Chrysanthemum morifolium Ramat extract and probiotics combination ameliorates metabolic disorders through regulating gut microbiota and PPARα subcellular localization

Abstract

Background: Chrysanthemum morifolium Ramat, a traditional Chinese medicine, has the effects on liver clearing, vision improving, and anti-inflammation. C. morifolium and probiotics have been individually studied for their beneficial effects on metabolic diseases. However, the underlying molecular mechanisms were not completely elucidated. This study aims to elucidate the potential molecular mechanisms of C. morifolium and probiotics combination (CP) on alleviating nonalcoholic fatty liver disease (NAFLD) and the dysregulation of glucose metabolism in high-fat diet (HFD)-fed mice.

Methods: The therapeutic effect of CP on metabolism was evaluated by liver histology and serum biochemical analysis, as well as glucose tolerance test. The impact of CP on gut microbiota was analyzed by 16S rRNA sequencing and fecal microbiota transplantation. Hepatic transcriptomic analysis was performed with the key genes and proteins validated by RT-qPCR and western blotting. In addition, whole body Pparα knockout (Pparα^-/-) mice were used to confirm the CP-mediated pathway.

Results: CP supplementation ameliorated metabolic disorders by reducing body weight and hepatic steatosis, and improving glucose intolerance and insulin resistance in HFD fed mice. CP intervention mitigated the HFD-induced gut microbiota dysbiosis, which contributed at least in part, to the beneficial effect of improving glucose metabolism. In addition, hepatic transcriptomic analysis showed that CP modulated the expression of genes associated with lipid metabolism. CP downregulated the mRNA level of lipid droplet-binding proteins, such as Cidea and Cidec in the liver, leading to more substrates for fatty acid oxidation (FAO). Meanwhile, the expression of CPT1α, the rate-limiting enzyme of FAO, was significantly increased upon CP treatment. Mechanistically, though CP didn't affect the total PPARα level, it promoted the nuclear localization of PPARα, which contributed to the reduced expression of Cidea and Cidec, and increased expression of CPT1α, leading to activated FAO. Moreover, whole body PPARα deficiency abolished the anti-NAFLD effect of CP, suggesting the importance of PPARα in CP-mediated beneficial effect.

Conclusion: This study revealed the hypoglycemic and hepatoprotective effect of CP by regulating gut microbiota composition and PPARα subcellular localization, highlighting its potential for therapeutic candidate for metabolic disorders.

Keywords: Chrysanthemum morifolium Ramat; Gut microbiota; NAFLD; PPARα; Probiotics; T2DM.

Fig. 1

Chrysanthemum morifolium Ramat extract and probiotics combination (CP) ameliorates high-fat diet-induced weight gain and liver steatosis. A Schematic representation of CP intervention in HFD-fed C57BL/6 mice and body weight change (n = 6 per group). B Representative images of liver and epididymal adipose tissue as well as H&E staining. C Liver weight, TG, and steatosis score. D Epididymal adipose tissue (eWAT) weight. E Serum ALT, TC, TG level and LDL levels. F Fasting blood glucose, intraperitoneal glucose tolerance test (ipGTT), and intraperitoneal insulin tolerance test (ipITT) results with area under the curve (AUC) calculation and. All data are shown as the Mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 2

CP regulates gut microbiota composition in HFD-fed mice. A Alpha diversity analysis (Chao index, Shannon index and Simpson index) on OUT level. B Beta diversity analysis using the unweighted unifrac method. C Venn plot of the number of OTUs in the control, HFD, and HFD_CP. D Relative abundance of bacteria at phylum level. E Linear discriminant analysis effect size (LEfSe) analysis of the characteristic genera of the gut microbiota. Distribution histogram based on LDA; a higher LDA score represents greater importance of the bacteria. F Heatmap showing the top 10 genera significantly altered by CP in HFD-fed mice. All data are shown as the Mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 3

The correlation of bacteria with mouse phenotype and function predication. A Correlation analysis of the gut microbiota and mouse phenotypes in three groups. B The prediction of gut microbial function based on 16S rRNA sequencing by PICRUSt2. *p < 0.05, **p < 0.01

Fig. 4

Glucose metabolism is improved by fecal transplantation from CP-treated mice to HFD-fed mice. A Schematic diagram of FMT experiment and body weight (n = 5 per group). B The relative abundance of representative genus in the recipient mice. C Fasting blood glucose, intraperitoneal glucose tolerance test (ipGTT), and intraperitoneal insulin tolerance test (ipITT) results with area under the curve (AUC) calculation. D Serum ALT, AST, TC, and TG levels. All data are shown as the Mean ± SEM. *p < 0.05, **p < 0.01

Fig. 5

Hepatic transcriptomic analysis of livers from CP treated mice. Mice were treated as in Fig. 1. A Volcano plot based on the changed genes of the HFD group compared with the Control group; the HFD_CP group compared with the HFD group. B Venn diagram of regulated genes by HFD or by HFD_CP. C Heatmap of the significantly changed genes between HFD and HFD_CP groups. D KEGG enrichment analysis of the reversed genes by CP. E The changes of lipid metabolism-related genes reversed by CP based on hepatic transcriptomic analysis. All data are shown as the Mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 6

CP improves HFD-induced NAFLD by activating PPARα. A The reversed pathways by CP, which was enriched with gene set enrichment analysis (GSEA) (adjusted p < 0.05). B The fatty acid metabolism, mTORC1 signaling, and PI3K-AKT-mTOR signaling pathways which are from the GSEA analysis of the liver gene profiling. Mice were treated as in Fig. 1. C mRNA expression of Cidea and Cidec in the liver. D The expression of total proteins of FASN, PPARα, PGC1α and CPT1α in the liver. E The protein level of nuclear PPARα and nuclear PGC1α in the liver. All data are shown as the Mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 7

PPARα deficiency abolishes HDCA-mediated anti-NAFLD effect. A Schematic representation of CP intervention in HFD-fed Pparα^−/−mice (n = 6 per group). B Body weight and epididymal adipose tissue (eWAT) weight. C Representative images of liver and epididymal adipose tissue as well as H&E staining. D Liver weight, TG, and steatosis score. E Serum ALT, TC, TG level

Fig. 8

Schematic diagram of the potential mechanisms of CP on ameliorating metabolic disorders