[The Role of Nrf2 Pathway Activation in Hippocampal Neuron Injury of Neonatal Rats with Bilirubin Encephalopathy].

OBJECTIVE: To explore the effect of nuclear factor-erythroid 2-related factor (Nrf2) pathway activation on hippocampal neuron damage in neonatal rats with bilirubin encephalopathy. METHODS: Neonatal rats were randomly assigned to a control group (Control), a model group (Model) and an Nrf2 activator TBHQ (tert-Butylhydroquinone) group (TBHQ), with 20 rats in each group. Bilirubin solution was injected through the cerebellomedullary cistern to establish the neonatal rat model of bilirubin encephalopathy. Neurobehavioral changes were observed in rats and the water content of the brain tissue was measured. Nissl staining was done to observe the damage of hippocampal neurons. TUNEL staining was used to observe the apoptosis of hippocampal neurons. Colorimetric analysis was done to determine the Caspase-3 activity in the hippocampus. The content of malondialdehyde (MDA) and reduced glutathione (GSH) and the activity of superoxide dismutase (SOD) in the hippocampus were examined by chemical analysis. qRT-PCR and Western blot were done to measure the expression of Nrf2 and heme oxygenase-l (HO-1) mRNA and proteins in the hippocampus. RESULTS: After injection of bilirubin into the cerebellomedullary cistern, the young rats in the Model group and the TBHQ group showed different degrees of neurological abnormalities, while those in the control group showed no significant neurobehavioral abnormalities. Compared with the Control group, the Model group had severe neuronal damage, and the water content of brain tissue, the apoptosis of hippocampal neurons, the activity of Caspase-3 and the content of MDA content significantly increased ( P<0.01), while the SOD activity, GSH content, the expression of Nrf2 and HO-1 mRNA and proteins significantly decreased ( P<0.05). Compared with the Model group, neuronal damage was improved in the TBHQ group, and the water content of brain tissue, apoptosis of hippocampal neurons, activity of Caspase-3 and MDA content were all significantly reduced ( P<0.01), while SOD activity, GSH content and the expression of Nrf2 and HO-1 mRNA and proteins were significantly increased ( P<0.05). CONCLUSION: Activation of the Nrf2 pathway can improve hippocampal neuronal damage in neonatal rats with bilirubin encephalopathy and inhibit neuronal apoptosis and the oxidation reaction.

SXBX pill suppresses homocysteine-induced vascular smooth muscle cells dedifferentiation by inhibiting NLRP3 inflammasomes activation via ERK/p38 MAPK pathways.

The dedifferentiation of vascular smooth muscle cells (VSMCs) is a key event in the pathogenesis of vascular remodeling-related disease. The present study aimed to investigate the effects of shexiangbaoxin (SXBX) pill, a traditional Chinese medicinal formula on VSMCs dedifferentiation and its potential mechanisms. High-fat diet (HFD) was introduced to lipoprotein receptor-deficient (LDLR-/-) mice to generate hyperhomocysteinemia (HHcy), and plasma Hcy and lipid levels were analyzed. The phenotype of VSMCs was assessed in mice with the treatment of low (45 mg/kg/d) or high (90 mg/kg/d) SXBX pill by measuring the contractile protein α-SMA, SM22α and synthetic proteins OPN using RT-qPCR, western blotting and immunofluorescence assay. In vitro, the proliferation, migration and dedifferentiation of VSMCs were measured by MTT, Edu incorporation, wound healing and western blotting assay. Small interfering RNA technology was used to examine the role of NLRP3 in the effects of SXBX pill on dedifferentiation. The results indicated that although SXBX pill had no influence on HFD-induced HHcy and hyperlipidaemia, it reversed HHcy-induced dedifferentiation of VSMCs in vivo. SXBX pill significantly inhibited proliferation, migration and dedifferentiation of Hcy-treated VSMCs. In addition, we found that Hcy activated NLRP3 inflammasomes in VSMCs and SXBX pill could attenuate NLRP3 inflammasomes activation. Moreover, subsequent analysis suggested that SXBX pill inhibited NLRP3 inflammasomes activation through regulation of ERK1/2 and p38 MAPK pathway. Knockdown of NLRP3 reversed the inhibitory effects of SXBX pill in VSMCs. In conclusion, SXBX pill inhibited Hcy-induced proliferation, migration and dedifferentiation of VSMCs by suppressing NLRP3 inflammasomes activation via of ERK/p38 MAPK pathway.

Hyperhomocysteinemia independently causes and promotes atherosclerosis in LDL receptor-deficient mice.

BACKGROUND: Hyperhomocysteine is an independent risk factor of coronary heart disease (CHD). However, whether hyperhomocysteine affects the progression of atherosclerosis is unclear. In the present study, we examined the effect of hyperhomocysteine on the formation of atherosclerosis in low-density lipoprotein receptor-deficient (LDLr(-/-)) mice. METHODS: Forty-eight 7-week-old LDLr(-/-) mice were assigned to the following groups: mice fed a standard rodent diet (control group), mice fed a high-methionine diet (high-methionine group), mice fed a high-fat diet (high-fat group), and mice fed a diet high in both methionine and fat (high-methionine and high-fat group). At the age of 19, 23, and 27 weeks, four mice at each interval in every group were sacrificed. RESULTS: At the end of the study, mice did not show atherosclerotic lesions in the aortic sinus and aortic surface until 27 weeks old in the control group. However, atherosclerotic lesions developed in the other three groups at 19 weeks. The amount of atherosclerotic lesions on the aortic surface was lower in the high-methionine group than in the high-fat group (P < 0.001). Atherosclerotic lesions on the aortic surface in the high-methionine and high-fat group were the most severe. The mean area of atherosclerotic lesions in the aortic sinus compared with atherosclerotic lesions on the aortic surface was lower in the high-methionine group than in the high-fat group (P < 0.001). Atherosclerotic lesions in the aortic sinus in the high-methionine and high-fat group were the most severe. CONCLUSIONS: Homocysteinemia accelerates atherosclerotic lesions and induces early atherosclerosis independently in LDLr(-/-) mice. Reducing the level of homocysteinemia may be beneficial for prevention and treatment of CHD.

Effects of rosuvastatin on the production and activation of matrix metalloproteinase-2 and migration of cultured rat vascular smooth muscle cells induced by homocysteine.

OBJECTIVE: To test the influence of homocysteine on the production and activation of matrix metalloproteinase-2 (MMP-2) and tissue inhibitors of matrix metalloproteinase-2 (TIMP-2) and on cell migration of cultured rat vascular smooth muscle cells (VSMCs). Also, to explore whether rosuvastatin can alter the abnormal secretion and activation of MMP-2 and TIMP-2 and migration of VSMCs induced by homocysteine. METHODS: Rat VSMCs were incubated with different concentrations of homocysteine (50-5000 µmol/L). Western blotting and gelatin zymography were used to investigate the expressions and activities of MMP-2 and TIMP-2 in VSMCs in culture medium when induced with homocysteine for 24, 48, and 72 h. Transwell chambers were employed to test the migratory ability of VSMCs when incubated with homocysteine for 48 h. Different concentrations of rosuvastatin (10(-9)-10(-5) mol/L) were added when VSMCs were induced with 1000 µmol/L homocysteine. The expressions and activities of MMP-2 and TIMP-2 were examined after incubating for 24, 48, and 72 h, and the migration of VSMCs was also examined after incubating for 48 h. RESULTS: Homocysteine (50-1000 µmol/L) increased the production and activation of MMP-2 and expression of TIMP-2 in a dose-dependent manner. However, when incubated with 5000 µmol/L homocysteine, the expression of MMP-2 was up-regulated, but its activity was down-regulated. Increased homocysteine-induced production and activation of MMP-2 were reduced by rosuvastatin in a dose-dependent manner whereas secretion of TIMP-2 was not significantly altered by rosuvastatin. Homocysteine (50-5000 µmol/L) stimulated the migration of VSMCs in a dose-dependent manner, but this effect was eliminated by rosuvastatin. CONCLUSIONS: Homocysteine (50-1000 µmol/L) significantly increased the production and activation of MMP-2, the expression of TIMP-2, and the migration of VSMCs in a dose-dependent manner. Additional extracellular rosuvastatin can decrease the excessive expression and activation of MMP-2 and abnormal migration of VSMCs induced by homocysteine.