| Part1:Establish and improvement in an elastase-induced abdominal aortic aneurysm rat modelObjective:To establish a stable and efficient model of abdominal aortic aneurysm.Methods:First,50 S-D rats were divided into 5 groups which were received aortic infusion of saline and different concentrated (0.1U/μL、1 U/μL、10 U/μL、100 U/uL)for 30min to made abdominal aortic aneurysm. They were scanned by ultro-sound and compared with formation and mortality. Then 30 S-D rats were divided into 3 groups which were received the same treatment of the best concentration for different time (lmin, 10min,30min). They were scanned and compared via the same method.Results:The survival rate of saline group was 100% and the rest were 90%. The aneurysmal rate of saline and 0.1U/μL groups were 0 while the rest were 100%. There was no significant relevant between infusion concentration and aorta enlargement within successful aneurysmal groups (P=0.136 & P=0.261). Then in the second experiment of aortic infusion with IU/μL elastase,30 S-D rats were divided into 3 groups:Infusion for lmin, 10min,30min. The survival rate of 30min group was 90% and the rest were 100%, The aneurysmal rate of lmin groups were 0 while the rest were 100%. The longer infusion time was, the larger abdominal aorta became (P=0.000 & P=0.007)Conclusion:It is a stable and efficient method to establish an abdominal aortic aneurysm model for rats with elastase infusion in 1U/μL for 10min.Part2:Inhibition of experimental abdominal aortic aneurysm in a rat model via the Tanshinone ⅡAObjective:The purpose of this study was to investigate whether Tanshinone ⅡA (Tan II A), one of the major lipophilic components of Salvia miltiorrhiza Bunge (SM), could inhibit the development of elastase-induced experimental abdominal aortic aneurysms (AAAs).Methods:Male Sprague-Dawley rats (n=12/each group) were devided into three groups:Tan ⅡA-, control-, and sham-. Rats of Tan ⅡA and control groups underwent intra-aortic elastase perfusion to induce AAAs, while rats of sham-group were perfused with saline. Only Tan Ⅱ A-group received Tan ⅡA treatment (2mg/per rat.d-1). The maximum luminal diameter of the abdominal aorta was measured 7 days before and after perfusion (the 0th,7th,14th,21th and 28th day). Systolic blood pressure was measured twice by tail-cuff technique before administration and sacrifice. Aortic tissue samples were harvested on the 28th day and evaluated by Enzyme Linked ImmunoSorbent Assay (ELISA), Real-time reverse transcription-polymerase chain reaction(RT-PCR), Western blot, Immunohistochemistry (IHC) and Miller’s elastin-Van Gieson’s (EVG) staining.Results:Tan ⅡA attenuated development of AAA, decreasing maximal aortic diameter by 25.8%(Tan ⅡA vs control,0.23±0.023 vs 0.31±0.038, t=5.62, p<0.001) and preserving elastin lamellae (Tan ⅡA vs control,14.6±3.3 vs 7.6±2.6, t=5.09, p<0.001). Tan ⅡA also decreased interleukin (IL)-6, monocyte chemotactic protein (MCP)-1, Inducible Nitric Oxide Synthase (iNOS),8-hydroxyguanine (8-OHdG, a marker of oxidative damage to DNA) and 8-isoprostane content (a marker of oxidative stress) in aortic tissues (Tan ⅡA vs control, MCP-1,0.68±2.45 vs 0.39±1.55, t=9.67,p<0.001;IL-6,48.3±9.7 vs 144.2±13.9,t=17.59,p<0.001; iNOS,0.52±3.24 vs 1.22±4.37,t=12.19,p<0.001; 8-OHdG,1.60±0.21 vs 2.46±0.27, t=7.83, p<0.001; 8-isoprostane, Tan ⅡA vs Control,13.38±3.27 vs 28.84±5.53, t=7.51, p<0.001;). The proteins of matrix metalloproteinase (MMP)-2 and MMP-9 were markedly down-regulated (Tan ⅡA vs control, MMP-2,1.85±0.27 vs 3.61±0.31, t=13.18, p< 0.001; MMP-9,2.28±0.23 vs 3.84±0.28, t=13.30, p<0.001), accompany with notably reduced mRNA expression of tumor necrosis factor (TNF)-α, MMP-2/9 and IL-1β(Tan ⅡA vs control, MMP-2,1.52±0.44 vs 2.14±0.54 t=2.76,p=0.007; MMP-9, 1.35±0.44 vs 2.76±0.77,t=5.48, p<0.001; Tan ⅡA vs control, IL-Ⅱβ,0.73±0.19 vs 1.35±0.22, t=6.59, p<0.001; TNF-α,0.68±0.19 vs 2.07±0.36, t=10.69, p<0.001). However, mRNA of tissue inhibitors of metalloproteinase (TIMP)-1 and TIMP-2 were both significantly up-regulated in Vitamin C group(Tan ⅡA vs control, TIMP-1, 0.93±0.26 vs 0.48±0.18, t=4.34, p<0.001; TIMP-2,3.11±0.58 vs 0.41±0.17, t=13.42, p<0.001).Vitamin C treatment had no significant effect on systolic blood pressure (p=0.226,0.287,0.304, respectively).Conclusion:Tan Ⅱ A could inhibit the development of elastase-induced experimental AAAs by suppressing proteolysis, inflammation, oxidative stress and preserving VSMCs. It may be a new pharmacological therapy of AAAs.Part3:Tanshinone ⅡA attenuates Elastase-Induced AAA in Rats via inhibition of MyD88-dependent TLR-4 SignalingObjective:The purpose of this study was to determine whether myeloid differentiation factor88 (MyD88)-dependent toll-like receptor-4 (TLR-4) signaling contributed to the inhibition of abdominal aortic aneurysm (AAA) by Tanshinone Ⅱ A (Tan Ⅱ A).Methods:Male Sprague-Dawley rats (n=12/group) were devided into three groups randomly:Tan ⅡA, control, and sham. The rats from Tan ⅡA and control groups underwent intraaortic elastase perfusion to induce AAAs, and those in the sham group were perfused with saline. Only the Tan ⅡA group received Tan ⅡA (2 mg/rat/d). Aortic tissue samples were harvested at 24 d after perfusion and evaluated using reverse transcriptase-polymerase chain reaction, Western blot, immunohistochemistry and immunofluorescence.Results:The over-expression of TLR-4, MyD88, phosphorylated nuclear factor κB (pNF-KB) and phosphorylated IκBα (plκBα) induced by elastase perfusion were significantly decreased by Tan IIA treatment (TLR-4,0.82316±0.22 vs 1.14120±0.29, t=-2.62, p=0.017; MyD88,0.63919±0.26 vs 0.99909±0.22, t=-3.17, p=0.006; pNF-KB,1.51225±0.40 vs 2.92694±0.49, t=-6.71, p<0.001; pIκBα,1.07369±0.16 vs 1.31090±0.18, t=-2.95, p=0.008).Conclusion:Tan Ⅱ A attenuates elastase-induced AAA in rats possibly via the inhibition of MyD88-dependent TLR-4 signaling, which may be one potential explanation of why Tan ⅡA inhibits AAA development through multiple effects. |
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