Application of a selective TNFR1 antagonist peptide SN61-4G 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 SN61‑4G in the treatment of non-alcoholic fatty liver disease. The selective TNFR1 antagonist peptide SN61‑4G has an amino acid sequence as shown in SEQ ID NO: 1. The present invention preliminarily verified its selective binding to TNFR1 and in vitro anti-inflammatory activity through in vitro experiments. The results showed that SN61‑4G may exhibit good anti-inflammatory activity by selectively binding to TNFR1 and inhibiting the interaction between TNF‑α and TNFR1. The present invention applies it to a mouse non-alcoholic fatty liver disease model induced by a high-fat and high-sugar diet (HFD), which is of great significance for the development of therapeutic drugs for non-alcoholic fatty liver disease.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the application of a selective TNFR1 antagonist peptide SN61-4G in non-alcoholic fatty liver disease. Background Art

[0002] Non-alcoholic fatty liver disease (NAFLD) is a common chronic liver disease. It is an acquired metabolic stress-induced liver injury characterized by excessive fat deposition in hepatocytes after excluding alcohol and other clear liver injury-causing factors. It is closely related to insulin resistance and genetic susceptibility. The disease spectrum includes non-alcoholic fatty liver disease, non-alcoholic steatohepatitis (NASH), and its related cirrhosis and liver cancer. According to an analysis of the global incidence of NAFLD, the incidence of NAFLD is approximately 32.4%, and with the increase in the number of obese patients, this number continues to rise (it was only 25.5% before 2015) (Younossi ZM, Koenig AB, Abdelatif D, et al. Global epidemiology of nonalcoholic fatty liver disease—meta-analytic assessment of prevalence, incidence, and outcomes [J]. MJH, 2016, 64(1): 73-84.). Due to a lack of treatment and the growing global obesity epidemic, the prevalence of NAFLD is increasing, leading to a simultaneous annual rise in the incidence of NAFLD-related cirrhosis and liver cancer. Currently, it has become the fastest-growing cause of cirrhosis and liver cancer incidence and their associated mortality. However, to date, there is no specific drug to treat NAFLD, and treatment options are limited to lifestyle interventions.

[0003] Tumor necrosis factor alpha (TNF-α) is an important cytokine that can affect physiological and pathological processes such as immune regulation, inflammation, shock, apoptosis, and autoimmunity. With the continuous deepening of the understanding of NAFLD, TNF-α has been confirmed to play an important role in NAFLD and is closely related to the progression of NAFLD (Younossi, Koenig, Abdelatif, Fazel, Henry and Wymer, 2016). The biological effects of TNF-α are mainly mediated by its two downstream receptors: tumor necrosis factor receptor 1 (TNFR1) signals mediate the inflammatory pathway, while tumor necrosis factor receptor 2 (TNFR2) mediates the protective pathway (Aggarwal BB. Signaling pathways of the TNFsuperfamily: a double-edged sword [J]. Nature reviews immunology, 2003, 3 (9): 745-756.). TNF-α antagonists, while antagonizing the pro-inflammatory effects of TNF-α, also affect its other biological functions, inhibiting not only the inflammatory pathway mediated by TNFR1 but also the immune regulation and repair functions mediated by TNFR2. At the same time, studies have shown that TNF-α inhibitors fail to achieve therapeutic endpoints in the treatment of NAFLD. Selectively blocking the signaling pathways transmitted by TNFR1 to block the biological functions of TNF-α, neutralize TNF-α-mediated liver damage, and simultaneously maintain the protective pathways of regeneration, cell survival, and immune response regulation mediated by TNFR2 is a possible treatment approach. Therefore, in recent years, therapeutic targets have gradually shifted to a single TNFR1. Currently, no highly selective TNFR1 antagonist is available on the market, but TNFR1-selective neutralizing antibodies have entered Phase I clinical trials.

[0004] Chinese patent document CN115073553A discloses that SN61-4G can treat inflammatory bowel disease associated with TNF-α. It also discloses that SN61-4G directly interacts with TNFR1, with a binding affinity of approximately 446 nM. It also binds exclusively to TNFR1, not to TNF-α or TNFR2, and can competitively inhibit the interaction between TNF-α and TNFR1. Based on this, the present invention focuses on the therapeutic effects of SN61-4G on non-alcoholic fatty liver disease. Summary of the Invention

[0005] The present invention aims to provide a selective TNFR1 antagonist peptide, SN61-4G, for use in non-alcoholic fatty liver disease. The drug used in this invention has been disclosed in Chinese patent document CN115073553A as being able to treat inflammatory bowel disease associated with TNF-α. The present invention provides a new indication for SN61-4G: non-alcoholic fatty liver disease.

[0006] The inventors' team has obtained a dodecapeptide, SN61-4G (SDPGASHLTGIR), and preliminarily verified its selective binding to TNFR1 and its anti-inflammatory activity in vitro through in vitro experiments. The results indicate that SN61-4G exhibits good anti-inflammatory activity, possibly by selectively binding to TNFR1 and inhibiting the interaction between TNF-α and TNFR1. The present invention applies this peptide to a high-fat, high-sugar diet (HFD)-induced non-alcoholic fatty liver disease (NAFLD) mouse model, which has important implications for the development of therapeutics for NAFLD.

[0007] The main technical solution of the present invention is: by establishing an HFD-induced non-alcoholic fatty liver disease animal model, it is demonstrated that SN61-4G has TNFR1 target selectivity and is used to treat non-alcoholic fatty liver disease.

[0008] The present invention provides use of a selective TNFR1 antagonist peptide SN61-4G in preparing a medicament for treating non-alcoholic fatty liver disease.

[0009] Furthermore, the amino acid sequence of the selective TNFR1 antagonist peptide SN61-4G is shown in SEQ ID NO: 1.

[0010] Furthermore, the drug for treating non-alcoholic fatty liver disease is: a pharmaceutical composition containing the selective TNFR1 antagonist peptide SN61-4G as the sole active ingredient, or comprising the selective TNFR1 antagonist peptide SN61-4G.

[0011] Furthermore, the pharmaceutical composition and conventional pharmaceutical excipients in pharmacy are prepared into pharmaceutical preparations.

[0012] Furthermore, the pharmaceutical preparation is a tablet, granule, dispersant, capsule, pill, injection, powder injection or aerosol, etc.

[0013] Furthermore, the drug for treating non-alcoholic fatty liver disease selectively antagonizes TNFR1.

[0014] The present invention uses an HFD-induced non-alcoholic fatty liver disease model to observe the therapeutic effects of the drug provided by the present invention. The results showed that after intraperitoneal injection of SN61-4G, SN61-4G can improve the increase in liver weight of NAFLD mice; significantly reduce the levels of triglycerides (TG) and total cholesterol in the serum of NAFLD mice.

[0015] The disease indicators of NAFLD mice were as follows: (TC), low-density lipoprotein cholesterol (LDL-C), alanine aminotransferase (ALT) and aspartate aminotransferase (AST); improved the high-density lipoprotein cholesterol (HDL-C) index in the serum of NAFLD mice; significantly reduced the activity of caspase-3 and the pro-inflammatory factors interleukin-6 (IL-6), TNF-α, interleukin-1β (IL-1β), transforming growth factor-β1 (TGF-β1), monocyte chemoattractant protein-1 (MCP-1), SN61-4G significantly reduced the mRNA expression of type I collagen α1 (COL1α1) and matrix metalloproteinase inhibitor-1 (TIMP-1); significantly inhibited the phosphorylation of JNK (Junction-N-terminal kinase), extracellular regulated protein kinase (ERK), p38, and IκB; and alleviated insulin resistance in NAFLD mice. Liver tissue analysis using Oil Red O and H&E staining showed that SN61-4G significantly reduced fat accumulation and improved inflammatory infiltration around adipocytes, liver tissue damage, hepatocyte vacuolation, and inflammatory cell infiltration in the hepatocyte portal area. Furthermore, the results showed that SN61-4G achieved a better therapeutic effect than infliximab (IFX).

[0016] The above research results show that the selective TNFR1 antagonist peptide SN61-4G has a good effect in treating non-alcoholic fatty liver disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the HPLC analysis result of SN61-4G.

[0018] Figure 2 This is the MS analysis result of SN61-4G.

[0019] Figure 3 The effect of SN61-4G on body weight and liver weight in the HFD-induced non-alcoholic fatty liver disease model in mice. Figure 4 The effect of SN61-4G on lipid accumulation in the liver of a HFD-induced non-alcoholic fatty liver disease model in mice; light microscopic image of tissue sections stained with Oil Red O (200x).

[0020] Figure 5 The effect of SN61-4G on the TG and TC levels in the serum of HFD-induced non-alcoholic fatty liver disease model in mice.

[0021] Figure 6 The effect of SN61-4G on the serum HDL-C and LDL-C levels in the HFD-induced non-alcoholic fatty liver disease model in mice.

[0022] Figure 7 Effects of SN61-4G on abdominal fat in a HFD-induced nonalcoholic fatty liver disease model in mice; light microscopic image of H&E-stained tissue sections (200x).

[0023] Figure 8 The effect of SN61-4G on liver histopathological damage in the HFD-induced non-alcoholic fatty liver disease model in mice is shown in light microscopy images of H&E-stained tissue sections (200x).

[0024] Figure 9 The effect of SN61-4G on serum ALT and AST in HFD-induced non-alcoholic fatty liver disease model in mice.

[0025] Figure 10 The effect of SN61-4G on IL-6, TNF-α, and IL-1β in the liver of HFD-induced non-alcoholic fatty liver disease model in mice.

[0026] Figure 11 The effect of SN61-4G on the NF-κB and MAPK pathways in the liver of a HFD-induced non-alcoholic fatty liver disease model in mice.

[0027] Figure 12 The effect of SN61-4G 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 (200x).

[0028] Figure 13 The effect of SN61-4G on TGF-β1, MCP-1, COL1α1, and TIMP-1 in the liver of a HFD-induced mouse non-alcoholic fatty liver disease model.

[0029] Figure 14 The effect of SN61-4G on caspase-3 activity in the liver of a HFD-induced non-alcoholic fatty liver disease model in mice; light microscopic image of immunohistochemical staining of tissue sections (200x).

[0030] Figure 15 The effect of SN61-4G on fasting blood glucose and fasting insulin in serum of HFD-induced non-alcoholic fatty liver disease model in mice. DETAILED DESCRIPTION

[0031] The specific implementation methods provided by the present invention are described in detail below with reference to the examples.

[0032] The experimental methods in the following examples are conventional methods unless otherwise specified.

[0033] The experiment of Example 2 was carried out using SN61-4G prepared in Example 1 and infliximab (IFX) powder purchased from Cilag AG.

[0034] Example 1: Synthesis and Detection of Selective TNFR1 Antagonist Peptide SN61-4G

[0035] The peptide SN61-4G was synthesized by Shanghai Qiangyao Biotechnology Co., Ltd. using solid phase peptide synthesis technology and analyzed by HPLC ( Figure 1 ) and MS( Figure 2 ) The purity and molecular weight of the product were analyzed, and the results showed that the purity was >98% and the molecular weight was 1210.32 g / mol.

[0036] Example 2: Treatment and Mechanism of Action of SN61-4G on HFD-Induced Non-Alcoholic Fatty Liver Disease Model in Mice

[0037] The specific implementation steps are:

[0038] 1. C57BL / 6 mice aged 6-8 weeks and weighing 18-20 g were selected and randomly divided into a control group (Control), a model group (Model), an IFX group, and a SN61-4G group. The control group ate a healthy diet without any treatment; the model group used a high-fat, high-sugar diet (HFD) to induce a non-alcoholic fatty liver disease model in mice for 24 consecutive weeks; after 16 weeks of continuous HFD feeding in the IFX group, IFX (4 mg / kg) was intraperitoneally injected once daily for 8 weeks, and HFD feeding was continued during the administration period; after 16 weeks of continuous HFD feeding in the SN61-4G group, SN61-4G (400 μg / kg) was intraperitoneally injected once daily for 8 weeks, and HFD feeding was continued during the administration period. The HFD formula was: 60% kcal high-fat feed (Synergy Biological, XTHF60) and 42 g / L mixed sugar water (55% fructose and 45% sucrose). The weight changes of mice were recorded every 7 days during the model establishment period and every day during the treatment period. Figure 3 As shown in A: During the model establishment period, the weight increase trend of mice in the model group, IFX group and SN61-4G group was significantly higher than that in the control group; treatment with IFX or SN61-4G could not reduce the weight of HFD-induced model mice.

[0039] 2. After the drug administration, all mice were fasted and deprived of water for 12 hours. Blood, liver tissue, and abdominal fat tissue were collected for analysis, and the liver tissue was weighed. The results showed that:

[0040] (1) Liver weight results are as follows Figure 3 As shown in B: Treatment with SN61-4G can reduce the liver weight of model mice, while IFX treatment has a weak effect on reducing the liver weight of model mice.

[0041] (2) Oil red O staining was used to observe lipid accumulation in the liver of model mice. Figure 4 As shown: The liver morphology of mice in the control group was normal without fat vacuoles; a large number of fat vacuoles were observed in the model group, with obvious fat accumulation; fat accumulation was significantly reduced in the IFX group and SN61-4G group, and the effect of SN61-4G was better than that of IFX.

[0042] (3) The blood biochemical index kit was used to detect the expression of TG, TC, HDL-C, LDL-C, ALT, and AST in the serum of model mice to study the effects of drugs on liver damage and dyslipidemia in mice. The results are as follows: Figure 5 、 6 As shown in Figures 9 and 10: Compared with the control group, the levels of TG, TC, LDL-C, ALT, and AST in the serum of the model group were significantly increased, and the level of HDL-C was significantly decreased, indicating that lipid accumulation in the liver of the model mice was increased and liver cell damage was very likely to occur; Compared with the model group, the levels of TG, TC, LDL-C, ALT, and AST in the serum of the SN61-4G group were significantly decreased, and the level of HDL-C was significantly increased. The above 6 indicators were close to the data of the control group, indicating that SN61-4G can alleviate HFD-induced liver lipid accumulation in the model mice and improve the liver cell damage that may occur in the model mice; Except for AST, the above indicators in the IFX group showed significant improvement; Overall, the effect of SN61-4G was better than that of IFX.

[0043] (4) H&E staining was used to observe the morphological changes of abdominal fat tissue, inflammatory cell infiltration, and inflammatory cell infiltration and damage in liver tissue of model mice. Figure 7 、 8As shown, compared with the control group, the model group mice showed significantly increased adipose tissue diameter, more inflammatory cell infiltration around adipocytes, increased inflammatory cell infiltration in liver tissue, and diffuse and extensive hepatocyte cytoplasmic vacuolation. Neither the SN61-4G group nor the IFX group improved the increased adipose tissue diameter in the model mice, but they did improve inflammatory infiltration around adipocytes, liver tissue damage, hepatocyte vacuolation, and inflammatory cell infiltration in the hepatocyte portal area. The NAFLD Activity Score (NAS) is an indicator of NAFLD severity. The NAS score, which ranges from 0 to 8, assesses the severity of liver inflammation, necrosis, and fibrosis through microscopic analysis of liver tissue samples. Based on the score, NAFLD is categorized into three levels: mild (0-2), moderate (3-4), and severe (5-8). The model group had an NAS score of 5-6; the SN61-4G and IFX groups had NAS scores of 3-4, indicating a reduction in NAFLD severity from severe to moderate. Therefore, the SN61-4G group and the IFX group can improve liver tissue inflammation and liver damage in model mice.

[0044] (5) Real-time fluorescence quantitative PCR was used to detect the mRNA expression levels of IL-6, TNF-α, IL-1β, TGF-β1, MCP-1, COL1α1, and TIMP-1 in the liver of mice to observe the in vivo anti-inflammatory effect and fibrosis inhibition of SN61-4G. The results are as follows Figure 10 、 13 As shown: Compared with the model group, the mRNA expression levels of IL-6, TNF-α, IL-1β, TGF-β1, MCP-1, COL1α1, and TIMP-1 in the liver of mice in the SN61-4G group and IFX group were significantly decreased.

[0045] (6) Sirius red staining was used to observe the fibrosis of liver tissue in model mice. Figure 12 As shown: The livers of mice in the model group were filled with a large number of fat vacuoles accompanied by increased connective tissue; the livers of mice in the SN61-4G group and IFX group had fewer vacuoles, and the fibrosis area was significantly improved compared with the model group.

[0046] (7) Immunohistochemistry was used to determine the activity of caspase-3 in the liver of model mice and to study the mechanism by which SN61-4G inhibited liver injury and liver fibrosis. Figure 14 As shown: Compared with the control group, the number of caspase-3 positive cells in the liver of mice in the model group was significantly increased; compared with the model group, the activity of caspase-3 in the liver of mice in the SN61-4G group and IFX group was significantly decreased.

[0047] (8) Western blot experiments were performed to detect the phosphorylation levels of key proteins in the TNF-TNFRs downstream nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways in liver tissue, to observe the anti-inflammatory therapeutic effect of SN61-4G and to explain the in vivo anti-inflammatory mechanism of SN61-4G. Figure 11 As shown in the figure: Compared with the model group, the SN61-4G group and the IFX group could significantly inhibit the phosphorylation of JNK, ERK, p38, and IκB, and the effect of the SN61-4G group was better than that of the IFX group.

[0048] (9) 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 drugs on insulin resistance in model mice. The results are as follows: Figure 15 As shown, compared with the control group, the fasting blood glucose (FBG) and fasting insulin (FINS) of the model mice were significantly increased, leading to a greater increase in HOMA-IR. The fasting blood glucose, fasting insulin, and HOMA-IR of the model mice in the SN61-4G group were significantly improved compared with the model group. The fasting blood glucose, fasting insulin, and HOMA-IR of the model mice in the IFX group were not significantly different from those in the model group. SN61-4G can alleviate insulin resistance in NAFLD mice, while IFX has no such effect.

[0049] The above results indicate that SN61-4G can effectively treat the HFD-induced non-alcoholic fatty liver disease model in mice.

[0050] 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. A use of a selective TNFR1 antagonist peptide SN61-4G in the preparation of a drug for treating non-alcoholic fatty liver disease, wherein the amino acid sequence of the selective TNFR1 antagonist peptide SN61-4G is shown in SEQ ID NO:

1.

2. The use according to claim 1, characterized in that The drug for treating non-alcoholic fatty liver disease is: a pharmaceutical composition with the selective TNFR1 antagonist peptide SN61-4G as the sole active ingredient, or containing the selective TNFR1 antagonist peptide SN61-4G.

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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