Different Propylene Shikimic Acid On Endothelial Cell Activation And Its Mechanism

Studies consist of six parts:1 Effects of ISA on the viability of HUVEC and the release of NO and 6-keto-PGFia in HUVECAIM: To examine the effect of 3,4-oxo-isopropylidene-shikimic acid (ISA) on the viability of human umbilical vein endothelial cells ( HUVEC ) and the release of NO and 6-keto-PGFIa in HUVEC. METHODS: Cell viability was assessed by MTT assay and the release of NO was measured by Nitrate reductase assay. Radioimmunoassay was used to assess 6-keto-prostaglandin Fia ( 6-keto-PGFia). RESULTS: The viability and the release of NO in HUVEC were unchanged 24 h after treatment with ISA. The release of 6-keto-PGFia in HUVEC was increased after treatment with ISA (1(T4 mol-L'1) for 24 h. CONCLUSION: ISA increased PGI2 release of HUVEC.2 Protective effects of ISA on hydrogen peroxide damaging HUVECAIM: To study the effects of ISA on hydrogen peroxide damaging HUVEC. METHODS: Morphological change was observed microscopically. Cell viability was measured by MTT assay. The release of intracellular lactate dehydrogenase ( LDH ) and NO was assessed by Nitrate reductase assay.. Radioimmunoassay was used to assess 6-keto-prostaglandin F,a ( 6-keto-PGFia ). RESULTS: The cell viability of HUVEC was decreased in a dose - dependent manner after incubation with H2O2 ( 50, 100 and 200 umol-L"1 ) for 4 h. At the concentration of 10? 10"4umol-L"', ISA prevented the inhibitory effect on cell viability and LDH release induced by H2O2 ( 200 jamol-L"1 ). ISA increased the level of NO and 6-keto-PGFla in H2O2- injured HUVEC. Pretreatment with ISA for 6 h also alleviated the morphological damage of H2O2 - injured HUVEC. CONCLUSION: ISA exerted protective effect on H2O2 damaging HUVEC.3 Effect of ISA on H2O2 - induced apoptosis in HUVECAIM: To observe the effect of ISA on H2O2 - induced apoptosis in HUVEC and explore its mechanism. METHODS: Annexin V-PI staining and TUNEL assay were used to examine the apoptotic rate. The expression of Bel - 2 was assessed by immunohistochemistry. RESULTS: As demonstrated by Annexin V-PI staining, H2O2 (100, 200 jamol-L"1 ) treatment of HUVEC for 6 h increased the early stage apoptosis from 8.38 % for control to 13.23 %, 19.39 % respectively, and also increased the terminal stage apoptosis and necrosis ( % ). But the early stage apoptosis was decreased to 10.27 % and the terminal stage apoptosis and necrosis was increased afer H2O2 ( 400 jamol-L"1 ) treatment. Preincubation with ISA (10"3, 10" jamol-L"1 ) for 6 h could reduced the early stage apoptosis and the terminal stage apoptosisAbstractand necrosis ( % ) induced by H202 ( 200 (imol-L"1 ). As demonstrated by TUNEL assay, Pretreatment with ISA (10"3 umol-L"1 ) for 6 h could decrease TUNEL-positive cells induced by H2O2 ( 100 umol-L"1 ), from 15.9 % to 11.5 %. In addition, Bcl-2 was not detected in normal HUVEC, which was not affected by treatment with ISA for 12 h. CONCLUSION: ISA inhibited H2O2 - inducing apoptosis in HUVEC, which was not related to up-regulating Bcl-2.4 Studies on anti-oxidation of ISA in vitroAIM: To study the anti-oxidation of ISA in vitro. METHODS: The scavenging rate on superoxide anion radicals in xanthine oxidase system and OH radicals produced by Fenton reaction were tested in vitro. The concentration of malonaldehyde ( MDA) in HUVEC was detected by thiobarbituric acid ( TBA ) method, and the activities of superoxide dismutase ( SOD ) and catalase ( CAT ) were also measured by colorimetric assay. RESULTS: ISA ( 10"5-10"3 mol-L"1 ) could dose dependency decrease the superoxide anion radicals, with the scavenging rate of 9.5 %, 20.0 %, 34.5 % respectively. ISA also scavenged OH radicals at 10"3 mol-L'1. Treatment HUVEC with H2O2 ( 200 umol-L'1 )for 6 h increased MDA production and reduced the activities of SOD and CAT. Preincubation with ISA ( 10"5, 10"4umol-L"1 ) for 6 h could alleviate all the above effects. The activities of SOD and CAT were unchanged after incubation with ISA for 12 h. CONCLUSION: ISA could scavenge the free radicals and had no direct effect on the a...