Application of the combined drug of mecobalamin and Shuilinjia in the treatment of non-alcoholic steatohepatitis

Abstract

The present invention discloses the application of the combined drug of mecobalamin (MeCbl) and Shuilinjia (SC) in the treatment of non-alcoholic steatohepatitis (NASH). The present invention firstly proposes that oral MeCbl can effectively intervene in and treat the progression of NASH disease, and firstly proposes that the combined drug of oral MeCbl and SC can synergistically enhance the treatment of NASH, as well as liver injury, inflammation, liver fibrosis and hepatic lipid deposition on the basis of monomeric drugs. The MeCbl / SC composition in the present invention has a potent effect on the treatment of NASH, provides a new therapeutic drug for such refractory liver diseases clinically, while reducing the single-drug use dose of SC, and reducing the incidence of adverse reactions and treatment costs of patients. The pharmacokinetics and safety data of the drug molecules involved in the present invention are relatively detailed, and the development of new indications can quickly enter clinical evaluation, saving development costs while shortening the R & D cycle.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to the application of a combined drug of mecobalamin and Shuilinjia in the treatment of non-alcoholic steatohepatitis. Background Art

[0002] Non-alcoholic steatohepatitis (NASH) is hepatic steatosis with both hepatocyte injury and inflammatory response. 35%-50% of NASH patients will progress to cirrhosis and hepatocellular carcinoma, which is one of the main causes of end-stage liver disease and liver transplantation. Clinically, there is a lack of causative treatment drugs (Pathogenesis and treatment of non-alcoholic steatohepatitis and its fibrosis. Clin Mol Hepatol, 2023, 29:77-98.). Currently, the research on NASH treatment drugs mainly focuses on multiple potential targets such as metabolism, inflammation, and fibrosis, including nuclear receptor agonists, PPAR agonists, chemokine receptor inhibitors, thyroid hormone receptor-β agonists, and GLP-1, FGF21, SGLT2 inhibitors, etc. (Novel therapeutic targets for cholestatic and fatty liver disease. Gut, 2022, 71:194-209. Efficacy of peroxisome proliferator-activated receptor agonists, glucagon-like peptide-1 receptor agonists, or sodium-glucose cotransporter-2 inhibitors for treatment of non-alcoholic fatty liver disease: a systematic review. Lancet Gastroenterol Hepatol, 2022, 7:367-378. FGF21 protects against hepatic lipotoxicity and macrophage activation to attenuate fibrogenesis in nonalcoholic steatohepatitis. Elife, 2023, 12). However, phase III clinical studies have shown that only the FXR agonist obeticholic acid has certain therapeutic effects, but the risk of drug use in NASH patients with liver fibrosis grades 2-3 is too high, so the FDA has refused to use it for NASH clinical indications.Clinical trial progress shows that new drug trials for NASH have repeatedly failed. The main reason is that the pathogenesis of NASH is complex, and drugs targeting a single target have problems such as insufficient efficacy and even obvious hepatotoxic side effects. Therefore, drug combinations targeting multiple NASH disease targets may be a potential solution for treating NASH.

[0003] Shuilinjia (SC) is a compound preparation composed of silybin and lecithin, which has the effects of anti-lipid peroxidation, stabilizing and repairing damaged cell membranes. As the main active ingredient of Shuilinjia, silybin can prevent liver damage caused by toxic substances and drugs, etc., and promote the regeneration and repair of hepatocytes. It is known as a "natural liver protectant". Lecithin is an amphiphilic molecule that can increase the liposolubility of silybin and accelerate the transport of silybin to the liver. After the two are made into a preparation, the in vivo absorption and bioavailability of silybin are significantly improved (Silibinin Capsules improves high fat diet-induced nonalcoholic fatty liver disease in hamsters through modifying hepatic de novo lipogenesis and fatty acid oxidation. J Ethnopharmacol, 2017, 208: 24-35), but there are still a series of problems such as gastrointestinal side effects caused by too high clinical drug doses and insignificant efficacy at low doses (A Randomized Trial of Silymarin for the Treatment of Nonalcoholic Steatohepatitis. Clin Gastroenterol Hepatol, 2017, 15(12): 1940-1949.e8. Silymarin in non-cirrhotics with non-alcoholic steatohepatitis: A randomized, double-blind, placebo controlled trial, PLoS One, 2019, 14(9): e0221683). Mecobalamin (MeCbl) is an endogenous vitamin B12, which is currently mainly used to treat peripheral nervous system diseases, including treating trigeminal neuralgia, facial spasm and polyneuritis, etc., and has now been made into tablets and injections and widely used in clinical. However, there is currently no relevant report on the combination of mecobalamin and Shuilinjia in the intervention of NASH. Summary of the Invention

[0004] Objective of the Invention: Aiming at the problems existing in the prior art, the present invention provides the use of mecobalamin (MeCbl) or the combination of mecobalamin (MeCbl) and Shuilingjia (SC) in the preparation of a drug for the treatment of non-alcoholic steatohepatitis (NASH) and its evolving diseases. This combination drug can exert a significant synergistic effect in the treatment of NASH based on the single drugs of Shuilingjia and mecobalamin. At the same time, the combination drug can effectively reduce the dosage of Shuilingjia, reduce the treatment cost of NASH and the incidence of adverse reactions, and provide a new combination drug for the treatment of NASH.

[0005] The present invention also provides a pharmaceutical composition for the treatment of non-alcoholic steatohepatitis and its evolving diseases, and the use of the combination drug in the preparation of drugs for the treatment of various acute and chronic tissue and organ injuries or failures.

[0006] Technical Solution: To achieve the above objective, the present invention provides the use of mecobalamin or the combination of mecobalamin and Shuilingjia in the preparation of a drug for the treatment of non-alcoholic steatohepatitis and its evolving diseases.

[0007] Among them, the chemical structural formula of silybin, the main active ingredient of Shuilingjia, is shown as Formula I below, and the chemical structural formula of mecobalamin is shown as Formula II below:

[0008]

[0009] Among them, the use of mecobalamin or the combination of mecobalamin and Shuilingjia in the preparation of a drug for the treatment of non-alcoholic steatohepatitis and its evolving diseases is achieved by including reducing liver injury, inflammation, liver fibrosis and liver lipid deposition.

[0010] Among them, the evolving diseases include acute liver failure, acute-on-chronic liver failure, chronic liver failure and liver failure complicated with infection.

[0011] Furthermore, in addition to NASH, the diseases also include liver failure caused by the end stage of NASH and multiple organ failures caused by complication, including renal failure, lung failure and heart failure.

[0012] Among them, mecobalamin alone can treat non-alcoholic steatohepatitis, including reducing liver injury, inflammation, liver fibrosis and liver lipid deposition; compared with mecobalamin used alone, the combination drug of mecobalamin and Shuilingjia can synergistically and potently intervene in liver injury, inflammation, liver fibrosis and liver lipid deposition of non-alcoholic steatohepatitis.

[0013] Among them, the mass composition ratio range of mecobalamin and Shuilingjia is (1 - 10):50.

[0014] Preferably, the mass composition ratio range of mecobalamin and Shuilingjia is (1 - 3):50.

[0015] Furthermore, the mass composition ratio range of mecobalamin and Shuilinjia is 1:50; the optimal mass ratio of mecobalamin and Shuilinjia in the pharmaceutical composition for oral intervention of NASH is 1:50.

[0016] The pharmaceutical composition for treating non-alcoholic steatohepatitis and its evolving diseases according to the present invention comprises mecobalamin and Shuilinjia as active ingredients, and a pharmaceutically acceptable carrier.

[0017] Among them, the pharmaceutical composition comprises mecobalamin and Shuilinjia with a mass ratio of (1-10):50.

[0018] Preferably, the pharmaceutical composition comprises mecobalamin and Shuilinjia with a mass ratio of (1-3):50.

[0019] Among them, the composition comprises various pharmaceutical preparations prepared from the composition, specifically including capsules, powders, tablets, granules, pills, injections, syrups, oral liquids, inhalants, ointments, suppositories or patches.

[0020] Among them, a quick-release and sustained-release bilayer tablet is required for drug release. After the mecobalamin and Shuilinjia pharmaceutical composition is orally administered into the body in the dosage form, Shuilinjia is instantaneously released, and mecobalamin is released with a delay of 6-8 hours, producing a significant synergistic effect.

[0021] Furthermore, the research of the present invention shows that there is no obvious synergistic therapeutic effect when mecobalamin and Shuilinjia are administered simultaneously in combination. When Shuilinjia is administered first and then mecobalamin is administered after an interval of 6-8 hours, a significant synergistic effect will be produced.

[0022] Furthermore, the present invention provides a dosage form of a pharmaceutical composition of mecobalamin and Shuilinjia. First, Shuilinjia is administered, and then mecobalamin is administered after an interval of 6-8 hours. The pharmaceutical composition comprises mecobalamin and Shuilinjia with a mass ratio of (1-3):50; or the pharmaceutical composition of mecobalamin and Shuilinjia is made into a sustained-release preparation to ensure that after entering the body, Shuilinjia is instantaneously released and mecobalamin is released with a delay of 6-8 hours.

[0023] Preferably, the application of the combination of mecobalamin and Shuilinjia in the preparation of a drug for treating non-alcoholic steatohepatitis and its evolving diseases, wherein after the drug enters the body, Shuilinjia is instantaneously released, mecobalamin is released with a delay of 6-8 hours, and at the same time, the mass ratio of mecobalamin and Shuilinjia is (1-3):50.

[0024] Furthermore, the pharmaceutical composition of mecobalamin and Shuilinjia further comprises pharmaceutically acceptable derivatives, including but not limited to: pharmaceutically acceptable prodrugs, salts, ester salts, or any other derivatives that can be directly or indirectly administered according to the needs of animals.

[0025] The present invention proposes a new NASH drug treatment strategy, namely the application of Silibinin Soft Capsules combined with Mecobalamin, which can be used to treat NASH, belonging to the new use of old drugs. The active ingredient Mecobalamin in the combined drug can treat NASH alone by oral administration, including reducing liver injury, inflammation, liver fibrosis and hepatic lipid deposition. After combination with Silibinin Soft Capsules and administered orally, compared with the use of Mecobalamin or Silibinin Soft Capsules alone, it can further synergistically enhance the effect and strongly intervene in NASH. The pharmacokinetics and safety data of the drug molecules involved in the present invention are relatively detailed, and it is expected to develop a new therapeutic drug for NASH and liver failure and tissue and organ failure caused by NASH while shortening the R & D cycle.

[0026] The application of Mecobalamin (MeCbl) and the pharmaceutical composition of MeCbl and Silibinin Soft Capsules (SC) in the preparation of drugs for the treatment of non-alcoholic steatohepatitis (NASH). In the NASH model induced by methionine- and choline-deficient high-fat diet (MCDHFD), it is first proposed that oral administration of MeCbl can effectively intervene in the progression of NASH, and it is first proposed that the combined drug of oral MeCbl and SC can synergistically enhance the treatment of liver injury, inflammation, liver fibrosis and hepatic lipid deposition on the basis of monomer drugs. The MeCbl / SC composition in the present invention has a strong effect on the treatment of NASH, providing a new therapeutic drug for such refractory liver diseases clinically.

[0027] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0028] The present invention first discloses a combined drug of Mecobalamin and Silibinin Soft Capsules and its medical use, and first discloses that oral administration of Mecobalamin alone and the combined administration of Mecobalamin and Silibinin Soft Capsules can intervene in liver injury, inflammation, liver fibrosis and hepatic lipid deposition in NASH, playing a role in treating NASH. After forming a combined drug, compared with the single administration of Silibinin Soft Capsules and Mecobalamin, Mecobalamin and Silibinin Soft Capsules can further synergistically enhance the intervention and treatment of NASH significantly, proving that the composition has a strong effect on the treatment of NASH, and is expected to provide a new therapeutic drug for such refractory liver diseases clinically, while reducing the clinical use dose of Silibinin Soft Capsules, reducing the incidence of its adverse reactions and the treatment cost of patients. The present invention is the new use of old drugs, the pharmacokinetic data of the combined drug molecules are relatively detailed, safe and reliable with very mild side effects, and the development of new indications can quickly enter clinical evaluation, shortening the R & D cycle and saving the development cost. Description of the Drawings

[0029] Figure 1Effects of MeCbl, SC alone, and two combination methods of MeCbl and SC (separate gavage combination on the same day vs. gavage combination after mixing the two drugs) on serum biochemical indexes of liver injury in MCDHFD model mice: aspartate aminotransferase (AST) (A - mixed administration, C - administration at different times) and alanine aminotransferase (ALT) (B - mixed administration, D - administration at different times). The abscissa in the figure represents the NASH model and each group of oral administration: Chow, MCDHFD Chow, MCDHFD Chow + SC (50 mg / Kg), MCDHFD Chow + MeCbl (1 mg / Kg), MCDHFD Chow + MeCbl (3 mg / Kg), MCDHFD Chow + MeCbl (10 mg / Kg), MCDHFD Chow + SC (50 mg / Kg) + MeCbl (1 mg / Kg), MCDHFD Chow + SC (50 mg / Kg) + MeCbl (3 mg / Kg), MCDHFD Chow + SC (50 mg / Kg) + MeCbl (10 mg / Kg). The ordinate in the figure represents the levels of ALT and AST in mouse serum. *p < 0.05, **p < 0.01, ***p < 0.001, vs MCDHFD Chow.

[0030] Figure 2 Effects of MeCbl, SC alone, and MeCbl combined with SC on the expression of genes related to liver lipid synthesis and transport in MCDHFD model mice: experimental result graphs of the relative mRNA expression levels of acetyl-CoA carboxylase (Acca) (A), stearoyl-CoA desaturase (Scd) (B), and liver fatty acid binding protein (L-fabp) (C); the abscissa in the figure represents the NASH model and each group of oral administration (the same as Figure 1 above), and the ordinate represents the relative mRNA expression levels of genes related to liver lipid synthesis and transport in mice. *p < 0.05, **p < 0.01, ***p < 0.001, vs MCDHFD Chow.

[0031] Figure 3 Effects of MeCbl, SC alone, and MeCbl combined with SC on the expression of genes related to liver fibrosis in MCDHFD model mice: experimental result graphs of the relative mRNA expression levels of α1-type I collagen (Col1a1) (A), α1-type II collagen (Col1a2) (B), transforming growth factor-β (Tgfb) (C), and smooth muscle actin 2 (Acta2) (D); the abscissa in the figure represents the NASH model and each group of oral administration (the same as Figure 1The same), and the vertical axis represents the relative expression levels of mRNA of genes related to liver fibrosis in mouse livers. *p < 0.05, **p < 0.01, ***p < 0.001, vs MCDHFD Chow.

[0032] Figure 4 Effects of MeCbl, SC alone, and MeCbl combined with SC on the expression of genes related to pro-inflammatory factors in the livers of MCDHFD model mice: Tumor necrosis factor-α (Tnfa) (A), Interleukin-1β (Il1b) (B), Interleukin-6 (Il6) (C). The horizontal axis in the figure represents the NASH model and each group administered orally (the same as Figure 1 The same), and the vertical axis represents the relative expression levels of mRNA of genes related to pro-inflammatory factors in mouse livers. *p < 0.05, **p < 0.01, ***p < 0.001, vs MCDHFD Chow.

[0033] Figure 5 Effects of MeCbl, SC alone, and MeCbl combined with SC on serum biochemical indices of liver injury and the expression of genes related to NASH disease in MCDHFD model mice. The grouping column in the table shows the gavage doses and groups of MeCbl, SC alone, and MeCbl combined with SC. AST / ALT is used to indicate the condition of liver injury in mice, Relative Acca, Scd, L-fabp mRNA level is used to indicate the condition of lipid deposition in mouse livers, Relative Col1a1, Col1a2, Tgfb, Acta2 mRNA level is used to indicate the condition of liver fibrosis in mouse livers, Relative Tnf-a, Il-1b, Il-6mRNA level is used to indicate the inflammatory response condition in mouse livers, and the combination index (CI) of the combined drugs is used to indicate the strength of the synergistic effect of the combined drugs in intervening in the pathological development of NASH.

[0034] Figure 6 Effects of MeCbl, SC alone, and MeCbl combined with SC on liver injury (H&E staining) (A), fibrosis (Masson staining) (B), and lipid deposition (Oil Red O staining) (C) in the livers of MCDHFD model mice. The scale bar in the figure is 100 μm.

[0035] Figure 7The effects of MeCbl, SC alone, and MeCbl combined with SC on the NAS score of liver tissue in MCDHFD model mice: NAS score (A), NAS score synergy index (B). (A) The horizontal axis in the figure is the NASH model and each oral administration group, and the vertical axis is the NAS score value of the mouse. (B) The synergy index (CI) of the combination drug in the figure is used to indicate the strength of the synergistic effect of the combination drug intervention on the NAS score. **p<0.01, ***p<0.001, vs MCDHFD Chow. DETAILED DESCRIPTION

[0036] The present invention will be further described below in conjunction with the accompanying drawings and examples. The present invention is not limited by these examples. Those skilled in the art can refer to the contents of this article and appropriately improve the process parameters to achieve. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention.

[0037] The raw materials and equipment used in the specific embodiments of the present invention are all known products, which are purchased from the market. The experimental method is a conventional method.

[0038] 1. Experimental Materials

[0039] 1.1 Instruments and Equipment

[0040] Various range pipettes (Eppendorf, Germany), Allegra 64R high-speed refrigerated benchtop centrifuge (BECKMAN, USA), BSA223S, PRACTUM224-1CN and BT125D electronic analytical balances (Sartorius, Germany); microplate constant temperature oscillator and vortex mixer (Qilin Bell, China); Milli-Q pure water system (Millipore, USA); Forma TM 900 Series-86℃ ultra-low temperature refrigerator (Thermo Fisher Scientific, USA); Synergy H1 full-function microplate reader (BioTek, USA); Leica upright microscope (DM6B) (Leica, Germany); Nanodropone ultra-micro-volume spectrophotometer (Thermo Fisher Scientific, USA); sterile 1mL syringe (Kantel, China);

[0041] 1.2 Reagents

[0042] Shuilinjia (Tianjin Tasly Pharmaceutical Co., Ltd., National Medicine Approval No. H20040299, China), Methylcobalamin (MCE, HY-B0586, USA); AST detection kit (Nanjing Jiancheng Bioengineering Institute, C010-2-1, China) and ALT detection kit (Nanjing Jiancheng Bioengineering Institute, C009-2-1, China); MCDHFD diet (Shanghai Parker Bio, Research Diets_A06071302, China); Universal tissue fixative (Biosharp, BL539A, China); Sodium carboxymethyl cellulose (CMC-Na) (Aladdin, C104984, China); Other chemical reagents were all purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).

[0043] Reagents required for RT-PCR include: RNA extraction kit (Takara, 9108, Japan); HiScript III RT SuperMix for qPCR (+gDNA wiper) (Vazyme, R323-01, China) and ChamQ SYBR qPCR MasterMix (Vazyme, Q331-02 / 03, China); DEPC H2O (DNase, RNase free) (Beyotime Biotechnology Co., Ltd., R0022, China); Ethanol, chloroform, etc. used in the process of extracting RNA were all purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).

[0044] 1.3 Experimental animals

[0045] About 25 g of SPF-grade C57 / BL6J male mice at 7 weeks of age were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. License number: SCXK (Shanghai) 2022-0004.

[0046] 2. Experimental methods

[0047] 2.1 Solution and drug preparation

[0048] 0.5% CMC-Na solution: Weigh 5 g of CMC-Na powder, add it to 1 L of ddH2O, stir magnetically at 40 °C for 4 h until the powder is completely dissolved, store at room temperature. Before use, add Tween-80 to 0.5% CMC-Na according to the volume ratio of Tween-8 and 0.5% CMC-Na of 1:20, and vortex to mix evenly.

[0049] SC (50 mg / Kg mouse body weight) gavage liquid: Take 1 SC (containing 35 mg of silybin), pour the powder into a dry and clean glass mortar, add 5.6 mL of 0.5% CMC-Na in small amounts several times, and grind carefully into a homogeneous suspension, prepared freshly before use.

[0050] MeCbl (1 / 3 / 10 mg / Kg mouse body weight) intragastric administration solution: Weigh 6 mg of MeCbl powder respectively, add 4.8 mL of 0.5% CMC-Na, and vortex to prepare the MeCbl intragastric administration solution of 10 mg / Kg mouse body weight. Take another two EP tubes, label them as 3 mg / Kg MeCbl and 1 mg / Kg MeCbl respectively. Add 3.5 mL of 0.5% CMC-Na to the 3 mg / Kg MeCbl tube and 3 mL of 0.5% CMC-Na to the 1 mg / Kg MeCbl tube. Then take 1.5 mL of the 10 mg / Kg MeCbl intragastric administration solution from the 10 mg / Kg MeCbl tube and add it to the 3 mg / Kg MeCbl tube, vortex and mix well. Then take 1.5 mL of the 3 mg / Kg MeCbl intragastric administration solution from the 3 mg / Kg MeCbl tube and add it to the 1 mg / Kg MeCbl tube, vortex and mix well. Prepare the solution freshly before use and operate under light protection.

[0051] SC + MeCbl mixed drug: On the basis of the above-mentioned SC and MeCbl drug preparation scheme, keep the solvent volume unchanged, double the mass of the drug powder, then vortex and mix well. Mix SC and MeCbl with different concentrations in a volume ratio of 1:1 to make the mixed drug. Prepare the solution freshly before use and operate under light protection. At the same time, the single drug also needs to be added with an equal volume of solvent.

[0052] 2.2 Construction of MCDHFD model

[0053] 54 six-week-old SPF-grade male wild-type C56BL / 6J mice, purchased from Shanghai Slack Experimental Animal Co., Ltd., were raised in the clean-grade breeding room of the Experimental Animal Center of China Pharmaceutical University, with constant temperature and humidity, and circadian rhythm control. The breeding temperature was about 23 - 24 °C, and the humidity was about 60 ± 10%. After one week of adaptive feeding with maintenance feed, the C57BL / 6J mice were randomly grouped, and the model was established and drugs were administered simultaneously as follows:

[0054] ① Chow group: Given maintenance feed and normal drinking water daily; ② MCDHFD group: Given MCDHFD feed and normal drinking water daily; ③ SC(50mg / Kg) group: Given MCDHFD feed and normal drinking water daily, and gavaged with 50mg / Kg SC at 9:00 every day; ④ MeCbl(1mg / Kg) group: Given MCDHFD feed and normal drinking water daily, and gavaged with 1mg / Kg MeCbl at 16:00 every day; ⑤ MeCbl(3mg / Kg) group: Given MCDHFD feed and normal drinking water daily, and gavaged with 3mg / Kg MeCbl at 16:00 every day; ⑥ MeCbl(10mg / Kg) group: Given MCDHFD feed and normal drinking water daily, and gavaged with 10mg / Kg MeCbl at 16:00 every day; ⑦ SC(50mg / Kg)+MeCbl(1mg / Kg) group: Given MCDHFD feed and normal drinking water daily, gavaged with 50mg / Kg SC at 9:00 every day and gavaged with 1mg / Kg MeCbl at 16:00 every day; ⑧ SC(50mg / Kg)+MeCbl(3mg / Kg) group: Given MCDHFD feed and normal drinking water daily, gavaged with 50mg / Kg SC at 9:00 every day, and gavaged with 3mg / Kg MeCbl at 16:00 every day; ⑨ SC(50mg / Kg)+MeCbl(10mg / Kg) group: Given MCDHFD feed and normal drinking water daily, gavaged with 50mg / Kg SC at 9:00 every day, and gavaged with 10mg / Kg MeCbl at 16:00 every day. The simultaneous gavage administration of SC and MeCbl after mixing is basically the same as the above single-drug experimental operation, only gavaging single drugs and mixed drugs at 9:00 every day, and ensuring that the dosage of the two drugs in the mixed drug is the same as the dosage of the drugs administered at different times in the above groups ⑦⑧⑨.

[0055] The mice were given drug treatment according to the above administration methods and dosages every day until the end of the 6-week model. After 6 weeks of modeling, blood was collected from the orbital venous plexus of the mice, and the whole liver was taken. The large liver lobes were fixed with 4% paraformaldehyde, and the remaining liver was stored frozen.

[0056] 2.3 Detection of the activities of AST / ALT enzymes in mouse serum

[0057] The whole blood collected from each group of mice was left to stand and coagulate at room temperature for 1 - 2 h, and then centrifuged at 3500 rpm for 10 minutes on a small centrifuge. The whole blood could be separated into the upper pale yellow serum and the lower dark red blood clot. The supernatant was aspirated into a new EP tube and stored in a -80°C ultra-low temperature refrigerator. When in use, the serum was taken out and thawed for standby. At the same time, the AST detection kit and the ALT detection kit were taken out of the 4°C refrigerator and placed at room temperature to equilibrate the temperature to room temperature. Two 96-well enzyme-linked immunosorbent assay (ELISA) plates were taken, one for detecting AST and the other for detecting ALT. The AST substrate solution and the ALT substrate solution were added to the corresponding wells of the 96-well plates, 20 μL per well, for a total of 54 wells. 10 μL of the serum from each sample was taken and added to a new EP tube, and then 40 μL of normal saline was added to obtain a 5-fold diluted serum. The diluted serum was added to the above-mentioned 96-well plates. The two 96-well plates were placed on a microplate shaker and shaken at 350 rpm and 37°C for 30 min. After the shaking was completed, the plates were taken out, and 20 μL of phenylhydrazine solution was added to each reaction well, and then shaken at 350 rpm and 37°C for 20 min. Additionally, at this time, the standard curves of AST and ALT were tested synchronously, and the liquid addition method of the required reagents was carried out according to Table 1. After the shaking was completed, the plates were taken out, and 20 μL of 0.4 M NaOH solution (the kit provides 4 M NaOH solution, which is diluted with ddH2O at a volume ratio of 1:10 when in use) was added to each reaction well. The plate was gently shaken manually horizontally for 2 min and then left to stand for 15 minutes. The absorbance value at 510 nm was read using an enzyme-linked immunosorbent assay (ELISA) reader; the standard curves of AST and ALT were plotted using Excel and the standard curve equations were obtained, so as to convert the AST and ALT enzyme activities (U / L) of each reaction well.

[0058] Table 1 Preparation of AST and ALT standard curves

[0059]

[0060]

[0061] 2.4 Detection of relative mRNA expression levels of NASH-related genes in mouse liver

[0062] Approximately 30 mg of small pieces of the liver stored at -80°C were placed in a homogenization tube for standby. The operations of cutting and weighing the liver were carried out with the liver on ice throughout the process. Then the following RT-Real time PCR operations were carried out:

[0063] 2.4.1 Total RNA extraction

[0064] Add 2 - 3 homogenizing beads and 1 mL of Trizol (Takata) into the homogenizing tube. Use a cryogenic grinder to grind according to the running time of 30 seconds, interruption time of 10 seconds, and grinding frequency of 60 HZ for a total of 5 times until complete homogenization without particles. Transfer the homogenate to a new EP tube, centrifuge at 12000g at 4°C for 5 min, and aspirate the supernatant and transfer it to a new EP tube. Add 200 μL of chloroform to the above homogenate supernatant, shake vigorously for 15 seconds, let it stand for 5 min after full emulsification without stratification. Centrifuge at 12000g at 4°C for 15 min, aspirate 200 μL of the supernatant and transfer it to a new centrifuge tube. Do not aspirate the middle white protein layer and the bottom red Trizol layer. Add an equal volume of pre-cooled isopropanol to the above supernatant, gently invert the centrifuge tube up and down to mix well, and then let it stand for 20 min to precipitate RNA. Centrifuge at 12000g at 4°C for 10 min, discard the supernatant, slowly add 1 mL of pre-cooled 75% DEPC ethanol along the centrifuge tube wall, gently invert the centrifuge tube up and down to wash the centrifuge tube wall, centrifuge at 12000g at 4°C for 5 min and discard the ethanol. Dry the precipitate at room temperature to ensure complete removal of ethanol, add 20 μL of DEPC H2O to dissolve RNA, and perform RNA quantification using a nanodrop one ultra-micro spectrophotometer. The concentration unit shown on the instrument is ng / μL. Finally, use DEPC water to adjust the RNA concentration to 500 ng / μL.

[0065] 2.4.2 Reverse Transcription

[0066] The kit used for reverse transcription is Vazyme's HiScript III RT SuperMix for qPCR (+gDNAwiper). Prepare the reverse transcription system according to the following ratio (the system volume required for a single reaction)

[0067]

[0068] Set the reverse transcription temperature as: 37°C for 15 min, 85°C for 5 s, and finally store the reverse transcribed cDNA at 4°C.

[0069] 2.4.3 Real-time PCR Reaction

[0070] The reagents used for PCR are purchased from Vazyme's ChamQ SYBR qPCR Master Mix. The specific system formula is as follows:

[0071]

[0072]

[0073] After the premixed system is completely premixed in the Bio-Rad PCR plate, it is placed in a vertical PCR plate centrifuge to centrifuge all the system solutions to the bottom of the well plate. The PCR temperature control conditions are: high-temperature denaturation: 95°C, 30 s; PCR reaction: 95°C, 5 s; annealing temperature (T m ) stays for 15 s; 72°C, 30 s. A total of 40 temperature control cycles are carried out. Melting curve: within 20 min, the temperature drops from 95°C to 70°C and stays at 95°C for 15 s.

[0074] 2.4.4 Real time-qPCR data processing

[0075] During the experiment, the internal reference GAPDH should be set as a control at the same time. The amplification rates of all groups should be uniformly close to 100% with a deviation within 5%. The Livak method is used to determine the relative differences in the expression of the same target gene in different samples: the CT values of the internal reference gene are used to normalize the CT values of the target gene for all samples.

[0076] ΔCT(test) = ΔCT(target, test) - ΔCT(ref, test)

[0077] ΔCT(calibrator) = ΔCT(target, calibrator) – ΔCT(ref, calibrator)

[0078] Then, the ΔCT of the calibration sample is used to normalize the test sample

[0079] ΔΔCT = ΔCT(test) - ΔCT(calibrator)

[0080] Finally, the logarithmic value is calculated to obtain the expression level

[0081] 2 -ΔΔCT = relative expression value. The primer sequences used are shown in Table 2 below

[0082] Table 2 RT-PCR primers

[0083]

[0084]

[0085] 2.4.5 Calculation of the synergy index of the relative expression levels of mouse serum transaminases and NASH-related genes mRNA after co-administration of mecobalamin and Shuilinjia

[0086] Based on the Chou-Talalay combined index method and CompuSyn software to analyze the synergy index of drug combinations, the average synergy effect index (Combination Index, CI value) range of compound combinations and the evaluation basis for interactions are shown in Table 3 below:

[0087] Table 3 Synergy Index

[0088]

[0089] 2.5 MCDHFD-NASH mouse liver pathological section microscopy

[0090] 2.5.1 H&E staining

[0091] After washing the liver tissue with PBS, blotting the surface moisture with filter paper, fixing it in 4% paraformaldehyde, and embedding it in paraffin sections. Process the embedded paraffin tissue sections, bake the sections (overnight at 56°C / 15 min at 85°C), and rehydrate conventionally (using the process of 20 min in xylene, 20 min in xylene, 10 min in 100% ethanol, 10 min in 100% ethanol, 10 min in 95% ethanol, 10 min in 80% ethanol, 5 min in 50% ethanol, 5 min in 30% ethanol, and 5 min of running water rinsing). Stain by hematoxylin for 5 - 10 min, running water rinsing for 5 min, hydrochloric acid differentiation solution for 3 s, running water rinsing for 5 min, bluing solution for 10 - 30 s, running water rinsing for 5 min, eosin for 5 min, and running water rinsing for 5 min. After conventional dehydration and cover slipping operations, observe through a pathological microscope (10 s in 50% ethanol, 10 s in 80% ethanol, 10 s in 90% ethanol, 10 s in 100% ethanol, 10 s in 100% ethanol, 10 s in xylene, 10 s in xylene, place in a fume hood for 1 min and then cover slip with neutral balsam), and place the sections under an Olympus microscope for observation and photography.

[0092] 2.5.2 Masson staining

[0093] After the liver tissue was washed clean with PBS, the surface moisture was blotted dry with filter paper, and it was fixed in 4% paraformaldehyde and embedded in paraffin sections. The embedded paraffin tissue sections were processed, baked (overnight at 56°C / 15 min at 85°C), and rehydrated conventionally (using the procedure of 20 min in xylene, 20 min in xylene, 10 min in 100% ethanol, 10 min in 100% ethanol, 10 min in 95% ethanol, 10 min in 80% ethanol, 5 min in 50% ethanol, 5 min in 30% ethanol, and 5 min of rinsing with running water). Through hematoxylin for 5 - 10 min, rinsing with running water for 5 min, hydrochloric acid differentiating solution for 3 s, rinsing with running water for 5 min, bluing solution for 10 - 30 s, rinsing with running water for 5 min, staining with Ponceau acidic fuchsin solution for 5 - 10 min, and quickly rinsing with distilled water. Treatment with 1% phosphomolybdic acid aqueous solution for about 3 - 5 min. Without washing with water, directly counterstain with aniline blue solution for 5 min. Treat with 1% glacial CH3COOH for 1 min. After conventional dehydration and coverslipping operations, it was observed under a pathological microscope (10 s in 50% ethanol, 10 s in 80% ethanol, 10 s in 90% ethanol, 10 s in 100% ethanol, 10 s in 100% ethanol, 10 s in xylene, 10 s in xylene, placed in a fume hood for 1 min and then coverslipped with neutral balsam, and the sections were placed under an Olympus microscope for observation and photography).

[0094] 2.5.3 Oil Red O Staining

[0095] After the liver tissue was washed clean with PBS, the surface moisture was blotted dry with filter paper, and frozen sections with a thickness of 6 - 10 μm were prepared and washed with water after being fixed with 4% paraformaldehyde. The sections were immersed in 60% isopropanol for 2 min, then added to the modified Oil Red O staining solution and stained in a sealed manner for 10 - 15 min, and pay attention to avoiding light during this process. Added 60% isopropanol for color separation until the background was colorless, added and slightly washed once with ice-cold distilled water, and counterstained the nucleus with Mayer hematoxylin staining solution for 5 min. Then washed once with ice-cold distilled water, the slides were blotted dry with filter paper, and coverslipped with glycerin gelatin. The sections were placed under an Olympus microscope for observation and photography.

[0096] 2.5.4 NAS Pathological Scoring

[0097] For liver cell fatty degeneration, it is scored from 0 - 3 points, ballooning degeneration from 0 - 2 points, and inflammation from 0 - 3 points, with a total score of 8 points.

[0098] (1) Hepatocyte fatty degeneration: 0 points (<5%); 1 point (5% - 33%); 2 points (34% - 66%); 3 points (>66%).

[0099] (2) Inflammation within the lobule (counting necrosis foci under a 20 - fold microscope): 0 points, none; 1 point (<2); 2 points (2 - 4); 3 points (>4).

[0100] (3) Ballooning degeneration of hepatocytes: 0 point, none; 1 point, rare; 2 points, common.

[0101] Add up the scores of the above three items: fatty degeneration of hepatocytes, ballooning degeneration of hepatocytes, and inflammation in the hepatic lobule, and the NAS score can be obtained.

[0102] Example 1

[0103] Construct a NASH model with MCDHFD diet to determine the inhibitory effect of orally administered MeCbl / SC alone and in combination on the serum transaminase activity of mice ( Figure 1 ).

[0104] Experimental protocol: Mice in the NASH model group were fed with MCDHFD diet freely for 6 weeks, and mice in the control group were fed with normal maintenance feed. When the mice were modeled, they were given MeCbl (1 / 3 / 10 mg / kg) or SC (50 mg / kg) or MeCbl (1 / 3 / 10 mg / kg) + SC (50 mg / kg) by gavage every day. Note that the administration methods were gavage with the mixture of SC and MeCbl at 9 am or gavage with SC and MeCbl at 9 am and 4 pm respectively. After 6 weeks of modeling, the mice were sacrificed and serum was collected to measure the levels of ALT and AST (the specific process was carried out according to Section 2.3 of the above experiment).

[0105] Experimental results: It can be seen from Figure 1 that oral administration of low, medium, and high doses (1 / 3 / 10 mg / kg) of MeCbl or 50 mg / kg of SC alone can reduce the serum ALT and AST levels to varying degrees compared with the model group; administration of low, medium, and high doses of MeCbl in combination with SC cannot synergistically reduce the serum ALT or AST levels of mice compared with single administration; while after separate gavage and combination, low, medium, and high doses of MeCbl combined with SC can significantly synergistically reduce the serum ALT level of mice compared with single administration, and the synergy indices are 0.107, 0.575, and 0.219 respectively; but only low-dose MeCbl (1 mg / kg) combined with SC can significantly synergistically reduce the serum AST level of mice compared with single administration, and the synergy index is 0.522. For the detailed index values and synergy indices, see Figure 5 .

[0106] This example proves that there may be a compatibility taboo when SC and MeCbl are mixed and administered in combination, and there is no obvious synergistic therapeutic effect compared with single drugs. However, when administered in combination at different time points, within the dose range of 50 mg / kg SC + 1 - 10 mg / kg MeCbl orally in mice, it can synergistically reduce the serum ALT level in NASH model mice. When orally administered 50 mg / kg SC + 1 mg / kg MeCbl, it can synergistically reduce the serum AST level in NASH model mice and thus intervene in the increase of liver transaminases induced by MCDHFD diet. Among them, the synergistic effect of 1 mg / kg MeCbl combined with 50 mg / kg SC is the strongest, and is significantly stronger than that of MeCbl alone and SC alone.

[0107] Example 2

[0108] Effects of oral administration of MeCbl / SC single drugs and combined drugs on the expression levels of genes related to liver lipid synthesis and transport, liver fibrosis, and inflammatory factors in NASH model mice ( Figure 2 ; Figure 3 ; Figure 4 ).

[0109] Experimental protocol: Sacrifice the mice according to the method in Example 1 and take the whole liver. Fix part of the large liver lobe with 4% paraformaldehyde, and freeze the remaining liver for storage. Take about 30 mg of small pieces of the liver stored at -80 °C and place them in a homogenization tube for standby, operating on ice. Then perform the RT-qPCR operation procedure (the specific process adopts Section 2.4 of the above experiment) to detect the expression levels of disease-related genes in the liver.

[0110] Experimental results: From Figures 2-4It can be seen that in the MCDHFD chow group, the relative expression level of Acca mRNA related to lipid synthesis in the mouse liver increased, the relative expression level of Scd mRNA decreased, and the relative expression level of L-fabp mRNA increased. The relative expression levels of fibrosis-related Col1a1, Col1a2, Tgfb, and Acta2 mRNAs were significantly higher than those in the normal control group. The relative expression levels of inflammatory factors Tnfa, Il1b, and Il6 mRNAs were significantly higher than those in the normal control group. Oral administration of 1 - 10 mg / Kg MeCbl or 50 mg / Kg SC alone could reversely regulate the mRNA expression of the above NASH-related genes to a limited extent. However, compared with the MCDHFD chow group, the combination of 1 - 10 mg / Kg MeCbl and 50 mg / Kg SC could reduce the relative expression level of Acca mRNA and increase the relative expression level of Scd mRNA. Only the combination of 10 mg / Kg MeCbl and 50 mg / Kg SC could strongly synergistically inhibit the relative expression level of Acca mRNA compared with single drug administration (synergy index 0.604); the combination of 1 or 3 mg / kg MeCbl and 50 mg / Kg SC could strongly synergistically increase the relative expression level of Scd mRNA compared with single drug administration (synergy indices 0.317, 0.753); the combination of 3 or 10 mg / kg MeCbl and 50 mg / Kg SC could strongly synergistically inhibit the relative expression level of L-fabp mRNA compared with single drug administration (synergy indices 0.478, 0.640); the combination of 1 - 10 mg / Kg MeCbl and 50 mg / Kg SC could strongly synergistically inhibit the relative expression levels of Col1a1, Col1a2, Tgfb, Acta2, Tnfa, and Il1b mRNAs in the mouse liver compared with single drug administration, and there were extremely significant statistical differences, but it could not reduce the relative expression level of Il6 mRNA. The detailed index values and synergy indices are shown in Figure 5 。

[0111] This example proves that: within the dose range of oral administration of 50 mg / kg SC + 1 - 10 mg / kg MeCbl in mice, it can synergistically reverse the changes in gene expression related to lipid synthesis and transport, liver fibrosis, and inflammatory factors in the liver of NASH model mice to different degrees, thereby intervening in NASH liver fat deposition, liver fibrosis, liver injury, and inflammation induced by MCDHFD; while oral administration of 1 - 10 mg / kg MeCbl or 50 mg / kg SC alone has a limited reverse effect on the NASH-related genes in the above liver.

[0112] Example 3

[0113] Intervention effect of single drugs and combined drugs of MeCbl / SC orally administered to NASH model mice on pathological liver injury in mice.

[0114] Experimental protocol: Mice were sacrificed according to the method of Example 1, and the whole liver was taken. After the liver tissue was washed clean with PBS, the surface moisture was blotted dry with filter paper, fixed with 4% paraformaldehyde, and subjected to H&E, Masson, and Oil Red O staining and mounting (the specific process was carried out according to Section 2.6 of the above experiment). The sections were placed under an Olympus microscope for observation and photography, and scored according to the NAS pathological scoring criteria.

[0115] Experimental results: As Figure 6 can be seen, compared with the diet control group, the liver injury in the MCDHFD Chow group was severe, with a large number of lipid droplets and cellular vacuolar degeneration between tissues, and a large number of neutrophil aggregations; the liver fibrosis in the MCDHFD Chow group was severe, with a large amount of collagen fibers filling the tissue, indicating a tendency of liver fibrosis and the evolution of NASH to a critical state of liver fibrosis; the liver lipid deposition in the MCDHFD Chow group was severe, with a large number of fat granules in the tissue. Although the single use of MeCbl or SC could alleviate the above liver lesions to a certain extent, the combination of low, medium, and high doses of MeCbl and SC could significantly synergistically improve liver fat deposition, liver fibrosis, liver injury, and inflammatory lesions in NASH model mice compared with the single drug administration group, and showed a concentration dependence. NAS pathological scoring of tissue sections found that the combination of SC and 3 mg / kg or 10 mg / kg MeCbl had a significant improvement compared with the model group. The oral administration of 50 mg / kg SC + 1 - 10 mg / kg MeCbl dose range compared with the single drug NAS score showed that the combination had a significant synergistic effect in reducing the NAS score ( Figure 7 ).

[0116] This example proves that within the dose range of 50 mg / kg SC + 1 - 10 mg / kg MeCbl orally administered to mice, it can synergistically inhibit pathological changes such as liver fat deposition, liver fibrosis, liver injury, and inflammation to varying degrees and show a concentration dependence, manifested as a significant decrease in NAS scoring.

[0117] In summary, based on the MCDHFD-induced NASH model, by co-administering SC with MeCbl at different doses, it was found that MeCbl, as a classical drug for treating peripheral neuropathy, could reduce the serum transaminase activity, inhibit lipid synthesis, and the expression of inflammatory factors and liver fibrosis-related genes in NASH model mice to a certain extent when orally administered alone. On this basis, by orally administering the combination of MeCbl and Shuilingjia, it was found that the combination of SC and MeCbl could significantly synergistically intervene in the development of NASH in mice on the basis of the single-drug efficacy. The mass composition ratio range of the combined drug to produce a synergistic therapeutic effect was MeCbl:SC = (1-10):50, and the mass composition ratio range to exert a strong synergistic intervention effect was (1-3):50. In particular, the combination of 50 mg / Kg SC and 1 mg / Kg MeCbl had the best combined efficacy, which could strongly synergistically reduce the activities of AST and ALT enzymes, as well as the expression of genes related to liver fibrosis, partial liver lipid synthesis and inflammation, effectively reduce the degree of liver tissue damage, steatosis and fibrosis lesions, indicating that 1:50 as the optimal mass ratio could almost synergistically reverse all NASH indicators. Furthermore, the application of the combination of mecobalamin and low-dose Shuilingjia in the preparation of drugs for treating non-alcoholic steatohepatitis and its evolving diseases was proposed. It should be noted that the present invention also found that SC and MeCbl could not be directly combined, and there might be incompatibility in the mixed administration scheme and no synergistic therapeutic effect could be produced, and they needed to be co-administered in separate doses within a certain time range.

Claims

1. Use of mecobalamin in combination with Shuilinjia in the preparation of a medicament for treating non-alcoholic steatohepatitis; the mass composition ratio range of the mecobalamin and Shuilinjia is (1-10):

50.

2. The application according to claim 1, characterized in that, The use of mecobalamin in combination with Shuilinjia in the preparation of a medicament for treating non-alcoholic steatohepatitis is by acting to reduce liver injury, inflammation, liver fibrosis and hepatic lipid deposition.

3. The application according to claim 1, wherein The mass composition ratio range of the mecobalamin and Shuilinjia is (1-3):

50.

4. A pharmaceutical composition for treating non-alcoholic steatohepatitis, characterized in that, It includes an active ingredient and a pharmaceutically acceptable carrier, and the active ingredient is mecobalamin and Shuilinjia with a mass ratio of (1-10):

50.

5. The pharmaceutical composition according to claim 4, characterized in that, The mass ratio of the mecobalamin and Shuilinjia is (1-3):

50.

6. The pharmaceutical composition according to claim 4, wherein The composition includes various pharmaceutical preparations prepared from the composition, and the dosage form of the pharmaceutical preparation is a capsule, powder, tablet, granule, pill, injection, syrup, oral liquid, inhalant, ointment, suppository or patch.

7. The pharmaceutical composition according to claim 6, characterized in that, The dosage form is an immediate-release and sustained-release bilayer tablet. After the mecobalamin and Shuilinjia pharmaceutical composition is orally administered into the body in this dosage form, Shuilinjia is instantaneously released, and mecobalamin is released after a delay of 6-8 hours.

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