NAFLD is a metabolic related syndrome characterized as dysfunctional hepatic lipid metabolism and insulin resistance [52–53], which has been considered the fastest growing cause of HCC [54] and strongly associated with the increasing risks of type 2 diabetes, cardiovascular disease, chronic kidney disease and hypertension [55–57]. Currently, no pharmacological therapies were approved by FDA in the treatment of NAFLD and seeking an effective agent is urgently desiderated. TCM has been proved to treat NAFLD with the unique advantages of holistic concept and differentiation treatment, as well as mechanisms of multi-target and multi-channel action [27–58–59](34344394; 31558860; 33639194). Shugan Xiaozhi (SGXZ) decoction was proposed and designed with insights gained from two classic prescriptions of TCM - “Sini decoction” and “Yinchenhao decoction”, and showed a good efficacy for the treatment of NAFLD [32–60]. In addition, both of Sini decoction and Yinchenhao decoction exerted therapeutic effect for NAFLD and NASH [61–63]. Nevertheless, the underlying therapeutic mechanisms of SGXZ decoction remained to be further elucidated.
In the present study, active compounds of SGXZ decoction with corresponding targets were firstly identified, and the overlapping targets between SGXZ decoction and NAFLD were considered to be the main targets these compounds acted on. Moreover, compounds including series of phenolic acids and flavonoids were selected as the key bioactive ingredients for the treatment of SGXZ decoction on NAFLD based on their contributions in the compound-target-disease network (Figure2, Table 1). Previous study indicated that the intake of phenolic acids alleviated hepatic steatosis, reduced fibrosis and the insulin resistance in NAFLD patients [64]. Gallic acid, a simple polyphenol, was reported to reduce lipid accumulation that is related to β-oxidation and ketogenesis [65]. Specifically, the hepatoprotective effect of gallic acid attributed to the repression of inflammatory signaling pathways including nuclear factor-κB (NF-κB)/TNF-α/IL-6 and ROS/NF-κB /TNF-α in NAFLD rats [66–67]. Chlorogenic acid, a natural polyphenol extracted from Artemisiae Scopariae (Yin-Chen in Chinese) [68] and Lonicera japonica (Jin-Yin-Hua) [69], could ameliorate HFD-induced hepatic steatosis and inflammation via inhibition of TNF-α and IL-6 in liver, which was associated with regulation of gut microbiota and an increase of Glucagon-like peptide-1 secretion [70–71]. Isochlorogenic acid A was suggested to possess properties of hepatoprotective and anti-hepatitis B through suppressing oxidation [72]. Moreover, it was indicated that isochlorogenic acid A exerted a protective effect on liver fibrosis through inhibiting inflammation via HMGB1/TLR4/NF-kB signaling pathways [73]. In addition, flavonoids are natural products widely distributed in plants, exhibiting not only antidiabetic and hypoglycemic activities but also anti-inflammatory and antioxidant properties, and were recognized to have protective effect in the treatment of NAFLD [74–76]. As a kind of flavonoid, liquiritin could ameliorate cyclophosphamide-induced liver injury and inflammation by inhibiting the elevated MMP-9 expression, hepatic infiltration of neutrophils, myeloperoxidase activity, IL-6 mRNA expression and NF-κB activation [77]. Naringin is a flavanone glycoside isolated from Aurantii Fructus Immaturus (Zhi-Shi) [78] and has been proved to improve lipid metabolism disorders through reducing hepatic lipid accumulation in tissue-engineered NAFLD model [79]. Neohesperidin could induce the PGC-1α expression through activating AMP-activated protein kinase (AMPK), increasing hepatic mitochondrial biogenesis and fatty acid oxidation in NAFLD mice [80]. To sum up, three phenolic acids (gallic acid, chlorogenic acid, isochlorogenic acid A) and three flavonoids (liquiritin, naringin, neohesperidin) with hepatoprotective effect could serve as the potentially main active ingredients of SGXZ decoction for the treatment of NAFLD.
Together with key targets in compound-target-disease network (Figure 3, Table 2), 10 targets with highest degree in the PPI network were as well recognized as core targets of SGXZ decoction treating NAFLD (Figure 4). The target protein level of VEGFA, FGF1, IL-2, LGALS3, SLC5A1 and IL-6 were excessively expressed in compound-target-disease network. Moreover, IL-6 possessed the highest degree in PPI network, followed by VEGFA, ESR1, HIF1A, MMP9, HSPA5, RELA and IL-2. Particularly, IL-6, IL-2 and VEGFA contributed significantly to the construction of both compound-target-disease and PPI networks, indicating their critical role among the targets. IL-6 has been considered to cause inflammatory properties and a vital factor associated with the pathology of metabolic disorders [81]. Numerous studies have highlighted that increased IL-6 level promoted hepatic insulin resistance [82] and impaired the lipid metabolism [83]. IL-6 deficiency ameliorated the hepatic inflammation and injury in NASH mice fed with methionine and choline-deficient diet [84]. Similarly, upregulated IL-2 potentially leads to the increased insulin resistance as well as several other metabolic inflammatory markers in obese population [85]. VEGFA is a major proangiogenic cytokine regulating angiogenesis. Increased VEGFA was reported in liver of animal model and serum of NASH patients [86–87], which accelerates angiogenesis and therefore drives hepatic inflammation and fibrosis in NAFLD [87].
Hepatic steatosis and lipid accumulation could produce a hypoxic proangiogenic microenvironment, while the respond to this condition was mediated by hypoxia-inducible factors (HIFs) [88–89]. Hypoxia upregulated the HIF1A expression in liver, leading to the aggravated steatosis by suppression of fatty acid (FFA) β-oxidation and by induction of FFA uptake and inflammatory factors [89–90]. Furthermore, HIF1A promoted liver fibrosis in NAFLD by activating PTEN/ NF-κB p65 signaling pathway [91]. RELA, also known as p65, is one of the five members in NF-κB family and is a pivotal transcription factor regulating inflammatory molecules [92]. Inhibition of NF-κB signaling alleviated hepatic lipid accumulation and hepatic inflammation in NAFLD [93–94]. Moreover, abnormal lipid deposition as well as insulin resistance in NAFLD often leads to endoplasmic reticulum (ER) stress, which further triggers the unfolded protein response and thereby causes inflammation in hepatocytes [95]. GRP78 (HSPA5) is a chaperone heat shock protein playing the central role in maintaining ER proteostasis under excessive stress [96]. Decreased expression of ER stress molecule GRP78 contributed to reducing fasting glucose and lipid profile, and attenuating NAFLD [97–99]. LGALS3 has been shown to participate in glucose intolerance and lipid metabolism disorders, which is an essential regulator of insulin resistance, fibrosis and inflammation cytokines including TNF-α, IL-6 and IL-1β [100–101]. FGF1 exerts a protective role in series of metabolic disorders [102]. Investigations revealed that FGF1 could reduce blood glucose and ameliorate hepatic steatosis, inflammation and fibrosis through modulation of oxidative stress and ER stress [103–104]. The onset of hepatic inflammation caused the fibrogenesis in NAFLD, which was manifested by deposited extracellular matrix (ECM) proteins including collagens, elastin and fibronectin [105–106]. MMP-9 performs the vital role in modulating and degrading gelatins, collagens and other ECM compounds [107–108]. A decreased MMP-9 was associated with more advanced fibrosis and serum liver injury indices (AST, GGT) in NAFLD patients, while increased MMP-9 activity could precede the clearance of fibrotic matrix [109–110]. SLC5A1 encodes the sodium glucose cotransporter 1 (SGLT1), and inhibition of SGLT1 not only contributed to regulate hepatic glucose metabolism but also mitigate development and progression of NAFLD [111–112]. To conclude, it was assumed that SGXZ decoction might perform comprehensive regulations of anti-inflammation, anti-lipid deposition, anti-insulin resistance, anti-fibrosis, and anti-ER stress on NAFLD through these key targets.
After that, GO functional enrichment analysis revealed that the targets in PPI network mainly involved oxidation, positive regulation of angiogenesis, metabolic process, hypoxia, extracellular matrix and insulin-like factor binding, and other biological processes (Figure 5). The results of GO analysis coincided with the indispensable contribution of aforementioned key targets among all targets in PPI network. In addition, KEGG enrichment analyses indicated that the action pathway mainly included insulin resistance, amino acid metabolism, cancer-related pathways, inflammation-related pathways, estrone signaling pathway and T cell receptor pathway (Figure 6A, Table 3). As shown in Figure. 6B, the ‘target-pathway’ interaction network suggested that bioactive compounds in SGXZ decoction performed therapeutic role in regulation of NAFLD through multi-targets and multi-pathways. Through literature retrieval, six signaling pathways including PI3K-Akt, MAPK, insulin resistance, HIF-1, Tryptophan metabolism and Estrogen signaling pathway were recognized as core pathways involved in the treatment of SGXZ decoction on NAFLD. The six core pathways and their interaction were cited from KEGG database and displayed in Figure 9. Among the core pathways, PI3K-Akt, MAPK and HIF-1 signaling pathway were associated with inflammation that is the driving force for the development and evolution of NAFLD [113–114]. Researches provided evidence that regulation of PI3K-Akt signaling mediators could improve hepatocyte damage, hepatic gluconeogenesis and lipid disorder [115–117]. Moreover, inhibiting apoptosis and promoting autophagy via the reactive oxygen species (ROS)/MAPK pathway significantly decreased total cholesterol and triglyceride levels of both plasma and liver in high fat diet-fed mice [118]. In addition, increased ROS formation, triglyceride and lipid accumulation in hepatocyte leaded to excessive oxidative stress and inflammatory responses that further produced a hypoxic microenvironment [88–119](33272734, 33804956). HIF-1 signaling acts as the key response to hypoxia. Loss of HIF-1α (HIF1A) activates the oxidative stress, promoted the lipid deposition and the secretion of pro-inflammatory IL-6 and TNF-α, suggesting its regulatory role in the progression of NAFLD [119–120](33272734, 30242180). Indeed, increased FFAs and lipogenesis in hepatocytes, release of proinflammatory cytokines from adipose tissue such as IL-6 and TNF-α, together with altered gut microbiota gave rise to the insulin resistance, which predisposes to the development of NAFLD and progression to NASH [121–123]. Tryptophan is a kind of amino acid that could produce indoles by bacterial enzyme trytophanase A in intestinal epithelial cell. Indoles promote intestinal barrier function and could translocate from the intestinal to the liver to modulate hepatic lipid metabolism and inflammation to protect against NAFLD [124]. Moreover, the hepatic metabolism and inflammation in the onset and progression of NAFLD showed a sex difference, which was potentially due to different estrogen signaling activity [125–126]. Estrogen deficiency increased risk for liver fibrosis, and postmenopausal women were more likely to develop NAFLD than men [127–128]. In general, the above six core pathways and their interaction were correlated to NAFLD and might be acted on by SGXZ decoction in the treatment of NAFLD.
In the end, molecular docking (Table 4, Figure 8) showed that most of the key compounds could be docked into the key protein targets. Moreover, bioactive compounds of isochlorogenic acid A, chlorogenic acid and gallic acid could bind best with all of the key targets, especially with HSPA5, RELA, MMP9, SLC5A1, HIF1A and FGF1. The results of molecular docking simulations provided a putative pharmacological activity for the treatment of SGXZ decoction on NAFLD. Our present study applied network pharmacology combined with molecular docking to predict the key active ingredients and pathways of SGXZ decoction in NAFLD treatment, which still need to be verified by future experimental validations.