Application of recombinant Glypican 1 protein in treatment of non-alcoholic steatohepatitis

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to application of recombinant Glypican 1 protein in treatment of non-alcoholic steatohepatitis. The Glypican 1 protein can treat non-alcoholic steatohepatitis for the first time, experiments prove that the Glypican 1 protein can improve liver lipid deposition, inflammation, fibrosis and other key pathological characteristics, and particularly, after the serum GPC1 level is increased, the Glypican 1 protein can inhibit high-fat and high-cholesterol diet induced mouse body weight increase, optimize the ratio of liver tissue weight to body weight, and improve the non-alcoholic steatohepatitis. The glucose tolerance and the insulin sensitivity are improved, so that the metabolic disorder is integrally improved; besides, the GPC1 accelerant can directly target macrophages, inhibit expression of pro-inflammatory genes (such as Tnf and Nos2) and relieve cascade reaction of liver inflammation, the anti-inflammatory effect is achieved by inhibiting an ERK1 / 2 phosphorylation pathway in mechanism, an innovative, efficient and flexible solution is provided for NASH treatment on the whole, and the defects of current treatment means are overcome.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to application of recombinant Glypican 1 protein in treatment of non-alcoholic steatohepatitis. BACKGROUND

[0002] Non-alcoholic steatohepatitis (NASH) related to metabolic disorders is the most common chronic liver disease at present, and NASH has become a public health problem that needs to be solved urgently. For the treatment of NASH, once inflammation is activated, single regulation of a certain target cannot completely control the progression of NASH. However, some molecular targets may delay the progression of NASH to a certain extent by controlling the flow and accumulation of active lipids in the liver. Improving the function of adipose tissue and some peripheral metabolic organs to improve liver lipid flux is also one of the treatment methods. At present, many studies focus on lifestyle intervention (diet and exercise intervention) and drug treatment. Obesity and T2DM are associated with the incidence of NASH, so treatment measures for obesity and T2DM can also reduce the risk of NASH. The degree of weight loss is crucial to the control of NASH. Controlling weight by reasonably limiting energy intake is a popular and researched topic in recent years. Low-carbohydrate, low-fat, low-glycemic index and Mediterranean diets can all improve NASH in a short follow-up time, but various factors will cause weight control to be poor to a certain extent.

[0003] At present, the treatment of NASH mainly includes lifestyle intervention (including diet and exercise intervention) and drug treatment.

[0004] Lifestyle intervention: Low-carbohydrate + diet can reduce liver triglyceride content more rapidly than low-fat, high-carbohydrate diet within 48 hours, but this difference is no longer obvious after 2-3 months. Therefore, according to personal preferences and adherence to diet control, it may be more important. In addition, regular exercise (aerobic exercise and resistance training) can reduce liver fat. More than 250 minutes of moderate to high intensity physical activity per week can greatly improve the level of liver steatosis in obese patients, and exercise time is related to metabolic improvement. However, it is difficult for most people to adhere to a certain lifestyle intervention method. Therefore, it may be necessary to use auxiliary drug treatment.

[0005] Drug intervention: Vitamin E (VitE) is an antioxidant that has been recommended by US and European guidelines as a treatment for NASH. A 96-week treatment with VitE at a dose of 800 IU daily significantly improved steatosis, lobular inflammation, and hepatocellular ballooning in NASH liver tissue, but not fibrosis, compared with placebo.

[0006] Pioglitazone is a Peroxisome proliferator-activated receptor-γ (PPARγ) agonist that increases adipocyte fatty acid storage and reduces circulating fatty acid levels. It is another treatment for NASH. Pioglitazone at a dose of 30 mg daily significantly improved histology in 34% of patients with NASH compared with placebo, but this was not significant after adjustment for VitE and placebo. Activation of these nuclear receptors leads to favorable metabolic and anti-inflammatory effects in preclinical studies.

[0007] In the GOLDEN-505 study, 19% of patients with NASH regressed without worsening fibrosis after 1 year of treatment with elafibranor (a dual agonist of Peroxisome proliferator-activated receptors alpha and delta) at a dose of 120 mg daily, compared with 12% of patients treated with placebo.

[0008] Glucagon-like peptide-1 (GLP-1) agonists such as liraglutide and semaglutide, which are used to treat T2DM, can reduce food intake by increasing insulin, reducing glucagon secretion, delaying gastric emptying, and reducing appetite, among other effects. Studies have shown that overweight patients with NASH treated with liraglutide at a dose of 1.8 mg daily for 48 weeks had a higher proportion of improvement in steatosis and hepatocellular ballooning and fewer patients with worsening fibrosis compared with placebo. However, the dose of liraglutide still needs to be adjusted due to its gastrointestinal side effects.

[0009] In a study of obeticholic acid (OCA), an agonist of the farnesoid X receptor (FXR), for the treatment of NASH, 45% of patients treated with OCA and 21% of patients treated with placebo achieved a reduction in NASH score of ≥2 points and no worsening of fibrosis from baseline over 72 weeks. In addition to obeticholic acid, the intestinal selective FXR receptor agonist GS-9674 (Cilofexor) can up-regulate the FXR target proteins FGF19 and SHP, reduce total cholesterol, low-density lipoprotein cholesterol and serum apolipoprotein B, and increase liver LDL receptor expression, and can significantly reduce liver steatosis and blood bile acid levels after 24 weeks at a daily dose of 100 mg. In general, the current treatment for NASH is mainly through reducing energy intake and increasing energy consumption, and early lifestyle interventions such as intermittent fasting to reduce intake and exercise to increase consumption, but there is no good treatment for patients who have progressed to the late stage of NASH and liver cirrhosis except surgery. There is also a lack of treatment for patients with complications such as T2DM and cardiovascular disease. Therefore, many current interventions are still effective for early-stage NASH, but the treatment for patients who have progressed to the middle and late stages with complications is not perfect, which is a difficult problem to be solved at the moment. SUMMARY

[0010] To solve the above technical problems, the purpose of the present application is to provide the use of a Glypican 1 promoter in the preparation of a drug for treating non-alcoholic steatohepatitis.

[0011] In order to achieve the above-mentioned purpose of the present application, the technical solution adopted by the present application is: The present application provides the use of a Glypican 1 promoter in the preparation of a drug for treating non-alcoholic steatohepatitis, The Glypican 1 promoter includes at least one of a1) to a4): a1) a substance that enhances the activity of Glypican 1 protein; a2) a substance that increases the content of Glypican 1 protein; a3) a substance that enhances the expression of Glypican 1 gene; a4) a substance that promotes the transcription of Glypican 1 gene.

[0012] In some embodiments of the present application, the amino acid sequence of the Glypican 1 protein includes at least one of b1) to b3): b1) the amino acid sequence shown in SEQ ID NO: 1; b2) a nucleotide sequence of SEQ ID NO: 1 with substitution of one or several amino acids and / or deletion and / or addition, and having the same function as the protein shown in SEQ ID NO: 1; or b3) an amino acid sequence having 95%, 96%, 97%, 98% or above homology with SEQ ID NO: 1, and having the same function as the protein shown in SEQ ID NO: 1.

[0013] In some embodiments of the present application, the protein having the same function in b2) is generally considered as conservative substitution, such as substitution of each other among the aliphatic amino acids Ala, Val, Leu and Ile, interchange of hydroxyl residues Ser and Thr, exchange of acidic residues Asp and Glu, substitution between amide residues Asn and Gin, exchange of basic residues Lys and Arg, and substitution between aromatic residues Phe and Tyr.

[0014] In some embodiments of the present application, b3) means that the given amino acid sequence shares at least 95% identity with the reference sequence, such as 95%, 96%, 97%, 98%, 99%. Alternatively, it means that the given amino acid sequence has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 differences with the reference sequence, which is preferably substitution or deletion of amino acids for polypeptide. Here the difference in identity can come from conservative substitution of amino acids, or introduction of tag protein, or introduction of nucleotide sequence, or introduction of signal peptide sequence, modification. For example, addition of His, GFP, HA, etc. tag at the C-terminus of SEQ ID NO: 1, resulting in change of identity, or addition of signal peptide at the N-terminus of SEQ ID NO: 1, or addition of conventional nucleotide sequence at the N-terminus, resulting in change of identity.

[0015] In some embodiments of the present application, the nucleotide sequence of the Glypican 1 gene comprises at least one of c1)~c2); c1) a nucleotide sequence encoding the protein of b1)~b3); c2) a nucleotide sequence as shown in SEQ ID NO: 2.

[0016] In some embodiments of the present application, the substance for increasing the expression amount of Glypican 1 protein comprises Glypican 1 protein.

[0017] Glypican-1 (GPC1) is a cell surface heparan sulfate proteoglycan anchored to the cell membrane by a glycosylphosphatidylinositol. It is composed of a core protein and three heparan sulfate chains and plays a regulatory role in a variety of cell signaling pathways, including fibroblast growth factor, vascular endothelial growth factor, transforming growth factor-beta, Wnt, Hedgehog, and bone morphogenetic protein signaling pathways.

[0018] In some embodiments of the present application, the Glypican 1 protein includes pharmaceutically acceptable salts or pharmaceutically acceptable chemical modifications thereof.

[0019] In some embodiments of the present application, the pharmaceutically acceptable modification includes at least one of phosphorylation, sulfation, acylation, glycosylation, ubiquitination, acetylation, methylation, sulfation, phosphatidylation, halogenation.

[0020] In some embodiments of the present application, the pharmaceutically acceptable salt includes acid addition salts and base addition salts.

[0021] A "pharmaceutically acceptable acid addition salt" means a salt with an inorganic acid and an organic acid, such as but not limited to hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and such as but not limited to acetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, camphoric acid, camphor 10 sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfic acid, ethane 1,2 disulfonic acid, ethanesulfonic acid, 2 hydroxyethanesulfonic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, 2 oxoglutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methane sulfonic acid and the like, which retains the biological effectiveness and properties of the free base, is not biologically or otherwise undesirable, and is formed with inorganic acids and organic acids.

[0022] "Pharmaceutically acceptable base addition salt" refers to those salts which retain the biological effectiveness and properties of the free acids and which are not biologically or otherwise undesirable. These salts are prepared from addition of inorganic or organic acids to the free base. Salts derived from inorganic acids include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Preferred inorganic salts are the ammonium, sodium, potassium, calcium, and magnesium salts. Salts derived from organic acids include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, dicyclohexylamine, lysine, arginine, histidine, caffeine, ethylenediamine, glucosamine, methylglucosamine, theobromine, triethanolamine, tromethamine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins and the like. Particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine.

[0023] In some embodiments of the present application, the medicament comprises a pharmaceutically acceptable excipient.

[0024] In some embodiments of the present application, the pharmaceutically acceptable excipient comprises at least one of a propellant, a colorant, a binder, a disintegrant, a filler, a lubricant, a wetting agent, an osmotic pressure adjusting agent, a stabilizer, a glidant, a flavoring agent, a preservative, a suspending agent, a coating material, a fragrance, an anti-adherent, a penetration enhancer, a pH adjusting agent, a buffer, a plasticizer, a surfactant, a foaming agent, an antifoaming agent, a thickening agent, an inclusion agent, a humectant, an absorbent, a diluent, a flocculating agent and a deflocculating agent, a filter aid, a release retarder.

[0025] The above pharmaceutically acceptable excipients are generally recognized as suitable for this use in the art and are non-active ingredients of the medicament. A compendium of pharmaceutically acceptable excipients can be found in the Handbook of Pharmaceutical Excipients, 2ndEdition, Edited by A. Wade and P.J. Weller; Published by American Pharmaceutical Association, Washington and The Pharmaceutical Press, London, 1994; the Compendium of Chinese Pharmacopoeia-Pharmaceutical Excipients, and the like.

[0026] In some embodiments of the present application, the dosage form of the product comprises one of a powder, a tablet, a granule, a capsule, a sustained release, a solution, a dry suspension, an effervescent tablet, an emulsion, a suspension, a syrup, a drop, a chewable tablet.

[0027] In some embodiments of the present application, the administration mode of the product comprises gastrointestinal administration or non-gastrointestinal administration.

[0028] In some embodiments of the present application, the gastrointestinal administration comprises one of oral administration, sublingual administration, rectal administration.

[0029] In some embodiments of the present application, the non-gastrointestinal administration comprises one of intravenous injection, subcutaneous injection, mucosal administration.

[0030] In some embodiments of the present application, the administration subject of the product is a mammal; including but not limited to: human, mouse, rat, pig, cow, sheep, horse, monkey, rabbit.

[0031] In some embodiments of the present application, the mammal includes human.

[0032] The beneficial effects of the present application are: The present application first proposes that Glypican 1 protein can treat non-alcoholic fatty hepatitis, and experiments prove that it can improve key pathological characteristics such as liver lipid deposition, inflammation and fibrosis. Specifically, after increasing the serum GPC1 level, it can inhibit the weight gain of mice induced by high-fat high-cholesterol diet, optimize the liver tissue weight to body weight ratio, and improve glucose tolerance and insulin sensitivity, thereby improving metabolic disorders as a whole; in addition, the GPC1 promoter can directly target macrophages, inhibit the expression of pro-inflammatory genes (such as Tnf, Nos2), and reduce the liver inflammation cascade reaction. Mechanistically, it realizes anti-inflammatory effect by inhibiting the ERK1 / 2 phosphorylation pathway, and overall provides an innovative, efficient and flexible solution for NASH treatment, making up for the shortcomings of current treatment methods. BRIEF DESCRIPTION OF DRAWINGS

[0033] The present application will be further described below in conjunction with the drawings and examples, wherein: Figure 1To increase the GPC1 level can inhibit the NASH progression results of HFHC mice, wherein: A is the body weight graph of Gpc1 adeno-associated virus injection mice fed with HFHC; B is the liver tissue weight to body weight ratio of Gpc1 adeno-associated virus injection mice fed with HFHC; C is the serum GPC1 content change of Gpc1 adeno-associated virus injection mice fed with HFHC; D-F are ITT and GTT experiments and area under the curve statistics of Gpc1 adeno-associated virus injection mice fed with HFHC; G-I are liver tissue H&E staining and statistics and F4 / 80 immunofluorescence staining of Gpc1 adeno-associated virus injection mice fed with HFHC; J is liver Masson staining and a-SMA immunohistochemical staining and statistics of Gpc1 adeno-associated virus injection mice fed with HFHC; K is the serum ALT and AST content change of Gpc1 adeno-associated virus injection mice fed with HFHC; L is the liver fibrosis gene change of Gpc1 adeno-associated virus injection mice fed with HFHC.

[0034] Figure 2 To inhibit the expression of pro-inflammatory genes in macrophages by GPC1, wherein: A is the determination of triglyceride content of the MAFL model of primary hepatocytes treated with GPC1 recombinant protein and OAPA; B is the gene expression of the M1 pro-inflammatory model of the LPS-induced macrophage cell line treated with GPC1 recombinant protein; C is the gene expression of the M1 pro-inflammatory model of primary macrophages induced by LPS+INFγ treated with GPC1 recombinant protein.

[0035] Figure 3 To inhibit the phosphorylation of ERK1 / 2 by GPC1, wherein: A is the phosphorylation level of ERK1 / 2 of BMDM cells induced by LPS+INFγ treated with GPC1 recombinant protein at different times; B is the phosphorylation level of ERK1 / 2 of BMDM cells induced by LPS+INFγ treated with GPC1 recombinant protein at different concentrations; C is the phosphorylation level of ERK1 / 2 of Raw264.7 cells induced by LPS treated with GPC1 recombinant protein at different times; D is the phosphorylation level of ERK1 / 2 of Gpc1 knockout mice fed with HFD; E is the phosphorylation level of ERK1 / 2 of fat knockout mice fed with HFD; F is the phosphorylation level of ERK1 / 2 of fat knockout mice fed with HFHC; G-H are the phosphorylation levels of ERK1 / 2 of Gpc1 adeno-associated virus injection mice fed with HFD and HFHC. DETAILED DESCRIPTION

[0036] The concept and technical effects of the present application will be described below in conjunction with the embodiments to fully understand the purpose, features and effects of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, not all embodiments, and other embodiments obtained by those skilled in the art based on the embodiments of the present application without creative labor are within the scope of protection of the present application.

[0037] Example 1 GPC1 inhibits the progression of NASH The serum GPC1 level of model mice was increased by injecting adeno-associated virus (AAV-Gpc1) through the tail vein, and the mice were induced with HFHC to establish a NASH model, so as to determine the therapeutic effect of GPC1 on NASH.

[0038] Among them, the amino acid sequence of the human Gpc1 protein is: MELRARGWWLLCAAAALVACARGDPASKSRSCGEVRQIYGAKGFSLSDVPQAEISGEHLRICPQGYTCCTSEMEENLANRSHAELETALRDSSRVLQAMLATQLRSFDDHFQHLLNDSERTLQATFPGAFGELYTQNARAFRDLYSELRLYYRGANLHLEETLAEFWARLLERLFKQLHPQLLLPDDYLDCLGKQAEALRPFGEAPRELRLRATRAFVAARSFVQGLGVASDVVRKVAQVPLGPECSRAVMKLVYCAHCLGVPGARPCPDYCRNVLKGCLANQADLDAEWRNLLDSMVLITDKFWGTSGVESVIGSVHTWLAEAINALQDNRDTLTAKVIQGCGNPKVNPQGPGPEEKRRRGKLAPRERPPSGTLEKLVSEAKAQLRDVQDFWISLPGTLCSEKMALSTASDDRCWNGMARGRYLPEVMGDGLANQINNPEVEVDITKPDMTIRQQIMQLKIMTNRLRSAYNGNDVDFQDASDDGSGSGSGDGCLDDLCSRKVSRKSSSSRTPLTHALPGLSEQEGQKTSAASCPQPPTFLLPLLLFLALTVARPRWR (SEQ ID NO: 1); The nucleotide sequence (coding sequence) of the human Gpc1 gene is:

[0039] AAV vector is H29035 pcAAV-Alb-ZsGreenl-WPRE.

[0040] Human and mouse Glypican 1 proteins have high homology, and the human protein is directly used for experiments in this embodiment.

[0041] 1. Experimental method High-fat / high-cholesterol diet (HFHC) mouse model construction: HFHC feed contains 40% fat, 20% fructose and 2% cholesterol, all purchased from Beijing Boaipaike Company. The control normal feed was provided by the Experimental Animal Center of South China Agricultural University. The fat, carbohydrate and protein energy in the normal chow diet (NCD) accounted for 12%, 67.4% and 20.6%, respectively. Before modeling, the mice adapted to 1-2 weeks of feeding were randomly allocated according to body weight, and the blood glucose value of the mice was detected and recorded at the beginning of feeding. Then one cage was fed with high-fat feed, and the other cage was fed with normal feed. During the high-fat feeding period, the body weight of the mice was detected every week, and the blood glucose changes of the mice in the two groups were detected at 12, 14 and 16 weeks of feeding, respectively, to confirm the success of model construction.

[0042] The ITT / GTT experiment was performed on mice at about 12-14 weeks. Before the experiment, the cage and bedding were changed and the mice were fasted. The insulin and glucose doses for the experiment were prepared by appropriately diluting the physiological saline according to the body weight and number of mice (the glucose solution needs to be filtered with a 0.22 um filter after preparation and left overnight). After weighing the mice after fasting, the required injection dose for each mouse was calculated according to the body weight (injection unit insulin: 1U / kg body weight; glucose: 1g / kg body weight). In order to avoid the deviation of the monitoring value caused by repeatedly grabbing the mice during injection and subsequent blood glucose monitoring, the mice were marked on the tail according to the number of mice during weighing, which facilitated insulin, glucose injection and blood glucose determination. First, a small part of the mouse tail tip was cut off with scissors, and the fasting blood glucose value after fasting was monitored, i.e. 0 min blood glucose. After all the mice were detected, insulin or glucose was injected intraperitoneally according to the order of the previously marked mouse tails. The time was started after the first mouse was injected, and the insulin injection interval was calculated according to the number of mice. Then the blood glucose was detected at 30 min, 60 min, 90 min and 120 min after injection, and the blood glucose value of each mouse at each time point was recorded. Then the blood glucose change value was plotted and the area under the curve was calculated. During the experiment, the mice should be handled gently and avoid repeated forceful handling. After the experiment, the corresponding feed before fasting was supplemented for continued feeding.

[0043] The cages and bedding were changed the night before sampling and the mice were fasted overnight. The next day, the mice were weighed and given an appropriate dose of anesthetic by intraperitoneal injection. After the mice were successfully anesthetized, the following sampling was performed in order.

[0044] 1) Orbital blood collection: After the mouse whiskers were cut (to avoid hemolysis during blood collection due to touching the hair), the mouse eyeball was quickly removed with an ophthalmic forceps, and the mouse number was dripped into a 1.5 ml EP tube prepared in advance. The dripping process should be suspended as much as possible to avoid touching the tube wall. Each mouse can take about 0.5-1 ml of blood. After the blood of the last mouse was collected, all blood collection tubes were placed at room temperature for 0.5-1 hour, then centrifuged at 3000 rpm for 10 minutes in a 4°C centrifuge. After centrifugation, the clear light yellow liquid on the top of the solution was carefully sucked into an 8-way tube and then aliquoted. The number was labeled for easy detection of serological indicators or stored at -80°C.

[0045] 2) Liver: After blood collection, the mouse was decapitated and disinfected with alcohol. Then the skin, peritoneum and other external tissues were cut with scissors to expose the liver. After removing the ligaments around the liver, the liver tissue was separated, photographed and weighed. Then part of the largest lobe of the liver was fixed in 4% paraformaldehyde for subsequent tissue embedding, sectioning and staining. The sampling position of each mouse should be as consistent as possible. The remaining liver tissue was cut and aliquoted for freezing according to the subsequent experimental requirements. All tissues were stored in liquid nitrogen for a short time and then frozen at -80°C.

[0046] 3) Fat tissue sampling: After the abdominal cavity was exposed, the mouse epididymal white adipose tissue (Epididymal White Adipose Tissue, eWAT) could be observed. The epididymis and vas deferens structures attached to the eWAT tissue were carefully removed and weighed. Then the middle section of the eWAT was fixed as the liver tissue, and the appropriate amount of tissue was taken for subsequent RNA and protein extraction experiments. The remaining part was stored in the tissue freezing tube. The inguinal WAT (Inguinal WAT, iWAT) was located under the skin of the mouse. After the skin was cut, the iWAT tissue could be seen symmetrically distributed on both sides of the mouse thigh. The tissue was carefully cut along the edge of the skin with scissors and weighed, and the lymph nodes were separated. The lymph nodes were cut to the size of a soybean and fixed in 4% paraformaldehyde for staining preparation. The appropriate amount of tissue was used for RNA and protein extraction experiments. The remaining tissue was frozen in the tissue freezing tube. The mouse brown adipose tissue (Brown Adipose tissue, BAT) was located in the scapula region. After separating the BAT and its peripheral white adipose tissue, the peripheral white adipose tissue was carefully removed. After separation, the tissue was weighed, and a small amount of tissue was cut and fixed in 4% paraformaldehyde, and part was used for RNA and protein extraction experiments, and the rest was frozen.

[0047] 4) Biochemical index detection Serum and liver triglyceride (TG) and total cholesterol (TC) detection: 1) Sample preparation: After blood sampling, centrifuge the supernatant and place it on ice for testing. When taking the liver tissue sample, weigh an appropriate amount of liver and record the weight. For high-fat samples, use anhydrous ethanol as the solvent, and for normal samples, use PBS or normal saline as the solvent. Add about 9 times the weight of the liver sample in solvent and 2 grinding beads, and homogenize in a pre-cooled tissue homogenizer. Conditions: 60 Hz, grind for 30 s, pause for 20 s, repeat for 4-6 times until no visible tissue pieces are left. Then centrifuge at 2000 rpm for 10 min and collect the supernatant for testing. 2) TG and TC detection: Add 2.5 μl of each sample prepared above to the enzyme-labeled plate, use TG or TC standard as positive control, and use ddH2O as negative control. Then add the prepared TG or TC working solution, 250 μl per well, and incubate in a 37°C constant temperature incubator for 10 min. Then use an enzyme-labeled instrument to detect the absorbance OD value of each tube. 3) Protein concentration of tissue and cell samples also needs to be determined; 4) Calculation method: Serum sample: TG / TC (mmol / L) = (sample OD value - blank OD value) / (calibration OD value - blank OD value) x calibration concentration (mmol / L). The protein concentration of tissue and cell samples also needs to be divided by the corresponding protein concentration to obtain the result.

[0048] Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) determination: 1) Serum sample as described in the sampling step, after serum separation, place it on ice for testing; 2) Add 0.1 mL of the sample to be tested in the enzyme-labeled plate, then add 0.5 mL of preheated substrate solution to the test tube, and only add 0.5 mL of substrate solution to the control tube, then mix gently and incubate in a 37°C incubator for 30 min. Then add 0.5 mL of 2,4-dinitrophenylhydrazine solution to the test tube, and add 0.5 mL of sample to the control tube, mix gently, and incubate in a 37°C incubator for 20 min. Then add 5 mL of 0.4 mmol / L sodium hydroxide solution to each well, mix well, and place it at room temperature for 5 min. Use an enzyme-labeled instrument to detect the absorbance OD value at wavelength 505 nm. The absolute OD value = test tube OD value - control tube OD value, and the corresponding ALT / AST activity value is obtained by referring to the standard curve.

[0049] 2. Experimental results The results are as follows Figure 1As shown, the NASH model was induced by feeding mice with HFHC diet, and the serum GPC1 level was increased by tail vein injection of Adeno-associated virus (AAC). The results showed that after 10 weeks of HFHC diet feeding, increasing the serum GPC1 level could inhibit the body weight of mice, and the ITT and GTT experiments showed that increasing the GPC1 level could significantly improve the glucose tolerance and insulin sensitivity of the model mice. The GPC1 content in the blood was also significantly increased compared with the control. After increasing the GPC1 level, the liver weight to body weight ratio of the mice was reduced to some extent, and the fat weight to body weight ratio was not significantly changed. The detection of the changes of serum and liver TG and TC contents showed that increasing the GPC1 level could reduce the TG level in the liver induced by obesity. The appearance of the liver and the results of H&E staining of the histology showed that increasing the GPC1 level could improve the lipid deposition in the liver. In addition, F4 / 80 immunofluorescence staining and the change of the expression level of inflammatory gene transcription showed that GPC1 increase could reduce the expression of inflammation in the liver induced by obesity. Therefore, the above results confirmed that increasing the GPC1 level could inhibit the degree of liver steatosis and inflammation expression induced by HFD diet.

[0050] In addition, histological staining, F4 / 80 immunofluorescence results, and inflammatory gene expression also showed that the liver inflammation level of the model mice was reduced after increasing the GPC1 level. Further detection of liver damage related serum ALT and AST indicators showed that GPC1 increased to some extent could inhibit liver damage. Masson staining and fibrosis index a-SMA immunohistochemical staining also suggested that increasing the GPC1 level could improve the fibrosis degree of HFHC mice.

[0051] Example 2 Recombinant GPC1 protein inhibits M1 macrophage inflammatory gene expression Detect whether the expression of inflammation related genes changes after adding recombinant GPC1 protein treatment in LPS and INFγ treated bone marrow derived macrophage (BMDM) induced M1 type pro-inflammatory model 1. Experimental method Cell model construction conditions: Cells: Mouse BMDM cells were cultured using complete DMEM (10% FBS, 1% P / S) medium without M-CSF, and when the cell density reached about 80%, LPS (100 ng / m) L and IFNγ (20 ng / mL) were used to induce for 24 h, which was the differentiation of BMDM cells into M1 inflammatory phenotype macrophages.

[0052] GPC1 protein treatment conditions: GPC1 protein was treated at a concentration of 100 ng / ml. GPC1 protein was added for treatment at the beginning of BMDM induction.

[0053] 2. Experimental results The results, as shown in Figure 2 Figure 6, showed that GPC1 could significantly inhibit the expression of proinflammatory genes such as Tnfα and Nos2 induced by LPS and INFγ. In combination with the previous finding that liver is the target organ of GPC1, we speculated that circulating GPC1 might regulate the inflammatory cascade by affecting the function of liver macrophages, and thus affect lipid deposition and fibrosis in the liver. Therefore, we further used LPS to induce the M1 proinflammatory cell model in the RAW264.7 cell line, and observed that the expression of proinflammatory genes was inhibited after adding recombinant GPC1 protein. The above results showed that the circulating GPC1 secreted protein could target the liver to inhibit the expression of inflammatory genes in the liver during obesity, and thus improve the degree of steatosis and weaken the degree of fibrosis, thereby delaying the progression of NASH.

[0054] Example 3 GPC1 inhibits ERK1 / 2 phosphorylation in macrophages 1. Experimental method The same as Example 2.

[0055] 2. Experimental results The results, as shown in Figure 3 Figure 6, showed that GPC1 could significantly inhibit the expression of proinflammatory genes such as Tnfα and Nos2 induced by LPS and INFγ. In combination with the previous finding that liver is the target organ of GPC1, we speculated that circulating GPC1 might regulate the inflammatory cascade by affecting the function of liver macrophages, and thus affect lipid deposition and fibrosis in the liver. Therefore, we further used LPS to induce the M1 proinflammatory cell model in the RAW264.7 cell line, and observed that the expression of proinflammatory genes was inhibited after adding recombinant GPC1 protein. The above results showed that the circulating GPC1 secreted protein could target the liver to inhibit the expression of inflammatory genes in the liver during obesity, and thus improve the degree of steatosis and weaken the degree of fibrosis, thereby delaying the progression of NASH.

[0056] The embodiments of the application are described in detail above with reference to the drawings, but the application is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made to the embodiments without departing from the spirit of the application. In addition, the embodiments of the application and the features in the embodiments can be combined with each other without conflict.

Claims

1. Application of Glypican 1 promoter in the preparation of drugs for the treatment of non-alcoholic steatohepatitis.

2. The application according to claim 1, characterized in that: The Glypican 1 accelerator includes at least one of a1) to a4): a1) Substances that enhance the activity of Glypican 1 protein; a2) Substances that increase the content of Glypican 1 protein; a3) Substances that enhance Glypican 1 gene expression; a4) Substances that promote the transcription of the Glypican 1 gene.

3. The application according to claim 2, characterized in that: The amino acid sequence of the Glypican 1 protein includes at least one of b1) to b3); b1) The amino acid sequence as shown in SEQ ID NO: 1; b2) A nuclear sequence of SEQ ID NO: 1 with one or more amino acid substitutions and / or deletions and / or additions, having the same function as the protein shown in SEQ ID NO: 1; or b3) Has 95%, 96%, 97%, 98% or more homology with SEQ ID NO: 1 and has the same amino acid sequence as the protein shown in SEQ ID NO:

1.

4. The application according to claim 2, characterized in that: The substance that increases the expression level of Glypican 1 protein includes Glypican 1 protein.

5. The application according to claim 4, characterized in that: The Glypican 1 protein includes pharmaceutically acceptable salts or pharmaceutically acceptable chemical modifications thereof.

6. The application according to claim 5, characterized in that: The pharmaceutically acceptable salts include acid addition salts and base addition salts; and / or The pharmaceutically acceptable modifications include at least one of phosphorylation, sulfonation, acylation, glycosylation, ubiquitination, acetylation, methylation, sulfation, phospholipidation, and halogenation.

7. The application according to claim 1, characterized in that: The drug includes pharmaceutically acceptable excipients and / or any one or more other active ingredients.

8. The application according to claim 7, characterized in that: The pharmaceutically acceptable excipients include at least one of the following: solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, binding agents, penetration enhancers, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, encapsulating agents, humectants, absorbents, diluents, flocculants and anti-flocculation agents, filter aids, release inhibitors, and carriers.

9. The application according to claim 8, characterized in that: The dosage form of the drug includes at least one of the following: suspension, granules, capsules, powders, tablets, emulsions, solutions, pellets, injections, oral preparations, suppositories, enemas, aerosols, patches, or drops.

10. The application according to claim 9, characterized in that: The route of administration of the drug includes at least one of the following: intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, oral administration, sublingual administration, nasal administration, nebulized administration, or transdermal administration.

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