KR20190129765A - Method for diagnosing rheumatoid arthritis by using metabolomics 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 of a normal individual. In addition to more accurate diagnosis of liver cancer, it can be usefully used in gender specific biomarker compositions and kits.

Description

METHOD FOR DIAGNOSING RHEUMATOID ARTHRITIS BY USING METABOLOMICS AND BIOMARKER COMPOSITION}

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

Alcohol consumption is known to be a major cause of human cancer such as breast cancer, stomach cancer, colon cancer, liver cancer (Connor, 2017). Alcohol-induced cancer accounts for 5.8% of global cancer deaths in total cancer deaths. Responsiveness to alcohol is known to differ between genders (Agabio et al., 2016), because the amount and activity of alcohol dehydrogenase (ADH) involved in alcohol metabolism differs between men and women. . ADH activity of females in liver is 70% higher than that of males, resulting in higher levels of acetaldehyde in women (Quintanilla et al., 2007). Alcohol-induced cancer mechanisms result in increased production of free radicals due to polymorphism of alcohol dehydrogenase (ADH) enzymes related to alcohol metabolism, folate degradation, and increased expression of CYP2E1 due to increased alcohol intake, and thus DNA Damage and DNA adduct production, increased cytokine expression related to inflammatory responses, increased absorption of carcinogens due to tissue damage, and increased angiogenesis have been suggested. However, there are no mechanisms to explain gender differences, including mechanism studies on cancer incidence from alcohol consumption.

Acetaldehyde, a major metabolite of alcohol, is conjugated with dopamine present in peripheral tissues by salsolinol synthase, and salsolinol (6,7-dihydroxy-1-methyl-). 1,2,3,4-tetrahydroisoquinoline; salsolinol; SAL). SAL is a dopamine-derived catechol isoquinoline (dopamine-derived catechol isoquinoline) that has been raised as a pathological factor associated with Parkinson's disease and alcoholism (DeCuypere et al., 2008). In recent years, research has focused on SALs causing brain tissue damage, and although SALs induce cell proliferation in breast cancer (Murata et al., 2013), these are only basic findings.

Liver disease or liver cancer, which is most affected by alcohol, often causes symptoms after the cancer has progressed so that the treatment is often missed, leading to death. For liver disease or liver cancer, the timing of treatment and appropriate treatment methods are important. Therefore, research is being conducted on techniques for increasing the accuracy of liver disease or liver cancer diagnosis and applying an effective treatment method.

Patent document 1 related to a biomarker for diagnosing liver cancer for improving the accuracy and effective treatment of liver disease or liver cancer diagnosis 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 Biomarker composition comprising at least one exosomal miRNA selected from the group consisting of Accession Number: LM378756), Mir-26b-5p (GenBank Accession Number: LM378770) and Mir-22-3p (GenBank Accession Number: LM378764). 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 limit in diagnosing liver cancer substantially derived from alcohol.

Accordingly, the present inventors have made diligent research to overcome the problems of the prior art, and when diagnosing liver disease or liver cancer through the alcohol metabolism salsolinol analysis, not only alcoholic liver disease or liver cancer can be diagnosed more accurately. As a result, it has been confirmed that the present invention can be used as a gender specific biomarker, thereby completing the present invention.

KR 10-2018-0029936 A

Accordingly, a main object of the present invention is to provide a method for providing information for diagnosing liver disease or liver cancer which can more accurately diagnose gender-specific liver disease or liver cancer derived from alcohol.

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

According to one aspect of 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; and providing an information providing method for diagnosing liver disease or liver cancer.

In order to confirm the effect of the alcohol metabolism Salsolinol in liver cells, salsolinol was treated in normal hepatocytes to confirm the expression of inflammatory factors, hormonal changes, free radicals and cell death. As a result, salsolin increased the expression of inflammatory genes, hepatic fibrosis related genes and hormone receptor factors, produced free radicals and induced cell death (see Experimental Examples 1 to 4). These results suggest that salsolin is a factor capable of inducing damage to hepatocytes or liver tissues to cause liver disease or liver cancer. Accordingly, the present inventors have attempted to provide information for diagnosing liver disease or liver cancer and to diagnose liver disease or liver cancer by analyzing salsolinol, which is an alcohol metabolite.

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

In order to confirm the effects of alcohol and salsolinol in males and females, the inventors confirmed that salsolinol was produced in male and female alcohol models, and that salsolinol was produced in males more than females. (See Experimental Example 5). In addition, the expression of antioxidant enzymes increased in males but decreased in females, whereas the expression of hormone receptor factor was expressed only in males (see Experimental Example 6). These results suggest that the production of salsolinol according to alcohol is gender specific, ie male specific, and that 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 of step (a) may be a biological sample capable of measuring the concentration of salsolin, and preferably, tissue, urine, blood, It is characterized in that the serum, plasma or body fluids.

In the method for providing information for diagnosing liver disease or liver cancer of the present invention, the concentration measurement in step (a) may be performed using a measuring device that can obtain information such as organic, inorganic, high molecular compound, structure and composition of biological molecules. Dual mass spectrometers, spectrophotometers, nuclear magnetic resonance spectroscopy, chromatography, ultraviolet spectroscopy, infrared spectroscopy, fluorescence spectroscopy, and enzyme-linked immunosorbent assay (ELISA) capable of measuring the concentration of salsolin And at least one device selected from the group consisting of mass spectrometers.

In the method for providing information for diagnosing liver disease or liver cancer of the present invention, when the salsolinol concentration of step (a) in step (b) is higher 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 for providing information for diagnosing liver disease or liver cancer of the present invention, the liver disease is at least one disease selected from the group consisting of hepatitis, hepatotoxicity, fatty liver, alcoholic liver disease, liver abscess and liver atrophy, Preferably it is characterized by alcoholic liver disease.

According to one experimental example of the present invention, as a result of confirming the effect of salsolinol in the liver cancer cell line, when Salsalin was treated to a human-derived liver cancer cell line, the male specifically showed cytotoxicity (see Experimental Example 7), and male Ho-1, an antioxidant enzyme, was specifically produced (see Experimental Examples 8 and 9), and Akt was specifically phosphorylated in males to induce the expression of HO-1 (see Experimental Example 10). Phosphorylation of phosphorus STAT3 (see Experimental Example 11) was confirmed. These results suggest that salsolinol is a factor that negatively affects hepatocytes specifically for males, and thus the salsolinol analysis may provide information for diagnosing liver disease or liver cancer.

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, male-specific liver tissue was damaged (see Experimental Example 12), male-specific liver tissue inflammation factor, Expression of hepatic fibrosis factor and epidermal mesenchymal metastasis factor was increased (see Experimental Examples 13 and 14), and antioxidant enzymes were produced specifically for males (see Experimental Examples 15 and 16). It was confirmed that the expression of the receptor is increased (see Experimental Examples 17 and 18). These results suggest that salsolinol is a male-specific factor that negatively affects liver tissue, and the analysis of salsolinol may provide information for diagnosing 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, which comprises an agent for quantifying salsolinol.

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

Formulation of salsolinol may include any substance known to measure the concentration of salsolinol.

The biomarker composition may further include a detection reagent. The detection reagent may be a conjugate labeled with a detector such as a chromophore, a fluorescent substance, a radioisotope or a colloid. The chromophore can be peroxidase, alkaline phosphatase or acidic phosphatase, and the fluorescent material can be fluorescein carboxylic acid (FCA), fluorescein isothiocyanate (FITC), fluorescein thiourea (FTH), 7-ace Toxycoumarin-3-yl, Fluorescein-5-yl, Fluorescein-6-yl, 2 ', 7'-dichlorofluorescein-5-yl, 2', 7'-dichlorofluorescein-6 -Yl, dihydro tetramethyllosamine-4-yl, tetramethyldamin-5-yl, tetramethyldamin-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 that can specifically bind to the detection reagent. The ligand may be a conjugate labeled with a detector such as a chromophore, a fluorescent substance, a radioisotope or a colloid, and a ligand treated with streptavidin or avidin.

In addition to the detection reagents described above, the composition of the present invention may include distilled water or a buffer to stably maintain their structure.

According to another aspect of the present invention, the present invention provides a kit for diagnosing liver disease or liver cancer, comprising a quantitative preparation 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 analyzer is composed of a dual mass spectrometer, spectrophotometer, nuclear magnetic resonance spectrometer, chromatography, ultraviolet spectrometer, infrared spectrometer, fluorescence spectrometer, ELISA (enzyme-linked immunosorbent assay) and mass spectrometer It is characterized in that the at least one quantitative analysis device selected from the group.

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

Formulation of salsolinol quantitatively included in the kit may be included as long as it is known to measure the concentration of salsolinol.

When diagnosing liver disease or liver cancer using the kit according to the present invention, when the concentration of salsolinol is increased compared to normal individuals, it may be determined as a liver disease or liver cancer risk group.

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, which is a metabolite in a biological sample, and comparing it with the concentration of salsolinol in normal individuals. In addition, since salzolinol is male-specific, the agent for quantifying salsolinol may be usefully used in biomarker compositions and kits for diagnosing liver disease or liver cancer in males.

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

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

[Identification of Salsolinol Effect on Normal Cell Line]

In order to determine the effect of salsolinol in normal hepatocytes, various tests were performed by treating salsolin in normal hepatocytes. Specific experimental methods and results are as follows.

1) Cell Culture and Drug Treatment

NCTC1469 cells, which are mouse normal hepatocytes, were distributed from Korea Cell Line Bank (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 incubated at 37% incubator at 5% CO ₂ . Salsolinol was purchased from Abcam (Cambridge, Mass., USA).

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

Salzolinol was treated to NCTC1469 cells, a normal mouse liver cell line, according to time and concentration, and then dissolved by trizol, followed by vortexing with 0.2 ml of chloroform, and then 12,000 rcf. The supernatant was taken by centrifugation for 15 minutes at. 0.5 ml isopropanol was added thereto and allowed to stand at room temperature for 10 minutes, followed by centrifugation at 4 ° C and 12,000 rcf for 10 minutes to precipitate RNA. After removing the supernatant, the RNA was washed with 75% ethanol, and the RNA was dried at room temperature after centrifugation at 7,600 rcf. After quantifying the isolated total RNA, according to the method provided by the manufacturer, reverse transcription reaction synthesizes cDNA using oligo dT primer and RT enzyme. 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 a quantitative software. Products amplified by real time PCR were quantified using comparative cycle threshold (Ct) method, and inflammatory cytokines (Ccl2, Col1a1, Tgfβ1) and androgen receptor (Ar) were measured in normal hepatocytes, and in liver tissues of animals. Antioxidant enzymes (Nqo1) and hormone receptors (Ar, Erα) were measured. Each sample was corrected by the expression level of β-actin.

3) Western blot analysis

NCTC1469 cells, which are mouse normal hepatocytes, were cultured in 6 well plates by 4 × 10 ⁵ , and proteins were extracted and Western blotting was performed. After treatment with salsolinol at different concentrations in cultured cells, the cells were scraped with a scraper, and then centrifuged. The cell pellets were washed with PBS and lysed with lysis buffer for 1 hour. After the mixture was centrifuged at 13,000 rpm for 15 minutes at 4 ° C, the supernatant was extracted. After protein quantification, 20 μg of protein was electrophoresed on SDS-polyacrylamide gel and then transferred to PVDF membrane for 2 hours. Reactions are performed using relevant antibodies, including caspase-3, and detection using a detection solution with enhanced chemiluminascence (ECL).

4) Reactive oxygen species

NCTC1469 cells, which are mouse normal hepatocytes, are incubated daily at 2.5 X 10 ⁴ on a 4 chamber slide. Cultured cells were treated with 100 μM salsolin and 5 mM of N-acetyl-L-cysteine (NAC), an antioxidant, for 6 hours. After washing with PBS, Hank's Balanced Salt Solution (HBSS) (Gibco, Grand Island, NY, USA) was mixed with 10 μM of DCF-DA, a fluorescent substance that can detect free radicals, and added to the cells. Incubate for minutes. Fluorescently stained cells are identified using a confocal laser microscope.

5) Statistical Processing

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

Experimental Example 1: Confirmation of inflammation in hepatocytes

The effect of SAL on inflammatory cytokine gene expression in normal hepatocytes was carried out via qPCR, the results are shown in Figures 1 and 2.

As can be seen in Figures 1 and 2, after the treatment of salsolinol in normal hepatocytes NCTC1469 by concentration, the results of cell viability experiments, it was confirmed that induction of apoptosis in a concentration-dependent manner. In addition, SAL was confirmed to induce the expression of IL-1β and IL-6, which are inflammation-related genes, in normal hepatocytes in a concentration- and time-dependent manner, and it was confirmed that the expression of Col1a1, a fibrosis-related gene, was increased by SAL.

Experimental Example 2: Confirmation of Hormonal Change

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

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

Experimental Example 3: Measurement of Active Oxygen

To determine if SAL induces free radical production, cultured mouse normal cell lines were treated with 100 μM salsolinol and the antioxidant N-acetyl-L-cysteine (NAC) 5 mM for 6 hours to produce free radical species. It confirmed, and the result is shown in FIG.

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

Experimental Example 4: Confirmation of Apoptosis in Hepatocytes

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

As shown in FIG. 5, the expression level of caspase-3, which is known as an apoptosis mediator, was constant, but the expression level of cleaved caspase-3 was increased. SAL induced PARP-cleavage, leading to cell death in normal hepatocytes. It can be seen.

[Confirmation of Alcohol and Salsolinol in Male and Female Mice]

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

1) Animal testing and alcohol treatment

The animals used in the experiments were male and female C57 / BL6 mice of 7 weeks old from Orient Bio (ORIENT Bio, Gyeonggi-do Province, Korea) and were subjected to the experiment after a week of adaptation to a standard diet. The environmental conditions of the experimental animal room were maintained at a temperature of 22 ± 2 ° C. and a humidity of 55 ~ 60% with a contrast of 12 hours. This animal experiment was conducted in accordance with the regulations of the Experimental Animal Ethics Committee of Sungshin Women's University. Model 1 administered 63% alcohol (5 g / kg) shortly and Model 2 administered 20 alcohol (3 g / kg) for one month. After the experiment, the 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 centrifuged at 3,000 rpm for 15 minutes at 4 ° C., and then plasma was separated. Extracted blood and liver tissues were stored at -85 ° C until used in the experiment.

2) MS analysis

In order to measure salsalinol and plasma salsalin synthase in the liver, MS experiments were conducted in the laboratory of Professor Kim Kwang-pyo of Kyung Hee University.

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

Alanine Aminotransferase (ALT) and Aspartate Aminotransferase (AST), which are representative markers of liver function test in plasma, were measured in the animal laboratory at Seoul National University.

Experimental Example 5: Confirmation of Salsolinol Formation by Alcohol

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

As can be seen in Figure 6, more SAL was detected in the plasma of the male mouse compared to the female mouse, Salsolinol synthase (Salsolinol Synthase) activity was higher in the male mouse liver tissue.

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

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

As can be seen in FIG. 8, there were no changes in ALT and AST in both male and female mice.

Experimental Example 6: Confirmation of Antioxidant Enzyme and Hormone Changes

In order to confirm the antioxidant enzyme and hormone changes according to alcohol, 20% alcohol (3 g / kg) in the liver tissue of the mouse administered for one month, the results are shown in Figure 9.

As can be seen in Figure 9, the antioxidant enzyme Nqo1 was found to increase significantly in male mice, but rather decreased in the mouse mouse liver tissue, the expression of male hormone receptor (Ar) in male mice significantly increased.

[Effect of SAL on Liver Cancer Cell Line]

In order to confirm the effect of salsolin 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

Human-derived liver cancer cell lines, SK-Hep1 (male) and SNU387 (female) cells, were obtained from Korea Cell Line Bank and were required for the minimum required medium (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) . Cells were incubated at 37% incubator at 5% CO ₂ . Salsolinol was purchased from Abcam (Cambridge, Mass., USA).

2) MTT analysis

Tetrazolium-based colorimetric (MTT) assays can be easily read quickly for many samples and are used primarily for the study of cytotoxicity in cultured cells. Liver cancer cells are cultured in 48 well plates by 2 x 10 ⁵ cells, and salsalol is treated by concentration and time. MTT reagent is dissolved in warm PBS and incubated in the incubator for 1 hour. After removing the MTT reagent, add DMSO and measure the absorbance.

3) Western blot analysis

Liver cancer cell lines SK-Hep1 (male) and SNU387 (female) cells were cultured by 5 X 10 ⁵ in a 60 mm culture vessel and the protein was extracted and subjected to Western blotting. Salsolol was treated in two cultured cells at different times and concentrations, washed with PBS, scraped with a scraper and lysed with lysis buffer for 1 hour, followed by 15 minutes at 13,000 rpm at 4 ° C. The supernatant was extracted by centrifugation. After protein quantification, 20 μg of protein was electrophoresed on SDS-polyacrylamide gel and then transferred to PVDF membrane for 2 hours. Reactions are performed using relevant antibodies, including HO-1, and detection using a detection solution with enhanced chemiluminascence (ECL).

4) Immunocytochemistry

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

5) ARE luciferase assay

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 and then treated with salsolinol for 12 hours. Activity was measured using promega luciferase asssay systems and quantified after analysis using beta-galactosidase Enzyme assay system with Reporter lysis buffer.

Experimental Example 7: Cytotoxicity Test (MTT assay)

Cytotoxicity test was performed by treating SALs by time and concentration in human-derived liver cancer cell lines, and the results are shown in FIG. 10.

As can be seen in FIG. 10, SAL showed cytotoxicity in SK-Hep1 (male), a liver cancer cell line, but did not show toxicity in SNU387 (female) cells.

Experimental Example 8: Identification of Antioxidant Enzymes

The expression of the antioxidant enzymes NQO1, TRX, and ho-1 was confirmed by qPCR and Western blot analysis by treating SAL with concentration and time in human-derived liver cancer cell lines, 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 SK-Hep1 (male) cell line.

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

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

As can be seen in Figure 13 and Figure 14, SAL induced the expression of HO-1 in a concentration, time-dependent. In addition, as can be seen in Figures 15 and 16, the movement of Nrf2, a major transcription factor that regulates HO-1 expression, was induced into the nucleus.

As shown in FIG. 17, it was confirmed that ARE-driven luciferase activity was increased and Ho-1 expression was decreased when knocking down the Nrf2 gene.

Experimental Example 10: ARE luciferase assay

To examine the antioxidant effects of SAL, SK-Hep1 (male) cells, a human-derived 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. 12 hours of treatment with salsolinol is shown in FIG. 18.

19 and 20, Akt phosphorylation was induced by SAL among major regulatory proteins of Nrf2 (FIG. 19), and expression of Ho-1 and Nrf2 induced by SAL by Akt pharmacological inhibitors. This decrease was confirmed (FIG. 20A). In addition, SAL produced reactive oxygen species, it was confirmed that the suppression of Nrf2 and Ho-1 expression induced by SAL by the antioxidant (Fig. 20b).

Experimental Example 11: Confirmation of Tumor Gene (STAT3)

In order to confirm whether SAL induces the expression of tumor genes (STAT3), SAL was treated in SK-Hep1 and SNU-387, which are the hepatic cancer cell lines of male and female origin, and then Western blot analysis was used to confirm the expression of STAT3. The results are shown in FIG. 21.

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

[Checking the Effect of SAL in Male and Female Mice]

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

Experimental Example 12: Confirmation of Hepatic Tissue Damage

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

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

Experimental Example 13: Confirmation of liver tissue inflammation

To determine whether SAL expresses the inflammatory cytokines MCP-1 (Ccl2), Col1a1, and Tgfb1 genes in liver tissues, males and females (200 mg / kg) are treated with SAL three times a week for four or eight weeks. After intraperitoneal administration, mouse liver tissue was taken and qPCR was performed. The results are shown in FIG. 23.

Referring to FIG. 23, it was confirmed that expression of MCP-1 (Ccl2) was significantly reduced in males compared to females in mouse liver tissues treated with SAL for 8 weeks. Chronic liver injury progresses to hepatic fibrosis, which increases expression of collagen1, an extracellular matrix protein, and significantly increases in male mouse liver tissue treated with TGFβ1, a biomarker related to liver fibrosis, for 4 weeks in mouse livers treated with SAL. The expression of collagen1 gene, Col1a1, was significantly increased in mouse liver tissues administered 8 weeks. In contrast, there was no difference in the expression of these genes in female liver tissue.

Experimental Example 14 Epithelial Mesenchymal Metastasis Confirmation

To determine whether SAL induces epithelial-mesenchymal transition (EMT) response, male and female mice (200 mg / kg) were intraperitoneally administered three times per week for 8 weeks and then mouse liver tissues were injected. Expression changes of epithelial mesenchymal metastasis markers in the harvested cells were measured, and the results are shown in FIG. 24.

As shown in FIG. 24, the expression of Snai1 was significantly increased in the liver tissues of male mice administered SAL for 8 weeks, but there was no expression change in female mice.

Experimental Example 15: Antioxidant Enzyme Identification (1)

To determine whether SAL induces the expression of antioxidant enzymes, male and female mice (200 mg / kg) were intraperitoneally administered SAL three times a week for four or eight weeks, and then mouse liver tissues were used to perform qPCR. The results are shown in FIG. 25.

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

Experimental Example 16: Antioxidant Enzyme Identification (2)

To determine if SAL induces changes in the expression of enzymes involved in redox cycling, male and female mice (200 mg / kg) were intraperitoneally administered SAL three times a week for four or eight weeks, followed by taking mouse liver tissue. qPCR was performed. In addition, fluorescence staining was performed on γH2AX in mouse liver tissue administered 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 liver tissue of female mice. It was confirmed that the decrease. SAL significantly induced NOX4 expression in male liver tissue, but not in female mice. In other words, SAL increases the expression of NOX4 in the liver tissue of male mice and induces the formation of reactive oxygen species by participating in redox cycling, but not in the liver tissue of female mice. SAL increased the expression of COMT in the liver tissue of male mice, but did not affect the expression in female mice. It was also found that reactive oxygen species produced by SAL can induce mutations in the liver tissue of male mice.

Experimental Example 17 Confirmation of Expression of DNA Damage Related Genes

In order to confirm the expression changes of genes related to DNA damage in SAL-treated mouse liver tissues, male and female mice (200 mg / kg) were intraperitoneally administered three times a week for 8 weeks, and then liver tissues of mice. Hormone expression changes were observed in, 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)]

Experiments were performed by intraperitoneal injection of salsolinol once every two days for eight weeks in aged C57BL / 6 mice (females, males). After dissection, the collected liver tissue was added to Trizol to dissolve, and vortexed with 0.2 ml of chloroform, followed by centrifugation at 12,000 rcf for 15 minutes to obtain supernatant. 0.5 ml isopropanol was added thereto and allowed to stand at room temperature for 10 minutes, followed by centrifugation at 4 ° C and 12,000 rcf for 10 minutes to precipitate RNA. After removing the supernatant, the RNA was washed with 75% ethanol, and the RNA was dried at room temperature after centrifugation at 7,600 rcf. After quantifying the isolated total RNA, according to the method provided by the manufacturer, reverse transcription reaction synthesizes cDNA using oligo dT primer and RT enzyme. 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 a quantitative software. Products amplified by real time PCR were quantified using comparative cycle threshold (Ct) method. Tumor suppressor genes and related proteins (p21, CDK, p53, ATM, etc.) were measured in normal hepatocytes. Each sample was corrected by the expression level of β-actin.

[Western blot analysis]

The experiment was conducted by intraperitoneal injection of salsolinol once every two days for eight weeks in 6-week-old C57BL / 6 mice (females, males). Then, the protein of liver tissue was extracted and subjected to western blotting. After protein quantification, 20 μg of protein was electrophoresed on SDS-polyacrylamide gel and then transferred to PVDF membrane for 2 hours. Reactions are performed using relevant antibodies, including caspase-3, and detection using a detection solution with enhanced chemiluminascence (ECL).

As a result, referring to FIG. 28, SAL significantly increased the expression of ATM, and although the expression of its regulatory tumor suppressor gene Trp53 appears to be increased in the liver tissue of male mice, it is not statistically significant. Could confirm. SAL did not affect the expression of Trp53 in the liver tissue of female mice. SAL showed decreased expression of Cdkn1a in the liver tissue of male mice and it was confirmed that the expression of Cdk2 under its control was significantly increased. In addition, increased expression of Cdk4 was also observed, suggesting that ultimately SAL might induce cell proliferation in the liver tissue of male mice. However, SAL could be estimated to increase the expression of Cdkn1a in female mice and to be involved in cell cycle arrest.

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

Experimental Example 18: Hormone Confirmation

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

As a result, referring to Figure 30, it was confirmed that the expression level of ERα (estrogen receptor alpha) and Ar (androgen receptor) in the liver tissue of the male mouse significantly increased. However, it was found that the expression of these receptors was not observed in the liver tissue of female mice.

Claims (11)

(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; and providing information for diagnosing liver disease or liver cancer.
The method of claim 1,
The method of providing information for diagnosing liver disease or liver cancer, characterized in that the biological sample of step (a) is a male specific sample.
The method of claim 1,
Method of providing information for diagnosing liver disease or liver cancer, characterized in that the biological sample of step (a) is tissue, urine, blood, serum, plasma or body fluids.
The method of claim 1,
The concentration measurement of step (a) is selected from the group consisting of dual mass spectrometer, spectrophotometer, nuclear magnetic resonance spectrometer, chromatography, ultraviolet spectrometer, infrared spectrometer, fluorescence spectrometer, enzyme-linked immunosorbent assay (ELISA) and mass spectrometer Method for providing information for diagnosing liver disease or liver cancer, characterized in that measured by one or more devices.
The method of claim 1,
Method of providing information for diagnosing liver disease or liver cancer, characterized in that if the salsolinol concentration of step (a) in step (b) is higher than the salsolinol concentration of a normal individual, it is determined as a liver disease or liver cancer risk group. .
The method of claim 1,
The liver disease is at least one disease selected from the group consisting of hepatitis, hepatotoxicity, fatty liver, alcoholic liver disease, liver abscess and liver atrophy.
Biomarker composition for diagnosing liver disease or liver cancer comprising a formulation for quantifying salsolinol.
The method of claim 7, wherein
The biomarker composition is a biomarker composition for diagnosing disease or liver cancer, characterized in that male-specific.
A kit for diagnosing liver disease or liver cancer, comprising a formulation for quantifying salsolinol or a quantitative analysis device.
The method of claim 9,
The quantitative analysis device is one or more quantitative analysis selected from the group consisting of dual mass spectrometer, spectrophotometer, nuclear magnetic resonance spectrometer, chromatography, ultraviolet spectrometer, infrared spectrometer, fluorescence spectrometer, enzyme-linked immunosorbent assay (ELISA) and mass spectrometer Liver disease or liver cancer diagnostic kit, characterized in that the device.
The method of claim 9,
The kit for diagnosing disease or liver cancer, characterized in that the male specific.

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