Five Constituents in Psoralea corylifolia L. Attenuate Palmitic Acid-Induced Hepatocyte Injury via Inhibiting the Protein Kinase C-α/Nicotinamide-Adenine Dinucleotide Phosphate Oxidase Pathway

Psoralea corylifolia L. (PC) is a traditional Chinese herb used to treat yang deficiency of the spleen and kidney in pediatric disease. Our previous studies have found that PC can alleviate the liver oxidative stress of juvenile mice with nonalcoholic steatohepatitis (NASH), and its mechanism is related to the inhibition of the protein kinase C-α (PKC-α)/nicotinamide-adenine dinucleotide phosphate oxidase (NOX) signaling pathway. The aim of this study was to confirm the aforementioned drug target in vitro and to conduct preliminary screening for some effective compounds of PC on the treatment of NASH. A primary hepatocyte model of non alcoholic fatty liver disease was established by palmitic acid. The existence of Psoralen, Isopsoralen, Neobavaisoflavone, Isobavachalcone, and Bakuchiol were identified by ultra-performance liquid chromatography. Then, five PC compounds were administered. Intracellular triglyceride and total cholesterol content, the cell supernatant alanine aminotransferase and aspartate aminotransferase, and hepatocellular superoxide anion were examined. The changes of PKC-α/NOX signaling pathways in hepatocytes were also determined. Furthermore, PKC-α activator phorbol 12-myristate 13-acetate was administered for 4 h before Psoralen intervention was conducted again to detect the changes of PKC-α/NOX signaling pathways. Our data demonstrated that Psoralen, Isopsoralen, and Isobavachalcone decreased intracellular content of triglyceride while all five PC compounds improved hepatocellular total cholesterol accumulation and hepatocyte damage in palmitic acid-induced primary hepatocyte model of non alcoholic fatty liver disease. All five PC compounds could also reduce hepatocytic superoxide anion levels, nicotinamide-adenine dinucleotide phosphate/reduced nicotinamide-adenine dinucleotide phosphate ratio, NOX activity as well as p47phox protein expression and PKCα activation in hepatocytes. Psoralen exhibited the best efficacy but the effectiveness was lost when pre-stimulated by phorbol 12-myristate 13-acetate. The results suggest that Psoralen, Isopsoralen, and Isobavachalcone could improve hepatocyte steatosis; five PC compounds could ameliorate hepatocyte injury, relieve oxidative stress, and downregulate the PKC-α/NOX signaling pathway of hepatocytes. In addition, Psoralen exhibits the best efficacy and a prospective PKC-α inhibitor pharmaceutical activity.

Psoralea corylifolia L. (PC) is a traditional Chinese herb used to treat yang deficiency of the spleen and kidney in pediatric disease. Our previous studies have found that PC can alleviate the liver oxidative stress of juvenile mice with nonalcoholic steatohepatitis (NASH), and its mechanism is related to the inhibition of the protein kinase C-a (PKC-a)/ nicotinamide-adenine dinucleotide phosphate oxidase (NOX) signaling pathway. The aim of this study was to confirm the aforementioned drug target in vitro and to conduct preliminary screening for some effective compounds of PC on the treatment of NASH. A primary hepatocyte model of non alcoholic fatty liver disease was established by palmitic acid. The existence of Psoralen, Isopsoralen, Neobavaisoflavone, Isobavachalcone, and Bakuchiol were identified by ultra-performance liquid chromatography. Then, five PC compounds were administered. Intracellular triglyceride and total cholesterol content, the cell supernatant alanine aminotransferase and aspartate aminotransferase, and hepatocellular superoxide anion were examined. The changes of PKC-a/NOX signaling pathways in hepatocytes were also determined. Furthermore, PKC-a activator phorbol 12-myristate 13-acetate was administered for 4 h before Psoralen intervention was conducted again to detect the changes of PKC-a/NOX signaling pathways. Our data demonstrated that Psoralen, Isopsoralen, and Isobavachalcone decreased intracellular content of triglyceride while all five PC compounds improved hepatocellular total cholesterol accumulation and hepatocyte damage in palmitic acid-induced primary hepatocyte model of non alcoholic fatty liver disease. All five PC compounds could also INTRODUCTION Nonalcoholic fatty liver disease (NAFLD) is the most prominent cause of chronic liver disease worldwide (Younossi et al., 2019). Because 25% of the world's population is currently thought to have NAFLD, this epidemic has become a severe public health problem (Younossi et al., 2016;Doycheva et al., 2017). NAFLD may result in adverse outcomes of liver diseases (liver cirrhosis, hepatocellular carcinoma) and cardiovascular diseases. Nonalcoholic steatohepatitis (NASH), a severe form of NAFLD, induces a significant increase in all-cause mortality for NAFLD patients (Wu et al., 2016; National Workshop on Fatty Liver and Alcoholic Liver Disease, Chinese Society of Hepatology, Chinese Medical Association. Fatty Liver Expert Committee, Chinese Medical Doctor Association, 2018).
Either a two-hit theory or a multiple hit-process, oxidative stress is one of the key driving forces in the initiation and progression of NAFLD from simple steatosis to NASH (Spahis et al., 2017;Alhasson et al., 2018;Uchida et al., 2018). Oxidative stress refers to a shift in the balance toward increased intracellular reactive oxygen species (ROS) generation, compared to breakdown (Mann et al., 2017). Nicotinamideadenine dinucleotide phosphate oxidase (NOX) is considered the major cellular ROS source (Loffredo et al., 2016;Long et al., 2017;Rabelo et al., 2018). Its activation has been associated with hepatic injury (Matsumoto et al., 2018;Zheng et al., 2018) and also plays a very plausible role as the starting point of extrahepatic damage, leading to inflammation and fibrosis through activation of Kupffer cells and hepatic stellate cells (HSCs) (Das et al., 2015;, thus causing a self-perpetuating circle of ROS formation and ROS-mediated damage (Masarone et al., 2018;Zhong and Liu, 2018). Therefore, NOX-specific antioxidant therapies may be a promising intervention to prevent or to treat NASH.
Psoralea corylifolia L. (PC), a traditional Chinese herb, a member of Leguminosae plant family, is widely used to treat yang deficiency of the spleen and kidney in both adult and pediatric disease in China. PC plays a beneficial role in multiple diseases, especially cancer and osteoporosis (Xin et al., 2019a). PC also has a wide range of antioxidant activities (Alam et al., 2018). Our previous studies have found that PC can alleviate the liver oxidative stress of juvenile mice with NASH, and its mechanism is related to the inhibition of the PKC-a/NOX signaling pathway (Zhou et al., 2017). Therefore, the aim of this study was to confirm the aforementioned drug target via further cell study and to conduct preliminary screening for some effective compounds of PC on the treatment of NASH.

Ultra-Performance Liquid Chromatography
Ultra-performance liquid chromatography (UPLC) was performed using a Waters Acquity Ultra Performance LC system (Milford, MA, USA) equipped with a quaternary solvent delivery pump, an autosampler, a thermostated column compartment, a photodiode array detector, and an Empower 3 workstation. The analytes were separated on a Waters Acquity HSST3 column (100 × 2.1 mm, 1.8 um particle). Chromatographic separation was achieved through a 15-min gradient delivery of a mixture of A (acetontrile) and B (1% phosphoric acid aqueous) at a flow rate of 0.3 ml/min and a column temperature of 35°C, using a photodiode array detector (310 nm + full scan).

Primary Hepatocyte Extraction and Culture
Female C57BL6J mice, aged 24 to 32 days, were chosen for the extraction of hepatocytes. This study was approved by the animal ethics committee of Wuhan Medical & Health Center for Women and Children, Huazhong University of Science and Technology (no. 2014010). A two-step collagenase digestion method was used and cultivated following the steps published by our research group (Xiong et al., 2019). Extracted hepatocytes were cultured in Dulbecco's modified Eagle medium/F-12 medium supplemented with 10% fetal bovine serum, 100 units/ ml penicillin, and 100 mg/ml streptomycin in 5% CO 2 /95% air at 37°C. Immunofluorescence was used to detect CK-18 protein in cells to identify whether the extracted cells were mouse primary hepatocytes (Banaudha et al., 2010).

Primary Hepatocyte Model of Nonalcoholic Fatty Liver Disease Establishment
Hepatocytes were seeded into 96-well plates and cultured in an incubator overnight. Then, the medium was replaced by medium containing PA at the concentration gradient (0, 0.01, 0.02, 0.05, 0.08, 0.1 mmol/l) for 24 h. The viability of hepatocytes in wells containing different concentrations of PA was assessed by CCK-8 assay. Next, hepatocytes were seeded into 6-well plates and cultured in an incubator overnight. Then, the medium was replaced by medium containing PA at the same concentration gradient for 24 h. The cells were collected, and intracellular content of TG was determined using commercial reagent (GPO-PAP method). The screening concentration of PA for model establishment was 0.02 mmol/l (minimal cytotoxicity with significantly increased intracellular TG content).

Cytotoxic Effect of Psoralea corylifolia L. Chemical Compounds
Hepatocytes were seeded into 96-well plates and cultured in an incubator overnight. Then, the medium was replaced by medium containing 0.02 mmol/l of PA with 1 of each PC chemical compounds (Psoralen, Isopsoralen, Neobavaisoflavone, Isobavachalcone or Bakuchiol) for 24 h. The concentration gradient of all the PC chemical compounds was in turn: 0, 0.2, 2, 20, 50, and 100 mg/ml. The cytotoxic effects of Psoralen, Isopsoralen, Neobavaisoflavone, Isobavachalcone, and Bakuchiol were evaluated by the CCK-8 assay. The screening concentration of all five PC chemical compounds was 0.2 mg/ml (less cytotoxicity with maximum concentration). Primary hepatocyte inhibition ratio of five PC chemical compounds under the selected concentration is listed in Table 1.

Oil Red O Staining
Hepatocytes were seeded into 6-well plates. Cellular model establishment and intervention abiding by the method described in "item 2.4" and "item 2.6." After treatment, the cells were washed 3 times with phosphate-buffered saline (PBS) and immobilized with 4% paraformaldehyde for 15 min. After three rinses with distilled water, the cells were dyed with Oil red O at 37°C for 15 min and soon afterward decolorized in 60% isopropanol for 5 s. After 2 rinses with distilled water, the cells were then stained with hematoxylin for 30 s. After washing and the observation of the lipid droplets in hepatocytes, pictures were taken under optical microscope. Five high-power fields were counted, and the stained cell count in 1,000 cells of each highpower field was compared.

Biochemical Assessment
Hepatocytes were seeded into 6-well plates. Cellular model establishment and intervention followed the method described in "item 2.4" and "item 2.6." After treatment, the cells and cell supernatant in each group were collected. The cell supernatant level of ALT/AST, and intracellular content of TG/TC were measured using commercial reagents.

Measurement of Superoxide Anion Levels
For the measurement of ROS, dihydroethidium (DHE), an oxidant-sensitive probe, is widely used. Ethidium and 2hydroxyethidium, two products of DHE oxidation, bind to the nuclear DNA, forming a strong red fluorescent complex (Zhou et al., 2013;. Hepatocytes were seeded into 6-well plates. Cellular model establishment and intervention followed the method described in "item 2.4" and "item 2.6." After treatment, the cells were incubated with DHE (5 mmol/l) in a dark container at 37°C for 30 min, and then nuclei were counterstained with 4',6diamidino-2-phenylindole (DAPI) for 10 min. Staining was observed under an inverted microscope (IX51, Olympus, Japan).

Measurement of Nicotinamide-Adenine Dinucleotide Phosphate/Reduced Nicotinamide-Adenine Dinucleotide Phosphate Ratio
NADPH is essential for redox homeostasis, the exhaustion of NADPH resulted in oxidative stress induction . Hepatocytes were seeded into 6-well plates. Cellular model establishment and intervention followed the method described in "item 2.4" and "item 2.6." Hepatocyte NADP/NADPH ratio was measured using an NADP/NADPH Assay kit (Colorimetric) according to the manufacturer's instructions.

Measurement of Nicotinamide-Adenine Dinucleotide Phosphate Oxidase Activity
A lucigenin chemiluminescence assay in the presence of NADPH as the substrate was used for measurement of NOX activity (Sung et al., 2017). Hepatocytes were seeded into 6-well plates. Cellular model establishment and intervention followed the method described in "item 2.4" and "item 2.6." The treated cells were collected, washed with ice PBS twice and then added ice Jude Krebs buffer (119 mmol/l NaCl, 20 mmol/l Hepes, 4.6 mmol/l KCl, 1 mmol/l MgSO 4 , 0.15 mmol/l Na 2 HPO 4 2·H 2 O, 136.09 mmol/l KH 2 PO 4 , 84.01 mmol/l NaHCO 3 , 147mmol/l CaCl2, and 180.2 mmol/l glucose) containing proteinase inhibitor cocktail for lysis cell. The protein concentration was quantified using a bicinchoninic acid protein assay kit. Hepatocyte protein extraction (40 mg), lucigenin (10 mmol/l) and NADPH (100 mmol/l) were added into the 96-well plates to start the reaction. One value per minute was measured with a chemiluminescent instrument for a total of 10 min. Enzyme activity is expressed as the relative light unit of protein per milligram per minute.

Western Blot Analysis
After centrifugation and determination of total protein concentrations, hepatocyte protein extraction (40 mg) was mixed with sample buffer and boiled for 5 min. According to protein molecular weight, 8 or 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis gel was chosen (120 volts, 90 min). Separated proteins on the gel were transferred to 0.45 mm polyvinylidene fluoride membranes (activated with methanol before use for 3 min). The membranes were then blocked with 5% fat-free dry milk in Tris-buffered saline with Tween (TBST) or 0.5% bovine serum albumin at room temperature for 1 h, followed by incubation with antibodies ( Table 2) at 4°C overnight. The next day, after washing with TBST three times (5 min per time), the membranes were incubated with HRP-goat anti-rabbit/mouse antibody diluted 1:10,000 at room temperature for 30 min. After washing with TBST four times (5 min per time), immunoreactive proteins were detected using the chemiluminescence method (LiDE110, Canon, Japan). Finally, band densities were determined by AlphaEaseFC software and quantified as the ratio between the optical density (OD) value of the target band and that of GAPDH.

Immunofluorescence
After completion of the treatments of the aforementioned further grouping experiments section, cells attached on coverslips were fixed in 4% paraformaldehyde for 30 min. Then, they were incubated with membrane-breaking liquid at room temperature for 10 min and with 3% H 2 O 2 in the dark for 20 min, followed by incubation with primary antibodies ( Table 3) at 4°C overnight and secondary antibody ( Table 3) at 37°C for 50 min. After that, they were stained with DAPI at room temperature for 5 min. Staining was viewed under a laser scanning confocal microscope (LSM880, Zeiss, Germany).

Statistical Analysis
All outcomes represent at least six independent experiments. All of the results were analyzed using SPSS 19.0 statistical software. Statistical significance was assessed by one-way analysis of variance following Kolmogorov-Smirnov normality. With homogeneity of variances, significance between different groups was determined by the least standard deviation test;  alternatively, the Games-Howell test was used. A probability of less than 0.05 was considered to be statistically significant.

Ultra-Performance Liquid Chromatography
Profile of Psoralea corylifolia L.

Concentrated Granules
The major active chemical constituents of PC are coumarins, flavonoids, and meroterpenes. According to Chinese Pharmacopoeia for the criterion of PC, coumarins: Psoralen and Isopsoralen were selected as markers for assessing the quality of the herb and some apropos preparations. UPLC of PC granules from three batches are shown in Figure 1 and Effects of Psoralea corylifolia L. Chemical Compounds on Palmitic Acid-Induced Hepatocyte Steatosis and Damage

Intracellular Content of Triglycerides and Total Cholesterol
As shown in Figure 2, the intracellular content of TG and TC in hepatocytes cultured with PA markedly increased (p < 0.01). The TG contents decreased significantly with the treatment of Psoralen, Isopsoralen, and Isobavachalcone as well as Go6976 (p < 0.01). As shown in Figure 3, cellular lipid droplets in those treatment groups were diminished compared to the model group. However, the TC content decreased significantly with the treatment of all five PC chemical compounds and Go6976 (p < 0.01). Furthermore, a difference in cellular TG/TC content was identified between Psoralen and other PC chemical compounds, indicating a better improvement in TG/TC accumulation (p < 0.05, p < 0.01).

Cell Supernatant Level of Alanine Aminotransferase and Aspartate Aminotransferase
As shown in Figure 4, untreated hepatocytes cultured with PA presented with both higher cell supernatant ALT and AST levels (p < 0.01), which released from damaged hepatocytes. However, treatment with all five PC chemical compounds and Go6976 reversed the increase in cell supernatant ALT and AST levels (p < 0.05, p < 0.01). Psoralen-treated model hepatocytes presented lower cell supernatant ALT/AST levels than other PC chemical compound-treated model hepatocytes (p < 0.05, p < 0.01).

Psoralea corylifolia L. Chemical Compounds Prevent Palmitic Acid-Induced Hepatocyte Oxidative Stress
To verify hepatocyte oxidative stress, we estimated the superoxide anion production by DHE staining (Figure 5). Compared with the control hepatocytes, a significantly high level of DHE fluorescence was observed in model hepatocytes exposed to PA (p < 0.01), indicating increased superoxide anion production. However, treatment with all five PC chemical compounds and Go6976 reduced the level of DHE fluorescence in hepatocytes cultured with PA (p < 0.01).
Psoralen treatment, compared with other PC chemical compounds, presented the greatest decrease in peroxide anion production in hepatocytes cultured with PA (p < 0.01).

Psoralea corylifolia L. Chemical Compounds Inhibit the Liver Protein Kinase C-a/Nicotinamide-Adenine Dinucleotide Phosphate Oxidase Signaling Pathway in Hepatocytes Cultured With Palmitic Acid
Hepatocytic Nicotinamide-Adenine Dinucleotide Phosphate/Reduced Nicotinamide-Adenine Dinucleotide Phosphate Ratio As shown in Figure 6, the ratio of hepatocytic NADP/NADPH was much higher in hepatocytes cultured with PA (p < 0.01).
After the treatment with all five PC chemical compounds and Go6976, hepatocytic NADP/NADPH ratio was significantly decreased (p < 0.01). Furthermore, Psoralen treatment, compared with other PC chemical compounds, presented the best hepatocytic NADP/NADPH decreasing ratio in hepatocytes cultured with PA (p < 0.01). As shown in Figure 7, the ratio of hepatocytic NADP/NADPH was much higher in hepatocytes cultured with PA (p < 0.01). After the treatment with Psoralen, hepatocytic NADP/NADPH ratio was significantly decreased (p < 0.01). Furthermore, PMA prestimulated, compared with Psoralen treatment only, hepatocytic NADP/NADPH ratio was significantly increased (p < 0.01).

Hepatocytic Nicotinamide-Adenine Dinucleotide Phosphate Oxidase Activity
As shown in Figures 8 and 9, hepatocytic NOX activity was evidently enhanced in hepatocytes cultured with PA (p < 0.01). After the treatment with all five PC chemical compounds and Go6976, hepatocytic NOX activity was notably decline (p < 0.01). Psoralen-treated model hepatocytes presented lower hepatocytic NOX activity than other PC chemical compounds-treated model hepatocytes (p < 0.01). Furthermore, PMA pre-stimulated, compared with Psoralen treatment only, hepatocytic NOX activity was sharply ascend (p < 0.01). Besides lucigenin assay, dual-labeled (gp91 phox -green and p47 phox -red) immunofluorescence was carried out to see the activation of hepatocytic NOX. Classic NOX (NOX2), expressed on hepatocytes, is composed of several subunits, mainly gp91 phox (membrane subunit) and p47 phox (cytosolic subunit). NOX2 activation it leads to the alignment of the cytosolic subunit p47 phox to the membrane subunit gp91 phox (Alhasson et al., 2018). As shown in Figure 10, obviously visible gp91 phox /p47 phox co-localization (yellow) events appeared in hepatocytes cultured with PA. After the treatment with Psoralen, the yellow color became significantly lighter. Furthermore, compared with Psoralen treatment only, the yellow color got stronger in the PMA pre-stimulated group.

Hepatocytic p47 phox Protein Expression
As shown in Figure 11, hepatocytic p47 phox protein levels were significantly increased in hepatocytes cultured with PA (p < 0.01). However, there was a reduction in the expression of p47 phox protein in all five PC chemical compounds and Go6976 groups (p < 0.05, p < 0.01). Additionally, Psoralen   showed the best effect in decreasing p47 phox protein expression among PC chemical compounds treatments (p < 0.01). As shown in Figure 12, PA induced overexpression of p47 phox in protein (p < 0.01) abolished by Psoralen treatment (p < 0.01). However, compared with Psoralen treatment only, the expression of p47 phox protein was significantly elevated in the PMA pre-stimulated group (p < 0.01).

Hepatocytic Protein Kinase C-a Activity
Hepatocytic PKC-a activity was shown as the ratio of phosphorylated PKC-a to PKC-a (p-PKCa/PKCa) protein expression in hepatocytes. As shown in Figure 13, it exhibited a marked elevation in p-PKCa/PKCa in hepatocytes exposure to PA (p < 0.01). The ratio of p-PKCa/PKCa was significantly reduced after the treatment of all five PC chemical compounds and Go6976 (p < 0.01). Moreover, Psoralen intervention showed a reduction of p-PKCa/PKCa when compared with other PC chemical compounds (p < 0.05, p < 0.01).
As shown in Figure 14, it exhibited a marked elevation in p-PKCa/PKCa in hepatocytes cultured with PA (p < 0.01). The ratio of p-PKCa/PKCa was significantly reduced after the treatment of Psoralen (p < 0.01). Moreover, PMA pre-  stimulated Psoralen intervention showed an elevation of p-PKCa/PKCa when compared with Psoralen treatment only (p < 0.01).

DISCUSSION
The traditional Chinese medicinal herb Psoralea corylifolia L. (PC), a hotspot in modern pharmacological research, has many diverse ethnopharmacological and medicinal applications (Alam et al., 2018;Xin et al., 2019a). Thus far, a variety of pharmacological activities of PC have been discovered, including anti-oxidative, antibacterial, anti-inflammatory, antidepressive, and estrogenic-like effects (Xin et al., 2019b). Our previous studies have reported that PC can treat juvenile NASH mice, attenuate the liver oxidative stress, and inhibit the PKC-a/ NOX signaling pathway (Zhou et al., 2017). However, notably, hundreds of compounds have been separated from PC. They belong to various groups, and coumarins, flavonoids, and meroterpenes are more dominant . T h e r e f o r e , b a s e d o n t h e e x t e n s i v e c h e m i c a l a n d pharmacological research, the anti-oxidative efficacy PC compounds (coumarins: Psoralen and Isopsoralen; flavonoids: Neobavaisoflavone and Isobavachalcone; meroterpenes:  Bakuchiol) were selected in this study to do further drug target validation. The result of UPLC verified that PC granules meet the requirements of the Chinese Pharmacopoeia and confirmed the existence of the above five compounds in PC granules.
PA can successfully induce cell steatosis and damage of juvenile mice primary hepatocytes: hepatocytes in the model group showed a large number of lipid droplets, elevated TG and TC contents, and increased the stable intracellular enzymes (ALT, AST) activity of hepatocytes. Psoralen, Isopsoralen, and Isobavachalcone could relieve both intracellular TG and TC contents. Neobavaisoflavone and Bakuchiol could only relieve intracellular TC content, but all five PC compounds could attenuate hepatocytic injury, and both cell supernatant ALT and AST levels were reduced. Moreover, Psoralen was the best in the aforementioned effects than others PC compounds. These results suggest that both two coumarins (Psoralen and Isopsoralen) and one of the flavonoids (Isobavachalcone) of PC compounds can improve PA-induced juvenile mice primary hepatocytic steatosis and damage, and the therapeutic effect of Psoralen is superior.
As for progression of NAFLD/NASH, oxidative stress is the most crucial pathological event during NAFLD development  and the hallmark between simple steatosis and NASH manifestation (Spahis et al., 2017), contributing to the aggravation of the liver fibrosis process (Ratziu et al., 2010). Hence, according to our previous researches about antioxidative bioactivity of PC (Zhou et al., 2013;Zhou et al., 2017), this study further focused on investigating the changes of oxidative stress situation in different PC compound groups. As in our previous in vivo study (Zhou et al., 2017;, DHE staining was performed to calculate superoxide anion production of hepatocytes. The appearance of superoxide can change DHE into ethidium bromide, which binds to DNA and exhibits red fluorescence in the nucleus (Gracia-Sancho et al., 2008;Vazquez-Chantada et al., 2013). In this study, a massive increase in superoxide anion production was identified in PA-induced model hepatocytes with NASH, and an amelioration effect was shown after all five PC compounds intervention. However, Psoralen treatment, compared with other PC compounds, presented the maximum decreasing of superoxide anion production. These findings further confirm the beneficial effect of all three main categories of PC compounds (coumarins, flavonoids, and meroterpenes) on improving oxidative stress in in vitro study.
As mentioned, NOX plays a pivotal role in the development of NASH . In the liver, NOX is functionally expressed both in the classic phagocytic form and in the nonphagocytic form. The mammalian NOX family comprises seven isoforms: NOX1, NOX2, NOX3, NOX4, NOX5, DUOX1, and DUOX2. Hepatocytes express mRNAs for NOX2 and regulatory subunit p47 phox (Paik et al., 2014). Our previous studies also described that PC was effective in the treatment of NASH in juvenile female mice, and the underlying mechanism might be related to the attenuation of hepatic oxidative stress via PKC-a/NOX signaling pathway (Zhou et al., 2017). Thus, the examination of the NADP/NADPH ratio, the NOX activity and the gene expression of its regulatory subunit p47 phox and upstream PKC-a were continued in the current study. We found that all the five PC compounds significantly reduced NADP/NADPH ratio, inhibited NOX activity and p47 phox protein expression in PAinduced primary hepatocytes with NASH. PKC-a activity was also decreased after intervention of all five PC compounds. Psoralen was superior to others PC compounds in attaining the aforementioned outcomes. These results demonstrated that the inhibition of all three main categories of PC compounds (coumarins, flavonoids, and meroterpenes) on ROS production might be related to the downregulation of the PKC-a/NOX signaling pathway in hepatocytes.
Subsequently, we added activator of PKC-a to pre-stimulate model hepatocytes before Psoralen intervention for further testing the possibility of Psoralen as a candidate PKC-a inhibitor. In this part, we re-evaluated the changes of the NADP/NADPH ratio, the activity of NOX and the gene FIGURE 10 | Immunofluorescence of gp91 phox and p47 phox (×400). gp91 phox , green; p47 phox , red; DAPI, blue; co-localization of gp91 phox / p47 phox , yellow. Control: Control group; Model: Model group (0.02 mmol/L PA); Psoralen: Psoralen group (0.02 mmol/L PA + 0.2 mg/ml Psoralen); PMA + Psoralen: Psoralen/activator group (0.02 mmol/L PA + 100 nmol/L PMA + 0.2 mg/ml Psoralen).   Values are mean ± SD (n = 6). a p < 0.01 versus group A; b p < 0.01 versus group B; c p < 0.05, d p < 0.01 versus group D. Group A (control); group B (0.02 mmol/L PA); group C (0.02 mmol/L PA + 1 mmol/L Go6976); group D (0.02 mmol/L PA + 0.2 mg/ml Psoralen); group E (0.02 mmol/L PA + 0.2 mg/ml Isopsoralen); group F (0.02 mmol/L PA + 0.2 mg/ml Neobavaisoflavone); group G (0.02 mmol/L PA + 0.2 mg/ml Isobavachalcone); group H (0.02 mmol/L PA + 0.2 mg/ml Bakuchiol). p-PKCa, phosphorylated protein kinase C-a; PKCa, protein kinase C-a. expression of its regulatory subunit p47 phox and upstream PKCa. Additionally, observation of cytosolic subunit p47 phox translocated to the membrane for assembly of the active complex of NOX enzymes. We found that in the PKC-a activator pre-stimulated group vs. Psoralen alone treatment group, the trend of changes of re-estimated items were noticeably reversed, accomplished with the decrease of gp91 phox /p47 phox membrane translocation. The results demonstrated a negative effect on downregulation of the PKCa/NOX signaling pathway when PKC-a activator and Psoralen were combined.
In summary, we concluded that Psoralen, Isopsoralen, and Isobavachalcone could improve the steatosis of hepatocytes. Five PC compounds (coumarins: Psoralen and Isopsoralen; flavonoids: Neobavaisoflavone and Isobavachalcone; meroterpenes: Bakuchiol) could ameliorate hepatocyte injury, relieve oxidative stress, and downregulate the PKC-a/NOX signaling pathway of hepatocyte. In addition, Psoralen exhibits the best efficacy and a prospective PKC-a inhibitor pharmaceutical activity.
Before the end, we would like to raise two important problems for the research of PC in the further. Firstly, the chemical composition of PC has two-sided effect on liver. Thus, the side of hepatotoxicity should not be ignored. A systematic and comprehensive study of domestic and foreign literatures and information has been made for safety evaluation and risk control measures of PC (Tian et al., 2017). This result suggested that PC may lead to liver damage, but the toxic components and mechanisms of PC have not been clear at present due to its complicated ingredients. Therefore, strictly apply PC in clinic is emphasized (According to Chinese Pharmacopoeia, the upper limit of the dosage for human body is 10 g/d.) and safety related basic and clinical studies are urgently required so as to full exert the efficacy and avoid the PC risk. Secondly, it was reported that copper may play a key role in NAFLD pathogenesis. A tight link between copper homeostasis and lipid metabolism has been well recognized (Feldman et al., 2015;Tarantino et al., 2018). Resent study found that adipocyte-specific Atp7a, a P-type ATPase that transports copper across cell membranes, knockout mice present hepatic steatosis and upregulated peroxisome proliferator activated receptor g (PPARg) in gonadal white adipose tissue (Tao et al., 2019). Moreover, copper has a great redox potential and altered copper levels can promote oxidative stress occurrence. New research detected that Bavachinin (BNN), one of the main active ingredients of PC, exhibits anti-tumor activity against non-small cell lung cancer by activating PPARg induced ROS increasing (Ge et al., 2019). These studies provide a new direction for exploring the protective effect of PC on liver, which may be involved in its impact on copper and related oxidative system.
Finally, we are aware of the limitation of the present study. Lack of lentiviral infection and siRNA-mediated gene silencing, the PKC-a overexpression and downregulation hepatocyte model are missing. Therefore, whether Psoralen directly inhibit PKC-a to be further verify in upregulated and downregulated PKCa expression hepatocyte models.

DATA AVAILABILITY STATEMENT
All datasets generated for this study are included in the article.

ETHICS STATEMENT
The animal study for the extraction of hepatocytes was reviewed and approved by the animal ethics committee of Wuhan Medical & Health Center for Women and Children, Huazhong University of Science and Technology (No. 2014010).