NAFLDs progression and development are complicated processes involving multiple factors that alter the homeostasis of liver tissue. In the other hand, TCM contains of tens or thousands of compounds that can act on multiple target proteins through multiple pathways to exert their synergism or compatible pharmacological effects on complex diseases. Thus, TCM may have myriad advantages on the treatment of NAFLD. In this paper, we combined the HFD-induced rat model, network pharmacology, and molecular docking to explore the efficacy and mechanism of action of GF for the treatment of NAFLD.
After the HFD-induced rat model established, then supplemented with GF, the histopathological analysis exhibited that GF exerted potential protective effects against NAFLD. Specifically, we observed significant changes in serum glucose, insulin, the levels of lipids, liver function enzymes (ALT, AST, and LDH), and MDA Levels in serum and the mRNA expression of lipogenesis in liver tissue among different groups of rats. ALT, AST, and LDH are the important of biochemical indicators of liver function, they were widely used in clinic. As we expected, this study showed that they changed differently in the control, HFD-fed rats treated with or without GF and metformin. Although the detailed pathogenesis of NAFLD has not resolved completely, lipogenesis is believed to have a significant role in the triglyceride accumulation. Lipogenesis including SREBP-1c, FAS, SCD-1, CD36, PPAR-α, and CPT-1 have been implicated in the NAFLD pathogenesis, their mRNA expression levels of liver tissues were significantly decreased following the HFD-fed rats treated with GF compared the non-treated ones, also in a dose-dependent manner. Moreover, serum glucose, insulin resistance, dyslipidemia and metabolic syndromes have played important roles in the NAFLD [16], insulin resistance inhibited the anti-lipolytic activity of insulin in the adipose tissue and increased free fatty acids (FFAs) in the serum and liver, leading to mitochondrial dysfunction as well as cardiac fat accumulation. As expected, the serum glucose, TC, LDL-C, and TG levels and insulin were decreased in HFD-fed rats supplemented GF compared with those untreated ones [17]. Furthermore, oxidative stress is essential risk factors for NAFLD, and promotes the production of reactive oxygen species (ROS) that stimulate an inflammatory process in hepatic tissues [18]. Our research showed that the MDA levels in serum was decreased in the HFD-induced rats treated with GF compared with those without any supplement. Moreover, the high dosage (100 mg/kg) of GF showed similar effects to the positive control drug metformin on the treatment of rats with NAFLD. Consequently, GF may play an important role in preventing or slowing the progression of NAFLD by regulating multi-factors, such as, serum glucose, insulin resistance, oxidative stress, free fatty acids, liver enzyme, and lipogenesis.
Based on the network pharmacology analysis, the main active compounds in GF including quercetin, oleanolic acid, kaempferol, and geniposide played key roles in the treatment of NAFLD by targeted on genes including PPARα, PPARγ, and CA2. Moreover, these main protein genes exerted their effects concentrated on the PPAR pathway. Next, we will discuss their roles in NAFLD from three molecular levels, such as, compounds, protein genes, and pathways.
Firstly, quercetin and kaempferol are natural flavonoids widely distributed in herbal medicine, vegetable, and edible fruits, featuring a variety of biological functions, studied primarily for their potential roles to combat oxidative and inflammatory processes. They showed potential for the treatment of fatty liver. Previous studies showed that both single quercetin and contained in herbal medicine could ameliorate NAFLD in HFD-induced mice [19]. Moreover, mice treated with up to 3000 mg/kg quercetin did not show any toxic effects [20]. Kaempferol, chemical structural analogues of quercetin, with less than a hydroxyl, also exhibited hepatoprotective effect [21], and suppressed hepatic gluconegeonesis [22] and inhibited hepatocellular carcinoma cell [23]. Geniposide, a major characteristic constituent in GF, which exerted protective effects against hepatic steatosis in rats fed with a HFD, the underlying mechanism might be associated with its antioxidant actions or regulation of adipocytokine release and expression of PPARα [24]. Oleanolic acid attenuated the subsequent development of high fructose diet-induced non-alcoholic fatty liver disease in rats [25].
Secondly, we identified PPARα, CA2, and PPARγ as potential target genes. PPARγ was a regulator of adipocyte differentiation. Additionally, it had been implicated in the pathology of numerous diseases including obesity, diabetes, atherosclerosis, and cancer [26]. Previous study showed that PPARα could inhibit fatty liver disease by activating the periostin-dependent JNK signaling pathway and modulating fatty acid oxidation [27]. Peroxisome proliferator-activated receptors (PPARs) are binder-activated nuclear receptors that are involved in the transcriptional regulation of lipid metabolism, energy balance, inflammation, and atherosclerosis [28].
Thirdly, the functions of potential target genes identified from KEGG enrichment analysis were associated with multiple signal transduction pathways, the most important one is the PPAR pathway, which was consistent with the conclusion of the PPI analysis. Which contribute to the complex regulation of NAFLD progression, and was the therapeutic target of anti-NAFLD compounds. Recent research found that PPARs were closely related to metabolic syndrome and its relevant complications [29]. PPARs pathway had protective effect on the NAFLD development because it could regulate the lipid metabolism [30]. Therefore, our results suggested that these signal pathways might be coordinated during NAFLD progression, and the effects of GF could be mediated through multiple signaling pathways concentrated on PPAR.
Finally, computational docking exercises showed that the main ingredients from GF formed comparative interactions with their according predicted proteins compared to the native ligands with those. Furthermore, quercetin had a high response and affinity with PPARγ were validated in the SPR test with the Kd value of 71.5 µM. The regulatory mechanisms of the processes were likely to be valid targets for modulating lipid metabolism and inflammation in the treatment of NAFLD. However, further studies on the relevance to NAFLD are needed.