Small protein UFD1s and application thereof in prevention, treatment and diagnosis of non-alcoholic steatohepatitis

Abstract

The invention discloses small protein UFD1s and application of the small protein UFD1s in prevention, treatment and diagnosis of non-alcoholic steatohepatitis, the small protein UFD1s provided by the invention has the effects of regulating energy metabolism and resisting cell stress, and the UFD1s can be used as an auxiliary diagnosis marker of the non-alcoholic steatohepatitis (NASH). Meanwhile, the deletion or low expression quantity of the UFD1s is related to the occurrence and development of the NASH, the UFD1s are expressed in vivo through the constructed expression substance and method, the effect of preventing and / or treating the NASH can be played, and a new thought is provided for preventing and / or treating the NASH.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a small protein UFD1s related to non-alcoholic fatty liver disease, and also relates to the application of the small protein UFD1s in the prevention, treatment and diagnosis of non-alcoholic fatty liver disease. Background Art

[0002] With the changes in lifestyle and nutritional structure in various regions around the world, the incidence of various metabolic diseases caused by glucose and lipid metabolism disorders due to nutritional imbalance is increasing, such as type 2 diabetes, insulin resistance, hyperlipidemia, non-alcoholic fatty liver disease (NAFLD), etc. These metabolic diseases not only seriously threaten people's health, but also require lifelong diagnosis and treatment, with high drug dependence and high cost, and are also accompanied by serious complications. Therefore, it is urgent to study the complex pathogenesis of metabolic diseases caused by nutritional imbalance and find effective prevention and treatment strategies.

[0003] Non-alcoholic steatohepatitis (NASH) is an increasingly common chronic liver disease that is not related to alcohol consumption and is widely believed to be closely related to insulin resistance, obesity and metabolic syndrome. Non-alcoholic fatty liver disease (NAFLD) refers to the accumulation of excessive fat in the form of triglycerides in the liver in more than 5% of liver tissue cells (fatty degeneration). In addition to excessive fat, the liver of NASH patients also has lobular inflammation, ballooning of hepatocytes, and faster-developing fibrosis compared with NAFLD. In the short term, the degree of fatty degeneration alone in NAFLD has no obvious correlation with increased morbidity or mortality. However, once it develops into NASH, it significantly increases the risk of cirrhosis, liver failure and hepatocellular carcinoma (HCC), and is a potentially fatal disease.

[0004] Many NASH patients appear healthy and are unaware of their liver abnormalities, so they are not easily detected. At present, the liver function is mainly determined by testing the levels of triglycerides, cholesterol and transaminases (such as aspartate transaminase and alanine transaminase) in the serum. The only diagnostic method for NASH is liver biopsy, which is to confirm whether the liver has symptoms of inflammation or fibrosis through histopathological testing, but this method is complicated, time-consuming and expensive, and there is a lack of convenient biomarkers for auxiliary diagnosis of NASH in clinical practice. In addition, regarding the treatment of NASH, for some patients with early NASH, blood sugar is mainly controlled by diabetes drugs and lifestyle improvements, such as active exercise, sitting less and moving more, which can slow down or even reverse the process of fatty degeneration; however, for patients with advanced NASH, there is currently a lack of standard and clear treatment measures, so effective prevention and treatment measures are urgently needed. Summary of the invention

[0005] In view of this, the primary purpose of the present invention is to provide a small protein UFD1s, which is the first protein discovered by the inventors to be conserved in mammals and has the functions of regulating energy metabolism and resisting cell stress. It can play an important role in the diagnosis, prevention and treatment of NASH, and can provide a new method for the diagnosis, prevention and treatment of NASH.

[0006] In the present invention, the results of previous research have shown that the conserved small protein UFD1s in humans and mice has a protective effect in regulating energy metabolism and resisting cellular stress. Under normal circumstances, UFD1s deficiency shows lower energy consumption, less oxygen volume consumption and higher respiratory exchange rate (RER). In addition, the lack of UFD1s protein leads to abnormal liver morphology, including mild hydropic degeneration, swelling, loose hepatocyte cytoplasm and abnormal accumulation of lipid droplets in the liver, and this abnormal phenomenon is more serious after 24h of starvation treatment. More importantly, the present invention found through mouse experiments that the expression level of UFD1s RNA in the liver of NASH mice was significantly reduced, and the absence of UFD1s accelerated the progression of NASH disease, including high levels of lipids and transaminases in serum, severe fatty degeneration, lobular inflammation and fibrosis. Based on the above research results, it can be determined that the small protein UFD1s has a certain effect of treatment, stabilization, improvement or prevention in nutritional imbalance metabolic diseases such as NASH, and can be used as a biomarker for auxiliary diagnosis of NASH. Based on this, the present invention proposes the use of the small protein UFD1s in the diagnosis, prevention and treatment of nutritional imbalance metabolic diseases such as NASH.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides a small protein UFD1s having an energy metabolism regulating function. The small protein UFD1s has an open reading frame sequence as shown in SEQ ID NO.3 or SEQ ID NO.4.

[0009] The second aspect of the present invention provides the use of a reagent for detecting the expression level of UFD1s in the liver tissue of a subject in the preparation of a product for auxiliary diagnosis of non-alcoholic fatty hepatitis, wherein the UFD1s is the small protein UFD1s described above.

[0010] The third aspect of the present invention provides an open reading frame sequence of a small protein UFD1s, wherein the open reading frame sequence is shown as SEQ ID NO.3 or SEQ ID NO.4.

[0011] The fourth aspect of the present invention provides an expression product of the small protein UFD1s, wherein the expression product contains the open reading frame sequence described above.

[0012] A fifth aspect of the present invention provides a drug for preventing and / or treating non-alcoholic steatohepatitis, wherein the drug contains an agonist of the small protein UFD1s.

[0013] A sixth aspect of the present invention provides the use of an agonist of the small protein UFD1s in the preparation of a medicament for preventing and / or treating non-alcoholic steatohepatitis.

[0014] The inventor has been deeply involved in biomedicine for many years. Through a large number of preliminary research and experiments, he has searched for new transcripts with stress response and energy metabolism regulation functions, and found a highly conserved UFD1 splicing form in humans and mice. In the present invention, it is named UFD1s. This UFD1s has not been reported before. The present invention determines that the conserved UFD1s protein in humans and mice has the protective effect of regulating energy metabolism and resisting cell stress.

[0015] In the present invention, the small protein UFD1s has an open reading frame sequence as shown in SEQ ID NO.3 or SEQ ID NO.4.

[0016] Open reading frame (ORF) refers to a continuous sequence of bases in a DNA or RNA sequence, starting from the start codon (usually ATG) and ending with the stop codon (such as UAG, UGA, UAA). This sequence contains triplet codons that can be translated into amino acids, and therefore has the function of encoding proteins.

[0017] In the present invention, SEQ ID NO.3 is a human ORF sequence, and SEQ ID NO.4 is a mouse ORF sequence. The specific sequence information is as follows:

[0018] SEQ ID NO.3:

[0019] ATGTTCTCTTTCAACATGTTCGACCACCCTATTCCCAGGGTCTTCCAAAACCGCTTC TCCACACAGTACCGCTGCTTCTCTGTGTCCATGCTAGCAGGGCCTAATGACAGTAATTAT GCCACCCTCGGCCCTGGACCAACTCAGCCGACTTAA(153nt)

[0020] SEQ ID NO.4:

[0021] ATGTTTTTCTTTCAACATGTTTGACCACCCGATTCCCCGGGTCTTCCAGAACCGCTTCTCCACGCAGTACCGCTGCTTCTCCGTGTCCATGCTAGCAGGGCCTAATGACAGTAATTATGCCACCCTCAGCCCTCGATCAACTCAGCCGGCTCAACATTACCTATCCTATGCTGTTTAA(177nt)

[0022] Furthermore, the amino acid sequence of the small protein UFD1s encoded by the open reading frame sequence mentioned above is shown as SEQ ID NO.1 or SEQ ID NO.2.

[0023] SEQ ID NO.1:

[0024] MFSFNMFDHPIPRVFQNRFSTQYRCFSVSMLAGPND SNYATLGPGPTQPT (human ORF)

[0025] SEQ ID NO.2

[0026] MFSFNMFDHPIPRVFQNRFSTQYRCFSVSMLAGPND SNYATLSPRSTQPAQHYLSYAV (mouse ORF)

[0027] It is understandable that the small protein UFD1s can also be a derivative protein based on the amino acid sequence shown in SEQ ID NO.1 or SEQ ID NO.2. In this article, the derivative protein refers to a protein having the same biological function or activity as the original protein obtained by substitution, replacement or addition, modification, labeling, etc. on the basis of the original amino acid sequence. The derivative protein described in this article can be obtained by substitution, deletion or addition at one or more positions of the amino acid sequence of SEQ ID NO.1 or 2; it can also be obtained by modification at one or more positions of the amino acid sequence of SEQ ID NO.1 or 2 (for example, it can be at least one of phosphorylation, methylation, amination, sulfhydrylation, substitution of oxygen with sulfur, substitution of oxygen with selenium, isotopization, etc.); it can also be obtained by connecting a marker (such as a therapeutic substance, etc.) to the amino acid sequence of SEQ ID NO.1 or 2 by known physical or chemical methods. As long as these derivative proteins have the same biological function or activity as the protein with the amino acid sequence shown in SEQ ID NO.1 or SEQ ID NO.2, they also fall within the scope of protection of the present invention.

[0028] The small protein UFD1s can also be a protein having at least 25% identity with the amino acid sequence shown in SEQ ID NO.1 or SEQ ID NO.2, for example, it can be a protein having at least 25%, at least 35%, at least 45%, at least 55%, at least 65%, at least 75%, at least 85%, at least 95% identity with the amino acid sequence shown in SEQ ID NO.1 or SEQ ID NO.2. It is well known to those skilled in the art that the identity of an amino acid sequence refers to the degree to which the residues at the same site of two amino acid sequences are completely identical, which indicates that in the comparison of the two amino acid sequences, the number of completely matched amino acid residues at the same position accounts for the percentage of the total comparison length. If the two amino acid sequences have a high degree of identity, then the two proteins are considered to have similar or identical biological functions or activities. Therefore, in the present invention, proteins having at least 25% identity with the amino acid sequence shown in SEQ ID NO.1 or SEQ ID NO.2 and having the same biological function or activity as the protein shown in the aforementioned amino acid sequence also fall within the protection scope of the present invention.

[0029] In some embodiments of the present invention, the UFD1s described herein can be used as a biomarker for auxiliary diagnosis of non-alcoholic fatty liver disease. By detecting the expression level of UFD1s in the liver tissue of the subject, it can be used as an auxiliary indicator for diagnosing non-alcoholic fatty liver disease, and can be used in conjunction with other non-alcoholic fatty liver disease indicators to facilitate the diagnosis of non-alcoholic fatty liver disease. Compared with the current traditional liver biopsy, the detection method is simpler and less costly.

[0030] In the present invention, the product refers to an in vitro detection product, such as a preparation, a test paper, a chip or a kit, but is not limited thereto. Preferably, the product is a chip or a kit.

[0031] In the present invention, the reagent for detecting the expression level of UFD1s in the liver tissue of the subject is a reagent for detecting the protein expression of UFD1s in a biological sample, or a reagent for detecting the RNA expression level of UFD1s in the liver tissue of the subject. By detecting the protein expression level or RNA expression level of UFD1s in the liver tissue of the subject, it is assisted in judging whether the subject suffers from NASH. Specifically, if the protein expression level or RNA expression level of UFD1s is low, it indicates that the subject is likely to suffer from NASH. The specific judgment method can be determined by those skilled in the art based on known methods or experiments, and is not particularly limited here. For example, in some specific cases, the RNA expression level of UFD1s is used to assist in judgment. If the RNA expression level in the liver tissue of the subject is reduced by more than 50% compared with the average RNA expression level in the normal liver tissue of a given healthy population, it is considered that the RNA expression level of UFD1s is low, indicating that the subject is likely to suffer from NASH, and then by coordinating other indicators, the diagnosis of NASH can be achieved.

[0032] In the present invention, the subject refers to a mammal, such as a human, mouse, rabbit or sheep, etc. Preferably, the subject refers to a human or mouse. In some specific implementation cases, the subject is a mouse.

[0033] It is understood that the reagents for detecting the expression level of UFD1s in the liver tissue of the subject vary according to the detection target (protein or RNA) and the detection technology. The specific detection method can be well known to those skilled in the art, such as polymerase chain reaction (PCR), quantitative polymerase chain reaction (qPCR), enzyme-linked immunosorbent assay (ELISA), Western Blot, flow cytometry or immunohistochemistry staining technology (IHC), etc., but are not limited thereto. The reagents used can be, for example, antibodies, nucleic acid probes or primers that specifically bind to UFD1s.

[0034] In some specific implementation cases, the reagent for detecting the expression level of UFD1s in the liver tissue of the subject is a reagent for detecting the RNA expression level of UFD1s in the liver tissue of the subject. In some specific implementation cases, the subject is a human, and the reagent contains a primer pair with sequences as shown in SEQ ID NO.5 and 6; in other specific implementation cases, the subject is a mouse, and the reagent contains a primer pair with sequences as shown in SEQ ID NO.7 and 8.

[0035] In addition, it is understandable that the products described above include not only the reagents necessary for detecting the expression level of UFD1s, but also buffer solutions, washing solutions, etc. required for different detection technologies, which can be selected based on the needs in the field and are therefore not specifically limited.

[0036] In other embodiments of the present invention, the present invention has determined through animal experiments that the small protein UFD1s affects the course or progression of NASH disease, and the loss of UFD1s accelerates the course of NASH disease. By injecting the expression material of the small protein UFD1s into the subject's body and increasing the expression level of the small protein UFD1s in the subject's body, the effect of preventing and / or treating NASH can be achieved.

[0037] In the present invention, the expression refers to any expression vector or RNA in the art that can be specifically expressed in the liver. The expression contains an open reading frame sequence as shown in SEQ ID NO.3 or SEQ ID NO.4, so that UFD1s can be specifically expressed in the liver, the expression level of UFD1s in the liver is increased, and the effect of preventing and / or treating NASH is achieved. In the present invention, its specific type can be a recombinant plasmid or a circular RNA.

[0038] In some specific embodiments of the present invention, the expression product is a recombinant plasmid, which is constructed by inserting an open reading frame sequence as shown in SEQ ID NO.3 or SEQ ID NO.4 into a backbone vector. In the present invention, the backbone vector is any vector in the art that can be specifically expressed in the liver, for example, it can be pLIVE or pAlb-Luc, but it is not limited thereto.

[0039] The specific method for constructing the recombinant plasmid can be well known to those skilled in the art, and mainly includes the following steps:

[0040] The open reading frame sequence of the small protein UFD1s was cloned into the backbone vector and sequenced to obtain a recombinant plasmid.

[0041] In some specific implementation cases, the present invention uses pLIVE to construct a recombinant plasmid, named pLIVE-UFD1s plasmid.

[0042] In some other specific embodiments of the present invention, the expression product is circular RNA, and the circular RNA is UFD1s cyclized in vitro, and the in vitro cyclization can be performed by methods familiar to those skilled in the art without particular limitation. In some specific implementation cases, the in vitro cyclization of UFD1s mainly includes the following steps:

[0043] Linear RNA containing the open reading frame sequence of the small protein UFD1s was obtained by in vitro transcription, and circularized RNA, namely circUFD1s, was obtained through a cyclization reaction.

[0044] Furthermore, in the present invention, the drug for preventing and / or treating non-alcoholic steatohepatitis refers to an agonist containing an effective amount of the small protein UFD1s, which can increase the expression level of the small protein UFD1s in liver tissue.

[0045] In the present invention, the drug for preventing and / or treating non-alcoholic steatohepatitis has at least one of the following effects:

[0046] (1) reducing the level of serum lipids in a subject, including triglycerides and total cholesterol;

[0047] (2) reduce the level of serum aspartate aminotransferase in subjects;

[0048] (3) reduce the degree of liver steatosis in subjects;

[0049] (4) Improve the degree of liver inflammation in subjects;

[0050] (5) Improve the degree of liver fibrosis in subjects.

[0051] In the present invention, the agonist of the small protein UFD1s contains the open reading frame sequence of the small protein UFD1s described above or the expression product of the small protein UFD1s described above.

[0052] Specifically, by administering a drug containing an agonist of the small protein UFD1s to a subject in vivo or in vitro, the expression level of the small protein UFD1s in the subject's liver is increased, thereby achieving the effect of preventing and / or treating non-alcoholic fatty liver disease.

[0053] The effective amount here refers to the minimum dose that can produce the desired preventive and / or therapeutic effect, and this dose is within the safe range (i.e., no adverse reactions will occur). In the present invention, the desired preventive effect refers to increasing the expression level of UFD1s in the subject's liver, thereby avoiding the development of NASH; the desired therapeutic effect refers to increasing the expression level of UFD1s in the subject's liver, so that NASH patients are improved or even cured. The specific effective amount varies according to the type of subject, the degree of progression of the disease, the method of administration, the preparation, etc., and those skilled in the art can determine it through experiments based on known methods, so there is no special limitation. For example, in some specific cases of the present invention, the subject is a mouse, wherein the injection amount of the pLIVE-UFD1s plasmid is 15 μg / time, and the number of times is once; the injection amount of circUFD1s is 10 μg / time, and the number of times is twice.

[0054] It is understandable that the above-mentioned drugs also contain any pharmaceutically acceptable carriers and / or excipients, which may vary according to the specific drug dosage forms, and their main functions include but are not limited to improving the stability of the drug, improving the delivery efficiency of the drug, improving the absorption or metabolism of the drug by the subject, etc., so that the drug can better exert the effect of prevention and / or treatment. The carriers and / or excipients can be selected or adjusted based on methods known to those skilled in the art, so they are not specifically described here.

[0055] Furthermore, the drugs described above may also contain other therapeutic substances that prevent and / or treat non-alcoholic fatty liver disease. These therapeutic substances refer to any substances that can be combined with the expressions in the present application to further improve the effect of preventing and / or treating non-alcoholic fatty liver disease.

[0056] In the present invention, the drug dosage form is not particularly limited, and can be well known in the art such as granules, capsules, powders, tablets, oral liquids, injections, etc. Preferably, the drug is an injection.

[0057] Beneficial effects of the present invention:

[0058] The present invention determines that the highly conserved small protein UFD1s in humans and mice has a protective effect on regulating energy metabolism and resisting cellular stress. The loss of UFD1s accelerates the progression of NASH. The present invention determines that the expression level of UFD1s in NASH is significantly reduced, so UFD1s can be used as a biomarker for auxiliary diagnosis of NASH; at the same time, by expressing the small protein UFD1s in the liver, the occurrence and development of NASH can be effectively prevented and / or improved.

[0059] The present invention constructs related expressions based on the small protein UFD1s. By delivering the expressions into the subject, these expressions can make UFD1s efficiently and stably expressed in the subject's liver, thereby playing a role in treating NASH. By delivering the expressions, lipids and transaminases in serum are reduced, and fatty degeneration, the number of lipid droplets, fibrosis and inflammation of NASH are reduced. This provides a new idea for the development of preventive and / or therapeutic drugs for non-alcoholic fatty hepatitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 :Demonstrating CRISPR / cas9 technology-mediated UFD1s - / - Strategies for deficient mice ( Figure 1 A), RNA levels of UFD1s and its long transcript form (UFD1f) ( Figure 1 B) and protein levels ( Figure 1 C).

[0061] Figure 2 : Under normal conditions, total energy expenditure, oxygen consumption, and gas exchange rate of wild-type and UFD1s-deficient mice were analyzed by metabolic cages ( Figure 2 A). The liver morphology of UFD1s-deficient mice was observed by hematoxylin-eosin (H&E) under normal and starvation conditions; the lipid droplet content was detected by oil red staining (Oil red O), and the autophagy status was observed by immunohistochemistry (IHC) of the autophagy marker LC3 ( Figure 2 B).

[0062] Figure 3 :UFD1s RNA levels in normal and NASH mouse livers ( Figure 3 A), serum triglyceride and total cholesterol levels ( Figure 3 B), serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels ( Figure 3 C).

[0063] Figure 4 :H&E, Oil Red and Masson staining of normal and NASH model mouse livers were used to observe liver fatty degeneration, lipid droplet number and fibrosis degree, respectively.

[0064] Figure 5 :Process of expressing UFD1s protein using plasmid and circular RNA in the NASH mouse model induced by methionine-choline deficient diet (MCD) ( Figure 5 A), and the expression level of UFD1s in the liver was detected by IHC of UFD1s ( Figure 5 B).

[0065] Figure 6 :Levels of triglycerides and cholesterol in serum of NASH mice expressing UFD1s by plasmid and circular RNA Figure 6 A) and serum AST and ALT levels ( Figure 6 B).

[0066] Figure 7 : H&E, Oil Red and Masson staining of the livers of NASH mice expressing UFD1s by plasmid and circular RNA were used to observe hepatic steatosis, the number of lipid droplets and the degree of fibrosis, respectively.

[0067] Figure 8 :Inflammatory scores in the liver of NASH mice expressing UFD1s by two strategies ( Figure 8 A), and the mRNA expression levels of fibrosis marker (COLLA1) and inflammation marker (TNF) in the liver ( Figure 8 B). DETAILED DESCRIPTION

[0068] Embodiments of the present invention will be described in detail below. The following described embodiments are exemplary and are only used to explain the present invention, and should not be construed as a limitation to the present invention.

[0069] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which this invention belongs. The terms used in the description of the present invention in this specification are only for the purpose of describing specific embodiments, and are not intended to limit the present invention. Additionally, unless otherwise specified, the methods without specific recorded conditions or steps are conventional methods, and the reagents and materials used can be obtained from commercial sources.

[0070] Animal source and breeding environment: The C57BL / 6 wild-type mice involved in this article were all purchased from Spiff (Beijing) Biotechnology Co., Ltd. All mice were bred in a specific pathogen-free (SPF) environment with a 12 / 12 h light / dark rhythm cycle (light time: 8:00 - 20:00), 22 ± 2 °C, and 40% - 60% humidity. All animal procedures were approved by the Animal Protection and Use Committee of the University of Science and Technology of China, and the animal ethics number is USTCACUC202101048.

[0071] Example 1 Construction of UFD1s-deficient mice and their stress protection effects under starvation conditions

[0072] In this example, UFD1s-deficient mice were constructed using the CRISPR / cas9 system, and UFD1s was detected at the RNA and protein levels. The metabolic cages were used to monitor the metabolism of UFD1s-deficient mice. The livers of wild-type and UFD1s-deficient mice were subjected to H&E, LC3 IHC, and oil red staining after normal diet and 24-hour starvation treatment. All images were taken by an Olympus IX81 inverted fluorescence microscope, and the scale bar was 50 μm. The specific experimental procedures are as follows:

[0073] <Construction of UFD1s-deficient mice>

[0074] Cas9 mRNA and the corresponding sgRNA (SEQ ID NO.9) were transcribed using the mMESSAGE mMACHINETM T7 ULTRA transcription kit (Invitrogen, AM1345) and the MEGAshortscript TM T7 transcription kit (Invitrogen, AM1354) respectively, and then purified using the MEGAclearTM transcription purification kit (Invitrogen, AM1908).

[0075] The single-stranded oligodeoxynucleotide (ssODN) containing the mutated splice site sequence (AGGTCA to CGCAGC, a synonymous mutation of UFD1f) and the homologous sequences (119 nucleotides) on both sides (SEQ ID NO.10) was synthesized by Shanghai Sangon Biotech Co., Ltd.

[0076] Then, Cas9 mRNA / sgRNA and ssODN were microinjected into the embryos of C57BL / 6 mice. Two weeks after birth, all the mice were genotyped by PCR (using the primer pairs shown in SEQ ID NO.11 and 12) and verified by Sanger sequencing. Subsequently, for UFD1s - / - (UFD1s deletion) All related experiments were conducted with wild-type mice of the same litter, age, and sex as the control group.

[0077] <Metabolic cage>

[0078] Wild-type and UFD1s-deleted mice (4 mice / group) were placed in the breeding cages of the Promethion Metabolic Cage System (Sable Systems) and raised under normal conditions. After 24 hours of adaptation, data for 24 consecutive hours were collected and relevant data were analyzed, including total energy consumption, oxygen (O2), and respiratory exchange ratio (RER).

[0079] <qPCR detection of RNA>

[0080] 1. RNA extraction

[0081] (1) After washing the corresponding liver tissues with 1×PBS, 1 mL of Trizol (Invitrogen, 15596026) and grinding beads (Servicebio, G0204) were added, and the mixture was vigorously shaken in a tissue disruptor for mechanical disruption.

[0082] (2) 200 μL of chloroform was added to 1 mL of TRIzol to extract RNA. The centrifuge tube was shaken vigorously up and down 30 times and left to stand at room temperature for about 2 min.

[0083] (3) After standing, the mixture was centrifuged at 12,000 g for 15 min at 4°C in a centrifuge.

[0084] (4) After centrifugation, the liquid was seen to be divided into three layers (RNA layer, protein layer, and DNA layer in sequence). The centrifuge tube was tilted at 45°, and the clear liquid in the uppermost layer was transferred to another nuclease-free 1.5 mL centrifuge tube using a nuclease-free pipette tip (without touching the middle layer).

[0085] (5) Add an equal volume of isopropanol and mix thoroughly by inverting. Let stand at room temperature for 10 min or at -20°C for 20 min. It can also be stored in a -80°C refrigerator for a long time.

[0086] (6) After centrifugation at 12,000 g for 15 min at 4°C, discard the supernatant and add 1 mL of pre-cooled 80% ethanol. Mix thoroughly by inverting the tube and centrifuge at 7,500 g for 5 min.

[0087] (7) After discarding the ethanol, turn the centrifuge tube upside down and let it dry naturally.

[0088] (8) After the RNA is dried, add 39 μL of diethyl pyrocarbonate (DEPC H2O) to dissolve the RNA.

[0089] (9) Perform DNase digestion according to the following DNase I digestion system:

[0090] Reagents volume <![CDATA[DEPC H2O]]> 39μL DNase I 5μL DNase I buffer (10×) 5μL RNase inhibitor 1μL Total 50μL

[0091] (10) After adding DNase I digestion system, mix thoroughly and place in a 37°C water bath for 30 min.

[0092] (11) After DNase I digestion, add 5 μL stop buffer and heat at 70°C for 5 min to denature and inactivate DNase I.

[0093] (12) Add 45 μL of DEPC H2O, 250 μL of 4°C precooled anhydrous ethanol, and 10 μL of 3 M sodium acetate to the above system and mix thoroughly. Place the sample in a −80°C refrigerator to precipitate for 30 min.

[0094] (13) 4°C, 12,000 g, 15 min. After centrifugation, discard the liquid and add 1 mL of pre-cooled 80% ethanol. Invert the tube and centrifuge at 4°C, 7,500 g, 5 min.

[0095] (14) After discarding the ethanol, turn the centrifuge tube upside down to dry naturally, and dissolve the RNA precipitate with an appropriate volume of DEPC H2O (on ice).

[0096] (15) After sufficient dissolution, the RNA concentration and purity of each sample were measured. The purity can be determined by the ratio of optical density OD260 / OD280. The ratio of pure RNA should be no less than 2.0.

[0097] 2. Reverse transcription

[0098] (1) Using Promega GoScript TMReverse transcription kit (Promega, A5000), take 500ng~1μg of target RNA in a nuclease-free PCR tube, add 1μL oligo(dT) / Random primer, and fill it up to 10μL with nuclease-free H2O, mix well and place it in a 70℃ metal bath for denaturation for 5min, and immediately place it on ice for 2min after denaturation.

[0099] (2) During this period, prepare the following reverse transcription system:

[0100]

[0101] (3) Reverse transcription is performed using the reverse transcription system in step (2) according to the following PCR procedure:

[0102] Reaction temperature time 25℃ 5min 42℃ 60min 70℃ 15min 4℃ Hold

[0103] (4) After the reaction, the product can be diluted in an appropriate ratio for polymerase chain reaction (PCR) or real-time quantitative PCR (qPCR), and the product can be stored at -20°C.

[0104] 3. Real-time quantitative qPCR

[0105] (1) Prepare the system required for a sample according to the following reaction system:

[0106]

[0107]

[0108] The primer information involved in this system is as follows:

[0109]

[0110] (2) After the reaction mixture is thoroughly mixed, centrifuge briefly, and add 15 μL of the system in step (1) to the wells of a 96-well plate. Add three wells in total for three replicates.

[0111] (3) After all samples have been added, apply sealing film, scrape it flat with a scraper, and centrifuge (be careful not to touch the film with your hands during this process). Place the 96-well plate in the real-time fluorescence quantitative PCR instrument QuantStudio 3 and perform the reaction according to the following procedure:

[0112]

[0113] (4) After the reaction is completed, save the results, take out the product and shut down the instrument correctly.

[0114] (5) Data analysis: First, the plate layout should be correct according to the loading order, sample name, and gene name. The housekeeping gene ACTB mRNA is used as an internal reference, and the double Δ method is used to analyze the expression level of the target gene.

[0115] <Protein level detection by western blotting (WB)>

[0116] (1) Sample preparation: After washing the corresponding liver tissue with 1× PBS, add an appropriate volume of RIPA lysis buffer (50mM Tris-HCl, pH 8.0, 150mM NaCl, 5mM EDTA, 1mM DTT, 1% NP-40, 0.1% SDS, 1× protease inhibitor cocktail (Roche)) and grinding beads, shake in a tissue disruptor, and mechanically disrupt; after high-speed centrifugation at 4°C, take the supernatant and take a small portion of the volume for protein quantification ( BCA protein detection kit, thermo, 23227) was set aside, and the remaining was added with 5× SDS loading buffer to 1×, and 10mM DTT was added to mix well, and then denatured in a metal bath at 100℃ for 10min. After denaturation, centrifuge and place on ice for experiment or store in a -80℃ refrigerator.

[0117] (2) The prepared protein samples were separated on SDS-PAGE gel and transferred to nitrocellulose membrane (PALL) according to the conventional protein immunoblotting experimental procedures. Images were taken by ImageQuant LAS4000 biomolecular imager (GE Healthcare), and the grayscale values ​​of protein bands were counted using Image J software.

[0118] The antibody information involved in the protein immunoblotting experiment in step (2) is as follows:

[0119]

[0120] <Starvation treatment of mice>

[0121] The mice were kept in an environment of 23-25°C (normal lighting hours), with free access to water at 9 pm on the first day, without food, and starved for 24 hours. They continued to fast for 12 hours on the second day and were bled at 9 am on the third day. All control mice were fasted for 12 hours, bled at 9 am on the third day under isoflurane anesthesia, and the liver tissue was removed and fixed in 4% paraformaldehyde fixative for at least 24 hours (3 mice / group).

[0122] <Hematoxylin & Eosin (H&E) staining>

[0123] The fixed liver tissue was dehydrated with anhydrous ethanol and then embedded in paraffin. The embedded tissue was cut into 5 μm slices with an ultrathin slicer and mounted on a poly-lysine-coated slide, and H&E stained with a hematoxylin and eosin dye set (Servicebio, G1003). The tissue was observed and photographed under an Olympus IX81 inverted fluorescence microscope. The nuclear (Nuc) / cytoplasm (Cyto) area ratio (%) was determined by Image J.

[0124] <Immunohistochemistry (IHC) staining>

[0125] (1) Dewaxing of paraffin sections: Place the paraffin sections of mouse liver in dewaxing solution for 10 min, then wash with anhydrous ethanol for 5 min three times, and then wash with distilled water.

[0126] (2) Antigen repair: Use antigen repair solution to repair the sections, and prevent excessive evaporation of the buffer. After natural cooling, place the sections in PBS and wash three times on a shaker, each time for 5 minutes.

[0127] (3) Blocking endogenous peroxidase: Place the slices in 3% hydrogen peroxide and incubate in the dark for 25 min. Then, place the slices in PBS and wash three times on a shaker, each time for 5 min.

[0128] (4) Serum blocking: Add 3% BSA in the tissue circle until it covers the tissue and block at room temperature for 30 minutes.

[0129] (5) Incubation with primary antibody: Gently shake off the blocking solution, add diluted primary antibody of the corresponding protein (diluted in PBS at a certain ratio), and place the sections in a humidified chamber and incubate overnight at 4°C.

[0130] (6) Incubation with secondary antibody: Place the sections in PBS and wash three times on a shaker, 5 min each time. After drying the sections, add the corresponding secondary antibody according to the primary antibody and incubate at room temperature for 1 h.

[0131] (7) Diaminobenzidine (DAB) (Servicebio, G1212) color development: Place the slices in PBS and wash three times on a shaker, 5 min each time. After the slices are dried, add freshly prepared DAB color development solution and control the color development time under a microscope. Brown-yellow color indicates positive. Wash the slices with ultrapure water to stop the color development.

[0132] (8) Re-staining of cell nuclei: stain the cell nuclei with hematoxylin staining solution for about 3 minutes, rinse with ultrapure water, differentiate with hematoxylin differentiation solution for a few seconds, rinse with ultrapure water, reverse with hematoxylin anti-blue solution, and rinse with running water.

[0133] (9) Dehydration and sealing: Place the slices in 75% ethanol for 5 min → 85% ethanol for 5 min → anhydrous ethanol twice for 5 min → n-butanol for 5 min → xylene for dehydration and transparency for 5 min, then slice and air-dry and seal.

[0134] (10) Microscopic examination: The cells were observed and photographed under an Olympus IX81 inverted fluorescence microscope. The average optical density (AOD) was calculated using Image J to represent the IHC signal.

[0135] The antibody information involved in this step is as follows:

[0136]

[0137] <Oil red O staining>

[0138] (1) Frozen section fixation: The frozen sections of mouse liver tissue were rewarmed and dried, and then fixed in 4% paraformaldehyde fixative for 15 min, washed with ultrapure water, and dried naturally.

[0139] (2) Oil red staining: Place the sections in oil red staining solution (Servicebio, G1015) in the dark for 10 min.

[0140] (3) Background differentiation: The slices were placed in 60% isopropanol for differentiation twice, for 3 seconds and 5 seconds respectively. Then the slices were immersed in ultrapure water for 10 seconds each time.

[0141] (4) Hematoxylin nuclear staining: Remove the slices and place them in hematoxylin solution for counterstaining for 5 min; then rinse with ultrapure water three times, for 5 s, 10 s, and 10 s respectively; differentiate with differentiation solution for 5 s, then rinse with ultrapure water twice, for 10 s each; deblue with anti-blue solution for 1 s, and then rinse with ultrapure water twice, for 5 s and 10 s respectively.

[0142] (5) Microscopic examination: After the sections were sealed, they were placed under an Olympus IX81 inverted fluorescence microscope for microscopic examination and photography. The percentage of OilRed O-positive area was calculated using Image-Pro plus software.

[0143] Results and Analysis

[0144] Figure 1 Demonstrated CRISPR / Cas9-mediated UFD1s - / - Strategies for deficient mice ( Figure 1 A), RNA levels of UFD1s and its long transcript form (UFD1f) ( Figure 1 B) and protein levels ( Figure 1C). It can be seen that the synonymous mutation at the alternative splicing site of the UFD1 gene results in a significant decrease in the protein level of UFD1s without affecting the expression of the long transcript UFD1f protein.

[0145] Through Figure 2 It can be seen that compared with wild-type mice, UFD1s-deficient mice have reduced energy consumption, oxygen volume consumption, and increased respiratory exchange ratio (RER) ( Figure 2 A). In addition, the livers of UFD1s-deficient mice show abnormal morphology, reduced autophagy activity, and lipid droplet accumulation, and this phenomenon is more severe after starvation treatment ( Figure 2 B).

[0146] The above results indicate that UFD1s has a cytoprotective effect against stress.

[0147] Example 2 Study on the correlation between the deletion of UFD1s and the progression of non-alcoholic steatohepatitis (NASH)

[0148] In this example, a NASH model can be induced by feeding mice with a methionine-choline (MCD)-deficient diet for 4 weeks, which is also a model widely recognized internationally. After the NASH disease model is successfully induced, the occurrence and development process of NASH are determined by detecting the liver UFD1s RNA expression level, serum lipid and transaminase levels, steatosis score, oil red staining, and fibrosis staining. The specific experimental procedures are as follows:

[0149] <MCD-induced NASH model>

[0150] Mice were first subjected to overnight starvation (water was available ad libitum), and then fed a normal diet (normal group) or an MCD diet (NASH group) for a total of 4 weeks, and fresh feed was changed every 2 - 3 days during this period. No mice died during all model induction experiments. All mice were subjected to 12-hour starvation before sacrifice, with 6 mice / group.

[0151] <Determination of serum triglyceride, total cholesterol, aspartate aminotransferase (AST), and alanine aminotransferase (ALT)>

[0152] Mice were anesthetized with isoflurane, and blood was collected by orbital enucleation. The collected blood was centrifuged at 3000 rpm for 10 min at room temperature, and the supernatant was retained as serum. Subsequently, the levels of triglyceride, total cholesterol, AST, and ALT transaminases in the serum were measured using a triglyceride assay kit (NJJC, F001-1-1), a total cholesterol assay kit (NJJC, F002-1-1), an aspartate aminotransferase detection kit (Solarbio, BC1560), and an alanine aminotransferase assay kit (Solarbio, BC1555), respectively. All operations were carried out according to the kit instructions.

[0153] <Hepatic steatosis score>

[0154] To quantify the difference in the degree of hepatic steatosis among different experimental groups, H&E staining of liver tissues (scale bar: 50 μm) was performed and pathological evaluation of steatosis grades was carried out. The scoring was evaluated by pathological experts (Wuhan Sevier Biotechnology Co., Ltd.).

[0155] The steatosis grade (score: 0 - 3) was determined by the percentage of microvacuoles / steatosis. The observed field of view was 20×. The specific scoring criteria are as follows:

[0156]

[0157]

[0158] <Masson staining>

[0159] To observe the degree of liver fibrosis, Masson staining can present the degree of fibrosis through the depth of color. Similar to the tissue staining preparation steps in Example 1, paraffin sections with a thickness of 5 μm were prepared and stained with Masson staining solution (Servicebio, G1006). Finally, it was examined under an Olympus IX81 inverted fluorescence microscope and photographed, and the percentage of the Masson positive area was calculated by Image J. The scale bar was 50 μm.

[0160] All others were detected or stained using the same method as in Example 1, and will not be elaborated here one by one.

[0161] Results and Analysis

[0162] By Figure 3 It can be seen that compared with normal mice, the level of UFD1s RNA in the liver of NASH mice was significantly decreased ( Figure 3 A), indicating that UFD1s can be used as a new biomarker for the auxiliary diagnosis of NASH. More importantly, the levels of triglyceride and total cholesterol in the serum of mice lacking UFD1s in the normal group and the NASH group with UFD1s deficiency were both significantly increased ( Figure 3 B). As is well known to those skilled in the art, transaminase levels are used to evaluate the degree of liver injury. In both the normal group and the NASH group, the deficiency of UFD1s led to a significant increase in the serum AST level of mice, while the ALT level did not change significantly ( Figure 3 C). Figure 3 The results in

[0163] By Figure 4 It can be seen that, compared with the normal group, the livers of UFD1s-deficient mice in the NASH group showed more severe steatosis, more lipid droplet accumulation, and a higher degree of fibrosis. This indicates that the deletion of UFD1s accelerates the development of NASH.

[0164] Example 3 Study on the correlation between UFD1s and the NASH process

[0165] To explore the therapeutic potential of UFD1s, in this example, recombinant plasmids and circular RNAs containing the UFD1s open reading frame sequence were constructed, and the UFD1s protein was expressed in a NASH mouse disease model through two strategies: plasmid injection and circular RNA delivery. The therapeutic effect of UFD1s was comprehensively evaluated by detecting serum lipids, transaminase levels, assessing the grade of liver steatosis, the number of lipid droplets, the degree of fibrosis, and the inflammation score. All images were taken with an Olympus IX81 inverted fluorescence microscope, and the scale bar was 50 μm. 6 mice / group.

[0166] <Construction and hydrodynamic injection of the <pLIVE-UFD1s plasmid>

[0167] The mouse UFD1s ORF sequence (SEQ ID NO.4) was cloned into the <pLIVE> vector backbone (between the NheI and SacI restriction enzyme sites) (Mirus Bio, MIR 5420) and identified by Sanger sequencing to construct the <pLIVE-UFD1s plasmid>.

[0168] During the 4-week process of inducing the NASH disease model with MCD diet in Example 2, plasmid injection was performed in the second week ( Figure 5 A). To obtain specific expression of UFD1s in the liver, in this example, 15 μg of <pLIVE> empty vector or <pLIVE-UFD1s plasmid> was diluted with 2 mL of normal saline (0.9% NaCl), approximately 10% of the body weight. Subsequently, the corresponding plasmid was injected into the mouse blood through the tail vein using a 2.5 mL syringe and a 27-gauge needle, and this process needed to be completed quickly within 5 s. No abnormalities or lethality were observed in the mice after hydrodynamic injection.

[0169] <In vitro circularization (circUFD1s) and delivery of UFD1s>

[0170] 1. In vitro circularization

[0171] (1) The UFD1s ORF (SEQ ID NO. 4) and the linear RNA containing the CVB3 internal ribosome entry site (IRES) were transcribed in vitro using the TranscriptAid T7 High Yield Transcription Kit (Thermo, K0441). The ATG of UFD1s was mutated to ATT as a negative reference. That is, 1 μg of linearized template (Uohuan Biotechnology), 2 μL of T7 RNA polymerase, and 10 mM NTP were fully mixed (20 μL system) and incubated at 37°C for 4 h.

[0172] (2) Using the TIE circularization system, the in vitro transcription product was placed at 50°C for 30 min for circularization. After the circularization, DNase I was added and incubated at 37°C for 15 min to remove the DNA template.

[0173] (3) After the cyclization reaction, the product was digested with RNase R (Epicentre, RNR07250) to remove linear RNA.

[0174] (4) Separate the circularized RNA using 5% urea gel, cut out the corresponding gel strip of circUFD1s according to the fragment size, and dissolve it in elution buffer.

[0175] (5) circUFD1s was extracted and purified using the phenol / chloroform method (pH 4.5).

[0176] 2. Delivery of circUFD1s

[0177] In the 4-week process of the MCD diet-induced NASH disease model in Example 2, circUFD1s or circUFD1s were respectively performed at the 2nd and 3rd weeks. ATG-ATT Injection ( Figure 5 A). 10 μg of circRNA was mixed with 20 μL of Lipofectamine 3000 (Invitrogen, L3000015) (PBS dilution, total volume 100 μL) and allowed to stand at room temperature for 15 min. Subsequently, the circRNA-Lipofectamine 3000 mixture was injected into the mouse through the tail vein within 30 s using a 1 mL syringe and a 27-gauge needle. After the injection, there was no abnormality or lethality in the mouse.

[0178] <Inflammation score>

[0179] To test whether the in vivo expression of UFD1s has a therapeutic effect on NASH inflammation, H&E staining of liver tissue (scale bar 50 μm) and pathological evaluation of lobular inflammation were performed, and the score was evaluated by pathology experts (Wuhan Sevier Biotechnology Co., Ltd.). The degree of lobular inflammation (score: 0-3) was evaluated according to the number of inflammatory sites (foci), and the specific scoring criteria were as follows:

[0180] Number of inflammatory sites (foci) Degree of lobular inflammation No foci 0 <2 foci / 200× field of view 1 2-4 foci / 200× field of view 2 >4 foci / 200× field of view 3

[0181] <Level of liver fibrosis and inflammation>

[0182] The same staining method as above was used to evaluate liver fatty degeneration, lipid droplet number and degree of fibrosis. At the same time, the degree of liver fibrosis and inflammation was investigated by detecting the mRNA expression levels of fibrosis marker (COLLA1) and inflammation marker (TNF) in the liver (the qPCR method was the same as above). The specific information of the primers involved is as follows:

[0183]

[0184] Results and Analysis

[0185] pass Figure 5 It can be seen that compared with the corresponding control group, both the pLIVE-UFD1s and circUFD1s groups had high levels of UFD1s protein expression in the liver ( Figure 5 B), indicating that both strategies can achieve efficient and stable expression of UFD1s in the liver.

[0186] pass Figure 6 It can be seen that compared with the corresponding control group, the levels of triglyceride and cholesterol in the serum of NASH mice injected with pLIVE-UFD1s and circUFD1s were significantly reduced ( Figure 6 A); In addition, compared with the corresponding control group, the levels of AST and ALT transaminases in the mice injected with pLIVE-UFD1s were significantly reduced, and the AST level in the serum of the mice injected with circUFD1s was significantly reduced, while the ALT level did not change significantly. This indicates that the expression of UFD1s can significantly reduce the levels of lipids and transaminases (especially AST) in serum.

[0187] In addition, compared with the corresponding control group, liver steatosis, lipid droplet number and fibrosis degree of NASH mice expressing UFD1s were significantly reduced. At the same time, the inflammation score of the liver of NASH mice expressing UFD1s, and the mRNA expression levels of fibrosis marker (COLLA1) and inflammation marker (TNF) in the liver were significantly lower than those in the corresponding control group ( Figure 8A and Figure 8 B) indicates that expressing UFD1s in the liver of NASH mice can reduce fatty degeneration, lipid droplet number, fibrosis and inflammation in NASH mice.

[0188] Based on the above results, it can be concluded that UFD1s can alleviate the development of NASH, and corresponding drugs for the prevention and / or treatment of NASH can be developed based on UFD1s.

[0189] Based on the experimental results of Examples 1-3, it is shown that UFD1s has a protective effect against cellular stress, and its expression level is significantly reduced in mice with non-alcoholic fatty liver disease, which is related to the occurrence and development of non-alcoholic fatty liver disease, and can be used as a biomarker for the diagnosis of the disease; after UFD1s is expressed in vivo in mice using plasmids and circular RNA, respectively, it has a significant effect on the treatment of non-alcoholic fatty liver disease, and greatly inhibits the progression of metabolic diseases caused by nutritional imbalance, and therefore has a high medical value.

[0190] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0191] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A small protein UFD1s that regulates energy metabolism and resists cell stress, characterized in that: The small protein UFD1s has an open reading frame sequence as shown in SEQ ID NO.3 or SEQ ID NO.

4.

2. The small protein UFD1s having the functions of regulating energy metabolism and resisting cell stress according to claim 1, characterized in that: The small protein UFD1s is selected from at least one of the following a to c: a: a protein with an amino acid sequence as shown in SEQ ID NO.1 or SEQ ID NO.2; b: A derivative protein formed by substitution, deletion or addition of the amino acid sequence shown in SEQ ID NO.1 or SEQ ID NO.2 at one or more positions, and having the functions of regulating energy metabolism and resisting cell stress; c: A protein that has at least 25% identity with the amino acid sequence shown in SEQ ID NO.1 or SEQ ID NO.2 and has the functions of regulating energy metabolism and resisting cell stress.

3. Use of a reagent for detecting the expression level of UFD1s in the liver tissue of a subject in the preparation of a product for auxiliary diagnosis of non-alcoholic fatty hepatitis, wherein: The UFD1s is the small protein UFD1s according to claim 1 or 2; Preferably, the product is a chip or a kit; Preferably, the reagent for detecting the expression level of UFD1s in the liver tissue of the subject is a reagent for detecting the expression amount of UFD1s protein in the liver tissue of the subject, or a reagent for detecting the RNA expression level of UFD1s in the liver tissue of the subject; Preferably, the reagent for detecting the expression level of UFD1s in the liver tissue of a subject is a reagent for detecting the RNA expression level of UFD1s in the liver tissue of a subject, and the reagent contains one of the following primer pairs: (1) a primer pair having sequences as shown in SEQ ID NOs. 5 and 6; (2) A primer pair having sequences as shown in SEQ ID NOs. 7 and 8.

4. An open reading frame sequence of a small protein UFD1s, characterized in that: The open reading frame sequence is shown as SEQ ID NO.3 or SEQ ID NO.

4.

5. An expression product of a small protein UFD1s, characterized in that: The expression product contains the open reading frame sequence as claimed in claim 4; Preferably, the expression product is a recombinant plasmid or circular RNA.

6. A drug for preventing and / or treating non-alcoholic fatty hepatitis, characterized in that: The drug contains an agonist of the small protein UFD1s.

7. The drug according to claim 6, characterized in that The agonist of the small protein UFD1s contains the open reading frame sequence of the small protein UFD1s according to claim 4 or the expression product of the small protein UFD1s according to claim 5 .

8. The drug according to claim 6 or 7, characterized in that The drug has at least one of the following effects: (1) reducing the level of serum lipids in a subject, including triglycerides and total cholesterol; (2) reduce the level of serum aspartate aminotransferase in subjects; (3) reduce the degree of liver steatosis in subjects; (4) Improve the degree of liver inflammation in subjects; (5) Improve the degree of liver fibrosis in subjects.

9. Use of an agonist of the small protein UFD1s in the preparation of a drug for preventing and / or treating non-alcoholic steatohepatitis; Preferably, the agonist of the small protein UFD1s contains the open reading frame sequence of the small protein UFD1s according to claim 4 or the expression product of the small protein UFD1s according to claim 5.

10. The use according to claim 9, characterized in that The drug has at least one of the following effects: (1) reducing the level of serum lipids in a subject, including triglycerides and total cholesterol; (2) reduce the level of serum aspartate aminotransferase in subjects; (3) reduce the degree of liver steatosis in subjects; (4) Improve the degree of liver inflammation in subjects; (5) Improve the degree of liver fibrosis in subjects.

Images