Antioxidant and Antiapoptotic Properties of n-Butanol Fraction the Acanthopanax senticosus Extracts in H2O2-RAW264.7 Cells and CCl4-Induced Liver Injury in Mice

The Acanthopanax senticosus has been shown to have a wide range of pharmacological activities, which are associated with health benefits, such as antioxidant, anti-inflammatory, and antiapoptotic properties. A previous study has shown that the n-butanol fraction of A. senticosus extract had the strongest antioxidant effect in vitro. This study aimed to investigate the effects that the n-butanol fraction of A. senticosus extract could relieve oxidative stress damage through antioxidant and antiapoptotic in the H2O2-stimulated RAW264.7 macrophages and the CCl4-induced liver injury. The result showed that the n-butanol fraction extract could relieve damage by increasing the intracellular antioxidant enzymes (SOD) level, decreasing intracellular ROS and MDA levels, and regulating antioxidant and antiapoptotic-related gene expression levels. The morphological observation of HE, TUNE, and immunohistochemistry staining of liver tissue verified that the n-butanol fraction extract is though anti-oxidative and antiapoptotic to alleviate cellular oxidative damage. The RT-PCR assay showed that the Keap1-Nrf2-ARE and the Bax/Bcl-2 signaling pathway were related to the molecular mechanism of action. The experimental results show that Acanthopanax senticosus extract has a good effect in treating liver injury and enhancing the antioxidant capacity of the body.


Introduction
Oxidative stress can produce a large number of reactive oxygen species (ROS) in the cell, and their prevalence occurs due to unbalanced oxygen homeostasis, leading to the production of intracellular oxidative stress [1], which can cause diferent degrees of oxidative damage and even apoptosis in the body and accelerate the process of cell disease [2,3]. Studies have proved that the antioxidant capacity is inversely proportional to the ROS in vivo and that the strength of the antioxidant enzyme activity is closely related to the degree of oxidative stress. Te decreasing antioxidant activity may directly lead to apoptotic [4].
Macrophages are important immunity cells for the host defense system to eliminate microbial pathogens. Studies have verifed that macrophages, which can produce ROS as infected by some bacteria, are a suitable model for antioxidant research [5]. It was found that excess ROS are readily converted to H 2 O 2 in cells and difuse through cell membranes, undergoing a heme-catalyzed Fenton reaction to produce highly reactive and toxic hydroxyl radicals, which eventually trigger macrophage death. In contrast, the mouse macrophage cell line RAW264.7 is the most commonly used mouse macrophage cell line in medical research, so this study used RAW264.7 cells to establish an oxidative stress model. Te liver is the main metabolic organ that converts glucose, fatty acids, and amino acids from the blood into the essential metabolic fuel for extrahepatic tissues during exercise and starvation [6], and the liver also is a detoxifying organ; thus, the liver is prone to oxidative stress. CCl 4 , a compound, can cause chemical liver damage, including infammation, which is commonly used to induce acute and chronic liver injury in mice models [7].
Acanthopanax senticosus (Rupr. & Maxim.) Harms (AS), as traditional Chinese medicine, belongs to the genus Acanthopanax (Araliaceae), widely used in China, Korea, Japan, and Russia [8]. According to traditional Chinese medicine theory, AS can stimulate qi, strengthen the spleen, and nourish the kidneys [9]. Te medicine chemistry studies showed that AS contains many pharmacological chemical compounds, including polysaccharides, saponins, polyphenols, and favonoids [10]. Modern pharmacological experiments have demonstrated that A. senticosus possessed antistress, antioxidant, antiapoptotic, and hepatoprotective activities [11]. Previous works showed that the n-butanol fraction of A. senticosus extracts (NASE) has higher antioxidant activity in vitro [12]. As reported in the literature, nbutanol fractions of AS signifcantly prolong the swimming time of mice to exhaustion [13]. As a follow-up, this work aimed to explore the protective mechanism of NASE in the H 2 O 2 -induced RAW264.7 injury and the CCl 4 -induced liver injury in mice. Tis study provided theoretical support for NASE use in clinical settings.

Preparation of Extract of NASE.
Te NASE was prepared according to document 12. Briefy, the AS was extracted by a complex enzyme-assisted ultrasound method. Te extraction conditions are enzyme ratio (cellulase: pectinase (3 : 2)), the amount of complex enzyme added is 6,960 U·g −1 , hydrolysis time is 59.80 min, with a temperature of 53.70°C, and a pH value of 6.05. Te active components in the extract were enriched and purifed with diferent polar solvents. In the n-butanol fraction, the contents of total favonoids and polyphenols were 61.0 ± 0.344 mg·g −1 and 24.93 ± 0.234 mg·g −1 , being used for the following experiments. penicillin-streptomycin and maintained at 37°C in a 5% CO 2 humidifed incubator, and the media was refreshed once the other day and the cell was passaged once from two days to three days. When RAW264.7 cells were in a logarithmic growth phase reaching approx. 80% confuence and the cell was digested with pancreatic enzyme and washed twice with PBS (0.05 M, pH 7.2), the cells were prepared with a singlecell suspension of 1.8 × 10 5 cell/mL and seeded in 96-well tissue culture plates at 200 μL per well.

Cytotoxicity Assay of NASE.
Te cells were divided into a normal group and diferent treatment groups. Te treatment groups were treated with diferent concentrations of NASE (25,50,100,150,200, and 250 μg·mL −1 , which was diluted by 1640 medium). When the cells reached 80% confuence, the supernatant was removed and washed with PBS once, added 100 μL of serum-free medium and 10 μL of CCK-8 solution per well, continuing to maintain in the incubator for 2 h, the absorbance was determined at OD 450 nm using an ELISA reader and cell viability was calculated [14].

Hydrogen Peroxide (H 2 O 2 ) Toxicity Test.
Te H 2 O 2 toxicity assay on RAW264.7 cells was also evaluated by the CCK-8 assay. When the cells were reached the logarithmic growth phase, and the cell density was adjusted to 5 × 10 4 cells·mL −1 , seeded the cells in 96-well plates, continuing to culture for 24 h. Ten, the supernatant was discarded, by additional media with the fnal concentration of 100, 200, 300, 400, 500, 600, 700, 800, and 900 μmol·L −1 hydrogen peroxide in a 96-well plate, and allowed to interact for 2 hours. Te absorbance was determined at OD 450 nm according to the abovementioned method, and the cell survival rate was calculated.

Cytoprotective Experiments.
Te cells were divided into a normal group, an H 2 O 2 group, and three experimental groups, including the NASE low-dose group (25 μg·mL −1 ), NASE medium-dose group (50 μg·mL −1 ), and the NASE high-dose group (100 μg·mL −1 ). After being added NASE for 24 h, the cell was added medium with H 2 O 2 of a fnal concentration of 300 μmol·L −1 , continued to interact for 2 h, determined the absorbance at OD 450 nm according to the abovementioned method, and calculated the cell survival rate [15].

2.4.
Detection of Intracellular ROS. RAW264.7 intracellular ROS were monitored by a ROS assay kit with a fuorescent probe (DCFH-DA). RAW264.7 cells were inoculated in a 6well plate and treated as mentioned above. Te cells were washed three times using PBS (0.05 M, pH 7.2). Ten, the cells were stained with 1 : 1000 diluted DCFH-DA and incubated for 30 min at 37°C according to the manufacturer's instructions. DCF fuorescence was detected by an inverted fuorescence microscope with an excitation wavelength of 485 nm and an emission wavelength of 530 nm in the 2 Evidence-Based Complementary and Alternative Medicine laboratory. Te fuorescence intensity of each group was analyzed using the Image J software.

Giemsa
Staining. RAW264.7 cells were inoculated in a 6-well plate and treated as mentioned above, and the Giemsa staining method was carried out according to the manufacturer's instructions. Te apoptotic cells and apoptotic vesicles were dark purple-blue and were easily recognized under a light microscope. Tree felds of view were selected for each picture, and the number of positive cells was calculated in each feld among 100 cells, and the percentage of the number of positive cells was calculated as the positive index of apoptotic cells.
2.6. Flow Cytometry Assay. RAW264.7 cells were inoculated in a 6-well tissue culture plate and treated as mentioned above, apoptosis was detected according to the Annexin V-FITC/PI kit. Adding 10 μL of the Annexin V to each group of cells, incubated for 10 min, adding another 5 μL of PI, and after 5 min of incubation, fow cytometry was used to detect the apoptosis rate of each group. Te mice were randomized into fve groups: (normal group, model group, NASE low-dose group (20 mg·mL −1 ), NASE medium-dose group (40 mg·mL −1 ), and the NASE high-dose group (80 mg·mL −1 ).
Te model group and three NASE-treated groups were injected intraperitoneally into a soybean oil solution containing 20% CCl 4 at a dose of 1 mL·kg −1 three times a week for 2 weeks. Te normal group was injected intraperitoneally with soybean oil three times a week for two weeks. For the NASE-treated groups, NASE was given to mice once a day by Gavage with diferent dosages. For the normal and model groups, mice were given double distilled water to minimize the efects of the gavages' procedure [16]. Te mice were weighed daily. Two weeks after drug administration, the mice were randomly selected for dissection.
On day 14, the mice were killed by cervical dislocation. Te liver of the mouse was weighted and pathological changes were observed. Apart of the liver tissue was fxed in a 4% paraformaldehyde solution, and the remaining liver tissue was frozen in a −80°C refrigerator. Te liver index (liver weight/body weight) was calculated.

HE Staining, Immunofuorescence Staining, and TUNEL
Assay. Te liver parafn tissue sections were routinely made. Histological changes were examined by hematoxylin and eosin [H & E] staining. Te protein expression of Bcl-2 and Bax in liver tissue was detected, respectively, with an immunohistochemical assay. Te immunohistochemical streptavidin biotin complex (SABC) method was used under the instructions of the SABC kit. Te TUNEL staining was operated according to the colorimetric TUNEL apoptosis Assay. Te images were observed by light microscopy and an upright Olympus fuorescence microscope. Te staining intensity of immunohistochemistry was evaluated using the Image J software.

Te Content of ALT, AST, ALP, and LDH in Serum.
After the animal experiment, the mice were sacrifced, whole blood was obtained and placed at room temperature for 6 h and the blood was centrifuged at 5000 r/min for 5 min. Te serum was separated. Te contents of ALT, AST, ALP, and LDH in the serum were determined using the corresponding kit.

Te Content of SOD and MDA.
Te RAW264.7 cells and liver tissue were ground with PBS (0.05 M, pH 7.2), and the supernatant was isolated by centrifuging at 4000 × g for 10 min at 4°C, then the concentration of protein was determined by a BCA protein detection kit. Te MDA and SOD contents were determined using a kit based on the manufacturer's instructions.

Quantitative Real-Time Polymerase Chain Reaction.
Total RNA from RAW264.7 cells and liver tissues was extracted with TRIzol reagents (Invitrogen) according to the manufacturer's instructions. Te RNA concentration was determined by Nanodrop software. Reverse transcription was conducted using the TaKaRa kit. Te real-time PCR was carried out with a BeyoFast TMSYBR Green qPCR Mix kit, and the reaction parameters were as follows: 95°C 2 min, 95°C 15 s, and 56-60°C 30 s for a total of 30 cycles. RNA samples were conducted in triplicate and data were normalized to the GAPDH level for mRNA. Te gene expression levels for mRNA were quantifed by the delta-delta comparative cycle threshold (2 −ΔΔCt ) method. Te primer sequences are shown in Table 1.

Statistical Analysis.
Te SPSS 22 software was used to analyze the data. A one-way analysis of variance (ANOVA) test was applied to compare mean values, and p < 0.05 was considered statistically signifcant. GraphPad Prism 8 was used to plot. All data were expressed as mean ± standard deviation.

Cytotoxicity and Cytoprotective Experiments.
Te RAW264.7 cells were treated with NASE at various concentrations in a 96-well plate. After interacting for 24 h, the ratio of cell survival was determined by the CCK-8 assay. Te cytotoxicity of H 2 O 2 on RAW264.7 cells was also evaluated by the CCK-8 assay kit. As shown in Figure 1(b), when its concentration reached 900 μM, the survival rate of RAW264.7 cells was only 10.26 ± 1.23%, indicating that H 2 O 2 stimulated RAW264.7 cells leading to oxidative damage and death. Te IC 50 of H 2 O 2 on RAW264.7 cells was 290.7 μM. To improve the operability of the experiment, 300 μM was used as the subsequent treatment concentration.
Te cytoprotection efect of NASE on H 2 O 2 -induced RAW264.7 cells was evaluated by the CCK-8 assay. According to Figure 1(c), the cell survival rate was signifcantly higher in the NASE group (25, 50, and 100 g·mL −1 ) than in the model group (p < 0.01). It appears that NASE reduces the oxidative stress damage caused by H 2 O 2 and decreases cell mortality as a result. Tus, NASE could protect RAW264.7 cells against H 2 O 2 -induced oxidative stress damage.

Giemsa
Staining. Te picture of Giemsa staining RAW264.7 cells was observed under a light microscope. As shown in Figure 2(a), the cell morphology of the normal group was bright and long spindle-shaped, with and uniform size of nuclei (Figure 2(a)). In the model group, the cells were shrunken and round after hydrogen peroxide injury, the nuclei were shrunken and the cell shape was not uniform (Figure 2(b)). In the NASE treatment groups, the cell morphology was signifcantly improved (Figures 2(c)-2(e)). Apoptotic cells can also be see in the picture, and the number of apoptotic cells decreased through NASE treatment in the NASE groups more than that in the model group. Te number of apoptotic cells in the model group was signifcantly higher than that in the normal group by the analysis of apoptotic cell counts (p < 0.05), indicating that H 2 O 2 caused apoptosis in RAW264.7. Compared with the model group, the NASE drug-treated group was able to decrease the apoptosis rate, especially in the high-dose group, and the diference was signifcant in the apoptosis rate compared with the model group (p < 0.05) (Figure 2(b)). No dosedependent efects of NASE were observed in the Giemsa staining experiments, but diferent concentrations of NASE were found to have good efects.

Determination of Intracellular ROS Level.
ROS is inseparable from the generation of cellular oxidative stress. After H 2 O 2 stimulated RAW264.7 cells for 2 h, the DCFH-DA probe was added to 96-well plates and incubated for 30 minutes. Ten, the probe could enter the cells and react with ROS, and the green fuorescence could be observed under the blue channel of the fuorescence microscope. As shown in Figure 3(a), when RAW264.7 cells were induced with H 2 O 2 for 2 h, the green fuorescence was signifcantly increased in the model group, and the green fuorescence in the NASE-treated groups showed a decreasing trend compared with the model group. Te results of the quantitative analysis showed that the NASE-treated group reduced the production of ROS compared with the model group, and the low-dose group reached a signifcant diference compared with the model group (p < 0.05) (Figure 3(b)). Te diference in fuorescence density between the middle-dose and high-dose groups was not signifcant compared with the model group (p > 0.05). During the experiment of taking photos, we found there is a diference in the density of cells in each feld of view, which may lead to a diference in the density of fuorescence. Although we have taken the method of averaging multiple felds of view, the results may still be afected. It may be due to the error of the experimental method, the efects of the medium and highdose groups were lower than those of the low-dose group compared with the model group. When the ROS content of the environment of the cells is too high, it will lead to an imbalance in the original oxidative state of the cells, resulting in apoptosis. NASE-treated RAW264.7 cells can signifcantly improve the ability of the cell to scavenge ROS, thus

Flow Cytometry to Detect the Efect of NASE on Apoptosis.
To investigate how NASE inhibits the apoptosis of RAW264.7 cells, a fow cytometry assay was used to examine the cellular apoptosis status. In the experiment, H 2 O 2 stimulated RAW264.7 cells for 2 h to induce oxidative stress and apoptosis, to evaluate the apoptosis of NASE-treated cells under oxidative stress. As shown in Figure 4, compared with the normal group (apoptosis rate of 0.2% ± 0.84%), the apoptosis rate of the model group (17.02% ± 0.41%) was signifcantly increased (p < 0.01). After a period of NASE pretreatment, the apoptosis rate of cells decreased, respectively. Tere was a signifcant decrease in the apoptosis rate among cells treated with high doses of NASE with the model group (p < 0.01).

Te Body Weight and Liver
Index. Carbon tetrachloride (CCl 4 ) can induce chemical liver injury, which is used to test the therapeutic efect of drugs in experimental animals. Intraperitoneal injection of CCl4 was able to cause liver infammation, resulting in reduced food intake and weight loss in mice. Te mice showed liver enlargement and increased liver mass, which in turn manifested as a larger liver index. Te changes in the body weight of mice are shown in Figure 5. On days 1-11, the body weight of mice increased gradually in all groups. Te body weight of the normal group had the greatest weight gain compared to the other groups. Te greatest fuctuations in weight gain were observed in the model group, which may be related to CCl 4 administration. Te weight gain in the NASE-treated groups was lower than that in the normal group. Te weight loss on day 14 was related to the 12 h of fasting before the end of the experiment. Tere was no statistically signifcant diference in body weight between the model and NASE-treated groups (p > 0.05). Tis fnding indicated that the NASE within the measured dosage range had no obvious efect on the body weight of mice. Changes in the liver indexes of mice are listed in Table 2, the liver index of the model group was signifcantly higher than that of the normal group (p < 0.01), indicating that CCl 4 caused liver swelling in model mice.
Te liver index of the NASE-treated group was signifcantly lower than that of the model group (p < 0.01, p < 0.05), indicating that NASE decreased liver swelling. Tis result revealed that the liver injury induced by CCl 4 in mice was successfully modeled.  Evidence-Based Complementary and Alternative Medicine 3.6. HE Staining. Te basic principle of hematoxylin eosin staining (HE staining) is that hematoxylin is a basic stain that stains the nucleus blue and eosin is an acidic stain that stains the cytoplasm purplish red, thereby confrming the basic cell structure. In this study, HE staining was used to analyze the pathological sections of the liver of mice with liver injury. Te HE staining was observed and analyzed under a light microscope and it was shown that the liver cells of mice in the normal group were structurally complete, neatly arranged, and had no infammatory infltration. Te liver cells of mice in the model group were arranged in disorder, the cells were enlarged or deformed, and there were infammatory infltration and necrosis. Te hepatic cells of the mice in the NASE groups were relatively intact and arranged more neatly than those in the model group.
However, there was still some infltration of infammatory cells. Te overall condition and morphology of the cells improved with the increase in the NASE dosage, as shown in Figure 6.

TUNEL Staining Assay.
During apoptosis, chromosomal DNA breaks to form apoptotic vesicles, and TUNEL staining is a combined molecular biology and morphology research method that can stain in situ in intact individual apoptotic nuclei or apoptotic vesicles, which can accurately refect the typical biochemical and morphological characteristics of apoptosis. Terefore, TUNEL staining was performed on the liver tissues of mice in each group (Figure 7(a)) to observe the formation of apoptotic bodies  Evidence-Based Complementary and Alternative Medicine (tan granules) in liver cells. Te results showed that almost no apoptotic bodies were observed in the normal group. Compared with the normal group, the apoptotic bodies in the model group were signifcantly increased, while the apoptotic bodies in the liver cells of mice in diferent doses of NASE groups were signifcantly reduced (Figure 7(b)). Tis indicated that the apoptosis of liver cells induced by CCl 4 could be alleviated to a certain extent under the action of NASE.

Expression of Bcl-2, Bax Proteins in Liver
Tissue. Liver injury causing hepatic fbrosis is a pathological process in which persistent liver injury leads to increased synthesis of extracellular matrix and an imbalance in its degradation, resulting in the development of hepatic fbrosis. Troughout this process, the focus is on the activation of hepatic stellate cells (HSC). Tere is evidence that there is a class of apoptotic mediators in HSC: Bcl-2 and Bax, with Bax being the main regulator of Bcl-2 activity. Overexpression of Bax in hepatocytes can lead to disruption of mitochondrial integrity. If this action is prevented at the early stage of liver injury, it can play a role in protecting hepatocytes against liver injury. Terefore, in this study, the apoptosis of hepatocytes was regulated by modulating the Bcl-2/Bax pathway, as shown in Figure 8. By measuring the relative levels of the pair of apoptotic genes, Bcl-2 and Bax, it was found that diferent doses of NASE could reduce the expression of Bax during apoptosis, which could increase the

Te Content of ALT, AST, ALP, And LDH in Serum.
When hepatocytes are injured, their cell membrane permeability increases and the cytoplasm releases alanine  Evidence-Based Complementary and Alternative Medicine aminotransferase (ALT) and aspartate aminotransferase (AST), and the activity of both in serum will be signifcantly increased, which is an important index to evaluate the degree of liver injury [19]. Te production of cytotoxicity is another manifestation of hepatocyte injury, and lactate dehydrogenase (LDH) is a widely expressed enzyme whose release has been used as a marker of membrane damage. Alkaline phosphatase (ALP) is also widely used as a biochemical indicator of hepatocyte damage [20]. Te activity levels of ALT, AST, ALP, and LDH refect the degree of damage to liver cells. Te efects of NASE on serum ALT and AST activities of liver-injured mice are shown in Figures 9(a) and 9(b). Compared with the normal group, the serum ALT and AST activities of mice in the model group were signifcantly higher (p < 0.01), indicating that a model of CCl 4induced liver tissue injury had been established. Te efects of NASE on the serum ALP and LDH activities of mice with liver injury are shown in Figures 9(c) and 9(d). Te serum ALP and LDH activities of mice in the model group showed an increase compared with the normal group. Compared with the model group, the serum ALP and LDH activities of mice in diferent dose groups were signifcantly reduced (p < 0.01 or p < 0.05). Te results indicated that NASE treatment can alleviate hepatocyte damage caused by oxidative stress.

Te Contents of Intracellular SOD and MDA.
Superoxide dismutase (SOD) and malondialdehyde (MDA), as important regulators of oxidative stress, account for a large proportion of the evaluation of the body's antioxidants. SOD is the main enzyme and antioxidant for scavenging oxygen radicals in the body, and its activity and content can indirectly refect the ability of the body to scavenge free radicals and whether the oxidative status of the body is balanced. Te content of MDA can refect the degree of tissue lipid peroxidation in the body, thus indirectly refecting the degree of oxidative damage in tissue cells. As shown in Figures 10(a) and 10(b), after the cells were induced by H 2 O 2 , the MDA content in the model group increased signifcantly compared with the normal group, and all dose groups of NASE reduced the production of MDA under oxidative stress, and the diference was highly signifcant compared with the model group (p < 0.01). Meanwhile, the antioxidant enzyme activity of SOD in RAW264.7 cells in the model group was reduced compared with the normal group, and the diference was highly signifcant compared with the NASE dose groups (p < 0.01). As shown in Figures 10(c) and 10(d), the content of SOD activity in the liver tissues of mice in the model group was signifcantly reduced and the content of MDA increased compared with the normal group (p < 0.01). Compared with the model group, the SOD activity content in the liver tissue of mice in each dose group of NASE increased signifcantly and the MDA content decreased, with the most signifcant increase in the SOD activity content (56.4 U/mL −1 ) and the most signifcant decrease in the MDA content (6.3 nmol·mL −1 ) of mice in the high dose group of NASE (p < 0.01).

Quantitative Real-Time Polymerase Chain Reaction.
Te Nrf2/HO-1 pathway is an antioxidant signaling pathway that the body protects normal cells against oxidative stress, as well as exogenous damage. When hepatocytes are stimulated by CCl 4 , Nrf2 phosphorylates into the nucleus and binds to the DNA sequence of ARE, an antioxidant element in the nucleus, initiating the expression of ARE-regulated downstream phase II detoxifcation enzymes and antioxidant enzyme genes, thereby resisting oxidative stress-induced damage. Terefore, in this experiment, we analyzed the transcript levels of Nrf-2, HO-1, Keap-1, NQO-1, Bax, and Bcl-2 mRNA in RAW264.7 cells and liver tissues by RT-PCR to investigate the efect of NASE on the Nrf2/HO-1 pathway, and the results are shown in Figure 11. Accordingly, it was observed that Nrf-2, Keap-1, and NQO-1 expressions were downregulated, and HO-1 expressions were upregulated in RAW264.7 cells of NASE groups compared to the model group (p < 0.05). Keap-1 and NQO-1 expressions were downregulated, and Nrf-2 and HO-1 expressions were upregulated in the liver tissue of NASE groups compared to the model group (p < 0.05). Interestingly, the expression levels of Nrf-2 show the opposite trend in RAW264.7 and liver tissue. Bcl-2 and Bax's genes are apoptosis-related genes discovered in recent years, and they are closely related to the occurrence, development, and prognosis of cells. Te expression results of Bcl-2 and Bax's genes in each group are shown in Figure 10.   Compared with the normal group, the Bax gene expression level in the model group was signifcantly upregulated, and the Bcl-2 gene expression level in the model group was signifcantly downregulated in RAW264.7 cells and mice liver tissue (p < 0.05 ); compared with the model group, the Bax mRNA expression levels in the NASE groups were signifcantly downregulated, and the Bcl-2 gene expression level in the model group was signifcantly upregulated in RAW264.7 cells and mice liver tissue (p < 0.05). Te ratio between the Bcl-2/Bax proteins is a key factor in regulating cell apoptosis, and the increase in the Bcl-2/ Bax ratio can inhibit the occurrence of apoptosis. After NASE treatment, the Bcl-2/Bax ratios all were improved in RAW264.7 cell and liver tissue. Te inhibitory efect of NASE on the cell oxidative stress and apoptosis may be associated with the activation of the Nrf2/Keap1//HO-1 and Bax/Bcl-2 signaling pathways.

Discussion
When oxidative stress occurs in the body, the balance of cellular oxygen utilization is disrupted, resulting in the accumulation of ROS in vivo [21]. Excessive intracellular ROS will cause cellular damage, loss of function, and ultimately infammation and even apoptosis. Te liver is an important metabolic organ in the body and plays a role in various pathways such as macronutrient metabolism, immune response, and the decomposition of biological compounds [22]. CCl 4 is commonly used to establish an experimental model of acute liver injury [23] and CCl 4 is metabolized by the liver to generate the free radical trichloromethyl [24]. Te free radicals generated after CCl 4 are metabolized by the liver, as a result of the free radical chain reaction, lipid peroxidation of the cell membrane is directly induced, and the cell membrane is damaged, resulting in oxidative stress, degeneration, liver cell damage, and necrosis. Numerous experiments have demonstrated that AS has strong antioxidant and anti-infammatory activities in vivo [25][26][27][28]. Te NASE is the active ingredient from the rhizome of the A. senticosus. In this experiment, NASE can promote the proliferation of RAW264.7 cells, reduce intracellular ROS level and MDA content, and increase SOD activity. In CCl 4 -induced liver injury, the contents of ALT, AST, ALP, and LDH are an evaluation indicator of liver function. Te NASE can increase their levels in serum, which are agreeing with previous reports [29][30][31][32]. Te histopathological fndings confrm the experimental results. After NASE treatment, a large number of infammatory cells infltrated the lobular structure, and bleeding was signifcantly reduced. Tere has been previous research reporting that AS can alleviate liver injuries in many animal models, and the present results confrm that [33][34][35].
In the cells, Nrf-2 regulates the expression of genes relating to cell defense against oxidative stress [36]. In physiological conditions, Nrf-2 binds to Keap1 to remain in the cytoplasm until it is degraded by the proteasome to maintain its low levels of protein [37]. Te Nrf-2 protein is released from Keap1 upon oxidative stress and translocated into the nucleus. By binding to antioxidative response elements (AREs), Nrf-2 hetero-dimers induce the transcription of related antioxidant enzymes, such as SOD, HO-1, and NQO-1 [38]. Apoptosis of cells is   regulated by Bcl-2 and Bax, two signaling pathways within mitochondria [39]. Normally, Bcl-2 inhibits apoptosis, while Bax promotes apoptosis. When Bcl-2 is defcient, apoptosis may occur. In this work, the NASE can upregulate the gene expression of Bcl-2 and HO-1 (p < 0.05), and downregulate the gene expression of Bax, Keap-1, and NQO-1 (p < 0.05). Tus, NASE can inhibit the oxidative stress of cells through the Nrf2-related pathway Keap1-Nrf2-ARE, and reduce the apoptotic activity of cells through the Bax/Bcl-2 signal pathway.

Conclusions
In a word, current results verifed that NASE protected RAW264.7 cells from H 2 O 2 -induced oxidative stress and prominently ameliorated CCl 4 -induced liver damage. Terefore, NASE has the potential to be a therapeutic medicine for preventing and treating cell damage and liver damage caused by oxidative stress.

Data Availability
Te data sets used and/or analyzed during the present study are available from the corresponding author on reasonable request. Evidence-Based Complementary and Alternative Medicine 15