OBJECTIVETo elucidate the effect of hydroxysafflor yellow A ( HYSA) on the proliferation of vascular smooth muscle cells (VSMCs) and the related mechanism.

METHODSVSMCs derived from SD rats were treated with DMEC culture medium (Control), 10 ng/ml PDGF (PDGF group), pretreatment with HYSA at different doses (1, 5, 10, 20, 40, 60 µmol/L) for 24 h then cotreatment with PDGF. After 24 h, MTT assay, Western blot and immunohistochemical staining were performed to evaluate the inhibitory effects of HYSA on VSMCs proliferation.

RESULTSHYSA inhibited PDGF induced VSMCs proliferation in a dose-dependent manner, dowregulated proliferating cell nuclear antigen (PCNA) expression and blocked PDGF activated PDGFR-MEK-ERK1/2 signaling pathway.

CONCLUSIONSHYSA inhibits VSMCs proliferation possibly via downregulating the expression of PCNA and blocking MEK-ERK1/2 signal transduction in VSMCs.

Animals ; Cell Proliferation ; Cells, Cultured ; Chalcone ; analogs & derivatives ; MAP Kinase Signaling System ; Mitogen-Activated Protein Kinase 1 ; Mitogen-Activated Protein Kinase 3 ; Mitogen-Activated Protein Kinases ; Muscle, Smooth, Vascular ; Myocytes, Smooth Muscle ; Proliferating Cell Nuclear Antigen ; Quinones ; Rats ; Rats, Sprague-Dawley

Abstrac:Aim To study the effect of hydroxysafflor yellow A ( HYSA ) on the proliferation of vascular smooth muscle cells ( VSMCs) and the related molecu-lar mechanism. Methods The inhibitory effects of hydroxysafflor yellow A on VSMC proliferation was de-tected using cell culture, MTT assay, Western blot and immunohistochemical staining. Results The results showed that HYSA inhibited cell proliferation induced by PDGF in a dose-dependent (5,10,20,40 μmol· L-1 ) manner, reduced proliferating cell nuclear anti-gen ( PCNA ) expression and blocked PDGFR-MEK-ERK1/2 signaling pathway activated by PDGF in VSMCs. Conclusion HYSA inhibits VSMCs prolifer-ation via reducing the expression of PCNA and blocking signal transduction of MEK-ERK1/2 in VSMCs.

Objective:To investigate the regulatory effects of endogenous nitric oxide (NO) on the activity of superoxide dismutase-1 (SOD1) and apoptosis of human umbilical vein endothelial cells (HUVECs).Methods:HUVECs were taken as the research object.The endothelial NO synthase (eNOS) short hairpin RNA(shRNA) lentivirus was employed to transfect HUVECs to knock down eNOS.HUVECs were divided in 4 groups: the scramble group, the eNOS shRNA group, the eNOS shRNA + sodium nitroprusside(SNP) group and the eNOS shRNA+ SNP+ tris (2-carboxyethyl) phosphine hydrochloride (TCEP) group.The protein expressions of eNOS and SOD1 dimer/monomer in cells were detected by western blot.The activity of SOD was detected by the enzyme-linked immunosorbent assay.The NO content in cells was detected with NO fluorescence probe.The level of superoxide anion in HUVECs was detected with dihydropyridine (DHE). The terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) assay was adopted to detect the apoptosis of HUVECs in situ.Results:Compared with the scramble group, the endogenous NO content (2.690±0.420 vs.15.029±2.193, P<0.01), eNOS protein expression (1.000±0.778 vs.3.141±0.199, P<0.01), SOD1 dimer/monomer ratio (4.6±1.0 vs.7.6±2.0, P<0.05) and SOD activity [(0.432±0.254) Carmen′s unit/10 4 cell vs.(1.000±0.116) Carmen′s unit/10 4 cell, P<0.01] were significantly decreased, while the level of intracellular superoxide anion (11.180±1.560 vs.6.146±1.007, P<0.01) and HUVECs apoptosis [75.0 (55.0, 100.0)% vs.0 (0, 0)%, P<0.01] were significantly increased in the eNOS shRNA group.Compared with the eNOS shRNA group, the content of endogenous NO (16.705±0.116 vs.2.690±0.420, P<0.01), the ratio of SOD1 dimer/monomer (7.3±2.0 vs.4.6±1.0, P<0.05) and the activity of SOD [(0.737±0.060) Carmen′s unit/10 4 cell vs.(0.432±0.254) Carmen′s unit/10 4 cell, P<0.05] were significantly increased, while the level of superoxide anion (6.897±1.648 vs.11.180±1.560, P<0.01) and the HUVECs apoptosis [0 (0, 0)% vs.75.0 (55.0, 100.0)%, P<0.01] were significantly decreased in the eNOS shRNA+ SNP group.Compared with the eNOS shRNA + SNP group, the ratio of SOD1 dimer/monomer (4.4±0.9 vs.7.3±2.0, P<0.05) and the activity of SOD [(0.214±0.084) Carmen′s unit/10 4 cell vs.(0.737±0.060) Carmen′s unit/10 4 cell, P<0.01] were significantly decreased, while the level of superoxide anion (10.917±1.552 vs.6.897±1.640, P<0.01) and the apoptosis level of HUVECs[63.6 (55.0, 90.0)% vs.0 (0, 0)%, P<0.01] were significantly increased in the eNOS shRNA+ SNP+ TCEP group.However, there was no significant difference in the NO content (16.112±0.926 vs.16.705±0.116, P>0.05). Conclusions:Endogenous NO could effectively antagonize the apoptosis of endothelial cells by increasing the cysteine-dependent SOD1 dimer/monomer ratio, enhancing SOD activity and inhibiting the accumulation of reactive oxygen species.

Fear memory contextualization is critical for selecting adaptive behavior to survive. Contextual fear conditioning (CFC) is a classical model for elucidating related underlying neuronal circuits. The primary visual cortex (V1) is the primary cortical region for contextual visual inputs, but its role in CFC is poorly understood. Here, our experiments demonstrated that bilateral inactivation of V1 in mice impaired CFC retrieval, and both CFC learning and extinction increased the turnover rate of axonal boutons in V1. The frequency of neuronal Ca²⁺ activity decreased after CFC learning, while CFC extinction reversed the decrease and raised it to the naïve level. Contrary to control mice, the frequency of neuronal Ca²⁺ activity increased after CFC learning in microglia-depleted mice and was maintained after CFC extinction, indicating that microglial depletion alters CFC learning and the frequency response pattern of extinction-induced Ca²⁺ activity. These findings reveal a critical role of microglia in neocortical information processing in V1, and suggest potential approaches for cellular-based manipulation of acquired fear memory.
Mice ; Animals ; Primary Visual Cortex ; Extinction, Psychological/physiology* ; Learning/physiology* ; Fear/physiology* ; Hippocampus/physiology*