A method for evaluating liver damage risk of a bamboo leaf green liquor

Abstract

The application discloses a liver damage risk evaluation method of bamboo leaf green liquor, and comprises the following steps: calculating the 7d LC 50 of zebra fish in bamboo leaf green liquor base liquor; taking the 7d LC 50 as a chronic exposure alcohol concentration, and performing 4-12 weeks of chronic exposure on the zebra fish by taking the bamboo leaf green liquor base liquor as a medium to obtain a zebra fish alcoholic fatty liver model; based on the chronic exposure alcohol concentration, performing chronic exposure on the zebra fish alcoholic fatty liver model, then performing liver damage evaluation and liver damage molecular index detection, and evaluating the liver damage risk of the bamboo leaf green liquor. The application can shorten the modeling cycle and improve the stability and repeatability of the model by determining a reasonable chronic exposure dose through acute LC 50 determination, and can evaluate the improvement effect of the bamboo leaf green liquor on alcoholic fatty liver on the basis of the established zebra fish alcoholic fatty liver model by taking a liquor product as an exposure medium.

Description

Technical Field

[0001] This invention relates to the field of liver injury risk assessment technology, and more specifically, to a method for assessing liver injury risk using Zhuyeqing wine. Background Technology

[0002] Alcoholic liver disease ( alcoholic liver disease Alcoholic liver disease (ALD) is a liver-damaging disease caused by long-term excessive alcohol consumption. Its pathological process typically includes steatosis, alcoholic hepatitis, fibrosis, and even cirrhosis. Globally, ALD is one of the leading causes of chronic liver disease and liver-related death. Its pathogenesis involves multiple steps, including lipid metabolism disorders, increased oxidative stress, activation of inflammatory factors, and activation of hepatic stellate cells.

[0003] Among related technologies, the animal models used to evaluate the risk of liver damage from alcohol consumption are time-consuming, costly, and complex to operate, and cannot quantify the risk of damage.

[0004] It should be noted that the information disclosed in the background section above is only used to enhance the understanding of the background of this disclosure, and therefore may include information that does not constitute prior art known to those skilled in the art. Summary of the Invention

[0005] The technical objective of this application is to address the above-mentioned shortcomings by providing a method for assessing the risk of liver injury from Zhuyeqing wine. This application utilizes acute LC... 50 Determining a reasonable chronic exposure dose can stably induce an alcoholic fatty liver disease model in adult zebrafish within 4–12 weeks, shortening the modeling period and improving the model's stability and reproducibility. Using alcoholic beverages as the exposure medium, the ameliorative effect of Zhuyeqing wine on alcoholic fatty liver disease can be evaluated based on the established zebrafish model.

[0006] To achieve the above objectives, this application provides the following technical solution: According to one aspect of this application, a method for assessing the liver injury risk of Zhuyeqing liquor is provided, comprising: selecting healthy adult zebrafish, exposing the zebrafish to gradient dilutions of Zhuyeqing liquor base for 7 days, and calculating the 7-day LC50 of the zebrafish in the Zhuyeqing liquor base. 50 ; with the 7d LC 50 of ~ The alcohol concentration used as the chronic exposure alcohol concentration was used to chronically expose zebrafish to Zhuyeqing liquor as a medium for 4–12 weeks, resulting in a zebrafish alcoholic fatty liver model. Based on the chronic exposure alcohol concentration, the zebrafish alcoholic fatty liver model was chronically exposed to ethanol, Zhuyeqing liquor as a medium, and Zhuyeqing liquor as a medium for 4–12 weeks, respectively. Then, liver damage was evaluated and molecular indicators of liver damage were detected to assess the liver damage risk of Zhuyeqing liquor.

[0007] In some embodiments, the chronic exposure alcohol concentration is the 7-day LC. 50 of The chronic exposure period is 4 weeks.

[0008] In some embodiments, the temperature of the chronically exposed rearing environment is 28±1℃, and the photoperiod condition is 14L:10D.

[0009] In some embodiments, the alcohol concentration of the gradient dilution solution of the Zhuyeqing liquor base is 0.50%, 0.75%, 1.00%, 1.25%, and 1.50%.

[0010] In some embodiments, the liver injury assessment is performed by quantitative detection of the lipid droplet area stained with Oil Red O staining in the liver, and the molecular marker detection of liver injury is performed by real-time quantitative PCR detection of lipid metabolism, inflammation, and fibrosis-related genes.

[0011] In some embodiments, the lipid metabolism genes are one or more of acetyl-CoA carboxylase, HMG-CoA synthase 1, fatty acid binding protein 1b.1, triglyceride lipase, adiponectin receptor 1b, and peroxisome proliferator-activated receptor α; the inflammation and fibrosis-related genes are one or more of caspase-1, NLRP3 inflammasome, apoptosis-associated speckle-like protein, interleukin-1β, matrix metalloproteinase 2, and tissue inhibitor of metalloproteinases 2a.

[0012] In some embodiments, the base liquor of Zhuyeqing liquor is Fenjiu.

[0013] Compared with the prior art, the advantages and positive effects of this application are as follows: through acute LC 50 Determining a reasonable chronic exposure dose can stably induce an alcoholic fatty liver disease model in adult zebrafish within 4–12 weeks, shortening the modeling period and improving the model's stability and reproducibility. Using alcoholic beverages as the exposure medium, the ameliorative effect of Zhuyeqing wine on alcoholic fatty liver disease can be evaluated based on the established zebrafish model.

[0014] Furthermore, compared to traditional rodent alcoholic liver disease models, this method shortens the modeling time. In addition, the water exposure method eliminates the need for gavage or injection, making it simple to operate, low in cost, suitable for large-scale experiments, and easier to operate per experiment. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 The survival curves of zebrafish exposed to base liquor solutions of different concentrations for 7 days in Example 1 of this application are shown.

[0017] Figure 2 The dose-response curves of zebrafish exposed to base liquor solutions of different concentrations for 7 days in Example 1 of this application are shown.

[0018] Figure 3 This illustrates the effect of the base wine in Example 1 of this application on lipid deposition in zebrafish liver.

[0019] Figure 4 The results of quantitative PCR detection of lipid metabolism genes in Example 1 of this application are shown.

[0020] Figure 5 The results of quantitative PCR detection of inflammation and fibrosis-related genes in Example 1 of this application are shown.

[0021] Figure 6 This illustrates the effects of different types of alcohol on lipid deposition in zebrafish livers in Example 2 of this application.

[0022] Figure 7 This shows the expression of lipid metabolism gene mRNA in Example 2 of this application.

[0023] Figure 8 This illustrates the expression of inflammation and fibrosis-related gene mRNAs in Example 2 of this application. Detailed Implementation

[0024] To better understand the above-mentioned objectives, features, and advantages of this application, the application will be further described below with reference to the accompanying drawings and embodiments. It should be noted that, unless otherwise specified, the embodiments and features described in the embodiments of this application can be combined with each other.

[0025] The present application will be further described below with reference to the accompanying drawings and specific embodiments.

[0026] Example 1: Constructing a zebrafish alcoholic fatty liver disease (AFLD) model.

[0027] I. Laboratory Animals and Their Housing Conditions Healthy adult zebrafish aged 6-8 months were selected as experimental animals and housed in a constant-temperature, light-controlled incubator with a light intensity of 2800-3000 Lx, a density of 6 fish / tank, a total water volume of 1.6 L per tank, and a water temperature controlled at 28±1℃. The photoperiod was set to 14 h light: 10 h dark (14L:10D). After one week of acclimatization, their general condition was observed, and deformed or unhealthy individuals were removed, retaining healthy zebrafish for subsequent experiments. During the acclimatization period, brine shrimp were fed twice daily, and the water was changed daily to maintain water quality stability.

[0028] II. Preparation of Base Wine Exposure Solution and 7-Day LC 50 Measurement.

[0029] The aquaculture water without any added substances served as the control group. The other experimental groups (alcohol concentrations of 0.50%, 0.75%, 1.00%, 1.25%, and 1.50%) used aquaculture water to dilute the base liquor (Fenjiu) to prepare the exposure solution. Successfully tamed zebrafish were randomly assigned to groups of 6 fish per tank, with 3 replicates. Each group of zebrafish was continuously exposed to the corresponding concentration of base liquor solution for 7 days. During this period, they were fed brine shrimp at regular intervals. After feeding, they were transferred to an equal volume of fresh exposure solution, and the tanks were cleaned simultaneously. Mortality and behavioral status were recorded. The tanks were covered and continuously aerated throughout the process to minimize ethanol evaporation.

[0030] Survival curves were plotted based on the cumulative mortality rates of each concentration group over 7 days. Data was input into GraphPad software, and dose-response curves were fitted using Nonlin. The corresponding mortality rates were calculated, and dose-response curves were fitted to determine the 7-day median lethal concentration (LC50) of the base liquor for adult zebrafish. 50 .

[0031] Figure 1 The survival curves of zebrafish exposed to base liquor solutions of different concentrations for 7 days are shown in the embodiments of this application. Figure 2 The dose-response curves of zebrafish exposed to base liquor solutions of different concentrations over 7 days are shown in the embodiments of this application. Figure 1-2 As shown, all patients in the 1.50% group died by day 4, with a significantly faster mortality rate than the 1.25% group; the 7-day survival rate was >75% in the 1.00% group, >90% in the 0.75% group, and there were no deaths in the 0.50% group and the control group. This indicates that the lethal effect is sensitive to changes in the 1.00%–1.25% range. Based on the fitted dose-response curve, the 7-day LC50 can be calculated as follows: 50 The alcohol concentration is approximately 1.07%.

[0032] III. Constructing a zebrafish model of alcoholic fatty liver disease (AFLD).

[0033] Based on the above calculation of 7-day LC 50 After setting the alcohol concentration to 1.07%, 1 / 25 and 1 / 5 of this concentration were selected as chronic exposure doses. Using Zhuyeqing liquor (Fenjiu) as the exposure medium, LC500 solutions were prepared with alcohol volume fractions of [missing values]. 50 ×1 / 25 and LC 50 Two base liquor solutions, each at 1 / 5 of the original volume, were used, with aquaculture water as a control. Domesticated adult zebrafish were randomly divided into a control group and a low-dose group (1 / 25 LC). 50 ) and high-dose group (1 / 5 LC) 50 The animals were continuously exposed to the corresponding solution for 12 weeks, during which the water temperature, light, density, and feeding conditions were the same as described above, with the solution changed and the animals fed every 24 hours. Samples were taken at 4, 8, and 12 weeks of exposure, and livers were isolated for histological staining and quantitative PCR detection.

[0034] Histological staining was performed on the liver using Oil Red O staining. The specific method was as follows: the isolated liver was fixed, embedded, and sectioned, then stained with Oil Red O, and imaged under a microscope. The lipid droplet area ratio was then quantitatively analyzed using ImageJ.

[0035] Figure 3 This illustrates the effect of a light-aroma base liquor on lipid deposition in zebrafish liver in Example 1 of this application, wherein... Figure 3 A shows the oil red O staining pattern of zebrafish liver (scale bar: 50 μm, 200 ×). Figure 3 B represents quantitative analysis using Oil Red O staining. For example... Figure 3 As shown, almost no red lipid droplets were observed in the liver tissue of the control group at any time point; however, with prolonged exposure time, 1 / 25 LC... 50 and 1 / 5 LC 50 In the base wine group, lipid droplets in hepatocytes gradually increased and merged. Lipid deposition was significantly higher in the base wine group than in the control group at 8 and 12 weeks, with 1 / 25 LC... 50 The proportion of lipid droplet area at each time point in the group was higher than 1 / 5 LC. 50 The study suggests that long-term (chronic) exposure to low concentrations of Zhuyeqing liquor (Fenjiu) can stably induce significant hepatic lipid accumulation.

[0036] The specific method for quantitative PCR detection is as follows: Total RNA was extracted from the liver at each time point, reverse transcribed into cDNA, and the expression levels of lipid metabolism, inflammation, and fibrosis-related genes were detected using real-time quantitative PCR. The relative expression levels were calculated using the 2^-ΔΔCt method. Lipid metabolism includes acetyl-CoA carboxylase (…). acc ) 、 HMG-CoA synthase 1 (HMG-CoA synthase 1) hmgcs-1 ) 、Fatty acid binding protein 1b.1 ( fabp1b.1 ) 、 Fatty acid triglyceride lipase ( atgl ) 、 Adiponectin receptor 1b (Adiponectin receptor 1b) adipor1b ) 、 Peroxisome proliferator-activated receptor α (PrEP) ppar-α Inflammation and fibrosis-related genes include caspase-1 (…). caspase-1 ) 、 NLRP3 inflammasome ( nlrp3 ), apoptosis-associated spot-like protein ( asc ), interleukin-1β ( il1-β ), matrix metalloproteinase 2 ( mmp2 ), tissue inhibitor of metalloproteinases 2a ( timp2a ) 。

[0037]

[0038] Figure 4 The results of quantitative PCR detection of lipid metabolism genes in Example 1 of this application are shown. Figure 5 The results of quantitative PCR detection of inflammation and fibrosis-related genes in Example 1 of this application are shown. Figure 4-5 As shown, at 4 and 8 weeks, lipid synthesis-related genes in the two-dose base wine groups... acc , hmgcs-1 , fabp1b.1 Overall upward adjustment, accompanied by ppar-α , atgl , adipor1b Regulatory changes in lipid metabolism genes, of which 1 / 25 LC 50 The upregulation was more pronounced in the group at 8 weeks; inflammation and fibrosis marker genes nlrp3 , asc , il1-β , mmp2 , timp2a In 1 / 25 LC 50 The levels of these genes were significantly elevated in the early stage (4 weeks), followed by a decline in some genes between 8 and 12 weeks. These molecular changes were largely consistent with the degree of lipid accumulation observed in Oil Red O staining.

[0039] The combined results of Oil Red O staining and quantitative PCR show that: at 1 / 25 LC... 50 Continuous exposure to the base liquor of Zhuyeqing (Fenjiu) for 4 weeks can stably induce an alcoholic fatty liver phenotype in adult zebrafish, characterized by lipid accumulation in hepatocytes and an imbalance of lipid metabolism-related genes.

[0040] Example 2: Application of the model in the study of the health benefits and liver damage mechanism of Zhuyeqing wine.

[0041] In the above LC 50 Based on the long-term exposure model, 1 / 25 LC was selected. 50 The corresponding alcohol concentration of the base liquor of Zhuyeqing wine (Fenjiu) was used as the chronic exposure dose. Ethanol solutions, Zhuyeqing wine base liquor (Fenjiu) solutions, and Zhuyeqing wine (liqueur) solutions with equal volume fractions of alcohol were prepared, with the aquaculture water as the control.

[0042] Domesticated adult zebrafish were randomly divided into a control group, an ethanol group, a base liquor group, and a Zhuyeqing liquor group, and were continuously exposed to the corresponding solutions for 4 weeks, with other feeding conditions the same as in the previous embodiments. After the exposure, liver tissue was collected for Oil Red O staining and real-time quantitative PCR detection.

[0043] The Oil Red O staining and real-time quantitative PCR detection methods are the same as in Example 1, and will not be repeated here.

[0044] Figure 6 This illustrates the effects of different types of alcohol on lipid deposition in zebrafish liver in Example 2 of this application, wherein... Figure 6 A shows the oil red O staining pattern of zebrafish liver (scale bar: 50 μm, 200×; 20 μm, 400×). Figure 6 B represents quantitative analysis using Oil Red O staining. For example... Figure 6 As shown, the control group showed intact hepatocyte structure with only a few lipid droplets; the ethanol group showed a large accumulation of red lipid droplets in hepatocytes, indicating the heaviest lipid deposition; the base liquor group showed a decrease in the number and coverage of lipid droplets compared to the ethanol group; the Zhuyeqing liquor group showed clear hepatocyte boundaries, with the lowest number and area of ​​lipid droplets among the three alcohol-containing groups. ImageJ quantitative analysis showed that the proportion of lipid droplet area in the ethanol group was significantly higher than that in the control group, while the base liquor and Zhuyeqing liquor groups showed an increase, but both were significantly lower than those in the ethanol group, with the Zhuyeqing liquor group showing the smallest increase.

[0045] Figure 7 This shows the expression of lipid metabolism gene mRNA in Example 2 of this application. Figure 8 This illustrates the mRNA expression of inflammation and fibrosis-related genes in Example 2 of this application. For example... Figure 7-8 As shown, the expression of liver inflammation-related genes mRNA was generally upregulated in both the ethanol and base liquor groups, while it showed an overall downward trend in the Zhuyeqing liquor group. Regarding lipid metabolism, the ethanol group... ppar-α , acc and hmgcs-1 Significantly upregulated, suggesting activation of fatty acid and cholesterol synthesis; in the Zhuyeqing wine group acc and hmgcs-1 The lipid droplets were significantly reduced compared to the ethanol group, indicating that it has an inhibitory effect on lipid synthesis-related genes, which is consistent with the reduction of lipid droplets observed in Oil Red O staining.

[0046] Combined results from Oil Red O staining and quantitative PCR showed that, at the same alcohol concentration, Zhuyeqing liquor significantly reduced lipid accumulation and inflammatory response in zebrafish livers compared to ethanol and base liquor. Zhuyeqing liquor also had a mitigating effect on liver damage compared to Fenjiu. This indicates that the zebrafish water exposure model established in this embodiment can be used to evaluate the hepatoprotective and lipid-regulating effects of Zhuyeqing liquor and to compare the differences in liver damage among different types of alcohol under the same alcohol conditions.

[0047] Through the above specific embodiments, those skilled in the art can easily implement this application. However, it should be understood that this application is not limited to the specific embodiments described above. Based on the disclosed embodiments, those skilled in the art can arbitrarily combine different technical features to achieve different technical solutions.

Claims

1. A method for assessing the risk of liver damage from Zhuyeqing wine, characterized in that, include: Healthy adult zebrafish were selected and exposed to gradient dilutions of Zhuyeqing liquor base for 7 days. The 7-day LC50 of the zebrafish in the Zhuyeqing liquor base was calculated. 50 ; With the 7d LC 50 of ~ The concentration was used as the alcohol concentration for chronic exposure. Zebrafish were chronically exposed to Zhuyeqing liquor for 4–12 weeks, and a zebrafish alcoholic fatty liver model was obtained after chronic exposure. Based on the alcohol concentration of the chronic exposure, the zebrafish alcoholic fatty liver model was chronically exposed for 4 to 12 weeks using ethanol, Zhuyeqing wine base liquor, and Zhuyeqing wine of the same alcohol concentration as the exposure medium. Then, liver damage was evaluated and molecular indicators of liver damage were detected to assess the risk of liver damage from Zhuyeqing wine.

2. The method according to claim 1, characterized in that, The chronic exposure alcohol concentration is the 7-day LC. 50 of The chronic exposure period is 4 weeks.

3. The method according to claim 2, characterized in that, The temperature of the chronically exposed rearing environment was 28±1℃, and the photoperiod was 14L:10D.

4. The method according to claim 1, characterized in that, The alcohol concentrations of the gradient dilution solutions of the Zhuyeqing liquor base are 0.50%, 0.75%, 1.00%, 1.25%, and 1.50%.

5. The method according to claim 1, characterized in that, The liver injury assessment was performed by quantitative detection of lipid droplet area in the liver using Oil Red O staining, and the molecular indicators of liver injury were detected by real-time quantitative PCR of lipid metabolism, inflammation, and fibrosis-related genes.

6. The method according to claim 5, characterized in that, The lipid metabolism gene is acetyl-CoA carboxylase. 、 HMG-CoA synthase 1 、 Fatty acid-binding protein 1b.1 、 Fat triglyceride lipase 、 Adiponectin receptor 1b 、 One or more of the peroxisome proliferator-activated receptor α; the inflammation and fibrosis-related gene is caspase-1. 、 One or more of the following: NLRP3 inflammasome, apoptosis-associated speckle-like protein, interleukin-1β, matrix metalloproteinase 2, and tissue inhibitor of metalloproteinases 2a. 。 7. The method according to claim 1, characterized in that, The base liquor for the Zhuyeqing liquor is Fenjiu.

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