| Tea is the traditional drink in China and one of the most popular beverages in the world.It has showed that tea polyphenols have the function of anti-allergy,anti-oxidation,metabolic syndrome prevention,anti-tumor,regulation of intestinal micro-ecology and cardiovascular protection.At present,cardiovascular disease is the major cause of resident's death.So,the exploration of the mechanism of tea polyphenols on the prevention from anti-apoptosis and anti-inflammation will provide an important theoretical and clinical significance to disclose the beneficial cardiovascular effects of tea.We firstly prepared pure EGCG3 "Me,one of methylated EGCG from tea polyphenols,and then established a rat VSMCs apoptosis model induced by glucose oxidase(G.O.)and cell inflammation model induced by angiotensin II(Ang II).The effects of EGCG and EGCG3" Me on the function of anti-apoptosis and anti-inflammation in rat VSMCs were further explored.The main results are as follows:1.An EGCG3 "Me crude product with a purity of 11% was obtained with a yield of 2.8% and a recovery of 27% from tea polyphenols using preparative medium pressure liquid chromatography(p MPLC).Then the crude EGCG 3" Me was further purified by using polyamide column chromatography to produce its monomer at the purity of 95% with the yield of 4.8% and the recovery of 42%.2.The rat VSMCs apoptosis cell model induced by 20 U/L G.O.for 4 h was successfully established,which was validated by cell viability and apoptosis assay by MTT and flow cytometry,respectively.3.In the apoptotic rat VSMCs,the reactive oxygen species(ROS)was detected by DCFH-DA probe and the results showed that EGCG(20 ?M,40 ?M)and EGCG3 "Me(20 ?M,40 ?M)significantly reduced the ROS level in a dose-dependent manner.Moreover,EGCG(20 ?M,40 ?M)and EGCG3" Me(20 ?M,40 ?M)inhibited the expression of Nox4 and 40?M EGCG3 "Me has shown a better effect compared with 40?M EGCG.Those suggested that EGCG and EGCG3" Me could reduce the level of intracellular ROS in VSMCs by down regulating the expression of Nox4 to inhibit the apoptosis.4.It's known that the the apoptosis-related proteins,including Cleaved caspase-9,Cleaved caspase-3,Cleaved caspase-7,Cleaved PARP and Bcl-2 family including Bax,Bcl-x L,Cytochrome C protein play the key roles in the apoptosis.Our results from western blotting have proved that EGCG3 "Me(20 ?M,40?M)as similar as EGCG(20 ?M,40?M)could reducethe protein expression of Cleaved Caspase-3,Cleaved Caspase-7,Cleaved Caspase-9,Cleaved PARP and Bax and inhibit the expression of Cytochrome C on the mitochondrial in VSMCs.Those suggested that the molecular mechanism of EGCG and EGCG3" Me to prevent apoptosis is throught the down-regulating of Caspase and the pro-apoptotic Bcl-2 family proteins,up-regulating of the anti-apopotic Bcl-2 family proteins,and further reducing the release of Cytochrome C in rat VSMCs.5.The inflammation of VSMCs is one of incentive to cardiovascular disease.The Ang II was applied to induce cell inflammation model in rat VSMCs to test the effects of EGCG3"Me and EGCG on the prevention of inflammation.It was found that EGCG3"Me(20?M,40?M)and EGCG(20?M,40?M)inhibited Ang II-induced up-regulation of NF-?B signaling pathway-associated proteins as phosphorylated I?B?,phosphorylated IKK? and nuclear P65 protein in rat VSMCs.EGCG3"Me has shown a stronger inhibition effect on P-I?B? than EGCG.Apoptosis and inflammation are the two key factors to induce cardiovascular disordes.EGCG3 "Me has shown to exert its anti-apoptosis and anti-inflammation effects in rat VSMCs,similar to EGCG.Those results provide an approach to elaborate the cardiovascular prevention effects of EGCG3" Me and its molecular mechanism. |
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