Herbal medicine formula Huazhuo Tiaozhi granule ameliorates dyslipidaemia via regulating histone lactylation and miR-155-5p biogenesis

Abstract

Background: Huazhuo Tiaozhi granule (HTG) is a herbal medicine formula widely used in clinical practice for hypolipidaemic effects. However, the molecular mechanisms underlying dyslipidaemia treatment have not been well elucidated.

Results: A significant reduction in the levels of total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) was observed in the serum of patients with dyslipidaemia after HTG treatment, without disruption in the levels of aspartate transaminase (AST), alanine transaminase (ALT), urea nitrogen (BUN), and creatinine (Cr). The dyslipidaemia rat model was induced by a high-fat diet and treated with Xuezhikang (0.14 g/kg/d) or HTG (9.33 g crude herb/kg/day) by gavage for 8 weeks. Body weight and liver index were markedly decreased in dyslipidaemic rats after treatment with Xuezhikang or HTG. HTG administration markedly ameliorated hyperlipidaemia by decreasing the levels of TC and LDL-C in serum and hepatic lipid accumulation. In vitro, lipid accumulation in LO2 and HepG2 cells was alleviated by serum treatment with HTG. High lactylation was observed in 198 proteins, including lactylation of histone H2B (K6), H4 (K80). Deep sequencing of microRNAs showed that miR-155-5p was significantly downregulated.

Conclusions: This study demonstrates that HTG is an effective and safe formula for treating dyslipidaemia, which promotes lactylation in hepatocytes, and the retardation of miR-155-5p biogenesis.

Keywords: Dyslipidaemia; Herbal medicine formula; Histone; Huazhuo Tiaozhi granule; Lactylation; miR-155-5p.

Fig. 1

Effects of HTG on lipid profiles. a TC, TG, LDL-C, HDL-C levels of dyslipidaemia patients after 8 weeks HTG treatment. b Overview of the experimental design in Wistar rats. c Body weight of rats at different time points (0, 4, 12 weeks). d TC level in serum after HTG treatment for 8 weeks. e TG level in serum after HTG treatment for 8 weeks. f LDL-C level in serum after HTG treatment for 8 weeks. g HDL-C level in serum after HTG treatment for 8 weeks. h AST levels in serum. i ALT levels in serum. j Liver weight of rats after HTG treatment for 8 weeks. k Liver index of rats after HTG treatment for 8 weeks. l, m, n Histological changes in rats after HTG treatment for 8 weeks. Scale bars, 100 μm. *p < 0.05, **p < 0.01, versus HFD group

Fig. 2

Screening of different expressed genes (DEGs) in liver tissues of dyslipidaemia mouse with HTG treatment or not. a Volcano plots. b GO analysis shows the biological process of DEGs c The heatmap plot of DEGs (top 26) enriched in lipid metabolic process. d Heatmap plot of DEGs (top 25) enriched in regulation of transcription by RNA polymerase II. e KEGG pathway

Fig. 3

Effects of HTG on liver cells stimulated with FFA (free fatty acids). a Cell viability of LO2 cells stimulated with different concentrations of FFA (0, 0.25, 0.5, 1, 1.5 mM) for 24 h. b Cell viability of LO2 cells after HTG treatment (0%, 5%, 10%, 15%, 20%) for 24 h. c Integrated density per LO2 cell after red oil O staining. d, e TC, TG levels of LO2 cells after 20% HTG-medicated serum treatment for 24 h. f Red oil O staining of HepG2 cells after 10% HTG-medicated serum treatment for 24 h. Scale bars, 25 μm. g TG levels of HepG2 cells after HTG treatment for 24 h. h, i Pyruvate, lactate levels in LO2 cells after HTG treatment. j, k, l Extracellular acidification rate (ECAR), basal glycosis, and compensatory glycolysis in LO2 cells. *p < 0.05, **p < 0.01, versus FFA group

Fig. 4

Effects of HTG on protein lactylation. a Panlactylated protein expression. b Differentially lactylated proteins and modified sites. c Heat map of the modification sites near the amino acid. d Subcellular localization of lactylated proteins. e KEGG pathway analysis

Fig. 5

Effects of HTG on liver miRNAs expression. a Hierarchical cluster analysis of differentially expressed miRNAs between HFD group and HTG group in dyslipidaemia rats. b Relative expression of miR-155-5p detected by RT-PCR. c Relative expression of miR-155-5p in LO2 cells after miR-155-5p mimics intervention for 48 h by RT-PCR. d, e Effects of miR-155-5p mimics on TC, TG levels of LO2 cells stimulated with FFA after 20% HTG-medicated serum treatment for 24 h. *p < 0.05, **p < 0.01, versus HFD group or FFA group

Fig. 6

Effects of HTG on histone lactylation. a Number of lactylated proteins enriched in genetic information processing via KEGG pathway analysis. b Cluster of lactylated proteins involved in regulation of RNA splicing. c Cluster of lactylated proteins (top 20) involved in regulation of cellular metabolic process. d PPI analysis of H2BC13. e Histone lactylation. f Western blot analysis of H2B, H4 lactylation levels