Preparation method of medicine for treating non-alcoholic fatty liver

Abstract

The invention belongs to the technical field of medicines, and relates to a preparation method of a medicine for treating non-alcoholic fatty liver. The medicine for treating the non-alcoholic fatty liver disease contains psoralen. The concentration of the psoralen in the medicine for treating the non-alcoholic fatty liver disease is 0.3 mu M to 3.5 mu M. Wherein the content of psoralen is greater than or equal to 98%. A preparation method of the medicine solution containing 100 mM of psoralen for treating the non-alcoholic fatty liver disease comprises the following steps: weighing 0.0906 g of psoralen powder in 5 mL of DMSO (dimethylsulfoxide), and carrying out vortex dissolution to obtain the medicine solution. Research results show that the medicine for treating the non-alcoholic fatty liver disease can reduce lipid deposition of a human hepatocyte model, down-regulate protein expression and transcriptional level of lipid synthesis related genes FASN and SREBP1, up-regulate protein expression and transcriptional level of lipid decomposition related genes CPT1 alpha, increase protein expression of antioxidant enzymes SOD1 and SOD2, and can be used for treating the non-alcoholic fatty liver disease. The lipid deposition and the superoxide state of the L02 hepatocytes induced by sodium oleate are relieved.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a method for preparing a drug for treating non-alcoholic fatty liver disease. Background Art

[0002] The liver plays a key role in many physiological processes, including regulating glucose and lipid metabolism levels. Non-alcoholic fatty liver disease (NAFLD) is mainly caused by dysfunctional hepatic lipid metabolism. NAFLD is the most common chronic liver disease worldwide and is closely associated with insulin resistance (IR) and type 2 diabetes mellitus (T2DM). The mechanisms behind these diseases are not fully understood, but involve factors such as liver fat accumulation, energy metabolism, and oxidative stress. Lipid toxicity has been proposed to play an important role in NAFLD.

[0003] The main pathogenesis of NAFLD is the "two-hit theory". Excessive accumulation of lipids in liver cells leads to disordered synthesis and transport of triglycerides in cells, thereby aggravating the body's resistance to insulin. Reactive oxygen comes from the decomposition of lipids by cells. Excessive ROS can damage mitochondria, leading to energy supply disorders in cells. Cells die due to lack of energy supply, and then are degraded and eliminated through the proteasome pathway and cell autophagy pathway.

[0004] Several studies have shown that Chinese herbal medicine has developed new treatments for non-alcoholic fatty liver disease (NAFLD) based on its unique theoretical system and a large number of herbal therapies. Most of these Chinese herbal medicines have been shown to improve the biochemical and histological changes of NAFLD in vitro and in vivo. Psoralea corylifolia, a local medicinal material produced in Sichuan, is widely used in the treatment of vitiligo and osteoporosis. It is worth noting that psoralen, as an extract of psoralea corylifolia, has been used in the treatment of atopic dermatitis, lichen planus, cutaneous mastocytosis, actinic dermatitis and heliuria, etc. Psoralen has also been shown to have anti-cancer, anti-fungal, anti-inflammatory, antioxidant, anti-depressant and estrogen-like effects. In addition, psoralen also plays a role in reversing multidrug resistance and is closely related to cell cycle, apoptosis and calcium antagonism.

[0005] Therefore, it is of great significance to develop a preparation method for treating non-alcoholic fatty liver disease. Summary of the invention

[0006] The purpose of the present invention is to provide a method for preparing a drug for treating non-alcoholic fatty liver disease.

[0007] A method for preparing a drug for treating non-alcoholic fatty liver disease, wherein the drug for treating non-alcoholic fatty liver disease contains psoralen. The method for preparing a drug solution for treating non-alcoholic fatty liver disease containing 100 mM psoralen is as follows: weigh 0.0906 g of psoralen powder in 5 mL of DMSO, and vortex to dissolve.

[0008] In the preparation method of a drug for treating non-alcoholic fatty liver disease of the present invention, the drug for treating non-alcoholic fatty liver disease refers to a drug used to treat a human hepatocyte lipid deposition model established by sodium oleate treatment.

[0009] In the preparation method of a drug for treating non-alcoholic fatty liver disease of the present invention, the concentration of psoralen in the drug for treating non-alcoholic fatty liver disease is 0.3 μM to 3.5 μM, wherein the content of psoralen involved is ≥ 98%.

[0010] In the method for preparing a drug for treating non-alcoholic fatty liver disease of the present invention, the human hepatocyte lipid deposition model established by sodium oleate treatment is a human hepatocyte lipid deposition model obtained by inducing liver L02 cells with 125 μM sodium oleate for 24 hours.

[0011] The present invention systematically studies the alleviating effect of psoralen on a lipid deposition cell model established by sodium oleate treatment, and the results show that psoralen reduces lipid deposition in a human hepatocyte model, downregulates the protein expression and transcription levels of lipid synthesis-related genes FASN and SREBP1, and simultaneously upregulates the protein expression and transcription levels of lipid decomposition-related gene CPT1α, thereby alleviating the lipid deposition state of L02 hepatocytes induced by sodium oleate.

[0012] 1. The present invention firstly proves through Nile red staining experiment that the NAFLD cell model can be successfully established by inducing with 125 μM sodium oleate for 24 hours. Sodium oleate has better solubility in the culture medium and less toxicity to cells.

[0013] The liver is the most important metabolic organ in the human body. The liver transports lipid metabolites to peripheral tissues, or recycles and removes peripheral free lipids. In NAFLD, the liver increases fat uptake and de novo synthesis, and the insufficient fatty acid oxidation level causes the body to be insufficient to maintain normal metabolic levels. Therefore, the present invention uses free fatty acids to simulate the high fatty acid levels of NAFLD patients in vitro. By consulting the literature and previous experiments, it is proved that the use of sodium oleate is better than the traditional inducer-oleic acid. The former has better solubility in the culture medium and less toxicity to cells. The results of Nile red staining showed that the normal human liver L02 cells were induced for 24 hours using sodium oleate at a concentration of 125 μM, and the cells produced severe lipid deposition. The results of protein immunoblotting showed that the expression of lipid synthesis-related proteins in liver L02 cells increased after sodium oleate induction, and the expression of lipid decomposition-related genes decreased. The above results show that the NAFLD cell model was successfully established using sodium oleate to induce L02 cells.

[0014] 2. It was verified that this model can well reflect the metabolic level of the liver in NAFLD patients.

[0015] Protein immunoblotting was used to detect the expression changes of genes related to lipid synthesis and lipolysis in the induced group and the control group. The results showed that the lipid metabolism of hepatocytes was disordered after sodium oleate induction, which further verified that this model can well reflect the metabolic level of the liver in NAFLD patients.

[0016] 3. Studies have found that psoralen can reduce lipid deposition in liver L02 cells in a dose-dependent manner.

[0017] Nile red staining and Oil Red O staining revealed that psoralen can reduce lipid deposition in liver L02 cells in a dose-dependent manner. Similarly, protein immunoblotting results also confirmed that psoralen can reduce the transcription and translation of genes related to lipid synthesis and increase the transcription and translation of genes related to lipolysis, thereby dynamically regulating the lipid metabolism of liver L02 cells to maintain a balanced state. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 :Nile red staining of liver L02 cells after treatment with 125 μM sodium oleate for 24 hours

[0019] Figure 2 : Changes of lipid deposition in sodium oleate-induced hepatic L02 cells treated with psoralen.

[0020] (A) Statistics of relative fluorescence intensity values ​​of lipid droplets stained with Nile red (B).

[0021] Figure 3 : Changes of lipid deposition in sodium oleate-induced hepatic L02 cells treated with psoralen.

[0022] (A) Oil red O staining of lipid droplets (B) Statistics of relative IOD values ​​of lipid droplets

[0023] Figure 4 : Expression of lipid metabolism-related genes in sodium oleate-induced hepatic L02 cells treated with psoralen.

[0024] (A) Changes in protein expression of lipid metabolism-related genes FASN, SREBP1, and CPT1α in liver L02 cells induced by sodium oleate. (B) Quantitative analysis of FASN, SREBP1, and CPT1α proteins. (C) Changes in protein expression of lipid synthesis genes FASN and SREBP1 in liver L02 cells induced by sodium oleate treated with psoralen and quantitative analysis of FASN and SREBP1 proteins.

[0025] Figure 5 :Expression of lipid metabolism-related genes in liver L02 cells treated with psoralen

[0026] (A) Changes in the mRNA expression of lipid metabolism-related genes Fasn, Srebp1, and Cpt1α in liver L02 cells. (B) Quantitative statistical heat map analysis of Fasn, Srebp1, and Cpt1α mRNA. DETAILED DESCRIPTION

[0027] The present invention is further described in conjunction with specific embodiments, but these embodiments are only intended to illustrate the present invention and are not intended to limit the scope of the present invention.

[0028] 1. Preparation of main reagent solutions

[0029] (1) Preparation of 100 mM sodium oleate stock solution

[0030] Use an analytical balance to weigh 0.6089 g of sodium oleate solid, dissolve it in 20 mL of ultrapure water, place it in a 50 mL centrifuge tube and vortex it to dissolve it, filter the prepared solution using a 0.22 μm filter in an ultra-clean bench, and finally store it in a -80°C refrigerator.

[0031] (2) Preparation of 1 mM sodium oleate working solution

[0032] Dilute the 100 mM sodium oleate stock solution with complete medium to a final concentration of 1 mM.

[0033] (3) Preparation of a drug solution containing 100 mM psoralen for the treatment of non-alcoholic fatty liver disease

[0034] Weigh 0.0906 g of psoralen powder (psoralen content ≥ 98%, purchased from Yuanye Biotechnology Co., Ltd., batch number 20201117) and add it to 5 mL of DMSO, vortex to dissolve, and then dispense into 1.5 mL EP tubes, 1 mL per tube, store in a -80 °C refrigerator, and dilute with culture medium when used.

[0035] (4) Preparation of a working solution containing 1 mM psoralen for the treatment of non-alcoholic fatty liver disease

[0036] The 100 mM psoralen drug stock solution was diluted with culture medium to a final concentration of 1 mM.

[0037] (5) Preparation of Nile red dye solution

[0038] Weigh 0.0096 g of Nile red powder and dissolve it in 1 mL of DMSO to prepare a 30 mM stock solution, store it in a -20°C refrigerator, and use culture medium to dilute it to a working solution with a concentration of 5 μM when staining cells.

[0039] (7) Preparation of Oil Red O working fluid

[0040] 0.5% Oil Red O mother liquor was prepared with ultrapure water at a volume ratio of 3:2, filtered through a filter with a diameter of 0.22 μm, and reserved for later use.

[0041] 2. Experimental Methods

[0042] (1) Place a clean, sterile round glass slide in a 24-well plate and add 1× PBS solution to submerge the slide. Keep it ready for use.

[0043] (2) Aspirate the PBS solution, resuspend the cells, inoculate them into 24-well plates and 6-well plates in proportion, and then place them in a 37°C, 5% CO2 incubator;

[0044] (3) After the cells adhered, they were induced with 125 μM sodium oleate solution for 24 h;

[0045] 3. Drug treatment

[0046] Liver L02 cells were treated with 0 μM, 0.37 μM, 1.1 μM, and 3.3 μM psoralen for 24 h;

[0047] 4. Cell Nile Red Staining

[0048] (1) Transfer the 24-well plate to a clean bench, remove the culture medium, and wash twice with 1× PBS solution, 1 mL each time;

[0049] (2) Add 5 μM Nile red working solution and incubate at 37°C in the dark for 30 min;

[0050] (3) Remove the staining solution, wash twice with 1× PBS solution, and then add 4% PFA to fix at room temperature for 20 min;

[0051] (4) Remove 4% PFA and wash three times with 1× PBS solution, 1 mL each time;

[0052] (5) Add 0.5% Triton X-100 and permeabilize for 15 min;

[0053] (6) Aspirate the 0.5% Triton X-100 solution, add Hoechst dye solution diluted at a ratio of 1:3000, and incubate at room temperature for 3 min;

[0054] (7) Remove the staining solution, wash with 1× PBS solution three times, 1 min each time, and then wash with ultrapure water three times, 3 min each time;

[0055] (8) Seal the slides with anti-fluorescence quenching sealing medium and store at -20°C away from light;

[0056] (9) Laser confocal microscopy was used to photograph the TRITC channel using a 63× oil objective (wavelength of 561 nm).

[0057] (10) Image J was used to analyze the fluorescence intensity of Nile red staining.

[0058] 5. Oil Red O Staining of Cells

[0059] (1) Take out the 24-well plate inoculated with cells, discard the culture medium, and wash twice with 1× PBS solution, 1 mL each time;

[0060] (2) Fix with 4% PFA solution for 20 min, discard the fixative, and wash once with 1 mL 1× PBS solution;

[0061] (3) Stain with filtered Oil Red O working solution for 45 minutes;

[0062] (4) Wash away the floating color with 60% isopropyl alcohol solution quickly;

[0063] (5) Wash 2-5 times with 1 mL of 1× PBS solution until no floating color remains;

[0064] (6) Stain with hematoxylin for 5 min, then wash 2-5 times with 1× PBS solution until no floating color remains;

[0065] (7) Place in 1% hydrochloric acid ethanol solution for differentiation for a few seconds, turn blue in tap water for 2 minutes, and finally wash with ultrapure water 2-5 times;

[0066] (8) Seal the slides with glycerol and store at room temperature.

[0067] 6.RT-PCR

[0068] (1) Total RNA extraction: Total RNA from liver L02 cells was extracted using the TransZol Up kit.

[0069] (2) Reverse transcription: cDNA was generated using SuperM-MuLV reverse transcriptase (Sangon, Shanghai, China), oligo(dT)12-18 primers (Sangon, Shanghai, China), and RNase inhibitor (BBI, Shanghai, China). cDNA was amplified using the following PCR conditions: 50°C for 45 min and 80°C for 15 min.

[0070] (3) Amplification

[0071] Table 1 Amplification system

[0072]

[0073] Table 2 PCR reaction conditions

[0074]

[0075]

[0076] Table 3 Primer information

[0077]

[0078] (3) Gel running: 5 μL of the amplified product from the previous step was loaded onto a 1% agarose gel and electrophoresed at 110 V and 240 mA for 25 min.

[0079] (4) Take pictures: Finally, take images under ultraviolet excitation

[0080] 7. Experimental Results

[0081] 7.1 Establishment of lipid deposition model in liver L02 cells

[0082] The experimental group treated liver L02 cells with 125μM sodium oleate for 24h, and the control group treated the cells with complete medium for the same time, and then used Nile red staining to detect the aggregation of cell lipid droplets. When the cells were treated with 125μM sodium oleate, the fluorescence intensity of Nile red staining of liver L02 cells increased compared with the control group, and the lipid droplets in the cells were evenly distributed around the cell nucleus. The results showed that lipid deposition occurred in liver L02 cells after the use of 125μM sodium oleate to induce cells.

[0083] 7.2 Drugs containing psoralen for the treatment of non-alcoholic fatty liver disease reduce the deposition of lipid droplets in liver L02 cells

[0084] After inducing liver L02 cells with 125 μM sodium oleate for 24 h, the experimental group was treated with psoralen at concentrations of 0.37 μM, 1.1 μM, and 3.3 μM for 24 h, and the control group (0 μM) was treated with complete medium for 24 h. Then, Nile red staining was used to detect the changes in cell lipid droplets after drug treatment. The number of lipid droplets in liver L02 cells decreased under the action of psoralen at concentrations of 0.37 μM, 1.1 μM, and 3.3 μM in a dose-dependent manner ( Figure 2 A). Compared with the control group, the number of lipid droplets in the drug-treated group containing psoralen for the treatment of non-alcoholic fatty liver disease decreased by about 30%, 33%, and 40%, respectively, and there were significant differences in each group ( Figure 2 B).

[0085] The cells were treated in the same way, and Oil Red O staining was used to detect the changes in lipid droplets in L02 cells treated with psoralen. The results were similar to those of Nile Red staining. Figure 3 Compared with the control group (0 μM), the relative IOD value of lipid droplets was significantly reduced after the treatment of liver L02 cells with drug concentrations of 0.37 μM, 1.1 μM, and 3.3 μM ( Figure 3 A), decreased by 30%, 16%, 47%, and 54% respectively, with significant differences in each group ( Figure 3 B).

[0086] 7.3 Drugs containing psoralen for the treatment of non-alcoholic fatty liver disease regulate the expression of genes related to lipid metabolism

[0087] like Figure 4 As shown in A, the expression of FASN, SREBP1, and CPT1α proteins was detected by Western blotting. The results showed that compared with the control group, the expression levels of FASN and SREBP1 proteins in the sodium oleate-induced group increased by 23% and 35%, respectively ( Figure 4 B). In contrast, the lipid degradation-related gene CPT1α decreased by 34% in the sodium oleate-induced group compared with the control group, indicating that the lipid metabolism homeostasis of L02 cells induced by sodium oleate was disturbed and lipid deposition occurred in the cells. However, psoralen treatment of sodium oleate-induced liver L02 cells can reduce the expression levels of FASN and SREBP1 proteins compared with the control group ( Figure 4 C), and the protein expression levels after treatment with psoralen at concentrations of 0.37μM, 1.1μM, and 3.3μM decreased by 22%, 30%, and 36%, respectively. Similarly, the SREBP1 protein expression levels in the psoralen treatment groups at concentrations of 0.37μM, 1.1μM, and 3.3μM decreased by 21%, 30%, and 45%, respectively, compared with the control group. It can be seen that drugs containing psoralen for the treatment of non-alcoholic fatty liver disease are dose-dependent in regulating lipid metabolism homeostasis in NAFLD cell models.

[0088] The effects of psoralen on the expression of Fasn, Srebp1, and Cpt1α mRNA in liver L02 cells were detected by RT-PCR. Figure 5 As shown in the figure, after treating liver L02 cells with 0.37μM, 1.1μM and 3.3μM psoralen, compared with the control group (model group), the expression level of Fasn mRNA decreased significantly; the expression level of Srebp1 mRNA decreased, but not significantly; the expression level of Cpt1α mRNA increased significantly. In summary, drugs containing psoralen for the treatment of non-alcoholic fatty liver disease can reduce the transcription and translation of genes related to lipid synthesis, increase the transcription and translation of genes related to lipolysis, reduce the synthesis of lipids in liver L02 cells, accelerate the decomposition of lipids, and thus maintain normal lipid metabolism homeostasis.

[0089] 8 Discussions

[0090] The liver is the most important metabolic organ in the human body. The liver transports lipid metabolites to peripheral tissues, or recycles and removes peripheral free lipids. In NAFLD, the liver increases fat uptake and de novo synthesis, and the insufficient fatty acid oxidation level causes the body to be insufficient to maintain normal metabolic levels. Therefore, the present invention uses free fatty acids to simulate the high fatty acid levels of NAFLD patients in vitro. By consulting the literature and previous experiments, it is proved that the use of sodium oleate is better than the traditional inducer-oleic acid. The former has better solubility in the culture medium and less toxicity to cells. The results of Nile red staining showed that the normal human liver L02 cells were induced for 24 hours using sodium oleate at a concentration of 125 μM, and the cells produced severe lipid deposition. The results of protein immunoblotting showed that the expression of lipid synthesis-related proteins in liver L02 cells increased after sodium oleate induction, and the expression of lipid decomposition-related genes decreased. The above results show that the NAFLD cell model was successfully established using sodium oleate to induce L02 cells.

[0091] The present invention uses Nile red staining experiments to prove that the NAFLD cell model can be successfully established by inducing 24 hours with 125 μM sodium oleate. At the same time, protein immunoblotting technology is used to detect the expression changes of lipid synthesis and lipolysis-related genes in the induced group and the control group. The results show that the lipid metabolism of hepatocytes is disordered after sodium oleate induction, which further verifies that the model can well reflect the metabolic level of the liver of NAFLD patients. Secondly, Nile red staining and Oil Red O staining are used to prove that drugs for treating non-alcoholic fatty liver disease containing psoralen can reduce lipid deposition in liver L02 cells, and are dose-dependent. Similarly, protein immunoblotting results also confirm that psoralen can reduce the transcription and translation of genes related to lipid synthesis, increase the transcription and translation of genes related to lipolysis, thereby dynamically regulating the lipid metabolism of liver L02 cells to maintain a balanced state.

Claims

1. A method for preparing a drug for treating non-alcoholic fatty liver disease, characterized in that: The medicine for treating non-alcoholic fatty liver contains psoralen.

2. The method for preparing a drug for treating non-alcoholic fatty liver disease according to claim 1, characterized in that: The preparation method of the drug solution for treating non-alcoholic fatty liver disease containing 100 mM psoralen is as follows: weigh 0.0906 g of psoralen powder into 5 mL of DMSO, and vortex to dissolve.

3. A method for preparing a drug for treating non-alcoholic fatty liver disease according to claim 1 or 2, characterized in that: The drug for treating non-alcoholic fatty liver disease refers to a drug used to treat a human hepatocyte lipid deposition model established by sodium oleate treatment.

4. A method for preparing a drug for treating non-alcoholic fatty liver disease according to claim 1 or 2, characterized in that: The concentration of psoralen in the preparation of the drug for treating non-alcoholic fatty liver disease is 0.3 μM to 3.5 μM.

5. The method for preparing a drug for treating non-alcoholic fatty liver disease according to claim 3, characterized in that: The concentration of psoralen in the preparation of the drug for treating non-alcoholic fatty liver disease is 0.3 μM to 3.5 μM.

6. The method for preparing a drug for treating non-alcoholic fatty liver disease according to claim 4, characterized in that: The psoralen content involved is ≥98%.

7. The method for preparing a drug for treating non-alcoholic fatty liver disease according to claim 5, characterized in that: The psoralen content involved is ≥98%.

8. The method for preparing a drug for treating non-alcoholic fatty liver disease according to claim 3, characterized in that: The human hepatocyte lipid deposition model established by sodium oleate treatment is a human hepatocyte lipid deposition model obtained by inducing liver L02 cells with 125 μM sodium oleate for 24 hours.

9. The method for preparing a drug for treating non-alcoholic fatty liver disease according to claim 5, characterized in that: The human hepatocyte lipid deposition model established by sodium oleate treatment is a human hepatocyte lipid deposition model obtained by inducing liver L02 cells with 125 μM sodium oleate for 24 hours.

10. The method for preparing a drug for treating non-alcoholic fatty liver disease according to claim 7, characterized in that: The human hepatocyte lipid deposition model established by sodium oleate treatment is a human hepatocyte lipid deposition model obtained by inducing liver L02 cells with 125 μM sodium oleate for 24 hours.

Images