Sijunzi, Lizhong, and Fuzilizhong Decoction Alleviate Nonalcoholic Fatty Liver Disease through Activation of PPAR Pathway

Objective Sijunzi, Lizhong, and Fuzilizhong decoction were traditional Chinese classic formulations, which are widely used in clinical treatment, and the underlying mechanism is unclear. In this study, we aim to investigate the molecular mechanisms underlying the protective effects of Sijunzi, Lizhong, and Fuzilizhong on nonalcoholic fatty liver disease (NAFLD). Methods Male Wistar rats were fed a high-fat diet for four weeks to induce NAFLD and were thereafter administered Sijunzi (8 g/kg/d), Lizhong (10 g/kg/d), or Fuzilizhong (10 g/kg/d) by gavage for four weeks. Hepatic damage, lipid accumulation, inflammation, autophagy, and peroxisome proliferator-activated receptor-α signaling were evaluated. Results The high-fat diet-fed rats showed typical symptoms of NAFLD, including elevated levels of hepatic damage indicators, increased hepatic lipid deposition and fibrosis, severe liver inflammation, and prominent autophagy. Upon administration of Sijunzi, Lizhong, and Fuzilizhong, liver health was improved remarkably, along with ameliorated symptoms of NAFLD. In addition, NAFLD-suppressed peroxisome proliferator-activated receptor-α signaling was reactivated after treatment with the three types of decoctions. Conclusions The results collectively signify the effective therapeutic and protective functions of Sijunzi, Lizhong, and Fuzilizhong against NAFLD and demonstrate the potential of Chinese herbal medication in mitigating the symptoms of liver diseases. Novelty of the Work. Traditional Chinese herbal medicine has been used for centuries to treat various diseases, but the molecular mechanisms of individual ingredients have rarely been studied. The novelty of our work lies in elucidating the specific signaling pathways involved in the control of NAFLD using three common Chinese herbal decoctions. We suggest that natural herbal formulations can be effective therapeutic agents to combat against NAFLD.


Introduction
e prevalence of nonalcoholic fatty liver disease (NAFLD) is increasing as overnutrition and obesity have become common among the worldwide population [1,2]. NAFLD is characterized by necroinflammation and fat accumulation in the liver without significant alcohol consumption [3] and is closely associated with central abdominal obesity, diabetes, and dyslipidemia [4,5]. e accumulation of triglycerides in hepatocytes could lead to the development of hepatic steatosis, whereas cellular stresses, such as oxidative stress, endotoxin stimulation, lipid peroxidation, and secretion of inflammatory cytokines can contribute to NAFLD [6]. Although the exact pathogenesis of NAFLD is not well understood, increased lipid accumulation, inflammation, autophagy, and oxidative stress are known to play important roles therein [7].
Traditional Chinese herbal medicine has been highlighted in the treatment of hepatic diseases such as NAFLD, as the use of natural herbal extracts is advantageous in preventing or mitigating undesired side effects. e theory of traditional Chinese medicine proposes that spleen dysfunction, damp-heat, and phlegm and stasis are the key manifestations of NAFLD. A special characteristic of Chinese herbal medicine is the preparation of a formulation containing several herbs to improve a set of abnormal syndromes associated with a disease. Herbal extracts contain a variety of natural compounds that target specific pathological pathways and provide therapeutic effects through a series of actions [8]. For example, the Sijunzi decoction (SJZ) has the functions of tonifying qi and strengthening the spleen, whereas the Lizhong (LZ) and Fuzilizhong (FZLZ) decoctions promote the replenishment of the spleen and warming of Yang. Previous studies have reported that SJZ, LZ, and FZLZ alleviated lipid peroxidation, liver fibrosis, and inflammatory reaction [9][10][11]. Of the major components of SJZ, LZ, and FZLZ decoctions, Codonopsis pilosula (Franch.) Nannf., Atractylodes macrocephala Koidz., and Glycyrrhiza uralensis Fisch. have demonstrated protective effects on the liver. us, it is essential to investigate the molecular mechanisms underlying the hepatoprotective effect of SJZ, LZ, and FZLZ against NAFLD.
A variety of factors regulate the synthesis, storage, and export of hepatic triglycerides and may thereby affect the magnitude of NAFLD. Among them, peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors belonging to the nuclear receptor superfamily, with three known subtypes: PPAR-α, −c, and −β/δ [12]. PPAR-α is found mainly in the liver, kidney, and heart and controls the expression of genes involved in lipid and glucose metabolism [13]. e inhibition of PPAR-α was shown to increase cellular lipids and reduce the β-oxidation of fatty acids [14]. PPAR-α also negatively regulated acute phase response signaling pathways and proinflammatory reaction, as demonstrated in rodent models of NAFLD, atherosclerosis, systemic inflammation, and nonalcoholic steatohepatitis [15,16]. Other factors contributing to NAFLD include sterol regulatory element-binding proteins (SREBPs), which are master transcription factors belonging to the basic helix-loop-helix leucine zipper family [17]. e activation of SREBP-1c triggers the expression of genes responsible for encoding enzymes that mediate hepatic glycogen and triglyceride synthesis, suggesting the potential role of SREBP-1c in the progression of NAFLD [18].
is study investigates the therapeutic effects of the three abovementioned decoctions used in Chinese herbal medicine (SJZ, LZ, and FZLZ) in a rat model of NAFLD. e extent of NAFLD and related indicators of disease progression were evaluated, and the possible involvement of the PPAR-α signaling pathway and SREBP-1c in the regulation of NAFLD progression was examined.
For the preparation of SJZ and LZ, all ingredients were placed in a glass beaker and soaked in 400 mL of water for 30 min. e ingredients were then boiled at max heat, and the heat was reduced so that the decoction continued to simmer. After 20 min, the decoction was separated using a double-layer gauze sheet. e solid ingredients were then added to 350 mL of water, and the boiling steps were repeated.
e filtered liquid portions from the two boiling steps were combined and concentrated accordingly. For the preparation of FZLZ, processed Chinese aconite was first soaked in 150 mL of water for 30 min, boiled at max heat, and simmered in low heat for 30 min. e other ingredients were placed in a glass beaker and soaked in 400 mL of water for 30 min. After boiling in max heat, the processed Chinese aconite was added, and the mixture was simmered at low heat for another 20 min. e decoction was separated using a double-layer gauze sheet. e solid ingredients were then added to 350 mL of water, and the boiling steps were repeated. e filtered liquid portions from the two boiling steps were combined and concentrated accordingly.

Detection of Active Ingredients in SJZ, LZ, and FZLZ.
e active ingredients of SJZ, LZ, and FZLZ were detected using the ultrahigh performance liquid chromatography (UHPLC). A 100 μl of aliquot concentrated SJZ, LZ, and FZLZ was extracted using 300 μl of methanol via vortex mixing for 30 s. After ultrasound in an ice-water bath for 1 h, the specimen was centrifugated at 12000 rpm at room temperature for 10 min. en a 5 μl aliquot of the supernatant was harvested and analyzed by UHPLC (Agilent, CA, USA), equipped with a UPLC BEH C18 Column (1.7 μm, 2.1 × 100 mm, Waters, Massachusetts, USA). e peak strength is usually proportional to the compound content. In the current work, the compounds in SJZ, LZ, and FZLZ with LQ.POS.B (stands for the signal strength of the drug component) >10000 were selected as active substances.

Animals and Treatments.
All experimental procedures were approved by the Animal Ethics Committee of Wuhan Integrated TCM and Western Medicine Hospital (No. 42000600013948). Specific-pathogen-free male Wistar rats (8 weeks of age, 180-200 g) were obtained from the Animal Center of Hubei (Wuhan, China). e rats were maintained under conditions of constant temperature (21 ± 1°C) and humidity (50 ± 15%) in a 12 h/12h light/dark cycle, with free access to deionized water and fed irradiated disinfectant food. After one week of adaptive feeding, control rats (n � 8) were fed a standard chow diet and experimental rats (n � 32) were fed a high-fat diet for four weeks to induce NAFLD. e high-fat diet was composed of a mixture of two solutions, prepared as follows [19,20]. Solution A contained a mixture of 25 g axungia dissolved by heating, 10 g of cholesterol, and 1 g of propylthiouracil tablets. After stirring, 25 mL of Tween 80 was added and mixed until use. Solution B contained 20 mL of propylene glycol in 30 mL of water. e solution was heated to 60°C, after which 2 g of sodium deoxycholate was added and mixed until use. Solutions A and B were mixed and 100 mL of water was added. e rats were given the same amount of standard chow diet and highfat diet by gavage daily at 10 : 00 in the morning. ose fed the high-fat diet to induce NAFLD were randomly divided into four groups (n � 8 per group). Drug dosage was obtained based on clinical dose conversion according to the guide for dose conversion between animals and human [21] and treated as follows: no further treatment (NAFLD), SJZ (8 g/kg/d), LZ (8 g/kg/d), and FZLZ (10 g/kg/d). e medicine was administered by gavage for four weeks, and rats in the control and NAFLD groups were given the same volume of normal saline by gavage for four weeks.

Oil Red O Staining.
Fresh liver tissue was stored at −80°C for histopathological and molecular assays. Frozen sections were stained with Oil Red O (Bioswamp Life Science Lab, Wuhan, China) to investigate the architecture of the hepatic lipid droplets in the liver. Slides stained with Oil Red O were visualized with an Olympus microscope and images were captured with an Olympus digital camera.

Statistical Analysis.
All data were analyzed with the statistical software SPSS 19.0 and expressed as the mean-± standard deviation (SD). Multigroup comparisons of the means were carried out by one-way analysis of variance with post hoc contrasts assessed by the Student-Newman-Keuls test. P < 0.05 was considered to be statistically significant.

General Condition.
We observed the general situation of the mice throughout the experiment, and all groups of mice did not restrict the supply of food and water. e results showed that compared with the control group, the weight of mice in the NAFLD group increased significantly (P < 0.05), compared with the NAFLD group, the weight of mice in the SJZ, LZ, and FZLZ groups decreased, and the weight of mice decreased gradually with the extension of the intervention time (P < 0.05). No rats died during the whole experimental intervention, suggesting that SJZ, LZ, and FZLZ have good safety (Table 1).

SJZ, LZ, and FZLZ Attenuated Hepatic Levels of NAFLD
Indicators. Rats subjected to a high-fat diet showed higher levels of TC, TG, ALT, HDL-C, and LDL-C ( Figure. 1) in liver tissues compared to those fed a normal diet (P < 0.05), suggesting the successful induction of NAFLD-like symptoms model. Administration of SJZ, LZ, and FZLZ after NAFLD induction significantly reduced the hepatic levels of the abovementioned indicators to varying degrees (P < 0.05). In particular, FZLZ resulted in lower levels of ALT than did SJZ (P < 0.05) and led to further reduced TG content than did LZ (P < 0.05).

SJZ, LZ, and FZLZ Reduced Hepatic Lipid Accumulation.
As is shown in Figure 2, the liver tissue of rats subjected to the high-fat diet (NAFLD) showed a tremendous amount of lipid accumulation. e administration of SJZ, LZ, and FZLZ accordingly reduced the degree of hepatic lipid deposition, as demonstrated by the lighter Oil Red O staining. FZLZ seemed to have a more prominent effect in reducing lipid accumulation than those of SJZ and LZ.

SJZ, LZ, and FZLZ Suppressed Liver Fibrosis.
e degree of liver fibrosis was evaluated by Masson's trichrome and Sirius Red staining for collagen and connective tissues, respectively (Figure 3(a)). In the liver of rats fed a high-fat diet (NAFLD), Masson's trichrome staining revealed significant areas of blue staining indicative of distinct collagen deposition and fibrosis, while Sirius Red staining showed a similar phenomenon for connective tissues. When NAFLD-induced rats were treated with SJZ, LZ, and FZLZ, their livers showed remarkable reductions in fibrotic areas. Immunohistochemistry further demonstrated that the hepatic expression of β-catenin and Axin   (Figure 3(a)). In addition, NAFLD induced a significant increase in the mRNA expression of TGF-β1, PAI-1, collagen I, and α-SMA (Figure 3(b)) and promoted the secretion of PDGF, FGF-2, and VEGF (Table 3), whereas these indicators were all downregulated by SJZ, LZ, and FZLZ (P < 0.05), with FZLZ exhibiting the greatest effect in suppressing liver fibrosis (P < 0.05).

SJZ, LZ, and FZLZ Mitigated NAFLD-Associated Inflammation and Autophagy.
To determine the effect of SJZ, LZ, and FZLZ on liver autophagy, the expression of several proteins associated with inflammation (IL-6, IL-12, iNOS, TNF-α, and IL-1β) and autophagy (LC3B) was detected in liver tissues, as summarized in Table 2 and Figure 4. e expression levels of IL-6, IL-12, iNOS (Table 4), TNF-α, IL-1β (Figures 4(a) and 4(b)), and LC3B (Figures 4(c) and 4(d)) in the liver of NAFLD-induced rats were higher than those ese proteins were downregulated by the administration of SJZ, LZ, and FZLZ (P < 0.05) (except for SJZ, which did not seem to affect TNFα expression), with FZLZ exerting a greater effect than those of SJZ and LZ (P < 0.05). e results revealed that the three decoctions had suppressive effects on liver inflammation and autophagy induced by NAFLD.

Effect of SJZ, LZ, and FZLZ on PPAR Pathway.
e regulatory role of the PPAR pathway in NAFLD was examined by western blot detection (Figure 5(a)) and quantification ( Figure 5(b)) of proteins relevant to the PPAR signaling pathway. In rats fed a high-fat diet (NAFLD), the protein levels of PPAR-α, CPT-1, and CPT-2 were downregulated while that of SREBP-1c was elevated (P < 0.05), suggesting the inhibition of PPAR-α and activation of SREBP-1c. Upon administration of SJZ, LZ, and FZLZ, the expression of PPAR-α, CPT-1, and CPT-2 was upregulated  while that of SREBP-1c was reduced (P < 0.05) to various extents, indicating that the three decoctions ameliorated the symptoms of NAFLD by promoting PPAR-α and inhibiting SREBP-1c.

Discussion
e aim of the present study was to reveal the molecular mechanisms underlying the protective effects of SJZ, LZ, and FZLZ against NAFLD. NAFLD is identified by the overaccumulation of lipids, triglycerides, and cholesterol [22] and is associated with lipid metabolic disorders, inflammatory cytokine secretion, oxidative stress, autophagy, and changes in related signaling pathways [23]. Another pathological feature of NAFLD is hepatic fibrosis, which is considered to signify the end stage of chronic liver diseases and often leads to cirrhosis [24]. A variety of factors, including PDGF, FGF-2, VEGF, β-catenin, Axin 2, TGF-β1, PAI-1, collagen I, and α-SMA, are key mediators of hepatic fibrosis [25,26]. As expected, rats fed a high-fat diet for four weeks displayed a series of NAFLD-associated symptoms, including high hepatic TG, TC, ALT, HDL-C, and LDL-C levels (Figure 1), lipid accumulation (Figure 2), and fibrosis ( Figure 3) in liver tissues. e administration of SJZ, LZ, and FZLZ accordingly attenuated the levels of liver damage induced by NAFLD.
Recently, increasing evidence has shown the vital role of autophagy in the development and progression of NAFLD [27]. Autophagy, a cell survival mechanism, is essential for the regulation of hepatic lipid metabolism. Autophagic dysfunction may increase lipid accumulation in the liver, thus participating in the emergence and progression of NAFLD [28]. e upregulation of the autophagy-lysosomal pathway might promote lysosomal lipid absorption and eventually alleviate hyperlipidemia-associated symptoms [29]. Liu et al. reported that the hepatic levels of TNF-α, IL-1β, and LC3B in mice fed a high-fat diet were upregulated as  Evidence-Based Complementary and Alternative Medicine a result of hyperlipidemia. However, liver inflammation was alleviated by rutin as evidenced by the downregulation of TNF-α and IL-1β in liver tissues, and autophagy was reduced as demonstrated by the downregulation of LC3B in liver tissues [30]. ese observations are in agreement with the results of the current study, whereby SJZ, LZ, and FZLZ suppressed liver inflammation due to NAFLD, as suggested by the decrease in IL-6, IL-12, iNOS, TNF-α, and IL-1β expression in liver tissues (Table 2 and Figure 4). Furthermore, the expression of LC3B in liver tissues, which reflects hepatic autophagy, was upregulated by NAFLD but downregulated by SJZ, LZ, and FZLZ ( Figure 4). Lipid homeostasis is controlled by the delicate balance between lipid synthesis and metabolism. Increased uptake of fatty acids in the liver could lead to lipid accumulation, and lipid-related diseases often involve the dysregulation of lipid homeostasis. PPAR-α, which is associated with hepatic lipid accumulation, is strongly expressed in the heart, intestinal mucosa, kidney, brown fat, skeletal muscle, and liver [31]. It regulates the expression of genes involved in lipoprotein metabolism, fatty acid oxidation, cholesterol catabolism, ketogenesis, and gluconeogenesis [32]. e decrease in PPARα often results in hyperlipidemia, whereas its upregulation leads to increased β-oxidation activity of fatty acids and lower hepatic TG levels [33,34]. e present study showed that the downregulation of PPAR-α due to NAFLD was counteracted by SJZ, LZ, and FZLZ. In addition, NAFLD caused a decrease in the protein levels of CPT-1 and CPT-2 and an increase in that of SREBP-1c, whereas the three decoctions counteracted the effects of NAFLD. CPT-1 and CPT-2, two mitochondrial membrane-associated enzymes, together with control fatty acid influx into mitochondria where β-oxidation occurs [35]. Meanwhile, SREBP-1c plays a pivotal role in the development and pathogenesis of NAFLD [36], and the downregulation of SREBP-1c reportedly suppressed de novo fatty acid synthesis in fatty acid overload [37]. Herein, the reduction of hepatic lipid accumulation mediated by SJZ, LZ, and FZLZ was accompanied by the activation of PPAR-α signaling and the inhibition of SREBP-1c.
In this study, we found that SJZ, LZ, and FZLZ can effectively alleviate hepatic lipid accumulation and fibrosis and thus play a role in the treatment of NAFLD. According to the TCM theory, FZLZ is better than LZ in treating fatty liver with Yang deficiency (aversion to cold, pale complexion, etc.), while SJZ is better than LZ in treating fatty liver with qi deficiency (limb weakness, dizziness, pale complexion, etc.). e common active substances of SJZ, LZ, and FZLZ were Ononin, Licoisoflavone A, Codonopsine, and Liquiritigenin by HPLC analysis, suggesting that Ononin, Licoisoflavone A, Codonopsine, and Liquiritigenin may be effective active substances for the treatment of NAFLD. In this study, we mainly studied the effects of FZLZ, SJZ, and LZ on NAFLD and its potential mechanisms, and we will further study the effects of Ononin, Licoisoflavone A, Codonopsine, and Liquiritigenin on NAFLD in the next experiments.
Taken together, the results summarized here indicate that SJZ, LZ, and FZLZ exert protective effects against liver damage by attenuating hepatic lipid accumulation and fibrosis. e three decoctions also ameliorated the symptoms of NAFLD by mitigating liver autophagy and regulating PPAR-α and its downstream target enzymes.
Data Availability e datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.