18beta-glycyrrhetinic acid on high-fat diet-induced mitochondrial dysfunction-mediated liver injury research method

Abstract

The application discloses a research method of 18beta-glycyrrhetinic acid for liver injury mediated by mitochondrial function damage induced by high-fat diet, which comprises the following steps: S1 test design, S2 test sample collection, S3 index determination, S4 test data statistical analysis, and S5 result. The 18beta-glycyrrhetinic acid (18beta-glycyrrhetinic acid, GA) is first explored to reduce the aggregation of liver collagen fibers by down-regulating the TGFbeta1-Smad2 / 3 signal, thereby relieving liver fibrosis, and improving the liver injury of largemouth bass caused by high-fat diet (HF). Secondly, the GA can reduce the expression of calcium ion transport proteins in the endoplasmic reticulum and mitochondria by activating the Ampk-Pgc1alpha-Sirt3 signal pathway, relieve liver mitochondrial damage, reduce the content of ROS, thereby inhibit the activation of pro-fibrotic factors. The HF can up-regulate the activity of liver function-related enzymes of largemouth bass, increase the deposition of liver lipid droplets, and the aggregation of collagen. After adding the GA, the adverse effects caused by the HF can be obviously improved, thereby relieving the liver injury of largemouth bass induced by the HF.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of fish feed, in particular to a research method for 18beta-glycyrrhetinic acid on liver injury mediated by mitochondrial function injury induced by high-fat diet. BACKGROUND

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

[0003] Reasonably increasing the fat level of the feed not only promotes the growth of fish, but also helps to improve the breeding environment. However, in actual production, the fat level of the feed is usually increased to save cost, and long-term feeding of high-level fat will cause liver damage in largemouth bass. Therefore, in order to improve the influence of high-fat diet on liver damage of 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] 18beta-glycyrrhetinic acid (18beta-Glycyrrhetinic acid, GA) is the main component of licorice, which is a natural active compound of pentacyclic triterpenoids. It is formed by hydrolysis of glycyrrhizic acid to remove the sugar acid chain, and GA is produced in the metabolic process in vivo. GA is a widely recognized anti-inflammatory, antioxidant, fat deposition reducing and liver protecting drug.

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

[0006] In view of this, the present application is proposed. SUMMARY

[0007] The purpose of the present application is to provide a research method for 18beta-glycyrrhetinic acid on liver injury mediated by mitochondrial function injury induced by high-fat diet, to solve the problems raised in the background art.

[0008] To achieve the above object, the present application provides the following technical solutions: 18β-glycyrrhetinic acid is used to study the method for inducing liver injury mediated by mitochondrial function damage caused by high-fat diet, which comprises the following steps: S1 test design, S2 test sample collection, S3 index determination, S4 test data statistical analysis, and S5 results.

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

[0010] S11 test diet: the basic feed comprises three protein sources of fish meal, chicken meal and fermented soybean meal, and two carbohydrates of wheat flour and cassava starch, and is prepared 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 plus GA 0.5 mg / kg group (HFL) with a protein level of 47.48% and a fat level of 16.25%, a high-fat diet plus GA 1.0 mg / kg group (HFM) with a protein level of 47.48% and a fat level of 16.44%, and a high-fat diet plus GA 1.5 mg / kg group (HFH) with a protein level of 47.55% and a fat level of 16.44%. The vitamin and mineral premix is added in an amount of 1% and 1.5%, respectively. Vitamin D, vitamin C and selenium are added by the step-by-step expansion method, and are mixed with the bulk raw materials uniformly. After oil and water are added, the mixture is mixed again, and is granulated by a small puffed feed machine. After drying at 30°C, the granules are stored in sealed plastic bags. After the approximate nutrient analysis of the nutritional components of each feed, the granules are used for experiments.

[0011] S12 test fish and feeding management: the test fish is healthy largemouth bass. After 2 weeks of temporary cultivation, 750 healthy largemouth bass with an average initial weight of 17.39±0.09 g are randomly allocated to 15 square cement pools, with 50 fish in each cement pool. The cement pools are randomly divided into 5 treatment groups, and the fish are fed with NC group, HF group, HFL group, HFM group and HFH group feed, respectively. Each treatment has 3 replicates. The growth test lasts for 77 days. The fish are fed to satiation at 7:30 am and 18:00 pm every day. After 40 minutes of feeding, the remaining residual feed is fished out, dried and weighed. The weather, water temperature, feeding rate, residual feed amount and food intake are recorded every day. The test uses micro-flow aquaculture, and the water flow rate is maintained at 1.0 L / min. The water temperature during the test period is 25.0±3.0°C. The intermittent oxygenation is maintained, and the dissolved oxygen is greater than 5.0 mg / L.

[0012] Preferably, the S2 includes the following steps: after the end of the feeding test, the largemouth bass is fasted for 24 h, 12 fish with similar body weight are selected from each repetition, and 0.01% MS-222 is used for anesthesia, after the fish body is wiped dry, the body weight is weighed and recorded, and blood is taken from the tail vein with an anticoagulant-washed 1.5 mL disposable syringe, 9 of the 12 largemouth bass are separated from the body and viscera, and the liver is quickly separated from the viscera group, then washed thoroughly with physiological saline, photographed, weighed and recorded, then placed in a bag with corresponding labels, wrapped with tin foil, and immediately placed in liquid nitrogen for freezing, then quickly transferred to a-80℃ ultra-low temperature refrigerator for storage, and used for subsequent test index determination, the remaining 3 largemouth bass are used to measure the body length and weight, 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, the frozen section sample is placed in a 2 mL cryopreservation tube, labeled, wrapped with tin foil, and immediately placed in liquid nitrogen for freezing, and used for subsequent oil red O staining, and the electron microscope sample is fixed in a 2.5% glutaraldehyde solution for electron microscope section observation.

[0013] Preferably, the S3 includes the following steps:

[0014] S31 Liver-somatic index: according to the sampling statistical data, the liver-somatic index is calculated to determine the growth and development of the liver;

[0015] S32 Determination of blood and liver biochemical indexes: the blood is transferred to a microcentrifuge tube, 3000g room temperature centrifugation for 10 min to separate the plasma, and the activities of alanine aminotransferase ALT, aspartate aminotransferase AST and alkaline phosphatase AKP, and the contents of triglyceride TG, total cholesterol TC and hydroxyproline HYP are determined using a commercial kit, the sample is taken out from the-80℃ refrigerator, 10% tissue homogenate is prepared with a homogenizer, and the supernatant is obtained by centrifugation at 3000r / min for 10 min to determine the contents of malondialdehyde MDA, glutathione S-transferase GST, total protein TP and adenosine triphosphate ATP;

[0016] S33 H&E staining analysis of liver: the liver tissue is fixed with 10% formalin solution for 7 days, then dehydrated with gradient ethanol solution, then transparentized with xylene for subsequent wax immersion, and finally embedded in paraffin, the sample is sliced using a paraffin sectioning machine, the slice thickness is 5μm, and hematoxylin-eosin H&E staining is performed, and the specific steps of H&E staining are as follows:

[0017] 1Put the sample slide into a 65℃ constant temperature drying box and dry for 2h;

[0018] 2Dewaxing: place the sample slide in xylene for 10 min each time, a total of 2 times;

[0019] 3hydration: the sample slides were immersed in anhydrous ethanol, 95% ethanol, 80% ethanol, 70% ethanol, 2 min each, and distilled water, 2 min;

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

[0021] 5differentiated with 1% hydrochloric acid for 60 s, and removed after turning blue;

[0022] 6eosin staining for 4 min, and washing with water for 30 s;

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

[0024] 8neutral gum mounting;

[0025] 9observed under a light microscope and photographed (Olympus BX43);

[0026] S34 analysis of liver oil red O staining: the specific steps for preparing oil red O staining are as follows:

[0027] 1the liver sample in a-80°C refrigerator was taken out and transferred into a-20°C refrigerator for buffering for 30 min;

[0028] 2the liver tissue was cut into a proper size of square block, and embedded and frozen with OCT embedding agent;

[0029] 3the tissue block was cut into 8 μm frozen sections with a pre-cooled freezing microtome;

[0030] 4the cut sample was transferred onto a glass slide with a corresponding label, and placed at room temperature for 10 min for staining;

[0031] 5the sample was first covered with washing solution for 20 s, and stained with oil red O dye for 20 min;

[0032] 6after oil red O dye staining, the sample was washed with washing solution for 30 s, and immersed in distilled water for shaking for 20 s;

[0033] 7the cell nucleus was counterstained with hematoxylin for 3 min, and washed with water for 30 s; and glycerol gelatin mounting was performed;

[0034] 8microscopic examination (Olympus BX43);

[0035] S35 analysis of liver masson staining: the sample was pretreated, and deparaffinized and hydrated as in the H&E staining above. The specific staining steps are as follows:

[0036] 1the sample was first stained with prepared Weigert iron hematoxylin staining solution for 10 min;

[0037] 2Differentiate for 15s using acidic ethanol differentiation solution, wash with water;

[0038] 3Return blue for 5min using Masson blue solution, wash with water, wash with distilled water for 1min;

[0039] 4Stain for 10min using ponceau red staining solution;

[0040] 5Wash for 1min using weak acid working solution, the weak acid working solution is distilled water: weak acid solution = 2:1;

[0041] 6Then wash for 2min using phosphomolybdic acid solution, wash for 1min using weak acid working solution;

[0042] 7Directly place into aniline blue staining solution for 2min, wash for 1min using weak acid working solution;

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

[0044] 9Xylene transparency for 3 times, each time for 2min, neutral gum fixation, and observation;

[0045] S36 Liver transmission electron microscope analysis: take a small piece of liver 1x1x1mm 3 , place in 2.5% glutaraldehyde, fast fixation at 4°C for 2-4h, 1% osmium acid prepared with 0.1M phosphate buffer PB at room temperature for 2h, after dehydration, penetration, embedding, sectioning and staining, and observation under transmission electron microscope, image collection and analysis;

[0046] S37 Liver immunohistochemical analysis: sample pre-treatment, deparaffination same as above H&E staining, immerse the section in 10mmol / L citrate buffer pH6.0, heat to boiling in microwave oven, 20min; incubate in wet box with primary antibody anti-α-Sma, anti-Tgfβ1, Sirt3 and Mcu antibody overnight, the next day, rewarm, wash, add 25μL of horseradish peroxidase HRP labeled secondary antibody anti-rabbit / mouse to each section, incubate at 37°C for 40min, wash with PBST for 2 times, each time for 5min, add DAPI (0.2ug / ml) to the section, 5min, wash with PBST for 2 times, each time for 5min. Mount, observe and take pictures under inverted fluorescence microscope;

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

[0048] S39 Membrane potential MMP determination: tissue mitochondria isolation kit was used to isolate liver mitochondria, and JC-1 fluorescent probe method was used to detect MMP according to the instructions, then fluorescence microscope was used to observe mitochondria, and fluorescence microplate reader was used to detect absorbance value, and red-green light ratio was calculated, excitation wavelength was 485 nm, and emission wavelength was 580 nm;

[0049] S310 Real-time quantitative PCR detection: comprising 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 refrigerator, 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, dissolved in 50 μL of RNase-free water, 1.0% agarose gel electrophoresis was used for 120V, 12 min to detect the degradation degree of RNA sample, and NanoDrop 1000 spectrophotometer was used to detect its concentration, and the sample purity was judged according to A260 / A280 ratio;

[0051] S3102 Real-time fluorescence quantitative primer design: all gene sequences of largemouth bass were obtained from GenBank or referred to previous studies. Primer 5.0 was used to design primers;

[0052] Table 2.Primer sequences of genes selected for q-PCR

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

[0054]

[0055]

[0056] S3103 Real-time fluorescent quantitative detection of gene expression level: dilute each treated 3 cDNA samples by 1:1, then detect fibrosis related genes: Tgfβ1a, Smad2, Smad3a, α-Sma, Collagen I, Fibronectin and Mmp9; Mitochondrial calcium ion transport related genes: Ampkα1, Pgc1α, Sirt3, Ip3r1, Sig1r1, Casr, Grp75, Vdac1 and Mcu, adopt TB Green TM Premix Ex Taq TM II(Tli RNaseH Plus) kit for fluorescent quantitative PCR amplification, 10 times of positive template for continuous dilution as PCR template, then make standard curve, amplification efficiency is 90%-110%, R 2 >0.9, single peak of dissolution curve. Adopt 10 μL PCR amplification system: 3.2 μL DEPC water, 1 μL cDNA template, 0.4 μL of upstream primer and downstream primer, and 5 μL SYBR Premix Ex Taq II, reaction cycle condition: 95℃ preheating 2 min, 95℃ 10 s and suitable annealing temperature 30 s for 40 times of cycle, after reaction, product specificity is detected by dissolution curve;

[0057] S3104 Relative quantitative calculation: expression level is represented by threshold cycle Ct, which is calculated by drawing fluorescence signal of each cycle, mRNA concentration of target gene is normalized with mRNA concentration of internal reference gene β-actin and 18SrRNA, after verifying that amplification efficiency of primers reaches that of target gene and internal reference gene, 2 -ΔΔCT Method calculation result, according to 10 times of sequence dilution to generate standard curve of specific gene to calculate internal reference gene amplification efficiency;

[0058] S311 Western blotting: extract total protein of liver tissue with RIPA Lysis Buffer (Beyotime, Beijing, China), use Enhanced BCA Protein Assay Kit (Beyotime Biotechnology Co., Ltd., China) to determine sample protein concentration, add SDS-PAGE protein loading buffer 5X to sample, denature at 95℃ for 10 min, separate by SDS-PAGE, transfer to polyvinylidene fluoride PVDF membrane, block with 5% skim milk for 1.5 h, then incubate primary antibody at 4℃ overnight, wash and incubate secondary antibody at room temperature for 1.5 h, use ECL Kit (Beyotime) to detect target protein expression, and use Chemi Doc Imaging Systems / Image Lab software (Bio-Rad) to record, β-actin is internal reference with equal sample loading amount.

[0059] Preferably, S4 includes the following steps: using software to process data, using variance analysis and combining Duncan's multiple comparison to detect significance, data is expressed as mean ± standard error Mean ± SEM, P <0.05 indicates significant difference, and Image-ProPlus6.0 software is used to quantitatively analyze H&E, oil red O and Masson staining of the liver.

[0060] Preferably, the liver body ratio is calculated according to the sampling statistical data, and the liver body index is calculated as follows: liver body index % = liver weight / body weight x 100.

[0061] Preferably, S5 includes the following steps:

[0062] S51 Effect of GA on liver damage induced by HF in largemouth bass, including the following steps:

[0063] S511 Liver body ratio: By anatomical observation of the appearance of the liver and statistical analysis of the liver body ratio of largemouth bass, it was found that the liver surface of the NC group was smooth and red. While the liver of the HF group of largemouth bass showed most of the liver surface was pale, and GA treatment significantly reduced the pale surface of the liver and increased the red surface of the liver. Compared with the NC group, the liver body ratio of the HF group was significantly increased, and GA treatment reduced the liver body ratio;

[0064] S512 Blood and liver function parameters: In order to explore the effect of GA on liver function of largemouth bass induced by HF, we detected 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, and the AST, ALT and AKP activities were significantly reduced after GA treatment P <0.05. The TG and TC content in the serum of the HF group was the highest, and the TG and TC content was significantly reduced after GA treatment P <0.05. Compared with the NC group, the MDA content in the HF group was significantly increased, and the MDA content in the GA group was significantly reduced P <0.05, the GST activity in the HF group was the lowest P <0.05, and the TP content in the NC group was the lowest P <0.05. The above results show that GA reduces the AST, ALT and AKP activity, TG, TC content in serum and MDA content in liver induced by HF, and increases the GST activity, thereby alleviating the liver damage induced by HF;

[0065] S513 Liver histopathology: Compared with the NC group, the hepatocytes of the HF group were significantly swollen, and the nuclei showed marginal displacement, and even disappeared in some cases. Compared with the HF group, the liver steatosis gradually eased in the GA treatment group, and the number of nuclei gradually increased and was located in the center of the cells. Statistical analysis found that the relative area of lipid vacuoles in the HF group significantly increased compared with the NC group, and the relative area of lipid vacuoles in the HFM group was the lowest in the relief group compared with the HF group. 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 treatment group gradually decreased. The oil red O positive area of the HF group significantly increased, and the oil red O positive area decreased after GA treatment. The above results showed that GA reduced the liver lipid degeneration and lipid droplet deposition induced by HF;

[0066] 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 largemouth bass liver sections showed that muscle fibers were red and collagen fibers were blue. HF treatment caused a large number of collagen fibers to aggregate, while the addition of GA treatment significantly reduced the aggregation of liver collagen fibers, and the therapeutic effect of HFM and HFH was close to that of the NC group. In addition, the fibrosis area was quantitatively evaluated by IPP after Masson staining, and it was found that GA treatment significantly reduced liver fibrosis compared with the HF group. HYP content is a sensitive biochemical indicator reflecting changes in collagen fibers. To further study the effect of GA on HF-induced liver fibrosis in largemouth bass, we detected the content of HYP in serum, and found that the content of HYP in serum of largemouth bass in the HF group significantly increased compared with the NC group, while GA administration significantly reduced the content of HYP compared with the HF group;

[0067] S521 Effect of GA on transcription of pro-fibrotic markers related to HSC activation induced by HF: The mRNA expression of pro-fibrotic markers α-Sma, Collagen I, Fibrontein and Mmp9 was further detected. The results showed that compared with the NC group, HF significantly increased the mRNA expression of α-Sma, Collagen I and Fibrontein in the liver, while GA significantly reduced the mRNA expression of α-Sma, Collagen I and Fibrontein compared with the HF group. Western-blot method was used to detect the protein levels of α-Sma, Collagen I and Fibrontein, and it was found that the protein levels of α-Sma, Collagen I 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 the addition of GA treatment. In addition, the results of immunohistochemical staining confirmed that GA significantly inhibited the expression of α-Sma induced by HF. The above results showed that GA could effectively alleviate the liver fibrosis of largemouth bass induced by HF;

[0068] S522Effect of GA on Tgfβ1-Smad2 / 3 signaling pathway in liver of HF-induced largemouth bass: Tgfβ1a is the most effective fibrocyte factor in the liver, therefore, in order 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 expression of Tgfβ1a, Smad2 and Smad3a in the HF group was significantly increased, while the mRNA expression was significantly reduced after GA treatment. 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 the protein levels were reduced after GA treatment. Consistent with the protein and mRNA expression, immunohistochemical results showed that compared with the HF group, the expression of Tgfβ1 in the liver tissue of GA-treated largemouth bass was significantly reduced. These results indicated that GA improved liver fibrosis by regulating Tgfβ1-mediated Smad2 / 3 signaling pathway;

[0069] S53Effect of GA on mitochondria induced by HF in liver, which includes the following steps:

[0070] S531Effect of GA on reactive oxygen species induced by HF in liver: Mitochondrial membrane potential MMP was measured by JC-1, and it was found that JC-1 staining showed bright red fluorescence as JC-1 polymer, and green fluorescence as JC-1 monomer. JC-1 staining showed bright red fluorescence and weak green fluorescence in the NC group, while red fluorescence was observed to be converted to green fluorescence in the HF group. Compared with the HF group, bright red fluorescence was observed in the GA group. Fluorescence microplate reader detection found the same phenomenon, and the red-green fluorescence ratio of the NC group and the GA treatment group was significantly increased compared with the HF group P<0.05. At the same time, we detected the ATP content in the liver, and found that the ATP content was decreased in the HF group, while the ATP content was significantly increased after GA treatment. These results indicated that GA treatment could alleviate the mitochondrial damage in the liver of largemouth bass induced by HF;

[0071] S532Effect of GA on mitochondrial damage induced by HF in liver: The number of mitochondria in the NC group was abundant, the endoplasmic reticulum structure was normal, the mitochondrial cristae was arranged in order, the mitochondrial membrane was complete, and the gap between endoplasmic reticulum and mitochondria was appropriate. In the HF treatment group, the mitochondrial membrane was obviously broken, the mitochondrial cristae was arranged in disorder, the endoplasmic reticulum structure was abnormal, and the gap between endoplasmic reticulum and mitochondria was reduced. In the HFM treatment group, the mitochondrial membrane, mitochondrial cristae arrangement, endoplasmic reticulum structure and gap between endoplasmic reticulum and mitochondria were restored to be consistent with the NC group;

[0072] S54 Effect of GA on the expression of cytoplasm and endoplasmic reticulum calcium ion transporters induced by HF: The mRNA expression of Casr, Sig1r and Ip3r1 in the liver tissue of the HF group increased. However, after GA treatment, the mRNA expression of CASR, Sig1R and Ip3r1 in the liver tissue decreased significantly P<0.05. Consistent with the mRNA expression, the protein level of Ip3r1 was detected by Western-blot, and it was found that compared with the NC group, the protein level of Ip3r1 in the HF group was the highest, and the protein level of Ip3r1 decreased after GA treatment;

[0073] S55 Effect of GA on the mRNA expression and protein level of mitochondrial calcium ion transporters induced by HF: In order to explore the potential mechanism of GA to inhibit mitochondrial calcium ion transporters, we studied whether GA could reduce the mRNA expression and protein level of mitochondrial calcium ion 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] S56 Effect of GA on the Ampk-Pgc1a-Sirt3 signal pathway induced by HF: Compared with the NC group, the mRNA expression of Ampk, Pgc1a and Sirt3 in the HF group decreased, and the mRNA expression was significantly enhanced after GA treatment. We also found that, consistent with the mRNA expression, the protein level of Ampk, Pgc1a and Sirt3 in the liver of the HF group decreased significantly, and the GA group increased significantly.

[0075] Preferably, the S511 liver body ratio is observed by anatomically observing the appearance of the liver and counting the liver body ratio of the largemouth bass.

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

[0077] 1. The research method of 18beta-glycyrrhetinic acid on liver injury induced by mitochondrial function damage mediated by high-fat diet, for the first time, it is found that GA can reduce the aggregation of collagen fibers in the liver by regulating Tgf beta1-Smad2 / 3 signal to relieve liver fibrosis, thereby improving the liver injury of largemouth bass caused by HF. Secondly, GA can reduce the expression of calcium ion transporters in endoplasmic reticulum and mitochondria through Ampk-Pgc1a-Sirt3 signal pathway, relieve mitochondrial function damage and reduce the content of ROS, thereby inhibiting the activation of pro-fibrotic factors. HF can up-regulate the activity of liver function-related enzymes of largemouth bass, increase the deposition of liver lipid droplets and the aggregation of collagen fibers, and after adding GA, the activity of liver function-related enzymes is reduced, the liver steatosis, lipid droplet deposition and collagen fiber aggregation are reduced, thereby relieving the liver injury of largemouth bass induced by HF.

[0078] 2. The 18β-glycyrrhizin protects the liver of largemouth bass induced by HF, and the expression of pro-fibrosis markers is up-regulated after largemouth bass is fed with HF, while the expression of pro-fibrosis markers is reduced by down-regulating TGFβ1-mediated Smad2 / 3 signal after GA is added, thereby alleviating the liver fibrosis of largemouth bass induced by HF.

[0079] 3. The 18β-glycyrrhizin protects the liver of largemouth bass induced by HF, and the mitochondria of largemouth bass is damaged after largemouth bass is fed with HF, and the content of ROS is increased, while GA can protect the mitochondrial membrane, mitochondrial crista and endoplasmic reticulum from the mitochondrial damage caused by HF by up-regulating the content of mitochondrial MMP and ATP and down-regulating the content of ROS, thereby improving the mitochondrial function.

[0080] 4. The 18β-glycyrrhizin protects the liver of largemouth bass induced by HF, and the mRNA expression and protein level of calcium ion transport protein in the endoplasmic reticulum and mitochondria are increased after largemouth bass is fed with HF, while GA can reduce the mRNA expression and protein level of calcium ion transport protein in the endoplasmic reticulum and mitochondria by up-regulating the Ampk-Pgc1α-Sirt3 signal, thereby improving the increase of the expression of mitochondrial calcium ion transport protein caused by HF. BRIEF DESCRIPTION OF DRAWINGS

[0081] Figure 1 To protect the liver of largemouth bass induced by HF in the present application;

[0082] Figure 2 To protect the liver of largemouth bass induced by HF in the present application;

[0083] Figure 3 To protect the liver of largemouth bass induced by HF in the present application;

[0084] Figure 4 To protect the liver of largemouth bass induced by HF in the present application;

[0085] Figure 5 To protect the liver of largemouth bass induced by HF in the present application;

[0086] Figure 6 To protect the liver of largemouth bass induced by HF in the present application; 2+ To protect the liver of largemouth bass induced by HF in the present application;

[0087] Figure 7 To protect the liver of largemouth bass induced by HF in the present application; DETAILED DESCRIPTION

[0088] In order to make the technical features, objectives and effects of the present application more clearly understood, the specific embodiments of the present application will now be described with reference to the accompanying drawings.

[0089] Embodiments

[0090] The preferred embodiment of the research method for 18β-glycyrrhetinic acid to high-fat diet-induced mitochondrial dysfunction-mediated liver injury provided by the present application is shown as follows: Figures 1-7 The research method for 18β-glycyrrhetinic acid to high-fat diet-induced mitochondrial dysfunction-mediated liver injury is shown as follows: S1 test design, S2 test sample collection, S3 index determination, S4 test data statistical analysis, and S5 results.

[0091] S1 includes the following steps:

[0092] S11 test diet: the basic feed contains three protein sources of fish meal, chicken meal and fermented soybean meal, with soybean oil as the fat source, and two carbohydrates of wheat flour and cassava starch, respectively, to prepare 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 group (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 group (HFM) with a protein level of 47.48% and a fat level of 16.44%, and the high-fat diet plus GA 1.5 mg / kg group (HFH) with a protein level of 47.55% and a fat level of 16.44%. The addition amounts of vitamin and mineral premixes are 1% and 1.5%, respectively. Vitamins D, C and selenium are added by the step-by-step expansion method, and are mixed with the bulk raw materials uniformly. After adding oil and water, they are mixed again uniformly, and are pelleted by a small-scale puffed feed machine. After drying at 30°C, they are stored in sealed plastic bags. After the approximate nutrient analysis of the nutritional components of each feed, they are used for experiments.

[0093] S12 Test fish and feeding management: The test fish is healthy largemouth bass, after 2 weeks of temporary cultivation, 750 healthy largemouth bass with an average initial weight of 17.39±0.09 g are randomly allocated to 15 square cement pools, 50 in each cement pool, and the cement pools are randomly divided into 5 treatment groups, and the NC group, the HF group, the HFL group, the HFM group and the HFH group are fed respectively. Each treatment is set up in triplicate. The growth test lasts for 77 days, and the fish are fed at 7:30 am and 18:00 pm every day. After 40 minutes of feeding, the remaining residual feed is removed, dried and weighed. The weather, water temperature, feeding rate, residual feed amount and food intake are recorded every day. The test uses micro-flow aquaculture, and the water flow rate is maintained at 1.0 L / min. The water temperature during the test is 25.0±3.0℃, and intermittent aeration is used to maintain the dissolved oxygen to be greater than 5.0 mg / L.

[0094] S2 includes the following steps: after the end of the feeding test, the largemouth bass is fasted for 24 h, 12 fish with approximately similar body weight are selected from each replicate, and 0.01% MS-222 is used for anesthesia. After the fish body is dried, the body weight is weighed and recorded, and blood is taken from the tail vein with an anticoagulant-washed 1.5 ml disposable syringe. Among them, 9 largemouth bass are separated from the body and viscera, and the liver is quickly separated from the viscera mass. Then, the liver is washed thoroughly with physiological saline, photographed, weighed and recorded, and then placed in a bag with the corresponding label. Then, it is wrapped with tin foil and immediately placed in liquid nitrogen for freezing. Then, it is quickly transferred to a-80℃ ultra-low temperature refrigerator for storage, which is used for subsequent test index determination. The remaining 3 largemouth bass are used to measure the body length and weight, and then the liver tissue is separated and fixed with 4% neutral formalin for subsequent HE staining, Masson staining and IHC staining analysis. In the same way, the frozen section sample is placed in a 2 mL cryopreservation tube, labeled, wrapped with tin foil and immediately frozen in liquid nitrogen for subsequent oil red O staining, and the electron microscope sample is fixed in a 2.5% glutaraldehyde solution for electron microscope section observation.

[0095] S3 includes the following steps:

[0096] S31 Liver-somatic index: According to the sampling statistical data, the liver-somatic index is calculated to determine the growth and development of the liver;

[0097] S32 Determination of blood and liver biochemical indicators: the blood was transferred to a microcentrifuge tube, 3000g room temperature centrifugation for 10 minutes to separate the plasma, alanine aminotransferase ALT, aspartate aminotransferase AST and alkaline phosphatase AKP activity, triglyceride TG, total cholesterol TC and hydroxyproline HYP content determination using commercial reagent kit, after taking out the sample from-80℃ refrigerator, using homogenizer to prepare 10% tissue homogenate, then centrifuged at 3000r / min for 10min, to obtain the supernatant, determination of malondialdehyde MDA, glutathione S-transferase GST, total protein TP and adenosine triphosphate ATP content;

[0098] S33 H&E staining analysis of liver: liver tissue was fixed with 10% formalin solution for 7 days, then dehydrated with gradient ethanol solution, then the sample was transparent with xylene, so as to be immersed in wax later, finally the sample was embedded in paraffin, the sample was sliced using a paraffin microtome, the slice thickness was 5μm, and hematoxylin-eosin H&E staining was used, the specific steps of H&E staining were as follows:

[0099] 1 Put the sample slide into a constant temperature drying oven at 65℃ and dry for 2h;

[0100] 2 De-waxing: put the sample slide into xylene for 10min each time, for a total of 2 times;

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

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

[0103] 5 Differentiate with 1% hydrochloric acid for 60s, take out after turning blue;

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

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

[0106] 8 Neutral gum mounting;

[0107] 9 Observe under a light microscope and take a photo Olympus BX43 observation;

[0108] S34 Oil red O staining analysis of liver: the specific steps of oil red O staining preparation are as follows:

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

[0110] 2 The liver tissue is cut into a proper size of square and embedded with OCT embedding agent for freezing;

[0111] 3 The tissue block is cut into 8 μm frozen sections with a pre-cooled freezing microtome;

[0112] 4 The cut sample is transferred to a glass slide with a corresponding label, and is placed at room temperature for 10 min for staining;

[0113] 5 The sample is first covered with a washing solution for 20 s, and is dyed with oil red O dye for 20 min;

[0114] 6 After oil red O dye staining, the sample is washed with a washing solution for 30 s, and is immersed in distilled water for 20 s of shaking;

[0115] 7 The cell nucleus is counterstained with hematoxylin for 3 min, and is washed with water for 30 s; and is mounted with glycerol gelatin;

[0116] 8 The sample is observed under a microscope (Olympus BX43);

[0117] S35 Liver Masson staining analysis: The sample is pretreated, and is dewaxed and hydrated as in the H&E staining above. The specific staining steps are as follows:

[0118] 1 The sample is first dyed with prepared Weigert iron hematoxylin staining solution for 10 min;

[0119] 2 The sample is differentiated with an acidic ethanol differentiation solution for 15 s, and is washed with water;

[0120] 3 The sample is blued with Masson blue solution for 5 min, and is washed with water again, and is washed with distilled water for 1 min;

[0121] 4 The sample is dyed with ponceau red staining solution for 10 min;

[0122] 5 The sample is washed with a weak acid working solution for 1 min, and the weak acid working solution is distilled water: weak acid solution = 2:1;

[0123] 6 The sample is then washed with phosphomolybdic acid solution for 2 min, and is washed with a weak acid working solution for 1 min;

[0124] 7 The sample is directly dyed with aniline blue staining solution for 2 min, and is washed with a weak acid working solution for 1 min;

[0125] 895% ethanol is used for rapid dehydration for 3 s, and absolute ethanol is used for dehydration for 3 times, each for 10 s;

[0126] 9 Xylene is used for transparency for 3 times, each for 2 min, and the sample is mounted with neutral gum, and is observed;

[0127] S36 Liver transmission electron microscopy analysis: Take a small piece of liver 1x1x1mm3, put it in 2.5% glutaraldehyde, 4°C rapid fixation for 2-4h, 1% osmium acid prepared with 0.1M phosphate buffer PB light fixation for 2h at room temperature, after dehydration, penetration, embedding, sectioning and staining, and observed under transmission electron microscope, image analysis;

[0128] S37 Liver immunofluorescence analysis: sample pretreatment, deparaffinization same as above H&E staining, immerse the section in 10mmol / L citrate buffer pH6.0, heat to boiling in microwave oven, 20min; incubate with primary antibody anti-α-Sma, anti-Tgfβ1, Sirt3 and Mcu antibody in wet box overnight, the next day, rewarming, after washing, add 25μL HRP labeled secondary antibody anti-rabbit / mouse peritoneal per section, 37°C incubation for 40min, PBST washing 2 times, 5min each time, add DAPI (0.2ug / ml) to the section, 5min, PBST washing 2 times, 5min each time. Mounting, inverted fluorescence microscope observation and photography;

[0129] S38 Reactive oxygen species detection: take out 200mg liver at the same time, immediately measure the concentration of reactive oxygen species ROS, according to the preparation method of single cell suspension, prepare single cell suspension according to the operation steps, then add fluorescence probe, and use diluted DCFH-DA to suspend liver cell sediment, liver cell sediment is incubated in 27°C incubator for 30-60min, centrifuged at 1000r / min for 10min, remove supernatant, collect cells and deposit with PBS suspension and mixing, use confocal fluorescence microscope to take pictures, use fluorescence microplate to detect absorbance value at 525nm, calculate ROS content;

[0130] S39 Membrane potential MMP determination: use tissue mitochondria separation kit to separate liver mitochondria, according to the instruction, use JC-1 fluorescence probe method to detect MMP, then use fluorescence microscope to observe mitochondria, use fluorescence microplate reader to detect absorbance value, calculate red and green light ratio, excitation wavelength is 485nm, emission wavelength is 580nm;

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

[0132] S3101 RNA extraction and cDNA synthesis: use kit to extract total RNA, briefly, take liver tissue out of-80°C refrigerator, grind into powder in liquid nitrogen, take 0.1g liver sample, add 1mL reagent to extract total RNA from tissue, dissolve in 50μL RNase-free water, use 1.0% agarose gel electrophoresis 120V, 12min to detect RNA sample degradation, use NanoDrop1000 spectrophotometer to detect its concentration, and judge sample purity according to A260 / A280 ratio;

[0133] S3102 Real-time quantitative primer design: All gene sequences of Micropterus salmoides were obtained from GenBank or referenced previous studies. Primers were designed by Primer 5.0;

[0134] Table 2. Primer sequences of genes selected for q-PCR

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

[0136]

[0137] S3103 Real-time quantitative detection of gene expression level: Each of the three cDNA samples of each treatment was diluted 1:1, then the fibrosis-related genes: Tgfβ1a, Smad2, Smad3a, α-Sma, Collagen I, Fibronectin and Mmp9; Mitochondrial calcium ion transport-related genes: Ampkα1, Pgc1α, Sirt3, Ip3r1, Sig1r1, Casr, Grp75, Vdac1 and Mcu were detected by TB Green TM Premix Ex Taq TM II(Tli RNaseH Plus) kit for fluorescent quantitative PCR amplification, 10 times of serial dilution of positive template as PCR template, then standard curve, amplification efficiency in 90%-110%, R 2 >0.9, single peak of melting curve. 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, reaction cycle condition: 95℃ preheating for 2 min, 95℃ for 10 s and suitable annealing temperature for 30 s for 40 cycles, after the reaction was completed, the product specificity was detected by melting curve;

[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 mRNA concentration of the target gene is normalized to the mRNA concentration of the internal reference gene β-actin and 18S rRNA. After verifying the amplification efficiency of the primers to achieve the amplification efficiency of the target gene and the internal reference gene, the results were calculated by 2 -ΔΔCT method, according to the standard curve of specific genes generated by 10 times of sequence dilution to calculate the amplification efficiency of the internal reference gene;

[0139] S311 Western Blot: Total protein of liver tissue was extracted with RIPA Lysis Buffer (Beyotime, Beijing, China), and the sample protein concentration was determined using Enhanced BCA Protein Assay Kit (Beyotime Biotechnology Co., Ltd., China). The sample was added with SDS-PAGE protein loading buffer 5X, denatured at 95°C for 10 min, separated by SDS-PAGE, transferred to polyvinylidene fluoride (PVDF) membrane, blocked with 5% skim milk for 1.5 h, and then incubated with primary antibody at 4°C overnight, washed and incubated with secondary antibody at room temperature for 1.5 h. The expression of 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 dilutions

[0141] Table 3.Primary antibodies and dilutions

[0142]

[0143] S4 includes processing data using software, using variance analysis and combining Duncan’s multiple comparison to detect significance. The data is expressed as mean ± standard error (Mean ± SEM), and P < 0.05 indicates significant difference. Image-ProPlus 6.0 software was used to quantify and statistically analyze liver H&E, oil red O and Masson staining.

[0144] The liver body ratio was calculated according to the sampling statistical data, and the liver body index was calculated as follows: liver body index % = liver weight / body weight x 100.

[0145] S5 includes the following steps:

[0146] S51 Effect of GA on liver damage induced by HF in largemouth bass, including the following steps:

[0147] S511 Liver body ratio: The appearance of liver was observed by dissection, and the liver body ratio of largemouth bass was calculated. It was found that the liver surface of NC group was smooth and red. The liver surface of HF group was mostly pale, while the liver surface of GA-treated group was significantly reduced, and the red surface of liver was increased. Compared with the NC group, the liver body ratio of the HF group was significantly increased, and the GA treatment reduced the liver body ratio.

[0148] S512 Blood and liver function parameters: To explore the effect of GA on the liver function of largemouth bass induced by HF, we detected the hematological and liver function of largemouth bass. The serum AST, ALT and AKP activities of HF group were significantly higher than those of NC group, and the AST, ALT and AKP activities were significantly reduced after GA treatment P<0.05. The TG and TC contents in serum were the highest in HF group, and 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 was significantly increased, and the MDA content in the GA group was significantly reduced P<0.05, and 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 AST, ALT and AKP activities, TG and TC contents in serum and MDA content in liver induced by HF, and increased the GST activity, thereby alleviating the liver injury induced by HF;

[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, and the same superscript letters in the same row indicate no significant difference (P>0.05), and different letters indicate significant difference (P<0.05).

[0153] S513 Liver histopathology: Compared with the NC group, the hepatocytes in the HF group were obviously swollen, and the cell nuclei showed edge shift, and some even disappeared, and the number was less. Compared with the HF group, the liver steatosis in the GA treatment group gradually eased, and the number of cell nuclei gradually increased and located in the cell center. Statistical analysis found that compared with the NC group, the relative area of lipid vacuoles in the HF group was significantly increased, and compared with the HF group, the relative area of lipid vacuoles in the HFM group was the lowest in the alleviation group. 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, and the lipid droplets in the liver of the GA treatment group gradually decreased. The oil red O positive area in the HF group was significantly increased, and the oil red O positive area was reduced after GA treatment. The above results showed that GA reduced the liver steatosis and lipid droplet deposition induced by HF;

[0154] S52GA's effect on HF-induced liver fibrosis in largemouth bass: Masson staining is a specific staining method for detecting collagen fibers. In largemouth bass liver sections, Masson staining can see that muscle fibers are red and collagen fibers are blue. After HF treatment, a large amount of collagen fibers gathered, while the addition of GA treatment can significantly reduce the aggregation of liver collagen fibers, and the therapeutic effect of HFM and HFH is close to the NC group. In addition, the use of IPP to quantitatively evaluate the fibrosis area of Masson staining found that relative to the HF group, GA treatment significantly reduced liver fibrosis, and HYP content is a sensitive biochemical indicator reflecting the changes in collagen fibers. To further study the effect of GA on HF-induced liver fibrosis in largemouth bass, we detected the content of HYP in serum, and found that compared with the NC group, the content of HYP in serum of largemouth bass in the HF group increased significantly, while compared with the HF group, GA administration significantly reduced the content of HYP;

[0155] S521GA's effect on HF-induced transcription of pro-fibrotic markers related to HSC activation: Further detection of the mRNA expression of pro-fibrotic markers α-Sma, Collagen I, Fibrontein and Mmp9. The results showed that compared with the NC group, HF significantly increased the mRNA expression of liver α-Sma, Collagen I and Fibrontein, while compared with the HF group, GA significantly reduced the mRNA expression of α-Sma, Collagen I and Fibrontein. Western-blot method was used to detect the protein level of α-Sma, Collagen I and Fibrontein, and it was found that the protein level of α-Sma, Collagen I and Fibrontein was 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 α-Sma induced by HF. The above results showed that GA can effectively reduce the liver fibrosis induced by HF in largemouth bass;

[0156] S522Effect of GA on Tgfβ1-Smad2 / 3 signaling pathway in liver of HF-induced largemouth bass: Tgfβ1a is the most effective fibrocyte factor in the liver, therefore, in order 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 expression of Tgfβ1a, Smad2 and Smad3a in the HF group was significantly increased, while the mRNA expression was significantly reduced after GA treatment. 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 the protein levels were reduced after GA treatment. Consistent with the protein and mRNA expression, immunohistochemical results showed that compared with the HF group, the expression of Tgfβ1 in the liver tissue of GA-treated largemouth bass was significantly reduced. These results indicated that GA improved liver fibrosis by regulating Tgfβ1-mediated Smad2 / 3 signaling pathway;

[0157] S53Effect of GA on mitochondria induced by HF in the liver, which includes the following steps:

[0158] S531Effect of GA on reactive oxygen species induced by HF in the liver: Mitochondrial membrane potential MMP was measured by JC-1, and it was found that JC-1 staining showed bright red fluorescence as JC-1 polymer and green fluorescence as JC-1 monomer. JC-1 staining showed that bright red fluorescence and weak green fluorescence were observed in the NC group, while red fluorescence was observed in the HF group. Compared with the HF group, bright red fluorescence was observed in the GA group. Fluorescence microplate reader detection found the same phenomenon, and the red-green fluorescence ratio of the NC group and the GA treatment group was significantly increased compared with the HF group P<0.05. At the same time, we detected the ATP content in the liver, and found that the ATP content was decreased in the HF group, while the ATP content was significantly increased after GA treatment. These results showed that GA treatment could alleviate the mitochondrial damage in the liver of largemouth bass induced by HF;

[0159] S532Effect of GA on mitochondrial damage induced by HF in the liver: The number of mitochondria in the NC group was abundant, the endoplasmic reticulum structure was normal, the mitochondrial cristae was arranged in order, the mitochondrial membrane was complete, and the gap between endoplasmic reticulum and mitochondria was appropriate. In the HF treatment group, the mitochondrial membrane was obviously broken, the mitochondrial cristae was damaged, the endoplasmic reticulum structure was abnormal, and the gap between endoplasmic reticulum and mitochondria was reduced. In the HFM treatment group, the mitochondrial membrane, mitochondrial cristae arrangement, endoplasmic reticulum structure and the gap between endoplasmic reticulum and mitochondria were restored to be consistent with the NC group;

[0160] Effects of S54GA on HF-induced expression of calcium transporters in liver cytoplasm and endoplasmic reticulum: The mRNA expression levels of CaSr, Sig1r, and Ip3r1 were all increased in the HF group. However, after GA treatment, the expression of CaSr, Sig1r, and Ip3r1 mRNA in liver tissue significantly decreased (P<0.05), consistent with the mRNA expression levels. Western blot analysis of Ip3r1 protein levels revealed that the HF group had the highest Ip3r1 protein level compared to the NC group, while GA treatment reduced the Ip3r1 protein level.

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

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

[0163] In the S511 liver-to-body ratio study, the appearance of the liver was observed through dissection and the liver-to-body ratio of the largemouth bass was statistically analyzed.

[0164] Working principle:

[0165] 1. This study on the effects of 18β-glycyrrhetinic acid (GA) on liver injury mediated by high-fat diet-induced mitochondrial dysfunction is the first to explore how GA can alleviate liver fibrosis by regulating Tgfβ1-Smad2 / 3 signaling and reducing collagen fiber aggregation in the liver, thereby improving liver injury in largemouth bass induced by high-fat diets (HF). Secondly, GA can reduce the expression of calcium transport proteins in the endoplasmic reticulum and mitochondria through the Ampk-Pgc1α-Sirt3 signaling pathway, alleviating liver mitochondrial damage and reducing ROS levels, thus inhibiting the activation of pro-fibrotic factors. HF upregulates the activity of liver function-related enzymes in largemouth bass, increasing hepatic lipid droplet deposition and collagen aggregation. GA supplementation reduces the activity of liver function-related enzymes, decreasing hepatic steatosis, lipid droplet deposition, and collagen fiber aggregation, thereby alleviating HF-induced liver injury in largemouth bass.

[0166] 2. The 18β-glycyrrhetinic acid method for studying liver injury induced by mitochondrial dysfunction mediated by high-fat diet, feeding HF to largemouth bass can up-regulate the expression of fibrosis-promoting markers; while adding GA treatment can reduce the expression of fibrosis-promoting markers by down-regulating Tgfβ1-mediated Smad2 / 3 signaling, thereby alleviating HF-induced liver fibrosis in largemouth bass.

[0167] 3. The 18β-glycyrrhetinic acid method for studying liver injury induced by mitochondrial dysfunction mediated by high-fat diet, feeding HF to largemouth bass can cause mitochondrial damage and increase ROS content; while GA can protect mitochondrial membrane, mitochondrial cristae and endoplasmic reticulum from HF-induced mitochondrial damage by up-regulating mitochondrial MMP and ATP content and down-regulating ROS content, thereby improving mitochondrial function.

[0168] 4. The 18β-glycyrrhetinic acid method for studying liver injury induced by mitochondrial dysfunction mediated by high-fat diet, feeding HF to largemouth bass can increase mRNA expression and protein levels of calcium ion transport proteins in endoplasmic reticulum and mitochondria, and GA can reduce mRNA expression and protein levels of endoplasmic reticulum and mitochondrial calcium ion transport proteins by up-regulating Ampk-Pgc1α-Sirt3 signaling, thereby improving the increase in expression of mitochondrial calcium ion transport proteins caused by HF.

[0169] The above is only a specific embodiment of the present application, and is not intended to limit the scope of the present application. Any person skilled in the art, without departing from the concept and principles of the present application, makes equivalent changes and modifications, which should be within the scope of the present application. It should be noted that the components of the present application are not limited to the above overall application, and the technical features described in the specification of the present application can be selected for single use or combined use, therefore, the present application naturally covers other combinations and specific applications related to the present application.

Claims

A method for studying liver injury mediated by mitochondrial dysfunction induced by a high-fat diet, comprising the following steps, characterized in that: The S1 experimental design used a diet consisting of three protein sources: fishmeal, chicken meal, and fermented soybean meal, with soybean oil as the fat source, and two carbohydrates: wheat flour and tapioca starch. The diet was formulated as follows: a control group (47.75% protein, 9.05% fat), a high-fat diet group (47.31% protein, 16.29% fat), a high-fat diet supplemented with GA 0.5 mg / kg (47.48% protein, 16.25% fat), a high-fat diet supplemented with GA 1.0 mg / kg (47.48% protein, 16.44% fat), and a high-fat diet supplemented with GA 1.5 mg / kg (1.0 mg / kg protein, 16.44% fat). The protein level was 47.55%, the fat level was 16.44%, and the vitamin and mineral premix additions were 1% and 1.5%, respectively. Vitamin D, vitamin C, and selenium were added. All feed ingredients were crushed, passed through a 40-mesh sieve, weighed according to the formula, and trace components were added using a stepwise expansion method. The ingredients were mixed evenly with the bulk ingredients, and then mixed evenly again after adding oil and water. The mixture was pelleted using a small extruded feed machine, dried at 30°C, and stored in sealed plastic bags. The nutritional components of each feed were analyzed in a preliminary nutrient analysis and used in experiments. S12 Experimental Fish and Rearing Management: The experimental fish were healthy largemouth bass. After being temporarily held for 2 weeks, 750 healthy largemouth bass with an average initial weight of 17.39±0.09 g were randomly assigned to 15 square cement ponds, with 50 fish in each pond. The cement ponds were randomly divided into 5 treatment groups, fed with NC, HF, HFL, HFM and HFH feeds respectively. Each treatment was replicated in 3 times. The growth experiment lasted for 77 days. Feeding was carried out at 7:30 am and 6:00 pm every day. 40 minutes after feeding, the remaining feed was removed, dried and weighed. Weather, water temperature, feeding rate, amount of uneaten feed and feed intake were recorded every day. The experiment adopted a micro-flow aquaculture method with a water flow rate maintained at 1.0 L / min. The water temperature during the experiment was 25.0±3.0℃, and dissolved oxygen was intermittently aerated to maintain a dissolved oxygen greater than 5.0 mg / L. S2 sample collection: After the feeding trial, largemouth bass were fasted for 24 hours. For each replicate, 12 fish of approximately similar weight were selected and anesthetized with 0.01% MS-222. After the fish were dried, they were weighed and their weight recorded. Samples were then rinsed with an anticoagulant. Blood was collected from the tail vein using a disposable syringe. Nine largemouth bass were separated from their bodies and viscera. The livers were quickly separated from the visceral mass, thoroughly washed with saline, photographed, weighed, and their weight recorded. They were then placed in labeled bags, wrapped in aluminum foil, and immediately frozen in liquid nitrogen. Afterward, they were quickly transferred to a -80°C ultra-low temperature freezer for storage, to be used for subsequent experimental index determination. The remaining three largemouth bass had their liver tissue separated and fixed with 4% neutral formalin for subsequent HE staining, Masson staining, and immunofluorescence staining analysis. Similarly, frozen sections were placed in 2mL cryovials, labeled, wrapped in aluminum foil, and immediately frozen in liquid nitrogen for subsequent Oil Red O staining. For electron microscopy, pea-sized tissue samples were fixed in 2.5% glutaraldehyde solution for transmission electron microscopy section observation. S3 index measurement S31 liver-to-body ratio; S32 Determination of blood and liver biochemical indicators; S33 Liver H&E staining analysis; S34 liver oil red O staining analysis; S35 liver Masson staining analysis; Transmission electron microscopy analysis of S36 liver cells; S37 Liver Immunofluorescence Analysis; S38 reactive oxygen species detection; S39 membrane potential MMP measurement; S310 real-time quantitative PCR detection; S3101 RNA extraction and cDNA synthesis; S3102 Real-Time Fluorescent Quantitative Primer Design; S3103 Real-Time Quantitative PCR for Gene Expression Level Detection: Three cDNA samples from each treatment were diluted 1:1, and fibrosis-related genes (Tgfβ1a, Smad2, Smad3a, α-Sma, CollagenⅠ, Fibronectin, and Mmp9) and mitochondrial calcium transport-related genes (Ampkα1, Pgc1α, Sirt3, Ip3r1, Sig1r1, CaSR, Grp75, Vdac1, and Mcu) were detected. Quantitative PCR amplification was performed using the TBGreen™ Premix ExTaq™ IITliRNase HPlus kit. 10-fold serially diluted positive templates were used as PCR templates. A standard curve was plotted, with an amplification efficiency of 90%-110%, R²>0.9, and a single peak in the melting curve. A 10 μL PCR amplification system consisted of 3.2 μL DEPC water, 1 μL cDNA template, 0.4 μL upstream primer, 5 μL downstream primer, and 0.4 μL downstream primer. SYBRPremixExTaqⅡ, reaction cycling conditions: preheating at 95℃ for 2 min, cycling at 95℃ for 10 s for 40 times and annealing at a suitable temperature for 30 s. After the reaction is completed, the product specificity is detected by melting curve. S3104 Relative quantitative calculation; S311 protein immunoblotting; Statistical analysis of S4 test data; S5 Results 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 appearance of the liver and statistically analyzing the liver-to-body ratio of largemouth bass, it was found that the liver surface of the NC group was smooth and reddish. 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: Hematological and liver function of largemouth bass were detected. Serum AST, ALT and AKP activities in the HF group were significantly higher than those in the NC group. Compared with the NC group, MDA content in the HF group was significantly increased. GA reduced HF-induced serum AST, ALT and AKP activities, TG, TC content and MDA content in the liver, and increased GST activity, thereby alleviating HF-induced liver injury. S513 liver histopathology: Compared with the NC group, the hepatocytes in the HF group were significantly swollen, with marginal displacement of the nuclei, some of which even disappeared, and the number of nuclei was less. Compared with the HF group, the hepatic steatosis in the GA treatment group was gradually alleviated, the number of nuclei gradually increased, and they were located in the center of the cell. GA reduced HF-induced hepatic lipid degeneration and lipid droplet deposition. Effects of S52 GA on HF-induced liver fibrosis in largemouth bass: The content of HYP in serum was detected. It was found that the HYP content in the serum of largemouth bass in the HF group was significantly increased compared with the NC group, while the GA administration significantly reduced the HYP content compared with the HF group. The 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 examined, and the protein levels of α-SMA, CollagenI and Fibrontein were detected by Western blot. GA can effectively alleviate HF-induced liver fibrosis in largemouth bass. Effects of S522 GA on the Tgfβ1-Smad2 / 3 signaling pathway in the liver of largemouth bass induced by HF: The mRNA expression of Tgfβ1a and its target proteins Smad2 and Smad3a was detected, and the protein levels of Tgfβ1, p-Smad2 and p-Smad3 were detected by Western blot. GA improves liver fibrosis by regulating the Tgfβ1-mediated Smad2 / 3 signaling pathway. The effects of S53 GA on HF-induced liver mitochondria include the following steps: Effects of S531 GA on HF-induced liver reactive oxygen species: Further detection of mitochondrial function-related indicators MMP and ATP content, and ROS detection by DCFH-DA, showed that GA treatment could alleviate HF-induced liver mitochondrial damage in largemouth bass. Effects of S532GA on HF-induced liver mitochondrial damage: GA treatment restored these characteristics, very similar to the NC group; S54GA inhibits HF-induced liver cytoplasmic and endoplasmic reticulum calcium. 2+ Effects on the expression of calcium ion transporters: Western blot analysis of Ip3r1 protein levels revealed that the HF group had the highest Ip3r1 protein levels compared to the NC group, while GA treatment reduced Ip3r1 protein levels. Effects of S55GA on HF-induced expression and protein levels of liver mitochondrial calcium transporter mRNA: The expression of Grp75, Vdac1, and Mcu was evaluated, and further analysis of liver Vdac1 and Mcu protein levels showed that these protein levels were significantly increased in the HF group, while Grp75 protein levels were relatively unaffected in both the HF and GA treatment groups. Effects of S56GA on HF-induced Ampk-Pgc1α-Sirt3 signaling pathway: By detecting the mRNA expression of Ampk, Pgc1α and Sirt3, GA increased the liver Sirt3 level reduced by HF. GA reduced liver Mcu expression by regulating Sirt3 expression.

2. The method for studying liver injury mediated by mitochondrial dysfunction induced by a high-fat diet induced by 18β-glycyrrhetinic acid according to claim 1, characterized in that: S3 includes the following steps: Based on the sampling statistics, the liver body index is calculated to determine the growth and development of the liver. Blood was transferred to microcentrifuge tubes and centrifuged at 3000 g at room temperature for 10 min to separate plasma. The activities of alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase, as well as the contents of triglycerides, total cholesterol, and hydroxyproline, were determined using commercial kits. After the samples were taken out of the -80°C freezer, a 10% tissue homogenate was prepared using a homogenizer and then centrifuged at 3000 r / min for 10 min to obtain the supernatant. The contents of malondialdehyde, glutathione S-transferase, total protein, and adenosine triphosphate were then determined. Liver tissue was fixed in 10% formalin solution for 7 days, then dehydrated using a gradient of ethanol solutions. The samples were then cleared with xylene for subsequent paraffin embedding. Finally, the samples were embedded in paraffin and sectioned using a paraffin microtome to a thickness of 5 μm. The sections were then stained with hematoxylin-eosin (H&E). The specific steps for 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 slide in xylene for 10 minutes each time, for a total of 2 times; 3. Hydration: Immerse the sample slides in anhydrous ethanol, 95% ethanol, 80% ethanol, and 70% ethanol for 2 min each, and then in distilled water for 2 min.

4. Stain with hematoxylin for 10 min, then wash with water for 30 s; 5. Differentiate with 1% hydrochloric acid for 60 seconds, remove after turning blue; 6. Stain with eosin for 4 min, then wash with water for 30 s; 7. Immerse the sample slides in 95% ethanol twice for 5 min each time, 100% ethanol twice for 5 min each time, 100% ethanol: xylene 1:1 for 5 min each time, and xylene twice for 5 min each time.

8. Neutral resin mounting; Observe and photograph the Olympus BX43 under a light microscope; The specific steps for preparing Oil Red O staining are as follows:

1. Remove the liver sample from the -80℃ freezer and transfer it to the -20℃ freezer for a buffer of 30 min; 2. The liver tissue was trimmed into appropriately sized cubes and embedded and frozen using OCT embedding agent; 3. Use a pre-cooled cryostat to cut the tissue block into 8μm frozen sections; 4. Transfer the cut sample to a glass slide with the corresponding label, let it stand at room temperature for 10 minutes, and wait for staining; 5. First, cover the sample with washing solution for 20 seconds, then stain with Oil Red O dye for 20 minutes; 6. After removing the oil red O dye, wash with washing solution for 30 seconds, then immerse in distilled water and wash on a shaker for 20 seconds.

7. Counterstain cell nuclei with hematoxylin for 3 min, wash with water for 30 s; mount with glycerol gelatin; 8. Microscopic examination of Olympus BX43; Sample pretreatment, dewaxing, and hydration are the same as those described for H&E staining. The specific staining steps are as follows:

1. First, stain with the prepared Weigert iron hematoxylin staining solution for 10 min; 2. Differentiate using acidic ethanol differentiation solution for 15 seconds, then wash with water; 3. Perform blueing with Masson blueing solution for 5 minutes, then wash with water and distilled water for 1 minute; 4. Stain with Ponceau S and Magenta solution for 10 min; 5. Wash with a 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 min, and then wash with weak acid working solution for 1 min; 7. Immerse directly in aniline blue staining solution for 2 min, then wash with weak acid working solution for 1 min; 8. Rapid dehydration with 95% ethanol for 3 seconds, followed by dehydration with anhydrous ethanol three times, each time for 10 seconds; 9. Apply xylene to clear the solution three times, 2 minutes each time, then seal with neutral resin and observe. Take a small piece of liver, 1×1×1mm 3 The samples were placed in 2.5% glutaraldehyde and rapidly fixed at 4°C for 2-4 h. Then, they were fixed in 1% osmium tetroxide prepared with 0.1M phosphate buffer PB at room temperature in the dark for 2 h. After dehydration, infiltration, embedding, sectioning and staining, the samples were observed under a transmission electron microscope and images were collected for analysis. Sample pretreatment: Dewaxing was performed as described above for H&E staining. Sections were submerged in 10 mmol / L citrate buffer (pH 6.0) and heated to boiling in a microwave oven for 20 min. The sections were then incubated overnight in a humidified chamber with primary antibodies against anti-α-Sma, anti-Tgfβ1, Sirt3, and Mcu. The sections were warmed and washed the next day. After washing, 25 μL of horseradish peroxidase (HRP)-labeled secondary antibody against rabbit / mouse was added to each section and incubated at 37°C for 40 min. The sections were washed twice with PBST for 5 min each time. DAPI was added to the sections and washed twice with PBST for 5 min each time. The sections were then mounted and observed and photographed under an inverted fluorescence microscope. Simultaneously, 200 mg of liver was removed, and the concentration of reactive oxygen species (ROS) was immediately measured. According to the preparation method of single-cell suspension, a single-cell suspension was prepared according to the operation steps. Then, a fluorescent probe was added, and the hepatocyte precipitate was suspended with diluted DCFH-DA. The hepatocyte precipitate 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 precipitate were collected, resuspended and mixed with PBS precipitate, and photographed using a confocal fluorescence microscope. The absorbance value at 525 nm was detected using a fluorescence microplate, and the ROS content was calculated. Liver mitochondria were isolated using a tissue mitochondrial isolation kit, MMPs were detected using the JC-1 fluorescent probe method, mitochondria were observed using a fluorescence microscope, absorbance values ​​were detected using a fluorescent microplate reader, and the red-green light ratio was calculated. The excitation wavelength was 485 nm and the emission wavelength was 580 nm. Total RNA was extracted using a kit. In short, liver tissue was taken from a -80°C freezer, ground into powder in liquid nitrogen, and 0.1 g of liver sample was taken. 1 mL of RNAiso Plus reagent was added to extract total RNA from the tissue. The RNA was dissolved in 40 μL of RNase-free water, and the degree of RNA degradation was detected by 1.0% agarose gel electrophoresis at 120V for 15 min. The concentration was detected by NanoDrop1000 spectrophotometer, and the purity of the sample was determined according to the A260 / A280 ratio. All gene sequences of the largemouth bass were obtained from GenBank or referenced from previous studies, and primers were designed using Primer 5.0; Expression levels were represented by the threshold period Ct, which was calculated by plotting the fluorescence signal for each period. The target gene mRNA concentration was normalized to match the mRNA concentrations of the internal reference genes βactin and 18S rRNA. After verifying that the primer amplification efficiency reached the amplification efficiency of the target gene and the internal reference gene, a 2-1 ratio was used. −ΔΔCT The amplification efficiency of the internal reference gene was calculated based on the standard curve of a specific gene generated by a 10-fold sequence dilution. Total protein was extracted from liver tissue using RIPALysis Buffer. Protein concentration was determined using an enhanced BCA protein assay kit. SDS-PAGE protein loading buffer was added to the sample, and the mixture was denatured at 95°C for 10 min. After SDS-PAGE separation, the sample was transferred to a PVDF membrane, blocked with 5% skim milk for 1.5 h, and then incubated with primary antibody overnight at 4°C. After washing, the sample was incubated with secondary antibody at room temperature for 1.5 h. Target protein expression was detected using an ECL Kit and recorded using ChemiDoc Imaging Systems / Image Lab software. β-actin was used as an internal control with an equal sample loading.

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

4. The method for studying liver injury mediated by 18β-glycyrrhetinic acid-induced mitochondrial dysfunction in accordance with claim 2, characterized in that, The liver body weight ratio is calculated based on sampling statistics. The liver body weight index is calculated as: liver body weight % = liver weight / body weight × 100.

5. The method for studying liver injury mediated by mitochondrial dysfunction induced by a high-fat diet induced by 18β-glycyrrhetinic acid according to claim 1, characterized in that: The S511 liver-to-body ratio is determined by anatomical observation of liver appearance and statistical analysis of largemouth bass liver-to-body ratio.

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