Edited by: Jinyong Peng, Dalian Medical University, China
Reviewed by: Dapeng Chen, Dalian Medical University, China; Federico Salomone, Azienda Sanitaria Provinciale di Catania, Italy
*Correspondence: Huan Qin
Suqi Yan
This article was submitted to Gastrointestinal and Hepatic Pharmacology, a section of the journal Frontiers in Pharmacology
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Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease worldwide. NAFLD includes a broad spectrum of liver disease, ranging from simple steatosis to nonalcoholic steatohepatitis (NASH) and NASH can progress to liver cirrhosis and hepatocellular carcinoma. The epidemic of NAFLD is rapidly becoming a severe public health problem (Watanabe et al.,
Unfortunately, to date, there are no approved therapies for children and adolescents with NAFLD other than lifestyle advice on diet and exercise (Zelber-Sagi et al.,
Male (
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).
After adaptive feeding with a standard diet for 3 days, all female mice were randomized into the normal control group (
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.,
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
Real-time PCR primer sequences.
5′-TGAAGGGTGGAGCCAAAAG-3′ | 5′-AGTCTTCTGGGTGGCAGTGAT-3′ | |
5′-TCCCCAAAGGGATGAGAAGTT-3′ | 5′-GAGGAGGTTGACTTTCTCCTGG-3′ | |
5′-CTGGGAAATCGTGGAAATGAG-3′ | 5′-AAGGACTCTGGCTTTGTCTTTCT-3′ | |
5′-GGCCCAATTACTAACAGGTTCC-3′ | 5′-TGACTTCACTGGAGTCCCGTAG-3′ | |
5′-CCCTACTATTCCTGATGGCACT-3′ | 5′-CTATGAGAAACCCACCACATCTG-3′ |
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.,
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
First antibody details.
GAPDH | Rabbit | Abcam | 1:10,000 |
Phosphorylated NF-κB p65 (S276) | Rabbit | Abcam | 1:500 |
NF-κB p65 | Rabbit | Abcam | 1:1,500 |
PI3K p85 | Rabbit | CST | 1:3,000 |
Phosphorylated Akt (S473) | Rabbit | CST | 1:1,000 |
Akt | Rabbit | CST | 1:2,000 |
p47phox | Mouse | Santa | 1:500 |
Phosphorylated PKC-α (S657 + Y658) | Rabbit | Abcam | 1:500 |
PKC-α | Rabbit | Abcam | 1:500 |
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.,
HPLC chromatogram of PC granule from three batches and reference standards. There were three batches of PC granule supplied by Sanjiu Medical and Pharmaceutical Co. Ltd. (lot: 1407001W, 1601002S, and 1601003S).
As shown in Table
The effect of PC on the progression of NASH in juvenile mice.
Control | 43.92 ± 3.85 | 32.26 ± 3.02 | 2.02 ± 0.19 | 4.23 ± 0.28 | 3.69 ± 0.22 |
Model | 91.59 ± 5.98 |
58.33 ± 3.84 |
5.98 ± 0.13 |
7.43 ± 0.27 |
8.73 ± 0.48 |
PC-L | 72.41 ± 3.35 |
49.33 ± 2.75 |
4.86 ± 0.27 | 6.45 ± 0.26 |
7.08 ± 0.15 |
PC-H | 53.07 ± 3.52 |
36.62 ± 2.61 |
2.42 ± 0.26 |
4.62 ± 0.17 |
4.58 ± 0.56 |
VitE | 58.72 ± 2.55 |
39.78 ± 1.58 |
2.68 ± 0.48 |
5.10 ± 0.36 |
4.82 ± 0.48 |
Compared with the juvenile mice in the control group, model mice presented with markedly higher liver TG and TC contents (
Mice in the model group showed severe IR characterized by elevated HOMA-IR (
As shown in Figure
Morphologic photos of mice hepatic tissue. Hematoxylin and eosin (H&E) staining. Original magnification: ×200.
Assessment of juvenile mice NAFLD/NASH severity in the liver tissues.
Control | 0 | 0 | 0 | 0 | Normal |
Model | 2.33 | 2 | 2 | 6.33 | NASH |
PC-L | 1.33 | 1.33 | 1.67 | 4.33 | Moderate NAFLD |
PC-H | 0.33 | 0.67 | 1 | 2 | Mild NAFLD |
VitE | 0.67 | 1 | 1.33 | 3 | Moderate NAFLD |
To verify liver fibrosis, we observed the collagen deposition in the portal and perisinusoidal areas by sirius red-stained liver sections (Figure
Sirius red staining in portal area from the mice in different groups. Original magnification: ×50.
As shown in Figure
The effect of PC on the expression of liver inflammatory factors. Values are mean ± SD (
Hepatic NF-κB activity was shown as the ratio of phosphorylated nuclear factor-κB (NF-κB) p65 to NF-κB p65 (p-p65/p65) protein expression in liver tissues. As shown in Figure
The effect of PC on the p-p65/p65 ratio in hepatic tissues. Values are mean ± SD (
As shown in Figure
The effect of PC on the expression of PI3K p85 in hepatic tissues. Values are mean ± SD (
Hepatic Akt activity was shown as the ratio of phosphorylated Akt to Akt (p-Akt/Akt) protein expression in liver tissues. As shown in Figure
The effect of PC on the p-Akt/Akt ratio in hepatic tissues. Values are mean ± SD (
As shown in Figure
Dihydroethidium staining in the liver from the mice in different groups. Original magnification: ×40.
As shown in Figure
The effect of PC on the hepatic NADPH activity. Values are mean ± SD (
As shown in Figure
The effect of PC on the expression of p47phox in hepatic tissues. Values are mean ± SD (
Hepatic PKC-α activity was shown as the ratio of phosphorylated PKC-α to PKC-α (p-PKCα/PKCα) protein expression in liver tissues. As shown in Figure
The effect of PC on the p-PKCα/PKCα ratio in hepatic tissues. Values are mean ± SD (
The traditional Chinese medicinal herb
The NASH model of juvenile mouse can be successfully established with a maternal-offspring high-fat diet feeding method (Bruce et al.,
We also evaluated liver fibrosis by H&E staining and sirius red staining. We observed fibroplasia in the portal area of model juvenile mice. The coloration proved that the main type of collagen deposition might be positively related to type I collagen. However, the coloration level noticeably decreased after PC or VitE treatment. The high dose of PC showed the weakest light. These results indicate that PC may be effective on ameliorating liver fibrosis process in NASH.
The pathogenesis of NAFLD involves OS, IR, and inflammation. Each of these elements is fueled by the other and each promotes the other, thereby forming a vicious circle (Tilg and Moschen,
First, we evaluated the changes of the NF-κB signaling pathway to elucidate the anti-inflammation mechanisms of PC. We found that NF-κB activity was significantly increased in liver tissue of juvenile model mice accompanied by the increase in the expression of NF-κB downstream inflammatory factors in liver tissue. These inflammatory factors involved inflammatory cytokines (TNF-α and IL-6) and chemokines (IL-8 and MCP-1). Treatment with PC decreased NF-κB activity and TNF-α, IL-6, IL-8, MCP-1 mRNA expressions. The effects of high dose were also superior to that of low dose. These results suggest that the protection mechanisms of PC against juvenile NASH may be related to reducing inflammation by inhibiting hepatic NF-κB signaling pathway.
Second, to explicate the mechanism of PC at attenuating IR, we estimated the changes of the hepatic PI3K-Akt signaling pathway. It is well known that the upregulation of the PI3K-Akt pathway is of benefit to IR improvement. Nevertheless, our study validated that the protein expression of PI3K p85 was significantly increased in liver tissue of juvenile model mice, accompanied by the increase in Akt activity in liver tissue. Treatment with PC decreased hepatic PI3K p85 protein expression and p-Akt/Akt. Interestingly, this paradox has also been reported in some studies (McKee et al.,
Third, we investigated the changes of OS situation. Excessive ROS generated by OS induces oxidative damage by peroxidation of the biomacromolecule and interferes with hepatic cell signal transduction by serving as a second messenger (Brandes and Kreuzer,
It is widely believed that the mechanism of anti-oxidant activity in PC involves scavenging ROS and inhibiting ROS generation (Chopra et al.,
In summary, we concluded that PC could improve the progression of NASH and ameliorate liver fibrosis in juvenile mice. The mechanism may be related to reduce inflammation by the NF-κB signaling pathway as well as inhibiting cells proliferation by the PI3K-Akt signaling pathway and decreasing OS through the PKC-α/NADPH oxidase signaling pathway. In addition, PC exhibits a dose dependent efficacy.
LisZ and SY conceived and designed the study. LisZ, JT, XX, JH, SZ, LinZ, and HQ performed the experiments. LisZ and HQ wrote the paper. LisZ and HD reviewed and edited the manuscript. All authors read and approved the manuscript.
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.