Application of Hydrostatin-SN10, a selective TNFR1 antagonist peptide, in non-alcoholic fatty liver disease

Abstract

The present invention relates to the field of biomedicine, and specifically provides an application of a selective TNFR1 antagonist peptide Hydrostatin-SN10 in the treatment of non-alcoholic fatty liver disease. The selective TNFR1 antagonist peptide Hydrostatin-SN10 has an amino acid sequence as shown in SEQ ID NO: 2. The present invention verified its selective binding to TNFR1 and its anti-inflammatory activity in vitro and in vivo through in vitro experiments. The results showed that Hydrostatin-SN10 may exhibit good anti-inflammatory activity by selectively binding to TNFR1 and inhibiting the interaction between TNF-α and TNFR1. By establishing an animal model of non-alcoholic fatty liver disease induced by a high-fat and high-sugar diet (HFD), it was demonstrated that Hydrostatin-SN10 has the use of treating non-alcoholic fatty liver disease.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a selective TNFR1 antagonist peptide Hydrostatin-SN10 and its application in non-alcoholic fatty liver disease. Background Art

[0002] Non-alcoholic fatty liver disease (NAFLD) includes non-alcoholic fatty liver disease (NAFL), non-alcoholic steatohepatitis (NASH), and more severe cirrhosis and even liver cancer. It is the main cause of chronic liver disease and hepatocellular carcinoma. Big data analysis shows that NAFLD is associated with an increased risk of diabetes, cardiovascular events, heart failure, and extrahepatic cancer (Targher G, Lonardo A, Byrne CD. Nonalcoholic fatty liver disease and chronic vascular complications of diabetes mellitus [J]. Nature reviews endocrinology, 2018, 14(2): 99-114.). Globally, the prevalence of NAFLD in the general adult population is approximately 25%, while the prevalence of NAFLD in overweight and obese subjects is even higher, at approximately 40%-60%. The highest prevalence of nonalcoholic fatty liver disease (55%-70%) worldwide occurs in patients with diabetes (Chalasani N, Younossi Z, Lavine JE, et al. The diagnosis and management of nonalcoholic fatty liver disease: practice guidance from the American Association for the Study of Liver Diseases [J]. Hepatology, 2018, 67(1): 328-357.). As a result, NAFLD has become an epidemic, including obesity, diabetes, cardiovascular disease, and obesity- and diabetes-related cancers.

[0003] In view of the complex pathogenesis of NAFLD, the "multiple hit theory" proposes that the patient's dietary habits, living environment and genetic factors can lead to insulin resistance, obesity, proliferation of adipocytes and changes in intestinal flora, thereby leading to increased levels of free fatty acids and cholesterol in the blood, imbalance in fatty acid metabolism regulation and enhanced lipotoxicity (Rives C, Fougerat A, Ellero-Simatos S, et al. Oxidative stress in NAFLD: role of nutrients and food contaminants [J]. Biomolecules, 2020, 10 (12): 1702.); insulin resistance can increase the resynthesis of liver fat, induce fat decomposition to form more free fatty acids, promote the synthesis and secretion of adipokines and proinflammatory cytokines including tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6), and ultimately induce the formation of an inflammatory microenvironment in the liver (Safari Z, Gérard P. The links between the gut microbiome and non-alcoholic fatty liver disease (NAFLD) [J]. Cell Mol Life Sci, 2019, 76(8): 1541-1558.).

[0004] Tumor necrosis factor-α (TNF-α) is a type II transmembrane protein with a molecular weight of 26 kDa. It is a major mediator of apoptosis, inflammation, and immunity. It is involved in the pathogenesis of various human diseases, including sepsis, psoriasis, inflammatory bowel disease, and rheumatoid arthritis. Studies have shown that TNF-α is highly expressed in patients with nonalcoholic fatty liver disease, revealing that TNF-α is closely related to the occurrence and development of nonalcoholic fatty liver disease (Crespo J, Fern P, Hern M, et al. Gene expression of tumor necrosis factor [alpha] and TNF-receptors, p55 and p75, in nonalcoholic steatohepatitis patients [J]. Hepatology, 2001, 34(6): 1158-63.). The biological effects of TNF-α are primarily mediated through its downstream receptors, tumor necrosis factor receptor 1 (TNFR1) and tumor necrosis factor receptor 2 (TNFR2). TNFR1 plays a role in harmful pathways leading to inflammation, metabolic alterations, and cell death, while TNFR2 is primarily involved in protective pathways involving regeneration, cell survival, and regulation of immune responses. Given the importance of TNF-α in nonalcoholic fatty liver disease and the distinct roles of the two tumor necrosis factor receptors, therapeutic interventions should result in the resolution of nonalcoholic fatty liver disease while avoiding the common side effects of traditional TNF-α antagonists, such as infection.

[0005] In previous studies, a phage display library of sea snake venom glands was selected using TNFR1 as a specific target, and a target-specific snake venom active peptide, Hydrostatin-SN10, was obtained. Chinese patent documents CN107090023A, CN107056921A, and CN115154581A disclose that Hydrostatin-SN10 can treat three diseases related to TNF-α: rheumatoid arthritis, inflammatory bowel disease, and sepsis. They also disclose that Hydrostatin-SN10 (10AA) is target-specific and selective, binding only to TNFR1, but not to TNF-α or TNFR2; its binding capacity to TNFR1 is approximately 2.8 μM, and it can competitively inhibit the binding of TNFR1 to TNF-α. Based on this, the present invention focuses on studying the therapeutic effect of Hydrostatin-SN10 on non-alcoholic fatty liver disease. Summary of the Invention

[0006] The present invention aims to provide a selective TNFR1 antagonist peptide, Hydrostatin-SN10, derived from the sea snake (Hydrostatin cyanocephalus), and its use in treating non-alcoholic fatty liver disease. Hydrostatin-SN10, a drug used in this invention, has been previously disclosed in Chinese patent documents CN107090023A, CN107056921A, and CN115154581A for its ability to treat three TNF-α-related diseases: rheumatoid arthritis, inflammatory bowel disease, and sepsis. The present invention provides a new indication for Hydrostatin-SN10: non-alcoholic fatty liver disease.

[0007] The inventor's team obtained a selective TNFR1 antagonist peptide, Hydrostatin-SN10, from a sea snake (Hymenoptera: Aglaonema cyanobacteria). Through in vitro experiments, they verified its selective binding to TNFR1 and its anti-inflammatory activity in vitro and in vivo. The results indicate that Hydrostatin-SN10 exhibits excellent anti-inflammatory activity, likely by selectively binding to TNFR1 and inhibiting the interaction between TNF-α and TNFR1. The main technical solution of the present invention is to establish an animal model of non-alcoholic fatty liver disease induced by a high-fat, high-sugar diet (HFD), demonstrating that Hydrostatin-SN10 has therapeutic potential for non-alcoholic fatty liver disease.

[0008] The present invention provides use of a selective TNFR1 antagonist peptide Hydrostatin-SN10 in preparing a medicine for treating non-alcoholic fatty liver disease.

[0009] Furthermore, the amino acid sequence of the selective TNFR1 antagonist peptide Hydrostatin-SN10 is shown in SEQ ID NO: 2; the nucleotide sequence of its encoding gene is shown in SEQ ID NO: 1.

[0010] Furthermore, the selective antagonism of TNFR1 means that Hydrostatin-SN10 is target-specific and selective, and only binds to TNFR1, but not to TNF-α and TNFR2, and can competitively inhibit the binding of TNFR1 and TNF-α.

[0011] Furthermore, in the drug for treating non-alcoholic fatty liver disease, Hydrostatin-SN10 selectively antagonizes TNFR1.

[0012] Furthermore, the drug for treating non-alcoholic fatty liver disease is: a pharmaceutical composition containing the selective TNFR1 antagonist peptide Hydrostatin-SN10 as the sole active ingredient, or comprising the selective TNFR1 antagonist peptide Hydrostatin-SN10.

[0013] Furthermore, the pharmaceutical composition and conventional pharmaceutical excipients in pharmacy are prepared into pharmaceutical preparations.

[0014] Furthermore, the pharmaceutical preparation is a tablet, granule, dispersant, capsule, pill, injection, powder injection or aerosol, etc.

[0015] The present invention adopts HFD-induced non-alcoholic fatty liver disease model to observe the therapeutic effect of the drug provided by the present invention. The results show that after administration of Hydrostatin-SN10, Hydrostatin-SN10 can improve the liver weight increase of NAFLD mice; significantly reduce the disease indicators of triglyceride (TG), total cholesterol (TC), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the serum of NAFLD mice; significantly reduce the mRNA expression of proinflammatory factors interleukin-6 (IL-6) and TNF-α; alleviate the insulin resistance of NAFLD mice; analyze liver tissue by Oil Red O staining and H&E staining, Hydrostatin-SN10 can significantly reduce fat accumulation, improve liver tissue damage, hepatocyte vacuolation, and inflammatory cell infiltration in the hepatocyte portal area; observe the liver tissue fibrosis of model mice by picrosirius red staining, and the Hydrostatin-SN10 group can improve liver fibrosis.

[0016] The above research results show that the selective TNFR1 antagonist peptide Hydrostatin-SN10 has a good effect in treating non-alcoholic fatty liver disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Effects of Hydrostatin-SN10 on body weight in a HFD-induced nonalcoholic fatty liver disease model in mice.

[0018] Figure 2 Effects of Hydrostatin-SN10 on liver weight in a HFD-induced nonalcoholic fatty liver disease model in mice. Figure 3 This is the effect of Hydrostatin-SN10 on lipid accumulation in the liver of the HFD-induced non-alcoholic fatty liver disease model in mice; Oil red O stained light microscopy image of tissue sections (200 times).

[0019] Figure 4 The effect of Hydrostatin-SN10 on the TG and TC levels in the liver of HFD-induced non-alcoholic fatty liver disease mouse model.

[0020] Figure 5The effect of Hydrostatin-SN10 on liver histopathological damage in the HFD-induced non-alcoholic fatty liver disease model in mice is shown in the light micrograph of H&E staining of tissue sections (200x).

[0021] Figure 6 The effect of Hydrostatin-SN10 on serum ALT and AST in HFD-induced non-alcoholic fatty liver disease model in mice.

[0022] Figure 7 The effect of Hydrostatin-SN10 on IL-6 and TNF-α in the liver of HFD-induced non-alcoholic fatty liver disease model in mice.

[0023] Figure 8 This is the effect of Hydrostatin-SN10 on liver fibrosis in the HFD-induced non-alcoholic fatty liver disease model in mice; light microscopic image of tissue sections stained with picrosirius red (200 times).

[0024] Figure 9 The effect of Hydrostatin-SN10 on fasting blood glucose and fasting insulin in serum of HFD-induced non-alcoholic fatty liver disease model in mice. DETAILED DESCRIPTION

[0025] The specific implementation methods provided by the present invention are described in detail below with reference to the examples.

[0026] The experimental methods in the following examples are conventional methods unless otherwise specified.

[0027] The experiment of Example 2 was carried out using Hydrostatin-SN10 prepared in Example 1.

[0028] Example 1: Synthesis and Detection of the Selective TNFR1 Antagonist Peptide Hydrostatin-SN10

[0029] The peptide Hydrostatin-SN10 was commissioned to Shanghai Qiangyao Biotechnology Co., Ltd. for synthesis using solid-phase peptide synthesis technology. Its purity and molecular weight were analyzed by HPLC and MS, with a molecular weight of 1250.29 Daltons and a purity of ≥98%.

[0030] Example 2: Therapeutic Effects of Hydrostatin-SN10 on HFD-Induced Non-alcoholic Fatty Liver Disease in Mice and Its Mechanism of Action

[0031] The specific implementation steps are:

[0032] 1. C57BL / 6 mice aged 6-8 weeks and weighing 18-20g were selected and randomly divided into a control group (Control), a model group (Model) and an SN10 group. The control group ate a healthy diet without any treatment; the model group used a high-fat and high-sugar diet (HFD) to induce a non-alcoholic fatty liver disease model in mice for 24 consecutive weeks; the SN10 group was treated with Hydrostatin-SN10 (800μg / kg) by intraperitoneal injection after 16 consecutive weeks of HFD feeding, once a day, for 8 weeks. During the treatment period, the mice were still fed with HFD. Among them, the HFD formula is: 60% kcal high-fat feed (Synergy Biological, XTHF60) and 42g / L mixed sugar water (55% fructose and 45% sucrose). The weight changes of mice were recorded every 7 days during the establishment of the model, and the weight changes of mice were recorded every day during the treatment. The weight results are as follows Figure 1 As shown: During the model establishment period, the weight increase trend of mice in the model group and SN-10 group was significantly higher than that in the control group; treatment with Hydrostatin-SN10 could not reduce the body weight of HFD-induced model mice.

[0033] 2. After the administration of Hydrostatin-SN10, all mice were fasted and deprived of water for 12 hours. Blood and liver tissue were collected for analysis, and the liver tissue was weighed. The results showed that:

[0034] (1) Liver weight results are as follows Figure 2 As shown: the liver weight of mice in the control group was the lightest, the liver weight of mice in the model group was the heaviest, and the liver weight of mice in the SN10 group was in the middle. Treatment with Hydrostatin-SN10 can reduce the liver weight of HFD-induced model mice.

[0035] (2) Oil red O staining was used to observe lipid accumulation in the liver of model mice. Figure 3 As shown: The control group mice showed no pathological changes in the liver; the model group showed significant fat accumulation, typically characterized by ballooning; and the SN10 group showed a significant reduction in fat accumulation, alleviating lipid accumulation in the livers of the model mice. The model group mice had approximately 40% fat deformation, while the SN10 group had less than 20% fat deformation, significantly reducing fat content. Hydrostatin-SN10 effectively alleviated HFD-induced lipid accumulation in the livers of the model mice.

[0036] (3) The biochemical indicator kit was used to detect the content of TG and TC in the liver of the model mice to study the effect of Hydrostatin-SN10 on the lipid accumulation in the liver of mice. The results are as follows: Figure 4The results show that the levels of TG and TC in the livers of the model group mice were significantly higher than those in the control group, indicating increased lipid accumulation in the livers of the model group mice. Compared with the model group, the levels of TG and TC in the livers of the SN10 group were significantly lower. Therefore, Hydrostatin-SN10 can alleviate HFD-induced lipid accumulation in the livers of the model mice.

[0037] (4) H&E staining was used to observe the inflammatory cell infiltration and damage in the liver tissue of the model mice. The NAFLD Activity Score (NAS) is an indicator for assessing the severity of NAFLD. The NAS score is a microscopic analysis of liver tissue samples to assess the severity of liver inflammation, necrosis, and fibrosis. The score range is 0-8 points. According to the score results, the NAS score divides NAFLD into three levels: mild (0-2 points), moderate (3-4 points), and severe (5-8 points). The results are as follows. Figure 5 As shown, compared with the control group, the model group showed increased inflammatory cell infiltration in the liver tissue of mice, diffuse and extensive hepatocyte cytoplasmic vacuolation, and a NAS score of 5-6 points in the model group, indicating that the liver tissue of the model group mice was damaged, the inflammation symptoms were severe, and the NAFLD condition was severe. Compared with the model group, the SN10 group showed ameliorated liver tissue inflammation. SN10 can improve liver tissue damage, hepatocyte vacuolation, and inflammatory cell infiltration in the hepatocyte portal area. The NAS score of the SN10 group was 3-4 points, and the NAFLD condition was reduced from severe to moderate. Therefore, Hydrostatin-SN10 can improve liver tissue inflammation and liver damage in model mice.

[0038] (5) The biochemical indicator kit was used to detect the expression of ALT and AST in the serum of model mice to study the effect of Hydrostatin-SN10 on liver damage in mice. The results are as follows: Figure 6 As shown in the results: Compared with the control group, the ALT and AST levels in the serum of the mice in the model group were significantly increased, indicating that the mice in the model group were very likely to suffer from liver cell damage; compared with the model group, the ALT and AST levels in the serum of the mice in the SN10 group were significantly decreased, indicating that the SN10 group mice were effectively improved by the treatment with Hydrostatin-SN10 drug, which may cause liver cell damage in the model mice induced by HFD.

[0039] (6) Real-time fluorescence quantitative PCR was used to detect the mRNA expression of IL-6 and TNF-α in mouse livers to observe the anti-inflammatory effect of Hydrostatin-SN10 in vivo. Figure 7As shown in the results, compared with the control group, the mRNA expression levels of IL-6 and TNF-α in the liver of mice in the model group were significantly increased; compared with the model group, the mRNA expression levels of IL-6 and TNF-α in the liver of mice in the SN10 group were significantly decreased. The selective TNFR1 antagonist peptide Hydrostatin-SN10 selectively binds to TNFR1 but not to TNF-α or TNFR2, competitively inhibiting the interaction between TNFR1 and TNF-α. Therefore, Hydrostatin-SN10 can selectively block the TNF-α / TNFR1 signaling pathway by binding to TNFR1, thereby inhibiting the release of IL-6 and TNF-α inflammatory factors and exerting an anti-inflammatory effect.

[0040] (7) Sirius red staining was used to observe the fibrosis of liver tissue in model mice. Figure 8 As shown: The livers of mice in the model group were filled with a large number of fat vacuoles accompanied by connective tissue hyperplasia; the livers of mice in the SN10 group had fewer vacuoles and the fibrosis area was significantly improved compared with the model group.

[0041] (8) Enzyme-linked immunosorbent assay (ELISA) was used to detect the blood glucose and insulin levels in the serum of model mice, and the insulin resistance index (HOMA-IR) curve was drawn to study the effect of Hydrostatin-SN10 on insulin resistance in model mice. The results are as follows: Figure 9 Results showed that compared with the control group, the fasting blood glucose (FBG), fasting insulin (FINS), and HOMA-IR of the model group mice were significantly increased; compared with the model group, the fasting blood glucose, fasting insulin, and HOMA-IR of the model mice in the SN10 group were significantly improved; Hydrostatin-SN10 can alleviate the insulin resistance of NAFLD mice. Combined with the results of IL-6 and TNF-α mRNA expression in mouse livers, because Hydrostatin-SN10 reduces the insulin resistance of NAFLD mice, it reduces the synthesis and secretion of pro-inflammatory cytokines such as IL-6 and TNF-α produced by insulin resistance.

[0042] The above results indicate that Hydrostatin-SN10 can effectively treat the HFD-induced non-alcoholic fatty liver disease model in mice.

[0043] The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the described embodiments. Those skilled in the art may make various equivalent modifications or substitutions without departing from the spirit of the present invention. These equivalent modifications or substitutions are all included in the scope defined by the claims of this application.

Claims

1. Use of a selective TNFR1 antagonist peptide Hydrostatin-SN10 in the preparation of a medicament for treating non-alcoholic fatty liver disease, wherein the amino acid sequence of the selective TNFR1 antagonist peptide Hydrostatin-SN10 is shown in SEQ ID NO:

2.

2. The use according to claim 1, characterized in that The drug for treating non-alcoholic fatty liver disease is: a drug composition containing the selective TNFR1 antagonist peptide Hydrostatin-SN10 as the sole active ingredient, or a drug composition containing the selective TNFR1 antagonist peptide Hydrostatin-SN10.

3. The use according to claim 2, characterized in that The pharmaceutical composition and conventional pharmaceutical excipients in pharmacy are prepared into pharmaceutical preparations.

4. The use according to claim 3, characterized in that The pharmaceutical preparation is tablet, granule, capsule, dripping pill, injection or aerosol.

5. The use according to claim 4, characterized in that The pharmaceutical preparation is a powder injection.

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