Method for researching 18 beta-glycyrrhetinic acid on liver injury mediated by mitochondrial function injury induced by high-fat ration

Abstract

The invention discloses a research method of 18beta-glycyrrhetinic acid on high-fat daily ration induced mitochondrial function injury mediated liver injury, which comprises the following steps: S1, test design, S2, test sample collection, S3, index determination, S4, test data statistical analysis, and S5, results, and the research method for 18beta-glycyrrhetinic acid (18beta-glyrhetinic acid, 18beta-glyrhetinic acid, 18beta-glyrhetinic acid, 18beta-glyrhetinic acid, 18beta-glyrhetinic acid, 18beta-glyrhetinic acid, 18beta-glyrhetinic acid, 18beta-glyrhetinic acid, 18beta- The liver injury of the micropterus salmoides caused by high fat ration (HF) can be improved by lowering TGF beta1-Smad2 / 3 signals to reduce aggregation of liver collagenous fibers, and the GA can activate Ampk-Pgc1alpha-Sirt3 signal pathways to reduce expression of calcium ion transporters in endoplasmic reticulum and mitochondria, relieve liver mitochondrial injury and reduce the content of ROS (reactive oxygen species), so that the liver injury of the micropterus salmoides caused by high fat ration (HF) can be relieved, and the liver injury of the micropterus salmoides caused by high fat ration (HF) can be relieved. And the HF can increase the activity of liver function related enzymes of the micropterus salmoides and increase liver lipid droplet deposition and collagen aggregation, and after the GA is added, the adverse effect caused by the HF can be obviously improved, so that the HF-induced liver injury of the micropterus salmoides can be relieved.

Description

Technical Field

[0001] The invention relates to the technical field of fish feed, in particular to a method for studying liver damage mediated by mitochondrial function damage induced by 18β-glycyrrhetinic acid in a high-fat diet. Background Art

[0002] Largemouth bass is a warm-water carnivorous fish. It is loved by consumers for its rapid growth, delicious meat and strong disease resistance. It is also one of the important economic fish cultivated in my country. The protein content in the feed of largemouth bass is extremely high, reaching 35% to 50%, which also means that its feed cost accounts for up to 60% of the total breeding cost. In order to improve the efficiency of aquaculture production and reduce costs, scientists and feed companies have taken a variety of measures, such as using carbohydrates and fats to replace part of the protein, thereby improving the nutrient absorption rate of fish. Carnivorous fish have low efficiency in the utilization of carbohydrates, so they will save protein in the form of fat.

[0003] Reasonable increase in feed fat level not only promotes fish growth, but also helps improve the breeding environment. However, in actual production, feed fat level is usually increased to save costs. Long-term feeding of high levels of fat can cause liver damage in largemouth bass. Therefore, in order to improve the effect of high-fat diet on liver damage in fish in aquaculture, it is urgent to develop a liver-protecting feed additive to solve the problem of liver damage caused by high-fat diet in aquaculture.

[0004] 18β-Glycyrrhetinic acid (GA) is the main component of licorice. It is a pentacyclic triterpenoid natural active compound formed by the hydrolysis of glycyrrhizic acid and the removal of the sugar acid chain. It produces GA during its metabolism in the body. GA is a widely recognized anti-inflammatory, antioxidant, fat-reducing and liver-protecting drug.

[0005] The liver is an important immune and metabolic organ in fish, and plays a key role in various metabolisms and detoxification, immune defense, maintenance of immune homeostasis, and immune surveillance in fish. Damage to the liver will affect the growth of fish. In actual production, it was found that long-term feeding of largemouth bass with a high-fat diet would induce liver damage. Therefore, adding GA to the feed can alleviate the adverse effects of high-fat diet on the liver, which has important production application value.

[0006] In view of this, the present invention is proposed. Summary of the invention

[0007] The purpose of the present invention is to provide a method for studying the effect of 18β-glycyrrhetinic acid on liver damage mediated by mitochondrial dysfunction induced by a high-fat diet, so as to solve the problems raised in the above background technology.

[0008] To achieve the above-mentioned purpose, the present invention provides the following technical scheme: A method for studying the effect of 18β-glycyrrhetinic acid on liver damage mediated by mitochondrial dysfunction induced by a high-fat diet, comprising the following steps: S1 experimental design, S2 experimental sample collection, S3 index determination, S4 experimental data statistical analysis, and S5 results.

[0009] Preferably, the S1 includes the following steps:

[0010] S11 Experimental diets: The basal feed contained three protein sources, fish meal, chicken meal and fermented soybean meal, soybean oil as the fat source, and two carbohydrates, wheat flour and cassava starch, and was formulated into a control group (NC) with a protein level of 47.75% and a fat level of 9.05%, a high-fat diet group (HF) with a protein level of 47.31% and a fat level of 16.29%, a high-fat diet with GA 0.5 mg / kg (HFL) with a protein level of 47.48% and a fat level of 16.25%, a high-fat diet with GA 1.0 mg / kg (HFM) with a protein level of 47.48% and a fat level of 16.44%, and a high-fat diet with GA 1.5mg / kg (HFH), protein level is 47.55%, fat level is 16.44%, vitamin and mineral premix addition amount is 1% and 1.5% respectively, vitamin D, vitamin C, selenium, feed raw materials are all crushed, passed through 40 mesh sieve, weighed according to the ratio, trace components are added by step-by-step expansion method, and mixed with bulk raw materials, and then mixed again after adding oil and water, pelletized by small extruded feed machine, dried at 30℃, stored in sealed plastic bags, and used for experiments after nutrient analysis of each feed;

[0011] S12 Experimental fish and feeding management: The experimental fish were healthy largemouth bass. After 2 weeks of temporary feeding, 750 healthy largemouth bass with an average initial weight of 17.39±0.09g were randomly assigned to 15 square cement pools, with 50 fish in each cement pool. The cement pools were randomly divided into 5 treatment groups, which were fed with NC group, HF group, HFL group, HFM group and HFH group feeds, respectively, with 3 replicates for each treatment. The growth experiment lasted for 77 days, and the fish were fed at 7:30 am and 18:00 pm every day. After 40 minutes of feeding, the remaining bait was fished out, dried and weighed. The weather, water temperature, feeding rate, amount of residual bait and food intake were recorded every day. The experiment adopted micro-flow aquaculture, and the water flow rate was maintained at 1.0L / min. The water temperature during the experiment was 25.0±3.0℃, and intermittent aeration was performed to keep the dissolved oxygen greater than 5.0mg / L.

[0012] Preferably, S2 includes the following steps: after the feeding experiment is completed, the largemouth black bass are fasted for 24 hours, 12 fish with roughly similar weight are selected for each repetition and anesthetized with 0.01% MS-222, after the fish body is wiped dry, the weight is weighed and recorded, and blood is collected from the tail vein with a 1.5mL disposable syringe rinsed with an anticoagulant. After the body and viscera of 9 largemouth black bass are separated, the liver is quickly separated from the visceral mass, and then thoroughly washed with physiological saline, photographed, weighed and recorded, and then put into a bag with a corresponding label, and then wrapped with tin foil, immediately frozen in liquid nitrogen, and then quickly transferred to a -80°C ultra-low temperature refrigerator for storage for subsequent test index determination. The remaining 3 largemouth black bass are first used to measure individual body length and weigh, and then the liver tissue is separated, and the liver tissue is fixed with 4% neutral formalin for subsequent HE staining, Masson staining and IHC staining analysis. In the same way, frozen section samples were placed in 2 mL cryovials, labeled, wrapped in tin foil and immediately frozen in liquid nitrogen for subsequent Oil Red O staining. For electron microscopy samples, soybean-sized tissues were fixed in 2.5% glutaraldehyde solution for electron microscopy section observation.

[0013] Preferably, S3 includes the following steps:

[0014] S31 Liver-to-body ratio: Based on the sampling statistical data, the liver-to-body index is calculated to determine the growth and development of the liver;

[0015] S32 Determination of blood and liver biochemical indices: The blood was transferred to a microcentrifuge tube and centrifuged at 3000g for 10 min at room temperature to separate plasma. The activities of alanine aminotransferase ALT, aspartate aminotransferase AST and alkaline phosphatase AKP, triglyceride TG, total cholesterol TC and hydroxyproline HYP were determined using commercial kits. After taking the sample out of the -80℃ refrigerator, a 10% tissue homogenate was prepared using a homogenizer, and then centrifuged at 3000r / min for 10 min to obtain the supernatant, and the malondialdehyde MDA, glutathione S-transferase GST, total protein TP and adenosine triphosphate ATP contents were determined.

[0016] S33 Liver H&E staining analysis: The liver tissue was fixed with 10% formalin solution for 7 days, then dehydrated with gradient ethanol solution, and then the sample was made transparent with xylene for subsequent wax immersion. Finally, the sample was embedded in paraffin and sliced ​​with a paraffin slicer with a thickness of 5 μm, and stained with hematoxylin-eosin H&E. The specific steps of H&E staining are as follows:

[0017] 1 Place the sample slide in a 65℃ constant temperature drying oven and dry for 2 hours;

[0018] 2 Dewaxing: Place the sample slides in xylene for 10 minutes each time, twice in total;

[0019] 3 Hydration: Immerse the sample slides in anhydrous ethanol, 95% ethanol, 80% ethanol, 70% ethanol for 2 min each, and distilled water for 2 min;

[0020] 4Hematoxylin staining for 10 min, washing with water for 30 s;

[0021] 5. Differentiate with 1% hydrochloric acid for 60 seconds and take out after it turns blue;

[0022] 6. Eosin staining for 4 min, washing with water for 30 s;

[0023] 7 Immerse the sample slides in 95% ethanol twice for 5 min each, 100% ethanol twice for 5 min each, 100% ethanol: xylene 1:1 for 5 min, and xylene twice for 5 min each;

[0024] 8. Seal the slide with neutral gum;

[0025] 9. Observe and take photos under optical microscope Olympus BX43 observation;

[0026] S34 Liver Oil Red O Staining Analysis: The specific steps for Oil Red O staining preparation are as follows:

[0027] 1. Take out the liver sample from the -80℃ refrigerator and transfer it to a -20℃ refrigerator for buffering for 30 minutes;

[0028] 2. Cut the liver tissue into square blocks of appropriate size and embed and freeze them with OCT embedding medium;

[0029] 3. Cut the tissue block into 8 μm frozen sections using a pre-cooled cryostat;

[0030] 4. Transfer the cut samples to the slides with corresponding labels and let them stand at room temperature for 10 minutes before staining;

[0031] 5. Cover the sample with washing solution for 20 seconds and then stain with Oil Red O dye for 20 minutes;

[0032] 6 After removing the Oil Red O dye stain, wash with washing solution for 30 seconds, immerse in distilled water and wash on a shaker for 20 seconds;

[0033] 7. Counterstain the cell nucleus with hematoxylin for 3 min, wash with water for 30 s, and seal the slides with glycerol gelatin;

[0034] 8. Microscopic examination: Olympus BX43;

[0035] S35 liver Masson staining analysis: Sample pre-treatment, dewaxing and hydration are the same as the above H&E staining. The specific staining steps are as follows:

[0036] 1. Stain with prepared Weigert iron hematoxylin staining solution for 10 minutes;

[0037] 2 Use acidic ethanol differentiation solution for differentiation for 15 seconds and wash with water;

[0038] 3. Use Masson bluing solution to return to blue for 5 minutes, then wash with water and distilled water for 1 minute;

[0039] 4. Stain with Ponceau fuchsin staining solution for 10 min;

[0040] 5 Wash with weak acid working solution for 1 min. The weak acid working solution is distilled water: weak acid solution = 2:1;

[0041] 6 Then wash with phosphomolybdic acid solution for 2 minutes and weak acid working solution for 1 minute;

[0042] 7 Directly put it into aniline blue staining solution for 2 minutes, and wash it with weak acid working solution for 1 minute;

[0043] 895% ethanol rapid dehydration for 3 seconds, anhydrous ethanol dehydration 3 times, each time for 10 seconds;

[0044] 9. Xylene transparent 3 times, 2 min each time, neutral gum sealing, observation;

[0045] S36 Transmission electron microscopy analysis of liver: Take a small piece of liver 1×1×1mm 3 , placed in 2.5% glutaraldehyde, quickly fixed at 4°C for 2-4h, fixed with 1% osmium acid prepared in 0.1M phosphate buffer PB at room temperature in the dark for 2h, dehydrated, infiltrated, embedded, sectioned and stained, and observed under a transmission electron microscope, and collected images for analysis;

[0046] S37 liver immunohistochemistry analysis: Sample pre-treatment, dewaxing and H&E staining as above, submerge slices with 10mmol / L citrate buffer pH6.0, heat to boiling in a microwave oven for 20min; incubate with primary antibodies anti-α-Sma, anti-Tgfβ1, Sirt3 and Mcu antibodies in a wet box overnight, rewarm the next day, wash, add 25μL of horseradish peroxidase HRP-labeled secondary antibody anti-rabbit / mouse to each slice, incubate at 37℃ for 40min, wash twice with PBST, 5min each time, add DAPI (0.2ug / ml) to the slices, 5min, wash twice with PBST, 5min each time. Seal the slices, observe and take pictures under an inverted fluorescence microscope;

[0047] S38 Reactive oxygen detection: 200 mg of liver was taken out at the same time, and the reactive oxygen ROS concentration was measured immediately. According to the preparation method of single cell suspension, single cell suspension was prepared according to the operation steps, and then the fluorescent probe was added, and the liver cell pellet was suspended with diluted DCFH-DA. The liver cell pellet was incubated in a 27°C incubator for 30-60 min, centrifuged at 1000 r / min for 10 min, the supernatant was removed, and the cells and sediment were collected and suspended and mixed with PBS sediment. The confocal fluorescence microscope was used to take pictures, and the absorbance value at 525 nm was detected with a fluorescent microplate to calculate the ROS content;

[0048] S39 Membrane potential MMP assay: liver mitochondria were isolated using a tissue mitochondrial isolation kit, and MMP was detected using the JC-1 fluorescent probe method according to the instructions. Mitochondria were then observed using a fluorescence microscope, and the absorbance was detected using a fluorescent microplate reader. The red-green light ratio was calculated, with an excitation wavelength of 485 nm and an emission wavelength of 580 nm.

[0049] S310 real-time quantitative PCR detection: including the following steps:

[0050] S3101 RNA extraction and cDNA synthesis: Total RNA was extracted using a kit. Briefly, liver tissue was taken out from a -80°C freezer, ground into powder in liquid nitrogen, 0.1 g of liver sample was taken, 1 mL of reagent was added to extract total RNA from the tissue, and dissolved in 50 μL of RNase-free water. The degradation degree of RNA sample was detected by 1.0% agarose gel electrophoresis at 120 V for 12 min. The concentration was detected by NanoDrop 1000 spectrophotometer, and the purity of the sample was determined based on the A260 / A280 ratio.

[0051] S3102 Real-time fluorescence quantitative primer design: All gene sequences of largemouth bass were obtained from GenBank or referenced from previous studies. Primers were designed using Primer5.0;

[0052] Table 2 Primers used for fluorescence quantification

[0053] Table 2.Primer sequences of genes selected for q-PCR

[0054]

[0055]

[0056] S3103 real-time fluorescence quantitative detection of gene expression levels: the three cDNA samples of each treatment were diluted 1:1, and then the fibrosis-related genes: Tgfβ1a, Smad2, Smad3a, α-Sma, CollagenⅠ, Fibronectin and Mmp9; mitochondrial calcium ion transport-related genes: Ampkα1, Pgc1α, Sirt3, Ip3r1, Sig1r1, Casr, Grp75, Vdac1 and Mcu were detected using TB Green TM Premix Ex Taq TM II (Tli RNaseH Plus) kit was used for fluorescence quantitative PCR amplification. The positive template diluted 10 times continuously was used as the PCR template, and then a standard curve was made. The amplification efficiency was between 90% and 110%, and R 2 >0.9, the melting curve is a single peak. A 10μL PCR amplification system was used: 3.2μL DEPC water, 1μL cDNA template, 0.4μL upstream primer and downstream primer, and 5μL SYBR Premix Ex TaqⅡ. The reaction cycle conditions were: 95℃ preheating for 2min, 40 cycles of 95℃ for 10s and appropriate annealing temperature for 30s. After the reaction was completed, the melting curve was used to detect the product specificity.

[0057] S3104 relative quantitative calculation: The expression level is represented by the threshold cycle Ct, which is calculated by plotting the fluorescence signal of each cycle. The target gene mRNA concentration is normalized to be consistent with the mRNA concentration of the internal reference gene β-actin and 18SrRNA. After verifying that the primer amplification efficiency reaches the target gene and internal reference gene amplification efficiency, 2 -ΔΔCT The amplification efficiency of the internal reference gene was calculated based on the results of the method and the specific gene standard curve generated by 10-fold serial dilution;

[0058] S311 protein immunoblotting: Total liver tissue protein was extracted with RIPA Lysis Buffer (Biyuntian, Beijing, China), and the sample protein concentration was determined using Enhanced BCA Protein Assay Kit (Biyuntian Biotechnology Co., Ltd., China). SDS-PAGE protein loading buffer 5X was added to the sample, and the samples were denatured at 95°C for 10 min. After SDS-PAGE separation, the membranes were transferred to polyvinylidene fluoride PVDF membranes and blocked with 5% skim milk for 1.5 h. The membranes were then incubated with primary antibodies overnight at 4°C on a shaker. After washing, the membranes were incubated with secondary antibodies at room temperature for 1.5 h. The expression of target proteins was detected using ECL Kit (Beyotime) and recorded using Chemi Doc Imaging Systems / Image Lab software (Bio-Rad). β-actin was used as an internal reference with equal sample loading.

[0059] Preferably, S4 includes using software to process data, using variance analysis combined with Duncan's multiple comparison to detect significance, the data are expressed as mean ± standard error Mean ± SEM, P < 0.05 indicates a significant difference, and using Image-ProPlus6.0 software to perform quantitative statistics on liver H&E, Oil Red O and Masson staining.

[0060] Preferably, the liver-to-body ratio is calculated based on sampling statistical data to obtain a liver-to-body index: liver-to-body index % = liver weight / body weight × 100.

[0061] Preferably, S5 comprises the following steps:

[0062] The effect of S51GA on HF-induced liver injury in largemouth bass includes the following steps:

[0063] S511 Liver-to-body ratio: By dissecting and observing the liver appearance and calculating the liver-to-body ratio of largemouth bass, it was found that the liver surface of the NC group was smooth and ruddy. The liver of largemouth bass in the HF group showed that most of the liver surface was pale, and GA treatment significantly reduced the pale surface of the liver and increased the ruddy surface of the liver. Compared with the NC group, the liver-to-body ratio of the HF group was significantly increased, and GA treatment reduced the liver-to-body ratio;

[0064] S512 Blood and liver function parameters: To explore the effect of GA on HF-induced liver function of largemouth bass. We tested the hematology and liver function of largemouth bass. The serum AST, ALT and AKP activities of the HF group were significantly higher than those of the NC group. After adding GA treatment, the AST, ALT and AKP activities were significantly reduced P<0.05. The HF group had the highest serum TG and TC contents, while the TG and TC contents were significantly reduced after GA treatment P<0.05. Compared with the NC group, the MDA content in the HF group increased significantly, while the MDA content in the GA group decreased significantly P<0.05. The GST activity was the lowest in the HF group P<0.05, and the TP content was the lowest in the NC group P<0.05. The above results showed that GA reduced the HF-induced AST, ALT and AKP activities in serum, TG, TC contents and MDA content in the liver, and increased GST activity, thereby alleviating HF-induced liver damage;

[0065] S513 Liver histopathology: Compared with the NC group, the hepatocytes in the HF group were significantly swollen, the nuclei showed marginal displacement, some even disappeared, and the number was small. Compared with the HF group, the fatty degeneration of the liver in the GA-treated group was gradually alleviated, the number of nuclei gradually increased, and was located in the center of the cell. Statistical analysis found that compared with the NC group, the relative area of ​​lipid vacuoles in the HF group increased significantly, and compared with the HF group, the relative area of ​​lipid vacuoles in the HFM group in the relief group was the lowest. The results of Oil Red O staining showed that compared with the NC group, a large number of lipid droplets appeared in the liver of the HF group, while the lipid droplets in the liver of the GA-treated group gradually decreased. The Oil Red O-positive area in the HF group increased significantly, and the Oil Red O-positive area decreased after GA treatment. The above results indicate that GA reduces HF-induced liver lipid degeneration and lipid droplet deposition;

[0066] Effect of S52GA on HF-induced liver fibrosis in largemouth bass: Masson staining is a specific staining method for detecting collagen fibers. Masson staining of liver sections of largemouth bass showed that muscle fibers were red and collagen fibers were blue. HF treatment resulted in the aggregation of a large number of collagen fibers, and the addition of GA treatment significantly reduced the aggregation of liver collagen fibers. The therapeutic effects of HFM and HFH were close to those of the NC group. In addition, the quantitative evaluation of the fibrosis area by Masson staining using IPP showed that GA treatment significantly reduced liver fibrosis relative to the HF group. HYP content is a sensitive biochemical indicator reflecting changes in collagen fibers. In order to further study the effect of GA on HF-induced liver fibrosis in largemouth bass, we detected the HYP content in serum and found that compared with the NC group, the HYP content in the serum of largemouth bass in the HF group was significantly increased, while compared with the HF group, GA administration significantly reduced the HYP content;

[0067] Effect of S521GA on the transcription of HF-induced profibrotic markers associated with HSC activation: The mRNA expression of profibrotic markers α-Sma, CollagenI, Fibrontein and Mmp9 was further detected. The results showed that compared with the NC group, HF significantly increased the mRNA expression of α-Sma, CollagenI and Fibrontein in the liver, while compared with the HF group, GA significantly reduced the mRNA expression of α-Sma, CollagenI and Fibrontein. The protein levels of α-Sma, CollagenI and Fibrontein were detected by Western-blot method. It was found that the protein levels of α-Sma, CollagenI and Fibrontein were the highest in the HF group, and the mRNA expression of Mmp9 was the highest in the HF group. Compared with the HF group, the mRNA expression of Mmp9 was significantly reduced after adding GA treatment. In addition, the results of immunohistochemical staining confirmed that GA significantly inhibited the expression of HF-induced α-Sma. The above results show that GA can effectively alleviate HF-induced liver fibrosis in largemouth bass;

[0068] Effects of S522GA on HF-induced Tgfβ1-Smad2 / 3 signaling pathway in the liver of largemouth bass: Tgfβ1a is the most potent fibroblast factor in the liver. Therefore, to explore the inhibitory mechanism of GA on HF-induced liver fibrosis, we detected the mRNA expression of Tgfβ1a and its target proteins Smad2 and Smad3a. Compared with the NC group, the mRNA expressions of Tgfβ1a, Smad2, and Smad3a in the HF group were significantly increased, while GA treatment significantly reduced their mRNA expressions. The Western-blot method was used to detect the protein levels of Tgfβ1, p-Smad2, and p-Smad3, and it was found that the protein levels of Tgfβ1, p-Smad2, and p-Smad3 in the HF group were significantly increased, and their protein levels were reduced after GA treatment, which was consistent with the protein and mRNA expressions. The immunohistochemical results showed that compared with the HF group, the expression of Tgfβ1 in the liver tissue of largemouth bass treated with GA was significantly reduced. These results indicate that GA improves liver fibrosis by regulating the Smad2 / 3 signaling pathway mediated by Tgfβ1.

[0069] The effects of S53GA on HF-induced liver mitochondria include the following steps:

[0070] Effect of S531GA on HF-induced liver reactive oxygen species: Mitochondrial membrane potential MMP was measured using JC-1, and it was found that JC-1 staining showed bright red fluorescence for JC-1 polymers, and green fluorescence for JC-1 monomers. JC-1 staining showed that bright red fluorescence and weak green light were visible in the NC group. On the contrary, a transition from red fluorescence to green fluorescence was observed in the HF group. Compared with the HF group, bright red fluorescence was visible in the GA group. Fluorescence microplate reader detection revealed the same phenomenon. The red-green fluorescence ratio of the NC group and the GA-treated group was significantly increased compared with the HF group, P<0.05. At the same time, we detected the liver ATP content and found that the HF group reduced the ATP content, while GA treatment significantly increased the ATP content. These results indicate that GA treatment can alleviate HF-induced mitochondrial damage in the liver of largemouth bass;

[0071] Effects of S532GA on HF-induced liver mitochondrial damage: In the NC group, the number of liver mitochondria was abundant, the endoplasmic reticulum structure was normal, the mitochondrial cristae were neatly arranged, the mitochondrial membrane was intact, and the gap between the endoplasmic reticulum and mitochondria was appropriate, while in the HF-treated group, the mitochondrial membrane was obviously ruptured, the mitochondrial cristae were disordered, the endoplasmic reticulum structure was abnormal, and the gap between the endoplasmic reticulum and mitochondria was reduced. In the HFM-treated group, the mitochondrial membrane, mitochondrial cristae arrangement, endoplasmic reticulum structure, and the gap between the endoplasmic reticulum and mitochondria were restored to be consistent with the NC group;

[0072] Effect of S54GA on the expression of HF-induced liver cytoplasmic and endoplasmic reticulum calcium transporters: The mRNA expression of Casr, Sig1r, and Ip3r1 in the HF group increased. However, after GA treatment, the expression of CASR, Sig1R, and Ip3r1 mRNA in liver tissues decreased significantly (P<0.05), which was consistent with the mRNA expression. Western-blot detected the protein level of Ip3p1 and found that compared with NC, the protein level of Ip3r1 was the highest in the HF group, while the protein level of Ip3r1 decreased after GA treatment.

[0073] Effects of S55GA on mRNA expression and protein levels of mitochondrial calcium transporters in the liver induced by HF: To explore the potential mechanism of how GA inhibits mitochondrial calcium transporters, we investigated whether GA could reduce the mRNA expression and protein levels of mitochondrial calcium transporters Grp75, Vdac1, and Mcu induced by HF. Compared with the NC group, the mRNA expression of Grp75, Vdac1, and Mcu in the HF group increased (P<0.05);

[0074] Effect of S56GA on HF-induced Ampk-Pgc1α-Sirt3 signaling pathway: Compared with the NC group, the mRNA expression of Ampk, Pgc1α and Sirt3 in the HF group was decreased, while GA treatment significantly enhanced their mRNA expression. We also found that, consistent with the mRNA expression, the protein levels of Ampk, Pgc1α and Sirt3 in the liver of the HF group were significantly decreased, while those in the GA group were significantly increased.

[0075] Preferably, the S511 liver-to-body ratio is determined by anatomically observing the appearance of the liver and calculating the liver-to-body ratio of largemouth bass.

[0076] Compared with the prior art, the present invention has the following beneficial effects:

[0077] 1. The research method of 18β-glycyrrhizic acid on liver damage mediated by mitochondrial dysfunction induced by high-fat diet explored for the first time that GA can alleviate liver fibrosis by regulating Tgfβ1-Smad2 / 3 signaling to reduce the aggregation of liver collagen fibers, thereby improving HF-induced liver damage in largemouth bass. Secondly, GA can reduce the expression of calcium ion transporters in the endoplasmic reticulum and mitochondria through the Ampk-Pgc1α-Sirt3 signaling pathway, alleviate liver mitochondrial dysfunction and reduce the content of ROS, thereby inhibiting the activation of pro-fibrotic factors. HF will upregulate the activity of liver function-related enzymes in largemouth bass, increase liver lipid droplet deposition and collagen aggregation, while the addition of GA will reduce the activity of liver function-related enzymes, reduce liver fatty degeneration, lipid droplet deposition and collagen fiber aggregation, thereby alleviating HF-induced liver damage in largemouth bass.

[0078] 2. The research method of 18β-glycyrrhizic acid on liver damage mediated by mitochondrial dysfunction induced by high-fat diet. HF feeding will upregulate the expression of pro-fibrotic markers in largemouth bass; and after adding GA treatment, it can reduce the expression of pro-fibrotic markers by downregulating TGFβ1-mediated Smad2 / 3 signaling, thereby alleviating HF-induced liver fibrosis in largemouth bass.

[0079] 3. The research method of the effect of 18β-glycyrrhizic acid on liver damage mediated by mitochondrial function damage induced by high-fat diet. HF feeding of largemouth bass will cause mitochondrial damage and increase ROS content; while GA can improve mitochondrial function by upregulating mitochondrial MMP and ATP content, downregulating ROS content, protecting mitochondrial membrane, mitochondrial cristae and endoplasmic reticulum from mitochondrial damage caused by HF.

[0080] 4. The research method of 18β-glycyrrhizic acid on liver damage mediated by mitochondrial dysfunction induced by high-fat diet. HF feeding of largemouth bass increased the mRNA expression and protein level of calcium ion transporters in the endoplasmic reticulum and mitochondria. GA can reduce the mRNA expression and protein level of endoplasmic reticulum and mitochondrial calcium ion transporters by upregulating Ampk-Pgc1α-Sirt3 signaling, thereby improving the increase in the expression of mitochondrial calcium ion transporters caused by HF. BRIEF DESCRIPTION OF THE DRAWINGS

[0081] Figure 1 The glycyrrhizic acid GA of the present invention protects the liver damage of largemouth bass induced by HF;

[0082] Figure 2 are the hematology and liver function parameters of the present invention;

[0083] Figure 3 The GA of the present invention inhibits HF-induced liver fibrosis in largemouth bass;

[0084] Figure 4 The effect of GA on HF-induced liver Tgfβ1-Smad2 / 3 pathway in the present invention;

[0085] Figure 5 The GA of the present invention alleviates the HF-induced mitochondrial function damage in the liver of largemouth bass;

[0086] Figure 6 The effect of GA on HF-induced Ca 2+ Effects of excessive translocation from the ER to mitochondria;

[0087] Figure 7 This is the effect of GA of the present invention on the Ampk-Pgc1α-Sirt3 pathway in the liver of largemouth bass induced by HF. DETAILED DESCRIPTION

[0088] In order to have a clearer understanding of the technical features, purposes and effects of the present invention, specific embodiments of the present invention are now described with reference to the accompanying drawings.

[0089] Example

[0090] A preferred embodiment of the research method of the present invention for the effect of 18β-glycyrrhetinic acid on liver damage mediated by mitochondrial dysfunction induced by high-fat diet is as follows: Figure 1-7 As shown: The research method of 18β-glycyrrhizic acid on liver damage mediated by mitochondrial function damage induced by high-fat diet includes the following steps: S1 experimental design, S2 experimental sample collection, S3 index determination, S4 experimental data statistical analysis, S5 results.

[0091] S1 includes the following steps:

[0092] S11 experimental diets: The basal feed contained three protein sources, fish meal, chicken meal and fermented soybean meal, soybean oil as the fat source, and two carbohydrates, wheat flour and cassava starch, and was formulated into the control group NC with a protein level of 47.75% and a fat level of 9.05%, the high-fat diet group (HF) with a protein level of 47.31% and a fat level of 16.29%, the high-fat diet plus GA 0.5 mg / kg (HFL) with a protein level of 47.48% and a fat level of 16.25%, the high-fat diet plus GA 1.0 mg / kg (HFM) with a protein level of 47.48% and a fat level of 16.44%, and the high-fat diet plus GA 1.5mg / kg (HFH), protein level is 47.55%, fat level is 16.44%, vitamin and mineral premix addition amount is 1% and 1.5% respectively, vitamin D, vitamin C, selenium, feed raw materials are all crushed, passed through 40 mesh sieve, weighed according to the ratio, trace components are added by step-by-step expansion method, and mixed with bulk raw materials, and then mixed again after adding oil and water, pelletized by small extruded feed machine, dried at 30℃, stored in sealed plastic bags, and used for experiments after nutrient analysis of each feed;

[0093] S12 Experimental fish and feeding management: The experimental fish were healthy largemouth bass. After 2 weeks of temporary rearing, 750 healthy largemouth bass with an average initial weight of 17.39±0.09g were randomly assigned to 15 square cement pools, 50 in each cement pool. The cement pools were randomly divided into 5 treatment groups, which were fed with NC group, HF group, HFL group, HFM group and HFH group feeds, respectively, with 3 replicates for each treatment. The growth experiment lasted for 77 days, and the fish were fed at 7:30 am and 18:00 pm every day. After 40 minutes of feeding, the remaining bait was fished out, dried and weighed. The weather, water temperature, feeding rate, amount of residual bait and food intake were recorded every day. The experiment adopted micro-flow aquaculture, and the water flow rate was maintained at 1.0L / min. The water temperature during the experiment was 25.0±3.0℃, and intermittent aeration was performed to keep the dissolved oxygen greater than 5.0mg / L.

[0094] S2 includes the following steps: after the feeding experiment is completed, the largemouth black bass are fasted for 24 hours, and 12 fish with approximately similar weights are selected from each replicate and anesthetized with 0.01% MS-222. After the fish bodies are wiped dry, they are weighed and the weights are recorded, and blood is collected from the tail vein with a 1.5 ml disposable syringe moistened with an anticoagulant. After the bodies and viscera of 9 largemouth black bass are separated, the livers are quickly separated from the visceral mass, and then thoroughly washed with physiological saline, photographed, weighed and the liver weights are recorded, and then put into bags with corresponding labels, and then wrapped with tin foil, immediately frozen in liquid nitrogen, and then quickly transferred to a -80°C ultra-low temperature refrigerator for storage for subsequent test index determination. The remaining 3 largemouth black bass are first used to measure individual body length and weigh, and then the liver tissues are separated, and the liver tissues are fixed with 4% neutral formalin for subsequent HE staining, Masson staining and IHC staining analysis. In the same way, frozen section samples were placed in 2 mL cryovials, labeled, wrapped in tin foil and immediately frozen in liquid nitrogen for subsequent Oil Red O staining. For electron microscopy samples, soybean-sized tissues were fixed in 2.5% glutaraldehyde solution for electron microscopy section observation.

[0095] S3 includes the following steps:

[0096] S31 Liver-to-body ratio: Based on the sampling statistical data, the liver-to-body index is calculated to determine the growth and development of the liver;

[0097] S32 Determination of blood and liver biochemical indices: The blood was transferred to a microcentrifuge tube and centrifuged at 3000g for 10 minutes at room temperature to separate plasma. The activities of alanine aminotransferase ALT, aspartate aminotransferase AST and alkaline phosphatase AKP, triglyceride TG, total cholesterol TC and hydroxyproline HYP were determined using commercial kits. After taking the sample out of the -80℃ refrigerator, a 10% tissue homogenate was prepared with a homogenizer, and then centrifuged at 3000r / min for 10 minutes to obtain the supernatant, and the malondialdehyde MDA, glutathione S-transferase GST, total protein TP and adenosine triphosphate ATP contents were determined.

[0098] S33 Liver H&E staining analysis: The liver tissue was fixed with 10% formalin solution for 7 days, then dehydrated with gradient ethanol solution, and then the sample was made transparent with xylene for subsequent wax immersion. Finally, the sample was embedded in paraffin and sliced ​​with a paraffin slicer with a thickness of 5 μm, and stained with hematoxylin-eosin H&E. The specific steps of H&E staining are as follows:

[0099] 1 Place the sample slide in a 65℃ constant temperature drying oven and dry for 2 hours;

[0100] 2 Dewaxing: Place the sample slides in xylene for 10 minutes each time, twice in total;

[0101] 3 Hydration: Immerse the sample slides in anhydrous ethanol, 95% ethanol, 80% ethanol, 70% ethanol for 2 min each, and distilled water for 2 min;

[0102] 4Hematoxylin staining for 10 min, washing with water for 30 s;

[0103] 5. Differentiate with 1% hydrochloric acid for 60 seconds and take out after it turns blue;

[0104] 6. Eosin staining for 4 min, washing with water for 30 s;

[0105] 7 Immerse the sample slides in 95% ethanol twice for 5 min each, 100% ethanol twice for 5 min each, 100% ethanol: xylene 1:1 for 5 min, and xylene twice for 5 min each;

[0106] 8. Seal the slide with neutral gum;

[0107] 9. Observe and take photos under optical microscope Olympus BX43 observation;

[0108] S34 Liver Oil Red O Staining Analysis: The specific steps for Oil Red O staining preparation are as follows:

[0109] 1. Take out the liver sample from the -80℃ refrigerator and transfer it to a -20℃ refrigerator for buffering for 30 minutes;

[0110] 2. Cut the liver tissue into square blocks of appropriate size and embed and freeze them with OCT embedding medium;

[0111] 3. Cut the tissue block into 8 μm frozen sections using a pre-cooled cryostat;

[0112] 4. Transfer the cut samples to the slides with corresponding labels and let them stand at room temperature for 10 minutes before staining;

[0113] 5. Cover the sample with washing solution for 20 seconds and then stain with Oil Red O dye for 20 minutes;

[0114] 6 After removing the Oil Red O dye stain, wash with washing solution for 30 seconds, immerse in distilled water and wash on a shaker for 20 seconds;

[0115] 7. Counterstain the cell nucleus with hematoxylin for 3 min, wash with water for 30 s, and seal the slides with glycerol gelatin;

[0116] 8. Microscopic examination: Olympus BX43;

[0117] S35 liver Masson staining analysis: Sample pre-treatment, dewaxing and hydration are the same as the above H&E staining. The specific staining steps are as follows:

[0118] 1. Stain with prepared Weigert iron hematoxylin staining solution for 10 minutes;

[0119] 2 Use acidic ethanol differentiation solution for differentiation for 15 seconds and wash with water;

[0120] 3. Use Masson bluing solution to return to blue for 5 minutes, then wash with water and distilled water for 1 minute;

[0121] 4. Stain with Ponceau fuchsin staining solution for 10 min;

[0122] 5 Wash with weak acid working solution for 1 min. The weak acid working solution is distilled water: weak acid solution = 2:1;

[0123] 6 Then wash with phosphomolybdic acid solution for 2 minutes and weak acid working solution for 1 minute;

[0124] 7 Directly put it into aniline blue staining solution for 2 minutes, and wash it with weak acid working solution for 1 minute;

[0125] 895% ethanol rapid dehydration for 3 seconds, anhydrous ethanol dehydration 3 times, each time for 10 seconds;

[0126] 9. Xylene transparent 3 times, 2 min each time, neutral gum sealing, observation;

[0127] S36 liver transmission electron microscopy analysis: a small piece of liver (1×1×1 mm3) was taken and placed in 2.5% glutaraldehyde at 4°C for 2-4 hours, and then fixed with 1% osmium acid prepared in 0.1 M phosphate buffer PB at room temperature in the dark for 2 hours. After dehydration, infiltration, embedding, sectioning and staining, it was observed under a transmission electron microscope and image analysis was collected;

[0128] S37 liver immunofluorescence analysis: Sample pre-treatment, dewaxing and H&E staining as above, submerge slices with 10mmol / L citrate buffer pH6.0, heat to boiling in a microwave oven for 20min; incubate with primary antibodies anti-α-Sma, anti-Tgfβ1, Sirt3 and Mcu antibodies in a wet box overnight, rewarm the next day, wash, add 25μL of horseradish peroxidase HRP-labeled secondary antibody anti-rabbit / mouse to each slice, incubate at 37℃ for 40min, wash twice with PBST, 5min each time, add DAPI (0.2ug / ml) to the slices, 5min, wash twice with PBST, 5min each time. Seal the slices, observe and take pictures under an inverted fluorescence microscope;

[0129] S38 Reactive oxygen detection: 200 mg of liver was taken out at the same time, and the reactive oxygen ROS concentration was measured immediately. According to the preparation method of single cell suspension, single cell suspension was prepared according to the operation steps, and then the fluorescent probe was added, and the liver cell pellet was suspended with diluted DCFH-DA. The liver cell pellet was incubated in a 27°C incubator for 30-60 min, centrifuged at 1000 r / min for 10 min, the supernatant was removed, and the cells and sediment were collected and suspended and mixed with PBS sediment. The confocal fluorescence microscope was used to take pictures, and the absorbance value at 525 nm was detected with a fluorescent microplate to calculate the ROS content;

[0130] S39 Membrane potential MMP assay: liver mitochondria were isolated using a tissue mitochondrial isolation kit, and MMP was detected using the JC-1 fluorescent probe method according to the instructions. Mitochondria were then observed using a fluorescence microscope, and the absorbance was detected using a fluorescent microplate reader. The red-green light ratio was calculated, with an excitation wavelength of 485 nm and an emission wavelength of 580 nm.

[0131] S310 real-time quantitative PCR detection: including the following steps:

[0132] S3101 RNA extraction and cDNA synthesis: Total RNA was extracted using a kit. Briefly, liver tissue was taken out from a -80°C freezer, ground into powder in liquid nitrogen, 0.1 g of liver sample was taken, 1 mL of reagent was added to extract total RNA from the tissue, and dissolved in 50 μL of RNase-free water. The degradation degree of RNA sample was detected by 1.0% agarose gel electrophoresis at 120 V for 12 min. The concentration was detected by NanoDrop 1000 spectrophotometer, and the purity of the sample was determined based on the A260 / A280 ratio.

[0133] S3102 Real-time fluorescence quantitative primer design: All gene sequences of largemouth bass were obtained from GenBank or referenced from previous studies. Primers were designed using Primer5.0;

[0134] Table 2 Primers used for fluorescence quantification

[0135] Table 2.Primer sequences of genes selected for q-PCR

[0136]

[0137] S3103 real-time fluorescence quantitative detection of gene expression levels: the three cDNA samples of each treatment were diluted 1:1, and then the fibrosis-related genes: Tgfβ1a, Smad2, Smad3a, α-Sma, CollagenⅠ, Fibronectin and Mmp9; mitochondrial calcium ion transport-related genes: Ampkα1, Pgc1α, Sirt3, Ip3r1, Sig1r1, Casr, Grp75, Vdac1 and Mcu were detected using TB Green TM Premix Ex Taq TM II (Tli RNaseH Plus) kit was used for fluorescence quantitative PCR amplification. The positive template diluted 10 times continuously was used as the PCR template, and then a standard curve was made. The amplification efficiency was between 90% and 110%, and R 2 >0.9, the melting curve is a single peak. A 10μL PCR amplification system was used: 3.2μL DEPC water, 1μL cDNA template, 0.4μL upstream primer and downstream primer, and 5μL SYBR Premix Ex TaqⅡ. The reaction cycle conditions were: 95℃ preheating for 2min, 40 cycles of 95℃ for 10s and appropriate annealing temperature for 30s. After the reaction was completed, the melting curve was used to detect the product specificity.

[0138] S3104 relative quantitative calculation: The expression level is represented by the threshold cycle Ct, which is calculated by plotting the fluorescence signal of each cycle. The target gene mRNA concentration is normalized to be consistent with the mRNA concentration of the internal reference gene β-actin and 18SrRNA. After verifying that the primer amplification efficiency reaches the target gene and internal reference gene amplification efficiency, 2 -ΔΔCT The amplification efficiency of the internal reference gene was calculated based on the results of the method and the specific gene standard curve generated by 10-fold serial dilution;

[0139] S311 protein immunoblotting: Total liver tissue protein was extracted with RIPA Lysis Buffer (Biyuntian, Beijing, China), and the sample protein concentration was determined using Enhanced BCA Protein Assay Kit (Biyuntian Biotechnology Co., Ltd., China). SDS-PAGE protein loading buffer 5X was added to the sample, and the samples were denatured at 95°C for 10 min. After SDS-PAGE separation, they were transferred to polyvinylidene fluoride PVDF membrane, blocked with 5% skim milk for 1.5 h, and incubated with primary antibody at 4°C overnight on a shaker. After washing, the secondary antibody was incubated at room temperature for 1.5 h. The expression of the target protein was detected using ECL Kit (Beyotime), and Chemi Doc Imaging Systems / Image Lab software (Bio-Rad) was used. β-actin was an internal reference with equal sample loading.

[0140] Table 3. Primary antibodies and dilution ratios.

[0141] Table 3.Primary antibodies and dilutions

[0142]

[0143] S4 includes the use of software to process the data, using analysis of variance combined with Duncan's multiple comparisons to detect significance, and the data are expressed as mean ± standard error Mean ± SEM. P < 0.05 indicates a significant difference. Image-ProPlus 6.0 software was used to perform quantitative statistics on liver H&E, Oil Red O, and Masson staining.

[0144] The liver-to-body ratio was calculated based on the sampling statistical data: liver-to-body index (%) = liver weight / body weight × 100.

[0145] S5 includes the following steps:

[0146] The effect of S51GA on HF-induced liver injury in largemouth bass includes the following steps:

[0147] S511 Liver-to-body ratio: By dissecting and observing the liver appearance and calculating the liver-to-body ratio of largemouth bass, it was found that the liver surface of the NC group was smooth and ruddy. The liver of largemouth bass in the HF group showed that most of the liver surface was pale, and GA treatment significantly reduced the pale surface of the liver and increased the ruddy surface of the liver. Compared with the NC group, the liver-to-body ratio of the HF group was significantly increased, and GA treatment reduced the liver-to-body ratio;

[0148] S512 Blood and liver function parameters: To explore the effect of GA on HF-induced liver function of largemouth bass. We tested the hematology and liver function of largemouth bass. The serum AST, ALT and AKP activities of the HF group were significantly higher than those of the NC group. After adding GA treatment, the AST, ALT and AKP activities were significantly reduced P<0.05. The HF group had the highest serum TG and TC contents, while the TG and TC contents were significantly reduced after GA treatment P<0.05. Compared with the NC group, the MDA content in the HF group increased significantly, while the MDA content in the GA group decreased significantly P<0.05. The GST activity was the lowest in the HF group P<0.05, and the TP content was the lowest in the NC group P<0.05. The above results showed that GA reduced the HF-induced AST, ALT and AKP activities in serum, TG, TC contents and MDA content in the liver, and increased GST activity, thereby alleviating HF-induced liver damage;

[0149] Table 4 Biochemical parameters of serum and liver of largemouth bass

[0150] Table 4.Biochemical parameters of serum and liver of Largemouth bass

[0151]

[0152] Note: Data are expressed as mean ± standard error. The same superscript letters in the same row indicate no significant difference (P>0.05), while different superscript letters indicate significant difference (P<0.05).

[0153] S513 Liver histopathology: Compared with the NC group, the hepatocytes in the HF group were significantly swollen, the nuclei showed marginal displacement, some even disappeared, and the number was small. Compared with the HF group, the fatty degeneration of the liver in the GA-treated group was gradually alleviated, the number of nuclei gradually increased, and was located in the center of the cell. Statistical analysis found that compared with the NC group, the relative area of ​​lipid vacuoles in the HF group increased significantly, and compared with the HF group, the relative area of ​​lipid vacuoles in the HFM group in the relief group was the lowest. The results of Oil Red O staining showed that compared with the NC group, a large number of lipid droplets appeared in the liver of the HF group, while the lipid droplets in the liver of the GA-treated group gradually decreased. The Oil Red O-positive area in the HF group increased significantly, and the Oil Red O-positive area decreased after GA treatment. The above results indicate that GA reduces HF-induced liver lipid degeneration and lipid droplet deposition;

[0154] Effect of S52GA on HF-induced liver fibrosis in largemouth bass: Masson staining is a specific staining method for detecting collagen fibers. Masson staining of liver sections of largemouth bass showed that muscle fibers were red and collagen fibers were blue. HF treatment resulted in the aggregation of a large number of collagen fibers, and the addition of GA treatment significantly reduced the aggregation of liver collagen fibers. The therapeutic effects of HFM and HFH were close to those of the NC group. In addition, the quantitative evaluation of the fibrosis area by Masson staining using IPP showed that GA treatment significantly reduced liver fibrosis relative to the HF group. HYP content is a sensitive biochemical indicator reflecting changes in collagen fibers. In order to further study the effect of GA on HF-induced liver fibrosis in largemouth bass, we detected the HYP content in serum and found that compared with the NC group, the HYP content in the serum of largemouth bass in the HF group was significantly increased, while compared with the HF group, GA administration significantly reduced the HYP content;

[0155] Effect of S521GA on the transcription of HF-induced profibrotic markers associated with HSC activation: The mRNA expression of profibrotic markers α-Sma, CollagenI, Fibrontein and Mmp9 was further detected. The results showed that compared with the NC group, HF significantly increased the mRNA expression of α-Sma, CollagenI and Fibrontein in the liver, while compared with the HF group, GA significantly reduced the mRNA expression of α-Sma, CollagenI and Fibrontein. The protein levels of α-Sma, CollagenI and Fibrontein were detected by Western-blot method. It was found that the protein levels of α-Sma, CollagenI and Fibrontein were the highest in the HF group, and the mRNA expression of Mmp9 was the highest in the HF group. Compared with the HF group, the mRNA expression of Mmp9 was significantly reduced after adding GA treatment. In addition, the results of immunohistochemical staining confirmed that GA significantly inhibited the expression of HF-induced α-Sma. The above results show that GA can effectively alleviate HF-induced liver fibrosis in largemouth bass;

[0156] Effects of S522GA on HF-induced Tgfβ1-Smad2 / 3 signaling pathway in the liver of largemouth bass: Tgfβ1a is the most potent fibroblast factor in the liver. Therefore, to explore the inhibitory mechanism of GA on HF-induced liver fibrosis, we detected the mRNA expression of Tgfβ1a and its target proteins Smad2 and Smad3a. Compared with the NC group, the mRNA expressions of Tgfβ1a, Smad2, and Smad3a in the HF group were significantly increased, while GA treatment significantly reduced their mRNA expressions. The Western-blot method was used to detect the protein levels of Tgfβ1, p-Smad2, and p-Smad3, and it was found that the protein levels of Tgfβ1, p-Smad2, and p-Smad3 in the HF group were significantly increased, and their protein levels were reduced after GA treatment, which was consistent with the protein and mRNA expressions. The immunohistochemical results showed that compared with the HF group, the expression of Tgfβ1 in the liver tissue of largemouth bass treated with GA was significantly reduced. These results indicate that GA improves liver fibrosis by regulating the Smad2 / 3 signaling pathway mediated by Tgfβ1.

[0157] The effects of S53GA on HF-induced liver mitochondria include the following steps:

[0158] Effect of S531GA on HF-induced liver reactive oxygen species: Mitochondrial membrane potential MMP was measured using JC-1, and it was found that JC-1 staining showed bright red fluorescence for JC-1 polymers, and green fluorescence for JC-1 monomers. JC-1 staining showed that bright red fluorescence and weak green light were visible in the NC group. On the contrary, a transition from red fluorescence to green fluorescence was observed in the HF group. Compared with the HF group, bright red fluorescence was visible in the GA group. Fluorescence microplate reader detection revealed the same phenomenon. The red-green fluorescence ratio of the NC group and the GA-treated group was significantly increased compared with the HF group, P<0.05. At the same time, we detected the liver ATP content and found that the HF group reduced the ATP content, while GA treatment significantly increased the ATP content. These results indicate that GA treatment can alleviate HF-induced mitochondrial damage in the liver of largemouth bass;

[0159] Effects of S532GA on HF-induced liver mitochondrial damage: In the NC group, the number of liver mitochondria was abundant, the endoplasmic reticulum structure was normal, the mitochondrial cristae were neatly arranged, the mitochondrial membrane was intact, and the gap between the endoplasmic reticulum and mitochondria was appropriate, while in the HF-treated group, the mitochondrial membrane was obviously ruptured, the mitochondrial cristae were damaged, the endoplasmic reticulum structure was abnormal, and the gap between the endoplasmic reticulum and mitochondria was reduced. In the HFM-treated group, the mitochondrial membrane, mitochondrial cristae arrangement, endoplasmic reticulum structure, and the gap between the endoplasmic reticulum and mitochondria were restored to be consistent with the NC group;

[0160] Effect of S54GA on HF-induced expression of liver cytoplasmic and endoplasmic reticulum calcium transporters: The mRNA expression of Casr, Sig1r, and Ip3r1 in the HF group increased. However, after GA treatment, the expression of Casr, Sig1r, and Ip3r1 mRNA in liver tissues decreased significantly (P<0.05), which was consistent with the mRNA expression. Western-blot detected the protein level of Ip3r1 and found that compared with NC, the protein level of IP3R1 was the highest in the HF group, while the protein level of Ip3r1 decreased after GA treatment.

[0161] S55 Effect of GA on mRNA expression and protein level of mitochondrial calcium transporters in liver induced by HF: To explore the potential mechanism of how GA inhibits mitochondrial calcium transporters, we investigated whether GA could reduce the mRNA expression and protein level of mitochondrial calcium transporters Grp75, Vdac1, and Mcu induced by HF. Compared with the NC group, the mRNA expression of Grp75, Vdac1, and Mcu in the HF group increased (P<0.05);

[0162] Effect of S56GA on HF-induced Ampk-Pgc1α-Sirt3 signaling pathway: Compared with the NC group, the mRNA expression of Ampk, Pgc1α and Sirt3 in the HF group was decreased, while GA treatment significantly enhanced their mRNA expression. We also found that, consistent with the mRNA expression, the protein levels of Ampk, Pgc1α and Sirt3 in the liver of the HF group were significantly decreased, while those in the GA group were significantly increased.

[0163] In S511 liver-to-body ratio, the liver appearance was observed by dissection and the liver-to-body ratio of largemouth bass was calculated.

[0164] Working principle:

[0165] 1. The research method of 18β-glycyrrhizic acid on liver damage mediated by mitochondrial function damage induced by high-fat diet explored for the first time that GA can alleviate liver fibrosis by regulating Tgfβ1-Smad2 / 3 signaling to reduce the aggregation of liver collagen fibers, thereby improving HF-induced liver damage in largemouth bass. Secondly, GA can reduce the expression of calcium ion transporters in the endoplasmic reticulum and mitochondria through the Ampk-Pgc1α-Sirt3 signaling pathway, alleviate liver mitochondrial damage and reduce the content of ROS, thereby inhibiting the activation of pro-fibrotic factors. HF will upregulate the activity of liver function-related enzymes in largemouth bass, increase liver lipid droplet deposition and collagen aggregation, while the addition of GA will reduce the activity of liver function-related enzymes, reduce liver fatty degeneration, lipid droplet deposition and collagen fiber aggregation, thereby alleviating HF-induced liver damage in largemouth bass.

[0166] 2. The research method of 18β-glycyrrhizic acid on liver damage mediated by mitochondrial dysfunction induced by high-fat diet. HF feeding will upregulate the expression of pro-fibrotic markers in largemouth bass; after adding GA treatment, it can reduce the expression of pro-fibrotic markers by downregulating Tgfβ1-mediated Smad2 / 3 signaling, thereby alleviating HF-induced liver fibrosis in largemouth bass.

[0167] 3. The research method of the effect of 18β-glycyrrhizic acid on liver damage mediated by mitochondrial function damage induced by high-fat diet. HF feeding of largemouth bass will cause mitochondrial damage and increase ROS content; while GA can improve mitochondrial function by upregulating mitochondrial MMP and ATP content, downregulating ROS content, protecting mitochondrial membrane, mitochondrial cristae and endoplasmic reticulum from mitochondrial damage caused by HF.

[0168] 4. The research method of 18β-glycyrrhizic acid on liver damage mediated by mitochondrial dysfunction induced by high-fat diet. HF feeding of largemouth bass increased the mRNA expression and protein level of calcium ion transporters in the endoplasmic reticulum and mitochondria. GA can reduce the mRNA expression and protein level of endoplasmic reticulum and mitochondrial calcium ion transporters by upregulating Ampk-Pgc1α-Sirt3 signaling, thereby improving the increase in the expression of mitochondrial calcium ion transporters caused by HF.

[0169] The above description is only an illustrative specific implementation mode of the present invention and is not intended to limit the scope of the present invention. Any equivalent changes and modifications made by any technician in the field without departing from the concept and principle of the present invention should fall within the scope of protection of the present invention. It should also be noted that the various components of the present invention are not limited to the above-mentioned overall application. The various technical features described in the specification of the present invention can be selected one by one or multiple ones can be selected and used in combination according to actual needs. Therefore, the present invention should naturally cover other combinations and specific applications related to the invention point of this case.

Claims

1. 18 The research method of the effect of β-glycyrrhetinic acid on liver damage mediated by mitochondrial dysfunction induced by high-fat diet comprises the following steps, characterized in that: S1 Experimental design; S2 test sample collection; S3 index determination; S4 Statistical analysis of experimental data; S5 Results.

2. The method for studying the effect of 18β-glycyrrhetinic acid on liver damage mediated by mitochondrial dysfunction induced by high-fat diet according to claim 1, characterized in that: The S1 includes the following steps: S11 Experimental diets: The basal feed contained three protein sources, fish meal, chicken meal, and fermented soybean meal, with soybean oil as the fat source, and two carbohydrates, wheat flour and cassava starch, and was formulated into the control group (NC) with a protein level of 47.75% and a fat level of 9.05%, the high-fat diet group (HF) with a protein level of 47.31% and a fat level of 16.29%, the high-fat diet plus GA0.5 mg / kg (HFL) with a protein level of 47.48% and a fat level of 16.25%, the high-fat diet plus GA1.0 mg / kg (HFM) with a protein level of 47.48% and a fat level of 16.44%, and the high-fat diet plus GA1.5 mg / kg (HFM) with a protein level of 47.75% and a fat level of 9.05%. mg / kg (HFH), protein level was 47.55%, fat level was 16.44%, vitamin and mineral premix addition was 1% and 1.5% respectively, vitamin D, vitamin C, selenium, all feed raw materials were crushed, passed through a 40-mesh sieve, weighed according to the ratio, trace components were added by step-by-step expansion method, and mixed evenly with bulk raw materials, added oil and water and mixed evenly again, pelleted with a small extruded feed machine, dried at 30°C, stored in sealed plastic bags, and used for experiments after a rough nutrient analysis of the nutritional components of each feed; S12 Experimental fish and feeding management: The experimental fish were healthy largemouth bass. After 2 weeks of temporary rearing, 750 healthy largemouth bass with an average initial weight of 17.39±0.09g were randomly assigned to 15 square cement pools, 50 in each cement pool. The cement pools were randomly divided into 5 treatment groups, which were fed with NC group, HF group, HFL group, HFM group and HFH group feeds, respectively, with 3 replicates for each treatment. The growth experiment lasted for 77 days, and the fish were fed at 7:30 am and 18:00 pm every day. After 40 minutes of feeding, the remaining bait was fished out, dried and weighed. The weather, water temperature, feeding rate, amount of residual bait and food intake were recorded every day. The experiment adopted micro-flow aquaculture, and the water flow rate was maintained at 1.0L / min. The water temperature during the experiment was 25.0±3.0℃, and intermittent aeration was performed to keep the dissolved oxygen greater than 5.0mg / L.

3. The method for studying the effect of 18β-glycyrrhetinic acid on liver damage mediated by mitochondrial dysfunction induced by high-fat diet according to claim 1, characterized in that: The S2 includes the following steps: after the feeding experiment is completed, the largemouth bass are fasted for 24 hours, 12 fish with roughly similar weights are selected for each repetition and anesthetized with 0.01% MS-222, after the fish body is wiped dry, the weight is weighed and recorded, and blood is collected from the tail vein with a 1.5mL disposable syringe rinsed with an anticoagulant. After the body and viscera of 9 largemouth bass are separated, the liver is quickly separated from the visceral mass, and then thoroughly washed with physiological saline, photographed, weighed and recorded, and then put into a bag with a corresponding label, and then wrapped with tin foil, immediately frozen in liquid nitrogen, and then quickly transferred to a -80°C ultra-low temperature refrigerator for storage for subsequent test index determination. The remaining 3 largemouth bass, liver tissue is separated, and the liver tissue is fixed with 4% neutral formalin for subsequent HE staining, Masson staining and immunofluorescence (IF) staining analysis. In the same way, frozen section samples were placed in 2 mL cryovials, labeled, wrapped in tin foil and immediately frozen in liquid nitrogen for subsequent Oil Red O staining. For electron microscopy samples, soybean-sized tissues were fixed in 2.5% glutaraldehyde solution for transmission electron microscopy section observation.

4. The method for studying the effect of 18β-glycyrrhetinic acid on liver damage mediated by mitochondrial dysfunction induced by high-fat diet according to claim 1, characterized in that: The S3 includes the following steps: S31 Liver-to-body ratio: Based on the sampling statistical data, the liver-to-body index is calculated to determine the growth and development of the liver; S32 Determination of blood and liver biochemical indices: The blood was transferred to a microcentrifuge tube and centrifuged at 3000g for 10 min at room temperature to separate plasma. The activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (AKP), and the contents of triglycerides (TG), total cholesterol (TC) and hydroxyproline (HYP) were determined using commercial kits. After taking the sample out of the -80℃ refrigerator, a 10% tissue homogenate was prepared using a homogenizer, and then centrifuged at 3000r / min for 10 min to obtain the supernatant, and the malondialdehyde (MDA), glutathione S-transferase (GST), total protein (TP) and adenosine triphosphate (ATP) contents were determined; S33 Liver H&E staining analysis: The liver tissue was fixed with 10% formalin solution for 7 days, then dehydrated with gradient ethanol solution, and then the sample was made transparent with xylene for subsequent wax immersion. Finally, the sample was embedded in paraffin and sliced ​​with a paraffin slicer with a thickness of 5 μm. The sample was stained with hematoxylin-eosin H&E. The specific steps of H&E staining are as follows: 1 Place the sample slide in a 65℃ constant temperature drying oven and dry for 2 hours; 2 Dewaxing: Place the sample slides in xylene for 10 minutes each time, twice in total; 3 Hydration: Immerse the sample slides in anhydrous ethanol, 95% ethanol, 80% ethanol, 70% ethanol for 2 min each, and distilled water for 2 min; 4Hematoxylin staining for 10 min, washing with water for 30 s; 5. Differentiate with 1% hydrochloric acid for 60 seconds and take out after it turns blue; 6. Eosin staining for 4 min, washing with water for 30 s; 7 Immerse the sample slides in 95% ethanol twice for 5 min each, 100% ethanol twice for 5 min each, 100% ethanol: xylene 1:1 for 5 min, and xylene twice for 5 min each; 8. Seal the slide with neutral gum; 9. Observe and take photos under optical microscope Olympus BX43 observation; S34 Liver Oil Red O Staining Analysis: The specific steps for Oil Red O staining preparation are as follows:

1. Take out the liver sample from the -80℃ refrigerator and transfer it to a -20℃ refrigerator for buffering for 30 minutes; 2. Cut the liver tissue into square blocks of appropriate size and embed and freeze them with OCT embedding medium; 3. Cut the tissue block into 8 μm frozen sections using a pre-cooled cryostat; 4. Transfer the cut samples to the slides with corresponding labels and let them stand at room temperature for 10 minutes before staining; 5. Cover the sample with washing solution for 20 seconds and then stain with Oil Red O dye for 20 minutes; 6 After removing the Oil Red O dye stain, wash with washing solution for 30 seconds, immerse in distilled water and wash on a shaker for 20 seconds; 7. Counterstain the cell nucleus with hematoxylin for 3 min, wash with water for 30 s, and seal the slides with glycerol gelatin; 8. Microscopic examination: Olympus BX43; S35 liver Masson staining analysis: Sample pre-treatment, dewaxing and hydration are the same as the above H&E staining. The specific staining steps are as follows:

1. Stain with prepared Weigert iron hematoxylin staining solution for 10 minutes; 2 Use acidic ethanol differentiation solution for differentiation for 15 seconds and wash with water; 3. Use Masson bluing solution to return to blue for 5 minutes, then wash with water and distilled water for 1 minute; 4. Stain with Ponceau fuchsin staining solution for 10 min; 5 Wash with weak acid working solution for 1 min. The weak acid working solution is distilled water: weak acid solution = 2:1; 6 Then wash with phosphomolybdic acid solution for 2 minutes and weak acid working solution for 1 minute; 7 Directly put it into aniline blue staining solution for 2 minutes, and wash it with weak acid working solution for 1 minute; 895% ethanol rapid dehydration for 3 seconds, anhydrous ethanol dehydration 3 times, each time for 10 seconds; 9. Xylene transparent 3 times, 2 min each time, neutral gum sealing, observation; S36 Transmission electron microscopy analysis of liver: Take a small piece of liver 1×1×1mm 3 , placed in 2.5% glutaraldehyde, quickly fixed at 4°C for 2-4h, fixed with 1% osmium acid prepared in 0.1M phosphate buffer PB at room temperature in the dark for 2h, dehydrated, infiltrated, embedded, sectioned and stained, and observed under a transmission electron microscope, and collected images for analysis; S37 liver immunofluorescence analysis: Sample pre-treatment, dewaxing and H&E staining as above, submerge slices with 10mmol / L citrate buffer pH6.0, heat to boiling in a microwave oven for 20min; incubate with primary antibodies anti-α-Sma, anti-Tgfβ1, Sirt3 and Mcu antibodies in a wet box overnight, rewarm the next day, wash, add 25μL of horseradish peroxidase HRP-labeled secondary antibody anti-rabbit / mouse to each slice, incubate at 37℃ for 40min, wash twice with PBST, 5min each time, add DAPI (0.2ug / ml) to the slices, 5min, wash twice with PBST, 5min each time. Seal the slices, observe and take pictures under an inverted fluorescence microscope; S38 Reactive oxygen detection: 200 mg of liver was taken out at the same time, and the reactive oxygen ROS concentration was measured immediately. According to the preparation method of single cell suspension, single cell suspension was prepared according to the operation steps, and then the fluorescent probe was added, and the liver cell pellet was suspended with diluted DCFH-DA. The liver cell pellet was incubated in a 27°C incubator for 30-60 min, centrifuged at 1000 r / min for 10 min, the supernatant was removed, and the cells and sediment were collected and suspended and mixed with PBS sediment. The confocal fluorescence microscope was used to take pictures, and the absorbance value at 525 nm was detected with a fluorescent microplate to calculate the ROS content; S39 Membrane potential MMP assay: liver mitochondria were isolated using a tissue mitochondrial isolation kit, and MMP was detected using the JC-1 fluorescent probe method according to the instructions. Mitochondria were then observed using a fluorescence microscope, and the absorbance was detected using a fluorescent microplate reader. The red-green light ratio was calculated, with an excitation wavelength of 485 nm and an emission wavelength of 580 nm. S310 real-time quantitative PCR detection: including the following steps: S3101 RNA extraction and cDNA synthesis: Total RNA was extracted using a kit. Briefly, liver tissue was taken out from a -80°C freezer, ground into powder in liquid nitrogen, 0.1 g of liver sample was taken, 1 mL of RNAiso Plus reagent was added to extract total RNA from the tissue, and the total RNA was dissolved in 40 μL of RNase-free water. The degradation degree of RNA sample was detected by 1.0% agarose gel electrophoresis at 120 V for 15 min. The concentration was detected by NanoDrop1000 spectrophotometer, and the purity of the sample was determined based on the A260 / A280 ratio. S3102 Real-time fluorescence quantitative primer design: All gene sequences of largemouth bass were obtained from GenBank or referenced from previous studies. Primers were designed using Primer 5.0; S3103 real-time fluorescence quantitative detection of gene expression levels: the three cDNA samples of each treatment were diluted 1:1, and then fibrosis-related genes: Tgfβ1a, Smad2, Smad3a, α-Sma, CollagenⅠ, Fibronectin and Mmp9; mitochondrial calcium ion transport-related genes: Ampkα1, Pgc1α, Sirt3, Ip3r1, Sig1r1, Casr, Grp75, Vdac1, Mcu were detected using TBGreen TM PremixExTaq TM IITliRNaseHPlus kit was used for fluorescence quantitative PCR amplification. The positive template diluted 10 times continuously was used as the PCR template, and then a standard curve was made. The amplification efficiency was between 90% and 110%, and R 2 >0.9, the melting curve is a single peak. A 10 μL PCR amplification system was used: 3.2 μL DEPC water, 1 μL cDNA template, 0.4 μL upstream primer and downstream primer, and 5 μL SYBR Premix Ex Taq II. The reaction cycle conditions were: 95 °C preheating for 2 min, 40 cycles of 95 °C for 10 s and appropriate annealing temperature for 30 s. After the reaction was completed, the melting curve was used to detect the product specificity. S3104 relative quantitative calculation: The expression level is represented by the threshold cycle Ct, which is calculated by plotting the fluorescence signal of each cycle. The target gene mRNA concentration is normalized to be consistent with the mRNA concentration of the internal reference gene βactin and 18SrRNA. After verifying that the primer amplification efficiency reaches the target gene and internal reference gene amplification efficiency, 2 -ΔΔCT The amplification efficiency of the internal reference gene was calculated based on the results of the method and the specific gene standard curve generated by 10-fold serial dilution; S311 protein immunoblotting: Total liver tissue protein was extracted with RIPALysis Buffer (Biyuntian, Beijing, China), and the sample protein concentration was determined using an enhanced BCA protein concentration assay kit (Biyuntian Biotechnology Co., Ltd., China). SDS-PAGE protein loading buffer (5X) was added to the sample, and the samples were denatured at 95°C for 10 min. After SDS-PAGE separation, the membranes were transferred to polyvinylidene difluoride PVDF membranes and blocked with 5% skim milk for 1.5 h. The primary antibody was incubated overnight at 4°C on a shaker. After washing, the secondary antibody was incubated at room temperature for 1.5 h. The expression of the target protein was detected using an ECL Kit (Beyotime) and recorded using Chemi Doc Imaging Systems / Image Lab software (Bio-Rad). β-actin was an internal reference with equal sample loading.

5. The method for studying the effect of 18β-glycyrrhetinic acid on liver damage mediated by mitochondrial dysfunction induced by high-fat diet according to claim 1, characterized in that: The S4 includes the use of software to process the data, using analysis of variance combined with Duncan's multiple comparisons to detect significance, and the data are expressed as mean ± standard error Mean ± SEM. P < 0.05 indicates a significant difference. Image-ProPlus 6.0 software is used to perform quantitative statistics on liver H&E, Oil Red O and Masson staining.

6. The method for studying the effect of 18β-glycyrrhetinic acid on liver damage mediated by mitochondrial dysfunction induced by high-fat diet according to claim 4, characterized in that: The liver-to-body ratio is calculated based on sampling statistical data to obtain a liver-to-body index: liver-to-body index % = liver weight / body weight × 100.

7. The method for studying the effect of 18β-glycyrrhetinic acid on liver damage mediated by mitochondrial dysfunction induced by high-fat diet according to claim 1, characterized in that: The S5 comprises the following steps: The effect of S51GA on HF-induced liver injury in largemouth bass includes the following steps: S511 Liver-to-body ratio: By dissecting and observing the liver appearance and calculating the liver-to-body ratio of largemouth bass, it was found that the liver surface of the NC group was smooth and ruddy. The liver of largemouth bass in the HF group showed that most of the liver surface was pale, and GA treatment significantly reduced the pale surface of the liver and increased the ruddy surface of the liver. Compared with the NC group, the liver-to-body ratio of the HF group was significantly increased, and GA treatment reduced the liver-to-body ratio; S512 Blood and liver function parameters: To explore the effect of GA on HF-induced liver function of largemouth bass. We tested the hematology and liver function of largemouth bass. The serum AST, ALT and AKP activities of the HF group were significantly higher than those of the NC group. After adding GA treatment, the AST, ALT and AKP activities were significantly reduced P<0.

05. The HF group had the highest serum TG and TC contents, while the TG and TC contents were significantly reduced after GA treatment P<0.

05. Compared with the NC group, the MDA content in the HF group increased significantly, while the MDA content in the GA group decreased significantly P<0.

05. The GST activity was the lowest in the HF group P<0.05, and the TP content was the lowest in the NC group P<0.

05. The above results showed that GA reduced the HF-induced AST, ALT and AKP activities in serum, TG, TC contents and MDA content in the liver, and increased GST activity, thereby alleviating HF-induced liver damage; S513 Liver histopathology: Compared with the NC group, the hepatocytes in the HF group were significantly swollen, the nuclei showed marginal displacement, some even disappeared, and the number was small. Compared with the HF group, the fatty degeneration of the liver in the GA-treated group was gradually alleviated, the number of nuclei gradually increased, and was located in the center of the cell. Statistical analysis found that compared with the NC group, the relative area of ​​lipid vacuoles in the HF group increased significantly, and compared with the HF group, the relative area of ​​lipid vacuoles in the HFM group in the relief group was the lowest. The results of Oil Red O staining showed that compared with the NC group, a large number of lipid droplets appeared in the liver of the HF group, while the lipid droplets in the liver of the GA-treated group gradually decreased. The Oil Red O-positive area in the HF group increased significantly, and the Oil Red O-positive area decreased after GA treatment. The above results indicate that GA reduces HF-induced liver lipid degeneration and lipid droplet deposition; S52 Effect of GA on HF-induced liver fibrosis in largemouth bass: Masson staining is a specific staining method for detecting collagen fibers. Masson staining of liver sections of largemouth bass showed that muscle fibers were red and collagen fibers were blue. HF treatment resulted in the aggregation of a large number of collagen fibers, and the addition of GA treatment significantly reduced the aggregation of liver collagen fibers. The therapeutic effects of HFM and HFH were close to those of the NC group. In addition, the quantitative evaluation of the fibrosis area by Masson staining using IPP showed that GA treatment significantly reduced liver fibrosis compared with the HF group. HYP content is a sensitive biochemical indicator reflecting changes in collagen fibers. In order to further study the effect of GA on HF-induced liver fibrosis in largemouth bass, we detected the HYP content in serum and found that compared with the NC group, the HYP content in the serum of largemouth bass in the HF group was significantly increased, while compared with the HF group, GA administration significantly reduced the HYP content; Effect of S521GA on the transcription of HF-induced profibrotic markers associated with HSC activation: The mRNA expression of profibrotic markers α-SMA, CollagenI, Fibrontein and Mmp9 was further detected. The results showed that compared with the NC group, HF significantly increased the mRNA expression of α-Sma, CollagenI and Fibrontein in the liver, while compared with the HF group, GA significantly reduced the mRNA expression of α-Sma, CollagenI and Fibrontein. The protein levels of α-SMA, CollagenI and Fibrontein were detected by Western-blot method. It was found that the protein levels of α-Sma, CollagenI and Fibrontein were the highest in the HF group, and the mRNA expression of Mmp9 was the highest in the HF group. Compared with the HF group, the mRNA expression of Mmp9 was significantly reduced after adding GA treatment. In addition, the results of immunofluorescence staining confirmed that GA significantly inhibited the expression of HF-induced α-Sma. The above results show that GA can effectively alleviate HF-induced liver fibrosis in largemouth bass; S522 Effect of GA on HF-induced Tgfβ1-Smad2 / 3 signaling pathway in the liver of largemouth bass: TGFβ1a is the most potent fibroblast factor in the liver. Therefore, to explore the inhibitory mechanism of GA on HF-induced liver fibrosis, we detected the mRNA expression of Tgfβ1a and its target proteins Smad2 and Smad3a. Compared with the NC group, the mRNA expressions of Tgfβ1a, Smad2, and Smad3a in the HF group were significantly increased, while GA treatment significantly reduced their mRNA expressions. The Western-blot method was used to detect the protein levels of Tgfβ1, p-Smad2, and p-Smad3, and it was found that the protein levels of Tgfβ1, p-Smad2, and p-Smad3 in the HF group were significantly increased, and their protein levels were reduced after GA treatment, which was consistent with the protein and mRNA expressions. The immunofluorescence results showed that compared with the HF group, the expression of Tgfβ1 in the liver tissue of largemouth bass treated with GA was significantly reduced. These results indicate that GA improves liver fibrosis by regulating the Smad2 / 3 signaling pathway mediated by Tgfβ1. The effects of S53 GA on HF-induced liver mitochondria include the following steps: S531 Effect of GA on HF-induced hepatic reactive oxygen species: Sirt3 is the main deacetylase in mitochondria, regulating all aspects of mitochondrial health and function. Virtual docking analysis of GA and Sirt3 was performed, and the binding energy was -9.0 kcal / mol. This indicates that GA can spontaneously bind to Sirt3 protein. The mitochondrial function-related indicators MMP and ATP content were further detected. Mitochondria are the main site of ATP formation. The results showed that compared with the NC group, the ATP content in the liver of the HF group was reduced, and the ATP content in the liver of the GA group was significantly increased (P<0.05). Mitochondrial dysfunction is associated with the production of reactive oxygen species (ROS), which contributes to the development of liver fibrosis. ROS is a key indicator for evaluating mitochondrial function. DAPI detected the nucleus (blue fluorescence), and DCFH-DA detected ROS (green fluorescence). Compared with the NC group, the green fluorescence in the HF group increased significantly. However, GA treatment effectively reduced the green fluorescence, indicating a decrease in ROS content. At the same time, the quantitative evaluation of ROS content confirmed that the ROS level was increased in the HF group, while the ROS level in the GA-treated group was significantly reduced (P<0.05). The cationic dye JC-1 showed changes in mitochondrial membrane potential (MMP). The HF group showed an increase in JC-1 monomers and a decrease in aggregates, while the GA-treated group showed an increase in JC-1 aggregates and a decrease in monomers, indicating the restoration of MMP. This observation was supported by the quantification of the MMP ratio (red / green fluorescence intensity), with an increase in MMP in the GA-treated group compared with the HF group. These results suggest that GA treatment can alleviate HF-induced mitochondrial damage in the liver of largemouth bass; Effect of S532GA on HF-induced liver mitochondrial damage: Ultrastructural analysis showed that the livers of the NC group had abundant mitochondria, clear endoplasmic reticulum structure, neatly arranged mitochondrial cristae, intact mitochondrial membrane, and appropriate gaps between the endoplasmic reticulum and mitochondria. In contrast, the HF group showed mitochondrial membrane rupture, damaged mitochondrial cristae arrangement, abnormal ER structure, and reduced ER-mitochondrial gap. GA treatment restored these characteristics, which were very similar to the NC group; Effect of S54GA on HF-induced hepatic cytosolic and endoplasmic reticulum Ca 2+ Effects on the expression of calcium ion transporters: The mRNA expression of Casr, Sig1r, and Ip3r1 in the HF group increased. However, after GA treatment, the expression of Casr, Sig1r, and Ip3r1 mRNA in liver tissues decreased significantly (P<0.05), which was consistent with the mRNA expression. Western-blot detected the protein level of Ip3r1 and found that compared with NC, the protein level of IP3R1 in the HF group was the highest, while the protein level of IP3R1 decreased after GA treatment. Effects of S55GA on mRNA expression and protein level of liver mitochondrial calcium transporter induced by HF: In order to further study the effect of GA on mitochondrial Ca 2+ Overload mechanism, we evaluated the expression of Grp75, Vdac1, and Mcu. The HF group had increased Grp75, Vdac1, and Mcu mRNA expression, while the GA group had significantly decreased Grp75, Vdac1, and Mcu mRNA expression (P<0.05). Further analysis of the hepatic Vdac1 and Mcu protein levels showed that these protein levels were significantly increased in the HF group. Notably, GA treatment resulted in a decrease in these levels (P<0.05). However, Grp75 protein levels were relatively unaffected in both the HF group and the GA-treated group; Effect of S56GA on HF-induced Ampk-Pgc1α-Sirt3 signaling pathway: By detecting the mRNA expression of Ampk, Pgc1α and Sirt3, it was found that GA significantly increased the mRNA expression of Ampk, Pgc1α and Sirt3 (P<0.05). Similar trends were seen in the protein levels of p-ampk, Pgc1α and Sirt3, which were significantly reduced in the HF group and significantly enhanced in the GA group (P<0.05). Consistent with the reduction in mRNA and protein expression, immunofluorescence staining found that GA significantly reduced the expression of Mcu in the liver induced by HF. In addition, we also found that GA increased the level of Sirt3 in the liver, which was reduced by HF. Collectively, these data suggest that GA reduces the expression of Mcu in the liver by regulating the expression of Sirt3.

8. The method for studying the effect of 18β-glycyrrhetinic acid on liver damage mediated by mitochondrial dysfunction induced by high-fat diet according to claim 7, characterized in that: The S511 liver-to-body ratio was determined by dissecting and observing the liver appearance and calculating the liver-to-body ratio of largemouth bass.

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