Abstract: BACKGROUND: Trichosanthes kirilowii Maxim. possesses antioxidant and anti-inflammatory properties and is commonly used in the treatment of cardiovascular diseases. However, the therapeutic efficacy of Trichosanthes kirilowii Maxim. is often limited by its complex composition and low bioavailability.
OBJECTIVE: To investigate the biological mechanisms underlying the effects of exosome-like vesicles derived from Trichosanthes kirilowii Maxim. in the prevention and treatment of atherosclerosis.
METHODS: (1) Extracellular vesicles were extracted from Trichosanthes kirilowii Maxim. using density gradient centrifugation. These vesicles were identified by transmission electron microscopy, nanoparticle size analyzer, and nanoparticle tracking analysis. Confocal laser scanning microscopy was used to observe the uptake of exosome-like vesicles derived from Trichosanthes kirilowii Maxim. by THP-1 cells. (2) THP-1 derived macrophages were randomly divided into the blank group, model group, and low-, medium-, and high-dose exosome-like vesicles derived from Trichosanthes kirilowii Maxim. groups. The blank group was cultured under normal conditions; the model group was treated with 50 mg/L oxidized low-density lipoprotein, and the exosome-like vesicles derived from Trichosanthes kirilowii Maxim. groups were treated with 50 mg/L oxidized low-density lipoprotein and 5, 10, or 20 mg/L of exosome-like vesicles derived from Trichosanthes kirilowii Maxim. After 24 hours of intervention, the expression levels of NLRP3 and Cleaved-Caspase-1 in the cells were assessed by western blot assay. (3) Thirty-three Apoe-/- mice were randomly divided into the blank group, model group, and exosome-like vesicles derived from Trichosanthes kirilowii Maxim. group. The blank group was fed a standard diet, while the other two groups were fed a high-fat diet for 16 weeks. Afterward,  the exosome-like vesicles derived from Trichosanthes kirilowii Maxim. group received intraperitoneal injections of exosome-like vesicles derived from Trichosanthes kirilowii Maxim. The blank and model groups received intraperitoneal injections of physiological saline, every other day for 4 weeks. Oil Red O staining was used to assess aortic plaque area. Hematoxylin-eosin staining was performed to observe aortic tissue pathological changes. Enzyme-linked immunosorbent assay was used to measure serum interleukin-1β and interleukin-18 levels. A reagent kit was used to assess blood lipid levels. Immunohistochemistry was employed to detect the expression of NLRP3 and Cleaved-Caspase-1 in the aorta. (4) High-performance liquid chromatography coupled with mass spectrometry and network pharmacology were used to predict the potential therapeutic targets of exosome-like vesicles derived from Trichosanthes kirilowii Maxim. in the treatment of coronary heart disease.
RESULTS AND CONCLUSION: (1) The exosome-like vesicles derived from Trichosanthes kirilowii Maxim. exhibited a tea-tray-like shape, with a size range of 50–150 nm, and were effectively internalized by THP-1 derived macrophages. (2) Cell experiments showed that compared to the model group, the expression levels of NLRP3 and Cleaved-Caspase-1 in the low-, medium-, and high-dose exosomes derived from Trichosanthes kirilowii Maxim. groups were reduced in a dose-dependent manner. (3) Mouse experiments revealed that compared to the model group, the exosome-like vesicles derived from Trichosanthes kirilowii Maxim. group showed reduced aortic plaque area, less inflammatory cell infiltration, decreased triglycerides, total cholesterol, and low-density lipoprotein cholesterol, increased high-density lipoprotein cholesterol, and reduced expression of NLRP3 and Cleaved-Caspase-1 proteins. Additionally, serum interleukin-1β and interleukin-18 levels were decreased. (4) Key pathways identified and enriched included the HIF-1 signaling pathway, PI3K-Akt signaling pathway, metabolic pathways, lipid metabolism, and atherosclerosis pathways. These results suggest that exosome-like vesicles derived from Trichosanthes kirilowii Maxim. can effectively alleviate atherosclerosis in mice, and their mechanism of action may be related to the inhibition of NLRP3 inflammasome-mediated inflammatory responses.

Key words: Trichosanthes kirilowii Maxim., exosome-like vesicle, NLRP3 inflammasome, atherosclerosis, inflammatory response, high performance liquid chromatography-mass, network pharmacology, coronary heart disease