Abstract:Aim To observe whether the proliferation and apoptosis of mouse vascular smooth muscle cells(VSMC) were induced by stretch stress (SS) via PKCα phosphorylation and to investigate the role of berberine (BBR) and related mechanism on the process. Methods The experiment was divided into six groups:normal control(NC) group, BBR group, PKCα inhibitor (Go6976) group, SS group, SS+BBR group and SS+Go6976 group. VSMC were pretreated with BBR or Go6976 for 1 h, and stimulated for 15 min with SS at 10% amplitude. PKCα and MAPK (ERK, JNK, p38) phosphorylation were measured by Western blot. After pretreated by BBR or Go6976, and SS stimulation for 1 h, the proliferation (Ki67) and apoptosis (TUNEL) of VSMC were measured by immunofluorescence. Results Compared with the NC group, SS could increase the phosphorylation levels of PKCα and MAPK(ERK, JNK, p38) (P＜0.05), and increase the levels of VSMC proliferation and apoptosis (P＜0.05). BBR could inhibit the phosphorylation levels of PKCα and MAPK (P＜0.05), while inhibiting VSMC proliferation and apoptosis (P＜0.05); Go6976 could inhibit PKCα, ERK and JNK phosphorylation (P＜0.05), but had no effect on the phosphorylation increase of p38, while inhibiting VSMC proliferation and apoptosis (P＜0.05). Conclusion BBR inhibits the phosphorylation of PKCα and MAPK (ERK, JNK, p38) induced by SS, and further inhibits the proliferation and apoptosis of VSMC.

Abstract:Aim To observe whether mechanical stretch stress (SS) induces the proliferation and migration of mouse vascular smooth muscle cells (VSMC) through PKCδ activation, and to further explore the effect and mechanism of berberine (BBR) on the proliferation and migration of VSMC. Methods Mouse VSMC in vitro were divided into six groups:negative control group (NC), berberine group (BBR), PKCδ inhibitor Sotrastaurin group (Sotras), SS group, SS+BBR group and SS+Sotras group. The cultured VSMC in each group,which were pretreated with BBR or Sotrastaurin or ddH2O for 1h, followed by SS (10% tensile strength) for different time or no treatment for control, were collected for the detection of PKCδ phosphorylation, proliferation and migration by Western blot, immunofluorescence and scratch assay respectively. Results Immunofluorescence and cell scratch test results showed that compared with NC group, SS stimulation significantly increased Ki67 positive level in VSMC by 469%(P＜0.05, n＝3), and reduced scratch width by 54.9%(P＜0.05, n＝3),while BBR and Sotrastaurin could significantly inhibit the changes caused by SS, Ki67 positive level decreased by 66.9% and 80.2%(P＜0.05, n＝3), and scratch width increased by 79.4% and 120.1%(P＜0.05, n＝3)compared with SS group, respectively. Meanwhile, Western blot results showed that SS stimulation induced a time-dependent increase in PKCδ phosphorylation compared with NC group, with the most significant increase of 97.5%(P＜0.05,n＝3) at 30 min, which also inhibited by berberine in a concentration-dependent manner, and the effect was the most significant at 200 μmol/L, with a decrease of about 37.6% (P＜0.05, n＝3). Conclusion Berberine inhibits SS-induced vascular smooth muscle cell proliferation and migration by inhibiting PKCδ phosphorylation.

Abstract:Aim To investigate the effect of simvastatin on reactive oxygen species(ROS) expression in stretch stress(SS) induced macrophage cells. Methods RAW264.7 macrophage cells were cultured in vitro, treated with SS, or pretreated with simvastatin for 60 min and then stimulated with SS, the expression of ROS was detected by Hoechst33342 and H2DCFDA fluorescent probe, the fluorescence intensity of ROS was detected and the positive ratio of ROS was analysed by SPSS statistical software. The expression of NADPH oxidase 1(NOX1) was evaluated by Western blot.Results SS increased the expression of ROS in a time dependent manner, and the expression of ROS reached the most significant at 60 min(P<0.05). Simvastatin could inhibit the effect induced by SS in a concentration dependent manner, the inhibition effect of simvastatin was the most significant in 0.3 μmol/L(P<0.05). The expression of NOX1 was significantly inhibited by simvastatin after SS. Conclusion Simvastatin repressed ROS expression of RAW264.7 macrophage cells induced by SS through inhibiting the expression of NOX1.

Abstract:Aim To investigate the effects of calcium blocker nifedipine and diuretic hydrochlorothiazide on mechanical stretch stress mediated phosphorylation of ERK1/2 （p-ERK1/2） and expression of Ki67 in vascular smooth muscle cells （VSMC）. Methods Cultured quiescent VSMC were pretreated with nifedipine and hydrochlorothiazide respectively and subjected to treatment with mechanical stretch stress. Level of p-ERK1/2 in the treated cells was detected by Western blot and meanwhile Ki67 expression was detected by immunofluorescent staining. Results Compared with the negative control group, nifedipine or hydrochlorothiazide had no effects on p-ERK1/2 and Ki67 expression in quiescent VSMC, while mechanical stretch stress stimulation significantly increased levels of p-ERK1/2 and Ki67 expression, which was inhibited by nifedipine in a concentration-dependent manner, and synergistically enhanced by hydrochlorothiazide. Conclusions Hydrochlorothiazide synergistically promotes increased p-ERK1/2 and Ki67 expression in VSMC induced by mechanical stretch stress, which can be inhibited by nifedipine in a concentration-dependent manner. These results provide novel mechanisms for traditional antihypertensive drugs.

Abstract:Aim To explore effects of oxidized low density lipoprotein(ox-LDL) and mechanical stretch stress(SS) on activation of extracellular regulated kinase(ERK1/2) in macrophage RAW264.7 cells. Methods The cultured RAW264.7 cells were identified via ink staining.Agarose gel electrophoresis was employed to identify and quantitate ox-LDL which was oxidated from n-LDL with copper sulfate.The identified RAW264.7 cells were subjected to treatment with ox-LDL and SS,respectively or jointly,for different dose/elongation and time duration,and then the phosphorylation of ERK1/2 in the macrophages was detected by Western blotting. Results RAW264.7 cells could phagocytize ink,forming cloudy ink plaque or black particles in cytoplasm.N-LDL could be oxidized into ox-LDL by copper sulfate,since a single lane about ox-LDL could be seen in agarose gel electrophoresis,and the electrophoretic mobility of ox-LDL was higher than that of n-LDL,indicating successful ox-LDL preparation.Ox-LDL and SS could induce ERK1/2 phosphorylation,respectively,in a time and dose dependent manner,and dramatical increase of ERK1/2 phosphorylation was observed when the cells were co-treated with SS and ox-LDL. Conclusions Ox-LDL and SS could induce ERK1/2 phosphorylation,and combined treatment of ox-LDL and SS could synergistically promote ERK1/2 phosphorylation in macrophages.This study could provide useful information for exploring the roles of macrophages and its mechanism in hypertension-related atherosclerosis.