Molecular Mechanism Of TanshinoneⅡA Anti-atherosclerosis Based On Inflammasome Activation

Atherosclerosis (AS) is one of important diseases threatening human health, which isthe most common pathogenic factor for many cardiovascular diseases. With thedevelopment of modern medical science, the study on AS pathogenesis is focusing on thecellular and molecular levels instead of pathological anatomy, now the AS has beenconsidered as an multifactor-involved sterile inflammation rather than originalunderstanding of the AS pathogenesis characterized by endothelial injury, lipid infiltrationand degenerative pathological process. It has been demonstrated that there are manyinflammatory cells and pro-inflammatory cytokines in AS plaque tissues, playing animportant role inAS occurrence and development.TanshinoneⅡA (TanⅡA) is one of the important effective components in Chinesetraditional medicine salviae miltiorrhizae, which has been widely used to prevent and treatthe cardiovascular diseases due to its functions of anti-inflammation, microcirculationimprovement, coronary expansion, platelet aggregation inhibition and anti-thrombus. TanⅡA is a main fat soluble component of the alviae miltiorrhizae, possessing the anti-ASfunction. Recently, many researches on AS pharmacology have been extensivelyperformed, however, the molecular mechanism of TanⅡAanti-AS is not fully elucidated.Recent studies proved that the low density lipoprotein cholesterin (LDL-Ch) mediatedAS was related to its cholesterol crystal (ChC)-induced the inflammasome activation ofmacrophages. The inflammasome identified in the last decade to be existed inmono/macrophages, is a protein complex composed of pattern recognition receptors andcaspase-1, the former sensing exogenous pathogen and endogenous danger signals, thelater cleaving pro-IL-1β and pro-IL-18into mature IL-1β and IL-18. IL-1β is an importantcytokine mediating tissue inflammation and also an important pro-inflammatory cytokinefor AS occurrence and development. It was reported that ChC could activate macrophageNLRP3inflammasome and consequently released IL-1β was considered to be a significantfactor triggering AS. Whether the TanⅡA anti-AS function is related to its inhibition onChC-induced macrophage inflammasome activation is unknown.To identify the anti-AS effect of TanⅡA in vivo, we fed ApoE gene-defect（ApoE-/-）mice to establish AS mouse model and then treated those mice by TanⅡA to analyze itseffects on AS plagues formation and blood lipid metabolism. Our results show that TanⅡAcan significantly reduce lipid deposition on ApoE-/-mouse aortic wall and inhibit ASplagues formation. Meanwhile TanⅡA reduced the LDL-Ch levels in ApoE-/-mouse serum, indicating that TanⅡAhas the strong effect of anti-ChC induced AS.To investigate the molecular mechanism of TanⅡA anti-ChC induced AS, we shouldmake sure whether Tan Ⅱ A inhibits ChC-induced inflammasome activation. Thus, wetreated mouse B6macrophages by TanⅡA and LPS successively, stimulated them by ChCand detected the IL-1β in the cell culture medium by ELISA and the mature caspase-1inthe cells by Western blot. The results demonstrated that the level of intracellular maturecaspase-1and IL-1β in the medium significantly decreased after TanⅡA treatmentand TanⅡA significantly inhibited ChC-induced inflammasome activation, therefore displaying itsanti-AS activity.It is reported that ChC can activate NLRP3inflammasome, which is associated withcathepsin B releasing due to ChC-induced endo-lysosome membrane damage. To furtheridentify that ChC can activate NLRP3inflammasome in macrophage, we use differentinflammasome pathway-associated gene-defect (NLRP3-/-, AIM2-/-, NLRC4-/-, ASC-/-andcaspase-1-/-) mouse B6macrophages to analyze the ChC effect on inflammasomeactivation. The results showed that ChC could stimulate AIM2-/-, NLRC4-/-B6cells, notNLRP3-/-, ASC-/-and caspase-1-/-(NLRP3pathway associated proteins) B6cells, to releaseIL-1β, illustrating that ChC can activate macrophage NLRP3inflammasome. To identifywhether ChC-induced inflammasome activation is associated with the cathepsin B releasefrom lysosome induced by ChC and to explore whether Tan Ⅱ A inhibition oninflammasome activation is related to its inhibition on lysosomal membrane damage andcathepsin B release, we stained TanⅡA-treated ChC-induced B6cell and cathepsin B-/-B6cells by fluorescent-conjugated antibodies and detected them by laser scanning confocalmicroscope to analyze the effects of ChC on lysosomal cathepsin B release and theinhibition of TanⅡA on the release. Results showed that ChC can significantly increasedcathepsin B level in the cytosol of wild-type B6cells (not cathepsin B-/-B6cells), while theTanⅡA significantly inhibited ChC-induced lysosomal cathepsin B release. Thus it can beseen from the results that ChC-induced NLRP3inflammasome activation is related to itseffect to trigger lysosomal cathepsin B release and the TanⅡA inhibitory function onChC-induced inflammasome activation is associated with its inhibition on lysosomalcathepsin B release.It was reported that Mitochondria-derived ROS was considered as one of theimportant activators of NLRP3inflammasome. Some particulates have been shown toinduce ROS production and subsequently activate inflammasome. However, whether ChCparticulate can induce ROS production and whether TanⅡA inhibits ChC-induced ROSproduction to restrain infflammasome activating remain open. To clarify these questions,we used2’,7’-Dichlorofluorescin diacetate(DCFH-DA), a fluorescence detector of ROS, todetect ROS generation in macrophages upon ChC treatment and to investigate theinhibitory effects of TanⅡAon ChC-induced ROS generation.In conclusion, by inhibiting ChC-induced lysosomal cathepsin B release and ROSgeneration, TanⅡA can inhibit ChC-induced NLRP3inflammasome activation to retard inflammasome-mediated AS occurrence and development. This study provided theimportant data for further investigating the mechanism of TanⅡA anti-AS and a newperspective for investigating the therapeutic function of effective components in salviaemiltiorrhizae.