| Objective: The present study was designed to investigate the proliferation promotion effect of the trilobatin(TLB)and the angiogenesis effect of TLB on middle cerebral artery occlusion(MCAO)-induced cerebral ischemic-reperfusion injury(CI/RI)in rats and explore its potential mechanisms.Methods:1.Study of the proliferation promotion effect of TLB on b End.3 cells in vitro: the safe concentration range and the optimum time of TLB on b End.3 cells were measured by MTT assay;The proliferation promotion effect of TLB on b End.3 cells within72 h were detecting using EDU fluorescence staining and Brd U ELISA assay;cellular morphologic variation was observed by phase contrast microscopy;Cell cycle was analyzed by flow cytometry,and the expression of cycle related proteins(cyclin D1,CDK4),Sir2 family proteins(SIRT1-7),and VEGFA protein expression were determined by western blot analysis;Furthermore,the affinity between TLB and SIRT7 were explored by molecular docking and surface plasmon resonance(SPR),respectively.2.Pharmacodynamic study of TLB on angiogenesis after CI/RI in vivo: Male SD rats(200-220 g)were adaptive feeding for one week.Then,CI/RI rat model were induced by MCAO,and reperfusion was performed after 2 h of focal cerebral ischemia.Thereafter,the rats were randomly divided into four groups: sham,sham+TLB 20 mg/kg,MCAO,MCAO+TLB 20 mg/kg.Sham+TLB 20 mg/kg and MCAO+TLB 20 mg/kg groups were treated with TLB 20 mg/kg by gavage twice a day for day 1,3,7,14,28,respectively.While,animals in sham and MCAO groups were given volume-matched saline.The laser doppler flowmetry was used to monitor cerebral blood flow(CBF)during modeling process.Then,neurological impairment in rats were scored according to the Longa 5 method;The levels of PECAM-1(CD31),angiogenesis marker,Brd U and tomato lectin were assessed by immunofluorescence staining;Furthermore,ELISA was adopted to measure the contents of VEGFA and VEGFR-2 in the cerebral ischemia penumbra and the expression of SIRT7 was detected by western blot analysis.Results:1.TLB promoted proliferation of b End.3 cells in vitro: The results showed that TLB promoted the proliferation of b End.3 cells in a concentration dependent manner,as well as increased the proportion of cells in S phase;Moreover,TLB not only increased the expressions of cyclin D1 and CDK4,but also elevated SIRT6 and SIRT7 expressions.In addition,the expression of VEGFA was also enhanced by TLB;Of note,TLB might directly bind to SIRT7 as evidenced by molecular docking and SPR.2.TLB promoted angiogenesis in rats after CI/RI in vivo: the CBF of MCAO rats decreased to less than 20% of the base value after insert the filament,and the CBF restored to 80% of the base value after 2 h of focal cerebral ischemia,indicating that the MCAO induced CI/RI model was successfully established.Then,the results showed that neurological scores of rats in MCAO group were significantly increased,however,the scores of rats in MCAO+TLB 20 mg/kg group were significantly reduced at days 3,7,14 and 28 after TLB treatment than that of MCAO group;Moreover,TLB also enhanced post-stroke neovascularization and newly formed functional vessels in cerebral ischemic penumbra after CI/RI;Additionally,TLB also up-regulated the contents of pro-angiogenic factor VEGFA and its receptor VEGFR-2 at days 3,7,14 and 28,as well as the protein expression of SIRT7 at day 28.Conclusion: TLB promotes b End.3 cells proliferation in a concentration dependent manner and improves long-term neurological recovery in rats after CI/RI,and its possible underlying mechanism is,at least partly,related to promoting angiogenesis via regulating SIRT7/VEGFA signaling pathway. |
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