Liver Protection of a Low-Polarity Fraction from Ficus pandurata Hance, Prepared by Supercritical CO2 Fluid Extraction, on CCl4-Induced Acute Liver Injury in Mice via Inhibiting Apoptosis and Ferroptosis Mediated by Strengthened Antioxidation

Ficus pandurata Hance (FPH) is a Chinese herbal medicine widely used for health care. This study was designed to investigate the alleviation efficacy of the low-polarity ingredients of FPH (FPHLP), prepared by supercritical CO2 fluid extraction technology, against CCl4-induced acute liver injury (ALI) in mice and uncover its underlying mechanism. The results showed that FPHLP had a good antioxidative effect determined by the DPPH free radical scavenging activity test and T-AOC assay. The in vivo study showed that FPHLP dose-dependently protected against liver damage via detection of ALT, AST, and LDH levels and changes in liver histopathology. The antioxidative stress properties of FPHLP suppressed ALI by increasing levels of GSH, Nrf2, HO-1, and Trx-1 and reducing levels of ROS and MDA and the expression of Keap1. FPHLP significantly reduced the level of Fe2+ and expression of TfR1, xCT/SLC7A11, and Bcl2, while increasing the expression of GPX4, FTH1, cleaved PARP, Bax, and cleaved caspase 3. The results demonstrated that FPHLP protected mouse liver from injury induced by CCl4 via suppression of apoptosis and ferroptosis. This study suggests that FPHLP can be used for liver damage protection in humans, which strongly supports its traditional use as a herbal medicine.


Introduction
According to a retrospective study in China, the annual incidence of liver injury is 23 per 100,000, which inevitably leads to a substantial socioeconomic burden [1]. A series of factors, such as alcohol, drugs, viruses, and being overweight, can cause ALI (acute liver injury) [2]. The common symptoms of ALI include weakness, fatigue, nausea, swollen abdomen, itching, and jaundice [3], and the clinical symptoms include abnormal levels of alanine transaminase (ALT) and aspartate aminotransferase (AST), which increase the risk of liver fibrosis, cirrhosis, and even acute hepatic failure [4,5].
It was previously reported that the pathological mechanisms of liver injury involve cytochrome P450 (CYP450) metabolism disorder, inflammation, oxidative stress, and cell

Antioxidation Effects of FPHLP
The antioxidation properties of FPHLP were evaluated by DPPH radical scavenging activity and T-AOC assays, as well as ROS detection via flow cytometry. As a result, FPHLP exhibited good DPPH radical scavenging activity in a concentration-dependent manner, with the highest clearance rate of 141.5% at a concentration of 40 mg/mL ( Figure 1A). The T-AOC activity of FPHLP was measured according to the ferric reducing antioxidant power (FRAP) method, and the results showed that FPHLP exhibited good antioxidant activity in a dose-dependent manner ( Figure 1B). The flow cytometry results also showed that the ROS level was increased in the H 2 O 2 -treated group, indicating that the model of H 2 O 2 -induced oxidative stress in HepG2 cells was successfully established. After FPHLP treatment (6,8, and 10 µg/mL), the high levels of ROS induced by H 2 O 2 were significantly reversed ( Figure 1C,D). These results suggested that FPHLP had good antioxidative stress potency. dant activity in a dose-dependent manner ( Figure 1B). The flow cytometry results also showed that the ROS level was increased in the H2O2-treated group, indicating that the model of H2O2-induced oxidative stress in HepG2 cells was successfully established. After FPHLP treatment (6,8, and 10 µ g/mL), the high levels of ROS induced by H2O2 were significantly reversed ( Figure 1C,D). These results suggested that FPHLP had good antioxidative stress potency.  Figures A and B, vs. 0 mg/mL group, * p < 0.05, ** p < 0.01, *** p < 0.001; in Figure C, vs. control group, *** p < 0.001, vs. H2O2-induced group, ## p < 0.01, ### p < 0.001.

FPHLP Protects against CCl4-Induced Liver Injury
To explore the protective effects of FPHLP against liver injury, C57BL6/J mice were intraperitoneally injected with CCl4 to establish the acute liver injury (ALI) model and intragastrically administered FPHLP. As a result, the appearance of the liver tissue obviously changed after CCl4 induction; most of the hepatic lobules appeared yellow and granular lesions were observed. Meanwhile, the appearance of injury in the liver tissues of the FPHLP-and silibinin-treated groups was alleviated ( Figure 2B), showing that FPHLP protected against ALI induced by CCl4. Furthermore, as shown in Figure 2C,D, the levels of ALT and AST in the serum of the model group were sharply enhanced after CCl4 induction and the level of LDH in the liver homogenate of the model group was significantly Results are expressed as the mean ± SEM (n = 3). In (A,B), vs. 0 mg/mL group, * p < 0.05, ** p < 0.01, *** p < 0.001; in (C), vs. control group, *** p < 0.001, vs. H 2 O 2 -induced group, ## p < 0.01, ### p < 0.001.

FPHLP Protects against CCl 4 -Induced Liver Injury
To explore the protective effects of FPHLP against liver injury, C57BL6/J mice were intraperitoneally injected with CCl 4 to establish the acute liver injury (ALI) model and intragastrically administered FPHLP. As a result, the appearance of the liver tissue obviously changed after CCl 4 induction; most of the hepatic lobules appeared yellow and granular lesions were observed. Meanwhile, the appearance of injury in the liver tissues of the FPHLP-and silibinin-treated groups was alleviated ( Figure 2B), showing that FPHLP protected against ALI induced by CCl 4 . Furthermore, as shown in Figure 2C,D, the levels of ALT and AST in the serum of the model group were sharply enhanced after CCl 4 induction and the level of LDH in the liver homogenate of the model group was significantly higher than that of the control group ( Figure 2E), indicating that the liver injury mouse model was successfully established. Meanwhile, the ALT, AST, and LDH levels of the FPHLP-and silibinin-treated groups were significantly reversed, suggesting that FPHLP and silibinin had good protective effects against liver injury in mice. Additionally, the H&E staining assay showed that the liver tissues of the model group had typical features of liver injury, such as inflammatory infiltration and enlarged vacuoles. After FPHLP and silibinin treatments, the classical features of pathological changes in the liver tissues were remarkably ameliorated ( Figure 2F). These data indicated that FPHLP could significantly alleviate the acute liver injury induced by CCl 4 in mice.
FPHLP-and silibinin-treated groups were significantly reversed, suggesting that FPHLP and silibinin had good protective effects against liver injury in mice. Additionally, the H&E staining assay showed that the liver tissues of the model group had typical features of liver injury, such as inflammatory infiltration and enlarged vacuoles. After FPHLP and silibinin treatments, the classical features of pathological changes in the liver tissues were remarkably ameliorated ( Figure 2F). These data indicated that FPHLP could significantly alleviate the acute liver injury induced by CCl4 in mice. . Results are expressed as the mean ± SEM. vs. control group, *** p < 0.001; vs. CCl4-treated only group, ## p < 0.01, ### p < 0.001.

FPHLP Protects Liver Injury via Inhibiting Apoptosis
Liver injury can cause hepatocyte death, which is irreversible. Hence, the protective effect of FPHLP against hepatocyte apoptosis was evaluated via western blotting and IHC . Results are expressed as the mean ± SEM. vs. control group, *** p < 0.001; vs. CCl 4 -treated only group, ## p < 0.01, ### p < 0.001.

FPHLP Protects Liver Injury via Inhibiting Apoptosis
Liver injury can cause hepatocyte death, which is irreversible. Hence, the protective effect of FPHLP against hepatocyte apoptosis was evaluated via western blotting and IHC assays. The results showed that the expression of cleaved PARP and Bax in the liver tissues of mice was upregulated, while that of Bcl2 was reduced after CCl 4 injection. However, after FPHLP treatment, the expression of cleaved PARP, Bax, and Bcl2 was significantly reversed, suggesting that FPHLP effectively suppressed hepatocyte apoptosis ( Figure 3A,B). Meanwhile, the expression of cleaved caspase-3 in the liver tissues was detected via IHC assay, and the results showed that the expression of cleaved caspase-3 was increased in the CCl 4 -treated group compared to that in the control group, while that in the FPHLP-treated groups was sharply suppressed ( Figure 3C). These results suggested that FPHLP effectively protected against apoptosis. ever, after FPHLP treatment, the expression of cleaved PARP, Bax, and Bcl2 was significantly reversed, suggesting that FPHLP effectively suppressed hepatocyte apoptosis (Figure 3A,B). Meanwhile, the expression of cleaved caspase-3 in the liver tissues was detected via IHC assay, and the results showed that the expression of cleaved caspase-3 was increased in the CCl4-treated group compared to that in the control group, while that in the FPHLP-treated groups was sharply suppressed ( Figure 3C). These results suggested that FPHLP effectively protected against apoptosis.

FPHLP Protects Liver Injury via Strengthening Antioxidative Activity
Oxidative stress has been recognized as an important mechanism underlying the pathophysiology of ALI [20]. In the present study, the influence of FPHLP on oxidative stress in the liver tissues of CCl4-induced ALI model mice was investigated via ELISA assays, western blotting, and IHC assays. As shown in Figure 4A-D, the levels of SOD and GSH, two important antioxidation parameters, were significantly decreased and the levels of ROS and MDA were significantly increased after CCl4 induction (p < 0.05 vs. control group), while those of the FPHLP-treated groups were significantly reversed (p < 0.05 vs. model group). Additionally, the effect of FPHLP on the expression of oxidative stress-related proteins, including Keap1, Nrf2, HO-1, and Trx-1, was assessed. The results

FPHLP Protects Liver Injury via Strengthening Antioxidative Activity
Oxidative stress has been recognized as an important mechanism underlying the pathophysiology of ALI [20]. In the present study, the influence of FPHLP on oxidative stress in the liver tissues of CCl 4 -induced ALI model mice was investigated via ELISA assays, western blotting, and IHC assays. As shown in Figure 4A-D, the levels of SOD and GSH, two important antioxidation parameters, were significantly decreased and the levels of ROS and MDA were significantly increased after CCl 4 induction (p < 0.05 vs. control group), while those of the FPHLP-treated groups were significantly reversed (p < 0.05 vs. model group). Additionally, the effect of FPHLP on the expression of oxidative stressrelated proteins, including Keap1, Nrf2, HO-1, and Trx-1, was assessed. The results showed that CCl 4 markedly increased the expression of Keap1 and decreased the expression of Nrf2 and its downstream factor HO-1 as well as Trx-1, while FPHLP treatment strongly reversed these effects ( Figure 4E,F), indicating that FPHLP effectively suppressed oxidative stress in the liver tissues. The IHC assay was also used to detect the expression of Nrf2, the critical factor of the Keap1/Nrf2/HO-1 pathway, in the liver tissues of ALI model mice, and the results showed that FPHLP enhanced the expression of Nrf2 in mice with CCl 4 -induced ALI ( Figure 4G). Altogether, these data demonstrated that FPHLP strongly alleviated the acute liver injury in mice by enhancing the antioxidative capacity. strongly reversed these effects ( Figure 4E,F), indicating that FPHLP effectively suppressed oxidative stress in the liver tissues. The IHC assay was also used to detect the expression of Nrf2, the critical factor of the Keap1/Nrf2/HO-1 pathway, in the liver tissues of ALI model mice, and the results showed that FPHLP enhanced the expression of Nrf2 in mice with CCl4-induced ALI ( Figure 4G). Altogether, these data demonstrated that FPHLP strongly alleviated the acute liver injury in mice by enhancing the antioxidative capacity. Results are expressed as the mean ± SEM. vs. control group, * p < 0.05, ** p < 0.01; vs. CCl4-treated only group, # p < 0.05, ## p < 0.01, ### p < 0.001.

FPHLP Protects Liver Injury via Inhibiting Ferroptosis
In our study, the effect of FPHLP on ferroptosis in ALI model mice was evaluated via western blotting and biochemical assays. The results showed that the level of Fe 2+ in the . Results are expressed as the mean ± SEM. vs. control group, * p < 0.05, ** p < 0.01; vs. CCl 4 -treated only group, # p < 0.05, ## p < 0.01, ### p < 0.001.

FPHLP Protects Liver Injury via Inhibiting Ferroptosis
In our study, the effect of FPHLP on ferroptosis in ALI model mice was evaluated via western blotting and biochemical assays. The results showed that the level of Fe 2+ in the liver tissue of the CCl 4 -treated group was markedly elevated compared to that of the control group, while those in the FPHLP-treated groups were significantly decreased ( Figure 5A). Additionally, the effect of FPHLP on ferroptosis-related protein expression, including GPX4, xCT, FTH1, and TfR1, was assessed via western blotting and the results showed that CCl 4 markedly downregulated the expression of GPX4 and FTH1, two important proteins that chelate Fe 2+ , and upregulated the expression of xCT and TfR1, while FPHLP remarkably reversed the effect ( Figure 5B,C). In all, our findings suggested that FPHLP significantly alleviated ALI via suppression of ferroptosis in mice. trol group, while those in the FPHLP-treated groups were significantly decreased ( Figure  5A). Additionally, the effect of FPHLP on ferroptosis-related protein expression, including GPX4, xCT, FTH1, and TfR1, was assessed via western blotting and the results showed that CCl4 markedly downregulated the expression of GPX4 and FTH1, two important proteins that chelate Fe 2+ , and upregulated the expression of xCT and TfR1, while FPHLP remarkably reversed the effect ( Figures 5B,C). In all, our findings suggested that FPHLP significantly alleviated ALI via suppression of ferroptosis in mice.

Materials and Reagents
The whole plant was collected from Dajin, Kaiping, Guangdong, China. It was iden-

Materials and Reagents
The whole plant was collected from Dajin, Kaiping, Guangdong, China. It was iden-

FPHLP Preparation
FPHLP supercritical fluid extraction of FPH was performed by Nantong Huaan Supercritical Extraction Co., Ltd. (Nangtong, China) using a supercritical fluid extractor (HA220-40-11, Nangtong, China). Briefly, 2212.5 g FPH was placed into the extraction tank and carbon dioxide was pressurized by a pressurizing pump. The conditions were as follows: 45 • C extraction temperature, 55 • C separation temperature, 28 MPa extraction pressure, and 8 MPa separation pressure [26]. The extraction time was set at 3 h and the extracted essential oil was collected every 1 h. A total of 6.5 g of paste was obtained with a yield of 0.294%. According to ref. [27], the daily dosage of FPH is 100~400 g for adults, which was converted to a mice dosage of 12~50 g/kg (calculated based on the quantity of crude material). According to the yield of FPHLP (0.3%), crude drug dosages of 12 and 50 g/kg were converted into extract dosages of 36 and 150 mg/kg. We selected 50 and 100 mg/kg as the testing dosages for the animal study after a pilot study.

DPPH Radical Scavenging Activity Assay
A DPPH assay kit (G0128W) obtained from Suzhou Grace Biotechnology Co., Ltd. (Suzhou, China) was used to evaluate the DPPH scavenging ability of FPHLP. Briefly, 150 µL of FPHLP (40, 20, 10, 5, 2.5, and 1.25 mg/mL) was added to DPPH working solution (150 µL) and 80% methanol served as the control, according to the manufacturer's instructions. The mixtures were co-incubated for 30 min at room temperature, then centrifuged at 5000× g for 5 min. A 200 µL aliquot of supernatant was transferred into a 96-well plate to measure the absorbance at 517 nm, and the DPPH scavenging rate was calculated according to the following equation: DPPH scavenging rate = 1 − (OD of measurement group − OD of control group) of blank group × 100 %

Total Antioxidant Capacity (T-AOC) Analysis
According to the instructions of the T-AOC detection kit (#BC1315, Biobox, Beijing, China) [28], different concentrations of FeSO 4 were used to establish a standard curve. The FPHLP samples were prepared in DMSO to obtain 10, 8, 6, 4, 2, and 1 mg/mL solution, and VC at 400 µg/mL was used as the positive control. The process was performed following the instructions of the T-AOC commercial kit. The blank group contained 180 µL of mixed working solution (control group) and 24 µL of distilled water. The measurement group containing 180 µL of mixed working solution, 6 µL of sample solution, and 18 µL of distilled water. The samples were co-incubated for 10 min after gentle mixing and 200 µL of the mixture was transferred to a 96-well plate to measure the absorbance of Fe 2+ -TPTZ at 592 nm. According to the standard curve, the absorbance of Fe 2+ -TPTZ was substituted to calculate the corresponding iron ion concentration. The final T-AOC was determined as follows: T-AOC (µM) = 34 × corresponding to the concentration of iron ions.
The concentration of T-AOC was regarded as the total antioxidant capacity of FPHLP.

Cell Lines and Culture
HepG2 cells were purchased from the Cell Bank of Shanghai Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China). Cells were cultured in Dulbecco's Modi-fied Eagle Medium (DMEM) (#8120364, Gibco) containing 10% fetal bovine serum (FBS) (#2176404, Gibco) and 1% penicillin-streptomycin and maintained in an incubator under 5% CO 2 at 37 • C. The logarithmically growing cells were used for the following experiments.

ROS Detection
HepG2 cells were used to evaluate the effect of FPHLP on ROS production according to ref. [29]. FPHLP was dissolved in DMSO and diluted with DMEM. Briefly, HepG2 cells were incubated with different concentrations of FPHLP (10, 8, and 6 µg/mL) for 24 h and then treated with 400 µM of H 2 O 2 for 4 h to induce oxidative stress. The ROS level was determined using a commercial ROS assay kit (#S0033S, Beyotime Biotechnology, Shanghai, China). The cells were dyed with DCFH-DA solution and the fluorescence was detected via flow cytometry.

Animal Experiment Design
The animal experiment was approved by the Animal Ethics and Welfare Committee (AEWC) of the Zhongshan Hospital of Traditional Chinese Medicine. The experimental procedures and animal treatments were carried out strictly following the principle of Laboratory Animal Care and the guidelines of the AEWC of Zhongshan Hospital of Traditional Chinese Medicine (AEWC-2021026). Eight-week-old C57BL/6 male mice were provided by Zhuhai BesTest Bio-Tech Company Limited (SYXK2020-0109). All mice had access to food and water ad libitum and were kept under a 12 h light/dark cycle. The mice were randomly divided into 5 groups: control group, 0.2% CCl 4 group, 0.2% CCl 4 + silibinin (120 mg/kg) group, 0.2% CCl 4 + FPHLP high dosage group (FPHLP-H, 100 mg/kg), and 0.2% CCl 4 + FPHLP low dosage group (FPHLP-L, 50 mg/kg). The FPHLP samples and silibinin were prepared in 0.5% CMC-Na and the control group was given 0.5% CMC-Na orally from days 8 to 14. Silibinin was given orally from days 8 to 14. FPHLP was given orally from days 8 to 14, twice a day. Mice were injected with 0.2% CCl 4 on day 13 and sacrificed after the FPHLP treatment was terminated. The experimental design is shown in Figure 2A.

Sample Collection
After treatment, mice were anesthetized with pentobarbital to collect blood samples and then sacrificed for dissection [30]. Blood was collected and serum was obtained by centrifugation at 4000 rpm at 4 • C for 10 min. The livers were collected and photographed. Part of the liver tissue was fixed in 4% paraformaldehyde (PFA) solution for hematoxylin and eosin (H&E) staining, and the rest was stored at −80 • C.

Enzyme-Linked Immunosorbent Assay (ELISA) and Biochemical Analyses
Biochemical parameters, including the concentrations of ALT and AST in serum, were determined using commercial kits according to the manufacturer's instructions. Fresh liver tissue (50 mg) was weighed and homogenized with cold phosphate-buffered saline (0.01 M, pH 7.4, w/v, 1: 10). The samples were then centrifuged at 5000× g for 10 min at 4 • C, and the supernatants were collected for biochemical analysis. The levels of LDH, ROS, MDA, SOD, and GSH in liver tissues were evaluated following the manufacturer's instructions.
The transferred membranes were blocked with QuickBlock TM solution (P0252, Beyotime Biotechnology, Shanghai, China) for 15 min at room temperature, washed in PBST (0.1% Tween-20 in PBS), and incubated with primary antibodies of cleaved PARP, Bcl2, Bax, Keap1, HO-1, Trx-1, Nrf2, GPX4, xCT, FTH1, TfR1, and GAPDH overnight at 4 • C. After being washed with PBST 3 times, the membranes were then incubated with secondary antibodies conjugated with horseradish peroxidase (HRP) for 2 h at room temperature. The protein blots were detected using an enhanced chemiluminescence (ECL) kit (KF8003, Affinity Biosciences, Cincinnati, OH, USA). All analyses of the protein blots were performed using Image J software.

Hematoxylin and Eosin (H&E) Staining
Histopathological examination was performed according to the reference with minor modifications [32]. Briefly, liver tissues were fixed in 4% PFA for 24 h, dehydrated by gradient ethanol, paraffin-embedded, sectioned (~4 µm), stained with H&E, and mounted with neutral gum. The morphological changes in tissues were observed under an optical microscope (Nikon Corporation, ECLIPSE Ti2-A, Tokyo, Japan) and photos were taken (magnification, 200×).

Immunohistochemistry (IHC) Study
IHC analysis was performed to examine the protein expression of Nrf2 and cleaved caspase-3 in the liver tissues [33]. Briefly, the paraffin-embedded samples were cut into sections (~4 µm) and sealed with 3% H 2 O 2 at room temperature to inactivate the enzyme, then boiled in 10 mM sodium citrate buffer (pH 6.0) for 10 min and cooled at room temperature. The sections were blocked with normal goat serum, incubated with anti-Nrf2 and anti-cleaved caspase-3 (1:200) overnight at 4 • C, and then with corresponding secondary antibodies for 1 h. The expression of Nrf2 and cleaved caspase-3 in the liver tissues was evaluated under an optical microscope and photos were taken (magnification, 200×).

GC/MS Analysis
To analyze the compounds, FPHLP was dissolved in chloroform for GC/MS analysis. The extraction process consisted of vortexing for 10 min, ultrasound for 30 min at room temperature, and centrifugation at 5000× g at 4 • C for 10 min. The characteristic components of FPHLP were analyzed using an Agilent 7890A-5975C GC/MS. The analytes were separated on a DB-5MS capillary column (60 m × 250 µm × 0.25 µm, HP-5MS, Agilent Technologies) coated with phenyl arylene polymer. The chromatographic temperature conditions used for separation were selected according to the reference, with the instrument parameters of 310 • C for the injection port temperature, 310 • C for the gas chromatography-mass spectrometer interface temperature, and N 2 drying gas at a flow rate of 1.5 mL/min [34]. Conditions for the GC-MS analysis: 70 eV of electron energy of electron impact (EI), 35~550 of the scanning range of mass charge ratio, and 230 • C ion source temperature. MSD ChemStation software was used to process data.

Statistical Analysis
All data were expressed as the mean ± standard error of the mean (SEM). Statistical differences between the two groups were compared by Student's t-test. Differences at p < 0.05 were considered statistically significant.

Discussion
A series of factors in life can lead to liver injury, such as excessive drinking, toxins, and drugs. The CCl 4 -induced liver injury model is a classical model commonly used to study liver function and liver-injury protection [35]. CCl 4 is a potent toxin metabolized by the cytochrome P450 system that can be transformed into free radicals in the body, which disturbs the metabolism of lipids on the liver cell membrane, resulting in the production of high levels of ROS and MDA, leading to inflammation and oxidative stress [36], and eventually inducing apoptosis and necrosis of hepatocytes [37].
The treatment of liver injury mainly includes scavenging free radicals, detoxifying, reducing transaminase levels, and regulating immunity [38]. Chinese herbal medicines have been used to treat liver diseases with little side effects by suppressing inflammation and oxidative stress. For example, Coptis chinensis inflorescence extract exerts a hepatoprotective function by reducing ROS generation induced by CCl 4 in HepG2 cells [39]. FPH was reported to have heat-clearing, detoxifying, and anti-inflammatory effects in the Chinese medicinal literature [31]. Our previous studies reported that the aqueous extract of FPH protected against alcohol-induced acute liver injury and alleviated colitis accompanied by secondary liver injury induced by dextran sulfate sodium (DSS) in mice [24,33]. However, no study has reported the bioactivity of FPHLP. This study showed that the low-polarity components of FPH effectively alleviated the acute liver injury in mice induced by CCl 4 .
ALT and AST leak into the serum when hepatocytes or their cell membranes are destroyed [40]. LDH, an important enzyme in glycolysis, is another key indicator of impaired liver function [41]. Inflammation dysregulation drives the liver pathology associated with acute liver injury [42], during which proinflammatory factors, such as IL-6 and LPS, promote chronic inflammation in the liver [30]. Under the pathological conditions of liver injury, excessive LPS activates innate immune cells, including Kupffer cells, leading to the release of proinflammatory cytokines such as IL-6 and TNFα, thereby reinforcing the inflammatory response [43]. Hence, inhibiting the acute inflammatory response and retarding the evolution of chronic inflammation can prevent hepatic failure from occurring. In this study, the levels of AST, ALT, LDH, IL-6, and LPS were significantly increased in the ALI mouse model induced by CCl 4 , while they were significantly reduced after FPHLP treatment, suggesting that FPHLP showed excellent efficiency against liver injury and could be regarded as a therapeutic candidate for ALI.
The activation of apoptosis is closely connected to mitochondria [44]. The activation of pro-apoptotic genes, such as Bax, results in the enhancement of mitochondrial permeabilization and release of cytochrome c, which induces caspase-3 stimulation and hepatocyte apoptosis [45]. The expression of pro-apoptotic proteins, such as Bax, cleaved caspase-3, and cleaved PARP, would be increased and that of anti-apoptotic proteins, such as Bcl2, would be decreased in impaired liver tissues [46,47], which was verified in the CCl 4 -induced ALI mouse model. In our study, FPHLP significantly reversed these effects, suggesting that FPHLP showed good efficiency in alleviating ALI via suppression of apoptosis.
Oxidative stress has become an important factor in hepatotoxicity, and increasing evidence shows that oxidative stress can promote ALI [46]. Oxidative stress can cause the imbalance of cystine/glutamate antiporter system and decrease cystine intake, thus blocking GSH synthesis [48]. GSH depletion also leads to more extensive hepatotoxicity when ROS excessively accumulates [49]. Excessive MDA covalently modifies proteins, nucleic acids, and other lipids, resulting in destruction of structural integrity or cell death [50,51]. The Keap1/Nrf2/ARE signaling pathway plays a critical role in protecting cells from endogenous and exogenous oxidative stresses [52], among which the transcription factor Nrf2 takes charge of detoxification and antioxidation and regulates GSH metabolism, while Keap1, a highly redox-sensitive member of the BTB-Kelch family, is takes responsibility for Nrf2 degradation when oxidative stress occurs [39]. Nrf2 can evade Keap1-mediated degradation, translocate to the nucleus, and activate a series of ARE-dependent genes, such as the antioxidative and cytoprotective genes HO-1 and SOD [40]. In this study, FPHLP significantly increased the levels of SOD and GSH and enhanced the expression of Nrf2 and HO-1, while inhibiting the expression of Keap1 and reducing the levels of ROS and MDA, indicating that FPHLP could alleviate ALI by suppressing oxidative stress.
Ferroptosis, a kind of non-apoptotic cell death characterized by GSH depletion and iron overloading [53], can be induced by the accumulation of redox-active iron, glutathione depletion, and lipid peroxidation [54]. Ferroptosis is involved in the occurrence and development of a series of liver diseases, including acute or chronic liver injury and liver cancer [55]. GPX4 is the key antioxidant enzyme that quenches phospholipid hydroperoxide by reducing lipid hydroperoxide to nontoxic lipid alcohol directly in the membrane. Once GSH is depleted, the Xc − -glutathione (GSH)-GPX4-dependent antioxidant defense system will be inactivated, leading to the accumulation of lipid hydroperoxides [53,56]. In addition, ferrous iron catalyzes and enhances the occurrence of lipid peroxidation [39]. TfR1, an identified ferroptosis marker, controls cellular iron absorption by carrying transferrinbound iron into cells via receptor-mediated endocytosis, and then Fe 3+ can be reduced to Fe 2+ by STEAP3 [57]. The labile iron released from organelles upon various stresses is incorporated into enzymes, leading to production of excessive ROS that results in lipid peroxidation [40]. FTH1, a major iron storage protein harboring ferroxidase activity, also plays an important antioxidative role in maintaining the balance of the redox system [58], by taking responsibility for the oxidation of Fe 2+ to Fe 3+ and reducing the accumulation of Fe 2+ [59]. As the major sites of iron utilization and master regulators of oxidative metabolism, mitochondria are the main source of ROS, which would induce apoptosis and ferroptosis of cells. Ferroptosis is also reportedly associated with severe damage of mitochondrial morphology, bioenergetics, and metabolism [60]. Our study revealed that FPHLP significantly reduced the expression of TfR1 and accumulation of Fe 2+ and increased the expression of GPX4 and FTH1, suggesting that FPHLP effectively suppressed ferroptosis when treating ALI in mice. Furthermore, ferroptosis can also propagate lipid peroxidation chain reactions, leading to hepatocyte destruction and apoptotic death [61]. Injured hepatocytes not only jeopardize liver function, but they also activate Kupffer cells, resulting in proinflammatory and fibrogenic responses and the vicious cycle of liver damage [62].
Altogether, our study indicated that FPHLP significantly protected the liver from injury in mice with CCl 4 -induced ALI by suppressing oxidative stress, inflammation, ferroptosis, and apoptosis, suggesting that FPHLP may be regarded as a potential hepatoprotective drug. However, the key active ingredient and target of FPHLP remain undefined, which will be investigated in the follow-up study.

Conclusions
In conclusion, this is the first study to explore the suppression of ALI by FPHLP as well as the underlying mechanism. The in vitro data showed that FPHLP efficiently suppressed oxidative stress, and the in vivo study indicated that FPHLP showed excellent inhibition against CCl4-induced ALI in a mouse model. FPHLP improved liver function by reducing ALT and ALT levels in ALI model mice and improved the antioxidant capacity by increasing the levels of antioxidant enzymes, such as SOD and GSH. In addition, FPHLP reduced the accumulation of Fe 2+ by enhancing the iron transport mechanism (Figure 7). Our study provides experimental evidence for the therapeutic function of FPHLP against ALI, suggesting that FPHLP might serve as a novel candidate for the treatment of liver injury. Figure 7. The speculated mechanism of FPHLP against ALI in mice induced by CCl4. Note: ↑ increases; ↓ decreases.

Data Availability Statement:
The data presented in this study are available upon reasonable request from the corresponding author.

Acknowledgments:
The authors appreciate Haibo Huang (Department of Chinese Medicine Identification, Guangzhou University of Chinese Medicine) for identifying the plant material.

Conflicts of Interest:
The authors declare no conflict of interest. Figure 7. The speculated mechanism of FPHLP against ALI in mice induced by CCl 4 . Note: ↑ increases; ↓ decreases.

Data Availability Statement:
The data presented in this study are available upon reasonable request from the corresponding author.