Abstract:Aim To observe the effect of TSB2 inhibiting the combination of heat shock protein 90 (HSP90) and endothelial nitric oxide synthase (eNOS) on the formation of atherosclerosis. Methods Human umbilical vein endothelial cells (HUVEC) were treated with TSB2 and the combination between HSP90 and eNOS was detected by co-immunoprecipitation. C57BL/6 mice and low density lipoprotein receptor knockout (LDLR－/－) mice were fed with normal diet (ND) or high fat diet (HFD) for 12 weeks while injected with phosphate buffered saline (PBS) or TSB2 intraperitoneally. The mice were divided into four groups:C57BL/6＋ ND＋PBS group, LDLR－/－＋ND＋PBS group, LDLR－/－＋HFD＋PBS group, LDLR－/－＋HFD ＋TSB2 group. Then the aorta was isolated. The combination between HSP90 and eNOS in aorta was measured. The atherosclerotic plaque in aorta and aortic sinus were determined. The production of nitric oxide (NO) and superoxide anion (O2·－) were also detected. At the same time, L-monomethyl-arginine (L-NMMA), a competitive substrate of L-arginine, was used to determine the production of NO, and L-nitroarginine methyl ester (L-NAME), a nitric oxide synthase inhibitor, was used to determine the production of O2·－. Results Compared with control group, the combination between HSP90 and eNOS was decreased by 41.06% (P＜0.05) in cultured HUVEC treated with TBS2. Compared with LDLR－/－＋HFD＋PBS group,the combination between HSP90 and eNOS in the mouse aortas was decreased by 40.95% (P＜0.05) in LDLR－/－ ＋HFD＋TSB2 group, and the production of O2·－ was decreased by 63.73% (P＜0.05) (L-NAME significantly inhibited the production of O2·－ in LDLR－/－＋HFD＋PBS group), while the production of NO had no significant change in the mouse aortic endothelial cells (L-NMMA inhibited NO production in all groups), and the formation of atherosclerotic lesions in aortas and aortic sinus were significantly decreased by 59.39% and 68.86% (P＜0.05) respectively in LDLR－/－＋HFD＋TSB2 group. Conclusion TSB2 can reduce the O2·－ production of uncoupled eNOS in vascular endothelial cells by inhibiting the combination of HSP90 and eNOS in aortic endothelial cells, and finally inhibits the formation of atherosclerosis.

Abstract:Aim To investigate the effects of perinatal hypoxia on carotid artery endothelium-dependent relaxations of 6 week old rabbits and its molecular mechanism.Methods Ten pregnant New Zealand White rabbits were randomly divided into two groups: normoxic control group (21% O2, n5) and perinatal hypoxia group (12% O2, n5) during day 10～29 of pregnancy. Pregnant rabbits had spontaneous deliveries, and only one male offspring from each litter was selected randomly and breast-fed for six weeks. Carotid arteries were obtained from 6 week old rabbits, endothelium-dependent relaxation (EDR) and expression of endothelial nitric oxide synthase (eNOS) and heat shock protein 90 (HSP90) of carotid arteries were examined.Results Perinatal hypoxia significantly impaired carotid artery EDR in 6 week old rabbits (P<0.05). Perinatal hypoxia significantly decreased carotid artery HSP90 expression (P<0.05) and eNOS activities (P<0.05) in 6 week old rabbits.Conclusions Perinatal hypoxia can induce functional impairment in vascular endothelium from 6 week old rabbits. The molecular mechanism involved is that perinatal hypoxia decrease HSP90 expression and eNOS activities of rabbit artery.

Abstract:AimTo investigate whether the downregulation of heat shock protein 90 (HSP90) was involved in cardiac cell injury induced by serum-glucose deprivation (SGD).MethodsH9c2 cardiomyocytes (H9c2 cells) were treated with SGD to establish an in vitro model of ischemic cardiac cell injury.H9c2 cells were pretreated with 17-AAG (a selective inhibitor of HSP90) for 60 min before exposure of cells to SGD.Cell viability was detected by CCK-8.The intracellular level of free calcium was measured by Fluo-3AM staining and photofluorography.The expressions of HSP90 and glucose-regulated protein 78 (GPR78) were tested by Western blot assay.ResultsTreatment of H9c2 cells with SGD for 24 h significantly induced downregulation of HSP90 expression, overload of intracellular calcium, and upregulation of GRP78 expression, which is a marker of endoplasmic reticulum stress.Pretreatment with 17-AAG, a selective HSP90 inhibitor, not only aggravated SGD-induced decrease in the viability of H9c2 cells, but also enhanced SGD-induced overload of intracellular calcium and upregulation of GRP78 expression in H9c2 cells.ConclusionInhibition of HSP90 may be one of the key mechanisms, by which H9c2 cells were damaged by SGD.

Abstract:Aim Phosphorylation and nuclear translocation of extracellular signalregulated kinases(ERK1/2) are most important for proliferation of oxidativestressed vascular smooth muscle cells(VSMC) and heat shock protein 90(HSP90) is involved in this process.We investigate whether heat shock protein 90 participated in extracellular signal-regulated kinases 1/2 pathway as a molecular chaperon. Methods Exposure vascular smooth muscle cells to LY83583(6-Anilinoquinoline-5,8-quinolinedione,produce reactive oxygen species,1 μmol/L) for different time,then heat shock protein 90,extracellular signal-regulated kinases 1/2 and phospho-extracellular signal-regulated kinases 1/2 in cell lysates were measured by western blot.Vascular smooth muscle cells were incubated with geldanamycin (a special inhibitor of heat shock protein 90,5 μmol/L) or vehicle for 30 min,then with LY83583(1 μmol/L) for 120 min,heat shock protein 90 binding with extracellular signal-regulated kinases 1/2 and phospho-extracellular signal-regulated kinases 1/2 were quatified by immunoprecipitation and western blot.The nuclear translocation of phospho-extracellular signal-regulated kinases 1/2 were measured by immunofluorenscense. Results Heat shock protein 90 increased in a time-dependent manner.It got the peak at 120 min which corresponded to the second peak of phospho-extracellular signal-regulated kinases 1/2.Immunoprecipitation and western blot analysis showed that LY83583 increased the complex of heat shock protein 90-phospho-extracellular signal-regulated kinases 1/2 about 5.5 times(p<0.01) vs control,and phospho-extracellular signal-regulated kinases 1/2 in total cell lysis(about 6.1 times vs control,p<0.01) and nuclear increased too.Geldanamycin attenuated the effect of LY83583. Conclusions Heat shock protein 90 bound with phospho-extracellular signal-regulated kinases 1/2 and promoted their nuclear translocation in oxidative-stressed vascular smooth muscle cells.