| With the in-depth research, the inflammation and immunity doctrine of atherosclerosis (AS) has been gradually recognized. Toll-like receptor (TLR) is a bridge between the immune reaction and chronic inflammation, which plays an important role in the occurrence and development of AS, and is considered as a new target for anti-inflammation and immunotherapy. Nevertheless, due to the particularity of immunologic function, the effectiveness and safety of TLR-blocker in clinical applications has not been confirmed. Tanshinone ⅡA is a natural cardio-protective agent with pleiotropic effect. However, yet it has not been reported whether Tanshinone II A can play anti-inflammation and immune-regulation effects through intervening TLR signal transduction pathways, which lead to stabilization of vulnerable AS plaque. In this study, with the starting point of immunity and inflammatory pathway associated with TLR4/MyD88/NF-κB, and on the basis of a combined studies of in vivo and in vitro, we observe the effect of Tanshinone II A on aorta vulnerable AS plaque of ApoE-deficient mice, and its relationship with macrophage cells TLR4/MyD88/NF-κB pathway and the expression of downstream inflammatory cytokine including TNF-a, ICAM-1. We aim to explore Tanshinone ⅡA’s anti-inflammation and immune-regulation mechanism in stabilizing AS vulnerable plaque, and provide new ideas and interventions in anti-inflammation and immunotherapy for AS.Part I:Tanshinone ⅡA stabilizes vulnerable atherosclerotic plaque via TLR4/MyD88/NF-KB signaling pathway in apoE deficient miceObjectives:Tanshinone ⅡA (Tan ⅡA), a lipophilic constituent of Salvia miltiorrhiza Bunge, has shown apromising cardioprotective effect including anti-atherosclerosis (anti-AS). This study aims to investigate whether Tan ⅡA could play anti-inflammatory and immune-regulatingroles in stabilizing vulnerable atherosclerotic plaque in apoE deficient mice viaToll-like receptor 4 (TLR4)/myeloid differentiation factor88 (MyD88)/nuclearfactor-kappa B (NF-κB) signaling pathway.Methods:Male apoE deficient (ApoE-/-) mice were fed with a high fat diet for 12 weeksand then randomized to vehicle (MOD) or Tan ⅡA (high dose (HT):90 mg/kg/day, moderate dose (MT):30 mg/kg/day, low dose (LT):10 mg/kg/day) or atorvastatin(ATO) (5mg/kg/day) for 13 weeks. Male C57BL/6 mice were fed with normal chowdiet as control. The plaque stability was evaluated according to the morphologyand composition of atherosclerotic plaque in HE staining and Movat staining sections by calculating the area proportion of extracellular lipid, collagenous fiberand foam cells to the plaque. The expression of key genes of TLR4/MyD88/NF-KB signaling pathway in aorta fractions was determined by immunohistochemistry and real-time polymerase chain reaction (RT-PCR). The concentrations of tumornecrosis factor-a (TNF-a) and monocyte chemoattractant protein-1 (MCP-1) were detected by cytometric bead array.Results:Tan ⅡA stabilized aortic plaque with a striking reduction in the area proportion of extracellular lipid (ATO:13.15±1.2%, HT:12.2±1.64%, MT:13.93±1.59%, MOD:18.84±1.46%, P<0.05) or foam cells (ATO:16.05±1.26%, HT:14.88±1.79%, MT: 16.61±1.47%, MOD:22.08±1.69%, P<0.05) to the plaque, and an evident increase in content of collagenous fiber (ATO:16.22±1.91%, HT:17.58±1.33%, MT:15.71±2.26%, LT:14.92±1.65%, MOD:9.61±0.7%, P<0.05) to the plaque than that in the modelgroup, concomitant with down-regulation the protein expression of TLR4, MyD88 and NF-κB p65, serum level of MCP-1, TNF-a and mRNA of TLR4, MyD88 and intercellular cell adhesion molecule-1 (ICAM-1) in a dose-dependent manner.Conclusions:These results suggest Tan ⅡA could stabilize vulnerable AS plaque in ApoE-/- mice via TLR4/MyD88/NF-icB signaling pathway, which might be a potential candidate agent with anti-inflammatory and immune-regulating effect in the prevention and treatmentof AS.Part Ⅱ:Effect of tanshinone ⅡA on TLR4/MyD88/NF-κB signaling pathway of RAW264.7 macrophages induced by LPSObjectives:The aim of this study was to explore whether the anti-inflammatory and immune effects of Tan ⅡA associated with TLR4/MyD88/NF-κB pathway in RAW264.7 macrophages induced by lipopolysaccharide (LPS).Methods:Inflammatory cell model was built by LPS-simulated RAW264.7 macrophage. There were six groups, control group (CON), model group (MOD), high dose of Tan ⅡA group (HT), middle dose of Tan ⅡA group (MT), low dose of Tan ⅡA group (LT), pathway inhibitor group (TAK-242). CCK-8 was used to evaluate the cell viability. Western-blot analysis was used to detect protein expression of TLR4, MyD88, NF-κB. Serum levels of inflammatory cytokines TNF-a was measured by ELISA assay.Results:RAW264.7 cells secreted large amounts of TNF-a after stimulation of LPS. HT decreased concentration of TNF-a compared with MOD (P<0.05). TLR4 protein expression of MOD was significantly increased and was twice of CON (P<0.05). HT significantly reduced the TLR4 protein expression, decreased by 43.18%(P<0.05). MyD88 protein expression of MOD was 2.10 times higher than CON (P<0.05), HT reduced MyD88 protein expression, decreased by 34.10% compared with MOD (P<0.05). Expression of P-NF-κB of MOD was higher than CON (P<0.05), HT reduced the protein expression of P-NF-κB (P<0.05). TAK-242 partially inhibited anti-inflammatory and immune effect of Tan II A.Conclusions:Anti-inflammatory and immune mechanism of Tan ⅡA partially involves TLR4/MyD88/NF-κB pathway. |
PDF Full Text Request