KR102171012B1 - Method for diagnosing liver disease or liver cancer using salsolinol and biomarker composition - Google Patents

Abstract

The present invention relates to a method for gender-specific diagnosis of liver disease or liver cancer by measuring the concentration of salsolinol, which is a metabolite in a biological sample, and comparing it with the concentration of salsolinol in a normal subject. In addition to being able to diagnose liver cancer more accurately, it can be usefully used in gender-specific biomarker compositions and kits.

Description

Liver disease or liver cancer diagnosis method and biomarker composition using salsolinol {METHOD FOR DIAGNOSING LIVER DISEASE OR LIVER CANCER USING SALSOLINOL AND BIOMARKER COMPOSITION}

The present invention relates to a method for diagnosing liver disease or liver cancer using metabolite analysis, and to a biomarker composition for diagnosing the same, and more specifically, after measuring the concentration of salsolinol, a metabolite from a biological sample, the measured A method for diagnosing liver disease or liver cancer as a method of providing information for diagnosing liver disease or liver cancer by comparing the salsolinol concentration with the salsolinol concentration of a normal individual, and to a biomarker composition for diagnosing the same.

Alcohol consumption is known to be a major cause of human cancers such as breast cancer, stomach cancer, colon cancer, and liver cancer (Connor, 2017). Alcohol-induced cancer accounts for 5.8% of all cancer deaths worldwide. The reactivity to alcohol is known to differ between sexes (Agabio et al., 2016), because the amount and activity of alcohol dehydrogenase (ADH) involved in alcohol metabolism differ between men and women. . In liver tissue, ADH activity in females is 70% higher than that in males, and blood acetaldehyde levels are higher in women (Quintanilla et al., 2007). Increased production of free radicals due to increased expression of CYP2E1 increased by alcohol intake, polymorphism of alcohol dehydrogenase (ADH) enzyme related to alcohol metabolism due to alcohol-induced cancer, and DNA Damage and DNA adduct generation, increased expression of cytokines related to inflammatory response, increased absorption of carcinogens due to tissue damage, and increased angiogenesis have been suggested. However, there is still no mechanism to explain the gender differences, including studies on the mechanisms of cancer occurrence due to alcohol consumption.

Acetaldehyde, the major metabolite of alcohol, is conjugated with dopamine present in peripheral tissues by salsolinol synthase to form salsolinol (6,7-dihydroxy-1-methyl-). 1,2,3,4-tetrahydroisoquinoline; salsolinol; SAL). SAL is a dopamine-derived catechol isoquinoline, which has been suggested as a pathological factor associated with Parkinson's disease and alcoholism (DeCuypere et al., 2008). Recently, studies showing that SAL causes brain tissue damage is dominant, and there are studies that indicate that SAL induces cell proliferation in breast cancer (Murata et al., 2013), but this is only a very basic study result.

Liver disease or liver cancer, which is most affected by alcohol, shows symptoms after the cancer has progressed considerably, and therefore, often leads to death due to missed treatment time. For liver disease or liver cancer, the timing of treatment and the appropriate treatment method according to that are important. Therefore, research on technology for improving the accuracy of diagnosis of liver disease or liver cancer and applying an efficient treatment method is being conducted.

Patent Document 1 related to a biomarker for liver cancer diagnosis for improving the accuracy of liver disease or liver cancer diagnosis and for efficient treatment is Mir-101 (GenBank Accession Number: NR_029516), Mir-106b (GenBank Accession Number: NR_029831), Mir-122 (GenBank Accession Number: NR_029667), Mir-195 (GenBank Accession Number: NR_029712), Mir-150-5p (GenBank Accession Number: LM379069), Mir-342-3p (GenBank Accession Number: LM379258), Mir-16-5p (GenBank Accession Number: LM378756), Mir-26b-5p (GenBank Accession Number: LM378770) and Mir-22-3p (GenBank Accession Number: LM378764) at least one exosome (exosomal) miRNA selected from the group consisting of a biomarker composition It is disclosed to include. However, Patent Document 1 may be easy to diagnose primary liver cancer, but there is a problem in that there is a limitation in diagnosing liver cancer derived from alcohol.

Accordingly, as a result of the inventors' intensive research efforts to overcome the problems of the prior art, when diagnosing liver disease or liver cancer through analysis of salsolinol, an alcohol metabolite, not only can alcoholic liver disease or liver cancer be diagnosed more accurately. In addition, it was confirmed that it can be used as a gender-specific biomarker, and the present invention was completed.

KR 10-2018-0029936 A

Accordingly, the main object of the present invention is to provide a method of providing information for diagnosing liver disease or liver cancer that can more accurately diagnose liver disease or liver cancer induced by alcohol in a gender specific manner.

Another object of the present invention is to provide a biomarker composition for diagnosing a gender-specific liver disease or liver cancer for providing information for diagnosing the liver disease or liver cancer.

Another object of the present invention is to provide a gender-specific kit for diagnosing liver disease or liver cancer for providing information for diagnosing liver disease or liver cancer.

According to one aspect of the present invention, the present invention,

(a) measuring the concentration of salsolinol from the biological sample; And

(b) comparing the salsolinol concentration of step (a) with the salsolinol concentration of a normal individual; provides a method of providing information for diagnosing liver disease or liver cancer.

Salsolinol, an alcohol metabolite, was treated with salsolinol in a normal hepatocyte cell line to confirm the expression of inflammatory factors, hormone changes, free radical measurements, and cell death. As a result, salsolinol increased the expression of inflammatory genes, hepatic fibrosis-related genes, and hormone receptor factors, and produced reactive oxygen species and induced apoptosis (see Experimental Examples 1 to 4). These results suggest that salsolinol is a factor capable of inducing liver cells or liver tissue damage and causing liver disease or liver cancer. Accordingly, the present inventors tried to provide information for diagnosing liver disease or liver cancer through analysis of salsolinol, an alcohol metabolite, and to diagnose liver disease or liver cancer.

In the method of providing information for diagnosing liver disease or liver cancer of the present invention, the biological sample in step (a) is a gender-specific sample and is a male-specific sample.

In order to confirm the effect of alcohol and salsolinol in males and females, the present inventors confirmed the production of salsolinol in male and female alcohol models, and confirmed that significantly larger amounts of salsolinol were produced in males than in females. (See Experimental Example 5). In addition, the expression of antioxidant enzymes increased in males, but rather decreased in females, and expression of hormone receptor factors was expressed only in males (see Experimental Example 6). These results suggest that the production of salsolinol according to alcohol is gender-specific, that is, male-specific, and salsolinol can be used as a gender-specific biomarker.

In the method for providing information for diagnosing liver disease or liver cancer of the present invention, the biological sample in step (a) may be a biological sample capable of measuring the concentration of salsolinol, preferably tissue, urine, blood, It is characterized in that it is serum, plasma or body fluid.

In the method for providing information for diagnosing liver disease or liver cancer of the present invention, the concentration measurement in step (a) is performed by using a measurement device capable of obtaining information such as the structure and composition of organic, inorganic, polymeric compounds, and biomolecules. Double mass spectrometer, spectrophotometer, nuclear magnetic resonance spectrometer, chromatography, ultraviolet spectrometer, infrared spectrometer, fluorescence spectrometer, ELISA (enzyme-linked immunosorbent assay) capable of measuring the concentration of salsolinol. And it characterized in that the measurement with one or more devices selected from the group consisting of a mass spectrometer.

In the method of providing information for diagnosing liver disease or liver cancer of the present invention, when the salsolinol concentration of step (a) in the step (b) is increased than the salsolinol concentration of a normal individual, the risk group for liver disease or liver cancer It is characterized by determining.

In the method of providing information for diagnosing liver disease or liver cancer of the present invention, the liver disease is characterized in that at least one disease selected from the group consisting of hepatitis, hepatotoxicity, fatty liver, alcoholic liver disease, liver abscess and hepatic atrophy, Preferably, it is characterized in that alcoholic liver disease.

According to an experimental example of the present invention, as a result of confirming the effect of salsolinol in a liver cancer cell line, when salsolinol was treated with a human-derived liver cancer cell line, the male-specific cytotoxicity was exhibited (see Experimental Example 7), and the male Specifically, the antioxidant enzyme Ho-1 was produced (see Experimental Examples 8 and 9), and the expression of HO-1 was induced by phosphorylating Akt specifically in males (see Experimental Example 10). It was confirmed that phosphorylation of phosphorus STAT3 (see Experimental Example 11). These results suggest that since salsolinol is a factor that negatively affects hepatocytes specifically in males, it is possible to provide information for diagnosis of liver disease or liver cancer through the analysis of salsolinol.

According to an experimental example of the present invention, as a result of confirming the effect of salsolinol in male and female normal mouse models, liver tissue was specifically damaged in males (see Experimental Example 12), and male-specific liver tissue inflammatory factors, The expression of hepatic fibrosis factor and epithelial mesenchymal transfer factor was increased (see Experimental Examples 13 and 14), and antioxidant enzymes were produced specifically for males (see Experimental Examples 15 and 16), and DNA damage-related and hormones specifically for males. It was confirmed that the expression of the receptor was increased (see Experimental Examples 17 and 18). These results suggest that salsolinol is a factor that negatively affects liver tissues specifically in males, and that the analysis of salsolinol can provide information for diagnosis of liver disease or liver cancer.

According to another aspect of the present invention, the present invention provides a biomarker composition for diagnosing liver disease or liver cancer comprising a formulation for quantification of salsolinol.

In the biomarker composition of the present invention, the biomarker composition is male-specific.

The formulation for quantification of salsolinol may include any material known to be capable of measuring the concentration of salsolinol.

The biomarker composition may further include a detection reagent. The detection reagent may be a conjugate labeled with a detection agent such as a color developing enzyme, a fluorescent substance, a radioactive isotope, or a colloid. The coloring enzyme may be peroxidase, alkaline phosphatase, or acidic phosphatase, and the fluorescent substance is fluorescein carboxylic acid (FCA), fluorescein isothiocyanate (FITC), fluorescein thiourea (FTH), 7-acetase Toxicoumarin-3-yl, fluorescein-5-yl, fluorescein-6-yl, 2',7'-dichlorofluorescein-5-yl, 2',7'-dichlorofluorescein-6 -Yl, dihydro tetramethylrosamin-4-yl, tetramethylrhodamine-5-yl, tetramethylrhodamine-6-yl, 4,4-difluoro-5,7-dimethyl-4-bora- 3a,4a-diaza-s-indacene-3-ethyl or 4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-s-indacene-3-ethyl , Cy3, Cy5, poly L-lysine-fluorescein isothiocyanate (FITC), rhodamine-B-isothiocyanate (RITC), PE (Phycoerythrin) or rhodamine.

The detection reagent may further include a ligand capable of specifically binding to the detection reagent. The ligand may be a conjugate labeled with a detection agent such as a chromogenic enzyme, a fluorescent substance, a radioisotope or a colloid, and a ligand treated with streptavidin or avidin.

In addition to the detection reagents as described above, the composition of the present invention may contain distilled water or a buffer solution that stably maintains their structure.

According to another aspect of the present invention, the present invention provides a kit for diagnosing liver disease or liver cancer, including a formulation for quantitative analysis or a quantitative analysis device of salsolinol.

In the diagnostic kit of the present invention, the kit is male-specific.

In the diagnostic kit of the present invention, the quantitative analysis device is composed of a double mass spectrometer, a spectrophotometer, a nuclear magnetic resonance spectrometer, chromatography, an ultraviolet spectrophotometer, an infrared spectrometer, a fluorescence spectrometer, an enzyme-linked immunosorbent assay (ELISA), and a mass spectrometer. It is characterized in that at least one quantitative analysis device selected from the group.

The kit may be used to diagnose liver disease or liver cancer on the same principle as the method of providing information for diagnosing liver disease or liver cancer as described above.

The formulation for quantification of salsolinol included in the kit may be any material known to be capable of measuring the concentration of salsolinol.

In the case of diagnosing liver disease or liver cancer using the kit according to the present invention, when the concentration of salsolinol is increased compared to normal subjects, it can be determined as a risk group for liver disease or liver cancer.

As described above, the method according to the present invention can gender-specifically diagnose liver disease or liver cancer by measuring the concentration of salsolinol, a metabolite in a biological sample, and comparing it with the concentration of salsolinol in a normal subject. In addition, since salsolinol is specific to males, the formulation for quantification of salsolinol can be usefully used in biomarker compositions and kits for diagnosing liver disease or liver cancer in males.

1 and 2 are results showing changes in inflammatory cytokine gene expression by SAL in normal hepatocytes.
3 is a result showing changes in hormone expression by SAL in normal hepatocytes.
4 is a result of confirming the production of active oxygen by SAL in normal hepatocytes.
5 is a result of confirming apoptosis by SAL in normal hepatocytes.
6 is a result of confirming the presence or absence of SAL production and SAL synthase activity in male and female mouse acute alcohol models.
7 is a result of confirming the SAL synthase activity in the chronic alcohol model of male and female mice.
8 is a result showing changes in the expression of ALT and AST in male and female mouse alcohol models (20%).
9 is a result of confirming changes in antioxidant enzymes and hormones in male and female mouse alcohol models (20%).
10 is a result of confirming the cytotoxicity by SAL in a human-derived liver cancer cell line.
11 and 12 are results confirming the expression of antioxidant enzymes by SAL in human-derived liver cancer cell lines.
13 and 14 are results showing changes in the expression of HO-1 by SAL in human-derived liver cancer cell lines.
15 to 20 are results showing the antioxidant effect of SAL in human-derived liver cancer cell lines.
Fig. 21 is a result showing changes in expression of a tumor gene (STAT3) by SAL in a human-derived liver cancer cell line.
22 is a result of confirming cell damage by SAL in normal liver tissues of male and female mice.
23 is a result of confirming the change in the expression of inflammatory cytokines by SAL in normal liver tissues of male and female mice.
24 is a result of confirming the change in epithelial mesenchymal metastasis reaction by SAL in normal liver tissues of male and female mice.
25 is a result of confirming the change in the expression of antioxidant enzymes by SAL in normal liver tissues of male and female mice.
26 and 27 are the results of confirming changes in enzyme expression related to redox cycling by SAL in normal liver tissues of male and female mice.
28 to 29 are the results of confirming the changes in the expression of tumor suppressor genes and proteins by SAL in normal liver tissues of male and female mice.
30 is a result showing the change in hormone expression by SAL in normal liver tissues of male and female mice.

Hereinafter, the present invention will be described in more detail through examples. Since these examples are for illustrative purposes only, the scope of the present invention is not to be construed as being limited by these examples.

[Confirmation of the effect of salsolinol in normal cell lines]

In order to confirm the effect of salsolinol in normal hepatocyte cell lines, various tests were conducted by treating salsolinol in mouse normal hepatocyte cell lines, and specific experimental methods and results are as follows.

1) Cell culture and drug treatment

NCTC1469 cells, which are normal mouse hepatocytes, were pre-sold from the Korea Cell Line Bank and received minimal essential media (Dulbecco Modified Eagle Medium, DMEM) (Gibco BRL grand Island, NY, USA) and 10% FBS (HyClone Lab, Inc, Logan, Utah, USA). ), 100 U/ml penicillin and 100 μg/ml streptomycin (Gibco, Grand Island, NY, USA) were added and cultured. Cells were cultured in a 5% CO ₂ state in a 37°C incubator. Salsolinol was purchased from Abcam (Cambridge, MA, USA).

2) Real-time polymerase chain reaction (Quantitative RT-PCR)

After treating the NCTC1469 cells, which is a normal mouse hepatocyte line, with salsolinol according to the time and concentration, trizol was added to lysis, and 0.2 ml of chloroform was added to vortex and then 12,000 rcf. Centrifuged at 15 minutes to take a supernatant. 0.5 ml isopropanol was added thereto, allowed to stand at room temperature for 10 minutes, and then centrifuged at 4° C. and 12,000 rcf for 10 minutes to precipitate RNA. After removing the supernatant, the RNA was washed with 75% ethanol, centrifuged at 7,600 rcf, and the RNA was dried at room temperature. After quantifying the isolated total RNA, the reverse transcription reaction is performed using oligo dT primer and RT enzyme to synthesize cDNA according to the method provided by the manufacturer. Using the synthesized cDNA as a template, each primer and Bio-Rad SYBR mix were added, amplified using a Bio-Rad Real-Time PCR instrument, and analyzed using quantitative software. Products amplified by real time PCR are quantified using a comparative cycle threshold (Ct) method, and inflammatory cytokines (Ccl2, Col1a1, Tgfβ1) and androgen receptors (Ar) are measured in normal hepatocyte cell lines, and in animal liver tissues. Antioxidant enzymes (Nqo1) and hormone receptors (Ar, Erα) were measured. Each sample was corrected with the expression level of β-actin.

3) Western blot analysis

NCTC1469 cells, a normal mouse hepatocyte cell line, were cultured in a 6 well plate for 4 X 10 ⁵ and protein was extracted and Western blotting was performed. After the cultured cells were treated with salsolinol by concentration, the cells were scraped off with a scraper including the medium and centrifuged, and then the cell pellet was washed with PBS and dissolved in lysis buffer for 1 hour. After that, the supernatant was extracted by centrifuging for 15 minutes at 13,000 rpm at 4°C. After protein quantification, 20 μg of protein is subjected to electrophoresis on SDS-polyacrylamide gel and transferred to PVDF membrane for 2 hours. It is reacted using related antibodies including caspase-3 and detected using a detection solution with enhanced chemiluminascence (ECL).

4) Reactive oxygen species measurement

NCTC1469 cells, a normal mouse hepatocyte line, are incubated in a 4 chamber slide at 2.5 X 10 ⁴ for a day. The cultured cells were treated with 100 μM salsolinol and 5 mM of the antioxidant N-acetyl-L-cysteine (NAC) for 6 hours. After washing with PBS, 10 μM of DCF-DA, a fluorescent substance capable of detecting active oxygen in cells, was mixed in Hank's Balanced Salt Solution (HBSS) (Gibco, Grand Island, NY, USA), and then added to the cells. Incubate for minutes. Fluorescent stained cells are identified using a confocal laser scanning microscope (Confocal Microscope).

5) statistical processing

Experimental analysis results were expressed as mean ± SEM, and a T-test was performed for the significance between each experimental group, and a one-way ANOVA (analysis of analysis of Prism 7) (Graph-pad, San Diego, CA, USA) variance) was used to test the significance at *P<0.05, **P<0.01, ***P<0.001 levels after statistical processing.

Experimental Example 1: Confirmation of inflammation in hepatocytes

The effect of SAL on the expression of inflammatory cytokine genes in normal hepatocytes was performed through qPCR, and the results are shown in FIGS. 1 and 2.

As can be seen in FIGS. 1 and 2, after treatment with salsolinol at different concentrations in normal hepatocytes NCTC1469, cell viability test results confirmed that apoptosis was induced in a concentration-dependent manner. In addition, it was confirmed that SAL induces the expression of the inflammation-related genes IL-1β and IL-6 in a concentration and time dependent manner in normal hepatocytes, and it was confirmed that the expression of the fibrosis-related gene Col1a1 was increased by SAL.

Experimental Example 2: Confirmation of Hormonal Change

In order to confirm whether SAL induces a change in hormone expression, the mouse normal cell line was treated with SAL by time, and then the change in hormone expression was observed in the mouse cell line.

As can be seen in Figure 3, it was confirmed that the expression of Androgen receptor (Ar) was significantly increased by SAL.

Experimental Example 3: Measurement of active oxygen

In order to confirm whether SAL induces the production of reactive oxygen species, the production of reactive oxygen species was treated with 100 μM salsolinol and 5 mM of the antioxidant N-acetyl-L-cysteine (NAC) in cultured mouse normal cell lines for 6 hours. It was confirmed, and the results are shown in FIG. 4.

As can be seen in Figure 4, SAL generated reactive oxygen species in normal hepatocytes.

Experimental Example 4: Confirmation of apoptosis in hepatocytes

In order to confirm whether SAL induces apoptosis, the cultured mouse normal cell line was treated with salsolinol to confirm the expression pattern of genes related to apoptosis, and the results are shown in FIG. 5.

As a result, as can be seen in Fig. 5, the expression level of caspase-3, known as apoptosis mediator, is constant, but the expression level of cleaved caspase-3 is increased. As a result, SAL induces PARP-cleavage to induce apoptosis in normal hepatocytes. Can be seen.

[Confirmation of relationship between alcohol and salsolinol in male and female mice]

In order to confirm the relationship between alcohol and salsolinol in male and female mice, various tests were conducted by treating male and female mice with alcohol and salsolinol, and specific experimental methods and results are as follows.

1) Animal testing and alcohol treatment

As for the animals used in the experiment, 7-week-old male and female C57/BL6 mice were sold from Orient Bio (Gyeonggi-do Province, Korea) and had an adaptation period with a standard diet for 1 week, and then the experiment was conducted. The environmental conditions of the laboratory animal room were maintained at 22±2℃ and 55~60% humidity, and the brightness and darkness were adjusted every 12 hours. This animal experiment was conducted in accordance with the regulations of the Laboratory Animal Ethics Committee of Sungshin Women's University. Model 1 administered 63% alcohol (5 g/kg) for a short period of time, and Model 2 administered 20 alcohol (3 g/kg) for one month. After the experiment was completed, the experimental animals were fasted for 4 hours and sacrificed with CO ₂ gas to collect liver tissue. Blood collected from the heart was treated with heparin and then centrifuged at 3,000 rpm for 15 minutes at 4° C. and plasma was separated. The extracted blood and liver tissue were stored at -85°C until used in the experiment.

2) MS analysis

In order to measure salsolinol in plasma and salsolinol synthase in liver tissue, MS analysis was entrusted to the laboratory of Professor Kwang-Pyo Kim of Kyung Hee University to conduct the experiment.

3) Measurement of liver function test index (ALT, AST)

Alanine aminotransferase (ALT) and aspartate aminotransferase (AST), which are representative liver function test indicators in plasma, were measured in the animal laboratory of Seoul National University to obtain information on liver status.

Experimental Example 5: Confirmation of salsolinol production by alcohol

In order to confirm the relationship between alcohol and salsolinol, it was confirmed whether salsolinol was produced in liver tissue and plasma of mice administered with 65% alcohol (5 g/kg) for 1 to 2 hours. It is shown in 6.

As can be seen in FIG. 6, more SAL was detected in plasma of male mice compared to female mice, and Salsolinol Synthase activity was higher in liver tissue of male mice.

In addition, it was confirmed that changes in SAL activity and expression of ALT and AST were measured in liver tissues of mice administered with 20% alcohol (3 g/kg) for one month, and the results are shown in FIGS. 7 and 8.

As can be seen in Figure 7, the SAL synthase activity was high in male mouse liver tissue treated with 20% alcohol for one month.

In addition, as can be seen in Figure 8, there was no change in ALT and AST in both male and female mice.

Experimental Example 6: Confirmation of changes in antioxidant enzymes and hormones

In order to check the changes in antioxidant enzymes and hormones according to alcohol, 20% alcohol (3 g/kg) was administered in the liver tissue of mice for one month, and the results are shown in FIG. 9.

As can be seen in Figure 9, it was confirmed that the antioxidant enzyme Nqo1 significantly increased in male mice, but rather decreased in female mouse liver tissue, and the expression of male hormone receptor (Ar) was significantly increased in male mice.

[Confirmation of the effect of SAL in liver cancer cell lines]

In order to confirm the effect of salsolinol in liver cancer cell lines, various tests were performed by treating salsolinol in human-derived liver cancer cell lines, and specific experimental methods and results are as follows.

1) Cell culture and drug treatment

SK-Hep1 (male) and SNU387 (female) cells, which are human-derived liver cancer cell lines, were pre-sale from the Korea Cell Line Bank, and the minimum essential media (Dulbecco Modified Eagle Medium, DMEM) and (RPMI1640) (Gibco BRL grand Island, NY, USA) And 10% FBS (HyClone Lab, Inc, Logan, Utah, USA), 100 U/ml penicillin and 100 μg/ml streptomycin (Gibco, Grand Island, NY, USA) were added and cultured. . Cells were cultured in a 5% CO ₂ state in a 37°C incubator. Salsolinol was purchased from Abcam (Cambridge, MA, USA).

2) MTT analysis

The Tetrazolium-based colorimetric (MTT) test method is mainly used to study cytotoxicity in cultured cells because it can easily and quickly read many samples. Liver cancer cells are cultured in 48 well plates as much as 2 x 10 ⁵ cells and salsolinol is treated by concentration and time. Dissolve the MTT reagent in warm PBS and incubate in an incubator for 1 hour. After removing the MTT reagent, add DMSO and measure the absorbance.

3) Western blot analysis

The liver cancer cell lines SK-Hep1 (male) and SNU387 (female) cells were cultured in a 60 mm culture vessel for 5 X 10 ⁵ and protein was extracted and Western blotting was performed. After treating the two cultured cells with salsolinol at different times and concentrations, washing them with PBS, scraping the cells with a scraper, dissolving them with lysis buffer for 1 hour, and then at 4℃ for 15 minutes at 13,000 rpm. The supernatant was extracted by centrifugation. After protein quantification, 20 μg of protein is subjected to electrophoresis on SDS-polyacrylamide gel and transferred to PVDF membrane for 2 hours. It is reacted using related antibodies including HO-1 and detected using a detection solution with enhanced chemiluminascence (ECL).

4) Immunocytochemistry

SK-Hep1 (male) cells, a liver cancer cell line, were placed in 12 mm cover glasses in a 12 well plate and cultured for 2.5 X 10 ⁴ and treated with 25 μM salsolinol for 3 hours and 12 hours. After the drug treatment was completed, it was fixed with 2% formalin solution at 37°C for 30 minutes, and permeated for 10 minutes with 0.1% Triton solution. Blocked with 3% BSA solution for 30 minutes, washed 3 times with PBS, treated with HO-1 antibody and Nrf2 antibody diluted 1:100, and left overnight at 4°C. The next day, FITC-anti rabbit antibody was diluted 1:1000, left at room temperature for 1 hour, and mounted using prolong Gold antifade reagent with DAPI. Fluorescent stained cells are identified using a confocal laser scanning microscope (Confocal Microscope).

5) ARE luciferase assay

SK-Hep1 (male) cells, a liver cancer cell line, were transfected with pCMV-β-galactosidase and luciferase reporter gene fusion construct (pGL2) or ARE Luciferase reporter gene for 36 hours and then treated with salsolinol for 12 hours. The activity was measured using Promega luciferase assay systems, and was quantified after analysis using a beta-galactosidase enzyme assay system with Reporter lysis buffer.

Experimental Example 7: Cytotoxicity test (MTT assay)

A cytotoxicity test was performed by treating the human-derived liver cancer cell line by time and concentration, and the results are shown in FIG. 10.

As can be seen in FIG. 10, SAL exhibits cytotoxicity in SK-Hep1 (male), a liver cancer cell line, whereas SNU387 (female) cells do not exhibit toxicity.

Experimental Example 8: Identification of antioxidant enzymes

Human-derived liver cancer cell lines were treated with SAL by concentration and time to confirm the expression levels of the antioxidant enzymes NQO1, TRX, and ho-1 by qPCR and Western blot analysis, and the results are shown in FIGS. 11 and 12.

As can be seen in FIGS. 11 and 12, HO-1 expression was significantly increased in the SK-Hep1 (male) cell line.

Experimental Example 9: Heme oxygenase-1 (HO-1) confirmation

In order to confirm whether SAL induces the expression of Heme oxygenase-1 (HO-1), SK-Hep1 (male), a human-derived liver cancer cell line, was treated with SAL by concentration and time to determine the expression level of ho-1 by qPCR, Western It was confirmed through blot analysis and immunofluorescence, and the results are shown in FIGS. 13 and 14.

13 and 14, SAL induced the expression of HO-1 in a concentration and time dependent manner. In addition, as can be seen in FIGS. 15 and 16, the migration of Nrf2, a major transcription factor regulating HO-1 expression, into the nucleus was induced.

As can be seen in Figure 17, it was confirmed that the ARE-driven luciferase activity was increased, and the expression of Ho-1 was decreased when the Nrf2 gene was knocked down.

Experimental Example 10: ARE luciferase assay

To confirm the antioxidant effect of SAL, human-derived liver cancer cell line SK-Hep1(male) cells were transfected with pCMV-β-galactosidase and luciferase reporter gene fusion construct (pGL2) or ARE Luciferase reporter gene for 36 hours. Salsolinol was treated for 12 hours, and the results are shown in FIG. 18.

In addition, as can be seen in FIGS. 19 and 20, phosphorylation of Akt among the major regulatory proteins of Nrf2 was induced by SAL (FIG. 19 ), and expression of Ho-1 and Nrf2 induced by SAL by Akt pharmacological inhibitors It was confirmed that this decreased (FIG. 20A). In addition, it was confirmed that SAL produced reactive oxygen species, and the expression of Nrf2 and Ho-1 induced by SAL was inhibited by an antioxidant (FIG. 20B).

Experimental Example 11: Confirmation of oncogene (STAT3)

In order to confirm whether SAL induces the expression of the tumor gene (STAT3), the male and female origin liver cancer cell lines SK-Hep1 and SNU-387 were treated with SAL, and then the expression of STAT3 was confirmed using western blot analysis. And the results are shown in FIG. 21.

As a result, referring to FIG. 21, it was confirmed that phosphorylation of STAT3 was increased in the Sk-Hep1 cancer cell line, but not observed in the SNU387 cell line. The expression of proliferating-cell nuclear antigen (PCNA) and 15-PGDH, a molecular indicator of cell proliferation, were also increased by SAL in SK-Hep1 cell line compared to SNU387.

[Confirmation of the effect of SAL in male and female mice]

In order to confirm the effect of salsolinol in male and female mice, various tests were conducted by treating male and female mice with salsolinol, and specific experimental methods and results are as follows.

Experimental Example 12: Confirmation of liver tissue damage

To confirm whether SAL induces cell damage to normal liver tissue, male and female mice (200 mg/kg) were intraperitoneally administered SAL 3 times a week for 4 or 8 weeks, and then the degree of tissue damage was observed. In order to perform H & E tissue staining by taking mouse liver tissue, the results are shown in FIG. 22.

As can be seen in FIG. 22, it was confirmed that inflammatory cell aggregation was increased in male mice than in females in the group administered SAL for 8 weeks.

Experimental Example 13: Confirmation of liver tissue inflammation

To confirm whether SAL expresses the inflammatory cytokines MCP-1 (Ccl2), Col1a1, and Tgfb1 genes in liver tissue, male and female mice (200 mg/kg) were given SAL 3 times a week for 4 or 8 weeks. After intraperitoneal administration, the mouse liver tissue was taken and qPCR was performed, and the results are shown in FIG. 23.

Referring to FIG. 23, it was confirmed that the expression of MCP-1 (Ccl2) in mouse liver tissues treated with SAL for 8 weeks was significantly reduced in males compared to females. Chronic liver injury proceeds to liver fibrosis, which increases the expression of collagen1, an extracellular matrix protein, and significantly in male mouse liver tissue treated with TGFβ1, a biomarker related to hepatic fibrosis, in SAL-treated mouse liver tissues. The expression of the collagen1 gene, Col1a1, was significantly increased in the mouse liver tissue administered for 8 weeks. In contrast, there was no difference in the expression of these genes in female liver tissue.

Experimental Example 14: Confirmation of epithelial mesenchymal metastasis

To confirm whether SAL induces epithelial-mesenchymal transition (EMT) reactions, male and female mice (200 mg/kg) were intraperitoneally administered SAL 3 times a week for 8 weeks, and then mouse liver tissues were administered. Changes in the expression of epithelial mesenchymal metastasis markers were measured in the taken cells, and the results are shown in FIG. 24.

As can be seen in FIG. 24, it was confirmed that the expression of Snai1 was significantly increased in the liver tissues of male mice to which SAL was administered for 8 weeks, whereas there was no change in expression in female mice.

Experimental Example 15: Confirmation of antioxidant enzyme (1)

To confirm whether SAL induces the expression of antioxidant enzymes, male and female mice (200 mg/kg) were intraperitoneally administered SAL 3 times a week for 4 or 8 weeks, and then qPCR was performed with mouse liver tissue. , The results are shown in FIG. 25.

Referring to FIG. 25, it was confirmed that the expression of the antioxidant enzymes SOD1, SOD2, SOD3, CAT, and GPx genes was significantly increased in male mouse liver tissue treated with SAL for 8 weeks. However, it was confirmed that there was no change in the expression of the genes of these antioxidant enzymes in the liver tissues of female mice, and the SOD2 was rather significantly reduced.

Experimental Example 16: Antioxidant Enzyme Confirmation (2)

To confirm whether SAL induces changes in the expression of enzymes related to redox cycling, male and female mice (200 mg/kg) were intraperitoneally administered SAL 3 times a week for 4 or 8 weeks, and then mouse liver tissue was taken. qPCR was performed. In addition, fluorescence staining was performed using γH2AX on mouse liver tissues administered with SAL for 8 weeks, and the results are shown in FIGS. 26 and 27.

As a result, referring to FIGS. 26 and 27, the expression of CYP2E1, NQO1, NQO2 and COMT was significantly induced by SAL in the liver tissue of male mice, whereas the expression of NQO1 was rather significant in the liver tissue of female mice. It was confirmed that it was reduced to. It was confirmed that SAL significantly induced NOX4 expression in male liver tissues, but not in female mice. That is, it can be inferred that SAL increases the expression of NOX4 in the liver tissues of male mice, and participates in redox cycling to induce the production of reactive oxygen species, but it can be assumed that it will not be the case in the liver tissues of female mice. It was found that SAL increased the expression of COMT in the liver tissue of male mice, but did not affect the expression in female mice. In addition, it was found that reactive oxygen species generated by SAL can induce mutations in liver tissues of male mice.

Experimental Example 17: DNA damage-related gene expression confirmation

In order to confirm the change in the expression of genes related to DNA damage in the liver tissues of mice administered with SAL, male and female mice (200 mg/kg) were intraperitoneally administered SAL 3 times a week for 8 weeks, and then liver tissues of mice. The change in the expression of the hormone was observed, and the results are shown in FIGS. 28 and 29. The specific experimental method is as follows.

[Real-time polymerase chain reaction (Quantitative RT-PCR)]

The experiment was conducted by intraperitoneally injecting salsolinol once every two days for 8 weeks to week-old C57BL/6 mice (female, male). After dissection, the collected liver tissue was lysed with Trizol, and then 0.2 ml of chloroform was added to vortex and centrifuged at 12,000 rcf for 15 minutes to obtain a supernatant. 0.5 ml isopropanol was added thereto, allowed to stand at room temperature for 10 minutes, and then centrifuged at 4° C. and 12,000 rcf for 10 minutes to precipitate RNA. After removing the supernatant, the RNA was washed with 75% ethanol, centrifuged at 7,600 rcf, and the RNA was dried at room temperature. After quantifying the separated total RNA, the reverse transcription reaction is performed using oligo dT primer and RT enzyme to synthesize cDNA according to the method provided by the manufacturer. Using the synthesized cDNA as a template, each primer and Bio-Rad SYBR mix were added, amplified using a Bio-Rad Real-Time PCR instrument, and analyzed using quantitative software. Products amplified by real time PCR were quantified using the comparative cycle threshold (Ct) method, and tumor suppressor genes and related proteins (p21, CDK, p53, ATM, etc.) were measured in normal hepatocyte cell lines. Each sample was corrected with the expression level of β-actin.

[Western blot analysis]

The experiment was conducted by intraperitoneally injecting salsolinol once every two days for 8 weeks to 6-week-old C57BL/6 mice (females, males). Then, the liver tissue protein was extracted and Western blotting was performed. After protein quantification, 20 μg of protein is subjected to electrophoresis on SDS-polyacrylamide gel and transferred to PVDF membrane for 2 hours. It is reacted using related antibodies including caspase-3 and detected using a detection solution with enhanced chemiluminascence (ECL).

As a result, referring to FIG. 28, SAL significantly increased the expression of ATM, and although it appears that the expression of Trp53, its regulatory tumor suppressor gene, is increased in the liver tissue of male mice, it is not statistically significant. Was able to confirm. It was found that SAL did not affect Trp53 expression in the liver tissue of female mice. SAL showed a decrease in the expression of Cdkn1a in the liver tissues of male mice, and it was confirmed that the expression of Cdk2 under its control was significantly increased. In addition, an increase in the expression of Cdk4 was also observed. Ultimately, it was predicted that SAL could induce cell proliferation in the liver tissue of male mice. However, SAL could be presumed to be involved in cell cycle arrest by increasing the expression of Cdkn1a in female mice.

In addition, referring to FIG. 29, SAL significantly increases the expression of HPGD in the liver tissue of male mice, whereas the expression of COX2 decreases.

Experimental Example 18: Hormone confirmation

In order to confirm whether SAL induces changes in hormone expression, male and female mice (200 mg/kg) were intraperitoneally administered SAL three times a week for 8 weeks, and then the changes in hormone expression in the liver tissues of the mice were observed. The results are shown in FIG. 30.

As a result, referring to FIG. 30, it was confirmed that the expression levels of ERα (estrogen receptor alpha) and Ar (androgen receptor) were significantly increased in the liver tissue of male mice. However, it was found that no changes in the expression of these receptors were observed in the liver tissues of female mice.

Claims (11)

(a) measuring the concentration of salsolinol from the separated biological sample; And
(b) comparing the salsolinol concentration of the step (a) with the salsolinol concentration of a normal individual; as a method for providing information for diagnosis of liver disease or liver cancer,
The method of providing information for diagnosis of liver disease or liver cancer, wherein the separated biological sample in step (a) is a male-specific sample.
delete The method of claim 1,
The method for providing information for diagnosis of liver disease or liver cancer, wherein the biological sample separated in step (a) is tissue, urine, blood, serum, plasma, or body fluid.
The method of claim 1,
The concentration measurement in step (a) is selected from the group consisting of a double mass spectrometer, a spectrophotometer, a nuclear magnetic resonance spectrometer, a chromatography, an ultraviolet spectrometer, an infrared spectrometer, a fluorescence spectrometer, an enzyme-linked immunosorbent assay (ELISA), and a mass spectrometer. Information providing method for diagnosing liver disease or liver cancer, characterized in that measured with one or more devices.
The method of claim 1,
In the step (b), if the salsolinol concentration in step (a) is higher than the salsolinol concentration of a normal individual, the method for providing information for diagnosis of liver disease or liver cancer, characterized in that it is determined as a risk group for liver disease or liver cancer .
The method of claim 1,
The liver disease is one or more diseases selected from the group consisting of hepatitis, hepatotoxicity, fatty liver, alcoholic liver disease, liver abscess and liver atrophy.
A biomarker composition for diagnosing liver disease or liver cancer comprising a formulation for quantification of salsolinol, wherein the biomarker composition is male-specific.
delete A kit for diagnosing liver disease or liver cancer comprising a quantitative preparation or a quantitative analysis device of salsolinol, wherein the kit is male-specific.
The method of claim 9,
The quantitative analysis device includes at least one quantitative analysis selected from the group consisting of a double mass spectrometer, a spectrophotometer, a nuclear magnetic resonance spectrometer, chromatography, an ultraviolet spectrometer, an infrared spectrometer, a fluorescence spectrometer, an enzyme-linked immunosorbent assay (ELISA), and a mass spectrometer. Liver disease or liver cancer diagnosis kit, characterized in that the device.
delete

Images