Cockroach glycosylated protein PAG-N1, and preparation method and application thereof

Abstract

The present application relates to the technical field of medicine, and provide a cockroach glycosylated protein PAG-N1 and a preparation method and application thereof; the cockroach glyglycosylated protein PAG-N1 contains glycosylated Q9BPS0 and Q9U8M0, and the molecular weights are 215943 Da and 212728 Da respectively, 8 glycosylation sites are predicted on the Q9BPS0 protein, and the molecular weight of sugar chain ranges from 2400 to 3500 Da; 6 glycosylation sites are predicted on the Q9U8M0 protein, and the molecular weight of sugar chain ranges from 1800 to 3600 Da; the cockroach glycosylated protein PAG-N1 is extracted after the cockroach dried whole worm is crushed, and it is found for the first time that PAG-N1 can effectively slow down the increase of body weight and liver weight of MASLD rats, reduce the accumulation of white adipose tissue in MASLD rats, improve insulin resistance and dyslipidemia, relieve liver inflammation, lipid deposition and collagen fiber hyperplasia, enhance the antioxidant capacity of liver, reduce liver damage, improve the liver function of rats, and then play the pharmacological activity of treating MASLD; therefore, the cockroach glycosylated protein PAG-N1 can be used for improving the liver damage of medicine or health care product, especially MASLD.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a cockroach glycosylated protein PAG-N1 and its preparation method. Background Technology

[0002] Glycosylated proteins are proteins formed by the covalent bonding of one or more oligosaccharide chains to the polypeptide chain after glycosylation modification within the cell. The oligosaccharide chain is usually formed by 2 to 10 monosaccharide residues linked by glycosidic bonds. The linkage mode is mainly divided into two types: N-linking (the oligosaccharide chain is linked to the amide group of the protein asparagine residue) and O-linking (the oligosaccharide chain is linked to the hydroxyl group of the protein serine or threonine residue). This special structure endows glycosylated proteins with unique physicochemical properties and biological functions.

[0003] cockroach ( Periplaneta americana L.) belongs to the class Insecta, order Blattodea, family Blattodea, and is commonly known as cockroach or oil thief. As a traditional medicinal insect, its medicinal value has been recorded in folk medicine for a long time. In recent years, it has received widespread attention from scholars at home and abroad. Studies have shown that cockroaches have complex components, containing a variety of active substances such as glycoproteins, polypeptides, polysaccharides, and amino acids. However, there are currently no reports on cockroach glycosylated protein PAG-N1 and its activity in existing technologies.

[0004] Metabolic dysfunction-associated fatty liver disease (MASLD), formerly known as non-alcoholic fatty liver disease (NAFLD), is the most common chronic liver disease worldwide, affecting approximately one-third of the global population, and its incidence is increasing annually. Its pathogenesis is complex and not yet fully understood. Metabolic syndrome (including obesity, diabetes, and hypertension) is considered a major risk factor for the development and progression of MASLD. Metabolic dysfunction-associated steatohepatitis (MASH) is a highly progressive type of MASLD, which, if left untreated, may further develop into cirrhosis and hepatocellular carcinoma. However, current treatment strategies and drugs for MASLD are limited, with lifestyle interventions (healthy diet and physical exercise) being the primary treatment approach. Although the U.S. Food and Drug Administration (FDA) has approved resmetirom for the treatment of MASH, it suffers from poor efficacy, significant adverse reactions (such as diarrhea and nausea), and high costs. Therefore, there is an urgent need to develop safe and effective drugs or regimens to improve MASLD. Summary of the Invention

[0005] To solve or partially solve the problems existing in the related technologies, one of the objectives of the present invention is to provide a cockroach glycosylated protein PAG-N1, wherein the cockroach glycosylated protein PAG-N1 is composed of glycosylated proteins Q9BPS0 and Q9U8M0, wherein the sequence of protein Q9BPS0 is shown in SEQ ID NO:1 and the sequence of protein Q9U8M0 is shown in SEQ ID NO:2.

[0006] The glycosylated protein Q9BPS0 contains eight glycosylation sites, namely asparagine at positions 211, 290, 932, 1318, 1398, 1417, 1532 and 1719.

[0007] The glycosylated protein Q9U8M0 contains six glycosylation sites, namely asparagine at positions 290, 684, 981, 1413, 1420, and 1720.

[0008] The structure of its glycosylated Q9BPS0 protein is shown below. Figure 2 For the specific sugar chain structure, see Figure 3 The structure of the glycosylated Q9U8M0 protein is shown below. Figure 4 For the specific sugar chain structure, see Figure 5 .

[0009] The second objective of this invention is to provide a method for preparing the cockroach glycosylated protein PAG-N1, the specific steps of which are as follows: (1) After crushing the cockroach medicinal material, add water at a solid-liquid ratio of 1:(8~10), heat and reflux at 80~90℃ for 1~3h, filter with 1000~2000 mesh filter cloth, centrifuge the filtrate at 2000~4000rpm, collect the centrifuged filtrate, and concentrate it under reduced pressure to a specific gravity of about 1.1~1.3 to obtain concentrated cockroach extract.

[0010] (2) Add hot water at 80-90°C to the concentrated cockroach extract at a solid-liquid ratio of (1:2) to (1:4), mix evenly and let stand to cool, then use a disc / tube centrifuge to separate oil and water at 4000-5000 rpm, take the aqueous phase and let it stand for a while until the oil and water naturally separate into layers, then take the aqueous phase for later use.

[0011] (3) Place the aqueous phase obtained in step (2) into a 50-100kDa ultrafiltration centrifuge tube for ultrafiltration centrifugation, and take the supernatant to obtain the cockroach crude glycosylated protein solution.

[0012] (4) Add the crude glycosylated cockroach protein solution to an activated Sephadex G gel column, elute with 3-5 column volumes of pure water at a flow rate of 0.3-0.5 mL / min for 2-4 h, collect the eluent, concentrate under reduced pressure at 70-80 °C, freeze dry to obtain purified cockroach glycosylated protein PAG-N1 with an extraction rate of 0.3%-0.8%.

[0013] A third objective of this invention is to provide the application of the aforementioned cockroach glycosylated protein PAG-N1 in the preparation of a treatment for metabolic dysfunction-related fatty liver disease.

[0014] The beneficial effects of this invention are: (1) This invention is the first to discover, isolate and purify a novel cockroach glycosylated protein PAG-N1, whose chemical structure and biological function are reported for the first time, filling the existing gap.

[0015] (2) This invention is the first to confirm that cockroach glycosylated protein PAG-N1 has the pharmacological activity to improve MASLD, providing a new source of natural active substances for the prevention and treatment of MASLD.

[0016] (3) This invention clarifies that PAG-N1 can slow down the increase of body weight and liver weight in MASLD rats, reduce the accumulation of white fat in MASLD rats, improve insulin resistance and dyslipidemia, alleviate liver inflammation, lipid deposition and collagen fiber hyperplasia, enhance the antioxidant capacity of the liver, reduce liver damage, improve liver function in rats, and thus play a role in the treatment of MASLD. Attached Figure Description

[0017] Figure 1 The image shown is an SDS-PAGE electrophoresis image from Example 4; lane 1 is a solution of crude cockroach glycosylated protein, and lane 2 is a solution of cockroach glycosylated protein PAG-N1.

[0018] Figure 2 This is a structural diagram of the Q9BPS0 glycosylated protein in Example 4.

[0019] Figure 3 The glycan structures connected to each glycosylation site of the Q9BPS0 glycosylated protein in Example 4 are shown below. It is N-acetylglucosamine. Mannose, It is glucose. It is galactose. It is fucose. It is sialic acid.

[0020] Figure 4 This is a structural diagram of the Q9U8M0 glycosylated protein in Example 4.

[0021] Figure 5 The glycan structures connected to each glycosylation site of the Q9U8M0 glycosylated protein in Example 4 are shown below. It is N-acetylglucosamine. Mannose, It is glucose.

[0022] Figure 6 The table shows the changes in body weight of rats during weeks 1-8 in Example 5.

[0023] Figure 7 The figure shows the changes in body weight of rats from 9 to 22 weeks in Example 5.

[0024] Figure 8 This is a line graph showing the changes in blood glucose levels during the OGTT in rats in Example 5.

[0025] Figure 9 This refers to the effect of PAG-N1 on glucose tolerance in MASLD rats in Example 5; whereby... Figure 9 (a) is the OGTT AUC analysis chart. Figure 9 (b) represents the INS content. Figure 9 (c) represents the FBG content. Figure 9 (d) represents the HOMA-IR index of rats; compared with the CON group, # P <0.05, ## P <0.01; compared with the HFD group, * P <0.05,** P <0.01.

[0026] Figure 10 The effect of PAG-N1 on the morphology and quality of rat liver in Example 5; wherein Figure 10 (a) is a photograph of liver tissue morphology. Figure 10 (b) Liver weight; compared with the CON group, # P <0.05, ## P <0.01; compared with the HFD group, * P <0.05,** P <0.01.

[0027] Figure 11 Morphological image of rat EWAT tissue in Example 5 ( n =6).

[0028] Figure 12 The H&E staining results of rat EWAT in Example 5 (200×, n =3).

[0029] Figure 13 The effect of PAG-N1 on rat white adipose tissue in Example 5; wherein Figure 13 (a) represents the weight of rat EWAT ( n =6), Figure 13 (b) Rat PWAT mass ( n =6), Figure 13 (c) Calculation results of BFP (%) in rats n =6), Figure 13 (d) Area of ​​rat adipocytes stained with EWATH&E ( n =3); compared with group CON, # P <0.05, ## P <0.01; compared with the HFD group, * P <0.05,** P <0.01.

[0030] Figure 14 The effect of PAG-N1 on blood lipids in MASLD rats in Example 5 ( n =6); where Figure 14 (a) represents the TG content. Figure 14 (b) represents the TC content. Figure 14 (c) represents the LDL content. Figure 14 (d) represents HDL content; compared with the CON group, # P <0.05, ## P <0.01; compared with the HFD group, * P <0.05,** P <0.01.

[0031] Figure 15 The effect of PAG-N1 on liver function in MASLD rats in Example 5 ( n =6); where Figure 15 (a) shows the AST activity in rat serum. Figure 15 (b) Serum ALT activity in rats; compared with the CON group, # P <0.05, ## P <0.01; compared with the HFD group, * P <0.05,** P <0.01.

[0032] Figure 16 The effect of PAG-N1 on liver fibrosis in MASLD rats in Example 5 ( n =6); where Figure 16(a) shows the serum LN content in rats. Figure 16 (b) Serum HA content in rats; compared with the CON group, # P <0.05, ## P <0.01; compared with the HFD group, * P <0.05,** P <0.01.

[0033] Figure 17 The effect of PAG-N1 on liver lipid levels in MASLD rats in Example 5 ( n =6); where Figure 17 (a) represents the TG content in the liver. Figure 17 (b) represents the total TC content in the liver. Figure 17 (c) Liver NEFA content; compared with the CON group, # P <0.05, ## P <0.01; compared with the HFD group, * P <0.05,** P <0.01.

[0034] Figure 18 The effect of PAG-N1 on liver inflammation levels in MASLD rats in Example 5 ( n =6); where Figure 18 (a) represents the level of TNF-α in the liver. Figure 18 (b) shows the liver's IL-6 level. Figure 18 (c) Liver IL-1β levels; compared with the CON group, # P <0.05, ## P <0.01; compared with the HFD group, * P <0.05,** P <0.01.

[0035] Figure 19 The effect of PAG-N1 on liver oxidative stress in MASLD rats in Example 5 ( n =6); where Figure 19 (a) represents liver SOD activity. Figure 19 (b) represents liver GSH-PX activity. Figure 19 (c) represents liver CAT activity. Figure 19 (d) represents liver MDA levels; compared with the CON group, # P <0.05, ## P<0.01; compared with the HFD group, * P <0.05,** P <0.01.

[0036] Figure 20 The results of H&E staining of rat liver tissue in Example 5 ( n =6); where Figure 20 (a) H&E staining of rat liver (200×). Figure 20 (b) H&E staining NAS score; Note: Yellow arrow: round fat vacuoles; Green arrow: hepatic cords; Red arrow: inflammatory infiltration; Blue arrow: ballooning degeneration cells; Black arrow: lipid droplets; Compared with the CON group, # P <0.05, ## P <0.01; compared with the HFD group, * P <0.05,** P <0.01.

[0037] Figure 21 The results of Oil Red O staining of rat liver tissue in Example 5 ( n =6); where Figure 21 (a) Oil Red O staining of rat liver (200×). Figure 21 (b) Percentage of area positive for Oil Red O staining; Note: Black arrows indicate lipid droplets formed by lipid aggregation; Compared with the CON group, # P <0.05, ## P <0.01; compared with the HFD group, * P <0.05,** P <0.01.

[0038] Figure 22 Masson staining results of rat liver tissue in Example 5 ( n =6); where Figure 22 (a) Masson staining of rat liver (200×). Figure 22 (b) Results of Masson staining for collagen fiber quantification; compared with the CON group, # P <0.05, ## P <0.01; compared with the HFD group, * P <0.05,** P <0.01.

[0039] Figure 23 The results of Sirius red staining of rat liver tissue in Example 5 ( n =6); where Figure 23 (a) Sirius red staining of rat liver (200×). Figure 23 (b) Results of quantification of collagen fibers stained with Sirius red; compared with the CON group, # P <0.05, ## P <0.01; compared with the HFD group, * P <0.05,** P <0.01.

[0040] Figure 24 The effect of PAG-N1 on hepatocyte apoptosis in MASLD rats in Example 5 ( n =6); where Figure 24 (a) TUNEL fluorescence staining of rat liver (200×). Figure 24 (b) Results of TUNEL fluorescence staining and quantitative fluorescence analysis of liver tissue; compared with the CON group, # P <0.05, ## P <0.01; compared with the HFD group, * P <0.05,** P <0.01. Detailed Implementation

[0041] The present invention will be further described in detail below with reference to specific embodiments and accompanying drawings, but the scope of protection of the present invention is not limited to the content described therein; unless otherwise specified, all reagents used in the present invention are commercially available analytical grade reagents, and all raw materials used can be purchased through conventional commercial channels.

[0042] Example 1 Extraction of cockroach glycosylated protein PAG-N1 (1) After crushing the cockroach medicinal material (Yunnan Jingxin Biotechnology Co., Ltd., 20250103), add water at a solid-liquid ratio of 1:8, heat and reflux at 80°C for 1 hour, filter with a 1000-mesh filter cloth, centrifuge the filtrate at 2000 rpm, collect the centrifuged filtrate, and concentrate it under reduced pressure to a specific gravity of about 1.1 to obtain concentrated cockroach extract.

[0043] (2) Add hot water at 80°C to the concentrated cockroach extract at a ratio of 1:2, mix evenly and let stand to cool, use a disc / tube centrifuge to separate oil and water at 4000 rpm, take the aqueous phase and let it stand until the oil and water naturally separate into layers, then take the aqueous phase for later use.

[0044] (3) Place the above aqueous phase in a 50kDa ultrafiltration centrifuge tube for ultrafiltration centrifugation, and take the supernatant to obtain the cockroach crude glycosylated protein solution.

[0045] (4) The crude glycosylated protein solution of cockroach was added to a Sephadex G gel column that had been fully swollen and washed to remove impurities. The column was eluted with 3 column volumes of pure water at a flow rate of 0.3 mL / min for 2 h. The eluent was collected, concentrated under reduced pressure at 70 °C, and freeze-dried to obtain purified cockroach glycosylated protein PAG-N1 with an extraction rate of 0.3%.

[0046] Example 2 Extraction of cockroach glycosylated protein PAG-N1 (1) After crushing the cockroach medicinal material (Yunnan Jingxin Biotechnology Co., Ltd., 20250103), add water at a solid-liquid ratio of 1:9, heat and reflux at 85°C for 2 hours, filter with 1500 mesh filter cloth, centrifuge the filtrate at 3000 rpm, collect the centrifuged filtrate, and concentrate it under reduced pressure to a specific gravity of about 1.2 to obtain concentrated cockroach extract.

[0047] (2) Add hot water at 85°C to the concentrated cockroach extract at a ratio of 1:3, mix evenly and let stand to cool, use a disc / tube centrifuge to separate oil and water at 4500 rpm, take the aqueous phase and let stand until the oil and water naturally separate, then take the aqueous phase for later use.

[0048] (3) Place the above aqueous phase in a 70 kDa ultrafiltration centrifuge tube for ultrafiltration centrifugation, and take the supernatant to obtain the cockroach crude glycosylated protein solution.

[0049] (4) The crude glycosylated protein solution of cockroach was added to a Sephadex G gel column that had been fully swollen and washed to remove impurities. The column was eluted with 4 column volumes of pure water at a flow rate of 0.4 mL / min for 3 h. The eluent was collected, concentrated under reduced pressure at 75 °C, and freeze-dried to obtain purified cockroach glycosylated protein PAG-N1 with an extraction rate of 0.5%.

[0050] Example 3 Extraction of cockroach glycosylated protein PAG-N1 (1) After crushing the cockroach medicinal material (Yunnan Jingxin Biotechnology Co., Ltd., 20250103), add water at a solid-liquid ratio of 1:10 (g / mL), heat and reflux at 90℃ for 3h, filter with 2000 mesh filter cloth, centrifuge the filtrate at 4000rpm, collect the centrifuged filtrate, and concentrate it under reduced pressure to a specific gravity of about 1.3 to obtain concentrated cockroach extract.

[0051] (2) Add hot water at 90°C to the concentrated cockroach extract at a ratio of 1:4, mix evenly and let stand to cool, use a disc / tube centrifuge to separate oil and water at 5000 rpm, take the aqueous phase and let stand until the oil and water naturally separate, then take the aqueous phase for later use.

[0052] (3) Place the above aqueous phase in a 100kDa ultrafiltration centrifuge tube for ultrafiltration centrifugation, and take the supernatant to obtain the cockroach crude glycosylated protein solution.

[0053] (4) The crude glycosylated protein solution of cockroach was added to a Sephadex G gel column that had been fully swollen and washed to remove impurities. The column was eluted with 5 column volumes of pure water at a flow rate of 0.5 mL / min for 4 h. The eluent was collected, concentrated under reduced pressure at 80 °C, and freeze-dried to prepare cockroach glycosylated protein PAG-N1 with an extraction rate of 0.8%.

[0054] Example 4: Identification of cockroach glycosylated protein PAG-N1 PAG-N1 was separated using SDS-PAGE, and the target band was excised after Coomassie brilliant blue staining. The target band was then subjected to complete N-glycopeptide qualitative analysis using LC-MS / MS, and the data was analyzed using the complete N-glycopeptide database search engine gPeptide. The specific steps are as follows: (1) Take the crude glycosylated cockroach protein solution obtained in step (3) of Example 1 and the cockroach glycosylated protein PAG-N1 obtained in step (4) of Example 1 as protein samples, and dilute them in SDS-PAGE protein loading buffer (5×) (Jiangsu Biyuntian Biotechnology Research Institute, P0015L). The dilution ratio is protein sample: SDS-PAGE protein loading buffer (5×) = 4:1. Boil the diluted protein samples in a metal bath for 7 minutes to obtain the samples. Prepare a 12% separating gel. Add the separating gel along the edge of the glass plate with a pipette, leaving a height of 1 cm. After the separating gel solidifies, add the stacking gel and insert a comb. After the gel has completely solidified, remove the comb and load 8 μL of sample and protein marker (positioned in the wells in the following order: protein marker, cockroach crude glycosylated protein solution, and cockroach glycosylated protein PAG-N1). Electrophoresis is performed at a constant voltage of 80 V for 20 min until the sample is compressed into a thin line. The voltage is then adjusted to 120 V, and electrophoresis is performed at this constant voltage for 90 min until the sample reaches the bottom of the gel. The gel is then placed in Coomassie Brilliant Blue staining solution and stained at room temperature for 1 h. The Coomassie Brilliant Blue staining solution is then poured out, and destaining solution is added to cover the gel. Destaining continues until the blue background has mostly faded and the protein bands are clear and sharp. The Coomassie Brilliant Blue staining results are as follows: Figure 1 As shown, the crude glycosylated protein solution of cockroaches showed multiple protein bands on the gel, indicating the presence of other proteins; while the purified cockroach glycosylated protein PAG-N1 showed a single, clear, and well-defined target band at 73 kDa. The gel containing the target band was cut off for later use.

[0055] (2) The gel containing the target band obtained in step (1) was sent to Hannover Biotech (Suzhou) Co., Ltd., and detected using LC-MS / MS (Thermo Fisher Scientific) technology combining a Dionex Ultimate 3000 RSLCnano high performance liquid chromatography system and an Orbitrap Exploris 480 mass spectrometer: Chromatographic conditions: ①Chromatographic column system: Pre-column (trapped column): Phenomenex Jupiter C18 column (5 μm, 300 Å, 360 μod × 200 μid, 5 cm).

[0056] Analytical column: Phenomenex Jupiter C18 column (5 μm, 300 Å, 360 μod × 75 μid, 70 cm).

[0057] ②Mobile phase: Mobile phase A is a mixture of 99.9% water and 0.1% formic acid, and mobile phase B is a mixture of 90% acetonitrile, 9.9% water and 0.1% formic acid.

[0058] ③ Flow rate: 0.3 μL / min.

[0059] ④ The elution gradient is shown in Table 1: Table 1 Elution gradient of mobile phase in liquid chromatography The mass spectrometry conditions were as follows: negative ion mode was used for mass spectrometry data acquisition; the ion transfer tube temperature was 300℃; the spray voltage was 1900V; the mass-to-charge ratio range was 700~2000 m / z; the resolution was 60k (at 200 m / z); and the target value for automatic gain control was 3×10⁻⁶. 6 The maximum ion implantation time was 20 ms; the MS / MS resolution was 30 kJ (m / z 200), and data-dependent acquisition was performed using Top 20 datasets. The automatic gain control target value was 5 × 10⁻⁶. 5 The maximum ion implantation time was 250 ms, the parent ion trapping window was 1.4 m / z, the HCD step-normalized collision mode was adopted, and the collision energy was 30%.

[0060] After obtaining the data, the data was analyzed using the complete N-glycopeptide database search engine gPeptide. The results showed that in the cockroach crude glycosylated protein solution obtained in step (3) of Example 1, glycosylation sites were identified on Q9BPS0 and Q9U8M0 proteins, with molecular weights of 215943 Da and 212728 Da, respectively; the glycosylation structure of Q9BPS0 is as follows: Figure 2As shown, a total of 8 glycosylation sites were predicted on the Q9BPS0 protein, with glycan molecular weights ranging from 2400 to 3500 Da, and the glycan structures at each site are as follows. Figure 3 As shown, the specific details are as follows: 211 ( Figure 3 a) 290 ( Figure 3 b), 932 Figure 3 c), 1318 Figure 3 d), 1398 Figure 3 e), 1417 Figure 3 f), 1532 ( Figure 3 g) and 1719 ( Figure 3 h), the sugar chains are both high-mannose and complex types; the glycosylation structure of Q9U8M0 is as follows Figure 4 As shown, a total of 6 glycosylation sites were predicted on the protein, with glycan molecular weights ranging from 1800 to 3600 Da. The glycan structures at each site are shown in the figure. Figure 5 As shown, specifically: 290 ( Figure 5 a), 684 ( Figure 5 b), 981 Figure 5 c), 1413 Figure 5 d), 1420 Figure 5 e) and 1720 ( Figure 5 f), all glycans are of the high-mannose type; all glycans bind to the N (asparagine) site on the protein.

[0061] Example 5 The effects of PAG-N1 on autoimmune hepatitis in mice were investigated using the following steps: (1) Thirty-six rats were prepared for acclimatization. After one week, the rats were randomly divided into a control group (CON, n =6) and the high-fat diet group ( n =30); control group rats were fed normal diet and normal drinking water, while high-fat diet group rats were fed HFD (high-fat diet + 15% fructose water); at the end of week 8, the high-fat diet group rats were randomly divided into 5 groups ( n=6 groups, namely HFD group, PGZ group (pioglitazone hydrochloride dispersible tablets, 3 mg / kg / d), PAG-N1 low-dose group (PAG-N1-L, 60 mg / kg / d), PAG-N1 medium-dose group (PAG-N1-M, 120 mg / kg / d), and PAG-N1 high-dose group (PAG-N1-H, 240 mg / kg / d); among them, PAG-N1 low-dose group, PAG-N1 medium-dose group, PAG-N1 high-dose group and PGZ group were administered by gavage at a volume of 10 mL / kg, while CON group and HFD group rats were given an equal volume of physiological saline, and the administration was continued for 14 weeks; the MASLD model was established for a total of 22 weeks; the PAG-N1 dosage was calculated with reference to the clinical treatment dosage of Ganlong capsules and the results of the preliminary experiment, and the Pioglitazone hydrochloride dispersible tablets (PGZ) dosage was calculated with reference to the clinical treatment dosage.

[0062] (2) The effect of PAG-N1 on the body weight of MASLD rats, the specific steps are as follows: The weight changes of rats were monitored, with weight measured weekly. The results of the weight changes of rats over 1-8 weeks are as follows: Figure 6 As shown, compared with the CON group, feeding HFD to the rats in the high-fat diet group resulted in a significant increase in body weight ( P <0.01); intervention was initiated from week 9 by gavage administration of PGZ and PAG-N1. The weight change curve of rats from week 9 to week 22 is shown in the figure. Figure 7 As shown, compared with the HFD group, PGZ and PAG-N1 significantly slowed down the weight gain in rats ( P <0.01), and PAG-N1 showed a better inhibitory effect on rat weight gain than PGZ.

[0063] (3) The effect of PAG-N1 on glucose tolerance in MASLD rats, the specific steps are as follows: Before the experiment, rats were fasted for 8 hours but allowed free access to water and were weighed. A drop of blood was collected via the tail vein to measure and record the 0-minute basal blood glucose level. Rats were then administered a 50% glucose solution by gavage at a dose of 4 mL / kg. Blood glucose levels were measured and recorded at 30, 60, 90, 120, 150, and 180 minutes after gavage. The results are as follows: Figure 8 As shown, compared with the CON group, HFD significantly increased blood glucose levels in rats at 7 time points, while after 14 weeks of intervention with PAG-N1 and PGZ, blood glucose levels in MASLD rats were significantly reduced at 60, 90, 120, 150, and 180 min. P <0.01); the area under the curve (AUC) of OGTT blood glucose change is as follows: Figure 9 As shown in (a), compared with the CON group, HFD significantly increased the AUC of blood glucose in rats ( P<0.01) indicates that the glucose metabolism capacity of MASLD rats is impaired, while the AUC of rats treated with PAG-N1 and PGZ was significantly reduced compared with the HFD group. P <0.01), indicating that PAG-N1 and PGZ intervention enhanced the ability of MASLD rats to regulate blood glucose homeostasis; in addition, HFD also induced INS (insulin, Figure 9 (b) FBG (fasting blood glucose) Figure 9 (c) Insulin resistance levels and the HOMA-IR index (an index of insulin resistance assessed by a homeostasis model). Figure 9 (d) The significant increase indicates that HFD induces insulin resistance in rats, which is effectively reversed after intervention with PAG-N1 and PGZ, indicating that PAG-N1 and PGZ can effectively improve insulin resistance.

[0064] (4) At the end of week 22, the rats were fasted and deprived of water for 12 hours. After the rats were anesthetized by inhaling 4% isoflurane, blood was collected from the abdominal aorta and the serum was separated by centrifugation at 3500 rpm / min for 10 min and stored in a -80℃ freezer. The rats were then euthanized (inhaled isoflurane at a concentration of 4%). The liver tissue, epididymal white adipose tissue (EWAT), perirenal white adipose tissue (PWAT), and colon tissue of the rats were taken. The morphological changes of the tissues were observed and photographed. The tissues were aliquoted for subsequent detection and analysis. Part of the liver and colon tissues were flash-frozen in liquid nitrogen and stored in a -80℃ freezer. The other part was fixed in 4% paraformaldehyde solution for histopathological analysis. EWAT was stored in a special fixative for adipose tissue.

[0065] (5) Effects of PAG-N1 on rat liver morphology and quality, the specific steps are as follows: The morphology of rat livers was observed, and the results are as follows: Figure 10 As shown in (a), the livers of rats in the CON group were of normal size and regular shape, without edema, and had a smooth, reddish-brown surface. In contrast, the livers of rats in the HFD group showed significant enlargement, yellowing, roughness, granular texture, and blunting of the edges—significant pathological features of fatty liver. Compared to the HFD group, PAG-N1 and PGZ intervention significantly improved the morphological characteristics of the rat livers, including enlargement, blunting of the edges, and roughness with yellowing. Furthermore, the liver quality was statistically analyzed, and the results are as follows: Figure 10 As shown in (b), liver mass was reduced after intervention with PAG-N1 and PGZ, and the liver of rats in the PAG-N1-H group showed better efficacy; this result indicates that the PAG-N1 extracted in this invention can effectively improve the morphological lesions of the liver in MASLD rats.

[0066] (6) Effects of PAG-N1 on the morphology and quality of rat fat, the specific steps are as follows: The morphology of white adipose tissue (EWAT) in the rat epididymis was observed, and the results are as follows: Figure 11 As shown, compared with the CON group, the EWAT in the HFD group rats showed significant accumulation, increased volume, and irregular shape. However, the EWAT in rats treated with PAG-N1 and PGZ was significantly reduced. Rat EWAT was prepared into paraffin sections, dewaxed with xylene, hydrated with a gradient of ethanol, rinsed with running water, stained with hematoxylin for 5 min, differentiated with hydrochloric acid and ethanol, blued with ammonia, dehydrated with a gradient of ethanol, stained with eosin for 5 min, dehydrated with anhydrous ethanol, cleared with n-butanol and xylene, and mounted with neutral resin to obtain H&E stained sections of EWAT. Image analysis results are shown below. Figure 12 As shown, compared with the CON group, HFD feeding significantly increased the size of epididymal adipocytes in rats, while PAG-N1 and PGZ intervention reduced the volume of epididymal adipocytes. Furthermore, the mass of EWAT and perirenal white adipose tissue (PWAT) in rats was statistically analyzed, and the results are as follows: Figure 13 (a) and Figure 13 As shown in (b), HFD significantly increased the mass of EWAT and PWAT in rats, which was significantly reversed after intervention with PAG-N1 and PGZ.

[0067] In addition, the body fat percentage (BFP) of rats is calculated using the following formula: BFP = (PWAT + EWAT) / rat body weight × 100% (5.1) The result is as follows Figure 13 As shown in (c), the body fat percentage of rats was significantly reduced after intervention with PAG-N1 and PGZ.

[0068] The size of epididymal adipocytes is generally indicative of lipid accumulation. To further observe the effect of PAG-N1 intervention on epididymal adipocytes, the adipocyte area was calculated from H&E-stained sections of rat EWAT tissue. The results are as follows: Figure 13 As shown in (d), the cell area of ​​EWAT cells in the HFD group was significantly increased, and the cell area was significantly decreased after intervention with PAG-N1 and PGZ. P <0.05); the above results indicate that PAG-N1 can effectively reduce HFD-induced white fat deposition in rats.

[0069] (6) Effects of PAG-N1 on blood lipids in MASLD rats, the specific steps are as follows: Take an appropriate amount of serum separated in step (4) and perform the test according to the instructions of the triglyceride (TG) assay kit (Nanjing Jiancheng Bioengineering Research Institute Co., Ltd., A110-1-1), total cholesterol (TC) assay kit (Nanjing Jiancheng Bioengineering Research Institute Co., Ltd., A111-1-1), low-density lipoprotein cholesterol (LDL) assay kit (Nanjing Jiancheng Bioengineering Research Institute Co., Ltd., A113-1-1), and high-density lipoprotein cholesterol (HDL) assay kit (Nanjing Jiancheng Bioengineering Research Institute Co., Ltd., A112-1-1); the blood lipid level test results are as follows: Figure 14 (a)- Figure 14 As shown in (d), compared with the CON group, HFD increased the levels of TG, TC, and LDL in rat serum and decreased the level of HDL, indicating that HFD caused dyslipidemia in rats; while after PAG-N1 and PGZ intervention, the dyslipidemia level in MASLD rats was significantly reversed. P The value <0.05 indicates that PAG-N1 has a substantial effect on improving HFD-induced lipid metabolism disorders, and the improvement effect of the PAG-N1-H group is better than that of PGZ.

[0070] (7) Effects of PAG-N1 on liver function and liver fibrosis markers in MASLD rats: The specific steps are as follows: Take an appropriate amount of rat serum and perform the tests according to the instructions for the aspartate aminotransferase (AST / GOT) test kit (Nanjing Jiancheng Bioengineering Research Institute Co., Ltd., C010-2-1), alanine aminotransferase (ALT / GPT) test kit (Nanjing Jiancheng Bioengineering Research Institute Co., Ltd., C009-2-1), laminin (LN) test kit (Nanjing Jiancheng Bioengineering Research Institute Co., Ltd., H148-1-2), and hyaluronic acid (HA) test kit (Nanjing Jiancheng Bioengineering Research Institute Co., Ltd., H141-1-2); the liver function test results are as follows. Figure 15 (a) and Figure 15 As shown in (b), compared with the CON group, the serum AST activity of rats in the HFD group was significantly increased ( P <0.05, indicating hepatocyte damage in the HFD group rats; compared with the HFD group, serum AST and ALT activities in the PAG-N1 and PGZ groups were significantly reduced ( P <0.05), indicating that PAG-N1 and PGZ have a significant protective effect on the liver of MASLD rats; the results of liver fibrosis index detection are as follows. Figure 16 (a) and Figure 16As shown in (b), the serum levels of liver fibrosis markers LN and HA in the HFD group were significantly higher than those in the CON group, indicating that collagen fiber hyperplasia existed in the liver of MASLD rats. The serum levels of liver fibrosis markers in the PAG-N1 and PGZ groups were significantly lower than those in the HFD group. P The value <0.05 indicates that PAG-N1 and PGZ intervention improved collagen fiber proliferation in the liver of MASLD rats. These results suggest that PAG-N1 has a positive therapeutic effect on liver damage and fibrosis progression caused by HFD.

[0071] (8) Effects of PAG-N1 on TC, TG and NEFA in the liver of MASLD rats, the specific steps are as follows: An appropriate amount of rat liver tissue was taken and processed according to the instructions of the total cholesterol (TC) assay kit (Nanjing Jiancheng Bioengineering Research Institute Co., Ltd., A111-1-1), tissue factor (TF) assay kit (Nanjing Jiancheng Bioengineering Research Institute Co., Ltd., H560-1), and free fatty acid (NEFA) assay kit (Nanjing Jiancheng Bioengineering Research Institute Co., Ltd., A042-2-1); the results of lipid changes in rat liver tissue are as follows. Figure 17 As shown, compared with the CON group, HFD significantly increased the levels of TC, TF, and NEFA in the liver of MASLD rats (P<0.05); rats treated with PAG-N1 and PGZ for 14 weeks showed a significant increase in TG ( ) in the liver. Figure 17 (a) TC ( Figure 17 (b) NEFA ( Figure 17 (c) The content was significantly reduced (P<0.05); the results indicate that PAG-N1 and PGZ can effectively reduce the lipid level in liver tissue caused by HFD.

[0072] (9) The effect of PAG-N1 on liver inflammatory factors in MASLD rats, the specific steps are as follows: Take an appropriate amount of rat liver tissue and perform the tests according to the instructions for the tumor necrosis factor-α (TNF-α) test kit (Nanjing Jiancheng Bioengineering Research Institute Co., Ltd., H052-1-2), interleukin-6 (IL-6) test kit (Nanjing Jiancheng Bioengineering Research Institute Co., Ltd., H007-1-2), and interleukin-1β (IL-1β) test kit (Nanjing Jiancheng Bioengineering Research Institute Co., Ltd., H002-1-2); the results of the detection of inflammatory factors in rat liver tissue are as follows: Figure 18 (a)- Figure 18 As shown in (b), compared with the CON group, the levels of inflammatory markers TNF-α, IL-6, and IL-1β in the liver tissue of rats in the HFD group were significantly increased; the expression of inflammatory markers in the liver tissue of rats after PAG-N1 and PGZ intervention was significantly decreased compared with the HFD group. P<0.05); the results showed that PAG-N1 could significantly reverse the level of liver tissue inflammation caused by HFD, indicating that PAG-N1 has the ability to resist the liver inflammatory response caused by HFD.

[0073] (10) Effects of PAG-N1 on oxidative stress in the liver of MASLD rats, the specific steps are as follows: An appropriate amount of rat liver tissue was taken and processed according to the instructions of the following kits: Total Superoxide Dismutase (SOD) Assay Kit (Nanjing Jiancheng Bioengineering Research Institute Co., Ltd., A001-3-2), Glutathione Peroxidase (GSH-PX) Assay Kit (Nanjing Jiancheng Bioengineering Research Institute Co., Ltd., A005-1-2), Catalase (CAT) Assay Kit (Nanjing Jiancheng Bioengineering Research Institute Co., Ltd., A007-1-1), and Malondialdehyde (MDA) Assay Kit (Nanjing Jiancheng Bioengineering Research Institute Co., Ltd., A003-1-2). The results of liver tissue oxidative stress detection are as follows: Figure 19 (a)- Figure 19 As shown in (c), compared with the CON group, HFD significantly reduced the activities of SOD, GSH-PX, and CAT in the liver of rats. Figure 19 As shown in (d), compared with the CON group, HFD significantly increased the level of MDA in the liver of rats. P <0.05); After 14 weeks of treatment with PAG-N1 and PGZ, the activities of SOD, GSH-PX, and CAT in the liver of rats were significantly increased, while the level of MDA was significantly decreased. P The value was <0.05, indicating that PAG-N1 can effectively alleviate oxidative damage to the liver.

[0074] (11) H&E staining of rat liver tissue, the specific steps are as follows: Rat liver paraffin sections were prepared, dewaxed with xylene, hydrated with a gradient of ethanol, and rinsed with running water. Hematoxylin staining was performed for 3–5 min, followed by differentiation with hydrochloric acid and ethanol, blue reversion with ammonia, dehydration with a gradient of ethanol, eosin staining for 5 min, dehydration with anhydrous ethanol, clearing with n-butanol and xylene, and mounting with neutral resin. Image acquisition and analysis were conducted, and the results are as follows: Figure 20 As shown in (a), the hepatic lobule structure of the CON group was clear and intact, the hepatic cords were arranged radially around the central vein, the hepatic sinusoidal structure was clearly visible, and the hepatocytes were of uniform size. In the HFD group, the hepatocytes were significantly enlarged, with large areas of fatty degeneration forming fat vacuoles. Obvious hepatocyte ballooning degeneration, nuclear displacement, and inflammatory cell infiltration were observed. The hepatocytes were irregular in shape, and the hepatic cords were arranged disorderly. Compared with the HFD group, the PAG-N1 and PGZ groups showed significantly reduced hepatocyte fatty degeneration and the number of fat vacuoles. No hepatocyte ballooning degeneration or inflammatory infiltration was observed, the hepatic cord structure was clear, and the cells were arranged neatly. The pathological NAS score results are as follows: Figure 20As shown in (b), rats in the HFD group simultaneously exhibited hepatic steatosis, ballooning degeneration, and inflammation, suggesting that HFD had induced rats to develop MASH, a progressive disease of MASLD. Furthermore, the NAS score of rats in the HFD group was significantly higher than that in the CON group. PAG-N1 and PGZ interventions significantly reversed the liver NAS score of MASLD rats. The experimental results indicate that PAG-N1 can effectively alleviate pathological liver damage.

[0075] (12) Oil Red O staining of rat liver tissue, the specific steps are as follows: Frozen sections of rat liver were mounted on pre-chilled glass slides, air-dried at room temperature, fixed with 4% paraformaldehyde for 15 min, and washed with PBS. The livers were then pretreated with 60% isopropanol, followed by staining with freshly prepared and filtered Oil Red O working solution for 15 min. After differentiation with 60% isopropanol and washing with distilled water, the nuclei were counterstained with hematoxylin, washed with water to regain blue color, and finally mounted with glycerol gelatin for image acquisition. The results of Oil Red O (ORO) staining are shown below. Figure 21 As shown in (a), the quantitative results of the positive area area are as follows: Figure 21 As shown in (b), compared with the CON group, the liver tissue of rats in the HFD group showed a large amount of lipid deposition that was stained red, and the proportion of lipid droplet-positive areas was significantly increased. P <0.01), compared with the HFD group, the number of red lipid droplets in the liver tissue of rats treated with PAG-N1 and PGZ was significantly reduced, and PAG-N1-H had a more significant effect. The experimental results are as follows. Figure 21 (a) and Figure 21 As shown in (b), PAG-N1 can effectively reduce lipid deposition in hepatocytes caused by MASLD.

[0076] (13) Masson staining of rat liver tissue, the specific steps of which are as follows: Rat liver paraffin sections were dewaxed with xylene, hydrated with graded ethanol, and rinsed with running water. The sections were then soaked in potassium dichromate solution overnight, rinsed with running water, stained with iron hematoxylin for 3 min, differentiated with hydrochloric acid-ethanol, and then blued again with running water. They were stained with Ponceau S and acid fuchsin for 8 min, rinsed with running water, treated with phosphomolybdic acid aqueous solution for 3 min, discarded, and then directly immersed in aniline blue staining solution for 6 min, followed by rapid differentiation with 1% glacial acetic acid. The sections were dehydrated with anhydrous ethanol, cleared, mounted with neutral resin, and images were acquired and analyzed. The collagen fiber deposition in the rat liver tissue was assessed. Masson staining results are shown below. Figure 22 As shown in (a), HFD significantly increased collagen fiber deposition around the central vein in rat liver tissue. Intervention with PAG-N1 and PGZ significantly improved collagen fiber deposition in rat liver tissue. Masson staining quantitative analysis of collagen fibers showed the following results: Figure 22 As shown in (b), PAG-N1 and PGZ were found to effectively reduce HFD-induced collagen fiber deposition in rat livers. P<0.01); indicating that PAG-N1 can effectively improve HFD-induced liver lesions in MASLD rats and reverse the process of liver fibrosis.

[0077] (14) Sirius red staining of rat liver tissue, the specific steps are as follows: Rat liver paraffin sections were dewaxed with xylene, hydrated with graded ethanol, and rinsed with running water. The sections were stained with Sirius red for 8 min, rinsed with running water, cleared, mounted with neutral resin, and images were acquired and analyzed. To clarify the degree of liver fibrosis caused by HFD-induced MASLD in rats and the ameliorative effect of PAG-N1 on liver fibrosis, this study used Sirius red (PSR) staining to observe collagen deposition in the liver. The results are as follows: Figure 23 As shown in (a), in the CON group, only a small amount of red collagen fibers were observed in the vessel walls of the portal area, which is a normal distribution; in the HFD group, the collagen fibers in the portal area significantly extended into the surrounding parenchyma, and the red collagen deposition increased significantly, with local pseudolobular structures visible; after intervention with PGZ or PAG-N1, the red collagen deposition in the liver tissue was significantly reduced; quantitative analysis is as follows. Figure 23 As shown in (b), the proportion of collagen fiber-positive area in the HFD group was significantly higher than that in the CON group. P <0.01), while both the PGZ group and the PAG-N1 group showed significantly lower levels than the HFD group ( P <0.01); the Sirius red staining results were consistent with the aforementioned Masson staining results, indicating that PAG-N1 can effectively inhibit the deposition of collagen fibers in the liver and play a role in improving MASLD-related liver fibrosis.

[0078] (15) TUNEL fluorescence staining of rat liver tissue, the specific steps of which are as follows: Rat liver paraffin sections were dewaxed with xylene, hydrated with graded ethanol, and rinsed with running water. The hydrated rat liver tissue sections were washed with PBS and permeabilized with proteinase K. TUNEL reaction solution was then prepared according to the kit instructions, added to the tissue sections, and incubated at 37°C in the dark. After washing, DAB staining was performed, followed by hematoxylin counterstaining, dehydration, clearing, and mounting. Finally, images were acquired. The TUNEL fluorescence staining results are shown below. Figure 24 As shown in (a), green fluorescence indicates positive expression of hepatocyte apoptosis. Compared with the CON group, the HFD group showed significantly more positive expression, while the PGZ, PAG-N1-M, and PAG-N1-H groups showed significantly less positive expression than the HFD group. Quantitative analysis of the positive expression results are as follows: Figure 24 As shown in (b), compared with the CON group, the HFD group showed a significant increase in the positive expression of hepatocyte apoptosis. P <0.01); Compared with the HFD group, the positive expression levels of hepatocyte apoptosis in the PGZ group and each PAG-N1 group were reduced to varying degrees. PThe result showed that PAG-N1 effectively reduced apoptosis in the liver tissue of MASLD rats (<0.01), indicating that PAG-N1 reduced the degree of liver damage in MASLD rats and decreased hepatocyte apoptosis.

[0079] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A cockroach glycosylated protein PAG-N1, characterized in that, The cockroach glycosylated protein PAG-N1 is composed of glycosylated proteins Q9BPS0 and Q9U8M0, wherein the sequence of protein Q9BPS0 is shown in SEQ ID NO:1 and the sequence of protein Q9U8M0 is shown in SEQ ID NO:

2. Among them, the glycosylated protein Q9BPS0 contains 8 glycosylation sites, namely asparagine at positions 211, 290, 932, 1318, 1398, 1417, 1532 and 1719; The glycosylated protein Q9U8M0 contains six glycosylation sites, namely asparagine at positions 290, 684, 981, 1413, 1420, and 1720.

2. The method for preparing the cockroach glycosylated protein PAG-N1 according to claim 1, characterized in that, The specific steps are as follows: (1) Crush cockroach medicinal material and add water, heat and reflux, then filter, centrifuge and concentrate under reduced pressure to obtain concentrated cockroach extract; (2) Add hot water to the concentrated cockroach extract, mix, centrifuge and remove the oil phase, let stand for a while, and take the aqueous phase for later use after the oil and water naturally separate. (3) The aqueous phase obtained in step (2) is subjected to ultrafiltration and centrifugation, and the supernatant is taken to obtain the crude glycosylated protein solution of cockroach; (4) The crude glycosylated protein solution of cockroach was added to a gel column, and after elution, vacuum concentration and freeze drying, cockroach glycosylated protein PAG-N1 was obtained.

3. The method for preparing the cockroach glycosylated protein PAG-N1 according to claim 2, characterized in that, The water addition condition in step (1) is a solid-liquid ratio of 1:(8~10).

4. The method for preparing cockroach glycosylated protein PAG-N1 according to claim 2, characterized in that, The conditions for vacuum concentration in step (1) are to concentrate under vacuum until the specific gravity is 1.1 to 1.

3.

5. The method for preparing the cockroach glycosylated protein PAG-N1 according to claim 2, characterized in that, The condition for adding hot water in step (2) is a solid-liquid ratio of 1:(2~4).

6. The method for preparing the cockroach glycosylated protein PAG-N1 according to claim 2, characterized in that, The elution conditions described in step (4) are elution at a flow rate of 0.3~0.5 mL / min for 2~4 h.

7. The use of the cockroach glycosylated protein PAG-N1 as described in claim 1 in the preparation of drugs for liver injury.

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