CN111759833A - Application of gallic acid in preparing medicine for treating alcoholic liver disease - Google Patents

Abstract

The invention discloses an application of gallic acid in preparation of a medicine for treating alcoholic liver diseases, belonging to the technical field of medicines. According to the invention, an animal model and a cell model of the alcoholic liver disease are established through alcohol stimulation, the intervention effect of gallic acid on alcohol-induced hepatocyte damage and programmed necrosis is observed, and researches show that the gallic acid can remarkably reduce the alcohol-induced hepatocyte damage and inhibit the programmed necrosis of the hepatocyte in-vivo and in-vitro experiments, and a new thought is provided for preventing and treating the alcoholic liver disease.

Description

Application of gallic acid in preparing medicine for treating alcoholic liver disease

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to application of gallic acid in preparation of a medicine for treating alcoholic liver diseases.

Background

The alcoholic liver disease refers to liver disease caused by long-term alcoholism, and the initial stage of the alcoholic liver disease is manifested as alcoholic fatty liver. According to the statistics of the world health organization, the number of people drinking wine globally reaches 23 hundred million, and about 7500 million of people suffer from alcoholic liver diseases. In asia, the incidence of alcoholic liver disease is second in rank among various liver diseases, second only to viral hepatitis. Prolonged alcohol abuse often leads to massive hepatocyte death and a severe inflammatory response of the liver. Without timely and effective intervention measures, alcoholic hepatitis can gradually develop into alcoholic hepatic fibrosis, liver cirrhosis and even liver cancer. Therefore, the safe and effective anti-alcoholic liver disease medicine is imminent and has great significance.

Severe alcoholism often induces extensive hepatocyte death and inflammatory responses. Apoptosis is the core event driving the development of inflammation and is the pathological basis of alcohol-induced liver inflammatory reaction. Programmed necrosis is a high-precision regulation process related to a complex signal network, and is mainly started by tumor necrosis factor receptor 1(TNFR1), Toll-like receptor (TLR) families and the like, death signals are transmitted through receptor interaction protein kinase 1(RIPK1) and RIPK3, mixed series protein kinase-like domains (MLKL) are collected and phosphorylated, the phosphorylated MLKL is combined with each other to form homooligomers, and finally the programmed necrosis process is executed. Apoptotic cells release large amounts of their contents, known as damage-associated molecular patterns (DAMPs), including the high mobility group proteins B1(HMGB1), IL-1 α, IL-33, and the like. DAMPs activate immune cells, causing them to synthesize and secrete inflammatory factors, further triggering apoptosis, forming a vicious circle, severely impeding disease reversal. Programmed necrosis is a core factor for promoting the occurrence and development of alcoholic liver diseases. Finding a drug for inhibiting alcohol-induced programmed hepatocyte necrosis and further improving alcoholic liver disease is of great importance, and has attracted more and more attention at home and abroad.

At present, aiming at the occurrence and development mechanisms of alcoholic liver diseases, a series of medicines and methods for treating the alcoholic liver diseases exist, and the primary treatment measure of the alcoholic liver diseases clinically is to give up wine so as to reverse the early stage alcoholic liver diseases and reduce the death rate of the diseases. However, for patients with severe alcoholic liver disease, liver transplantation is the only fundamental treatment measure, but the clinical application of the liver transplantation is extremely limited due to complicated operation, high cost and scarce donors. Although clinical common medicines such as corticosteroid and pentoxifylline can relieve part of clinical symptoms, the wide application of the medicines is severely limited by factors such as obvious toxic and side effects, large individual difference of curative effect and the like. At present, the intervention of the worldwide medical fort of alcoholic liver disease still lacks strength, the search for a treatment method capable of effectively preventing or reversing the alcoholic liver disease is urgent, and the research and the development of safe and effective treatment medicaments have great significance.

In recent years, the traditional Chinese medicine and the active ingredients thereof have great advantages in the aspect of treatment of alcoholic liver diseases, and the active ingredients of the traditional Chinese medicine become an important treasury for development of new clinical medicines. Gallic acid is a natural plant phenolic compound with the chemical name 3,4, 5-trihydroxybenzoic acid, which is widely found in pomegranate, grapes, nuts, and green tea. It has been shown that gallic acid has effective protective effect on liver injury caused by different causes, such as paracetamol, carbon tetrachloride and methotrexate.

However, there has not been any report on the use of gallic acid in alcoholic liver disease.

Disclosure of Invention

The invention aims to provide application of gallic acid in preparation of a medicine for treating alcoholic liver diseases.

In order to achieve the purpose, the invention adopts the following technical scheme:

according to the invention, a mouse alcoholic liver disease model and an alcoholic liver cell injury model established by a human liver cell line LO2 are adopted, the influence of gallic acid on liver cell injury and programmed necrosis at the whole, cell and molecular level is researched, the anti-alcoholic liver disease activity and molecular mechanism of the gallic acid are determined, and an important basis is provided for preparing the gallic acid into the alcoholic liver disease treatment medicine.

Experimental results show that the gallic acid has excellent activity of resisting alcoholic liver diseases, remarkably improves the liver injury condition of alcoholic liver disease mice, inhibits the activity reduction of liver cells caused by alcohol in vitro, relieves the liver cell injury, and inhibits the programmed necrosis of the liver cells.

The active ingredient of the medicine for treating the alcoholic liver disease is gallic acid, and the medicine is prepared into a clinically practical medicine preparation according to a conventional medicine preparation process, and has obvious curative effects of relieving hepatocyte injury and inhibiting programmed necrosis and resisting the alcoholic liver disease.

Drawings

FIG. 1 is a graph showing the effect of gallic acid on the viability of alcohol-treated hepatocytes in example 2. Human hepatocyte line LO2 was intervened with alcohol (100mM) and/or gallic acid (15, 30, 60 μ M) for 24 hours, CCK-8 kit detects hepatocyte viability Ethanol: alcohol; GA: and (4) gallic acid. Data are presented as mean ± standard deviation. Compared with the control group^###P<0.001; compared with model group^*P<0.05，^**P<0.01，^***P<0.001。

FIG. 2 is a graph showing the effect of gallic acid on alcohol-treated hepatocyte damage in example 2. Human liver cell line LO2 was intervened in with alcohol (100mM) and/or gallic acid (15, 30, 60 μ M) for 24 hr, cell culture fluid was taken, and supernatant was centrifuged to detect liver cell injury index. Wherein: a is AST content, B is ALT content, and C is LDH content. Ethanol: alcohol; GA: and (4) gallic acid. Data are presented as mean ± standard deviation. Compared with the control group^###P<0.001; compared with model group^*P<0.05，^**P<0.01，^***P<0.001。

FIG. 3 shows the effect of gallic acid on alcohol-induced apoptosis of hepatocytes in example 2, human hepatocyte line LO2 was intervened with alcohol (100mM) and/or gallic acid (15, 30, 60. mu.M) for 24 hours, intracellular mRNA was extracted and subjected to Real-time PCR detection, where A is the mRNA expression of RIPK1 and B is the mRNA expression of RIPK3, intracellular total protein was extracted and subjected to Western blot detection, the protein expression (C) of RIPK1 and RIPK3 was detected, β -Actin was used as a reference for protein loading, gray-scale scanning and quantitative statistics (D and E) were performed, F was used as a cell culture solution, supernatant was centrifuged to detect the content of hepatocyte apoptosis index HMGB1, Ethanol was alcohol-induced necrosis of hepatocytes(ii) a GA: and (4) gallic acid. Data are presented as mean ± standard deviation. Compared with the control group^###P<0.001; compared with model group^*P<0.05，^**P<0.01，^***P<0.001。

Detailed Description

The invention will be further illustrated with reference to specific embodiments. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention; furthermore, it should be understood that various changes and modifications can be made by those skilled in the art after reading the disclosure of the present invention, and equivalents fall within the scope of the claims appended to the present application.

Example 1

Investigating the influence of gallic acid on the liver tissue damage of mice

1 materials of the experiment

1.1 Experimental animals

Healthy male ICR mice, weighing 20-25 g, were purchased from the university of southeast university laboratory animal center. Animals eat and drink water freely, and are fed with standard pellet feed in a standard illumination period (12 hours of illumination, 12 hours of night), at room temperature of 21-25 deg.C, and in a constant humidity environment, water is changed every day, and padding is changed every other day.

1.2 Experimental drugs and reagents

And (3) gallic acid: merck, Sigma Aldrich (Sigma-Aldrich), cat #: g7384 MSDS; sodium carboxymethylcellulose: national pharmaceutical group chemical reagents ltd, product number: 1010670204600. red star Erguotou: beijing Red Star corporation, Bar code: 6906785230868. chloral hydrate: national pharmaceutical group chemical reagents ltd, product number: 30037517. aspartate Aminotransferase (AST) kit: nanjing was established as a bioengineering institute, with a cargo number: c010-2-1. Glutamic-pyruvic transaminase (ALT) kit: nanjing was established as a bioengineering institute, with a cargo number: c009-2-1. Alkaline phosphatase (ALP): nanjing was established as a bioengineering institute, with a cargo number: a059-2-2. Lactate Dehydrogenase (LDH) kit: nanjing was established as a bioengineering institute, with a cargo number: a020-2-2. CCK-8 kit: nanjing was established as a bioengineering institute, with a cargo number: g021-1-1. HMGB1 ELISA kit: wuhan Huamei bioengineering, Inc., Cat number: CSB-E08225 m. IL-1. alpha. ELISA kit: abcam, cat # cat: ab 199076.

1.3 Molding and administration

The method comprises randomly dividing 60 mice into 4 groups, namely a normal control group, a model group, a gallic acid low dose group and a gallic acid high dose group, wherein each group comprises 15 mice, and the four groups except the normal control group are subjected to intragastric lavage by using 56% (v/v) alcohol, wherein the weight of the mice is 10mg/kg once a day and the time lasts for 4 weeks.

In addition, the gallic acid treatment group was administered with sodium carboxymethylcellulose solution of gallic acid by intragastric administration, and the gallic acid dose of the low dose group and the high dose group was 50mg/kg body weight and 100mg/kg body weight, respectively, once a day for 4 weeks. The normal group and the model group were gavaged with the corresponding amount of distilled water and/or 0.3% sodium carboxymethylcellulose (table 1). After the experiment, each mouse was weighed and anesthetized by intraperitoneal injection of 10% chloral hydrate (350mg/kg), and after blood was drawn from the orbit, the abdominal cavity was dissected open to isolate the complete liver. After the blood of the mouse is kept stand for 2 hours, the blood is centrifuged for 15 minutes at 3000 r/min, and the supernatant is taken and frozen at minus 20 ℃ for standby.

TABLE 1 Experimental groups and treatment protocols

2 contents of the experiment

2.1 serum liver function index (AST, ALT, ALP, LDH)

Detection was performed using a semi-automated biochemical analyzer.

2.2 serum indices for programmed necrosis (HMGB1, IL-1. alpha.)

Detection was performed using a semi-automated biochemical analyzer.

3 results of the experiment

3.1 Gallic acid can significantly reduce alcohol-induced liver injury in mice

During the development process of alcoholic liver disease, liver cells are continuously subjected to alcohol toxicity, and cell destruction and death are caused. AST, ALT, ALP and LDH synthesized and secreted by the hepatocytes are released from the hepatocytes into the blood through the cell membrane, so that the content in the blood is significantly increased. Therefore, serum levels of AST, ALT, ALP, and LDH may well reflect the degree of hepatocyte damage clinically.

Compared with a normal control group, the activity of the AST, ALT, ALP and LDH of the serum of the model group is obviously increased; the results of gallic acid reducing the serum levels of these three enzymes in alcoholic liver disease mice compared to the model group are shown in table 2.

TABLE 2 serum indices AST, ALT, ALP and LDH of each group of mice (mean. + -. standard deviation)

Note: compared with the normal group^###P<0.001, compared to model group^*P<0.05，^**P<0.01，^***P<0.001

3.2 Gallic acid can significantly reduce programmed necrosis of liver cell in mice with alcoholic liver disease

In the process of alcoholic liver disease progression, damage of liver cells is aggravated, programmed necrosis of liver cells is induced, and the necrotic liver cells release danger related molecular patterns, wherein non-inflammatory key genes HMGB1 and inflammatory factors IL-1 alpha are taken as representatives, so that the levels of the two proteins in serum are obviously increased.

Compared with a normal control group, the serum content of HMGB1 and IL-1 alpha in the model group is obviously increased; the results of gallic acid reducing the serum content of these two proteins in alcoholic liver disease mice compared to the model group are shown in table 3.

TABLE 3 serum hepatocyte necrosis index HMGB and IL-1. alpha. detection results (mean. + -. standard deviation) for each group of mice

Note: compared with the normal group^###P<0.001, compared to model group^*P<0.05，^**P<0.01

The results show that the liver injury indexes AST, ALT, ALP and LDH in the serum of the mice in the alcoholic liver disease model group and the procedural necrosis related indexes HMGB1 and IL-1 alpha are obviously increased compared with the normal group, so that the method has obvious significance; the gallic acid has good liver protection effect, and the liver injury index and the programmed necrosis index are reduced more obviously with the increase of the dosage of the gallic acid, and the gallic acid has significant difference.

4 small knot

The gallic acid can obviously improve liver injury and programmed hepatocyte necrosis in the alcoholic liver disease in vivo.

Example 2

Culturing human hepatocyte line LO2 in vitro, treating with alcohol to establish alcoholic hepatocyte injury model, and observing the influence of gallic acid on alcohol-induced hepatocyte activity, injury and programmed necrosis.

1 materials of the experiment

1.1 test cells

The human hepatocyte LO2 cell line was purchased from shanghai cell bank of the national academy of sciences of shanghai.

1.2 Experimental drugs and reagents

Dimethyl sulfoxide: national pharmaceutical group chemical reagents ltd, product number: ZA 3648001. Anhydrous ethanol: national pharmaceutical group chemical reagents ltd, product number: 10009259. dulbecco's Modified Eagle Medium (DMEM): gibico, Inc., Cat number: 12100-061. Fetal bovine serum: gibico, Inc., Cat number: 10099. penicillin G sodium salt: biosharp company, cat # No.: BS 215B. Streptomycin sulfate: biosharp company, cat # No.: BS 077B. Pancreatic enzyme cell digestive juice: biosharp company, cat # No.: BL 501A. Ethylene diamine tetraacetic acid: biosharp company, cat # No.: BS 039B. CCK-8: nanjing was established as a bioengineering institute, with a cargo number: g021-1-1. Primers for Real-time PCR experiments were designed and synthesized by Kinsley Biotechnology Ltd (Table 4). 75% of ethanol: national pharmaceutical group chemical reagents ltd, product number: 80176961. chloroform: national pharmaceutical group chemical reagents ltd, product number: 10006818. diethylpyrocarbonate water (DEPC): biosharp company, cat # No.: BL 510A. Trizol reagent: thermo Scientific, usa, cat #: 15596018. real-time PCR reverse transcription kit: japan Takara corporation, cat number: RR 037A. An amplification kit: japan Takara corporation, cat number: RR 430A. RIPA lysate: biosharp company, cat # No.: BL 504A. Phenylmethylsulfonyl fluoride (PMSF): biosharp company, cat # No.: BS 507A. And (3) skim milk powder: biosharp company, cat # No.: BS 075B. Bovine Serum Albumin (BSA): biosharp company, cat # No.: BS 043D. Phosphatase inhibitor cocktail: kakiky biotechnology limited, cat #: KGP 602. Pre-staining protein marker: U.S. ThermoScientific, cat number: 26616. bisquinolinecarboxylic acid (BCA) protein quantification kit: bi yun tian biotechnology, cat #: and (3) P0012. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) protein loading buffer (5 ×): bi yun tian biotechnology, cat #: P0015L. Methanol: national pharmaceutical group chemical reagents ltd, product number: 10014108. tween 20: national pharmaceutical group chemical reagents ltd, product number: 30189328. polyvinylidene fluoride membrane (PVDF): millpore, USA, Cat No.: IPFL 00010. ECL chemiluminescence kit: millpore, USA, Cat No.: 345818. RIPK1 antibody: santa Cruz Biotechnology (Santa Cruz Biotechnology), cat no: sc-133102. RIPK3 antibody: santa cruz biotechnology, cat #: sc-374639. Beta-actin antibodies: santa cruz biotechnology, cat #: sc-8432. HMGB1 ELISA kit: nanjing Senega Biotech, Inc., cat #: SBJ-H0321.

TABLE 4 primers used for Real-time PCR

2 contents of the experiment

2.1 cell culture

The hepatocytes were cultured in DMEM medium containing 10% fetal bovine serum, 100U/mL penicillin and 100. mu.g/mL streptomycin. The cell culture box was set at 37 ℃ and 5% CO₂。

2.2CCK-8 cell viability assay

Logarithmic phase cells were collected, cell suspension concentration was adjusted and divided into 96 well plates, 180. mu.L per well, approximately 3 × 10 per well⁴And (4) cells. Placing the cells in a culture medium to continue culturing until the cell fusion degree reaches 50-70%. Adding 100mM ethanolAnd/or gallic acid (15, 30, 60. mu.M), and culturing was continued for 24 hours. Add 10. mu.L CCK-8 solution to each well, continue to culture for 4 hours, detect the absorbance value at 450nm on an ELISA. In this experiment, both background (no cells, only medium) and control wells (cells, same concentration of drug solvent, medium, CCK-8) were set. Each group is provided with 5-6 multiple holes. The experiment was repeated 3 times.

2.3 Biochemical indicator detection of hepatocyte injury

Logarithmic phase cells were collected, cell suspension concentration was adjusted and divided into 96 well plates, 180. mu.L per well, approximately 3 × 10 per well⁴And (4) cells. Placing the cells in a culture medium to continue culturing until the cell fusion degree reaches 50-70%. 100mM ethanol and/or gallic acid (15, 30, 60. mu.M) were added thereto, and the culture was continued for 24 hours. Taking the culture medium, centrifuging and taking the supernatant, and detecting the activity of AST, ALT and LDH enzymes in the culture medium supernatant according to a kit instruction method.

2.4Real-time PCR

Extraction of RNA

LO2 cells were seeded in 10cm diameter cell culture dishes in logarithmic growth phase. Placing the cells in a culture medium to continue culturing until the cell fusion degree reaches 50-70%. 100mM ethanol and/or gallic acid (15, 30, 60. mu.M) were added thereto, and the culture was continued for 24 hours. 1mL of Trizol reagent was added to each dish, and the plate was blown with a pipette under ice-bath conditions and incubated at room temperature for 5 minutes to allow sufficient dissociation of the nucleic acid-protein complex. 0.2mL of chloroform was added to each 1mL of the Trizol reagent homogenate and the vial cap was closed. After shaking the tube manually vigorously for 15 seconds, it was incubated at room temperature for 3 minutes. Centrifugation was carried out at 12000 rpm at 4 ℃ for 15 minutes. After centrifugation, the upper colorless aqueous phase was carefully aspirated and transferred to a new centrifuge tube. 0.5mL of isopropyl alcohol was added, mixed well, incubated at room temperature for 10 minutes, and centrifuged at 12000 rpm at 4 ℃ for 10 minutes. The colloidal precipitate formed at the bottom and on the side walls of the tube was RNA. As much supernatant as possible was removed, 50. mu.L of 75% ethanol was added, and the RNA pellet was washed thoroughly. After shaking, the cells were centrifuged at 7500 rpm at 4 ℃ for 5 minutes. The ethanol solution was removed and the RNA pellet was dried in a fume hood. Resuspend RNA with DEPC and incubate in an oven at 55 ℃ for 10 minutes for adequate lysis. Detecting A260/A280 value, taking a sample between 1.8-2.1 for reverse transcription. RNA was subpackaged and stored in an ultra-low temperature freezer at-80 ℃.

Reverse transcription of RNA

The total volume of reverse transcription for each RNA sample was 20. mu.L, containing 4. mu.L of 5 × ipt reaction mix, 1. mu.L of ispriptreverse transcriptase, and appropriate amounts of RNA template and DEPC water. Fully mixing and centrifuging to ensure that the liquid does not hang on the wall. Reverse transcription was performed under the following conditions: 15 minutes at 42 ℃; 5 minutes at 95 ℃; 0-5 ℃ for 5 minutes, and the final system was maintained at 4 ℃. The cDNA was stored at-20 ℃.

cDNA amplification

The total reaction system was 20. mu.L, containing 20. mu.L supermix (TaKaRa), 2. mu.L primer (F) 10. mu.M, 2. mu.L primer (R) 10. mu.M, 2. mu.L (100ng) cDNA and 4. mu.L water. Mix well and centrifuge, PCR program set up (50 cycles), each cycle conditions at 94 ℃ for 5 minutes; 30 seconds at 94 ℃; 45 seconds at 55 ℃; 30 seconds at 72 ℃; 7 minutes at 72 ℃. The final system temperature was maintained at 4 ℃.

d. Result processing

By 2^-ΔΔCtThe method carries out statistical analysis on the result;

with reference to gene C_TValue of C of target gene_TNormalizing the value;

△ C for calibration of samples_TValue △ C of experimental sample_TNormalizing the value;

the relative expression levels of the various RNAs were calculated.

e. Matters of Experimental attention

The homogenizer is cleaned by tap water, soaked with acid overnight, washed by deionized water, cleaned by DEPC water once and dried for later use.

Cleaning metal products such as tweezers with deionized water, soaking in DEPC water, autoclaving, and oven drying.

In the operation process, the disposable mask and the gloves are worn, and once the gloves contact suspicious pollutants, the gloves are replaced by new gloves.

2.5Western blot：

a. Total protein extraction

After the cells in the six-well plate are treated by adding medicine for 24 hours, 150 mu L of RIPA protein lysate and PMSF mixed solution (100: 1) of protease inhibitor are added and fully ground. The homogenate was aspirated into a centrifuge tube, ice-washed for 30 minutes, centrifuged at 12000 rpm at 4 ℃ for 15 minutes, and the supernatant solution was aspirated by a pipette.

b. Protein concentration determination

Protein concentration was measured according to the BCA method, which was performed as follows:

(1) diluting the protein to be detected by 20 times and 40 times respectively in a pore plate by using deionized water;

(2) BCA reagent as A: b is 50: preparing a BCA working solution according to the proportion of 1, and fully mixing. Adding 200 mu L of working solution into each hole of a 96-hole plate;

(3) adding 1mg/mL standard protein solution into a standard substance well of a 96-well plate according to the volume of 0, 1, 2, 4, 8, 12, 16 and 20 muL, adding 1 muL of sample into the 96-well plate, and adding standard substance diluent to make up to 20 muL; at the same time, 20. mu.L of diluted protein to be tested was added to each well and incubated at 37 ℃ for 30 minutes.

(4) And (3) measuring the absorbance of the sample at 562nm by using the microplate reader, drawing a standard curve by using the OD value as an abscissa and the concentration as an ordinate, and calculating the protein concentration of the sample according to the standard curve.

c. Polyacrylamide gel electrophoresis

(1) The reagents and methods required for the preparation of the separation gel (two gels) are shown in Table 5.

TABLE 5 separation gel concentration and formulation

(2) The reagents and methods required for the preparation of 4% concentrated gum (two-piece gum) are shown in Table 6.

TABLE 64% concentrated gum formulation

Cleaning and drying the glass plates, building an experimental device, fixing the glass plates, filling the separation glue prepared according to the above table between the glass plates, sealing with deionized water, and polymerizing at room temperature. When observed under light, when a clear interface appears between the water seal layer and the separation glue layer, the separation glue is fully polymerized. The deionized water was poured off and blotted dry with filter paper, and then 4% concentrated gum was poured in and a comb was inserted. Preparing 1 Xelectrophoresis buffer solution, taking out the glass plate after the gel is solidified, building an electrophoresis device, adding the electrophoresis buffer solution and carefully removing a comb. Protein samples were denatured by adding 6 Xloading buffer and heating in boiling water for 10 min. The loading volume was converted to the loading amount of 50. mu.g/sample of protein, and an appropriate amount of protein was aspirated and electrophoresis was started after loading. Electrophoresis was performed at a constant current of 40mA per gel, and was terminated when the desert blue had just escaped.

d. Rotary film

1 Xtransmembrane buffer was prepared in advance and cooled to 4 ℃ (formulation: 100mL of 10 Xtransmembrane buffer, 200mL of methanol, 700mL of deionized water). The PVDF membrane was soaked in methanol for 5 minutes before the start of the membrane transfer. And (4) taking out the clamp after the electrophoresis is stopped, taking out the glass, and removing the redundant part of the concentrated gel. In the membrane transferring buffer solution, putting the sponge, the filter paper, the gel, the PVDF membrane, the filter paper and the sponge in sequence, paying attention to remove all bubbles, putting the membrane transferring buffer solution into a membrane transferring groove in a black-to-black direction, and pouring the membrane transferring buffer solution. The film is rotated for 90 minutes under the condition of ice bath at constant pressure of 100V. The protein was identified as being up-converted by ponceau staining (formula: 0.1g ponceau, 5mL glacial acetic acid, water to 100 mL). The PVDF membrane is marked to confirm the protein surface, and after dyeing, the membrane can be cut open according to the indication of a protein Marker and the size of a target protein so as to combine different antibodies. After dyeing, washing with deionized water until no dye color exists, and then sealing.

e. Immune response

TBS-T was used to prepare a 5% nonfat dry milk blocking solution, and the PVDF membrane was placed in the blocking solution and blocked for 2 hours. The PVDF membrane was removed from the blocking solution by forceps while avoiding the protein, washed with TBS-T, and then placed in a primary antibody containing the corresponding ratio, and bound overnight at 4 ℃. The preparation method of the primary antibody comprises the following steps: BSA was dissolved in TBS-T to a concentration of 5% and 10% NaN3 stock solution (for preservation) was added, diluted to 0.1% for use, and the corresponding amount of primary antibody was added at the primary antibody dilution ratio. Typically around 4mL is used for the formulation volume of the primary antibody.

The primary antibody is recovered and then the secondary antibody is bound. The PVDF membrane was rinsed three times in TBS-T from the primary antibody, each time for 10 minutes (small multiple times). The secondary antibody was diluted with 5% skim milk powder in TBS-T at the specified dilution ratio, added to the membrane and incubated on a shaker at room temperature for 2 hours.

f. Chemiluminescence, development and fixation

Putting a PVDF film on a preservative film, mixing a proper amount of equal-volume liquid A and liquid B in an ECL chemiluminescence kit, adding the mixture on the surface of the film after uniformly mixing, and imaging the strip by a gel imager.

3 results of the experiment

3.1 Gallic acid enhances hepatocyte viability with alcohol treatment

The alcohol treatment can cause the decrease of the activity of the liver cells, and the decrease of the cell activity indicates that the alcohol has cytotoxic effect on the liver cells. Compared with a normal control group, the cell activity of the liver cells of the model group is obviously reduced, and a more obvious cytotoxic effect is generated; gallic acid significantly enhanced the viability of the alcohol-treated hepatocytes compared to the model group, as shown in figure 1.

3.2 Gallic acid inhibits alcohol-induced liver cell injury

Alcohol exerts a continuous cytotoxic effect on hepatocytes, not only causes the decrease in the viability of hepatocytes, but also causes the destruction and death of cells. After the liver cells are damaged, enzymes such as AST, ALT and LDH in the cells are released to the outside of the cells through cell membranes, so that the enzyme content in a cell culture medium is increased, and the AST, ALT and LDH levels in the culture medium can well reflect the damage degree of the liver cells.

Compared with a normal control group, the AST, ALT and LDH activities of the serum of the model group are obviously increased; compared with the model group, gallic acid reduced the contents of these three enzymes in the serum of the alcoholic liver disease mice, as shown in fig. 2.

3.3 Gallic acid inhibits alcohol-induced apoptosis of hepatocytes

Real-time PCR results show that compared with a normal control group, the mRNA levels of RIPK1 and RIPK3 in the liver cells of the model group are obviously increased; mRNA levels of RIPK1 and RIPK3 were significantly reduced in gallic acid-treated hepatocytes compared to the model group, as shown in fig. 3A and 3B.

Western blot results show that the protein levels of RIPK1 and RIPK3 in the liver cells of the model group are obviously increased compared with those of a normal control group; the protein levels of RIPK1 and RIPK3 were significantly reduced in gallic acid-treated hepatocytes compared to the model group, as shown in fig. 3C-E.

ELISA results show that compared with a normal control group, the protein level of HMGB1 in the culture medium of the model group is obviously increased; compared to the model group, protein levels of HMGB1 were significantly reduced in the gallic acid-treated group medium, as shown in fig. 3F.

The results show that the alcohol treatment causes the significant decline of the activity of the liver cells, the liver cell damage indexes AST, ALT and LDH in the culture medium are significantly increased, the expression of procedural necrosis related indexes RIPK1 and RIPK3 in the liver cells and the release of HMGB1 and IL-1 alpha are significantly increased compared with the normal group, and the alcohol treatment has significant significance; the gallic acid has good effect of protecting liver cells, and the damage index and the programmed necrosis index of the liver cells are obviously reduced along with the increase of the dosage of the gallic acid, and the gallic acid has significant difference.

4. Small knot

The gallic acid can remarkably improve alcohol-induced hepatocyte injury and hepatocyte programmed necrosis in vitro.

Sequence listing

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Application of <120> gallic acid in preparation of medicine for treating alcoholic liver disease

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Claims (7)

1. Application of gallic acid in preparing medicine for treating alcoholic liver disease is provided.

2. Use according to claim 1, characterized in that: the gallic acid can reduce the content of AST, ALT, ALP and LDH in blood serum.

3. Use according to claim 1, characterized in that: the gallic acid can reduce HMGB1 and IL-1 alpha content in serum.

4. Use according to claim 1, characterized in that: the gallic acid can enhance cell activity of liver cells.

5. Use according to claim 1, characterized in that: the gallic acid can reduce release of AST, ALT and LDH by liver cells.

6. Use according to claim 1, characterized in that: the gallic acid can reduce the expression of RIPK1 and RIPK3 in hepatocytes.

7. Use according to claim 1, characterized in that: the gallic acid can reduce release of HMGB1 from hepatocytes.

Images