Metabolomic analysis has been used to characterize the effects and mechanisms of drugs for nonalcoholic fatty liver disease (NAFLD) at the metabolic level. Nuciferine is an active component derived from folium nelumbinis and has been demonstrated to have beneficial effects on a high-fat diet (HFD) induced hepatic steatosis model. However, the effect of the altered metabolites of nuciferine on NAFLD has not yet been elucidated. In this study, we established a NAFLD rat model using HFD and treated with nuciferine. The lipid content levels, pro-inflammatory cytokines, and oxidative stress were investigated to access the therapeutic effects of nuciferine. Additionally, the metabolic regulatory mechanisms of nuciferine on NAFLD were analyzed using untargeted metabolomics. Gene expression of the key enzymes related to the changed metabolic pathways following nuciferine intervention was also investigated. The results showed that nuciferine treatment significantly reduced the body weight, levels of lipids, and liver enzymes in the blood and improved the hepatic steatosis in the NAFLD rat model. Nuciferine treatment also increased the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) and decreased the levels of methane dicarboxylic aldehyde (MDA) in the liver. Nuciferine treatment decreased the serum levels of interleukin (IL)-6, IL-1β, and tumor necrosis factor-alpha (TNF-α) and upregulated the gene expression of IL-6, IL-1β, and TNF-α in the liver. Metabolomic analysis indicated a metabolism disorder in the NAFLD rat model reflected in a dysfunction of the glycerophospholipid, linoleic acid, alpha-linolenic acid, arginine and proline metabolism. Conversely, treatment with nuciferine improved the metabolic disorder in the NAFLD rat model. Nuciferine treatment also regulated the gene expression of key enzymes related to the glycerophospholipid, linoleic acid, and alpha-linolenic acid metabolism pathways in the liver. In conclusion, our study demonstrated an amelioration of the metabolic disorders following nuciferine treatment in NAFLD rat model. Our study contributes to the understanding of the effects and mechanisms of drugs for complex diseases using metabolomic analysis and experimental approaches.

The dysbiosis in gut microbiota could affect host metabolism and contribute to the development of nonalcoholic fatty liver disease (NAFLD). Da-Chai-Hu decoction (DCH) has demonstrated protective effects on NAFLD, however, the exact mechanisms remain unclear. In this study, we established a NAFLD rat model using a high fat diet (HFD) and provided treatment with DCH. The changes in gut microbiota post DCH treatment were then investigated using 16S rRNA sequencing. Additionally, serum untargeted metabolomics were performed to examine the metabolic regulations of DCH on NAFLD. Our results showed that DCH treatment improved the dyslipidemia, insulin resistance (IR) and ameliorated pathological changes in NAFLD model rats. 16S rRNA sequencing and untargeted metabolomics showed significant dysfunction in gut microbiota community and serum metabolites in NAFLD model rats. DCH treatment restored the dysbiosis of gut microbiota and improved the dysfunction in serum metabolism. Correlation analysis indicated that the modulatory effects of DCH on the arachidonic acid (AA), glycine/serine/threonine, and glycerophospholipid metabolic pathways were related to alterations in the abundance of Romboutsia, Bacteroides, Lactobacillus, Akkermansia, Lachnoclostridium and Enterobacteriaceae in the gut microflora. In conclusion, our study revealed the ameliorative effects of DCH on NAFLD and indicated that DCH’s function on NAFLD may link to the improvement of the dysbiosis of gut microbiota and the modulation of the AA, glycerophospholipid, and glycine/serine/threonine metabolic pathways.

Pi-Dan-Jian-Qing decoction (PDJQ) can been used in the treatment of type 2 diabetes mellitus (T2DM) in clinic. However, the protective mechanisms of PDJQ on T2DM remain unknown. Recent studies have shown that the changes in gut microbiota could affect the host metabolism and contribute to progression of T2DM. In this study, we first investigated the therapeutic effects of PDJQ on T2DM rats. 16S rRNA sequencing and untargeted metabolomics analyses were used to investigate the mechanisms of action of PDJQ in the treatment of T2DM. Our results showed that PDJQ treatment could improve the hyperglycemia, hyperlipidemia, insulin resistance (IR) and pathological changes of liver, pancreas, kidney, and colon in T2DM rats. PDJQ could also decrease the levels of pro-inflammatory cytokines and inhibit the oxidative stress. 16S rRNA sequencing showed that PDJQ could decrease the Firmicutes/Bacteroidetes (F to B) ratio at the phylum level. At the genus level, PDJQ could increase the relative abundances of Lactobacillus, Blautia, Bacteroides, Desulfovibrio and Akkermansia and decrease the relative abundance of Prevotella. Serum untargeted metabolomics analysis showed that PDJQ could regulate tryptophan metabolism, histidine metabolism, tricarboxylic acid (TCA) cycle, phenylalanine, tyrosine and tryptophan biosynthesis and tyrosine metabolism pathways. Correlation analysis indicated that the modulatory effects of PDJQ on the tryptophan metabolism, histidine metabolism and TCA cycle pathways were related to alterations in the abundance of Lactobacillus, Bacteroides and Akkermansia. In conclusion, our study revealed the various ameliorative effects of PDJQ on T2DM, including improving the liver and kidney functions and alleviating the hyperglycemia, hyperlipidemia, IR, pathological changes, oxidative stress and inflammatory response. The mechanisms of PDJQ on T2DM are likely linked to an improvement in the dysbiosis of gut microbiota and modulation of tryptophan metabolism, histamine metabolism, and the TCA cycle.

Evodiamine (EVO) is an alkaloid extracted from Evodia rutaecarpa and has various pharmacological activities, including hypolipidemic, anti-inflammatory, anti-infective, and antitumor effects. However, the therapeutic effects of EVO on type 2 diabetes mellitus (T2DM) and the possible mechanisms remain unknown. In this study, we used a T2DM rat model using a high-fat diet (HFD) combined with streptozotocin (STZ) injections followed by treatment with EVO. First, we evaluated the therapeutic effects of EVO on T2DM rats, following which we evaluated the anti-inflammatory and anti-oxidative effects of EVO on T2DM rats. Finally, we analyzed the metabolic regulatory mechanism of EVO in T2DM rats using an untargeted metabolomics approach. The results showed that EVO treatment alleviated the hyperglycemia, hyperlipidemia, insulin resistance (IR), and pathological changes of the liver, pancreas and kidneys in T2DM rats. Moreover, EVO treatment ameliorated the oxidative stress and decreased the serum levels of pro-inflammatory cytokines in T2DM model rats. Serum untargeted metabolomics analysis indicated that the EVO treatment affected the levels of 26 metabolites, such as methionine, citric acid, cholesterol, taurocholic acid, pilocarpine, adrenic acid, and other metabolites. These metabolites were mainly related to the amino sugar and nucleotide sugar metabolism, arginine biosynthesis, arginine and proline metabolism, glutathione metabolism, and tryptophan metabolism pathways. In conclusion, EVO can reduce blood glucose and improve oxidative stress and inflammatory response in T2DM rats. These functions are related to the regulation of amino sugar and nucleotide sugar metabolism, arginine biosynthesis, arginine and proline metabolism, glutathione metabolism, and tryptophan metabolism pathways.

Vasomotor dysfunction is one of the key pathological aspects of shock and heart failure (HF). Shenfu injection (SFI) has been widely used for the treatment of shock and HF in China. Pharmacological studies have suggested that SFI can reduce peripheral circulation resistance and improve microcirculation. However, whether it has a regulatory effect on macrovascular has not been elucidated. In this study, we used thoracic aorta rings isolated from Wistar rats and the human umbilical vein cell line (EA.hy926) to explore the vasodilative activity of SFI and its potential mechanisms. The relaxation due to SFI was measured after pre-treatment with selective soluble guanylate cyclase (sGC) inhibitor or cyclooxygenase (COX) inhibitor and compared with the vasodilation effect of SFI only treated with norepinephrine (NE). The contents of NO, endothelin-1 (ET-1), endothelial nitric oxide synthase (eNOS), COX-1, 6-K-PGF_1α, and caveolin-1 were evaluated respectively. Additionally, the level of eNOS mRNA and total eNOS and its phosphorylation were studied to investigate the potential mechanisms involved. Experimental results showed that SFI markedly attenuated NE-induced vasoconstriction but that this effect was significantly eliminated after pre-incubation with the selective sGC inhibitor 1-H-[1, 2, 4] oxadiazolo [4, 3-α] quinoxaline-1-one (ODQ), instead of the COX inhibitor indomethacin (INDO). SFI significantly increased the eNOS content and up-regulated the eNOS mRNA expression, while it did not affect the content of COX-1 and 6-K-PGF_1α. SFI also markedly increased NO content but significantly reduced the content of ET-1 and caveolin-1 in the cell supernatant. Furthermore, it promoted the expression of total eNOS and the phosphorylation of eNOS at serine (Ser) 1177 but inhibited the phosphorylation at threonine (Thr) 495, which was significantly reversed by PI3K-specific inhibitor LY294002. In conclusion, our study showed the vasodilation effect of SFI in thoracic aorta is mediated entirely by enhancing eNOS activity through the PI3K/Akt signaling pathway, providing novel knowledge on the effect of SFI on shock and HF for future clinical applications.

Hirudin, an extract from Hirudo spp., is an anticoagulant used to treat a variety of renal diseases, including diabetic nephropathy (DN). Currently, hirudin has to be used at high dosages to treat DN because it poorly targets the kidneys, although at high dosages it can have severe side effects. Developing a targeted drug delivery system for hirudin, then, could boost its positive therapeutic effects while lowering the risk of side effects. Liposomes have been demonstrated to have significant renal targeting potential, but here we show that a hirudin-loaded liposome is an effective delivery method for patients with DN. In this study, we prepared a hirudin/liposome complex and tested its efficacy by injecting it into a rat model. We then compared the renal accumulation of hirudin between complex-injected rat models and rat models that received injections of hirudin alone. We also investigated the mechanisms behind the complex’s effects. The hirudin/liposome complex increased the accumulation of hirudin in kidney tissues and relieved the renal injury in DN rat models. Moreover, the hirudin/liposome complex down-regulated the expression of TGF-β1 and VEGF in the kidneys. We demonstrated that a hirudin/liposome complex can have a significant positive effect on DN. The mechanism may be that the complex inhibits the expression of VEGF and TGF-β1.