Application method of recombinant hirudin in preparation of medicine for treating non-alcoholic fatty liver disease

Abstract

The invention provides an application method of recombinant hirudin in preparation of a medicine for treating non-alcoholic fatty liver disease, and belongs to the field of biotechnology research. The results of nutritional status, biochemical detection, liver histological detection and protein expression detection of six groups of mice are analyzed; from a comprehensive perspective, it is further verified that the recombinant hirudin can treat the non-alcoholic fatty liver disease by influencing liver lipid metabolism and inflammation pathway expression.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology research, and specifically relates to an application method of recombinant hirudin in preparing a drug for treating non-alcoholic fatty liver disease. Background Art

[0002] Hirudin is a natural active substance extracted from the salivary glands and secretions of blood-sucking leeches. It has multiple pharmacological effects such as anti-inflammatory, anticoagulant, antithrombotic, platelet aggregation inhibition, lipid-lowering, and anti-tumor. Studies have found that hirudin can significantly reduce liver index, effectively inhibit the enrichment of lipids in tissues, and can also reduce the levels of TC, TG, LDL-C, and HDL-C in the blood. Recombinant hirudin is an artificial product obtained through genetic engineering. It is similar to hirudin found in nature and has the same biochemical and pharmacological properties as natural hirudin. Recombinant hirudin is a potent direct thrombin inhibitor and is increasingly used to replace anticoagulation. With the expansion of clinical trials, it will be used to prevent and treat more diseases, and its development prospects are very broad.

[0003] Protease-activated receptor-1 is a thrombin receptor, and coagulation cascade proteases such as thrombin play a key role in inflammation and fibrosis in multiple organs such as the liver. In patients with chronic viral hepatitis and in mouse models of liver fibrosis, the progression of liver fibrosis is associated with a procoagulant state; plasminogen activator inhibitor is the main physiological inhibitor of tissue-type plasminogen activator in plasma. It has been reported in the literature that protease-activated receptor-1 is related to lipid metabolism and inflammation.

[0004] In current research, it has been found that recombinant hirudin can enhance liver function, but it has not yet been found whether recombinant hirudin has an effect on liver lipid metabolism and inflammatory pathway expression, and it has not been possible to combine histological and molecular biological conclusions to further verify whether there is a correlation between the two. Summary of the invention

[0005] In view of the above problems, the present invention provides a method for preparing a drug for treating non-alcoholic fatty liver disease using recombinant hirudin, successfully proves the positive therapeutic effect of recombinant hirudin as the main ingredient of the drug in treating non-alcoholic fatty liver disease, and makes further efficacy verification explanation in combination with the results of histological and biomolecular detection indicators.

[0006] The present invention is implemented by the following technical scheme: comprising a method for preparing a drug for treating non-alcoholic fatty liver disease using recombinant hirudin, specifically comprising the following steps:

[0007] S1. Preparation of recombinant hirudin, specifically comprising the following steps:

[0008] Step 1. Construction of high-copy recombinant hirudin yeast strain;

[0009] Step 2. The high-copy recombinant hirudin yeast strain obtained in step 1 is subjected to two-stage expansion culture to obtain a fermentation seed liquid;

[0010] Step 3. The fermented seed solution obtained in step 2 was inoculated into a 30L fermenter for high-density fermentation to obtain a fermentation solution containing recombinant hirudin:

[0011] Step 4. After the fermentation broth is centrifuged and filtered, it is purified by two steps of chromatography to obtain recombinant hirudin with a purity greater than 99%, and the recombinant hirudin lyophilized powder is obtained by the steps of detection, sterilization, dilution, filling, freeze-drying, capping, labeling, etc.;

[0012] S2 experimental process, specifically the following steps:

[0013] Step 5. Redissolving the recombinant hirudin lyophilized powder in physiological saline to obtain a drug solution;

[0014] Step 6: Grouping: Adaptive feeding of male C57BL / 6J mice, setting up blank control group; model group; low-dose treatment group of recombinant hirudin injection; medium-dose treatment group of recombinant hirudin injection; high-dose treatment group of recombinant hirudin injection; positive control group;

[0015] Step 7: Group intervention treatment:

[0016] Blank control group: fed with normal diet;

[0017] Model group: fed with a high-fat diet;

[0018] The low-dose recombinant hirudin injection treatment group was fed with a high-fat diet and subcutaneously injected with a low-dose recombinant hirudin injection;

[0019] The medium-dose recombinant hirudin injection treatment group was fed with a high-fat diet and subcutaneously injected with a medium-dose recombinant hirudin injection;

[0020] The high-dose recombinant hirudin injection treatment group: the mice were fed with a high-fat diet and subcutaneously injected with a high-dose recombinant hirudin injection;

[0021] Positive control group: fed with a high-fat diet and given vitamin E by oral gavage;

[0022] S3 drug efficacy evaluation scheme, specifically the following steps:

[0023] Step 8. Comprehensive nutritional status: Method: Monitor the weight changes of mice in each group;

[0024] Step 9. Biochemical detection: Method: TG, TC, ALT, AST, MDA, LDH, SOD, GSH concentrations in liver and serum;

[0025] Step 10. Histological examination: Methods: Observe liver tissue; H&E staining; Oil Red O staining; F4 / 80 immunofluorescence staining; MASSON staining; Target protein PAR-1 immunofluorescence staining;

[0026] Step 11. Detection of protein expression level: Method: Western blot;

[0027] S4 test result evaluation, including the following points:

[0028] Evaluation 1: According to the test scheme in step 8, the following results were obtained: The weight change curve of mice. Compared with the control group, the weight of the mice in the model group increased significantly after being given a high-fat diet. After being treated with recombinant hirudin and vitamin E, the weight of the mice gradually decreased. This evaluation result proves that recombinant hirudin can affect lipid metabolism from the perspective of nutritional status;

[0029] Evaluation 2: According to the test scheme of step 9, the following results were obtained: Compared with the control group, the TG and TC contents of the model group were significantly increased. After the administration of recombinant hirudin and vitamin E, the TG and TC contents were significantly decreased compared with the model group. Compared with the control group, the ALT and AST contents of the model group were significantly increased. After the administration of recombinant hirudin and vitamin E, their contents were significantly decreased. Compared with the control group, the MDA, LDH, and GSH contents in the model group were significantly increased, and the SOD content was significantly decreased. After the administration of recombinant hirudin and vitamin E, the MDA, LDH, and GSH contents gradually decreased, and the SOD content gradually increased. Compared with the control group, the ALT, AST, TG, TC, LDH, MDA, and GSH contents in the model group were significantly increased, and the SOD content was significantly decreased. In addition, the improvement of liver abnormalities by recombinant hirudin was dose-dependent. The above evaluation results prove that recombinant hirudin can increase lipid metabolism and protect the liver from the perspective of biochemical detection.

[0030] Evaluation 3: Based on the test plan evaluation in step 10, we can get:

[0031] Observation of liver tissue: The liver of mice in the blank control group was observed with the naked eye. The normal liver was tough, with sharp edges, smooth surface and dark red color, while the livers of the other groups were light in color, whitish and with blunt edges. The model group was the most obvious, with a yellowish-white color. The color of the liver after treatment with recombinant hirudin and vitamin E was between the control group and the model group, and with the increase of the dosage, the color and state of the liver gradually recovered, indicating that recombinant hirudin can accelerate liver lipid metabolism and fill the liver blood vessels;

[0032] H&E staining observations: The liver structure of mice in the blank control group was normal, the hepatocyte morphology was intact, the cell nucleus was clear, the cytoplasm was rich, there was no edema and fatty degeneration, and no obvious inflammatory cell infiltration was observed. The liver structure of the model group was severely abnormal, the cells were swollen, the cytoplasm was vacuolated, and there were a large number of inflammatory cell infiltrations. The administration of recombinant hirudin and vitamin E significantly improved the effect of liver lipid accumulation on liver tissue structure. This shows that recombinant hirudin can reduce liver damage and has a protective effect on the liver;

[0033] Observation of Oil Red O staining of mouse liver: Compared with the blank control group, the lipid droplets in the liver tissue of the model group mice accumulated in granular form. After the administration of recombinant hirudin, the lipid droplet aggregation around the mouse liver decreased in a dose-dependent manner, and the size of the lipid droplets also decreased with the increase of the dose of recombinant hirudin. At the same time, the positive control drug vitamin E also showed a protective effect, significantly inhibiting the aggregation of lipid droplets compared with the model group;

[0034] Observation of F4 / 80 immunofluorescence staining of mouse liver: The fluorescence intensity in the model group was significantly higher than that in the control group, indicating that the liver tissue contained a large number of macrophages; while in the treatment group and the positive control drug group, the fluorescence intensity was significantly reduced, and as the dose of recombinant hirudin increased, the fluorescence intensity weakened accordingly;

[0035] Observation of mouse liver MASSON staining: Compared with the control group, the model group mice had collagen fiber accumulation in the liver tissue, obvious fat vacuoles, and significant liver fibrosis. After treatment with recombinant hirudin, the blue staining area gradually decreased with the increase in the dosage, significantly improving the degree of liver fibrosis;

[0036] Observation of immunofluorescence staining of mouse liver target protein PAR-1: Compared with the control group, the fluorescence intensity of the model group increased significantly, indicating that PAR-1 expression increased. After the administration of recombinant hirudin, the expression of PAR-1 gradually decreased, which was consistent with the expression of vitamin E in the positive control group. This shows that recombinant hirudin can inhibit the expression of PAR-1 in liver tissue;

[0037] The above evaluation results prove from the perspective of histological detection that recombinant hirudin has the effect of inhibiting the aggregation and formation of lipid droplets, the accumulation of collagen fibers in liver tissue, alleviating liver damage, and inhibiting the expression of PAR-1 in liver tissue.

[0038] Evaluation 4: Based on the test plan evaluation in step 11, we can get:

[0039] In the model group, the protein expression of TNF and IL1B was significantly higher than that of the blank control group. After treatment with recombinant hirudin, the protein expression levels of these two inflammatory factors gradually decreased with the increase of the dosage, and the protein expression of the positive control group also decreased. In the model group, the expression of CD36, PPARG, and SREBP1C proteins was significantly higher than that of the control group, and decreased in a dose-dependent manner after the administration of hirudin, which was consistent with the protein expression trend of the positive control group;

[0040] In the model group, the protein expression levels of p-IKKB, p-P65, and p-IKBA were significantly higher than those in the control group, while the expression of IKBA was the opposite. After the administration of recombinant hirudin and VitE, the protein expression levels of p-IKKB, p-P65, and p-IKBA were significantly reduced, while the protein expression level of IKBA was significantly increased.

[0041] In the model group, the protein expression of PAR-1 and PAI-1 was significantly higher than that of the blank control group. After the administration of recombinant hirudin, the expression levels of these proteins gradually decreased with the increase of the dosage, and were consistent with the expression trend of the positive control group. From the perspective of protein expression level detection, it was proved that recombinant hirudin has the effect of inhibiting the expression of TNF, IL1B, and NF-κB inflammatory pathways.

[0042] According to the above evaluation, PAR-1 and PAI-1 were significantly inhibited after treatment with recombinant hirudin, and recombinant hirudin can inhibit the expression of lipid metabolism-related indicators CD36, SREBP1C, and PPARG, and also inhibit the expression of TNF, IL1B, and NF-κB inflammatory pathways. The results once again verified that recombinant hirudin affects liver lipid metabolism and inflammatory pathway expression.

[0043] Furthermore, step one is specifically as follows: synthesize the hirudin improved gene into the gene expression cassette of the yeast gene expression plasmid pMPT-07, use Bgl II linearization treatment to recover the plasmid by gel cutting, discard the original parts required for Escherichia coli, take 1 μg of the recovered product, and transfer it into the uracil auxotrophic Hansen yeast HPU electrocompetent cells by electrotransformation, and use SD-Ura auxotrophic plates to screen genetically engineered bacteria. The correctly screened strains are continuously subcultured to detect their genetic stability. Extract the genome of the genetically stable strain for copy number detection to obtain a high-copy recombinant hirudin yeast strain with a target gene copy number greater than 30;

[0044] Furthermore, the preparation process of the two-stage expansion culture medium in step 2 is as follows:

[0045] Preparation of culture medium:

[0046] Seed culture medium: 20 g / L glucose, 13.4 g / L YNB; 0.3 L of primary seed culture medium, 1.5 L of secondary seed culture medium, high pressure wet heat sterilization.

[0047] Fermentation medium: Fermentation starting volume 12L: NH4H2PO4 192g, MgS04·7H2O 36g, potassium hydrogen phthalate 128g, NaCl 4g, glucose 480g, plus 10mL defoamer, autoclave at 110-115℃ in a tank for 30min.

[0048] Feed solution: 5 L in total: 80 g NH4H2PO4, 15 g MgS04·7H2O, 53 g potassium hydrogen phthalate, 1.7 g NaCl. Dissolve the above raw materials in 1.25 L water and sterilize by high pressure. Take another 3 L 10% glucose solution and sterilize it at 115°C for 30 min.

[0049] Furthermore, the cultivation process of the two-stage expansion culture in step 2 is as follows: take a high-copy recombinant yeast strain seed (1 mL, OD600=100) stored at -196°C and inoculate it into 0.3L of primary seed culture medium, culture it at 35°C for 24h, transfer it to 1.5L of secondary seed culture medium for expansion culture, and culture it at 35°C for 24h until the cell OD600 reaches above 10 to obtain fermentation seeds.

[0050] Furthermore, the fermentation experiment of high-density fermentation in step 3 is as follows: 1.8L of fermentation seeds are placed in a sterilized fermentation tank containing 12L of fermentation medium to start fermentation, and the pH value of the fermentation tank is controlled to be set at 5.4-5.5: the fermentation temperature is controlled at 30°C: the tank pressure is 0.5Kg / cm 2 During the growth phase of the fermentation process, when the dissolved oxygen in the fermenter drops to 60%, connect the feed line and use a peristaltic pump to pump in about 385 mL of feed solution at a uniform speed;

[0051] When the dissolved oxygen level in the fermentation tank drops below 40%, the air flow rate is adjusted to 15 L / min, the stirring speed is adjusted to 700 rpm, the cell OD600 reaches above 80, and this state is maintained until the dissolved oxygen recovery phenomenon is observed. When the dissolved oxygen in the fermentation tank rises from the lowest point to above 60%, the peristaltic pump power switch is turned on, the pump speed is set to be linked to the dissolved oxygen, and the feed solution is started to be added so that the dissolved oxygen value of the fermentation tank fluctuates regularly between 30% and 60%. When the slope of the product peak growth rate curve in HPLC is less than 0.2, the fermentation is terminated to obtain a fermentation liquid containing recombinant hirudin.

[0052] Further, step 4 is specifically as follows: the fermentation broth containing recombinant hirudin obtained in step 3 is subjected to low-speed centrifugation to harvest the fermentation supernatant, and the fermentation supernatant is filtered through a 0.45 μm microporous filter for purification; the recombinant hirudin is purified by cation exchange chromatography and reverse phase chromatography, a chromatography column is filled with SP-550C cation exchange chromatography resin, and the recombinant hirudin is initially separated by the chromatography column, the sample is diluted with water to reduce its conductivity to below 5 ms / cm, and then the pH of the sample is adjusted to 3.0 with phosphoric acid, and the sample is loaded at a loading flow rate of 150 mL / min. After the loading is completed, it is washed with 2 column volumes of loading buffer, and linear gradient elution is performed with 10 column volumes of eluent (1M NaCl prepared with 20 mM pH 3.0 citric acid buffer), and each elution peak component is collected, and the purity of hirudin in each component is detected by HPLC;

[0053] The GC 300sd reverse phase chromatography resin is used to fill the chromatography column, and the recombinant hirudin is purified by the chromatography column. The chromatography column is equilibrated with 1 column volume of Buffer A (0.1% trichloroacetic acid), 1 column volume of Buffer B (0.1% trichloroacetic acid + 40% isopropanol) and 2 column volumes of Buffer A in sequence. After equilibration, the sample with the best SP chromatography is selected for loading at a flow rate of 20 mL / min. After the loading is completed, the chromatography column is linearly eluted from 100% Buffer A to Buffer B at a flow rate of 25 mL / min for 300 minutes, and the components are collected step by step. The components with a purity greater than 99% are collected and combined, and the residual organic reagents in the sample are removed by a vacuum concentrator. The sample concentration is detected by the Lowry method. After sterilization, sorbitol protective solution and diluent are added, and the sample is canned into a vial. After gradient cooling, the sample is freeze-dried, capped, and labeled. The freeze-dried powder is stored at 4-8°C.

[0054] The present invention conducts intervention experiments by setting up six groups of mice, and comprehensively verifies the effects of recombinant hirudin on liver lipid metabolism and inflammatory pathway expression by means of comprehensive nutritional status monitoring, biochemical detection, histological detection, and protein expression level detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 This is a weight change trend diagram of six groups of mice according to an embodiment of the present invention;

[0056] Figure 2 This is a bar graph showing the detection of TG, TC, AST, ALT, SOD, MDA, LDH and GSH indicators in the liver of six groups of mice according to one embodiment of the present invention;

[0057] Figure 3 This is a bar graph showing the detection of serum TG, TC, AST, ALT, SOD, MDA, LDH and GSH indicators in six groups of mice according to one embodiment of the present invention;

[0058] Figure 4 This is a picture of liver specimens of six groups of mice after being treated with recombinant hirudin according to an embodiment of the present invention;

[0059] Figure 5 This is an observation diagram of liver H&E staining of six groups of mice after treatment with recombinant hirudin according to an embodiment of the present invention;

[0060] Figure 6 This is an observation diagram of Oil Red O staining of the liver of six groups of mice after treatment with recombinant hirudin according to an embodiment of the present invention;

[0061] Figure 7 This is an observation diagram of liver F4 / 80 immunofluorescence staining of six groups of mice after treatment with recombinant hirudin according to an embodiment of the present invention;

[0062] Figure 8 This is a MASSON staining observation diagram of the liver of six groups of mice after being treated with recombinant hirudin in one embodiment of the present invention;

[0063] Fig. 9 This is an observation diagram of immunofluorescence staining of the liver target protein PAR-1 in six groups of mice after treatment with recombinant hirudin according to an embodiment of the present invention;

[0064] Fig.10 This is a diagram showing the expression of TNF and IL1B proteins detected by Western blot after six groups of mice were treated with recombinant hirudin according to an embodiment of the present invention;

[0065] Fig.11 This is a diagram showing the expression of CD36, PPARG and SREBP1C proteins detected by Western blot after the six groups of mice were treated with recombinant hirudin according to an embodiment of the present invention;

[0066] Fig.12 This is a diagram showing the expression of NF-κB validation pathway proteins detected by Western blot after six groups of mice were treated with recombinant hirudin according to an embodiment of the present invention;

[0068] Fig.13 This is a diagram showing the expression of PAR-1 and PAI-1 proteins detected by Western blot after six groups of mice were treated with recombinant hirudin according to an embodiment of the present invention. DETAILED DESCRIPTION

[0069] The present invention is implemented by the following technical scheme: comprising a method for preparing a drug for treating non-alcoholic fatty liver disease using recombinant hirudin, specifically comprising the following steps:

[0070] S1. Preparation of recombinant hirudin, specifically comprising the following steps:

[0071] Step 1. Construction of high-copy recombinant hirudin yeast strain;

[0072] Step 2. The high-copy recombinant hirudin yeast strain obtained in step 1 is subjected to two-stage expansion culture to obtain a fermentation seed liquid;

[0073] Step 3. The fermented seed solution obtained in step 2 was inoculated into a 30L fermenter for high-density fermentation to obtain a fermentation solution containing recombinant hirudin;

[0074] Step 4. After the fermentation broth is centrifuged and filtered, it is purified by two steps of chromatography to obtain recombinant hirudin with a purity greater than 99%, and the recombinant hirudin lyophilized powder is obtained by the steps of detection, sterilization, dilution, filling, freeze-drying, capping, labeling, etc.;

[0075] S2 experimental process, specifically the following steps:

[0076] Step 5. Redissolving the recombinant hirudin lyophilized powder in physiological saline to obtain a drug solution;

[0077] Step 6: Grouping: Male 8-week-old C57BL / 6J mice were adaptively fed for one week and randomly divided into six groups: blank control group; model group; low-dose recombinant hirudin injection treatment group; medium-dose recombinant hirudin injection treatment group; high-dose recombinant hirudin injection treatment group; positive control group, 10 mice in each group, a total of 60 mice.

[0078] Step 7: Group intervention treatment:

[0079] Blank control group: fed with normal diet for 12 weeks;

[0080] Model group: fed with a high-fat diet for 12 weeks;

[0081] The low-dose recombinant hirudin injection treatment group: the mice were fed a high-fat diet for 12 weeks and subcutaneously injected with a low-dose (0.3 mg / kg) recombinant hirudin injection;

[0082] The medium-dose recombinant hirudin injection treatment group: the mice were fed a high-fat diet for 12 weeks and subcutaneously injected with a medium-dose (0.6 mg / kg) recombinant hirudin injection;

[0083] The high-dose recombinant hirudin injection treatment group: the mice were fed a high-fat diet for 12 weeks and subcutaneously injected with a high-dose (1.2 mg / kg) recombinant hirudin injection;

[0084] Positive control group: fed with a high-fat diet and given vitamin E by oral gavage at a dose of 100 mg / kg for 12 consecutive weeks;

[0085] S3 drug efficacy evaluation scheme, specifically the following steps:

[0086] Step 8 Comprehensive nutritional status: Method: Monitor the weight changes of mice in each group;

[0087] Step 9 Biochemical test: Method: TG, TC, ALT, AST, MDA, LDH, SOD, GSH concentrations in liver and serum;

[0088] Step 10 Histological examination: Methods: Observe liver tissue; H&E staining; Oil Red O staining; F4 / 80 immunofluorescence staining; MASSON staining; Target protein PAR-1 immunofluorescence staining;

[0089] Step 11: Detection of protein expression level: Method: Western blot;

[0090] S4 test result evaluation, including the following points:

[0091] Evaluation 1: According to the test plan evaluation in step 8, we can get: Figure 1 As shown in the figure, the weight change curve of mice shows that compared with the control group, the weight of mice in the model group increased significantly after being fed a high-fat diet. After being treated with recombinant hirudin and vitamin E, the weight of mice gradually decreased. This evaluation result proves that recombinant hirudin can affect lipid metabolism from the perspective of nutritional status.

[0092] Evaluation 2: According to the test plan evaluation in step 9, we can get: Figure 2 As shown in the results, the detection of liver TG, TC, AST, ALT, SOD, MDA, LDH, and GSH indicators showed that TG and TC are indicators related to blood lipid levels. The higher the blood lipid content, the easier it is to form fatty liver. Compared with the control group, the TG and TC contents in the model group were significantly increased. After the administration of recombinant hirudin and vitamin E, the TG and TC contents were significantly decreased compared with the model group. ALT and AST are related indicators of liver cell damage. Compared with the control group, the ALT and AST contents in the model group were significantly increased. After the administration of recombinant hirudin and vitamin E, their contents were significantly decreased. Abnormal oxidative stress indicators are an early phenomenon of liver damage. Compared with the control group, the contents of MDA, LDH, and GSH in the model group were significantly increased, and the content of SOD was significantly decreased. After the administration of recombinant hirudin and vitamin E, the contents of MDA, LDH, and GSH gradually decreased, and the content of SOD gradually increased.

[0093] like Figure 3As shown in the results, the serum TG, TC, AST, ALT, SOD, MDA, LDH, and GSH indicators were tested. As shown, the changes in serum biochemical indicators of NAFLD are related to liver function indicators. Abnormal liver function will lead to liver damage, increased blood lipid levels, and oxidative stress, which will lead to increased serum ALT, AST, TG, TC, LDH, MDA, and GSH levels, and decreased SOD content. Compared with the control group, the ALT, AST, TG, TC, LDH, MDA, and GSH levels in the model group increased significantly, and the SOD content decreased significantly. In addition, the improvement of liver abnormalities by recombinant hirudin is dose-dependent. The above evaluation results prove from the perspective of biochemical detection that recombinant hirudin can increase lipid metabolism and protect the liver;

[0094] Evaluation 3: Based on the test plan evaluation in step 10, we can get:

[0095] Observe the liver tissue with naked eyes: Figure 4 As shown, the liver of mice in the blank control group was observed with naked eyes. The normal liver was tough, with sharp edges, smooth surface and dark red color, while the livers of the other groups were light in color, whitish and with blunt edges. The model group was the most obvious, with a yellowish-white color. The color of the liver after treatment with recombinant hirudin and vitamin E was between the control group and the model group, and with the increase of the dosage, the color and state of the liver gradually recovered, indicating that recombinant hirudin can accelerate liver lipid metabolism and fill the liver blood vessels;

[0096] H&E staining observation: Figure 5 As shown, the liver structure of mice in the blank control group was normal, the hepatocyte morphology was intact, the cell nucleus was clear, the cytoplasm was rich, there was no edema and fatty degeneration, and no obvious inflammatory cell infiltration was observed. The liver structure of the model group was severely abnormal, the cells were swollen, the cytoplasm was vacuolated, and there were a large number of inflammatory cells infiltrating. When recombinant hirudin and vitamin E were given, the effect of liver lipid accumulation on liver tissue structure was significantly improved. This shows that recombinant hirudin can reduce liver damage and has a protective effect on the liver;

[0097] Observation of Oil Red O staining of mouse liver: Figure 6 As shown in the figure, compared with the blank control group, the lipid droplets in the liver tissue of the model group mice accumulated in granular form. After the administration of recombinant hirudin, the lipid droplet aggregation around the liver of the mice decreased in a dose-dependent manner, and the size of the lipid droplets also decreased with the increase of the dose of recombinant hirudin. At the same time, the positive control drug vitamin E also showed a protective effect, significantly inhibiting the aggregation and formation of lipid droplets compared with the model group;

[0098] Observation of F4 / 80 immunofluorescence staining of mouse liver: Figure 7As shown, the fluorescence intensity in the model group was significantly higher than that in the control group, indicating that the liver tissue contained a large number of macrophages; while in the treatment group and the positive control drug group, the fluorescence intensity was significantly reduced, and as the dose of recombinant hirudin increased, the fluorescence intensity weakened accordingly;

[0099] Observation of mouse liver MASSON staining: Figure 8 As shown in the figure, compared with the control group, the liver tissue of the model group mice had collagen fiber accumulation, obvious fat vacuoles, and significant liver fibrosis. After treatment with recombinant hirudin, the blue-stained area gradually decreased with the increase in the dosage, significantly improving the degree of liver fibrosis;

[0100] Observation of mouse liver target protein PAR-1 immunofluorescence staining: Fig. 9 As shown, compared with the control group, the fluorescence intensity of the model group increased significantly, indicating that the expression of PAR-1 increased. After the administration of recombinant hirudin, the expression of PAR-1 gradually decreased, which was consistent with the expression of vitamin E in the positive control group. This shows that recombinant hirudin can inhibit the expression of PAR-1 in liver tissue;

[0101] The above evaluation results prove from the perspective of histological detection that recombinant hirudin has the effect of inhibiting the aggregation and formation of lipid droplets, the accumulation of collagen fibers in liver tissue, alleviating liver damage, and inhibiting the expression of PAR-1 in liver tissue.

[0102] Evaluation 4: According to the test plan evaluation of step 11, we can get: Fig.10 As shown in the figure, the protein expression of TNF and IL1B detected by Western blot in six groups of mice after treatment with recombinant hirudin was summarized as follows: in the model group, the protein expression of TNF and IL1B was significantly higher than that of the blank control group. After treatment with recombinant hirudin, the protein expression levels of these two inflammatory factors gradually decreased with the increase of the dosage, and the protein expression level of the positive control group also decreased;

[0103] like Fig.11 As shown, the protein expressions of CD36, PPARG, and SREBP1C detected by Western blot in the six groups of mice after treatment with recombinant hirudin were summarized as follows: in the model group, the expression of CD36, PPARG, and SREBP1C proteins was significantly higher than that in the control group, and decreased in a dose-dependent manner after administration of hirudin, which was consistent with the protein expression trend of the positive control group;

[0104] like Fig.12As shown in the figure, the protein expression of NF-κB validation pathway detected by Western blot in six groups of mice after treatment with recombinant hirudin was summarized as follows: In the model group, the protein expression levels of p-IKKB, p-P65, and p-IKBA were significantly higher than those in the control group, while the expression of IKBA was the opposite. After administration of recombinant hirudin and VitE, the protein expression levels of p-IKKB, p-P65, and p-IKBA were significantly reduced, and the protein expression level of IKBA was significantly increased;

[0105] like Fig.13 As shown in the figure, the protein expression of PAR-1 and PAI-1 in six groups of mice after treatment with recombinant hirudin was detected by Western blot. In the model group, the protein expression of PAR-1 and PAI-1 was significantly higher than that of the blank control group. After the administration of recombinant hirudin, the expression levels of these proteins gradually decreased with the increase of the dosage, and were consistent with the expression trend of the positive control group. From the perspective of protein expression level detection, it was proved that recombinant hirudin has the effect of inhibiting the expression of TNF, IL1B, and NF-κB inflammatory pathways.

[0106] According to the above evaluation, PAR-1 and PAI-1 were significantly inhibited after treatment with recombinant hirudin, and recombinant hirudin can inhibit the expression of lipid metabolism-related indicators CD36, SREBP1C, and PPARG, and also inhibit the expression of TNF, IL1B, and NF-κB inflammatory pathways. The results once again verified that recombinant hirudin affects liver lipid metabolism and inflammatory pathway expression.

[0107] In this embodiment, step one is specifically as follows: synthesize the hirudin improved gene into the gene expression cassette of the yeast gene expression plasmid pMPT-07, use Bgl II linearization treatment to recover the plasmid by gel cutting, discard the original parts required for Escherichia coli, take 1 μg of the recovered product, transfer it into uracil auxotrophic Hansen yeast HPU electrocompetent cells by electrotransformation, and use SD-Ura auxotrophic plates to screen genetically engineered bacteria. The correctly screened strain is continuously subcultured to detect its genetic stability. The genome of the genetically stable strain is extracted for copy number detection to obtain a high-copy recombinant hirudin yeast strain with a target gene copy number greater than 30;

[0108] In this embodiment, the preparation process of the two-stage expansion culture medium in step 2 is as follows:

[0109] Preparation of culture medium:

[0110] Seed culture medium: 20 g / L glucose, 13.4 g / L YNB; 0.3 L of primary seed culture medium, 1.5 L of secondary seed culture medium, high pressure wet heat sterilization.

[0111] Fermentation medium: Fermentation starting volume 12L: NH4H2PO4 192g, MgS04·7H2O 36g, potassium hydrogen phthalate 128g, NaCl 4g, glucose 480g, plus 10mL defoamer, autoclave at 110-115℃ in a tank for 30min.

[0112] Feed solution: 5 L in total: 80 g NH4H2PO4, 15 g MgS04·7H2O, 53 g potassium hydrogen phthalate, 1.7 g NaCl. Dissolve the above raw materials in 1.25 L water and sterilize by high pressure. Take another 3 L 10% glucose solution and sterilize it at 115°C for 30 min.

[0113] In this embodiment, the cultivation process of the two-stage expansion culture in step 2 is as follows: take a high-copy recombinant yeast strain seed (1 mL, OD600=100) stored at -196°C and inoculate it into 0.3L of primary seed culture medium, culture it at 35°C for 24h, transfer it to 1.5L of secondary seed culture medium for expansion culture, and culture it at 35°C for 24h until the cell OD600 reaches above 10 to obtain fermentation seeds.

[0114] In this embodiment, the fermentation experiment of high-density fermentation in step 3 is specifically as follows: 1.8L fermentation seeds are placed in a sterilized fermentation tank containing 12L fermentation medium to start fermentation, and the pH value of the fermentation tank is controlled to be set at 5.4-5.5: the fermentation temperature is controlled at 30°C: the tank pressure is 0.5Kg / cm 2 .

[0115] During the growth phase of the fermentation process, when the dissolved oxygen in the fermenter drops to 60%, connect the feed line and use a peristaltic pump to pump in about 385 mL of feed solution at a constant speed.

[0116] When the dissolved oxygen level in the fermentation tank drops below 40%, the air flow rate is adjusted to 15 L / min, the stirring speed is adjusted to 700 rpm, the cell OD600 reaches above 80, and this state is maintained until the dissolved oxygen recovery phenomenon is observed. When the dissolved oxygen in the fermentation tank rises from the lowest point to above 60%, the peristaltic pump power switch is turned on, the pump speed is set to be linked to the dissolved oxygen, and the feed solution is started to be added so that the dissolved oxygen value of the fermentation tank fluctuates regularly between 30% and 60%. When the slope of the product peak growth rate curve in HPLC is less than 0.2, the fermentation is terminated to obtain a fermentation liquid containing recombinant hirudin.

[0117] In this embodiment, step 4 is specifically as follows: the fermentation broth containing recombinant hirudin obtained in step 3 is subjected to low-speed centrifugation to harvest the fermentation supernatant, and the fermentation supernatant is filtered through a 0.45 μm microporous filter for purification; the recombinant hirudin is purified by cation exchange chromatography and reverse phase chromatography, a chromatography column is filled with SP-550C cation exchange chromatography resin, and the recombinant hirudin is initially separated by the chromatography column, the sample is diluted with water to reduce its conductivity to below 5 ms / cm, and then the pH of the sample is adjusted to 3.0 with phosphoric acid, and the sample is loaded at a loading flow rate of 150 mL / min. After the loading is completed, it is washed with 2 column volumes of loading buffer, and linear gradient elution is performed with 10 column volumes of eluent (1 M NaCl prepared with 20 mM pH 3.0 citric acid buffer), and each elution peak component is collected, and the purity of hirudin in each component is detected by HPLC;

[0118] The GC 300sd reverse phase chromatography resin is used to fill the chromatography column, and the recombinant hirudin is purified by the chromatography column. The chromatography column is equilibrated with 1 column volume of Buffer A (0.1% trichloroacetic acid), 1 column volume of Buffer B (0.1% trichloroacetic acid + 40% isopropanol) and 2 column volumes of Buffer A in sequence. After equilibration, the sample with the best SP chromatography is selected for loading at a flow rate of 20 mL / min. After the loading is completed, the chromatography column is linearly eluted from 100% Buffer A to Buffer B at a flow rate of 25 mL / min for 300 minutes, and the components are collected step by step. The components with a purity greater than 99% are collected and combined, and the residual organic reagents in the sample are removed by a vacuum concentrator. The sample concentration is detected by the Lowry method. After sterilization, sorbitol protective solution and diluent are added, and the sample is canned into a vial. After gradient cooling, the sample is freeze-dried, capped, and labeled. The freeze-dried powder is stored at 4-8°C.

[0119] The present invention conducts intervention experiments by setting up six groups of mice, and comprehensively verifies the effects of recombinant hirudin on liver lipid metabolism and inflammatory pathway expression by means of comprehensive nutritional status monitoring, biochemical detection, histological detection, and protein expression level detection.

Claims

1. A method for preparing a drug for treating non-alcoholic fatty liver disease using recombinant hirudin, characterized in that: The specific steps include: S1. Preparation of recombinant hirudin, specifically comprising the following steps: Step 1. Construction of high-copy recombinant hirudin yeast strain; Step 2. The high-copy recombinant hirudin yeast strain obtained in step 1 is subjected to two-stage expansion culture to obtain a fermentation seed liquid; Step 3. The fermented seed solution obtained in step 2 was inoculated into a 30L fermenter for high-density fermentation to obtain a fermentation solution containing recombinant hirudin: Step 4. After the fermentation broth is centrifuged and filtered, it is purified by two steps of chromatography to obtain recombinant hirudin with a purity greater than 99%, and the recombinant hirudin lyophilized powder is obtained by the steps of detection, sterilization, dilution, filling, freeze-drying, capping, labeling, etc.; S2 experimental process, specifically the following steps: Step 5. Redissolving the recombinant hirudin lyophilized powder in physiological saline to obtain a drug solution; Step 6: Grouping: Adaptive feeding of male C57BL / 6J mice, setting up blank control group; model group; low-dose treatment group of recombinant hirudin injection; medium-dose treatment group of recombinant hirudin injection; high-dose treatment group of recombinant hirudin injection; positive control group; Step 7: Group intervention treatment: Blank control group: fed with normal diet; Model group: fed with a high-fat diet; The low-dose recombinant hirudin injection treatment group was fed with a high-fat diet and subcutaneously injected with a low-dose recombinant hirudin injection; The medium-dose recombinant hirudin injection treatment group was fed with a high-fat diet and subcutaneously injected with a medium-dose recombinant hirudin injection; The high-dose recombinant hirudin injection treatment group: the mice were fed with a high-fat diet and subcutaneously injected with a high-dose recombinant hirudin injection; Positive control group: fed with a high-fat diet and given vitamin E by oral gavage; S3 drug efficacy evaluation scheme, specifically the following steps: Step 8. Comprehensive nutritional status: Method: Monitor the weight changes of mice in each group; Step 9. Biochemical detection: Method: TG, TC, ALT, AST, MDA, LDH, SOD, GSH concentrations in liver and serum; Step 10. Histological examination: Methods: Observe the liver tissue status; H&E staining; Oil Red O staining; F4 / 80 immunofluorescence staining; MASSON staining; Target protein PAR-1 immunofluorescence staining; Step 11. Detection of protein expression level: Method: Western blot; S4 efficacy results evaluation, including the following points: Evaluation 1: Evaluate the comprehensive nutritional status according to the efficacy evaluation scheme in step 8 to obtain the optimal group; Evaluation 2: Evaluate the biochemical test results according to the efficacy evaluation scheme in step 9 to obtain the optimal group; Evaluation 3: Evaluate the histological test results according to the efficacy evaluation scheme in step 10, as follows: Observe liver tissue: The optimal group was obtained by observing the liver tissue status of mice; H&E staining observation: The optimal group was obtained by evaluating the mouse liver tissue structure and cell morphology based on the H&E staining observation; Observation of Oil Red O staining of mouse liver: The optimal group was obtained by evaluating the aggregation of lipid droplets in mouse liver tissue based on the observation of Oil Red O staining of mouse liver; Observation of F4 / 80 immunofluorescence staining of mouse liver: The optimal group was obtained by evaluating the fluorescence intensity based on the observation of F4 / 80 immunofluorescence staining of mouse liver; Observation of MASSON staining of mouse liver: The optimal group was obtained by evaluating the liver fibrosis of mice based on the observation of MASSON staining of mouse liver; Observation of immunofluorescence staining of PAR-1 target protein in mouse liver: The expression of PAR-1 in liver tissue was evaluated based on the observation of immunofluorescence staining of PAR-1 target protein in mouse liver to obtain the optimal group; Evaluation 4: According to the detection scheme of step 11, the expression of lipid metabolism-related indicators CD36, SREBP1C, PPARG and TNF, IL1B, NF-κB inflammatory pathways were evaluated to obtain the optimal group.

2. The method for preparing a drug for treating non-alcoholic fatty liver disease using recombinant hirudin according to claim 1, characterized in that: Step 1 is as follows: synthesizing the hirudin modified gene into the gene expression cassette of the yeast gene expression plasmid pMPT-07, linearizing the plasmid with BglII, recovering the plasmid by gel cutting, discarding the original parts required by Escherichia coli, taking 1 μg of the recovered product, transferring it into uracil auxotrophic Hansenula HPU electrocompetent cells by electrotransformation, screening genetically engineered bacteria using SD-Ura auxotrophic plates, and continuously subculturing the correctly screened strains to detect their genetic stability; The genome of the genetically stable strain was extracted for copy number detection to obtain a high-copy recombinant hirudin yeast strain with a target gene copy number greater than 30.

3. The method for preparing a drug for treating non-alcoholic fatty liver disease using recombinant hirudin according to claim 1, characterized in that: The preparation process of the two-stage expansion culture medium in step 2 is as follows: Preparation of culture medium: Seed culture medium: 20g / L glucose, 13.4g / L YNB; 0.3L primary seed culture medium, 1.5L secondary seed culture medium, high pressure wet heat sterilization; Fermentation medium: Fermentation starting volume 12L: NH4H2PO4 192g, MgS04·7H2O 36g, potassium hydrogen phthalate 128g, NaCl 4g, glucose 480g, plus 10mL defoamer, autoclave at 110-115℃ for 30min in a tank; Feed solution: 5 L in total: 80 g NH4H2PO4, 15 g MgS04·7H2O, 53 g potassium hydrogen phthalate, 1.7 g NaCl. Dissolve the above raw materials in 1.25 L water and sterilize by high pressure. Take another 3 L 10% glucose solution and sterilize it at 115°C for 30 min.

4. The method for preparing a drug for treating non-alcoholic fatty liver disease using recombinant hirudin according to claim 1, characterized in that: The cultivation process of the two-stage expansion culture in step 2 is as follows: take a high-copy recombinant hirudin yeast strain seed (1 mL, OD600=100) stored at -196°C and inoculate it into 0.3L of primary seed culture medium, culture it at 35°C for 24h, transfer it to 1.5L of secondary seed culture medium for expansion culture, and culture it at 35°C for 24h until the cell OD600 reaches above 10 to obtain fermentation seeds.

5. The method for preparing a drug for treating non-alcoholic fatty liver disease using recombinant hirudin according to claim 1, characterized in that: The specific fermentation experiment of high-density fermentation in step 3 is as follows: 1.8L fermentation seeds are placed in a sterilized fermentation tank containing 12L fermentation medium to start fermentation. The pH value of the fermentation tank is controlled at 5.4-5.5: the fermentation temperature is controlled at 30℃: the tank pressure is 0.5Kg / cm 2 ; During the growth phase of the fermentation process, when the dissolved oxygen in the fermenter drops to 60%, connect the feed line and use a peristaltic pump to pump in about 385 mL of feed solution at a constant speed. When the dissolved oxygen level in the fermentation tank drops below 40%, the air flow rate is adjusted to 15 L / min, the stirring speed is adjusted to 700 rpm, the cell OD600 reaches above 80, and this state is maintained until the dissolved oxygen recovery phenomenon is observed. When the dissolved oxygen in the fermentation tank rises from the lowest point to above 60%, the peristaltic pump power switch is turned on, the pump speed is set to be linked to the dissolved oxygen, and the feed solution is started to be added so that the dissolved oxygen value of the fermentation tank fluctuates regularly between 30% and 60%. When the slope of the product peak growth rate curve in HPLC is less than 0.2, the fermentation is terminated to obtain a fermentation liquid containing recombinant hirudin.

6. The method for preparing a drug for treating non-alcoholic fatty liver disease using recombinant hirudin according to claim 1, characterized in that: Step 4 specifically comprises: subjecting the fermentation liquid containing the recombinant hirudin obtained in step 3 to low-speed centrifugation to harvest the fermentation supernatant, filtering the fermentation supernatant through a 0.45 μm microporous filter for purification; Purification of recombinant hirudin by cation exchange chromatography and reverse phase chromatography: using SP-550C cation exchange chromatography resin to fill the chromatography column, primary separation of recombinant hirudin by chromatography column, diluting the sample with water to reduce its conductivity to below 5ms / cm, adjusting the pH of the sample to 3.0 with phosphoric acid, loading, the loading flow rate is 150mL / min, after loading, washing with 2 column volumes of loading buffer, linear gradient elution with 10 column volumes of eluent (1M NaCl prepared with 20mM pH 3.0 citric acid buffer), collecting each elution peak fraction, and detecting the purity of hirudin in each fraction by HPLC; using GC 300sd reverse phase chromatography resin to fill the chromatography column, purifying recombinant hirudin by chromatography column, using 1 column volume of Buffer A (0.1% trichloroacetic acid), 1 column volume of Buffer B (0.1% trichloroacetic acid + 40% isopropanol) and 2 column volumes of Buffer A equilibrated chromatography column, after equilibration, the sample with the best SP chromatography was selected for loading at a flow rate of 20 mL / min. After loading, the chromatography column was linearly eluted from 100% Buffer A to Buffer B at a flow rate of 25 mL / min for 300 min, the components were collected step by step, the purity of hirudin in each component was detected by HPLC, the components with a purity greater than 99% were collected and combined, the residual organic reagent in the sample was removed by a vacuum concentrator, the sample concentration was detected by the Lowry method, sorbitol protective solution and diluent were added after sterilization, the sample was canned into a vial, freeze-dried, capped and labeled after gradient cooling, and the freeze-dried powder was stored at 4-8°C.

7. The method for preparing a drug for treating non-alcoholic fatty liver disease using recombinant hirudin according to claim 1, characterized in that: In step seven, the feeding time and feeding frequency of the low-dose recombinant hirudin injection treatment group, the medium-dose recombinant hirudin injection treatment group, and the high-dose recombinant hirudin injection treatment group were the same.

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