The Enhanced Pharmacological Effects of Modified Traditional Chinese Medicine in Attenuation of Atherosclerosis Is Driven by Modulation of Gut Microbiota

Abstract

Accumulating evidence indicated that gut microbiota-targeted therapy is a promising strategy to treat Cardiovascular Disease (CVD). Traditional Chinese Medicine (TCM) has been used in CVD treatments for over 2,000 years which is believed to result from the modulation of gut microbiota, yet the underlying mechanism remains elusive. According to the theoretical system of TCM, we developed an innovative formula of TCM named "TongMai ZhuYu (TMZY)" on top of one classic Chinese herbal formula ["XueFu ZhuYu (XFZY)"], which can more effectively alleviate CVD in the clinical practice. Here, we first systematically assessed the pharmacological effects of TMZY, XFZY, and atorvastatin on atherosclerosis (AS) induced by high-fat diet (HFD) in rats. TMZY typically outperformed others in alleviating AS rats by characterization of pathological morphology, immunohistochemistry, inflammatory cytokines. Remarkably, combining this modified TCM formula (TMZY) with atorvastatin can further help the alleviation of AS in rats by suppressing immune and inflammatory responses. Furthermore, to test whether TMZY alleviated AS symptoms by altering gut microbial compositions (dysbiosis), we employed 16S amplicon sequencing to investigate gut microbiota changes in the AS mice induced by high choline diet (HCD) using both TMZY and XFZY under antibiotic-treated and untreated conditions. TCM formulas induced consistent and remarkable changes in the phenotypes and microbiota in the HCD mice. TMZY modulated more changes in the gut microbiota to improve diseased phenotypes than XFZY. Notably, the TMZY-intervention effect on CVD in mice attenuated after the suppression of gut microbial activity by antibiotics. Collectively, we demonstrated that TCM herbals could effectively modulate the gut microbiota as a mechanism for altering the pathogenesis of cardiovascular disorders in mice/rats.

Keywords: atherosclerosis; cardiovascular disease; combination of Chinese and Western medicine; gut microbiota; traditional Chinese medicine (TCM).

Figure 1

The chromatographic profile of TMZY extracts and chemical structures of major pharmaco-active compounds identified. (A) Typical high-performance liquid chromatography (HPLC) chromatograms of TMZY extracts. mAU, milli-absorbance unit. (B) Chemical structures of the major pharmaco-active compounds identified. Peak No.: 2. Ursolic acid; 6. Hydroxyl-safflor yellow A; 11. Paeoniflorin; 12. Ferulic acid; 16. Naringin; 17. Hesperdin; 18. Neohesperidin.

Figure 2

HE staining of thoracic aortic reveals the interventional effect of TCM treatments on atherosclerotic rats. (A) HE staining of rat thoracic aorta in healthy control group. (B) HE staining of rat thoracic aorta in HFD group. (C) HE staining of rat thoracic aorta with HFD and atorvastatin intervention. (D) HE staining of rat thoracic aorta with HFD and XFZY intervention. (E) HE staining of rat thoracic aorta with HFD and TMZY intervention. (F) HE staining of rat thoracic aorta with HFD and the integrative treatment. We marked mild hyperplasia of vascular SMCs with arrows. (G–H) The boxplots of digitalized intima-media thickness of thoracic aorta (which were generated by ipp6.0) of rats from six treatment groups. The p values of significant associations are all marked on top of boxplots, while NS donates “non-significance”.

Figure 3

The expression of immune factors in thoracic aortic is associated with TCM treatments. (A) Expression of immune factors (e.g. TGF-β, VCAM1, HMGB1, and FOXP3) in healthy controls. (B) Expression of immune factors in HFD intervention group. (C) Expression of immune factors in HFD with atorvastatin treated group. (D) Expression of immune factors in HFD with XFZY treated group. (E) Expression of immune factors in TMZY with atorvastatin treated group. (F) Expression of immune factors in HFD with the integrative treatment. (G) The bar plot indicating the digitalized intima-media thickness of thoracic aorta (which were generated by ipp6.0) of rats from six groups. The p values of significant associations are all marked on top of boxplots, while NS donates “non-significance”.

Figure 4

The blood lipid indexes and expression of serum immune factors in atherosclerotic rats were greatly improved under TCM treatments. Blood lipid indexes [i.e. TC (mmol/L), TG (mmol/L), HDL-C (mmol/L), and LDL-C (mmol/L)] and expression of serum immune factors [i.e. hs-CRP (ng/L), IFN-r (ng/L), and IL-4 (ng/L)] were respectively compared across all six groups. The p values of significant associations are all marked on top of boxplots, while NS donates “non-significance”.

Figure 5

The phenotypic changes (weight-gain, concentration of TMAO, and platelet aggregation) of mice in the high-fat diet (HFD) by TCM treatments with or without the antibiotic pre-treatment. (A) Boxplots of weight-gain of mice from (1) control group, (2) HFD groups, (3) HFD with XFZY treatment, (4) HFD with TMZY treatment, (5) HFD with antibiotic intervention, (6) HFD with antibiotic intervention and XFZY treatment, (7) HFD with antibiotic intervention and TMZY treatment. (B) Boxplots of the TMAO level (μM) in mice among the seven groups. (C) Boxplots of the platelet aggregation (%) in mice among the seven groups.

Figure 6

The dysbiosis in the gut microbiota of HFD mice was perturbed and rehabilitated by TCM treatments. (A) Boxplots of Shannon diversity among (1) controls, (2) HFD groups, (3) HFD with XFZY treatment, (4) HFD with TMZY treatment, (5) HFD with antibiotic intervention, (6) HFD with antibiotic intervention and XFZY treatment, (7) HFD with antibiotic intervention and TMZY treatment. (B) PCoA plot of fecal microbiota from the seven groups based on Meta-Strom distance. The boxplots of PC1 and PC2 were presented as well. Statistical significance is marked as ** (p < 0.005). (C) Heat-map of differentially abundant genera between HFD group and healthy controls, where the relative abundance of those marker genera in other five groups were also presented as reference. The color indicates the log10-scaled relative abundance of a bacterial genus.

Figure 7

Microbial functional response in the gut microbiome to TCM treatments on HFD mice. In the left panel, a heatmap indicate the mean fold change of the relative abundance of a given pathway in a treatment group over the HFD group. All 42 pathways are significantly different between healthy controls from HFD group, which were designated as a “reference” microbial response to HDF that presumably corresponds to a full recovery in the gut microbiome from HFD (the leftmost column in the heatmap). The right panel showed the statistical significance (p value) of the fold changes of corresponding pathways in the left panel and its association directionality under a treatment as compared to HDF group. A black cell indicates a significant change in the corresponding pathway toward the healthy status under a treatment. A gray cell refers to a health-associated shift but no statistical significance was found. A white cell indicates neither a health-associated shift nor a statistical significance related to HFD was found in a pathway under a treatment.