Protective Effect of Fuzi Lizhong Decoction against Non-alcoholic Fatty Liver Disease via Anti-inflammatory Response through Regulating p53 and PPARG Signaling

Abstract

Fuzi Lizhong decoction (FLD) is derived from an ancient Chinese Pharmacopoeia and has been used in clinical treatment for years. The present study aimed to investigate the activities and underlying mechanisms of FLD against non-alcoholic fatty liver disease (NAFLD). Network pharmacology analysis demonstrated that FLD might affect NAFLD through regulating p53 and peroxisome proliferator activated receptor gamma (PPARG), which has been confirmed in vitro and in vivo. In vivo NAFLD was induced in rats by a high-fat diet, and in vitro studies were performed on HL-7702 cells treated with oleic acid and linoleic acid. We showed that FLD significantly improved NAFLD by regulating the immune system to induce the release of interleukin-10 (IL-10), interferon-α (IFN-α), and IFN-β through activating p53 signaling and inhibiting PPARG signaling in vivo and in vitro. P53 inhibition induced by NAFLD was recused by FLD, while PPARG overexpression induced by NAFLD was inhibited by FLD. In addition, NAFLD resulted in increased levels of total cholesterol, triglyceride, and blood glucose in the serum and free fatty acid in the liver, which were reduced by FLD treatment. Evidently, FLD exhibited potent protective effects against NAFLD via p53 and PPARG signaling. Our study could provide novel insights into the mechanisms of FLD as an anti-inflammatory candidate for the treatment of NAFLD in the future.

INTRODUCTION

The pathological process of non-alcoholic fatty liver disease (NAFLD) manifests a spectrum of diseases ranging from hepatocellular steatosis and steatohepatitis to fibrosis and irreversible cirrhosis.¹⁾ In NAFLD, ectopic fat accumulates in the form of triglycerides (TGs) in the liver, which accounts for more than 5% of the total liver weight. Abnormal TG accumulation in hepatocytes (steatosis) results in non-alcoholic steatohepatitis, which is characterized by the activation of inflammation response and tissue injury.^2,3) NAFLD development is closely associated with many pathogenic events, including the activation of the innate immune system, hepatic macrophage recruitment, and changes in lipid homeostasis.⁴⁾ Despite much advanced methods have been used for NAFLD treatment, the therapeutic effect are limited. Thus, it is imperative to develop effective therapeutic strategies for NAFLD treatment.

Fuzi Lizhong decoction (FLD) is a traditional Chinese herbal concoction extracted from dried roots of Codonopsis pilosula, Radix Aconiti Praeparata (Sub-root processed product of Aconitum carmichaelii Debx),⁵⁾ Radix Glycyrrhizae Preparata (honey-burn processed root of Glycyrrhiza uralensis),⁶⁾ dried rhizome of Atractylodes macrocephala Koidz, and Rhizoma zingiberis (derived from the dry rhizome of Zingiber officinale Rosc.).⁷⁾ This ancient prescription is primarily used to treat spleen asthenic syndrome, chronic digestive diseases, and chronic enteritis.⁸⁾ Previous studies have indicated that FLD regulated energy metabolism in vivo, reduced serum levels of TGs and blood sugar, improved the immune system, and mobilized the body’s inflammatory system.^9–11) In clinical practice, FLD has significant therapeutic effects against chronic obstructive lung disease, viscera inflammation, and myocardium symptoms.^12–14) Nevertheless, the effect of FLD in the treatment of liver diseases has not been clarified.

Herein, Network pharmacology analysis demonstrated that FLD might affect NAFLD through regulating p53 and peroxisome proliferator activated receptor gamma (PPARG). Furthermore, the effect of FLD on NAFLD and its regulatory effect on p53 and PPARG have been investigated in vitro and in vivo.

MATERIALS AND METHODS

Reagents and Materials

Dried roots of Codonopsis pilosula. (Batch No.: 201602010), Radix Aconiti Praeparata (201601015), Radix Glycyrrhizae Preparata (201602011), Dried rhizome of Atractylodes macrocephala. (201602011), and Rhizoma zingiberis (20160127) were purchased from Wuhan No. 1 Hospital affiliated to Tongji Medical College, Huazhong University of Science and Technology and processed and sold by Hubei Tianji Chinese Herbal Sliced Medicine Co., Ltd. in accordance with the processing standards of the Food and Drug Administration of the People’s Republic of China. Sample preservation and quality control for each batch of all herbs are the responsibility of the Pharmacy Department of Wuhan No.1 Hospital. Polyene phosphatidylcholine was purchased from Sanofi Beijing Pharmaceuticals Co., Ltd. (Cat. No. 5JD065B). Enzyme-linked immunosorbent assay (ELISA) kits for interleukin-10 (IL-10, RA20090), interferon (IFN)-α (RA20351), and IFN-β (RA20594) were purchased from Myhalic Biotechnology Co., Ltd. (Bioswamp, Wuhan, China). Penicillin, streptomycin, and antimycotics were obtained from Sigma-Aldrich (U.S.A.). Antibodies against toll-like receptor (TLR) 4 (ab13867, 1 : 1000 dilution), TRAF3 (ab36988, 1 : 1000 dilution), TRAM (ab219313, 1 : 1000 dilution), and IRF3 (ab25950, 1 : 1000 dilution) were purchased from Abcam (U.S.A.). Anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (2118, 1 : 10000 dilution) was purchased from CST (U.S.A.). Horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (Cat. No. W4011; 1 : 3000 dilution) was purchased from Promega Corporation (U.S.A.). The Nonesterified Free Fatty Acids Assay Kit was purchased from Nanjing Jiancheng (A042-1, China).

Preparation of FLD

The crude drugs of FLD are composed of Dried roots of Codonopsis pilosula (15.00 g), Dried rhizome of Atractylodes macrocephala (9.00 g), Radix Glycyrrhizae Preparata (6.00 g), Rhizoma zingiberis (9.00 g), and Radix Aconiti Praeparata (9.00 g). The water-based decoctions were prepared according to a traditional formula, and the decoctions were concentrated until use. Briefly, Radix Aconiti Praeparata was first soaked in 150 mL of water for 30 min, boiled at max heat, and simmered in low heat for 30 min. The other ingredients were placed in a glass beaker and soaked in 400 mL of water for 30 min. After boiling in max heat, the Radix Aconiti Praeparata was added and the mixture was simmered at low heat for another 20 min. The decoction was separated using a double-layer gauze sheet. The solid ingredients were then added to 350 mL of water, and the boiling steps were repeated. The filtered liquid portions from the two boiling steps were combined and concentrated accordingly (35 mL of concentrated decoction per 48 g of crude drug).

Detection of Active Ingredients in FLD

The active ingredients of FLD were detected using the Ultra high performance liquid chromatograph (UHPLC). A 100-µL of aliquot concentrated FLD was extracted using 300 µL of methanol via vortex mixing for 30 s. After ultrasound in ice-water bath for 1 h, the specimen was centrifugated at 12000 rpm at 4 °C for 10 min. Then a 5-µL aliquot of the supernatant were harvested and analyzed by UHPLC (Agilent, CA, U.S.A.), equipped with an ultra performance liquid chromatography (UPLC) BEH C18 Column (1.7 µm, 2.1 × 100 mm, Waters, MA, U.S.A.). The peak strength is usually proportional to the compound content. In the current work, the compounds in FLD with LQ.POS.A > 10000 were selected as active substances.

Prediction of Active Substances Targets for FLD and Gene Targets for NAFLD

The protein targets of the active substances in FLD were predicted using the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) (http://tcmspw.com/tcmsp.php). NAFLD-associated human genes were obtained from DiSGeNET (https://www.uniprot.org/database/DB-0218) and CooLGeN (https://ci.sum.edu.cn/Test/CoolGeN/) databases. The targets were then mapped with NAFLD using PharmGKB database (https://www.pharmgkb.org/), the therapeutic target database (TTD, http://bidd.nus.edu.sg/group/cjttd/), and OMIM database (https://www.omim.org/).

Animals and Experimental Groups

Forty-eight male Wistar rats were obtained from Hubei Provincial Academy of Preventive Medicine (Certification No. 42000600013948). The rats were housed in a specific pathogen-free facility at Wuhan First Hospital. Following an acclimatization period, the rats were randomly divided into the following groups (n = 8 per group): normal control (NC), NAFLD model (MOD), positive control (PC), high-dose FLD (HIG), medium-dose FLD (MID), and low-dose FLD (LOW). The rats in the NC group received a standard laboratory diet and those in the model group received a high-fat diet (standard laboratory diet +2% cholesterol + 10% lard + 2.5% vegetable oil).¹⁵⁾ The rats in the HIG, MID, and LOW groups received FLD treatment at 3.6, 7.2, and 14.4 mL/kg/d, respectively. The dose of 3.6 mL/kg/d was obtained according to the guide for dose conversion between animals and human.¹⁶⁾ The rats in the PC group were treated with 30 mg/kg/d polyene phosphatidylcholine, trade name Essentiale^®, which is often used in the clinical treatment of NAFLD.¹⁷⁾ The rats were maintained under a 12 h light–dark cycle at 23 ± 2 °C. This experiment is approved by the Institutional Review Board of Wuhan Myhalic Biotechnology Co., Ltd. based on the ethical Guidelines for Animal Care and Use of the Model Animal Research Institute

Cell Culture and Treatment

The human liver HL-7702 cell line was obtained from Procell (CL-0111). HL-7702 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (ThermoFisher, Waltham, U.S.A.), 100 U/mL penicillin, 100 mg/mL streptomycin, and 100 mg/mL antimycotics. The cells were grown in an incubator with a humidified atmosphere (95% air/5% CO₂ (v/v)) at 37 °C for 48 h until they reached 80% confluence. In vitro steatosis was induced by incubating the cells with a 1 : 1 (v/v) mixture of oleic (18 : 1) and linoleic acid (18 : 2) at 6 mmol/L (model, MOD group).¹⁸⁾ The cells were exposed to equal volumes of FLD prepared at different concentrations (0.36, 0.72, and 1.08 mL of FLD was added to 1 mL of serum-free DMEM for LOW, MID, and HIG, respectively) or 5 µmol/L polyene phosphatidylcholine (positive control, PC) for 48 h.

Histological and Serological Examination

Liver tissues were fixed in 2 mL of 4% paraformaldehyde. After 24 h, the fixative was aspirated and replaced with fresh 4% paraformaldehyde. Liver samples were embedded in paraffin wax and sections were stained with hematoxylin–eosin (H&E). Light microscopic examination was performed with a Nikon E400 microscope (Nikon Instrument Group, Japan). Serum levels of total cholesterol (TC), triglyceride (TG), and blood glucose (Glu) were detected using an automatic biochemical analyzer (model 7180, Tokyo, Japan). The free fatty acid (FFA) level was detected by the Nonesterified Free Fatty Acids Assay Kit.

ELISA

IL-10, IFN-α, and IFN-β levels in the serum, liver, and cell culture supernatant was evaluated by ELISA kits in accordance with the manufacturer’s protocols.

Total RNA Extraction and RT-Quantitative (q)PCR

Total RNA was extracted from liver tissue or HL-7702 cells using TRIzol reagent (TaKaRa Bio Inc., Dalian, China) and assessed using an UV spectrophotometer and 1% agarose electrophoresis. For each sample, 1 µg of RNA was reverse-transcribed to obtain first-strand cDNA using the PrimeScript^® RT reagent kit with gDNA Eraser (TaKaRa Bio, Inc.) following the manufacturer’s instructions. The reaction mixture (20 µL of total volume) contained 10 µL of 2 × SYBR Premix Ex Taq^™ (TaKaRa Bio Inc.), 0.5 µL of each primer, and 0.2 ± 0.02 µg of cDNA template. The following three-step qPCR reaction was performed: Pre-denaturation at 95 °C for 30 s, followed by 40 cycles of denaturation at 95 °C for 3 min, annealing at 60 °C for 20 s, and elongation at 72 °C for 20 s. The primers used are as follows: p53, F, 5′-TTT GAG GTT CGT GTT TGT G-3′, R, 5′-TGG GCA GTG CTC TCT TTG-3′; PPARG, F, 5′-ACT CCC ATT CCT TTG AC-3′, R, 5′-CGC ACT TTG GTA TTC TT-3′; GAPDH, 5′-CAA GTT CAA CGG CAC AG-3′, R, 5′-CCA GTA GAC TCC ACG ACA T-3′. Gene expression was calculated using the 2^-ΔΔCq method.¹⁹⁾ For each group, three samples were measured and three technical replicates of each measurement were obtained. GAPDH act as an internal control.

Western Blot

Protein expression levels were analyzed by Western blot conducted using standard methods with modification. Liver tissue samples were homogenized in radio immunoprecipitation assay (RIPA) lysis buffer containing protease inhibitor at 4 °C. For in vitro study, cells were washed twice with phosphate-buffered saline and lysed with RIPA buffer (Beyotime, China) containing protease inhibitor at 4 °C. Both cell and tissue lysates were centrifuged at 12000 × g for 15 min and the supernatants were collected. Protein concentration was detected using a bicinchoninic acid (BCA) kit (Bioswamp Life Science). Equal amounts of protein (30 µg) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred onto polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, U.S.A.). The membranes were blocked for 2 h at room temperature with 5% skim milk in Tris-buffered saline (20 mmol/L Tris, 500 mmol/L NaCl, and 0.05% Tween 20). Subsequently, the membranes were incubated at room temperature for 1 h with primary antibodies against p53, PPARG, and GAPDH (Bioswamp). GAPDH was used as an internal reference. The membranes were then washed with Tris-buffered saline and incubated with goat anti-rabbit secondary antibody for 2 h at room temperature. Immunoreactivity was visualized via a colorimetric reaction using enhanced chemiluminescent substrate buffer (EMD Millipore) and analyzed using a Gel Doc EZ imager. Bands were quantified using Quantity One 5.0 (Bio-Rad Laboratories, Hercules, CA, U.S.A.).

Statistical Analysis

The statistical differences of the experimental data were evaluated through one-way ANOVA followed by Turkey using SPSS 19.0 software package. Differences were considered as statistically significant at p < 0.05. All results are expressed as mean ± standard deviation (S.D.).

RESULTS

Identification of Active Ingredients in FLD

According to the results of UHPLC (Supplementary 1), we selected 11 compounds as the active ingredients in FLD (Area > 10000). They were Codonopsine, (S)-6-Gingerol, Delcosine, Ononin, (S)-8-Gingerol, Glyasperin C, Licoisoflavanone, Licuroside, Aconifine, Gancaonin B, (S)-10-Gingerol (Table 1).

Table 1. Candidate Active Substances in FLD

Description	m/z	Retention time (min)	Area
Codonopsine	268.15	2.000	244231
(S)-6-Gingerol	317.17	10.165	176871
Delcosine	454.28	3.818	173698
Ononin	431.13	6.960	32579
(S)-8-Gingerol	345.20	11.734	23434
Glyasperin C	357.17	10.573	22229
Licoisoflavanone	355.12	10.891	14754
Licuroside	551.18	6.619	14386
Aconifine	662.32	8.526	14237
Gancaonin B	369.13	10.437	11913
(S)-10-Gingerol	373.23	13.096	11716

Targets Identification of FLD on NAFLD

Among the 11 candidate active ingredients, 77 protein targets were collected from TCMSP database (shown in Supplementary 2). Seventy-six NAFLD-related human genes were obtained from DiSGeNET and CooLGeN, followed by mapping with pharmGKB, TTD, OMIM databases (shown in Supplementary 3). Finally, we found 4 targets (TP53, JUN, DPP4, and PPARG) of 11 candidate active ingredients in FLD were related with NAFLD, indicating that FLD might have a treatment effect on NAFLD by regulating TP53, JUN, DPP4, and PPARG. The effect of FLD on NAFLD and its regulatory effect on TP53 and PPARG have been investigated subsequently.

Effects of FLD on Serological Examination and Histological Changes in Rats with NAFLD

To evaluate the protective effect of FLD against NAFLD, the levels of TC, TG, and Glu in the serum and the level of FFA in liver tissues were compared in each group. Compared to the control, TC, TG, Glu, and FFA were increased in rats with NAFLD, but FLD treatment decreased their levels in model rats (Fig. 1A). Analysis of liver tissue damage by HE staining revealed that the livers from control rats had an intact lobular architecture with clear central veins (Black arrow in NC group) and radiating hepatic cords, whereas the livers of rats with NAFLD exhibited hepatic fatty degeneration with primarily fat vacuoles was visible (Black arrow in MOD group), destruction of liver architecture, and inflammatory cell infiltration (Red arrow in MOD group) (Fig. 1B). Inflammatory injury was significantly reduced in FLD-treated model rats.

Fig. 1. FLD Improved the Symptoms of NAFLD

A: Serum levels of TC, TG, and Glu and liver levels of FFA were detected by ELISA. B: Representative images of HE-stained liver sections. Black arrow in NC group indicates central veins; Black arrows in MOD group indicate hepatic fatty degeneration with primarily fat vacuoles; Red arrow in MOD group indicates inflammatory cell infiltration. All values are expressed as the mean ± standard deviation (S.D.) (n = 3). * p < 0.05 vs. NC; ^# p < 0.05 vs. MOD; ^▲ p < 0.01 vs. HIG.

FLD Treatment Up-Regulated Anti-inflammatory Factors in Serum and Liver of Rats

As shown in Fig. 2, the serum and liver levels of IL-10, IFN-α, and IFN-β in the model rats were markedly decreased compared with those in control rats, but were subsequently increased by FLD administration.

Fig. 2. FLD Induced the Production of IL-10, IFN-α, and IFN-β in Serum and Liver of Rats with NAFLD

All values are expressed as the mean ± S.D. (n = 3). * p < 0.05 vs. NC; ^#p < 0.05 vs. MOD; ^▲p < 0.01 vs. HIG.

FLD Treatment Activated p53 Signaling but Inactivated PPARG Signaling in Vivo

Compared with control rats, the NAFLD model rats showed decreased mRNA expression of p53 and increased mRNA of PPARG, which were reversed by FLD treatment (Fig. 3A). The protein levels of p53 and PPARG showed the same trend as mRNA expression (Fig. 3B). Together, these data indicated that FLD attenuated NAFLD via activation of p53 and inactivation of PPARG signaling.

Fig. 3. The Effect of FLD on the Activation of p53 and PPARG in Rats with NAFLD Was Evaluated

A: mRNA levels of p53 and PPARG in rat liver tissue were detected by RT-PCR. B: Protein levels of p53 and PPARG in rat liver tissue were detected by Western blot. All values are expressed as the mean ± S.D. (n = 3). * p < 0.05 vs. NC; ^# p < 0.05 vs. MOD; ^▲ p < 0.01 vs. HIG.

FLD Increased the Levels of IL-10, IFN-α, and IFN-β in HL-7702 Cells

To verify the treatment effect of FLD on NAFLD, the NAFLD model was established in HL-7702 cells. The cells were exposed to different doses of FLD and the levels of IL-10, IFN-α, and IFN-β in the cell supernatant were measured (Fig. 4). Compared with the non-treated control cells, NAFLD-induced model cells showed decreased levels of IL-10, IFN-α, and IFN-β, which were then increased by FLD treatment in the model cells.

Fig. 4. The Production of IL-10, IFN-α, and IFN-β in HL-7702 Cells Was Evaluated by ELISA

All values are expressed as the mean ± S.D. (n = 3). * p < 0.05 vs. NC; ^# p < 0.05 vs. MOD; ^▲ p < 0.01 vs. HIG.

FLD Induced the Activation of p53 Signaling but Inactivation of PPARG Signaling in HL-7702 Cells

The protein and mRNA levels of p53 and PPARG in HL-7702 cells were measured to evaluate the effect of FLD treatment on NAFLD-induced cells (Fig. 5). Compared to the control group, the mRNA and protein levels of p53 were decreased, whereas those of PPARG were increased in model cells. FLD treatment reversed these results by increasing the mRNA and protein levels of p53 and decreasing those of PPARG compared to those of cells in the model group.

Fig. 5. The Effect of FLD on p53 and PPARG Signaling in HL-7702 Cells Was Evaluated

A: mRNA levels of p53 and PPARG in HL-7702 cells were detected by RT-PCR. B: Protein levels of p53 and PPARG in HL-7702 cells were detected by Western blot. All values are expressed as the mean ± S.D. (n = 3). * p < 0.05 vs. NC; ^# p < 0.05 vs. MOD; ^▲ p < 0.01 vs. HIG.

DISCUSSION

NAFLD is one of the most common causes of liver diseases worldwide. It remains a major cause of morbidity and mortality because of rising levels of obesity, diabetes, and metabolic syndromes. Previous studies have illustrated that experimental animals fed a high-fat diet experienced simple steatosis at week 4, steatohepatitis at week 8, and hepatic fibrosis at week 16, depicting the full developmental process of NAFLD that may well represent the human disorder.²⁰⁾ In the present study, we successfully generated a rat model of NAFLD by imitating a high-fat diet and investigated the protective effect and potential mechanism of FLD against NAFLD.²¹⁾ Even though an accurate definition of NAFLD has not yet been established in rodents, the observed development of substantial steatosis with associated necroinflammatory and fibrotic changes in the setting of obesity, an elevated hepatosomatic index, and increased TG, TC, glucose, and FFA levels testify that this animal model is successful and useful for characterizing the molecular basis of NAFLD. Rats fed a high-fat diet exhibited increased serum levels of TC, TG, Glu, and FFA in addition to histological damage, but these changes were suppressed by FLD administration. We found that FLD exerted potent effects against NAFLD by suppressing PPARG and enhancing p53 signaling, increasing anti-inflammatory response, and alleviating histopathological changes, which were consistent with network pharmacology analysis.

As the ‘Guardian of the genome’ against cellular stresses, p53 was first described as a tumor suppression gene.^22,23) Recently, accumulating evidence demonstrated the central role of p53 in the development of NAFLD.^24,25) In particular, previous research have indicated that p53 was implicated in the molecular mechanisms of hepatocellular injury²⁶⁾ and lipid metabolism.²⁷⁾ Inflammation is an important physiological feature of NAFLD. It has been reported that p53 inhibition promoted the inflammatory response of mice.²⁸⁾ In addition, p53 attenuated activation of the nuclear factor-kappaB (NF-κB) pathway,²⁹⁾ in turn inhibited the inflammatory response through regulating ILs, IFNs and tumor necrosis factor.^30,31) Collectively, our current results demonstrated that FLD increased the expression of IL-10 and IFNs in NAFLD-induced model cells and rats, which might be associated with its activation of p53.

In addition, our current study confirmed the results of network pharmacology that FLD affect NAFLD through regulating PPARG. FLD inhibited the expression of PPARG in NAFLD-induced model cells and rats. PPARG is a member of peroxisome proliferator activated receptors, that can be act as a feature of steatotic liver.³²⁾ Previous study demonstrated that targeted deletion of PPARG in macrophages protects mice and in hepatocytes against diet-induced hepatic steatosis, suggesting a pro-steatotic role of PPARG both in parenchymal and non-parenchymal cells.³³⁾ Moreover, PPARG was highly expressed in NAFLD experimental models patients, thereby induced hepatic steatosis and triglyceride clearance.^34,35)

In conclusion, FLD treatment significantly suppressed PPARG and enhanced p53 signaling in the liver of rats with NAFLD and increased the expression of IL-10 and IFNs. This finding indicated that FLD can regulate PPARG and p53 signaling, which exhibited excellent protective effect against NAFLD.

Acknowledgments

This work is supported by Wuhan Municipal Science and Technology Bureau Applied Basic Research Project (No. 2017060201010224).

Author Contributions

JY and ZS designed this work; JY, WM, QM, JS, and LS were responsible for the experiments; JY, SZ, CL, LA, and ND analyzed the data; JY drafted the manuscript; ZS revised the manuscript; All authors reviewed and approved the final manuscript.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

The online version of this article contains supplementary materials.

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