Psoralea corylifolia L. Seed Extract Attenuates Nonalcoholic Fatty Liver Disease in High-Fat Diet-Induced Obese Mice

Nonalcoholic fatty liver disease (NAFLD), along with obesity, is increasing world-wide and is one of the major causes of chronic hepatic disease. The present study evaluated the ameliorative effect of extract of Psoralea corylifolia L. seed (PCS) on high fat diet-induced NAFLD in C57BL/6 mice after daily administration at 300 or 500 mg/kg for 12 weeks. Treatment with PCS extract significantly reduced body weight and blood glucose levels and improved glucose tolerance and insulin sensitivity. In addition, PCS extract treatment significantly attenuated lipid accumulation in liver and adipose tissue and reduced serum lipid and hepatic triglyceride levels. Furthermore, the expression of lipogenic genes and inflammatory genes were reduced, and the expression of fat oxidation-related genes was increased in the liver of PCS extract-treated mice compared with control mice. Our study suggests the therapeutic potential of PCS extract for NAFLD by inhibiting lipid accumulation and inflammation in liver.


Introduction
Metabolic disorders such as obesity and type 2 diabetes have increased worldwide [1]. More than 200 million people suffer from diabetes and more than 1 billion people are overweight or obese [2]. Nonalcoholic fatty liver disease (NAFLD) refers to the spectrum of pathological conditions characterized by fatty infiltration of the liver, ranging from simple lipid accumulation to nonalcoholic steatohepatitis and to the fibrosis and cirrhosis that occur in the absence of alcohol consumption, viral infection, or other specific etiologies [3]. Because of its strong association with obesity and type 2 diabetes mellitus, NAFLD is now widely considered as characteristic of the metabolic syndrome with insulin resistance. A high-fat diet (HFD) is known to be linked to NAFLD, and HFD-induced lipotoxicity induces hepatic insulin resistance, which plays a major role in the pathogenesis of type 2 diabetes [4,5]. Thus, there is a critical need to clarify the mechanisms that mediate the development and progression of NAFLD and type 2 diabetes, and to identify potential therapies for the disease.
Despite of prevalence of NAFLD, effective therapy for NAFLD is not fully established. The current therapy for NAFLD consists of managing body weight, oxidative stress, insulin resistance, and lipid profile. Insulin receptor sensitizing agents such as thiazolidinediones and antioxidants such as Vitamin E have been tested for the treatment of NAFLD [6]. Because NAFLD is associated with several metabolic disorders, targeting drug development for NAFLD may be effective against obesity, type 2 diabetes, dyslipidemia, and oxidative stress as well as for the improvement of NAFLD.

Serum Lipid Profile
After 12 weeks of PCS extract treatment, blood samples were collected after 4 h of food deprivation. Blood samples were centrifuged at 3000 g for 20 min, and serum was collected. Serum levels of total cholesterol, triglycerides, low-density lipoprotein (LDL)-cholesterol and high-density lipoprotein (HDL)-cholesterol were measured using Beckman Coulter AU680 chemistry analyzer (Beckman Coulter, Inc. Brea, CA, USA).

Oil Red O Staining
Liver pieces were embedded in optimal cutting temperature compound. Frozen liver sections were cut at 10 µm thickness, fixed with 10% buffered formalin, dehydrated with 100% propylene glycol, and stained with 0.5% Oil Red O for 30 min at 55˝C. Sections were washed repeatedly with 85% propylene glycol, followed by distilled water. Then, sections were stained with hematoxylin. Lipid droplets were stained red.

Hematoxylin and Eosin Staining
The adipose tissues were fixed in 10% neutral buffered formalin, embedded in paraffin, and stained with hematoxylin and eosin. For staining, slides were deparaffinized by incubation in xylene, hydrated in a series of ethanol (100%, 95%, 80%, and 70%), washed in distilled water and stained with hematoxylin (Sigma-Aldrich, St. Louis, MO, USA) and followed by eosin (Sigma-Aldrich). After washing, sections were rapidly dehydrated in an ethanol series. Finally, the sections were washed in xylene and mounted.

Quantification of Liver Triglyceride Content
Liver tissue (50 mg) was digested with ethanolic KOH (2 parts EtOH: 1 part 30% KOH) overnight, and then KOH and distilled water were added to the digested solution. After centrifugation (1000 g for 5 min), the supernatant was transferred into a new microtube and mixed with 1 M MgCl 2 . The sample was incubated for 10 min on ice and then centrifuged at 1000 g for 5 min. Triglyceride content was measured in the upper phase solution using a TG-S kit (Asan Pharmaceutical Company, Seoul, Korea).

Quantitative Real-Time RT-PCR (qRT-PCR) Analysis
The total RNA was extracted from the liver tissue using TRIZOL reagent (Invitrogen Corp., Carlsbad, CA, USA), following the manufacturer's instructions, and cDNA was synthesized using a PrimeScript 1st strand cDNA synthesis kit (Takara Bio Inc., Kyoto, Japan). qRT-PCR was performed using the SYBR Premix Ex Taq II, ROX plus (Takara Bio Inc.) and the Prism 7900HT sequence detection system (Applied Biosystems, Foster City, CA, USA). PCR was carried out for 40 cycles (2 min at 50˝C, 10 min at 95˝C, and 40 cycles of 10 s at 95˝C and 1 minute at 60˝C). The primer sequences used are shown in Table 1. The relative copy number was calculated using the threshold crossing point (Ct) as calculated by ∆∆Ct.

Gene
Forward/Reverse Primers

Statistical Analyses
All data are expressed as mean˘standard error of at least three independent experiments. Data were analyzed using Analysis of Variance followed by post-hoc analysis using the Tukey range test (SPSS 10.0 statistical software, SPSS Inc., Chicago, IL, USA). p-values less than 0.05 were considered statistically significant.

PCS Extract Treatment Decreased Hemoglobin A1c, Blood Glucose Levels, and Body Weight Gain in HFD-Fed Mice
PCS extract (300 mg/kg/day or 500 mg/kg/day) was administered into C57BL/6 mice for 12 weeks during HFD feeding, and we compared changes in HbA1c levels, blood glucose levels, and body weight. The mice fed with HFD showed significantly increased HbA1c ( Figure 1A), blood glucose levels ( Figure 1B) and body weights ( Figure 1C) compared with regular chow diet-fed control mice. Treatment with PCS extract in HFD-fed mice reduced HbA1c and blood glucose levels, and body weight gain dose dependently with a significant reduction at a dose of 500 mg/kg/day compared with vehicle-treated HFD-fed mice (Fig 1 A-C). There were no differences in food intake among the vehicle and PSC extract treatment groups in HFD-fed mice ( Figure 1D). 500 mg/kg/day compared with vehicle-treated HFD-fed mice (Fig 1 A-C). There were no differences in food intake among the vehicle and PSC extract treatment groups in HFD-fed mice ( Figure 1D).

PCS Extract Treatment Improved Insulin Sensitivity and Glucose Tolerance in HFD-Fed Mice
To address whether PCS extract treatment improved insulin sensitivity, we performed insulin tolerance tests after 12 weeks of PCS treatment. PCS extract-treated HFD-fed mice showed an enhanced reduction in glucose levels in response to exogenous insulin at 90 min following insulin injection compared with vehicle-treated HFD-fed mice, indicating that insulin sensitivity was improved by PCS extract treatment ( Figure 2A). To determine whether blood glucose levels are properly controlled in PCS extract-treated mice, we performed glucose tolerance tests after 12 weeks of PCS treatment. Intraperitoneal glucose tolerance tests showed that a glucose load given to the normal control group produced a rapid increase in blood glucose levels at 30 min which returned to baseline values within 120 min ( Figure 2B). All HFD-fed mice showed hyperglycemia above 400 mg/dL 30 min after glucose loading. However, treatment with PCS extracts improved glucose tolerance dose dependently and treatment with 500 mg/kg/day of PCS extract resulted in a significant improvement in glucose tolerance compared with the vehicle-treated HFD-fed mice ( Figure 2C).

PCS Extract Treatment Improved Insulin Sensitivity and Glucose Tolerance in HFD-Fed Mice
To address whether PCS extract treatment improved insulin sensitivity, we performed insulin tolerance tests after 12 weeks of PCS treatment. PCS extract-treated HFD-fed mice showed an enhanced reduction in glucose levels in response to exogenous insulin at 90 min following insulin injection compared with vehicle-treated HFD-fed mice, indicating that insulin sensitivity was improved by PCS extract treatment ( Figure 2A). To determine whether blood glucose levels are properly controlled in PCS extract-treated mice, we performed glucose tolerance tests after 12 weeks of PCS treatment. Intraperitoneal glucose tolerance tests showed that a glucose load given to the normal control group produced a rapid increase in blood glucose levels at 30 min which returned to baseline values within 120 min ( Figure 2B). All HFD-fed mice showed hyperglycemia above 400 mg/dL 30 min after glucose loading. However, treatment with PCS extracts improved glucose tolerance dose dependently and treatment with 500 mg/kg/day of PCS extract resulted in a significant improvement in glucose tolerance compared with the vehicle-treated HFD-fed mice ( Figure 2C).

Figure 2.
Effect of PCS extract on insulin tolerance and glucose tolerance in HFD-fed mice. Mice were fed with regular chow diet (CON) or HFD for 12 weeks. HFD-fed were treated with vehicle or PCS extract (300 or 500 mg/kg/day) from the first day of HFD feeding (n = 7-10/group). (A) Insulin tolerance tests (ITT) were performed after 12 weeks of PCS treatment. Blood glucose levels were measured at the indicated times after insulin injection (1 U/kg Intraperitoneal (i.p.)); (B) Glucose tolerance tests (GTT). Blood glucose levels were measured at the indicated times after glucose load (2 g/kg i.p.); (C) Area under the curve (AUC) of GTT graph. * p < 0.05 vs. vehicle-treated HFD-fed mice.

PCS Extract Treatment Decreased Plasma Lipid Profiles in HFD-Fed Mice
We next investigated whether there are any changes in serum lipid levels after PCS extract treatment. Serum triglyceride levels were not increased by HFD, but PCS extract treatment significantly decreased serum triglyceride levels at a dose of 300 or 500 mg/kg/day ( Figure 3A   Effect of PCS extract on insulin tolerance and glucose tolerance in HFD-fed mice. Mice were fed with regular chow diet (CON) or HFD for 12 weeks. HFD-fed were treated with vehicle or PCS extract (300 or 500 mg/kg/day) from the first day of HFD feeding (n = 7-10/group). (A) Insulin tolerance tests (ITT) were performed after 12 weeks of PCS treatment. Blood glucose levels were measured at the indicated times after insulin injection (1 U/kg Intraperitoneal (i.p.)); (B) Glucose tolerance tests (GTT). Blood glucose levels were measured at the indicated times after glucose load (2 g/kg i.p.); (C) Area under the curve (AUC) of GTT graph. * p < 0.05 vs. vehicle-treated HFD-fed mice.

PCS Extract Treatment Decreased Plasma Lipid Profiles in HFD-Fed Mice
We next investigated whether there are any changes in serum lipid levels after PCS extract treatment. Serum triglyceride levels were not increased by HFD, but PCS extract treatment significantly decreased serum triglyceride levels at a dose of 300 or 500 mg/kg/day ( Figure 3A). Total cholesterol ( Figure 3B), HDL-cholesterol ( Figure 3C), and LDL-cholesterol ( Figure 3D) levels were significantly increased in HFD-fed mice as compared with regular chow diet-fed control mice. PCS extract treatment at both doses (300 and 500 mg/kg/day) significantly inhibited this increase of total cholesterol and LDL-cholesterol. HDL-cholesterol levels were slightly, but significantly inhibited by treatment with 500 mg/kg PCS extract ( Figure 3B-D).

PCS Extract Treatment Decreased Plasma Lipid Profiles in HFD-Fed Mice
We next investigated whether there are any changes in serum lipid levels after PCS extract treatment. Serum triglyceride levels were not increased by HFD, but PCS extract treatment significantly decreased serum triglyceride levels at a dose of 300 or 500 mg/kg/day ( Figure 3A). Total cholesterol ( Figure 3B), HDL-cholesterol ( Figure 3C), and LDL-cholesterol ( Figure 3D) levels were significantly increased in HFD-fed mice as compared with regular chow diet-fed control mice. PCS extract treatment at both doses (300 and 500 mg/kg/day) significantly inhibited this increase of total cholesterol and LDL-cholesterol. HDL-cholesterol levels were slightly, but significantly inhibited by treatment with 500 mg/kg PCS extract ( Figure 3B-D).

PCS Extract Treatment Decreased Lipid Accumulation in Liver and Adipose Tissue in HFD-Fed Mice
The accumulation of hepatic lipid during a HFD is a major cause of NAFLD [20]. To investigate the effects of PCS extract on the development of NAFLD in HFD-fed mice, we assessed the lipid content in the liver. Lipid droplet accumulation was obviously increased in HFD-fed mice, whereas the accumulation of lipid droplets was reduced in 300 or 500 mg/kg PCS extract-treated mice compared with vehicle-treated mice ( Figure 4A). Hepatic triglyceride levels, which were increased in HFD-fed mice, were significantly lower in PCS extract-treated mice ( Figure 4B). Histological analysis of epididymal adipose tissue sections by hematoxylin and eosin staining also showed smaller adipocytes in PCS extract-treated HFD-fed mice than in vehicle-treated HFD-fed mice ( Figure 4A).

PCS Extract Treatment Decreased Lipid Accumulation in Liver and Adipose Tissue in HFD-Fed Mice
The accumulation of hepatic lipid during a HFD is a major cause of NAFLD [20]. To investigate the effects of PCS extract on the development of NAFLD in HFD-fed mice, we assessed the lipid content in the liver. Lipid droplet accumulation was obviously increased in HFD-fed mice, whereas the accumulation of lipid droplets was reduced in 300 or 500 mg/kg PCS extract-treated mice compared with vehicle-treated mice ( Figure 4A). Hepatic triglyceride levels, which were increased in HFD-fed mice, were significantly lower in PCS extract-treated mice ( Figure 4B). Histological analysis of epididymal adipose tissue sections by hematoxylin and eosin staining also showed smaller adipocytes in PCS extract-treated HFD-fed mice than in vehicle-treated HFD-fed mice ( Figure 4A).

PCS Extract Treatment Decreased mRNA and Protein Expression of Genes for Lipid Metabolism and Hepatic Inflammation in Liver of HFD-Fed Mice
To investigate de novo lipogenesis in PCS extract treated mice, we measured the expression of sterol regulatory element binding protein (SREBP)-1c, stearoyl-coenzyme A desaturase (SCD) 1 and fatty acid synthase (FAS) mRNA, which are involved in lipogenesis. We found that SREBP1c and SCD1 mRNA levels were significantly decreased in PCS extract-treated HFD-fed mice as compared with vehicle-treated HFD-fed mice ( Figure 5A,B). FAS mRNA levels declined in PCS extract-treated HFD-fed mice but this was not significant ( Figure 5C).

PCS Extract Treatment Decreased mRNA and Protein Expression of Genes for Lipid Metabolism and Hepatic Inflammation in Liver of HFD-Fed Mice
To investigate de novo lipogenesis in PCS extract treated mice, we measured the expression of sterol regulatory element binding protein (SREBP)-1c, stearoyl-coenzyme A desaturase (SCD) 1 and fatty acid synthase (FAS) mRNA, which are involved in lipogenesis. We found that SREBP1c and SCD1 mRNA levels were significantly decreased in PCS extract-treated HFD-fed mice as compared with vehicle-treated HFD-fed mice ( Figure 5A,B). FAS mRNA levels declined in PCS extract-treated HFD-fed mice but this was not significant ( Figure 5C). A previous in vitro study showed that PCS extract could induce activation of mitochondrial function and synthesis [18]. Therefore, we examined the effects of PCS extracts on the mRNA and protein expression of PGC1α and CPT1α, which are involved in fatty acid oxidation, in the liver of PCS extract-treated mice. mRNA expression of PGC1α ( Figure 5D) and CPT1α ( Figure 5E) was not changed by PCS extract treatment, but protein levels of PGC1α and CPT1α were increased in the liver of PCS-treated mice compared with vehicle-treated mice ( Figure 5F).
The proinflammatory cytokines are associated with the pathogenesis of NAFLD and contribute to the increased risk for nonalcoholic steatohepatitis and liver cirrhosis [21]. To investigate whether PCS extract treatment affects liver inflammation, we measured the expression of inflammatory molecules such as interleukin (IL)-1β, monocyte chemoattractant protein (MCP) 1 and suppressor of cytokine signaling (SOCS) 3. We found that mRNA expression of IL-1β ( Figure 6A), MCP1 ( Figure 6B) and SOCS3 ( Figure 6C) was significantly decreased in PCS extract-treated HFD-fed mice as compared with vehicle-treated HFD-fed mice. Figure 5. Effect of PCS extract on mRNA and protein expression of lipid metabolism in liver. Mice were fed with regular chow diet (CON) or HFD for 12 weeks. HFD-fed mice were treated with vehicle (´) or PCS extract (300 or 500 mg/kg/day) from the first day of HFD feeding (n = 7-10/group). After 12 weeks of PCS extract treatment, total RNA was extracted from the liver tissue and qRT-PCR analysis was performed for (A) sterol regulatory element binding protein (SREBP1); (B) stearoyl-coenzyme A desaturase (SCD1); (C) fatty acid synthase (FAS); (D) proliferator-activated receptor γ coactivator (PGC1α); and (E) carnitine palmitoyltransferase (CPT1α); (F) Total protein was prepared and Western blotting analysis was carried out for CPT1 and PGC1. * p < 0.05 vs. vehicle-treated HFD-fed mice.
A previous in vitro study showed that PCS extract could induce activation of mitochondrial function and synthesis [18]. Therefore, we examined the effects of PCS extracts on the mRNA and protein expression of PGC1α and CPT1α, which are involved in fatty acid oxidation, in the liver of PCS extract-treated mice. mRNA expression of PGC1α ( Figure 5D) and CPT1α ( Figure 5E) was not changed by PCS extract treatment, but protein levels of PGC1α and CPT1α were increased in the liver of PCS-treated mice compared with vehicle-treated mice ( Figure 5F).
The proinflammatory cytokines are associated with the pathogenesis of NAFLD and contribute to the increased risk for nonalcoholic steatohepatitis and liver cirrhosis [21]. To investigate whether PCS extract treatment affects liver inflammation, we measured the expression of inflammatory molecules such as interleukin (IL)-1β, monocyte chemoattractant protein (MCP) 1 and suppressor of cytokine signaling (SOCS) 3. We found that mRNA expression of IL-1β ( Figure 6A), MCP1 ( Figure 6B) and SOCS3 ( Figure 6C) was significantly decreased in PCS extract-treated HFD-fed mice as compared with vehicle-treated HFD-fed mice.

Discussion
Excessive energy consumption induces fat accumulation in adipose tissue and the liver, leading to obesity and fatty liver disease [22], which are emerging health problems. Natural products including green tea extract, coffee and several extracts of medical plants have been hypothesized to prevent NAFLD or its progression via several mechanisms, such as sensitizing insulin effects, activating adiponectin expression, and down-regulating pro-inflammatory cytokines, by antioxidant effects or by anti-dyslipidemic properties [23][24][25].
The seeds of Psoralea corylifolia (PCS), commonly known as "Boh-Gol-Zhee" in Korea, have been used in herbal and traditional medicine. In a previous study, we found that PCS extract has anti-oxidative effects in hepatocytes and pancreatic β-cells [17,18]. NAFLD patients' lowered antioxidant capacity has led to the idea that PCS extract might have beneficial effects on NAFLD.
According to the "multihit" hypothesis, disrupted lipid metabolism and insulin resistance are the first step towards NAFLD development [26]. In our study, HFD-fed mice showed a significant increase of serum lipid and blood glucose levels, impaired glucose tolerance and insulin resistance. However, hyperglycemia, increase of HbA1c levels, glucose intolerance and insulin resistance associated with HFD were ameliorated by PCS extract treatment. High levels of serum lipid mediates lipotoxicity by inducing lipid over-accumulation through insulin resistance [27]. In our study, PCS extract treatment significantly lowered the HFD-induced rise in serum lipid levels, indicating that the decrease of serum lipid might have contributed to ameliorating insulin resistance, resulting in lowered free fatty acids (FFA) influx to liver.
The liver plays an important role in whole-body energy homeostasis, and thus, its functional disorder has relevance for metabolic syndrome and diabetes. The liver not only takes up FFA from the diet and adipose tissue, but also participates in the de novo synthesis of FFA by their conversion into triglycerides through esterification. Also, hepatic triglycerides can be released again into circulation as very low-density lipoproteins, and excess FFA in liver which are not synthesized into triglycerides are used by β -oxidation [28]. NAFLD occurs when this regulation is disrupted in the liver, leading to hepatic steatosis [29]. SREBP1c, a key player in hepatic lipogenesis, activates nearly all genes required for de novo synthesis of fatty acid and triglyceride synthesis [30], and SCD1 is the rate-limiting enzyme involved in the biosynthesis of monounsaturated fatty acids [31]. Unexpectedly, HFD significantly decreased the gene expression of SREBP1c and SCD1 compared with regular chow diet, possibly because excessive fat was already present and there was no need Figure 6. Effect of PCS extract on mRNA and protein expression of hepatic inflammation. Mice were fed with regular chow diet (CON) or HFD for 12 weeks. HFD-fed were treated with vehicle (´) or PCS extract (300 or 500 mg/kg/day) from the first day of HFD feeding (n = 7-10/group). After 12 weeks of PCS extract treatment, total RNA was extracted from the liver tissue qRT-PCR analysis was performed for (A) interleukin-1β (IL-1β); (B) monocyte chemoattractant protein 1 (MCP1); and (C) suppressor of cytokine signaling 3 (SOCS3). * p < 0.05 vs. vehicle-treated HFD-fed mice.

Discussion
Excessive energy consumption induces fat accumulation in adipose tissue and the liver, leading to obesity and fatty liver disease [22], which are emerging health problems. Natural products including green tea extract, coffee and several extracts of medical plants have been hypothesized to prevent NAFLD or its progression via several mechanisms, such as sensitizing insulin effects, activating adiponectin expression, and down-regulating pro-inflammatory cytokines, by antioxidant effects or by anti-dyslipidemic properties [23][24][25].
The seeds of Psoralea corylifolia (PCS), commonly known as "Boh-Gol-Zhee" in Korea, have been used in herbal and traditional medicine. In a previous study, we found that PCS extract has anti-oxidative effects in hepatocytes and pancreatic β-cells [17,18]. NAFLD patients' lowered antioxidant capacity has led to the idea that PCS extract might have beneficial effects on NAFLD.
According to the "multihit" hypothesis, disrupted lipid metabolism and insulin resistance are the first step towards NAFLD development [26]. In our study, HFD-fed mice showed a significant increase of serum lipid and blood glucose levels, impaired glucose tolerance and insulin resistance. However, hyperglycemia, increase of HbA1c levels, glucose intolerance and insulin resistance associated with HFD were ameliorated by PCS extract treatment. High levels of serum lipid mediates lipotoxicity by inducing lipid over-accumulation through insulin resistance [27]. In our study, PCS extract treatment significantly lowered the HFD-induced rise in serum lipid levels, indicating that the decrease of serum lipid might have contributed to ameliorating insulin resistance, resulting in lowered free fatty acids (FFA) influx to liver.
The liver plays an important role in whole-body energy homeostasis, and thus, its functional disorder has relevance for metabolic syndrome and diabetes. The liver not only takes up FFA from the diet and adipose tissue, but also participates in the de novo synthesis of FFA by their conversion into triglycerides through esterification. Also, hepatic triglycerides can be released again into circulation as very low-density lipoproteins, and excess FFA in liver which are not synthesized into triglycerides are used by β-oxidation [28]. NAFLD occurs when this regulation is disrupted in the liver, leading to hepatic steatosis [29]. SREBP1c, a key player in hepatic lipogenesis, activates nearly all genes required for de novo synthesis of fatty acid and triglyceride synthesis [30], and SCD1 is the rate-limiting enzyme involved in the biosynthesis of monounsaturated fatty acids [31]. Unexpectedly, HFD significantly decreased the gene expression of SREBP1c and SCD1 compared with regular chow diet, possibly because excessive fat was already present and there was no need for lipid synthesis. In fact, similar results have been reported [32,33] and it was reported that HFD did not induce lipogenic gene expression, despite fatty liver induction [34,35]. These differences in lipogenic gene expression might be dependent on the duration of HFD. Regardless, the expression of SREBP1c and SCD1 mRNA was further decreased by PCS extract treatment.
CPT1 is associated with the mitochondrial outer membrane and regulates energy production from the main oxidative substrates [36]. PGC1α controls many aspects of lipid β-oxidation, mitochondrial biogenesis and respiration [37]. Protein levels of CPT1 and PGC1α were increased by PCS extract treatment. These results suggest that PCS extract treatment improves NAFLD through regulation of overall hepatic lipid metabolism.
It is known that PCS extract and bakuchiol, which is the main component of PCS, have anti-inflammatory effect in macrophages [38]. Hepatic inflammation is a critical event in the progression of NAFLD and may exacerbate lipid-mediated injuries [39]. Because NAFLD is strongly associated with hepatic inflammation, the gene expression of inflammatory markers were measured in the liver of PCS extract-treated mice given a HFD. As in the case of lipogenic gene expression, the HFD did not induce the mRNA expression of IL-1βand MCP1 in the present study, which agrees with the study of Lei Zhao et al. [40]. It is possible that a HFD was not enough to induce nonalcoholic steatohepatitis and hepatic inflammation in the fatty liver. However, a HFD did induce the expression of SOCS3. PCS treatment reduced the gene expression of IL-1β, MCP1 and SOCS3.

Conclusions
In conclusion, we showed the ameliorative effects of PCS extract on HFD-induced NAFLD in mice. PCS extract treatment decreased body weight gain and serum lipid levels in HFD-fed mice. Lipid accumulation in liver and adipose tissue was decreased probably due to the decrease of lipogenic gene expression and increase of lipid β-oxidation related gene expression. In addition, PCS extract treatment reduced inflammatory gene expression. Thus, these results provide insights into the therapeutic potential of PCS extract in the management of NAFLD.