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

Abstract

Provided are a small protein UFD1s and use thereof in the prevention, treatment, and diagnosis of non-alcoholic steatohepatitis. The provided small protein UFD1s can regulate energy metabolism and combat cell stress, and UFD1s can be used as an auxiliary diagnostic marker for non-alcoholic steatohepatitis (NASH). Meanwhile, the deletion or low expression level of UFD1s is related to the development and progression of NASH. By expressing UFD1s in vivo by means of a constructed expression vector and a method, its effect in preventing and / or treating NASH can be exerted, thereby providing a new idea for the prevention and / or treatment of NASH.

Description

Small protein UFD1s and its application in the prevention, treatment and diagnosis of non-alcoholic steatohepatitis Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a small protein UFD1s associated with non-alcoholic steatohepatitis (NAH), and also to the application of this small protein UFD1s in the prevention, treatment and diagnosis of NHA. Background Technology

[0002] Non-alcoholic steatohepatitis (NASH) is a common chronic liver disease unrelated to alcohol consumption and is widely believed to be closely associated with insulin resistance, obesity, and metabolic syndrome. NAFLD is characterized by excessive fat accumulation in the liver, in the form of triglycerides, in more than 5% of liver tissue cells (steatodegeneration). In addition to excessive fat, the livers of NASH patients exhibit lobular inflammation, ballooning hepatocyte degeneration, and faster-progressing fibrosis compared to NAFLD. In the short term, the degree of steatosis alone in NAFLD is not significantly correlated with increased morbidity or mortality; however, once it progresses to NASH, it significantly increases the risk of cirrhosis, liver failure, and hepatocellular carcinoma (HCC), making it a potentially fatal disease.

[0003] Many NASH patients appear healthy without realizing their liver abnormalities, making it difficult to detect. Currently, liver function is primarily assessed by measuring serum triglyceride, cholesterol, and transaminase levels (such as aspartate aminotransferase and alanine aminotransferase). The only diagnostic method for NASH is a liver biopsy, which uses histopathological examination to confirm the presence of inflammation or fibrosis. However, histopathological examination is complex, time-consuming, and expensive, and there is a lack of readily available biomarkers for the clinical diagnosis of NASH. Furthermore, regarding the treatment of NASH, for some early-stage patients, the main approach is to control blood sugar with diabetes medications and improve lifestyle habits, such as regular exercise and reducing sedentary activity, which can slow down or even reverse the process of fatty degeneration. However, for late-stage NASH patients, there is currently a lack of standard and well-defined treatment measures, thus highlighting the urgent need for effective prevention and treatment measures. Summary of the Invention

[0004] In view of this, the primary objective of the present invention is to provide a small protein, UFD1s, which is the first protein conserved in mammals that has the functions of regulating energy metabolism and resisting cellular 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.

[0005] In this invention, preliminary research revealed that the conserved small protein UFD1s in humans and mice plays a protective role in regulating energy metabolism and resisting cellular stress. Under normal conditions, UFD1s deficiency manifests as lower energy consumption, less oxygen volume consumption, and a higher respiratory exchange rate (RER). Furthermore, UFD1s protein deficiency leads to abnormal liver morphology, including mild hydropic degeneration, swelling, loose hepatocyte cytoplasm, and abnormal accumulation of lipid droplets in the liver, which is exacerbated after 24 hours of starvation. More importantly, this invention, through mouse experiments, found that UFD1s RNA expression levels in the liver of NASH mice were significantly reduced, and UFD1s deficiency accelerated the progression of NASH disease, including high levels of lipids and transaminases in serum, severe steatosis, lobular inflammation, and fibrosis. Based on these findings, it can be determined that the small protein UFD1s has certain therapeutic, stabilizing, improving, or preventative effects in nutritional imbalance metabolic diseases such as NASH, and can serve as a biomarker for the auxiliary diagnosis of NASH. Based on this, the present invention proposes the use of small protein UFD1s in the diagnosis, prevention and treatment of nutritional imbalance metabolic diseases such as NASH.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] The first aspect of the present invention discloses a small protein UFD1s that has the functions of regulating energy metabolism and resisting cellular stress, the amino acid sequence of which is shown in SEQ ID NO.1 or SEQ ID NO.2.

[0008] A second aspect of the present invention discloses an expression vector containing a nucleotide sequence encoding the small protein UFD1s, the nucleotide sequence being shown in SEQ ID NO.3 or SEQ ID NO.4.

[0009] A third aspect of the present invention discloses a medicament for the prevention and / or treatment of non-alcoholic steatohepatitis, the medicament containing the small protein UFD1s or the expression vector described above.

[0010] The fourth aspect of the present invention discloses applications as follows:

[0011] (1) The application of reagents for detecting the expression level of UFD1s in the liver tissue of subjects in the preparation of products for the auxiliary diagnosis of non-alcoholic steatohepatitis, wherein the UFD1s are the small protein UFD1s mentioned above;

[0012] (2) The use of the small protein UFD1s or the expression vector described above in the preparation of drugs for the prevention and / or treatment of non-alcoholic steatohepatitis.

[0013] Through extensive preliminary research and experiments, the applicant sought novel transcripts with stress response and energy metabolism regulation functions. In this study, a highly conserved cleavage form of the ubiquitin fusion degradation protein UFD1 in humans and mice was discovered and named UFD1s in this invention. This UFD1s protein had not been previously reported, and this invention confirms that the conserved UFD1s protein in humans and mice has protective functions in regulating energy metabolism and resisting cellular stress.

[0014] In this invention, the amino acid sequence of the small protein UFD1s is shown in SEQ ID NO.1 or SEQ ID NO.2.

[0015] SEQ ID NO.1 (Human Origin):

[0016] MFSFNMFDHPIPRVFQNRFSTQYRCFSVSMLAGPNDSNYATLGPGPTQPT.

[0017] SEQ ID NO.2 (Mouse-borne):

[0018] MFSFNMFDHPIPRVFQNRFSTQYRCFSVSMLAGPNDSNYATLSPRSTQPAQHYLSYAV.

[0019] In this invention, the nucleotide sequence encoding the small protein UFD1s is shown in SEQ ID NO.3 or SEQ ID NO.4.

[0020] SEQ ID NO.3 (Human Origin):

[0021] ATGTTCTCTTCAACATGTTCGACCACCCTATTCCCAGGGTCTTCCAAAACCGCTTCTCCACACAGTACCGCTGCTTCTCTGTGTCCATGCTAGCAGGGCCTAATGACAGTAATTATGCCACCCTCGGCCCTGGACCAACTCAGCCGACTTAA (153 nt).

[0022] SEQ ID NO.4 (Mouse source):

[0023] ATGTTTTTCTTTCAACATGTTTGACCACCCGATTCCCCGGGTCTCCAGAACCGCTTCTCCACGCAGTACCGCTGCTTCTCCGTGTCCATGCTAGCAGGGCCTAATGACAGTAATTATGCCACCCTCAGCCCTCGATCAACTCAGCCGGCTCAACATTACCTATCCTATGCTGTTTAA (177 nt).

[0024] In some embodiments of the present invention, the UFD1s described herein can be used as a biomarker to assist in the diagnosis of non-alcoholic steatohepatitis (NAH). By detecting the expression level of UFD1s in the liver tissue of the subject, it can serve as an auxiliary indicator for the diagnosis of NHA. In conjunction with other NHA indicators, it facilitates the diagnosis of NHA. Compared with the current traditional histopathological examination, the detection method is simpler and less costly.

[0025] In this invention, the product refers to an in vitro diagnostic product, such as a formulation, test strip, chip, or reagent kit, but is not limited thereto. Preferably, the product is a chip or reagent kit.

[0026] In this invention, the reagent used to detect the expression level of UFD1s in the liver tissue of a subject is either a reagent for detecting the protein expression level of UFD1s in a biological sample or a reagent for detecting the RNA expression level of UFD1s in the liver tissue of a subject. By detecting the protein or RNA expression level of UFD1s in the liver tissue of a subject, the diagnosis of NASH can be aided. Specifically, if the protein or RNA expression level of UFD1s is low, it suggests that the subject is likely to have NASH. The specific method of judgment 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 aid in the diagnosis. If, compared with the average RNA expression level in normal liver tissue (such as adjacent non-cancerous tissue) of a given population, the RNA expression level in the subject's liver tissue is reduced by more than 50%, then the RNA expression level of UFD1s is considered low, suggesting that the subject is likely to have NASH. By combining this with other indicators, the diagnosis of NASH can be achieved.

[0027] In this invention, the subject refers to a mammal, such as a human, mouse, rabbit, or sheep. Preferably, the subject refers to a human or mouse. In some specific implementations, the subject is a mouse.

[0028] It is understood that the reagents used to detect the expression level of UFD1s in the liver tissue of the subject vary depending on the target (protein or RNA) and the detection technology. Specific detection methods may be those 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 blotting, bleed cytology, or immunohistochemical staining (IHC), but are not limited to these. The reagents used may include, for example, antibodies, nucleic acid probes, or primers that specifically bind to UFD1s.

[0029] In some specific implementation examples, 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 examples, the subject is a human, and the reagent contains primer pairs with sequences as shown in SEQ ID NO. 5 and 6; in other specific implementation examples, the subject is a mouse, and the reagent contains primer pairs with sequences as shown in SEQ ID NO. 7 and 8.

[0030] Furthermore, it is understood that the products mentioned above include not only the reagents necessary for detecting UFD1s expression levels, but also buffer solutions, washing solutions, etc., required for different detection technologies. These can be selected based on the needs of the field and are therefore not specifically limited.

[0031] In other embodiments of the present invention, cell experiments and animal experiments have determined that the small protein UFD1s affects the course or progression of NASH disease. The absence of UFD1s accelerates the progression of NASH disease. By injecting the expression vector of the small protein UFD1s into the subject, the expression level of the small protein UFD1s in the subject can be increased, thereby achieving the effect of preventing and / or treating NASH.

[0032] In this invention, the expression vector refers to any plasmid or RNA in the art capable of specific expression in the liver. The expression vector contains a nucleotide sequence encoding the small protein UFD1s, as shown in SEQ ID NO. 3 or SEQ ID NO. 4, thereby enabling specific expression of UFD1s in the liver, increasing the expression level of UFD1s in the liver, and thus achieving the effect of preventing and / or treating NASH. In this invention, the specific type can be a recombinant plasmid or a circular RNA.

[0033] In some specific embodiments of the present invention, the expression vector is a recombinant plasmid, which is constructed by inserting a nucleotide 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, such as pLIVE or pAlb-Luc, but is not limited thereto.

[0034] The specific methods for constructing recombinant plasmids can be those well known to those skilled in the art, and mainly include the following steps:

[0035] The nucleotide sequence encoding the small protein UFD1s was cloned into a backbone vector and identified by sequencing to obtain a recombinant plasmid.

[0036] In some specific implementation cases, the present invention uses pLIVE to construct recombinant plasmids, named pLIVE-UFD1s plasmid.

[0037] In other specific embodiments of the present invention, the expression vector is a circular RNA, and the circular RNA is used to circularize UFD1s in vitro. Specifically, methods familiar to those skilled in the art can be used for in vitro circularization, without particular limitation. In some specific embodiments, the in vitro circularization of UFD1s mainly includes the following steps:

[0038] Linear RNA containing a nucleotide sequence encoding the small protein UFD1s was obtained through in vitro transcription. After circulation, circular RNA, namely circUFD1s, was obtained.

[0039] Furthermore, in this invention, the drugs for preventing and / or treating non-alcoholic steatohepatitis refer to drugs containing an effective amount of small protein UFD1s or the expression carrier described above as an active ingredient, which can increase the expression level of small protein UFD1s in liver tissue.

[0040] In this invention, the drug for preventing and / or treating non-alcoholic steatohepatitis has at least one of the following effects:

[0041] (1) Reduce the level of serum lipids in the subject, including triglycerides and total cholesterol;

[0042] (2) Reduce the level of serum aspartate aminotransferase in the subjects;

[0043] (3) Reduced the degree of hepatic steatosis in the subjects;

[0044] (4) Improves the degree of liver inflammation in the subjects;

[0045] (5) Improved the degree of liver fibrosis in the subjects.

[0046] Specifically, by administering drugs containing small protein UFD1s or the expression vectors described above to subjects, the expression level of small protein UFD1s in the liver of the subjects is increased, thereby exerting the effect of preventing and / or treating non-alcoholic steatohepatitis.

[0047] The effective dose here refers to the minimum dose that produces the desired preventive and / or therapeutic effect, and this dose is within the safety range (i.e., does not produce adverse reactions). In this invention, the desired preventive effect refers to increasing the expression level of UFD1s in the liver of the subject, thereby preventing the development of NASH; the desired therapeutic effect refers to increasing the expression level of UFD1s in the liver of the subject, resulting in improvement or even cure of NASH patients. The specific effective dose varies depending on the type of subject, the degree of disease progression, and the method of administration, formulation, etc., and can be determined experimentally by those skilled in the art based on known methods, therefore there is no particular limitation. For example, in some specific cases of this invention, the subject is a mouse, wherein the injection dose of pLIVE-UFD1s plasmid is 15 μg / time, once; the injection dose of circUFD1s is 10 μg / time, twice.

[0048] It is understood that the aforementioned drugs also include any pharmaceutically acceptable carriers and / or excipients. These carriers and / or excipients may vary depending on the specific drug dosage form, and their main functions include, but are not limited to, improving drug stability, improving drug delivery efficiency, and improving the subject's ability to absorb or metabolize the drug, so that the drug can better exert its preventive and / or therapeutic effects. The carriers and / or excipients mentioned can be selected or modified based on methods known to those skilled in the art, and therefore will not be specifically described here.

[0049] Furthermore, the aforementioned drug may also contain other therapeutic substances that prevent and / or treat non-alcoholic steatohepatitis (NAH). These therapeutic substances refer to any substances that can be combined with the expression vector in this application to further enhance the efficacy of preventing and / or treating NHA.

[0050] In this invention, the drug dosage form is not particularly limited and can be any form well-known in the art, such as granules, capsules, powders, tablets, oral liquids, or injections. Preferably, the drug is an injection.

[0051] The beneficial effects of this invention are:

[0052] This invention identifies the highly conserved small protein UFD1s in humans and mice as having protective effects in regulating energy metabolism and resisting cellular stress. The loss of UFD1s accelerates the progression of NASH. This invention demonstrates that the expression level of UFD1s is significantly reduced in NASH, therefore UFD1s can serve as a biomarker for the auxiliary diagnosis of NASH; furthermore, expressing the small protein UFD1s in the liver can effectively prevent and / or improve the occurrence and development of NASH.

[0053] This invention constructs expression vectors based on the small protein UFD1s. By delivering these expression vectors to subjects, UFD1s can be efficiently and stably expressed in the liver, thereby exerting a therapeutic effect on NASH. Delivery of the expression vectors reduces serum lipids and transaminases, and decreases steatosis, lipid droplet number, fibrosis, and inflammation associated with NASH. This provides a new approach for the development of drugs for the prevention and / or treatment of non-alcoholic steatohepatitis. Attached Figure Description

[0054] Figure 1: Demonstration of CRISPR / Cas9 technology-mediated UFD1s - / - The strategy of defective mice (Figure 1A), the RNA level (Figure 1B) and protein level (Figure 1C) of UFD1s and its long transcript form (UFD1f).

[0055] Figure 2: Under normal conditions, total energy consumption, oxygen consumption, and gas exchange rate of wild-type and UFD1s-deficient mice were analyzed using a metabolic cage (Figure 2A). Liver morphology of UFD1s-deficient mice was observed under normal and starvation conditions using hematoxylin-eosin (HE); lipid droplet content was detected by Oil Red O staining; and autophagy was observed by immunohistochemistry (IHC) of the autophagy marker LC3 (Figure 2B).

[0056] Figure 3: UFD1s RNA levels in the liver of normal and NASH mouse models (Figure 3A), serum triglyceride and total cholesterol levels (Figure 3B), and serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels (Figure 3C).

[0057] Figure 4: HE, Oil Red, and Masson staining of livers in normal and NASH model mice, used to observe hepatic steatosis, number of lipid droplets, and degree of fibrosis, respectively.

[0058] Figure 5: The process of expressing UFD1s protein using plasmid and circular RNA strategies in a methionine-choline-deficient diet (MCD) induced NASH mouse model (Figure 5A), and the expression level of UFD1s in the liver was detected by IHC (Figure 5B).

[0059] Figure 6: Serum triglyceride and cholesterol levels (Figure 6A) and serum AST and ALT levels (Figure 6B) in NASH mice expressing UFD1s via plasmid and circular RNA.

[0060] Figure 7: H&E, Oil Red, and Masson staining of the livers of NASH mice expressing UFD1s via plasmids and circular RNA, used to observe hepatic steatosis, lipid droplet count, and degree of fibrosis, respectively.

[0061] Figure 8: Inflammation scores of NASH mice expressing UFD1s using two strategies (Figure 8A), and the expression levels of fibrosis marker (COLLA1) and inflammation marker (TNF) mRNA in the liver (Figure 8B).

[0062] Figure 9: Immunohistochemical staining analysis of the expression of small protein UFD1s in liver tissues of non-NASH and NASH clinical patients (N=8 cases) (Figure 9A); determination of triglyceride content in human HEK293T wild-type (WT) and UFD1s-deficient (UFD1s KO) cells (Figure 9B); Nile red staining to detect lipid droplet content in small protein UFD1s-deficient and overexpressed (UFD1s OE) cells (Figure 9C); Evaluation of fatty acid oxidation rate in small protein UFD1s-deficient and overexpressed cells by measuring the oxygen consumption rate (OCR) of cells with exogenous palmitic acid (PA) (Figure 9D and E). Embodiments of the present invention

[0063] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0064] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of this invention is for the purpose of describing particular embodiments only and is not intended to limit the invention. Furthermore, unless otherwise specified, methods not specifically describing conditions or steps are conventional methods, and the reagents and materials used are commercially available.

[0065] Animal source and rearing environment: All C57BL / 6 wild-type mice used in this article were purchased from Spiefer Biotech (Beijing) Co., Ltd. All mice were housed in a specific pathogen-free (SPF) environment with a 12 / 12h light / dark cycle (lighting time: 8:00~20:00), at 22±2℃ 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, with animal ethics number USTCACUC202101048.

[0066] Example 1: Construction of UFD1s-deficient mice and their stress-protective effect under starvation conditions

[0067] In this embodiment, UFD1s-deficient mice were constructed using the CRISPR / Cas9 system, and UFD1s was detected at both RNA and protein levels. Metabolic cages were used to monitor the metabolism of the UFD1s-deficient mice. Liver samples from wild-type and UFD1s-deficient mice were stained with HE, LC3 IHC, and Oil Red dye after normal diet and 24-hour starvation, respectively. All images were taken using an Olympus IX81 inverted fluorescence microscope, scale bar 50 μm. The specific experimental procedure is as follows:

[0068] 1.1 Construction of UFD1s-deficient mice

[0069] Cas9 mRNA and the corresponding sgRNA (nucleotide sequence TGCTAGCAGGGCCTAATGAC) were transcoded using the mMESSAGE mMACHINETM T7 ULTRA transcription kit (Invitrogen, AM1345) and MEGAshortscript, respectively. TM Transcription was generated using the T7 Transcription Kit (Invitrogen, AM1354) and then purified using the MEGAclear™ Transcription Purification Kit (Invitrogen, AM1908).

[0070] The single-stranded oligodeoxyribonucleic acid (ssODN) was synthesized by Sangon Biotech (Shanghai) Co., Ltd., which contains a mutated splice site sequence (AGGTCA to CGCAGC, a synonymous mutation of UFD1f) and homologous sequences on both sides (119 nucleotides) (SEQ ID NO.9).

[0071] Then, Cas9 mRNA / sgRNA and ssODN were microinjected into the embryos of C57BL / 6 mice. Two weeks after birth, all mice were genotyped by PCR (using the forward primer GATGACTTGCTATGTTAGTGCTTGG and the reverse primer CCATGCCTTAGGACAGTGACATT), and the results were verified by Sanger sequencing. Subsequent UFD1s... - / - (UFD1s deletion) All related experiments used wild-type mice of the same littermate, age and sex as the control group.

[0072] 1.2 Metabolic cage

[0073] Wild-type and UFD1s-deficient mice (n=4 each) were placed in Promethion Metabolic Cage System (Sable Systems) cages and housed under normal conditions. After 24 hours of acclimatization, data were collected over 24 consecutive hours and analyzed, including total energy expenditure, oxygen (O2), and respiratory exchange rate (RER).

[0074] 1.3 qPCR detection of RNA

[0075] 1.3.1 RNA extraction

[0076] (1) After washing the corresponding liver tissue with 1×PBS, add 1 mL of Trizol (Invitrogen, 15596026) and grinding beads (Servicebio, G0204), and mechanically disrupt the tissue by vigorous shaking in a tissue homogenizer.

[0077] (2) Add 200 μL of chloroform to 1 mL of TRIzol to extract RNA. Shake the centrifuge tube vigorously up and down 30 times and let it stand at room temperature for about 2 min.

[0078] (3) After standing, centrifuge at 12000 g speed for 15 min at 4℃.

[0079] (4) After centrifugation, you will see the liquid divided into three layers (RNA layer, protein layer and DNA layer in sequence). Tilt the centrifuge tube at 45° and use a nuclease-free pipette tip to transfer the clear liquid at the top to another nuclease-free 1.5 mL centrifuge tube (do not touch the middle layer).

[0080] (5) Add the same volume of isopropanol and mix thoroughly by inverting the container. Let it stand at room temperature for 10 min or at -20℃ for 20 min to precipitate. Alternatively, it can be stored in a -80℃ refrigerator for a long time.

[0081] (6) After centrifuging at 12000 g for 15 min at 4℃, discard the supernatant and add 1 mL of pre-cooled 80% ethanol. Mix thoroughly by inverting the mixture and then centrifuge at 7500 g for 5 min.

[0082] (7) After discarding the ethanol, invert the centrifuge tube and let it air dry naturally.

[0083] (8) After the RNA is dried, add 39 μL of water treated with diethyl pyrocarbonate (DEPC H2O) to dissolve the RNA.

[0084] (9) Perform DNA digestion using the DNase I digestion system as shown in Table 1:

[0085] Table 1 DNase I Digestion System

[0086]

[0087] (10) After adding the DNase I digestion system, mix thoroughly and then place in a 37°C water bath for 30 min to digest.

[0088] (11) After DNase I digestion is complete, add 5 μL stop buffer and heat at 70°C for 5 min to denature and inactivate DNase I.

[0089] (12) Add 45 μL DEPC H2O, 250 μL anhydrous ethanol pre-cooled at 4℃ and 10 μL 3M sodium acetate to the above system and mix thoroughly. Place the sample in a -80℃ refrigerator for 30 min to precipitate.

[0090] (13) 4℃, 12000 g, 15 min. After centrifugation, discard the liquid and add 1 mL of pre-cooled 80% ethanol. After inverting the mixture, centrifuge at 4℃, 7500 g, for 5 min.

[0091] (14) After discarding the ethanol, invert the centrifuge tube to air dry naturally, and dissolve the RNA precipitate with an appropriate volume of DEPC H2O (on ice).

[0092] (15) After dissolution, the RNA concentration and purity of each sample are measured. The purity can be judged by the ratio of optical density OD260 / OD280. The ratio of pure RNA should not be less than 2.0.

[0093] 1.3.2 Reverse transcription

[0094] (1) Using Promega GoScrip TMFor the reverse transcription kit (Promega, A5000), take 500 ng to 1 μg of target RNA in a nuclease-free PCR tube, add 1 μL of oligo(dT) / Random primer, and bring the volume up to 10 μL with DEPCH2O. Mix well and heat in a 70°C metal bath for 5 min to denature. Immediately after denaturation, place on ice for 2 min.

[0095] (2) Prepare the reverse transcription system as shown in Table 2 during the period:

[0096] Table 2 Reverse Transcription System

[0097]

[0098] (3) Reverse transcription was performed using the reverse transcription system from step (2) according to the PCR procedure shown in Table 3:

[0099] Table 3 PCR Procedure

[0100]

[0101] (4) After the reaction is completed, the product is diluted in an appropriate ratio and polymerase chain reaction (PCR) or real-time quantitative PCR (qPCR) is performed. The product is stored at -20℃.

[0102] 1.3.3 Real-time quantitative PCR (qPCR)

[0103] (1) Prepare the system required to make one sample according to the reaction system shown in Table 4:

[0104] Table 4 qPCR reaction system

[0105]

[0106] The primer information involved in this system is shown in Table 5:

[0107] Table 5 Primer information for real-time quantitative qPCR

[0108]

[0109] (2) After thoroughly mixing the reaction mixture, briefly centrifuge and add 15 μL of the system from step (1) to the wells of a 96-well plate. A total of three wells need to be added for three replicates.

[0110] (3) After all samples have been added, seal the plate with film, smooth it with a scraper, and centrifuge (be careful not to touch the film with your hands during this process). Place the 96-well plate into the QuantStudio 3 real-time fluorescence quantitative PCR instrument and perform the reaction according to the procedure shown in Table 6:

[0111] Table 6 qPCR reaction procedure

[0112]

[0113] (4) After the reaction is complete, save the results, take out the product and turn off the instrument correctly.

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

[0115] 1.4 Western blotting (WB) for protein level detection

[0116] (1) Sample preparation: After washing the corresponding liver tissue with 1×PBS, add an appropriate volume of RIPA lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 5 mM EDTA, 1 mM DTT, 1% NP-40, 0.1% SDS, 1×protease inhibitor cocktail (Roche, 05892970001)) and grinding beads, shake in a tissue homogenizer and mechanically homogenize; after high-speed centrifugation at 4℃, take the supernatant, take a small volume for protein quantification (Pierce® BCA protein assay kit, Thermo, 23227) for later use, add 5×SDS loading buffer to 1× for the remainder, add 10 mM DTT and mix thoroughly, denature in a metal bath at 100℃ for 10 min, after denaturation, centrifuge and place on ice to stand for experiment or store in a -80℃ freezer.

[0117] (2) The prepared protein samples were separated on an SDS-PAGE gel and transferred to a nitrocellulose membrane (PALL) according to the standard protein immunoblotting procedure. Images were captured using an ImageQuant LAS4000 biomolecular imager (GE Healthcare) and the grayscale values ​​of the protein bands were statistically analyzed using ImageJ software.

[0118] The antibody information involved in step (2) of the protein immunoblotting experiment is shown in Table 7:

[0119] Table 7 Antibody Information for Western Blotting of Proteins

[0120]

[0121] 1.5 Starvation treatment in mice

[0122] Mice were kept in an environment of 23-25°C (normal light exposure time). On the first night, they had free access to water but were not given food and were starved for 24 hours. On the second day, they were fasted for another 12 hours and bled to death at 9:00 AM on the third day. All control mice were fasted for 12 hours and were anesthetized with isoflurane and bled to death at 9:00 AM on the third day. The liver tissue was dissected and fixed in 4% paraformaldehyde fixative for at least 24 hours (3 mice / group).

[0123] 1.6 Hematoxylin-eosin (HE) staining

[0124] After fixation, liver tissue was dehydrated with anhydrous ethanol and then embedded in paraffin. The embedded tissue was then sliced ​​into 5 μm thick sections using an ultramicrotome and mounted on poly-L-lysine-coated slides. Hematoxylin and eosin staining was performed using a Servicebio, G1003 dye kit. The tissue was observed and photographed under an Olympus IX81 inverted fluorescence microscope. The nucleus / cytoplasm area ratio (%) was determined using ImageJ.

[0125] 1.7 Immunohistochemical (IHC) staining

[0126] (1) Dewaxing paraffin sections to water: The paraffin sections of mouse liver were placed in the dewaxing solution for 10 min, then in anhydrous ethanol for 5 min three times, and then washed with distilled water.

[0127] (2) Antigen retrieval: Antigen retrieval solution was used to retrieval the slides to prevent excessive evaporation of the buffer solution. After natural cooling, the slides were placed in PBS and washed three times on a shaker for 5 minutes each time.

[0128] (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 shake them three times on a shaker for 5 min each time.

[0129] (4) Serum blocking: Add 3% BSA to the tissue in the histochemistry zone and block at room temperature for 30 min.

[0130] (5) Incubate with primary antibody: Gently shake off the blocking solution, add the diluted primary antibody for the corresponding protein (diluted with PBS at a certain ratio), and place the slide in a humidified chamber and incubate overnight at 4°C.

[0131] (6) Incubation with secondary antibody: Place the slides in PBS and wash three times on a shaker for 5 min each time. After drying the slides, add the corresponding species of secondary antibody according to the primary antibody and incubate at room temperature for 1 h.

[0132] (7) Diaminobenzidine (DAB) (Servicebio, G1212) staining: Place the slide in PBS and shake it three times on a shaker for 5 minutes each time. After the slide is dried, add freshly prepared DAB staining solution and control the staining time under a microscope. Brownish-yellow indicates a positive result. Wash the slide with ultrapure water to stop further staining.

[0133] (8) Counterstaining of cell nuclei: stain 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 blue with hematoxylin blue solution, and rinse with running water.

[0134] (9) Dehydration and mounting: Place the sections in 75% ethanol for 5 min → 85% ethanol for 5 min → two times anhydrous ethanol for 5 min → n-butanol for 5 min → xylene for 5 min to dehydrate and clear. Finally, dry the sections and mount them.

[0135] (10) Microscopic examination: Observe and photograph under an Olympus IX81 inverted fluorescence microscope. The average optical density (AOD) was calculated from the image to represent the IHC signal.

[0136] The antibody information involved in this step is shown in Table 8:

[0137] Table 8 Antibody Information in IHC

[0138]

[0139] 1.8 Oil Red O Staining

[0140] (1) Fixation of frozen sections: The frozen sections of mouse liver tissue were rewarmed and dried, and fixed in 4% paraformaldehyde fixative for 15 min, washed with ultrapure water, and air-dried naturally.

[0141] (2) Oil red staining: Immerse the sections in oil red staining solution (Servicebio, G1015) for 10 min in the dark.

[0142] (3) Background differentiation: The sections were immersed in 60% isopropanol for differentiation twice, for 3 s and 5 s respectively. The sections were then immersed in ultrapure water for washing twice, for 10 s each time.

[0143] (4) Hematoxylin staining: Take out the section and place it in hematoxylin solution for counterstaining for 5 min; then rinse it three times with ultrapure water for 5 s, 10 s and 10 s respectively; after differentiating with differentiation solution for 5 s, rinse it twice with ultrapure water for 10 s each time; invert blue solution for 1 s, and then place the section in ultrapure water for 5 s and 10 s respectively.

[0144] (5) Mounting and microscopic examination: After mounting, the slides were examined and photographed under an Olympus IX81 inverted fluorescence microscope. The percentage of Oil Red positive area was calculated using Image-Pro plus software.

[0145] 1.9 Results and Analysis

[0146] Figure 1 illustrates CRISPR / Cas9 technology-mediated UFD1s - / - The strategy in defective mice (Figure 1A), RNA levels of UFD1s and its long transcript form (UFD1f) (Figure 1B), and protein levels (Figure 1C). It can be seen that synonymous mutations at alternative splicing sites in the UFD1 gene significantly reduce the protein level of UFD1s without affecting the expression of the long transcript UFD1f protein.

[0147] As shown in Figure 2, compared to wild-type mice, UFD1s-deficient mice exhibited decreased energy consumption, reduced oxygen volume consumption, and increased respiratory exchange rate (RER) (Figure 2A). Furthermore, UFD1s-deficient mice displayed abnormal liver morphology, decreased autophagy activity, and lipid droplet accumulation, with these phenomena becoming more pronounced after starvation treatment (Figure 2B).

[0148] These results indicate that UFD1s have a protective effect against stress.

[0149] Example 2: Correlation between the loss of murine UFD1s and the progression of non-alcoholic steatohepatitis (NASH)

[0150] In this embodiment, a NASH model was induced in mice by feeding them a methionine-choline (MCD)-deficient diet for 4 weeks, which is also a widely accepted model internationally. After successful induction of the NASH disease model, the development and progression of NASH were determined by detecting liver UFD1s RNA expression levels, serum lipid and transaminase levels, steatosis score, Oil Red staining, and fibrosis staining. The specific experimental procedure is as follows:

[0151] 2.1 MCD-induced NASH model

[0152] Mice were first subjected to overnight starvation (with free access to water), then fed a normal diet (normal group) or MCD diet (NASH group) for a total of 4 weeks, with fresh feed given every 2-3 days. No mice died during any of the model induction experiments. All mice were starved for 12 hours before sacrifice, with 6 mice per group.

[0153] 2.2 Determination of serum triglycerides, total cholesterol, aspartate aminotransferase (AST), and alanine aminotransferase (ALT)

[0154] Mice were anesthetized with isoflurane, and blood was collected by enucleation. The collected blood was centrifuged at 3000 rpm for 10 min at room temperature, and the supernatant was retained as serum. Serum triglyceride, total cholesterol, AST, and ALT levels were then measured using a triglyceride assay kit (NJJC, F001-1-1), a total cholesterol assay kit (NJJC, F002-1-1), an aspartate aminotransferase assay kit (Solarbio, BC1560), and an alanine aminotransferase assay kit (Solarbio, BC1555). All procedures were performed according to the kit instructions.

[0155] 2.3 Hepatic steatosis score

[0156] To quantify the differences in the degree of hepatic steatosis among different experimental groups, liver tissue was stained with hematoxylin and eosin (scale bar 50 μm) and pathologically assessed for the grade of steatosis. The scores were evaluated by pathology experts (Wuhan Saiweier Biotechnology Co., Ltd.).

[0157] The grade of fatty degeneration (score: 0-3) is determined by the percentage of microvacuoles / fatty degeneration. The field of view is 20×. The specific scoring criteria are shown in Table 9.

[0158] Table 9 Scoring Criteria for Fatty Degeneration

[0159]

[0160] 2.4 Masson staining

[0161] To observe the degree of liver fibrosis, Masson staining can be used to indicate the degree of fibrosis through the intensity of the color. Similar to the tissue staining preparation steps in Example 1, 5 μm thick paraffin sections were prepared and stained with Masson staining solution (Servicebio, G1006). Finally, the sections were examined and photographed under an Olympus IX81 inverted fluorescence microscope. The percentage of Masson-positive area was calculated using ImageJ, with a scale bar of 50 μm.

[0162] All other tests or staining were performed using the same methods as in Example 1, and will not be described in detail here.

[0163] 2.5 Results and Analysis

[0164] As shown in Figure 3, compared with normal mice, the UFD1s RNA level in the liver of mice with NASH was significantly reduced (Figure 3A), indicating that UFD1s can serve as a novel biomarker for the auxiliary diagnosis of NASH. More importantly, the serum triglyceride and total cholesterol levels were significantly elevated in both the normal group and the NASH group of mice lacking UFD1s (Figure 3B). As is well known to those skilled in the art, transaminase levels are used to assess the degree of liver damage. In both the normal and NASH groups, UFD1s deficiency led to a significant increase in serum AST levels, while ALT levels did not change significantly (Figure 3C). The results in Figure 3 indicate that UFD1s deficiency causes more severe liver damage.

[0165] As shown in Figure 4, compared with the normal group, the livers of mice with UFD1s deficiency in the NASH group exhibited more severe steatosis, greater lipid droplet accumulation, and a higher degree of fibrosis. This indicates that the absence of UFD1s accelerates the development of NASH.

[0166] Example 3: Correlation Study between Mouse-Derived UFD1s and NASH Process

[0167] To explore the therapeutic potential of UFD1s, recombinant plasmids and circular RNAs containing nucleotide sequences encoding UFD1s were constructed in this study. UFD1s protein was expressed in a NASH mouse model using both plasmid injection and circular RNA delivery strategies. The therapeutic effect of UFD1s was comprehensively evaluated by detecting serum lipid and transaminase levels, assessing the grade of hepatic steatosis, lipid droplet number, degree of fibrosis, and inflammation score. All images were taken using an Olympus IX81 inverted fluorescence microscope, scale bar 50 μm. Six mice per group.

[0168] 3.1 Construction and high-pressure injection of pLIVE-UFD1s plasmid

[0169] The nucleotide sequence (SEQ ID NO.4) encoding the mouse small protein UFD1s was cloned into the pLIVE vector backbone (between the NheI and SacI restriction sites) (Mirus Bio, MIR 5420), and the pLIVE-UFD1s plasmid was constructed by Sanger sequencing.

[0170] In the 4-week MCD diet-induced NASH disease model of Example 2, plasmid injection was performed in week 2 (Figure 5A). To obtain specific expression of UFD1s in the liver, 15 μg of pLIVE empty vector or pLIVE-UFD1s plasmid (approximately 10% of body weight) was diluted with 2 mL of physiological saline (0.9% NaCl). The plasmid was then injected into the mouse bloodstream via the tail vein using a 2.5 mL syringe and a 27-gauge needle; this process was to be completed rapidly within 5 seconds. No abnormalities or lethality were observed in the mice after the high-pressure injection.

[0171] 3.2 In vitro circularization (circUFD1s) and delivery of UFD1s

[0172] 1. In vitro cyclization

[0173] (1) The nucleotide sequence (SEQ ID NO.4) of the mouse small protein UFD1s 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). At the same time, the ATG of UFD1s was mutated to ATT as a negative reference. Specifically, 1 μg of linearized template (Urogene Biotech), 2 μL of T7 RNA polymerase and 10 mM NTP were thoroughly mixed (20 μL system) and reacted at 37℃ for 4 h.

[0174] (2) Using the TIE cyclization system, the in vitro transcription product was placed at 50℃ for 30 min to perform a cyclization reaction. After cyclization, DNase I was added and incubated at 37℃ for 15 min to digest and remove the DNA template.

[0175] (3) After the cyclization reaction is completed, the product is digested with RNase R (Epicentre, RNR07250) to remove linear RNA.

[0176] (4) Circular RNA was separated by 5% urea gel. According to the fragment size, the corresponding circUFD1s gel strip was cut and dissolved in elution buffer.

[0177] (5) circUFD1s were extracted and purified using the phenol / chloroform method (pH 4.5).

[0178] 2. Delivery of circUFD1s

[0179] During the 4-week process of the MCD diet-induced NASH disease model in Example 2, circUFD1s or circUFD1s were performed in weeks 2 and 3, respectively. ATG-ATTThe injection was performed (Figure 5, A). 10 μg of circRNA was thoroughly mixed with 20 μL of Lipofectamine 3000 (Invitrogen, L3000015) (diluted with PBS, total volume 100 μL) and allowed to stand at room temperature for 15 min. Subsequently, the circRNA-Lipofectamine 3000 mixture was injected into mice via the tail vein over 30 s using a 1 mL syringe and a 27-gauge needle. No abnormalities or lethality were observed in the mice after the injection.

[0180] 3.3 Inflammation score

[0181] To investigate the therapeutic effect of UFD1s expression on NASH inflammation, liver tissue was stained with hematoxylin and eosin (HE) (scale bar 50 μm) and pathologically assessed for lobular inflammation. Scoring was performed by a pathologist (Wuhan Saiwei Biotechnology Co., Ltd.). The degree of lobular inflammation (score: 0-3) was assessed based on the number of inflammatory sites (foci), as shown in Table 10.

[0182] Table 10 Scoring Criteria for the Severity of Lobular Inflammation

[0183]

[0184] 3.4 Degree of liver fibrosis and inflammation

[0185] The same staining methods as described above were used to assess hepatic steatosis, lipid droplet count, and fibrosis degree. Simultaneously, the degree of hepatic fibrosis and inflammation was assessed by detecting the expression levels of the fibrosis marker (COLLA1) and inflammation marker (TNF) mRNA in the liver (qPCR method as described above). Specific primer information is shown in Table 11.

[0186] Table 11 Primer Information

[0187]

[0188] 3.5 Results and Analysis

[0189] As shown in Figure 5, compared with the corresponding control group, both the pLIVE-UFD1s and circUFD1s groups showed high levels of UFD1s protein expression in the liver (Figure 5B), indicating that both strategies can achieve efficient and stable expression of UFD1s in the liver.

[0190] As shown in Figure 6, compared with the corresponding control groups, the serum triglyceride and cholesterol levels of NASH mice injected with pLIVE-UFD1s and circUFD1s were significantly reduced (Figure 6A). Furthermore, compared with the corresponding control groups, the levels of AST and ALT transaminases in mice injected with pLIVE-UFD1s were significantly reduced (Figure 6B), while the serum AST level in mice injected with circUFD1s was significantly reduced, while the ALT level did not change significantly. This indicates that UFD1s expression can significantly reduce serum lipid and transaminase (especially AST) levels.

[0191] Furthermore, compared with the corresponding control group, the liver steatosis, lipid droplet number, and fibrosis degree of NASH mice expressing UFD1s were significantly reduced (Figure 7). Simultaneously, the inflammation score of the liver of NASH mice expressing UFD1s, as well as the expression levels of fibrosis marker (COLLA1) and inflammation marker (TNF) mRNA in the liver, were significantly lower than those of the corresponding control group (Figure 8A and Figure 8B), indicating that expressing UFD1s in the liver of NASH mice can reduce steatosis, lipid droplet number, fibrosis, and inflammation degree in NASH mice.

[0192] Based on the above results, it can be concluded that UFD1s can alleviate the progression of NASH, and drugs for the prevention and / or treatment of NASH can be developed based on UFD1s.

[0193] Example 4: A study on the correlation between human UFD1s and NASH progression and the treatment of NASH.

[0194] 4.1 Immunohistochemistry of clinical samples

[0195] A total of 16 clinical samples were included (8 non-NASH controls and 8 NASH liver samples). These clinical samples were paraffin-embedded adjacent normal tissues of hepatocellular carcinoma, provided by the First Affiliated Hospital of the University of Science and Technology of China. All tissue samples were collected in accordance with the informed consent policy, and this study was approved by the Biomedical Ethics Committee of the First Affiliated Hospital of USTC, with biomedical ethics number 2024-RE-491.

[0196] Inclusion criteria for clinical samples:

[0197] 1. Patients with primary malignant liver tumors who have received treatment and undergone surgery at the First Affiliated Hospital of the University of Science and Technology of China after obtaining biomedical ethics certification, and whose diagnosis has been confirmed by pathology.

[0198] 2. Age 37-81 years old.

[0199] 3. Pathological scoring of adjacent tissues from the above-mentioned liver cancer patients was performed for NASH, and the diagnosis was made by pathology experts from the First Affiliated Hospital of the University of Science and Technology of China. The following criteria were used to diagnose NASH: ballooning degeneration (0 points, none; 1 point, rare; 2 points, common); fatty degeneration (0 points (<5%); 1 point (5%–33%); 2 points (34%–66%); 3 points (>66%)); and lobular inflammation (20x magnification counting of necrotic foci) (0 points, none; 1 point (<2); 2 points (2–4); 3 points (>4)). Patients must present all three criteria and have a total score (NAS score) ≥ 4 to be diagnosed with NASH; otherwise, they are considered non-NASH patients.

[0200] 4. Signed informed consent form, showed good compliance, and was willing to accept follow-up visits.

[0201] Exclusion criteria for clinical samples (samples meeting at least one of the following criteria will be excluded):

[0202] 1. Suffering from two or more malignant tumors at the same time, or having a malignant tumor lesion at the surgical site that is not the primary lesion.

[0203] 2. Women of childbearing age who have a positive blood pregnancy test or who have not undergone a pregnancy test, or women who are pregnant or breastfeeding.

[0204] 3. The patient participated in other therapeutic clinical trials during the course of their illness where the treatment measures were unclear or where treatment information could not be collected.

[0205] 4. Patients who cannot undergo surgery or obtain tumor tissue due to tumor spread.

[0206] 5. Comorbidities such as severe central nervous system diseases, respiratory diseases, autoimmune diseases, chronic renal insufficiency, long-term use of immunosuppressants, and severe uncontrolled infections.

[0207] Immunohistochemical staining (IHC) of small protein UFD1s was performed on clinical samples, and the specific steps can be referred to in Example 1.

[0208] 4.2 Cell Materials

[0209] 1. Human HEK293K (WT): ATCC, CRL-11268.

[0210] 2. Construction of UFD1s-deficient cells (UFD1s KO):

[0211] (1) Plasmid construction: Design an sgRNA targeting human UFD1s (see Table 12 for specific sequence information) and construct it into the px330-mCherry vector (Addgene, 98750); construct the UFD1s mutation splice site sequence (AGGTCA mutated to CGCAGC) and the homologous sequences on both sides into the pcDNA3.0 vector (Invitrogen, V79020).

[0212] (2) Cell transfection: 500,000 HEK293T wild-type cells were seeded in 6-well plates, and sgRNA plasmid (1 μg) expressing Cas9 mRNA and homologous plasmid (1 μg) were co-transfected into the seeded cells.

[0213] (3) Flow cytometry sorting: 48 h after cell transfection, single cells were sorted using the BD FACSAria™ III cell sorting system (Beckman, 5 laser), with sheath solution prepared in advance. Cells were digested with trypsin (Gibco, 25200072), washed twice with PBS, resuspended in PBS containing 10% serum, and filtered through flow cytometry tubes with a filter membrane. Simultaneously, 96-well cell culture plates were prepared for sorting, and finally, positive single cells were sorted based on red fluorescence.

[0214] (4) Cell expansion culture and identification: The sorted 96-well plates were placed in an incubator and cultured for about 10 days. They were observed under a microscope. Only wells that formed monoclonal cells were selected and the cells were transferred to 48-well plates for expansion culture. At the same time, a small number of cells were retained to extract the genome and perform PCR and Sanger sequencing for genotyping. The primer sequences for PCR identification are shown in Table 12: Human UFD1s KO F and Human UFD1s KO R.

[0215] Table 12 Sequence information involved in constructing UFD1s KO

[0216]

[0217] 3. Construction of UFD1s overexpressing cells (UFD1s OE):

[0218] Using human cDNA as a template, PCR amplification was performed to obtain the nucleotide sequence (SEQ ID NO.3) encoding the small protein UFD1s containing a 5' untranslated region (UTR). Using p3×FLAG-Myc-CMV24 (Sigma, E9283) as the vector backbone, a FLAG tag was constructed at the C-terminus of the UFD1s nucleotide sequence using homologous recombination (Vazyme, C112-02) to construct a human UFD1s overexpression plasmid, namely the UFD1s-FLAG plasmid. The constructed plasmid was verified by Sanger sequencing. Subsequently, UFD1s-FLAG was transfected into appropriate cells for plasmid overexpression (UFD1s OE) (specific steps can be found in the UFD1s KO cell transfection procedure), with the p3×FLAG-Myc-CMV24 vector plasmid used as an empty vector control (EV). The primer sequences for constructing the human UFD1s-FLAG plasmid are shown in Table 13.

[0219] Table 13 Primer information involved in constructing the UFD1s-FLAG plasmid

[0220]

[0221] All cell cultures were performed under standard conditions. The complete culture medium consisted of DMEM basal medium (Gibco, 11995065), 10% fetal bovine serum (CLARK, FB25015), and 1% penicillin / streptomycin antibiotics (Beyotime, C0222), and was cultured in a Thermo cell culture incubator at 37°C and 5% CO2. Cells were examined using PCR and DAPI staining to ensure they were free from mycoplasma contamination. All subsequent tests were performed 24 h after cell culture.

[0222] 4.3 Determination of intracellular triglyceride content

[0223] (1) Intracellular triglyceride levels were detected using a triglyceride assay kit (Nanjing Jiancheng Biotechnology, NJJC, F001-1-1).

[0224] (2) Collect cells and lyse them with RIPA lysis buffer for 30 min, then centrifuge at 4°C for 10 min and retain the supernatant. Take an equal volume of cell lysate to determine triglyceride levels.

[0225] (3) After thoroughly mixing the blank tube (0 μL), standard (3 μL), test tube (3 μL) and triglyceride test solution (300 μL), place them in a water bath at 37℃ for 5 min, zero the blank tube, and read the absorbance value of each tube at a wavelength of 546 nm.

[0226] (4) The triglyceride measurement value was normalized with the total cell protein amount (thermo, 23200) determined by the Bradford method protein quantification kit to obtain the final triglyceride content.

[0227] 4.4 Nile Red Staining

[0228] (1) After washing the cells planted on the slide twice with PBS solution, add 4% formaldehyde solution (prepared with PBS) and fix at room temperature for 10 min.

[0229] (2) After removing the fixative from the cells, wash twice with PBS and incubate with 0.05 μg / mL Nile Red solution (diluted with PBS) at room temperature for 20 min.

[0230] (3) Remove Nile Red staining solution, add 1 μg / mL Hoechst 33342 to stain cell nuclei, incubate at room temperature for 10 min, and wash twice with PBS.

[0231] (4) Microscopic examination. The lipid droplets were observed and photographed under a confocal microscope (Zeiss LSM 980), and the Nile Red signal was quantified using ImageJ software.

[0232] 4.5 Determination of Fatty Acid Oxidation

[0233] (1) The oxidation of fatty acids (FAO) in cells was analyzed using a cell energy metabolism analyzer (Seahorse XFe96, Agilent). FAO needs to be detected based on the cell oxygen consumption rate (OCR) experiment.

[0234] (2) 1.0 × 10 4 ~2.0×10 4 One cell per well was seeded into a 96-well XF cell culture plate and cultured in normal DMEM medium for 24 h.

[0235] (3) When detecting FAO, the cells need to be starved, that is, the culture medium is replaced with substrate-restricted DMEM (containing 0.5 mM glucose, 1 mM glutamine, 0.5 mM carnitine, and 1% FBS) for 12 h.

[0236] (4) After the starvation treatment, the substrate-limiting DMEM was removed and FAO experimental medium (KHB buffer: 111 mM NaCl, 4.7 mM KCl, 1.25 mM CaCl2, 2 mM MgSO4, 1.2 mM NaH2PO4, and 2.5 mM glucose, 0.5 mM carnitine, 5 mM HEPES, pH 7.4) was added and incubated in a CO2-free incubator at 37°C for 45 min.

[0237] (5) Then add the reagents from the Cell Mito Stress Test Kit (Agient, 103015-100), including oligomycin (1.5 μM), 4-trifluoromethoxyphenylhydrazone FCCP (1.0 μM) and rotenone / antimycin A (Rot / AA, 0.5 μM).

[0238] (6) Before the instrument is used for testing, add BSA or palmitic acid (PA) to the cell microwells (final concentration of 50 μM).

[0239] (7) Wave software was used to obtain all the data, and GraphPad Prism software was used to analyze the data.

[0240] 4.6 Results and Analysis

[0241] Figure 9 shows the experimental results from Example 4.

[0242] Figure 9A shows the expression of the small protein UFD1s in the liver tissues of non-NASH and NASH clinical patients (N=8 cases) using immunohistochemical staining. The results indicate that the small protein UFD1s is expressed at low levels in the liver tissues of NASH patients.

[0243] Figure 9B shows the determination of triglyceride content in human HEK293T wild-type (WT) and UFD1s-deficient cells (UFD1s KO). It was found that the deletion of UFD1s led to an increase in triglyceride levels in the cells.

[0244] Figure 9C shows the lipid droplet content in human HEK293T cells detected by Nile Red staining in cells lacking the small protein UFD1s (UFD1s KO) and those overexpressing it (UFD1s OE). The results show that the small protein UFD1s can inhibit the accumulation of lipid droplets in cells.

[0245] Figure 9 shows that D and E are used to assess the rate of fatty acid oxidation in cells with and without the small protein UFD1s by measuring the oxygen consumption rate (OCR) of cells through the addition of exogenous palmitic acid (PA). The results show that the small protein UFD1s can promote fatty acid oxidation.

[0246] The experimental results in Figure 9 show that the small protein UFD1s can promote fatty acid oxidation, inhibit lipid droplet accumulation, and reduce lipotoxicity, thus having a therapeutic effect on NASH.

[0247] The experimental results from Examples 1-4 demonstrate that UFD1s possesses a protective effect against cellular stress. Its expression level was significantly reduced in mice with non-alcoholic steatohepatitis (NASH), which is associated with the development and progression of NASH and can serve as a biomarker for the auxiliary diagnosis of this disease. In vivo expression of UFD1s in mice using both plasmid and circular RNA methods produced significant therapeutic effects on NASH, greatly inhibiting the progression of metabolic diseases caused by nutritional imbalances. Furthermore, analysis of clinical samples revealed that UFD1s expression was significantly lower in NASH patients than in non-NASH patients. Cellular experiments further confirmed that UFD1s also has a therapeutic effect on NASH in human cells. Therefore, the small protein UFD1s disclosed in this invention has high medical value.

[0248] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above 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.

[0249] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A small protein UFD1s having energy metabolism regulation and resistance to cellular stress effects, characterized by, The amino acid sequence of the small protein UFD1s is shown in SEQ ID NO.1 or SEQ ID NO.

2.

2. The small protein UFD1s having energy metabolism regulation and resistance to cellular stress effects according to claim 1, wherein, The nucleotide sequence encoding the small protein UFD1s is shown in SEQ ID NO.3 or SEQ ID NO.

4.

3. An expression vector, characterized by, The expression vector contains a nucleotide sequence encoding the small protein UFD1s as described in claim 1 or 2, as shown in SEQ ID NO. 3 or SEQ ID NO.

4.

4. The expression vector of claim 3, wherein, The expression vector is a recombinant plasmid or circular RNA.

5. A medicament for preventing and / or treating nonalcoholic steatohepatitis, characterized by, The drug contains the small protein UFD1s as described in claim 1 or 2, or the expression vector as described in claim 3 or 4.

6. The medicament according to claim 5, wherein The drug has at least one of the following effects: (1) Reduce the level of serum lipids in the subject, including triglycerides and total cholesterol; (2) Reduce the level of serum aspartate aminotransferase in the subjects; (3) Reduced the degree of hepatic steatosis in the subjects; (4) Improves the degree of liver inflammation in the subjects; (5) Improved the degree of liver fibrosis in the subjects.

7. Applications of any of the following: (1) The application of reagents for detecting the expression level of UFD1s in the liver tissue of subjects in the preparation of products for the auxiliary diagnosis of non-alcoholic steatohepatitis, wherein, The UFD1s is the small protein UFD1s described in claim 1 or 2; (2) The use of the small protein UFD1s as described in claim 1 or 2, or the expression vector as described in claim 3 or 4, in the preparation of a medicament for the prevention and / or treatment of non-alcoholic steatohepatitis.

8. Use according to claim 7, wherein the compound is ###0002### The product is a chip or a reagent kit.

9. The use according to claim 7, wherein the compound is ###00003### 8 or ###00004### 9. The reagent used to detect the expression level of UFD1s in the liver tissue of the subject is either a reagent for detecting the protein expression level of UFD1s 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.

10. The use according to claim 7, wherein the compound is ###00003### 8 The reagent used to detect 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, and the reagent contains one of the following primer pairs: (1) The nucleotide sequences are as shown in the primer pairs in SEQ ID NO.5 and 6; (2) The nucleotide sequences are as shown in the primer pairs in SEQ ID NO.7 and 8.

Images