As one of the most common liver diseases, the incidence of NAFLD is increasing gly rising worldwide and its prevalence is approximately 25.24% in liver disease. Patients with NAFLD are more likely to suffer from cardiovascular and tumor diseases and have a lower survival rate compared with ordinary patients [18]. To date, many studies have discovered that Chaihu and its prescription bring out positive therapeutic effects on NAFLD [19-20].In order to explore the multiple mechanisms of Xiaochaihu decoction, TCMSP database was used to obtain the effective active ingredients and potential targets in this study. Moreover, the NAFLD related disease targets were searched in the Gene cards database, TTD and the OMIM database. Common targets were obtained through gene mapping. The ‘ingredient-target-disease’ interaction network was constructed by Cytoscape software.
4.1 Analysis of Active Ingredients
In the network of ingredient-target-disease, key ingredients with higher degree includesquercetin, kaempferol, stigmasterol and beta-sitosterol. Quercetin is a widely existing flavonoid in nature. Recent research showed that it demonstrated anti-oxidant, anti-inflammatory and immunomodulatory activities in NAFLD treatment [21]. A clinical trial found that treatment with quercetin could change the hematological parameters of patients with NAFLD with increasing of RBC and decreasing of ferritin [22]. Kaempferol has a variety of pharmacological effects such as anti-cancer, anti-oxidation, anti-viral, anti-inflammatory, antibacterial activities. It can also enhance the immunity and attenuate liver fibrosis via inhibiting activin receptor-like kinase 5 [23-24]. Both beta-sitosterol and stigmasterol are components of phytosterols, have a wide range of pharmacological effects such as anti-inflammatory, antibacterial, analgesic, and anti-cancer. Moreover, beta-sitosterol and stigmasterol were proved to be effective in NAFLD mainly by regulating lipid metabolism [25].
4.2 Analysis of Potential Targets
The target with the highest degree was PTGS2. The following targets were ESR1, NOS2, PPARG in the network of ingredient-target-disease. PTGS2 is an oxidative stress-related factor related to prostaglandin biosynthesis related to inflammation and mitosis. He found that down-regulation of PTGS2 mRNA was related to the repair of drug-induced liver injury in mice [26]. ESR1 is an estrogen receptor expressed by hepatocytes. NOS2 is an isozyme of NOS which plays an important role in inflammation, tumor and cell damage. PPARG could not only inhibit the expression of inflammatory factors such as TNF-α and IL-1, but also regulate adipocyte differentiation as well as lipid metabolism. This activity might contribute to the prevention against NAFLD [27]. Above all, the key ingredients containing quercetin, kaempferol, stigmasterol and beta-sitosterol might be closely related to the regulation of PTGS2, ESR1, NOS2 and PPARG during Xiaochaihu decoction treating NAFLD.
4.3 Analysis of PPI Network
The target protein levels of AKT1, IL6, JUN, MAPK8 and STAT3 were excessively expressed in PPI network. This result indicated the series of targets of Xiaochaihu decoction treating NAFLD. An animal model research has proven that the decreased expression of AKT1 resulted in FoxO1 inhibition and further achieved the effect of ameliorating NAFLD in diabetic rat [38]. Another research also suggested that the inhibition of AKT1-mediated ROS production suppressed the process of NAFLD to liver fibrosis conversion [39]. IL-6 is an important pro-inflammatory factor and its production has been shown to be positively correlated with the occurrence and development of NAFLD [30]. JUN including JNK, JUN B and JUN C belongs to the AP-1 family of transcription factors. The research from Yan indicated that JNK participated in mediating the process of liver inflammation and fat accumulation during NAFLD [31].Moreover, MAPK8 was confirmed to be related to liver regeneration in mice [32]. It might contribute to the prevention against NAFLD. Yu’s study has found that up-regulation of the NF-kB and STAT3 signaling pathway could promote insulin resistance in liver cells which was potent for NAFLD prevention [33]. As a result, the comprehensive regulation of AKT1, IL6, JUN, MAPK8 and STAT3 was potential for the therapeutic effect on NAFLD with Xiaochaihu decoction.
4.4 Analysis of Signaling Pathways
DAVID database was used for conducting KEGG analysis and GO analysis. The result of GO indicated that potential targets were mainly enriched in response to nutrient levels in biological progress, cytokine receptor binding in molecular function and membrane raft in cellular components. In addition, the results of KEGG were directly related to the NAFLD pathway. We retrieved six pathways related to liver disease as core pathways from the top 20 pathways of KEGG enrichment analysis and divided them into three aspects: metabolism, oxidative stress and immunity. As shown in Figure 8, the ‘disease-pathway-target’ interaction network demonstrated that active ingredients in Xiaochaihu decoction played a multi-targets and multi-pathways role in regulation of NAFLD. Furthermore,Figure 9 demonstrates the distribution of the target proteins of Xiaochaihu decoction on the predicted pathways. The pathways related to metabolism included AGE-RAGE signaling pathway in diabetic complications, Fluid shear stress and atherosclerosis and Insulin resistance. According to previous statistics, NAFLD is a common complication of diabetes [34]. The accumulation of fat caused by NAFLD will increase insulin resistance, which will affect the liver's export of hepatic glycogen. This process will eventually lead to disorder of lipid metabolism and aggravate the condition. Asadipooya Kamyar found that AGE-RAGE interaction could promote the fat accumulation in the liver. It will cause many complications in NAFLD including inflammation, fibrosis and insulin resistance [35]. The pathological basis of atherosclerosis is the disorder of lipid metabolism. It often occurs together with NAFLD and makes the condition worsen. A study revealed that the occurrence of NAFLD was closely related to atherosclerosis [36]. The pathways related to oxidative stress is HIF-1 signaling pathway. The second-hit theory, a currently recognized theory revealing the pathogenesis of NAFLD, stated that oxidative stress could accelerate the conversion of NAFLD into NASH. A new research also found that patients with NAFLD had significantly higher expression of HIF-1 in peripheral blood and Th17 cell differentiation compared with healthy people. Moreover, the joint examination on HIF-1 and Th17 cell might be helpful for the early diagnosis of NAFLD [37].The pathways related to immunity include Th17 cell differentiation and IL-17 signaling pathway. A study from Su demonstrated that the levels of Th22 cell and Th17 cell were positively correlated with the degree of liver lesions in NAFLD model mice and involved in the occurrence as well as development of NAFLD [48]. Generally speaking, patients with infection-induced immunodeficiency are more susceptible to NAFLD. Overall, the above pathways and their interactions may be closed relevant to NAFLD.
In this study, network pharmacology systematically uncovered the key targets, active ingredients and the crucial signaling pathways of Xiaochaihu decoction for the treatment of NAFLD. It also provided a direction and evidence for future researches. Nonetheless, there still exists the limitation in this study. The source, performance and dosage of traditional Chinese medicine in actual application are not considered in this study. This is whatever so important during research. In addition, there are many other variable factors should be noticed including NAFLD models in different species. Therefore, more researches are ought to carry out in the future to further reveal the mechanisms of Xiaochaihu on NAFLD.