Abstract Acute liver injury (ALI) is a global health problem associated with high mortality and has attracted clinical attention. Ginkgo biloba extract (GBE) is an extract from dried Ginkgo leaves that has many pharmacological effects because of its various ingredients and has been shown to be hepatoprotective. We investigated the hepatoprotective effect of GBE on carbon tetrachloride (CCl[4])-induced acute liver injury in vitro. The components of Ginkgo biloba extract are analyzed by LC-MS, and the key targets of "liver injury-Ginkgo biloba" are identified based on bioinformatics analysis. The signaling pathways such as PI3K/AKT are mainly enriched with high correlation in KEGG. The results of in vitro experiments showed that compared with the Model group, except that the ALT activity level and MDA content in EGB-L group were not significantly decreased (P > 0.05), the activity of ALT, AST and MDA content in other EGB groups were significantly decreased (P < 0.05), and the activities of SOD and CAT were significantly increased (P < 0.05). The expression of inflammatory factors IL-1β, IL-6 and TNF-α were also detected. The results showed that compared with the Model group, the contents of IL-6 in EGB-L group were not significantly decreased (P > 0.05), while the contents of IL-1β, IL-6 and TNF-α in other EGB groups were significantly decreased (P < 0.05), indicating that EGB could reduce the level of cell inflammation. Western blot assay detected the protein expression levels of GF, RTK, PI3K, AKT and p-AKT in cells. The results showed that compared with the Model group, the protein expression levels of GF, RTK, PI3K, AKT and P-AKT were significantly increased after EGB treatment (P < 0.05), and the protein expression level of the EGB-H group was higher than the EGB-L group. Ginkgo biloba extract can inhibit the expression of downstream related genes by activating PI3K/AKT signaling pathway, and at the same time alleviate the inflammatory response of cells, reduce the level of inflammation, and protect the cell damage caused by CCl[4]. Keywords: Ginkgo biloba extract, Liver injury, HepG2 cells, PI3K/AKT signaling pathway Abbreviations LC-MS Liquid chromatography-mass spectrometry GO Gene Ontology KEGG The Kyoto Encyclopedia Genes and Genomes OMIM Online Mendelian Inheritance in Man DAVID The database for annotation visualization and integrated discovery SOD Superoxide dismutase MDA Malondialdehyde BP Biological process CC Cellular component MF Molecular function CON Blank group MOD Model group EGB-H High dose Ginkgo biloba extract group EGB-M Middle dose Ginkgo biloba extract group EGB-L Low dose Ginkgo biloba extract group RTK Receptor tyrosine kinase PI3K Phosphatidylinositol 3-kinase PIP2 Phosphatidylinositol diphosphate PIP3 Phosphatidylinositol triphosphate AKT Protein kinase B PTEN Protein Tyrosine Phosphatase 1. Introduction Liver disease is a global health problem, and acute liver injury in particular is associated with high mortality [[29]1,[30]2]. Acute liver injury (ALI) refers to sudden liver cell damage and liver dysfunction caused by various factors in a short period of time [[31]3]. Viral infection, drug poisoning, immune response and ischemia-reperfusion are the common predisposing factors of ALI [[32]4,[33]5]. Multiple signaling pathways are involved in the occurrence and development of ALI, including TGF-β/Smad, PI3K/AKT, Nrf2/HO-1 signaling pathways. The PI3K/AKT signaling pathway is involved in various physiological and pathological processes of cells, including apoptosis, proliferation, growth, secretion and chemotaxis. It not only plays an important role in various liver diseases such as chemical liver injury [[34]6], liver ischemia-reperfusion injury and cirrhosis [[35]7], but is also closely related to oxidative stress and inflammation [[36]8]. Studies have found that when inflammation occurs, the expression of PI3K signal will be down-regulated, thus inhibiting the secretion of pro-inflammatory factors in macrophages and dendritic cells and activating the secretion of anti-inflammatory factors [[37]9]. AKT, also known as protein kinase B, is an important effector downstream of PI3K. PI3K transduction signals can activate the expression of pro-inflammatory cytokines IL-6 and TNF-α downstream of AKT. In order to study its pathological mechanism and seek effective treatment, domestic and foreign scholars have established a series of ALI models, including CCl[4]-induced chemical liver injury models [[38]10]. The chemical acute liver injury caused by CCl[4] is the most typical. Carbon tetrachloride, as a classical chemical agent of hepatotoxicity, can accurately simulate the functional, metabolic and morphological changes of hepatocytes under the condition of liver injury in the induced liver injury model, and has many advantages such as stable model, good repeatability, simple and easy operation, and is commonly used to study the efficacy and mechanism of hepatoprotective drugs. HepG2 cells are similar in morphology and function to human liver tissue, and have the advantages of simple culture and stable genetic background. HepG2 cells are often used as research objects to construct models of oxidative stress, steatosis and insulin resistance. At present, there are many drugs used to treat and prevent liver injury, but these drugs all have relatively serious toxic side effects. Therefore, finding safe and efficient active ingredients from natural plants and animals for the prevention and treatment of such diseases has become the focus of researchers today [[39]11]. Ginkgo biloba Extract (EGB) is an effective component extracted from the dried leaves of Ginkgo biloba L. It contains a variety of antioxidant and neuroprotective components such as flavonoids, quercetin and terpenoid lactones, which have been widely used in clinical practice [[40]12]. Studies have found that EGB has many pharmacological effects, such as antioxidant, anti-platelet aggregation, regulating vascular activity, scavenging free radicals, and protecting nerves [[41]13]. It is mainly used in clinical treatment of coronary Atherosclerosis heart disease, senile dementia, cerebral insufficiency and other diseases [[42]14]. In recent years, domestic and foreign scholars have found that EGB also has a good effect in the treatment of liver injury, but the research on its mechanism of action is not comprehensive, which also limits its clinical use. At present, there are still few studies on the treatment of liver injury by Ginkgo biloba. Our study expects to establish a CCl[4]-induced HepG2 cell injury model to observe the effect of EGB on HepG2 cell injury. In addition, we hope to explore the protective mechanism of CCl[4]-induced injury of HepG2 cells through PI3K/AKT signaling pathway, so as to provide certain experimental basis for studying the protective effect of Ginkgo biloba on liver and its mechanism. 2. Materials and methods 2.1. Materials 2.1.1. Test drugs and reagents Ginkgo biloba leaves (Hebei Golden Leaf Pharmaceutical Co., ltd., Baoding, China); Acetonitrile (Fisher Chemical); Formic acid (Fisher Chemical); Methanol (Fisher Chemical); Ultrapure water (Millipore); CCl[4] (Beijing Chemical Plant, Beijing, China); HepG2 cells (CS-0009, Beijing Dingguo Changsheng Biotechnology Co., ltd., Beijing, China); Trypsin (GENVIEW); Fetal bovine Serum (Hangzhou Sijiqing Company, Hangzhou, China); MTT (American Sigma Chemical Reagents Co., ltd., USA); 1640 Medium (11011-8611, Zhejiang Tianhang Biotechnology Co., LTD); DMSO (Liaoning Quanrui Reagent Co., ltd., Jinzhou, China); TNF-α(YJ002095), IL-1β([43]JN301814), and IL-6([44]JN063159) ELISA kits (Shanghai Langton Biotechnology Co., ltd., Shanghai, China); ALT(C009-2-1), AST(C010-2-1), CAT(A007-1-1), SOD(A001-3-2), and MDA (A003-1-2) assay kits (Nanjing Jiancheng Bioengineering Research Institute, Nanjing, China). 2.1.2. Instruments AL204 electronic balance [Mettler-Toledo Instruments (Shanghai) Co., ltd., Shanghai, China]; RE-52AA Rotary evaporator (Shanghai Yarong Biochemical Instrument Factory, Shanghai, China); Chromatographic ultra-high performance liquid phase U3000 (Thermo Scientific); Mass spectrometer Triple TOF5600+ (AB SCIEX™); Vacuum centrifugal concentrator, Concentrator plus™ (Eppendorf, Germany); Handheld centrifuge D1008 (Scilogex, USA); Vortex meter MX-S(Scilogex, USA); BA-400 microscope (Macaudi Industrial Group Co., ltd, Xiamen, China); Galaxy 170 S Cell Incubator (Eppendor); Infinite M200 microplate reader (TECAN Group, Switzerland); Cryogenic high-speed centrifuge Microfuge 22R Centrifuge (Beckman Coulter, USA); 4 °C refrigerated refrigerator (Haier Group Co., ltd, Qingdao, China); BCD-206STPH low temperature refrigerator(Haier Group Co., ltd, Qingdao, China). 2.2. Methods 2.2.1. Extraction and component identification of Ginkgo biloba extract Under the solid-liquid ratio of 1∶43 (g: ml), the crude extract of Ginkgo biloba leaves was extracted by heating reflux extraction method. And then the crude extract of Ginkgo biloba leaves was separated and purified by macroporous adsorption resin method to EGB. Liquid chromatography-mass spectrometry (LC-MS) was used to determine the composition and molecular weight distribution of EGB. Weigh EGB 25 mg precisely, add 75 % methanol 25 mL precisely, weigh the total mass, ultrasonic in 37 °C water bath for 30 min, let it stand, wait for the stopper tube to return to room temperature, fill with 75 % methanol to reduce the weight, swirl and mix the sample, let it stand, centrifuge for 10 min to obtain supernatant, and then pass 0.22um microporous filter membrane. Filtrate 10 μL was taken as test material. Chromatographic conditions: Column: Waters BEH C18 Column (150 × 2.1 mm, 1.7 μm); Column temperature: 35 °C; Mobile phase A: 0.1% formic acid-water; Mobile phase B: acetonitrile; Flow rate: 0.3 mL/min; Analysis time of each component: 15 min ([45]Table 1). Mass spectrum conditions: Positive and negative ion modes of electrospray ionization (ESI) were used for detection ([46]Table 2). Table 1. Gradient of mobile phase. Time（min） Mobile phase 0.0 A:95% B:5% 8.0 A:50% B:50% 10.0 A:5% B:95% 12.0 A:5% B:95% 15.0 A:95% B:5% [47]Open in a new tab Table 2. Conditions of ESI source parameters. Parameters of MS Values of parameters TOF MS scan range 100–1200Da Ion Source Gas1(Gas 1) 50 Ion Source Gas2(Gas 2) 50 Curtain Gas（CUR） 25 Ion Sapary Voltage Floating (ISVF) 5500V（Positive ion）and 4400V（Negative ion） Source Tempreture 500 °C（Positive ion）and 450 °C（Negative ion） product ion scan range 50–1000Da TOF MS scan accumulation time 0.2s product ion scan accumulation time 0.01s Declustering potential(DP) ±60V Collision Energy 35 ± 15eV [48]Open in a new tab Finally, the Analysis Base File Converter software is used to process all the data, and the LC-MS information is summarized and analyzed to obtain the result. 2.2.2. Screening of active ingredients, targets, liver injury disease targets and co-regulatory targets of Ginkgo biloba leaves The chemical constituents of Ginkgo biloba and their SMILE numbers were analyzed by LC-MS, and the SMILE numbers were input into the SwissTargetPrediction database ([49]http://swisstargetprediction.ch/) to obtain the targets of EGB. The GeneCards database ([50]https://www.Genecards.org/) and Online Mendelian Inheritance in Man (OMIM) database ([51]https://www.omim.org/) were used to search the targets related to liver injury, and the duplicates were removed to obtain the disease targets of liver injury. The obtained data of potential targets of Ginkgo biloba leaves and targets related to liver damage were input into VennDiagram database to make a Venndiagram ([52]https://bioinformatics.psb.ugent.be/webtools/Venn/), and the co-regulatory targets of liver damage as the active components of Ginkgo biloba leaves were obtained. 2.2.3. Construction of the network of active ingredients and co-regulatory targets of Ginkgo biloba leaves, and construction of PPI network Cytoscape3.7.2 software was used to input co-regulatory targets and active components of Ginkgo biloba to construct the network of active components and co-regulatory targets of Ginkgo biloba. The co-regulatory targets were input into the String database (https://stringdb.org/) to construct the PPI network, the species was selected as Homo sapiens, the score was set to ≥ 0.9, and other parameters were set as default. The PPI network map between the active components of Ginkgo biloba and the common target proteins of liver injury was obtained. 2.2.4. GO functional annotation and KEGG pathway enrichment analysis The key targets of Ginkgo biloba treatment of liver injury were obtained through the Bioconductor database with R software installation package. Finally, the results of Gene Ontology (GO) functional analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of co-regulatory targets were presented by drawing graphs in R language. 2.2.5. Protective effect of EGB on CCl[4]-induced liver injury in vitro 2.2.5.1. Cell culture HepG2 cells were cultured in 1640 medium supplemented with 10% fetal bovine serum at 37 °C, 5% CO[2], and saturated humidity, and cells in the logarithmic growth phase were harvested. PBS(0.1 M): phosphate buffer: NaCl (8.00g) + KCl (0.20g) + Na[2]HPO[4] (1.44g) + KH[2]PO[4] (0.24g)→distilled water at constant volume to 1000 ml; Pancreatic enzyme (0.25%): trypsin (0.25g) + EDTA-2Na (0.02g)→PBS to 100 ml 2.2.5.2. Effects of EGB on normal HepG2 cells HepG2 cells in logarithmic growth phase, 100 μL per well, were seeded in 96-well plates and incubated in an incubator for 24 h at 37 °C in 5% CO[2]. Discard the supernatant and replace it with 1 diluted culture medium 1 μg/mL, 10 μg/mL, 100 μg/mL of PSP and each component was evenly distributed, and a blank control group (containing only cells and 1640 culture medium) and a zero adjustment well (containing only 1640 culture medium) were set, with 3 parallel samples in each group. After incubation in CO[2] incubator for 24 h, 20 μL MTT solution (5 mg/mL) was added to each well, incubated for 4 h, the supernatant was discarded, 150 μL DMSO was added to each well, and the absorbance was determined at the wavelength of 490 nm after shaking for 10 min. [MATH: Cellsurvivalrate(%)=A(experimentalgroup)–A(zeroadjustmentgroup)A(blankcontrolgroup)–A(zeroadjustmentgroup) :MATH] (1) 2.2.5.3. Preparation of CCl[4] injury solution 4.82 mL of CCl[4] and 5.18 mL of DMSO were prepared into 5 mol/L of CCl[4] solution, and then 5 mol/L of CCl[4] solution 100 μL, and diluted into 100 mmol/L of CCl[4] injury solution with cell culture medium. 2.2.5.4. Screening of CCl[4] injury concentration HepG2 cells of logarithmic growth stage were inoculated into 96-well plates with 100 μL per well and cultured in an incubator at 37 °C and 5% CO[2] for 24 h. After cell adhesion, CCl[4] injury solution prepared by 20 μL, 30 μL, 40 μL, 50 μL, 60 μL was added, and blank control hole and zero hole were set up, 3 parallel samples were set up in each group. After continued incubation for 24 h, the absorbance was measured at 490 nm according to the MTT method, and the survival rate was calculated according to the formula in 2.2.5.2. 2.2.5.5. Biochemical index measurement The cells in logarithmic growth phase were digested with trypsin and diluted to 1 × 10^5/mL, and 500 μL of each well were seeded in 24-well plates and incubated for 24 h at 37 °C in 5% CO[2]. After the cells were attached to the wall, the supernatant was discarded, and the PSP and its components were diluted by medium into concentrations of 1 μg/mL, 10 μg/mL, 100 μg/mL, and the complete medium was added into the blank hole. After 24 h of culture, except for the blank group and the zero setting well, 40 μL CCl[4] injury solution was added to the other wells, with three parallel wells in each group. And then after 24 h of culture, the supernatant was taken, and ALT, AST, MDA, SOD, CAT, TNF-α, IL-6, and IL-1β in the supernatant were detected according to the instructions of the kit. 2.2.6. Western Blot verification HepG2 cells of each group were taken and weighed, and 10 times the volume of protein cracking solution was added to each group, which was fully ground by homogenizer. After observation, the unorganized fragments were placed on ice for cracking for 1 h, centrifuged at 12000 r/min at 4 °C for 10 min, and the supernatant was obtained. Measure the protein content in the supernatant of different cell lysis groups using the BCA reagent kit, then add an appropriate amount of 5x Loading Buffer and boil in boiling water for 20 min before loading the sample. After electrophoresis for 2.5 h, the samples were transferred to PVDF membrane and blocked with 5% skim milk powder for 1 h at room temperature. The membranes were washed 3 times with TBS-T solution for 15 min each time, and the primary antibody dilutions (RTK, PI3K, AKT, p-AKT, β-actin) were added and incubated at 4 °C overnight. After the film was washed with TBS-T solution, the corresponding diluent of the second antibody was added and incubated at room temperature for 1 h. The film was washed three times by TBS-T for 15 min each time, and then developed and photographed with ECL luminescent solution. 2.2.7. Statistical analysis SPSS 19.0 was used for data processing. One-way ANOVA was used for comparison of samples between groups, and T-test was used for comparison between two groups. Data were represented by mean ± s (P < 0.05), indicating differences; P < 0.01, indicating a significant difference. 3. Results 3.1. Results of LC-MS A total of 63 components were obtained by LC-MS analysis, including 20 negative ion components and 43 positive ion components(S2). Among the 43 positive ion components, flavonoids, terpenoids and lipids were the most, and there were 15 flavonoids, such as Myricetin, Quercetin, Kaempferol, Isorhamnetin, Bilobetin. There are 5 terpenoids, such as Ophiopogonoside A, 2-(hydroxymethyl)-6-(6-hydroxy-6-methyl-3-propan-2-ylcyclohex-3-en-1-yl ) Oxyoxane-3,4,5-triol, 2-Butanone,3-Buten-2-one, 4-[4-(beta-D-glucopyranosyloxy)-2-hydroxy-2,6,6-trimethylcyclohexyliden e]-. Among the 20 negative ion components, flavonoids, organic acids and fatty acid compounds were the majority, and there were 7 flavonoids, such as Apigenin, Glycitein, afzelin, Daidzein, Isorhamnetin, etc. There are 5 organic acids, such as CAFFEIC acid, Benzoic acid+2O, O-Hex, P-Coumaric acid, 2-hydroxy-6-[(8Z,11Z) -PentadECa-8,11,14-trienyl] benzoic acid ([53]Fig. 1(A and B)). Fig. 1. [54]Fig. 1 [55]Open in a new tab Liquid chromatography-mass spectrometry (LC-MS) chromatography A: positive ion chromatography; B: negative ion chromatography. After processing by MS-DIAL software, 53 compounds with a Score of 80 or more were selected according to Total Score, among which flavonoids accounted for 43.4%, including isoflavones and diflavones. Other chemical components include polysaccharides, anthraquinones, ginkgolides and other components (S2). 3.2. Screening of active ingredients, targets, liver injury disease targets and co-regulatory targets of Ginkgo biloba leaves Based on the LC-MS analysis results and SwissTargetPrediction database, a total of 53 Ginkgo biloba leaf active ingredients, 167 Ginkgo biloba leaf active ingredient targets, and 2082 liver injury disease targets with correlation greater than 10 were obtained. The intersection of liver injury targets and active ingredient targets of Ginkgo biloba leaves resulted in 123 co-regulatory targets of Ginkgo biloba treatment of liver injury ([56]Fig. 2). Fig. 2. [57]Fig. 2 [58]Open in a new tab Intersection of active ingredient targets of Ginkgo biloba and liver injury related targets. 3.3. Construction of the network of active ingredients and co-regulatory targets of Ginkgo biloba leaves, and construction of PPI network The active ingredient of Ginkgo biloba-co-regulatory target network was constructed using Cytoscape3.7.2 software ([59]Fig. 3). Through the regulatory network, it was found that the co-regulatory targets were mainly related to Quercetin, Catechin, β-sitosterol and Kaempferol in Ginkgo biloba leaves, and they were regarded as the key active components. Fig. 3. [60]Fig. 3 [61]Open in a new tab Ginkgo biloba active ingredients-co-regulatory target network Orange arrow nodes represent active ingredients and blue nodes represent co-regulatory targets. (For interpretation of the references