ABSTRACT
This study aimed to investigate the mechanism of "Trichosanthis Fructus-Allii Macrostemonis Bulbus" (GX) on phlegm and blood stasis syndrome (PBSS) rats combining the methods of network pharmacology and experimental verification. Animal experiment ethical requirements were approved by the Ethical Committee Experimental Animal Center of Anhui University of Chinese Medicine (grant number: AHUCM-rats-2021070). Based on the HPLC-Q-TOF-MS analysis and database, 69 chemical constituents of GX and 163 targets of GX for the treatment of phlegm and blood stasis-related cardiovascular diseases were obtained. Then, key targets such as serine/threonine kinase 1 (Akt1), tumor necrosis factor (TNF), interleukin 6 (IL6), vascular endothelial growth factor A (VEGFA), cellular tumor antigen p53 (Tp53) were screened. Pathway analysis showed that the targets of GX in the treatment of phlegm and blood stasis-relate cardiovascular diseases were mainly involved in PI3K/Akt signaling pathway, sphingolipid metabolism, platelet activation, hypoxia inducible factor-1 (HIF-1), ras-proximate-1 (rap1) and other signaling pathways. In addition, molecular docking analysis showed that apigenin, cucurbitacin D, linolenic acid and kaempferol and other key components had potential binding ability with Akt1, TNF, IL6, VEGFA and Tp53. In the animal experiments, compared to the phlegm and blood stasis syndrome group, GX could significantly improve the traditional Chinese medicine syndrome score, blood lipid, vascular endothelial structure disorders and reduce serum endothelin-1 (ET-1) level, increase serum nitric oxide (NO) and endothelial nitric oxide synthase (eNOS) levels, which could restore aortic endothelial function. In addition, the expression of intercellular cell adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in aorta could be significantly reduced, which could improve the vascular endothelial injury of aorta. Western blot revealed that GX could significantly decrease the phosphorylation levels of phosphoinositide 3-kinase (PI3K) and Akt in aorta. This study revealed the mechanism of GX in treatment of phlegm and blood stasis-relate cardiovascular diseases is consistent with the characteristics of multiple ingredients, multiple targets and multiple pathways. In addition, this study also clarified that the reversal of pathological of phlegm and blood stasis syndrome rats may be related to GX inhibiting PI3K/Akt signaling pathway, which could improve vascular inflammation and vascular endothelial function injury.
ABSTRACT
Objective:To explore the effect of Trichosanthis Fructus-Allii Macrostemonis Bulbus medicine on the proliferation and autophagy levels of aortic plaque vascular smooth muscle cells （VSMCs） in ApoE<sup>-/-</sup> mice with atherosclerosis （AS）. Method:A total of 40 ApoE<sup>-/-</sup> mice were fed with high-fat diet to replicate AS animal models. They were randomly divided into model group， Trichosanthis Fructus-Allii Macrostemonis Bulbus group， rapamycin group and atorvastatin group， and 10 mice with normal diet C57BL/6J mice were the blank group. The blank group and the model groups were given normal saline by gavage， while Trichosanthis Fructus-Allii Macrostemonis Bulbus group， rapamycin group and atorvastatin group were given corresponding drugs by gavage for 8 weeks. After the experiment， the mice were sacrificed. Total cholesterol （TC）， triglyceride （TG）， high-density lipoprotein cholesterol （HDL-C） and low-density lipoprotein cholesterol （LDL-C） levels were detected by the Microplate reader， the ratio of the aortic plaque area to the total area was observed and measured by staining with aortic gross oil red O. Western blot method was used to detect the proliferation-related protein proliferating cell nuclear antigen （PCNA） and <italic>α</italic>-smooth muscle actin （<italic>α</italic>-SMA） levels of VSMCs in the aortic media. Transmission electron microscopy was used to observe the autophagosomes of VSMCs and detect the expressions of VSMCs autophagy-related proteins Beclin-1， light chain proteinⅡ （LC3Ⅱ） and p62. Result:Compared with the model group， the Trichosanthis Fructus-Allii Macrostemonis Bulbus group showed significant reduction in the aortic lipid accumulation and plaque area of AS mice and the levels of TC， TG， LDL-C （<italic>P</italic><0.01） and increase of HDL-C （<italic>P</italic><0.05）. Trichosanthis Fructus-Allii Macrostemonis Bulbus significantly reduced the levels of proliferation-related antigens PCNA and <italic>α</italic>-SMA in aortic VSMCs （<italic>P</italic><0.01）， and inhibited the excessive proliferation of VSMCs. Trichosanthis Fructus-Allii Macrostemonis Bulbus significantly up-regulated Beclin-1 and LC3Ⅱ in aortic VSMCs protein expression， decreased p62 accumulation （<italic>P</italic><0.01）， increased the expressions of VSMCs autophagosomes， and increased the autophagy level of VSMCs. Conclusion:Trichosanthis Fructus-Allii Macrostemonis Bulbus regulates blood lipid levels in AS mice， and inhibits the excessive proliferation of aortic VSMCs and plaque formation in the aorta of AS mice. The mechanism may be related to the up-regulation of the autophagy activity of VSMCs.
ABSTRACT
In this study, ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry(UPLC-Q-TOF-MS)-based liver metabolomics approach was used to explore the mechanism of "Trichosanthis Fructus-Allii Macrostemonis Bulbus" in improving atherosclerosis(AS) of mice with apolipoprotein E gene knockout(ApoE~(-/-)). AS mouse model was induced by high-fat diet. The pathological and biochemical indexes such as the histopathological changes, body weight, liver weight, blood lipid level and inflammatory factors in the liver of mice were determined. The metabolic profiling of mice liver samples was performed with UPLC-Q-TOF-MS. Multiple statistical analysis methods including partial least squares discriminant analysis(PLS-DA) and orthogonal partial least squares discriminant analysis(OPLS-DA) were employed to screen and identify biomarkers. The levels of related enzymes including LCAT, sPLA2, EPT1 and ACER1 were detected. The results showed that "Trichosanthis Fructus-Allii Macrostemonis Bulbus" significantly reduced the areas of aortic plaque and fat vacuoles of liver in AS mice and decreased the accumulation of lipid droplets and liver coefficient. "Trichosanthis Fructus-Allii Macrostemonis Bulbus" also regulated the levels of blood lipid and inflammatory injury in the liver. The metabolites of the control group, the model group and the "Trichosanthis Fructus-Allii Macrostemonis Bulbus" group could be distinguished significantly. Fifteen potential biomarkers related to AS were discovered and preliminarily identified, seven of which could be regulated by "Trichosanthis Fructus-Allii Macrostemonis Bulbus" in a trend of returning to normal. Metabolic pathway analysis screened out two major metabolic pathways. "Trichosanthis Fructus-Allii Macrostemonis Bulbus" obviously regulated the levels of LCAT, sPLA2, EPT1 and ACER1. It was inferred that "Trichosanthis Fructus-Allii Macrostemonis Bulbus" could play a major role in AS treatment by regulating glycerophospholipid and sphingolipid metabolism disorders in the liver, with the mechanism probably relating to the intervention of the expression of LCAT, sPLA2, EPT1 and ACER1.
Subject(s)
Animals , Mice , Apolipoproteins E/genetics , Atherosclerosis/genetics , Chromatography, High Pressure Liquid , Drugs, Chinese Herbal , Liver , MetabolomicsABSTRACT
Objective:Based on the hyperlipidemia rat model and network pharmacology technology, the mechanism of action of Trichosanthis Fructus-Allii Macrostemonis Bulbus herb pairs against hyperlipidemia was analyzed. Method:The levels of blood lipids and inflammatory factors were measured through prophylactic administration of low, medium and high-dose Trichosanthis Fructus-Allii Macrostemonis Bulbus herb pairs in hyperlipidemia rats. The active ingredients of Trichosanthis Fructus-Allii Macrostemonis Bulbus herb pairs were screened out through Traditional Chinese Medicine System Pharmacology Database and Analysis Platform (TCMSP) and text mining. The targets of active ingredients screened through the Swiss Target Prediction, Similarity ensemble approach (SEA), DrugBank database. The disease targets were collected through Therapeutic Target Database (TTD), Online Mendelian Inheritance in Man (OMIM), DrugBank, DisGeNET database. The targets of active ingredients and disease target were integrated, and screened through topological parameters to gain the main candidate targets of Trichosanthis Fructus-Allii Macrostemonis Bulbus herb pairs against hyperlipidemia. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis and the gene ontology (GO) functional enrichment analysis were conducted through ClueGO and Database for Annotation, Visualization and Integrated Discovery (DAVID), respectively. The traditional Chinese medicine-chemical ingredient-target network model, and the target-pathway network model were constructed through Cytoscape, and their crosstalk target and signal pathway were analyzed. Result:Animal experiments showed that the prophylactic administration of Trichosanthis Fructus-Allii Macrostemonis Bulbus herb pairs significantly reduced the levels of total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C) in serum of rats with hyperlipidemia, increased high-density lipoprotein (HDL-C) levels, and inhibited the expressions of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). According to the findings, 27 active ingredients, such as mandenol, diosmetin and α-spinasterol, might be the main active ingredients of Trichosanthis Fructus-Allii Macrostemonis Bulbus herb pairs, 16 crosstalk targets and 10 signal pathways might be the main therapeutic targets and pathways, main targeting apolipoprotein A1 (APOA1), apolipoprotein A2 (APOA2), apolipoprotein C3 (APOC3), lipoprotein lipase (LPL), low-density lipoprotein receptor (LDLR) and other crosstalk targets affected cholesterol metabolism, bile secretion, peroxisome proliferator activated receptor (PPAR) signaling pathway in regulating the lipid level, targeting tumor necrosis factor (TNF), IL-6, interleukin-1β (IL-1β), mitogen-activated protein kinase 1 (MAPK1), C-C motif chemokine 2 (CCL2) and other crosstalk targets affected tumor necrosis factor (TNF) signaling pathway, Toll-like receptor signaling pathway, interleukin-17 (IL-17) signaling pathway, and hypoxia inducible factor (HIF) signaling pathway in regulating the inflammatory factor level. The DAVID database for GO enrichment analysis showed that the hyperlipidemia was treated mainly through biological processes, such as inflammation, lipid localization, storage and lipid metabolism. Conclusion:These findings can predict the mechanism of action of Trichosanthis Fructus-Allii Macrostemonis Bulbus herb pairs against hyperlipidemia, and provide a theoretical basis for the material basis and clinical application of Trichosanthis Fructus-Allii Macrostemonis Bulbus herb pairs.