compared with the use of anoectochilus glycoside alone or the use of aspirin alone, it greatly alleviates liver lesions and liver damage, significantly reduces lipid droplet accumulation, reduces the infiltration of inflammatory cells, alleviates inflammation, and almost no collagen fiber hyperplasia is observed. In addition, peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) and silent information regulator 1 (SIRT1) play an important role in the metabolic regulation and anti-oxidative stress of MASH. PGC-1α is a key regulator of mitochondrial biogenesis and fatty acid oxidation, which can promote energy metabolism and improve liver lipid accumulation. SIRT1 is an upstream regulator of PGC-1α, which can participate in the regulation of energy metabolism, anti-oxidative stress and inflammatory response by regulating the activity of PGC-1α. Studies have shown that in MASH, the expression of PGC-1α and SIRT1 is usually inhibited, leading to impaired mitochondrial function, lipid metabolism disorders and aggravated oxidative stress. The combined use of roxburghii glycoside and aspirin significantly improved the loss of PGC-1α and SIRT1, effectively promoted the expression of PGC-1α and SIRT1, and PGC-1α was close to returning to normal. Furthermore, Ym-1 and Fizz1 are one of the hallmark molecules of M2 polarization of macrophages. In MASH, the expression of Fizz1 increases with the progression of the disease, and its overexpression even promotes the process of fibrosis. Ym-1 and Fizz1 are almost undetectable in normal mouse liver tissue, but significantly increased in the mouse MASH model. When anoectochilus glycosides and aspirin were used in combination, Ym-1 and Fizz1 were close to normal, with obvious improvement, indicating that the M2 polarization of mouse liver cells was alleviated after combined drug treatment, confirming that the combination of anoectochilus glycosides and aspirin has a significant synergistic effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] 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.

[0021] Figure 1 Gross view of mouse liver, CC represents the control group, HF represents the model group, FA represents the aspirin treatment group, FK represents the roxburghii glycoside treatment group, and FAK represents the roxburghii glycoside combined with aspirin treatment group;

[0022] Figure 2 H&E staining of paraffin sections of mouse liver tissue. In the figure, CC represents the control group, HF represents the model group, FA represents the aspirin treatment group, FK represents the roxburghii glycoside treatment group, and FAK represents the roxburghii glycoside combined with aspirin treatment group.

[0023] Figure 3 Masson staining of paraffin sections of mouse liver tissue. In the figure, CC represents the control group, HF represents the model group, FA represents the aspirin treatment group, FK represents the roxburghii glycoside treatment group, and FAK represents the roxburghii glycoside combined with aspirin treatment group.

[0024] Figure 4 is the serum alanine aminotransferase content of mice. In the figure, CC represents the control group, HF represents the model group, FA represents the aspirin treatment group, FK represents the anoectin treatment group, and FAK represents the anoectin combined with aspirin treatment group. “ns” represents no significant difference. " represents P ≤ 0.05," ” represents P ≤ 0.0001;

[0025] Figure 5 is the serum triglyceride content of mice. In the figure, CC represents the control group, HF represents the model group, FA represents the aspirin treatment group, FK represents the anoectin treatment group, and FAK represents the anoectin combined with aspirin treatment group. “ns” represents no significant difference. " represents P ≤ 0.05," " represents P ≤ 0.01," ” represents P ≤ 0.001;

[0026] Figure 6 is the content of serum low-density lipoprotein in mice. In the figure, CC represents the control group, HF represents the model group, FA represents the aspirin treatment group, FK represents the anoectin treatment group, and FAK represents the anoectin combined with aspirin treatment group. “ns” represents no significant difference. " represents P ≤ 0.05," ” represents P ≤ 0.0001;

[0027] Figure 7 is the relative mRNA level of tumor necrosis factor-α in mice. In the figure, CC represents the control group, HF represents the model group, FA represents the aspirin treatment group, FK represents the anoectin treatment group, and FAK represents the anoectin combined with aspirin treatment group. “ns” represents no significant difference. " represents P ≤ 0.05," ” represents P ≤ 0.01;

[0028] Figure 8 is the relative mRNA level of interleukin-1β in mice. In the figure, CC represents the control group, HF represents the model group, FA represents the aspirin treatment group, FK represents the anoectin treatment group, and FAK represents the anoectin combined with aspirin treatment group. “ns” represents no significant difference. " represents P ≤ 0.05, " " represents P ≤ 0.001," ” represents P ≤ 0.0001;

[0029] Fig. 9 The results of immunoblotting of mouse peroxisome proliferator-activated receptor γ coactivator-1α protein, CC represents the control group, HF represents the model group, FK represents the group treated with anoectin alone, and FAK represents the group treated with anoectin combined with aspirin;

[0030] Fig.10 is the relative expression of peroxisome proliferator-activated receptor γ coactivator-1α protein in mice. In the figure, CC represents the control group, HF represents the model group, FK represents the group treated with anoectin alone, and FAK represents the group treated with anoectin combined with aspirin. “ns” represents no significant difference, " represents P ≤ 0.05," ” represents P ≤ 0.01;

[0031] Fig.11 is the relative mRNA level of mouse peroxisome proliferator-activated receptor γ coactivator-1α. In the figure, CC represents the control group, HF represents the model group, FA represents the aspirin treatment group, FK represents the anoectin treatment group, and FAK represents the anoectin combined with aspirin treatment group. “ns” represents no significant difference. " represents P ≤ 0.05," " represents P ≤ 0.001," ” represents P ≤ 0.0001;

[0032] Fig.12 is the relative mRNA level of mouse silent information regulator 1. In the figure, CC represents the control group, HF represents the model group, FA represents the aspirin treatment group, FK represents the anoectin treatment group, and FAK represents the anoectin combined with aspirin treatment group. “ns” represents no significant difference. " represents P ≤ 0.05," ” represents P ≤ 0.0001;

[0033] Fig.13 The expression of PGC-1α was detected in paraffin sections of mouse liver tissue. In the figure, CC represents the control group, HF represents the model group, FA represents the aspirin treatment group, FK represents the anoectin treatment group, and FAK represents the anoectin combined with aspirin treatment group.

[0034] Fig.14 The results of immunoblotting of Ym-1 and Fizz1 proteins in mice. In the figure, CC represents the control group, HF represents the model group, FK represents the group treated with roxburghii glycoside alone, and FAK represents the group treated with roxburghii glycoside combined with aspirin. DETAILED DESCRIPTION

[0035] 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.

[0036] It should be understood that the terms described in the present invention are only for describing special embodiments 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. Each smaller range between the intermediate value in any stated value or stated range and 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.

[0037] 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.

[0038] 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 the skilled artisan. The present invention description and examples are exemplary only.

[0039] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0040] Sources of materials used in the present invention: Anoectochilus glycoside was purchased from MCE, and aspirin was purchased from Sigma.

[0041] Example 1

[0042] To verify the therapeutic effects of anointing agent and aspirin, a MASH model of C57 mice (8-week-old mice + 16-week Western diet) was established with a high-fat and high-sugar diet (HFD), and anointing agent 16 mg / kg and aspirin 2 mg / kg were gavaged for 25 days. Liver function, pathology, inflammatory factors and other related indicators were detected. The control group (CC) was a normal diet mouse group, the model group (HF) was a high-fat and high-sugar diet model mouse group, the aspirin treatment group (FA) was aspirin 4 mg / kg gavage treated mouse group, the anointing agent treatment group (FK) was a anointing agent 32 mg / kg gavage treated mouse group, and the aspirin combined with anointing agent treatment group (FAK) was aspirin 2 mg / kg + anointing agent 16 mg / kg gavage treated mouse group.

[0043] 1. Pathological changes

[0044] like Figure 1 As shown, in the mouse MASH model group (HF), it can be seen that the mouse's gross appearance shows that the liver has increased in volume, the capsule is tight and smooth, the edges are blunt, the texture is soft, the color is yellow, and it feels greasy. The liver of the MASH model mouse treated with aspirin alone (FA) was relieved to a certain extent compared with the MASH model, but the gross liver color was still yellow. The liver of the MASH model mouse treated with aureus glycoside alone (FK) and aureus glycoside + aspirin (FAK) was reddish brown, soft in texture, and normal in volume. The liver lesions in the aureus glycoside + aspirin co-treatment group (FAK) were greatly alleviated, and the combined treatment effect of the two drugs was significantly better than that of either alone.

[0045] like Figure 2As shown, H&E staining of liver tissue paraffin sections of mice in the model group (HF) showed that a large amount of lipids accumulated in hepatocytes to form lipid droplets and inflammatory cells infiltrated. The fatty degeneration of mice in the group treated with aspirin alone (FA) was partially improved compared with the HF group, but there were still some lipid droplets, and the improvement was not as good as that in the group treated with aureus glycoside alone (FK) and the group treated with aureus glycoside + aspirin (FAK). It can be observed that the accumulation of lipid droplets in the liver of mice in the group treated with aureus glycoside + aspirin (FAK) was significantly reduced compared with the model group (HF) and the groups treated with aspirin (FA) and aureus glycoside (FK) alone, and the infiltration of inflammatory cells was significantly reduced. The combined treatment effect of the two drugs was significantly better than that of the single drug.

[0046] like Figure 3 As shown in the figure, collagen fiber proliferation can be observed in Masson staining of liver tissue paraffin sections of mice in the model group (HF). The collagen fiber proliferation observed under Masson staining microscopy in mice treated with aspirin (FA) alone or anoectin (FK) alone was improved compared with the HF group, but compared with the anoectin + aspirin combined treatment group (FAK), the improvement of fibrosis was relatively poor. After aspirin + anoectin treatment, collagen fiber proliferation was almost not observed in the mouse liver.

[0047] 2. Serological testing

[0048] like Figure 4 As shown in the figure, serum alanine aminotransferase (ALT) detection showed that the ALT level of MASH model (HF) mice was significantly higher than that of normal mice (CC). However, the ALT level of MASH model mice decreased after aspirin (FA) or roxburghii glycoside (FK) treatment alone. It is worth noting that the ALT level almost returned to normal after the roxburghii glycoside + aspirin combined treatment group (FAK). This shows that the liver damage of mice in the roxburghii glycoside + aspirin treatment group (FAK) has been greatly alleviated, and the combined treatment effect of the two drugs is significantly better than that of either drug alone.

[0049] like Figure 5 and Figure 6 As shown, the detection of triglyceride (TG) and low-density lipoprotein (LDL-C) showed the same results as serum alanine aminotransferase (ALT). The levels of triglyceride (TG) and low-density lipoprotein (LDL-C) in mice in the aureofaunan glycoside + aspirin treatment (FAK) group were significantly decreased and almost returned to normal. The combined treatment effect of the two drugs was significantly better than that of either drug alone.

[0050] 3. Detection of changes in inflammatory factors

[0051] Tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) are key proinflammatory factors in the inflammatory response. TNF-α can induce the release of other inflammatory mediators, promote the recruitment and activation of inflammatory cells, and is an important initiator of the inflammatory cascade. IL-1β can enhance the inflammatory response and promote tissue damage. Both show abnormally high expression in MASH and are important driving factors of the inflammatory pathological process.

[0052] like Figure 7 and Figure 8 The qPCR results of mouse liver tissue shown in the figure show that the proinflammatory factors TNF-α and IL-1β in the model group (HF) mice were significantly higher than those in the normal group (CC), while after aspirin treatment alone (FA) and roxburghii glycoside treatment alone (FK), TNF-α and IL-1β were somewhat lower than those in the model (HF) group, and the TNF-α and IL-1β in the roxburghii glycoside + aspirin combined gavage treatment (FAK) group were significantly lower than those in the model (HF) group, indicating that the inflammation in the mice was relieved after drug treatment, and the combined treatment effect of the two drugs was significantly better than that of either drug alone.

[0053] 4. Mechanism of Action

[0054] At the specific mechanism level, the contents of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) and silent information regulator 1 (SIRT1) were detected. Both play an important role in the metabolic regulation and anti-oxidative stress of MASH. PGC-1α is a key regulator of mitochondrial biogenesis and fatty acid oxidation, which can promote energy metabolism and improve liver lipid accumulation. SIRT1 is an upstream regulator of PGC-1α, which can participate in the regulation of energy metabolism, anti-oxidative stress and inflammatory response by regulating the activity of PGC-1α. Studies have shown that in MASH, the expression of PGC-1α and SIRT1 is usually inhibited, leading to impaired mitochondrial function, lipid metabolism disorders and increased oxidative stress.

[0055] like Fig. 9 , Fig.10 , Fig.11 and Fig.12The results of the mouse liver tissue qPCR experiment showed that the PGC-1α and SIRT1 content of the model group (HF) mice was significantly lower than that of the control group (CC). After treatment with aurea glycoside (FK) alone, PGC-1α and SIRT1 recovered to a certain extent compared with the model group (HF), while the PGC-1α and SIRT1 of the mice in the aurea glycoside + aspirin combined gavage treatment (FAK) group were significantly improved compared with the model group (HF), and PGC-1α was close to normal. The above results show that after drug treatment, the loss of PGC-1α in mice was alleviated, among which the combined treatment effect of aurea glycoside + aspirin was significantly better than that of single use, and the recovery level was significant. Fig. 9 As shown, WB results showed the same conclusion.

[0056] The expression of PGC-1α was detected in paraffin sections of liver tissue of experimental mice. Fig.13 As shown, it can be observed that since PGC-1α is located in the cell nucleus, the cell nuclei of the model group (HF) are blue and negative, while those of the normal group (CC) are brown and positive. After aspirin treatment alone (FA) or roxburghii glycoside treatment alone (FK), some cell nuclei in the liver sections of mice are positive. The number of positive cells in mice treated with roxburghii glycoside + aspirin combined with oral administration (FAK) increased significantly, which is consistent with the above-mentioned mRNA and protein level detection results, further confirming that the combination of roxburghii glycoside + aspirin can more effectively promote the expression of PGC-1α, thereby exerting a synergistic effect.

[0057] 5. Detection of Macrophage M2 Polarization

[0058] Ym-1 and Fizz1 are one of the hallmark molecules of M2 polarization of macrophages. In MASH, the expression of Fizz1 increases with the progression of the disease, and its overexpression even promotes the process of fibrosis.

[0059] Mouse liver tissue WB experiment results are as follows Fig.14 As shown in the figure, Ym-1 and Fizz1 were almost undetectable in the liver tissue of normal mice (CC), but significantly increased in the model group (HF) mice. After treatment with aureus glycoside alone (FK), Ym-1 and Fizz1 showed a certain decrease compared with the HF group, but they had not completely disappeared. However, after aureus glycoside + aspirin combined gavage treatment (FAK), the Ym-1 and Fizz1 of mice were close to normal, with obvious improvement. The above results show that the M2 polarization of mouse hepatocytes was alleviated after drug treatment, among which the combined treatment of aureus glycoside + aspirin was significantly better than that of single use, and the recovery level was significant.

[0060] The above experimental results show that in the mouse MASH model, the use of aspirin alone or anoectin alone can reduce liver fatty lesions to a certain extent, while the combination of anoectin and aspirin can significantly reduce liver fat accumulation, can more efficiently exert the therapeutic effect, and significantly reduce liver lesions and inflammation. Therefore, the combined use of these two drugs provides a new potential treatment for MASH disease.

[0061] 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. Use of anoectochilus glycoside combined with aspirin in the preparation of a drug for preventing and / or treating metabolism-related fatty liver disease, characterized in that: The mass ratio of anoectochiloside to aspirin in the medicine is 8:

1.

2. A drug for preventing and / or treating metabolic-related fatty liver disease, characterized in that: The active ingredients of the medicine are composed of roxburghii glycoside and aspirin, and the mass ratio of roxburghii glycoside to aspirin in the medicine is 8:

1.

3. The drug according to claim 2, characterized in that: The drug also includes pharmaceutically acceptable excipients.

4. Use of anoectochilus glycoside in combination with aspirin in the preparation of a drug for preventing and / or treating metabolism-related fatty liver disease, wherein the mass ratio of anoectochilus glycoside to aspirin in the drug is 8:

1.

5. A drug for preventing and / or treating metabolic-related fatty hepatitis, characterized in that: The active ingredients of the medicine are composed of roxburghii glycoside and aspirin, and the mass ratio of roxburghii glycoside to aspirin in the medicine is 8:

1.

6. The drug according to claim 5, characterized in that: The drug also includes pharmaceutically acceptable excipients.

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