Male (n = 5) and female (n = 15) C57BL6J mice, age 56 to 63 days, obtained from the Institute of Laboratory Animal Sciences, CAMS&PUMC, were used in this study (Certification: no.11401300045242). The mice were maintained at ambient temperature (22 ± 1°C) with a 12:12 h light-dark cycle and free access to water and food (Barrier system, Center of Experimental Animals, Tongji Medical College, Huazhong University of Science and Technology. Certification: no.00142666). All experiments were approved by the animal ethics committee of Wuhan Medical & Health Center for Women and Children, Huazhong University of Science and Technology (no. 2014010).

The Laboratory Animal Center of Tongji Medical College of Huazhong University of Science and Technology provided a standard diet (35% flour, 20% soy meal, 20% corn meal, 15.5% bran, 0.5% bean oil, 5% fish meal, 2.5% bone meal, 1% dusty yeast, and 0.5% salt). High-fat diets D12451, 4.73 total kcal/g (45% kcal and 24% g fat, 20% kcal and 24% g protein, 35% kcal and 41% g carbohydrate), were purchased from Research Diets, Inc. (New Brunswick, NJ, USA).

Psoralea corylifolia L. concentrated granules were provided by Sanjiu Medical and Pharmaceutical Co. Ltd. (Shenzhen, China). One gram of PC granule equals to 20 g PC traditional Chinese herbals. High performance liquid chromatography (HPLC) was performed to identify the main chemical constituents in PC concentrated granules to assess the quality. Psoralen (P.N. 110739–201416) and Isopsoralen (P.N. 110738–201614) purchased from National Institutes for Food and Drug Control (Beijing, China), were taken as reference standards. HPLC was performed on Agilent 1260 system (USA) equipped with an Agilent ZORBAX Eclipse Plus C18 (250 × 4.6 mm, 5 μm). The mobile phase included methyl alcohol-waters (55:45, v/v) at a flow speed of 1.0 ml/min in condition of column temperature 30°C. The detection wavelength was 246 nm.

After adaptive feeding with a standard diet for 3 days, all female mice were randomized into the normal control group (n = 3) and model group (n = 12). All male mice were in the same randomization, with a ratio of 1:3 to female mice for mating. All male mice and female mice in the control group continued the standard diet. Female mice in four model groups were fed with high-fat diets 1 week before conception and during gestation and lactation. Litter size was standardized to four pups to guarantee that no litter was nutritionally biased. After weaning, the female offspring mice in the control group were selected and fed a standard diet: the normal control group (Control, n = 6). The female offspring mice in the model groups were selected and assigned to high-fat diets, generating four other groups: the juvenile NAFLD model group (Model, n = 6), low-dose PC treated group (PC-L, n = 6), high-dose PC treated group (PC-H, n = 6), vitamin E (Zhejiang Medicine Co., Ltd., Xinchang Pharmaceutical Factory, Shaoxing, China) treated group (VitE, n = 6). After 3 days, mice in the aforementioned treatment groups were administered corresponding therapy, PC-L (1.125 mg/g/d), PC-H (2.25 mg/g/d), or VitE (0.01 mg/g/d), intragastrically to 9 weeks of age. Oral gavage was performed once a day between 9:00 and 11:00 a.m. Mice in the normal control and juvenile NAFLD model group were administered the same volume of distilled water. Doses were adjusted to the body weight recorded once every 5 days.

All the offspring female mice were euthanized at the end of 9 weeks. Blood samples were obtained by orbital sinus puncture at the time of euthanization. The specimen serum was collected and stored at −20°C for analysis after centrifuging at 3,000 revolutions per minute for 30 min at 4°C. Livers were quickly removed by laparotomy and flushed with normal saline on ice. Samples from the left outside lobe of the liver were fixed in 4% paraformaldehyde solution for paraffin embedding. Samples from the left inside lobe of the liver were prepared for frozen sections. Samples from the right lobe and the caudate lobe of the liver were preserved at −80°C for the analysis.

Fasting blood glucose was measured by the hexokinase method using a GLU Kit (Shanghai Mind Bioengineering Co., Ltd., Shanghai, China). Fasting insulin was determined by enzyme-linked immunosorbent assay method using a Mouse Ins (Insulin) ELISA Kit (Elabscience Biotechnology Co. Ltd., Wuhan, China). Homeostatic model assessment of insulin resistance (HOMA-IR) was calculated according to the formula as follows: fasting blood glucose (mmol/L) × fasting insulin (mIU/L)/22.5.

Serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and liver content of triglycerides (TG), and total cholesterol (TC) were measured using commercial reagents (Jiancheng Bioengineering Institute, Nanjing, China).

The paraffin slides stained with hematoxylin and eosin (H&E) were observed under optical microscope to assess hepatocyte steatosis and cellular infiltrate. The paraffin slides stained with sirius red were observed under polarizing microscope (Axio Scope.1A, Carl Zeiss, Germany) to assess the collagen deposition of the liver. The following criteria were used for scoring hepatic histology. The Kleiner scoring system was used to evaluate the severity of NAFLD (Kleiner et al., 2005). An activity score was generated by adding the individual scores for three features: steatosis (<5% = 0, 5–33% = 1, 33–66% = 2, >66% = 3); ballooning (none = 0, few = 1, prominent = 2); and lobular inflammation (none = 0, <2 foci = 1, 2–4 foci = 2, >4 foci = 3). A score of <3 represents a mild nonalcoholic fatty liver; a score of 3 to 4 represents a moderate nonalcoholic fatty liver; and a score of 5 or more represents NASH.

Hepatic total RNA was extracted from the liver tissue with TRIzol reagent in accordance with the manufacturer's instructions. First, RNA purity and concentration were tested using a nucleic acid/protein analyzer (Thermo, USA). Second, the extracted total RNA (1 μg) was reverse transcribed using the PrimeScript™RRT Reagent Kit with gDNA Eraser (TaKaRa Company, Dalian, China) on a Mastercycler gradient PCR apparatus (Eppendorf Company, Germany). Next, the complementary DNA was kept at −20°C before PCR amplification. Next, real-time polymerase chain reaction (PCR) analyses were performed in 48-well optical PCR plates using SYBR® Premix Ex Taq™ (TaKaRa Company, Dalian, China) on an Applied Biosystems StepOne Real-Time PCR System (Thermo, USA). Finally, 2^−ΔΔCT was used for analyzing the data. Primer sequences are listed in Table 1.

Serving as an oxidant-sensitive probe, dihydroethidium (DHE) is widely used for the measurement of ROS. Ethidium and 2-hydroxyethidium, two products of DHE oxidation, bind to the nuclear DNA, thereby forming a strong red fluorescent complex (Zhou et al., 2013). Frozen sections of liver (6 μm) were incubated with DHE (5 mmol/L, Beyotime Institute of Biotechnology, Shanghai, China) in a dark container at 37°C for 30 min (Zhou et al., 2013) and then observed under an inverted microscope (IX51, Olympus, Japan).

Liver homogenate was lyzed in mammal tissue protein extraction reagent. The extracted protein was then supplemented with a protease inhibitor cocktail and phenylmethylsulfonylfluoride (PMSF) (Sinopharm Chemical Reagent Co. Ltd., Shanghai, China). Next, the samples were centrifuged at 12,000 revolutions per minute for 15 min at 4°C. The supernatant was collected to quantify the protein concentration using a bicinchoninic acid protein assay kit (ASPEN Biotechnology Co. Ltd., Wuhan, China). Liver NADPH activity was measured using an NADPH Activity Quantification Kit (Genmed Scientifics Inc., Shanghai, China).

Liver extracted proteins (40 μg) were mixed with sample buffer, boiled for 5 min, and subjected to 8 and 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis gel graded from maximum to minimum of protein molecular weight (120 volts, 60–90 min). Separated proteins on the gel were transferred to 0.45 μm polyvinylidene fluoride membranes. 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, the membranes were incubated with HRP-goat anti-rabbit/mouse antibody (ASPEN Biotechnology Co. Ltd., Wuhan, China) diluted 1:10,000 at room temperature for 30 min. After washing with TBST four times, immunoreactive proteins were detected using the chemoluminescence method (LiDE110, Canon, Japan). Finally, band densities were determined by AlphaEaseFC software and quantified as the ratio between OD-value of the target band to that of GAPDH.

All data were analyzed using SPSS19.0 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 least standard deviation (LSD) test; alternatively, the Games-Howell test was used. A probability of <0.05 was considered to be statistically significant.

The major active chemical constituents of PC are coumarins, flavonoids, and meroterpenes (Chopra et al., 2013; Zhang et al., 2016). According to Chinese Pharmacopoeia for the criterion of PC, coumarins: psoralea and isopsoralen were sleceted as markers for assessing the quality of the herb and some apropos preparations. HPLC of PC granules from three batches were shown in Figure 1. The result showed that the two main peaks were detected and identified by comparing the retention time with reference standards. Psoralea assay in three batches of PC granules were 7.035, 6.998, 6.730 mg/g, respectively. Isopsoralen assay in three batches of PC granules were 4.801, 4.858, 5.040 mg/g, respectively. The result verified that PC concentrated granules prepared from Chinese traditional medicinal PC and demonstrated that the three batch granules of testing data adhere to the Chinese Pharmacopoeia standard, as qualified samples can be used in the experiment.

To verify liver fibrosis, we observed the collagen deposition in the portal and perisinusoidal areas by sirius red-stained liver sections (Figure 3). For the mice in the model groups, collagen deposition mainly occurred in the portal area of liver tissues. It manifested as a bright yellow light on polarizing microscopy, indicating portal fibrosis with type I collagen deposition. For the mice in the treatment groups, the type I collagen also deposited in the portal area of liver tissues. However, treatment with different dose of PC and VitE significantly weakened the level of yellow color in liver tissues of juvenile mice. In addition, the PC-H group presented weaker light than that of the PC-L groups.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer DC and handling editor declared their shared affiliation.