Leonurine Attenuates Obesity-Related Vascular Dysfunction and Inflammation

Figure 1. LEO alleviates obesity and lipid deposition. (A) Experimental design and grouping. (B) Body weight of mice at the start and at 2, 4, 6, 8, 10, and 12 weeks after a normal diet (chow), a high-fat diet (HFD), or a high-fat diet with LEO (HFD + LEO). (C) Representative image of mice body. (D) Body weight of mice fed chow, HFD, or HFD + LEO after 12 weeks. (E) SWAT, EWAT, and BAT weight after 12 weeks. (F) The levels of total cholesterol and total triglycerides were measured, and statistical analysis was conducted. (G) Representative pictures of EWAT stained by H&E, scale bar = 100 µm. Quantitative analysis of the adipocyte area. (H) Representative pictures of liver stained by oil red, scale bar = 100 µm. Quantitative analysis of percentage of lipid droplet area. Data are shown as means ± S.E.M. n = 5 per group. * p < 0.05, ** p < 0.01 and **** p < 0.0001 between groups.

Figure 2. LEO improves inflammation. (A) Representative immunofluorescence pictures of the expression and nuclear translocation of F4/80 in three groups’ EWAT. (B) Quantitative analysis of the ratios of F4/80. (C–F) mRNA expression of TNF-α, IL-1β, MCP-1, and COX-2 in the EWAT; n = 5 mice per group. (G–I) mRNA expression of TNF-α, IL-1β, and MCP-1 in the RAW264.7 cells treated with 100 ng/mL of TNF-α with or without 20 μM of LEO for 8 h. Data are shown as means ± S.E.M. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 between groups.

Figure 3. LEO alleviates oxidative stress. (A) mRNA expression level of NOX4 in the EWAT; n = 5 mice per group. (B) mRNA expression level of NOX4 in the RAW264.7 cells. Data are shown as means ± S.E.M. * p < 0.05, ** p < 0.01, *** p < 0.001 between groups.

Figure 4. LEO improves vascular function and remodeling. (A) Endothelium-dependent relaxation (EDR) of mesenteric artery ring from groups of mice in response to ACH. * p < 0.05 between chow group and HFD group. (B) SNP-induced endothelium-independent relaxation of mesenteric artery ring from groups of mice. * p < 0.05 between chow group and HFD group. # p < 0.05 between HFD group and HFD + LEO group. (C) The structure of collagen fiber was observed by Masson’s trichrome staining. Collagen fibers are in blue and the smooth muscle fibers are in red; n = 5 mice per group. (D) The quantitative analysis of the percentages of collagen content. Data are shown as means ± S.E.M. * p < 0.05 and **** p < 0.0001 between groups.

Figure 5. LEO targets YTHDF1 protein. (A) The chemical constructions of LEO and the biotin-conjugated LEO. (B) Flowchart of LIP-MS analysis. Cells were incubated with LEO or biotin-conjugated LEO for 6 h. Then, proteins pulled down by streptavidin beads were used for LC-MS analysis. (C) The image of proteins pulled down by streptavidin beads by using sodium dodecyl sulfate polyacrylamide gel electrophoresis. (D) Mass spectrum peak of YTHDF1 by LC-MS analysis. (E) RAW264.7 cells were incubated with LEO-biotin or LEO, followed by pull-down assay by using streptavidin beads; representative blot from three independent experiments is shown. (F) Representative images of autodocking for LEO and YTHDF1. (G) The protein level of YTHDF1 and GAPDH were detected by western blot in cell lysates of TNF-α (100 ng/mL)-stimulated RAW264.7 cells, which were pretreated with or without LEO. Image J was used to analyze gray value. Representative blot from three independent experiments is shown. (H) RAW264.7 cells were transfected to express YTHDF1-HA and mRNA expression levels of YTHDF1 were measured by qPCR. (I,J) RAW264.7 cells were transfected to express YTHDF1-HA and mRNA expression level of COX2 and MCP-1…

Figure 6. Schematic diagram of the mechanism of LEO alleviating oxidative stress in adipose tissue and vascular dysfunction of obese mice.