Use of a ghrh antagonist for the preparation of a medicament for the treatment of nonalcoholic fatty liver disease

Abstract

This invention relates to the biomedical field and discloses the application of GHRH antagonists in the preparation of drugs for treating non-alcoholic fatty liver disease (NAFLD). This invention discovers that GHRH-ANT can significantly improve liver imaging and histopathological indicators in NAFLD, providing a new strategy for the treatment of NAFLD and possessing significant clinical therapeutic value. Furthermore, this invention also discovers that GHRH-ANT achieves the treatment of NAFLD by inhibiting the PKA / CREB1 / CD36 signaling pathway, providing important scientific theoretical basis for NAFLD drug development and also offering a new strategy for the regulation of the PKA / CREB1 / CD36 signaling pathway in the liver.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and more particularly to the application of GHRH antagonists in the preparation of drugs for treating non-alcoholic fatty liver disease. Background Technology

[0002] Nonalcoholic fatty liver disease (NAFLD) is a clinicopathological syndrome characterized by diffuse macrovesicular steatosis of hepatocytes, resulting from hepatic lipid metabolism disorders excluding alcohol and other clearly defined hepatotoxic factors. Patients typically have no history of excessive alcohol consumption, but their pathological changes are similar to those of alcoholic liver disease (ALD). The disease spectrum includes nonalcoholic simple fatty liver, nonalcoholic steatohepatitis, cirrhosis, and liver cancer. Due to increased social pressure, poor dietary habits, and emotional factors, the incidence of NAFLD is rising annually. Currently, there are no specific drugs for treating NAFLD; clinical treatment typically involves symptomatic relief using a combination of lipid-lowering and hepatoprotective drugs to slow or halt its progression. Therefore, developing drugs that can better block and reverse liver damage caused by NAFLD is a pressing issue in the treatment of NAFLD.

[0003] Growth hormone-releasing hormone (GHRH) is a neuroendocrine peptide composed of 44 amino acids, primarily secreted by the hypothalamus. Previous studies have found that this hormone regulates the release of growth hormone (GH) by binding to the anterior pituitary growth hormone receptor (GHRH-R). However, increasing research indicates that GHRH-R is present in other tissues, such as the lungs, heart, eyes, and placenta, suggesting that GHRH may have functions independent of the neuroendocrine system axis.

[0004] Growth hormone-releasing hormone antagonists (GHRH-ANTs) can highly target peripheral organs expressing GHRH-R, competitively binding to GHRH-R, and exhibit significant biological activity and a long half-life in vivo. GHRH-ANTs have been found to inhibit tumor cell division, reduce intraocular inflammation caused by endotoxins, and improve brain oxygenation during aging; however, no studies have yet shown that they can play a role in the treatment of NAFLD. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides the application of GHRH antagonists in the preparation of drugs for treating non-alcoholic fatty liver disease (NAFLD). This invention discovers that GHRH-ANTs can significantly improve liver imaging and histopathological indicators in NAFLD, providing a new strategy for the treatment of NAFLD and possessing significant clinical therapeutic value.

[0006] The specific technical solution of this invention is as follows: In a first aspect, the present invention provides the use of a growth hormone-releasing hormone antagonist in the preparation of a medicament for treating non-alcoholic fatty liver disease.

[0007] Our team has discovered that growth hormone-releasing hormone antagonists (GHRH-ANTs) have a therapeutic effect on non-alcoholic fatty liver disease (NAFLD). In vivo and in vitro experiments showed that GHRH-ANTs significantly improved liver imaging and histopathological parameters in mice with high-fat diet-induced NAFLD, reduced hepatic lipid deposition, and decreased mouse body weight. Furthermore, it reduced oleic acid-induced formation of intracellular lipid droplets in hepatocytes. This novel application of GHRH-ANTs provides new insights into its role and offers a new strategy for the treatment of NAFLD, possessing significant clinical implications.

[0008] Preferably, the drug treats non-alcoholic fatty liver disease by inhibiting the PKA / CREB1 / CD36 signaling pathway in hepatocytes.

[0009] Further research by the invention team revealed that the therapeutic effect of GHRH-ANT on NAFLD depends on the PKA / CREB1 / CD36 signaling pathway. That is, GHRH-ANT targets hepatocytes, inhibits the activation of the PKA / CREB1 signaling pathway in hepatocytes, downregulates CREB1 expression, and inhibits CD36 transcription, thereby weakening the liver's ability to take up long-chain fatty acids and reducing the excessive deposition of subsequent lipid droplets in hepatocytes, thus achieving the treatment of NAFLD.

[0010] Preferably, the drug comprises the following (a) and / or (b): (a) Growth hormone-releasing hormone antagonists; (b) Pharmaceutically acceptable salts and / or esters of growth hormone-releasing hormone antagonists.

[0011] Furthermore, the drug also includes one or more of a pharmaceutically acceptable carrier, excipient, solvent, and buffer solution.

[0012] Preferably, the dosage form of the drug is an injection, an oral preparation, a patch, or a spray, and the administration method of the drug is subcutaneous injection, intravenous injection, intramuscular injection, oral administration, or topical application.

[0013] Preferably, the growth hormone-releasing hormone antagonist is a GHRH antagonist from the MIA series, AVR series, MZ series, or JV series.

[0014] Further, the growth hormone-releasing hormone antagonist comprises the following amino acid sequence: PhAcAdaTyr-DArg-Asp-Ala-Ile-Phe(F)5-Thr-Ala-Har-Tyr(Me)-His-Orn-Val-Leu-Abu-Gln-Leu-Ser-Ala-His-Orn-Leu-Leu-Gln-Asp-Ile-Nle-DArg-Har-NH2.

[0015] The above sequence is PhAcAda-tyrosine—D-arginine—aspartic acid—alanine—isoleucine—phenylalanine—threonine—alanine—homarginine—tyrosine—histidine—ornithine—leucine—2-aminobutyric acid—glutamine—leucine—serine—alanine—histidine—ornithine—leucine—leucine—glutamine—aspartic acid—isoleucine—ortholeucine—D-arginine—homarginine-NH2. Here, PhAcAdaTyr indicates that the N-terminus of tyrosine is modified with PhAcAda, Phe(F)5 indicates perfluorinated substitution of the benzene ring in the phenylalanine side chain, Tyr(Me) indicates methylation of the phenolic hydroxyl group in the tyrosine side chain, and Har-NH2 indicates that the carboxyl group of horaginine is replaced by -CONH2. The structural formulas of the above residues are as follows:

[0016] .

[0017] Secondly, the present invention provides the application of a growth hormone-releasing hormone antagonist in the preparation of a drug for the prevention and treatment of non-alcoholic fatty liver disease-related diseases, wherein the non-alcoholic fatty liver disease-related diseases are excessive deposition of lipid droplets in hepatocytes, or cirrhosis, liver dysfunction, liver cancer, type 2 diabetes, and atherosclerosis caused by non-alcoholic fatty liver disease.

[0018] Nonalcoholic fatty liver disease (NAFLD) is characterized by excessive deposition of lipid droplets in hepatocytes. Studies have shown that growth hormone-releasing hormone (GHR) antagonists can improve this excessive deposition. Furthermore, NAFLD can induce related diseases such as cirrhosis, liver dysfunction, liver cancer, type 2 diabetes, and atherosclerosis. Therefore, GHR antagonists, by treating NAFLD, can help prevent the occurrence and progression of these related diseases.

[0019] Thirdly, this invention provides the application of growth hormone-releasing hormone antagonists in the preparation of inhibitors of the PKA / CREB1 / CD36 signaling pathway in hepatocytes.

[0020] Fourthly, this invention provides the application of growth hormone-releasing hormone antagonists in the preparation of CD36 or CREB1 expression inhibitors in hepatocytes.

[0021] Fifthly, the present invention provides the application of PKA / CREB1 / CD36 signaling pathway inhibitors in the preparation of drugs for treating non-alcoholic fatty liver disease.

[0022] This invention discovers that GHRH-ANT treats NAFLD by inhibiting the PKA / CREB1 / CD36 signaling pathway, suggesting that PKA / CREB1 / CD36 signaling pathway inhibitors may become a new treatment for NAFLD, providing important scientific theoretical basis for NAFLD drug development.

[0023] Preferably, the PKA / CREB1 / CD36 signaling pathway inhibitor includes a growth hormone-releasing hormone antagonist.

[0024] Compared with the prior art, the present invention has the following advantages: (1) This invention is the first to discover that GHRH-ANT has a therapeutic effect on non-alcoholic fatty liver disease, providing a new treatment strategy for NAFLD and having important clinical significance; (2) This invention is the first to discover that GHRH-ANT achieves the treatment of non-alcoholic fatty liver disease by inhibiting the PKA / CREB1 / CD36 signaling pathway, which provides an important scientific theoretical basis for the drug development of NAFLD and also provides a new strategy for the regulation of the PKA / CREB1 / CD36 signaling pathway in the liver. Attached Figure Description

[0025] Figure 1 These are the Micro-MRI results of mice in each group 6 months after drug administration. Figure 1 A is a micro-MRI image of a mouse liver; Figure 1 B represents the relative fat index of mouse liver.

[0026] Figure 2 This study investigated the effects of GHRH-ANT on body weight and body shape in mice induced by a high-fat diet. Figure 2 A represents the change in body weight of mice in each group during the 0-6 month period after drug administration; Figure 2 B represents the body length and width of mice in each group 6 months after administration.

[0027] Figure 3 These are the Micro-CT results of mice in each group 6 months after drug administration. Figure 3 A is a Micro-CT image of a mouse liver; Figure 3 B represents the relative CT value of the mouse liver.

[0028] Figure 4This study investigated the effects of GHRH-ANT on the histopathological characteristics of liver tissue in mice induced by a high-fat diet. Figure 4 A represents the liver size of mice in each group 6 months after drug administration; Figure 4 B represents the liver weight of mice in each group 6 months after administration; Figure 4 C shows liver sections (HE staining) of mice in each group 6 months after drug administration. Figure 4 D represents the relative area of ​​lipid droplets in the liver of mice in each group after 6 months of drug administration; Figure 4 E shows liver sections (Oil Red O staining) of mice in each group 6 months after drug administration. Figure 4 F represents the relative area of ​​Oil Red staining in the liver of mice in each group 6 months after drug administration.

[0029] Figure 5 This study investigated the effect of GHRH-ANT on oleic acid-induced lipid droplet formation in AML12 cells. Figure 5 A shows representative images of AML12 cells stained with Lyso-Tracker Red (red) and BodiPy 512 (green) in each group; Figure 5 B represents the relative area of ​​lipid droplets in each group of AML12 cells.

[0030] Figure 6 This relates to the effects of GHRH-ANT on CD36-related metabolic pathways. Among them, Figure 6 A represents the expression of GHRH-R in human hepatocytes of different immortalization processes; Figure 6 B is that GHRH-ANT significantly inhibits the expression of CD36 in hep3B cells; Figure 6 C is related to the metabolic signaling pathways affected by GHRH-ANT.

[0031] Figure 7 GHRH-ANT downregulates GHRH-R and CD36 in the liver of NAFLD mice. Figure 7 A shows immunofluorescence staining indicating that GHRH-ANT downregulates GHRH-R in mouse liver; Figure 7 B is Figure 7 Quantitative analysis of A; Figure 7 C is immunofluorescence staining showing that GHRH-ANT downregulates CD36 in mouse liver; Figure 7 D is Figure 7 Quantitative analysis of C.

[0032] Figure 8 GHRH-ANT downregulates GHRH-R and CD36 in N hepatocytes in vitro. Figure 8 A is immunofluorescence staining showing that GHRH-ANT downregulates GHRH-R in hepatocytes; Figure 8 B is an immunoblot showing that GHRH-ANT inhibits OA-induced upregulation of GHRH-R and CD36.

[0033] Figure 9 CREB1 is a transcription factor for CD36 in hepatocytes. Figure 9 A is a result from the CistromeD database showing that CREB1 has transcriptional activity against CD36 in hepatocytes; Figure 9 B is the possible binding site of CREB1 on CD36 as shown by JASPAR; Figure 9 C is the expression of CREB1 that enhances the transcriptional activity of CD36; Figure 9 D is the inhibition of OA-mediated CD36 transcriptional activation by GHRH-ANT; Figure 9 E is a stable overexpression of GHRH-R that further enhances the transcriptional activity of CD36.

[0034] Figure 10 GHRH-ANT inhibits CREB1 activation in the liver of NAFLD mice. Figure 10 A is GHRH-ANT inhibiting CREB1 nuclear translocation; Figure 10 B is GHRH-ANT inhibiting CREB1 phosphorylation.

[0035] Figure 11 GHRH-ANT inhibits the PKA / CREB1 signaling pathway in vitro. Figure 11 A shows that GHRH-ANT inhibits the activation of PKA (demonstrating that GHRH-ANT reduces the abundance of various phosphorylated substrates of PKA). Figure 11 B is the effect of GHRH-ANT on the in vitro inhibition of OA-induced CREB1 activation; Figure 11 C is GHRH-ANT that inhibits OA-mediated phosphorylation of CREB1.

[0036] Figure 12 The mechanism diagram shows that GHRH-ANT inhibits CD36 expression through the GHRH-R / PKA / CREB1 signaling pathway, thereby inhibiting the occurrence and development of NAFLD by reducing CD36-mediated fatty acid uptake. Detailed Implementation

[0037] The present invention will be further described below with reference to embodiments.

[0038] General Implementation Examples Application of growth hormone-releasing hormone antagonists in the preparation of drugs for treating non-alcoholic fatty liver disease (NAFLD). These drugs treat NAFLD by inhibiting the PKA / CREB1 / CD36 signaling pathway within hepatocytes.

[0039] Optionally, the drug comprises the following (a) and / or (b): (a) Growth hormone-releasing hormone antagonists; (b) Pharmaceutically acceptable salts and / or esters of growth hormone-releasing hormone antagonists.

[0040] Optionally, in addition to (a) and / or (b) above, the drug may also include one or more of a pharmaceutically acceptable carrier, excipient, solvent, and buffer solution.

[0041] Optionally, the dosage form of the drug is an injection, oral administration, patch, or spray, and the administration method is subcutaneous injection, intravenous injection, intramuscular injection, oral administration, or topical application.

[0042] Optionally, the growth hormone-releasing hormone antagonist is a GHRH antagonist from the MIA series, AVR series, MZ series, or JV series.

[0043] Optionally, the growth hormone-releasing hormone antagonist comprises the following amino acid sequence: PhAcAdaTyr-DArg-Asp-Ala-Ile-Phe(F)5-Thr-Ala-Har-Tyr(Me)-His-Orn-Val-Leu-Abu-Gln-Leu-Ser-Ala-His-Orn-Leu-Leu-Gln-Asp-Ile-Nle-DArg-Har-NH2.

[0044] The application of growth hormone-releasing hormone antagonists in the preparation of drugs for the prevention and treatment of non-alcoholic fatty liver disease (NAFLD), wherein NAFLD is defined as excessive deposition of lipid droplets in hepatocytes, or as cirrhosis, liver dysfunction, liver cancer, type 2 diabetes, or atherosclerosis caused by NAFLD.

[0045] Application of growth hormone-releasing hormone antagonists in the preparation of inhibitors of the PKA / CREB1 / CD36 signaling pathway in hepatocytes.

[0046] Application of growth hormone-releasing hormone antagonists in the preparation of CD36 expression inhibitors in hepatocytes.

[0047] Application of growth hormone-releasing hormone antagonists in the preparation of inhibitors of CREB1 expression in hepatocytes.

[0048] Application of PKA / CREB1 / CD36 signaling pathway inhibitors in the preparation of drugs for the treatment of non-alcoholic fatty liver disease.

[0049] Optionally, the PKA / CREB1 / CD36 signaling pathway inhibitor includes a growth hormone-releasing hormone antagonist. Specific Implementation The present invention will be described below through specific embodiments. The GHRH antagonist (GHRH-ANT) used in the embodiments is the MIA602 series, but the scope of protection of the present invention is not limited thereto. The embodiments used a high-fat diet to model non-alcoholic fatty liver in mice, but the scope of protection of the present invention is not limited thereto.

[0051] The amino acid sequence of MIA602 is as follows: PhAcAdaTyr-DArg-Asp-Ala-Ile-Phe(F)5-Thr-Ala-Har-Tyr(Me)-His-Orn-Val-Leu-Abu-Gln-Leu-Ser-Ala-His-Orn-Leu-Leu-Gln-Asp-Ile-Nle-DArg-Har-NH2 (i.e., PhAcAda-tyrosine—D-arginine—aspartic acid—alanine—isoleucine—phenylalanine—threonine—alanine—homarginine—tyrosine—histidine—ornithine—valine—leucine—2-aminobutyric acid—glutamine—leucine—serine—alanine—histidine—ornithine—leucine—leucine—glutamine—aspartic acid—isoleucine—ortholeucine—D-arginine—homarginine-NH2). Wherein, PhAcAdaTyr indicates that the N-terminus of tyrosine is modified with PhAcAda, Phe(F)5 indicates perfluorinated substitution of the benzene ring in the phenylalanine side chain, Tyr(Me) indicates methylation of the phenolic hydroxyl group in the tyrosine side chain, and Har-NH2 indicates that the carboxyl group of horaginine is replaced by -CONH2. The structural formulas of the above residues are as follows:

[0052] .

[0053] Example 1: Effects of GHRH-ANT on body weight and body shape in mice induced by a high-fat diet The experimental animals were C57BL / 6 mice (male, 8 weeks old, animal qualification certificate number: SCXK (Su) 2018-CN110859953A40008, purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.), which were divided into control diet group (CD), control diet + drug administration group (CD+GHRH-ANT), high-fat diet group (HFD) and high-fat diet + drug administration group (HFD+GHRH-ANT).

[0054] The control diet group (CD) and the control diet + drug administration group (CD+GHRH-ANT) were fed with the control diet (TP23300, Nantong Trofi Feed Technology Co., Ltd., China), while the high-fat diet group (HFD) and the high-fat diet + drug administration group (HFD+GHRH-ANT) were fed with the high-fat diet (TP23300, Nantong Trofi Feed Technology Co., Ltd., China). The feeding methods for the four groups were as follows: feed was placed in a location within the 8-week-old mouse cages where they could freely access food; each cage contained 2 mice; 7g of feed was placed at a time, and the feed was changed every 48 hours; any uneaten feed from the previous feeding was discarded; the entire feeding process lasted for 6 months.

[0055] Mice in both the control diet + drug group (CD + GHRH-ANT) and the high-fat diet + drug group (HFD + GHRH-ANT) received a single dose of 5 μg of the test drug. The test drug was prepared as follows: GHRH-ANT was dissolved in DMSO to prepare a stock solution with a concentration of 100 mg / mL, which was then diluted with 10% propylene glycol to a working concentration of 0.1 mg / mL (final DMSO concentration <1%) before use. The control diet group (CD) and the high-fat diet group (HFD) received an equal volume of physiological saline. All four groups of mice were administered the test drug (or physiological saline) subcutaneously once daily at the same time for 6 months.

[0056] During the 6-month administration period, mice were weighed weekly on a scale, and the readings were recorded. This was repeated three times, and the average value was taken. The results are shown in the table below. Figure 2 A. At the end of the experiment, after the mice reached a stable state under isoflurane vapor-induced anesthesia, they were placed on sterile cloths. The body length and width of the mice were measured using an L-shaped ruler and photographed. The results are shown below. Figure 2 B. From Figure 2 It can be seen that GHRH-ANT can inhibit the increase in body weight induced by a high-fat diet in mice and reduce the body length and width of mice fed a high-fat diet for 6 months.

[0057] Example 2: Effects of GHRH-ANT on imaging of liver tissue in mice induced by a high-fat diet In Example 1, after 6 months of drug administration, mice underwent Micro-CT and Micro-MRI, as detailed below: Mice in each group were anesthetized with isoflurane vapor using a small animal anesthesia machine until they reached a stable state. The mice were then fixed in a supine position to the scanning stage with medical tape and continuously ventilated with isoflurane vapor. The ambient temperature was maintained at approximately 22-24 °C. Micro-MRI and Micro-CT were performed, and the results are shown below. Figure 1 and Figure 3 (The accompanying pictures in the instruction manual cannot contain color.) Figure 3Red dots in a Micro-CT image are difficult to show, but are clearly visible in the original color image. From... Figure 1 and 3 It can be seen that after high-fat diet feeding, the liver volume increased, the relative fat index increased, the CT value of the liver parenchyma decreased in a scattered manner, and the CT value was similar to that of adipose tissue. The intrahepatic duct structure was disordered and not clearly displayed, suggesting the formation of NAFLD. In the HFD+GHRH-ANT group, the liver volume was normal, the increase in relative fat index was smaller, the decrease in CT value was not obvious, and the intrahepatic structure was clear, suggesting that GHRH-ANT improved the performance of high-fat diet induced NALD in mice to a large extent.

[0058] Example 3: Effects of GHRH-ANT on the histopathology of liver tissue in mice induced by a high-fat diet In Example 1, six months after drug administration, the size of the mouse liver was measured, and liver histology was observed by HE staining and Oil Red O staining, as detailed below: Liver size measurement: After anesthetizing mice in each group, liver tissue was removed by perfusion with physiological saline and then with 4% paraformaldehyde. The size and weight of the liver were measured, and the results are shown in the table below. Figure 4 A and Figure 4 B. From Figure 4 A and Figure 4 B shows that the liver volume and weight increased after feeding with a high-fat diet, while GHRH-ANT treatment reduced the increase in liver volume and weight in mice fed with a high-fat diet.

[0059] Liver histological HE staining: After anesthetizing mice, the livers were perfused successively with physiological saline and 4% paraformaldehyde. The livers were then harvested, cut into small cubes (edge ​​<8mm cubes), and fixed in 4% paraformaldehyde. Before staining, the tissue blocks were rinsed with tap water overnight, then dehydrated and permeated in a gradient manner, embedded in paraffin, sectioned (4 μm thick), spread, baked, and dried. During staining, the following steps were performed sequentially: gradient hydration, hematoxylin staining, differentiation, eosin staining, dehydration, permeation, and mounting. The results were then examined under a microscope and photographed. See attached table. Figure 4 C and Figure 4 D. From Figure 4 C and Figure 4As shown in Figure D, in the CD group and the CD+GHRH-ANT group, the hepatocyte cytoplasm was uniformly stained, the nuclei were of normal size and shape, there were no vacuoles in the hepatocytes, the liver lobules were intact and clear, the hepatocytes around the central vein showed no degeneration or necrosis and were arranged radially, there was no proliferation of fibrous tissue, and the liver plates were neatly arranged. In the HFD group, the hepatocyte cytoplasm was pale and loosely stained, the liver lobules were severely damaged and disordered, and swollen, necrotic, watery, and ballooning degeneration of hepatocytes and predominantly bullous fatty degeneration were visible. In the HFD+GHRH-ANT group, the liver lobules were partially damaged, the hepatocytes showed mild degeneration and necrosis, the liver structure was significantly improved, and the ballooning and bullous degeneration of hepatocytes was significantly reduced compared with the HFD group. These results suggest that GHRH-ANT can significantly improve the histological results of liver tissue examination in high-fat diet-induced non-alcoholic fatty liver disease.

[0060] Oil Red O staining for liver histology: Mice in each group were anesthetized and perfused successively with physiological saline and 4% paraformaldehyde. Liver tissue was harvested, cut into small cubes (edge ​​<8 mm cubes), and fixed in 4% paraformaldehyde. The tissue blocks were then placed in 20% and 30% sucrose solutions until they sank to the bottom. They were then frozen-embedded with OCT embedding agent and sectioned (10 μm thick) at -20 ℃. Sections were stored at -80 ℃. Before staining, sections were briefly rinsed in distilled water, immersed in 60% isopropanol solution for 1-2 seconds, stained with Oil Red O staining solution (prepared fresh) in a sealed container for 10 min, immersed in 60% isopropanol solution for 1-2 seconds, washed in distilled water for 1-2 seconds, counterstained with hematoxylin for 1-2 seconds, blotted dry with filter paper, mounted with glycerol gelatin, and examined under a microscope. Results are shown in the figure. Figure 4 E and Figure 4 F. From Figure 4 E and Figure 4 As shown in Figure F, hepatocytes in the CD and CD+GHRH-ANT groups were neatly arranged with a small amount of lipid deposition; hepatocytes in the HFD group showed ballooning and bullous degeneration, and were strongly positive for lipid staining; hepatocytes in the HFD+GHRH-ANT group were neatly arranged, ballooning and bullous degeneration disappeared, and lipid deposition was significantly improved. These results suggest that GHRH-ANT can significantly improve hepatic lipid deposition in high-fat diet-induced non-alcoholic fatty liver disease.

[0061] Example 4: Effect of GHRH-ANT on oleic acid-induced lipid droplet formation in AML12 cells The experiment was based on the mouse hepatocyte line AML12 and was divided into a control solvent group (VEH), a control solvent + drug administration group (VEH+GHRH-ANT), an oleic acid group (OA), and an oleic acid + drug administration group (HFD+GHRH-ANT).

[0062] The complete culture medium for the oleic acid group (OA) and the oleic acid + drug administration group (HFD + GHRH-ANT) contained 100 μM oleic acid. The GHRH-ANT used in the control solvent + drug administration group (VEH + GHRH-ANT) and the oleic acid + drug administration group (HFD + GHRH-ANT) was dissolved in DMSO to prepare a concentration of 1 × 10⁻⁶. -3 The stock solution of M was diluted with complete culture medium to a working concentration of 1×10⁻⁶ before use. - 6 M; the control solvent group (VEH) and the oleic acid group (OA) were given an equal volume of complete culture medium containing 0.1% DMSO. All four groups of cells were cultured for 24 hours.

[0063] Lyso-Tracker Red staining of hepatocytes: Remove the cell culture medium, add the prepared Lyso-Tracker Red staining working solution pre-incubated at 37°C, and incubate with the cells at 37°C for 30 minutes. Remove the Lyso-Tracker Red staining working solution and add fresh cell culture medium. Then observe using a fluorescence microscope. At this point, lysosomes will show bright, strong fluorescent staining. If the staining effect is unsatisfactory, the concentration of Lyso-Tracker Red in the Lyso-Tracker Red staining working solution can be increased, or the staining time can be appropriately extended within the recommended time range.

[0064] BodiPy 512 staining of hepatocytes: Remove the cell culture medium, add the prepared BodiPy staining working solution, and incubate with the cells at 37°C for 30 minutes. Remove the BodiPy staining working solution and add fresh cell culture medium. Observation is then typically performed using a fluorescence microscope.

[0065] Images of hepatocytes after Lyso-Tracker Red and BodiPy 512 staining are shown below. Figure 5 (Due to the lack of color in the accompanying diagrams, the red fluorescence is difficult to show; however, it is clearly visible in the original color image.) From Figure 5 It can be seen that a small number of scattered lipid droplets were observed in AML12 cells in the VEH group and the VEH+GHRH-ANT group; a large number of lipid droplets aggregated and fused in AML12 hepatocytes in the OA group; and lipid droplet formation and fusion were reduced in AML12 cells in the OA+GHRH-ANT group. These results suggest that GHRH-ANT can significantly improve oleic acid-induced lipid droplet formation in AML12 cells.

[0066] Example 5: Effects of GHRH-ANT on the PKA / CREB1 / CD36 signaling pathway in hepatocytes The experiment was based on mouse hepatocyte line AML12 and human hepatocyte line Hepa3b, and was divided into control solvent group (VEH), control solvent + drug group (VEH+GHRH-ANT), oleic acid group (OA) and oleic acid + drug group (HFD+GHRH-ANT).

[0067] The complete culture medium for the oleic acid group (OA) and the oleic acid + drug administration group (HFD + GHRH-ANT) contained 100 μM oleic acid. The GHRH-ANT used in the control solvent + drug administration group (VEH + GHRH-ANT) and the oleic acid + drug administration group (HFD + GHRH-ANT) was dissolved in DMSO to prepare a concentration of 1 × 10⁻⁶. -3 The stock solution of M was diluted with complete culture medium to a working concentration of 1×10⁻⁶ before use. - 6 M; the control solvent group (VEH) and the oleic acid group (OA) were given an equal volume of complete culture medium containing 0.1% DMSO. All four groups of cells were cultured for 24 hours.

[0068] The transcriptome expression profiles of 81 immortalized human hepatocyte cell lines (GSE97098) from the GEO database (https: / / www.ncbi.nlm.nih.gov / geo) were extracted and processed using R. Then, ggplot2 (R package) was used to plot a heatmap of GHRH-R related gene expression in the human hepatocyte cell line. The results showed that Hep3b cells expressed GHRH-R (… Figure 6 A). Hep3B cells were treated with GHRH-ANT and control solvents for 24 hours, respectively. RNA was extracted, quality controlled, library constructed, and transcriptome sequenced (Shanghai Zhongke New Life Technology Co., Ltd., China). Transcriptome sequencing results showed that GHRH-ANT treatment significantly inhibited the transcription of Hep3b CD36. Gene enrichment analysis using EnrichR (R package) showed that GHRH-ANT could interfere with Hep3b metabolism-related signaling pathways (…). Figure 6 B and 6C).

[0069] Immunofluorescence results showed that GHRN-ANT could reduce the increase in CD36 and GHRH-R abundance induced by a high-fat diet. Figure 7 Because the accompanying illustrations in the instruction manual cannot contain color, Figure 7 Some fluorescence in A and 7C is difficult to show or distinguish, but can be clearly observed in the original color image. In vitro, GHRH-ANT also downregulates CD36 (…). Figure 8 Because the accompanying illustrations in the instruction manual cannot contain color, Figure 8 Some fluorescence in A is difficult to show or distinguish, but can be clearly observed in the original color image.

[0070] Prediction of transcription factor binding sites: CD36 (species: homo sapiens, range: 10kb, cell type: hepacyte) was submitted to the cistrome DB database (http: / / cistrome.org / db / ). The results showed that CREB1 is a potential transcription factor of CD36 in hepatocytes. Figure 9 A). Using the online tool JASPAR (https: / / jaspar.genereg.net / ), predict the transcription factors of CD36 and their paired base sequences in the Vertebrata species. Figure 9 B). Luciferase reporter assays showed that overexpression of the CREB1 plasmid with lipo3000 enhanced CD36 transcriptional activity in hep3B cells. On the other hand, GHRH-ANT inhibited oleic acid-mediated CD36 transcriptional activity, and in hep3b cells overexpressing GHRH-R, CD36 transcriptional activity was further enhanced. Figure 9 C~9E).

[0071] Immunofluorescence results of mouse liver tissue showed that a high-fat diet increased the expression of CREB1 and P-CREB1 in mouse hepatocytes, while GHRH-ANT inhibited the activation of the high-fat diet-mediated CREB1 signaling pathway in mice. Figure 10 Because the accompanying illustrations in the instruction manual cannot contain color, Figure 10 Some fluorescence in the image is difficult to show or distinguish, but can be clearly observed in the original color image.

[0072] Western blotting results showed that acid could activate the CREB1 signaling pathway in Hep3b cells. Conversely, GHRH-ANT could inhibit oleic acid-mediated CREB1 signaling pathway activation via the PKA signaling pathway. Immunofluorescence staining of cells also showed similar experimental results. Figure 11 ).

[0073] In summary, it is hypothesized that GHRH-ANT inhibits lipid droplet formation by affecting the PKA / CREB1 / CD36 signaling pathway in hepatocytes. The mechanism of GHRH-ANT treatment for NAFLD may be as follows: Figure 12 As shown, GHRH-ANT inhibits CD36 expression through the GHRH-R / PKA / CREB1 signaling pathway, thereby inhibiting the occurrence and development of NAFLD by reducing CD36-mediated fatty acid uptake.

[0074] Unless otherwise specified, the raw materials and equipment used in this invention are all commonly used in the field; unless otherwise specified, the methods used in this invention are all conventional methods in the field.

[0075] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications, alterations, and equivalent transformations made to the above embodiments based on the technical essence of the present invention shall still fall within the protection scope of the present invention.

Claims

1. Application of the growth hormone-releasing hormone antagonist MIA602 in the preparation of drugs for the treatment of non-alcoholic fatty liver disease.

2. The application as described in claim 1, characterized in that, The drug treats non-alcoholic fatty liver disease by inhibiting the PKA / CREB1 / CD36 signaling pathway in hepatocytes.

3. The application as described in claim 1, characterized in that, The drug includes the following (a) and / or (b): (a) Growth hormone-releasing hormone antagonists; (b) Pharmaceutically acceptable salts and / or esters of growth hormone-releasing hormone antagonists.

4. The application as described in claim 3, characterized in that, The drug also includes a pharmaceutically acceptable carrier.

5. The application as described in claim 3, characterized in that, The drug also includes pharmaceutically acceptable excipients.

6. The application as described in claim 3, characterized in that, The drug also includes pharmaceutically acceptable solvents.

7. The application as described in claim 3, characterized in that, The drug also includes pharmaceutically acceptable buffer solutions.

8. The application as described in claim 1, characterized in that, The dosage form of the drug is injection, oral preparation, patch, or spray.

9. The application as described in claim 1, characterized in that, The drug can be administered via subcutaneous injection, intravenous injection, intramuscular injection, oral administration, or topical application.

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