Anti-apoptotic and neuroprotective effects of oxysophoridine on cerebral ischemia both in vivo and in vitro.

In this study, we investigated the neuroprotective effect of oxysophoridine on ischemia and ischemia-like insults. Protection by oxysophoridine was studied at the in vivo level using a model of middle cerebral artery occlusion in mice and at the in vitro level using primary rat hippocampal neuronal cultures exposed to oxygen-glucose deprivation, a model of ischemia-like injury. The behavioral test was performed by using the neurological scores. The infarction volume of brain was assessed in the brain slices stained with 2,3,5-triphenyl tetrazolium chloride. The neuron apoptosis was evaluated by Hoechst 33342 staining. The morphological change in the neurons was examined using a Transmission Electron Microscope (TEM or EM). To evaluate neuron apoptosis, caspase-3, -9, and - 8 activities were measured using assay kits with an ELISA reader. The Western blotting assay was used to evaluate the release of cytochrome c and expression of caspase-3, Bcl-2, and Bax proteins. The quantitative real-time PCR assay was used to evaluate the release of cytochrome c and the expression of caspase-3 mRNA. Oxysophoridine-treated groups (62.5, 125, 250 mg/kg) markedly reduced neurological deficit scores and infarct volumes. Treatment with oxysophoridine (5, 20, 80 µmol/L) significantly attenuated neuronal damage, with evidence of decreased cell apoptosis and decreased cell morphologic impairment. Furthermore, treatment with oxysophoridine could effectively downregulate the expression of cytochrome c and caspase-3 in both mRNA and protein levels, and Bax in the protein level, and induce an increase of Bcl-2 in the protein level. The caspase-3, -9, and -8 activities were also inhibited. These findings suggested that oxysophoridine may be a potential neuroprotective agent for cerebral ischemia injury.

Antinociceptive effects of matrine on neuropathic pain induced by chronic constriction injury.

CONTEXT: Sophora alopecuroides L. (Leguminosae) is a commonly used Chinese herbal drug that possesses antipyretic, anti-inflammatory and analgesic effects. Among various alkaloids isolated from S. alopecuroides, matrine has been identified as the major bioactive component contributing to a variety of pharmacological effects, and studies have also shown that matrine has an analgesic effect. OBJECTIVE: To investigate the antinociceptive effects of matrine on neuropathic pain induced by chronic constriction injury (CCI) in mice. MATERIALS AND METHODS: The von Frey, plantar, cold-plate, locomotor activity and rota-rod test were performed to assess the degree of mechanical, radiant, thermal, spontaneous locomotor activity and motor coordination changes respectively, at different time intervals, i.e., one day before surgery and 7, 8, 10, 12 and 14 days post surgery. Matrine was administered from the 8th day after the surgery for seven days. RESULTS: Our present study shows that matrine at the dose of 30 mg/kg i.p. increased the paw withdrawal threshold (0.88 ± 0.16), paw withdrawal latency (7.01 ± 0.11) and the counts of paw withdrawal (19.7 ± 1.15) from the day 8 for the nerve injured paw compared to the CCI group (0.18 ± 0.04, 4.62 ± 0.18, 44.3 ± 2.99, respectively). Matrine, in a dose-dependent effect, was also found to produce a protective role in both plantar and cold-plate tests. The analysis of the effect supports the hypothesis that matrine is useful in neuropathic pain therapy. DISCUSSION AND CONCLUSION: The results of this study suggest that matrine could be useful in the treatment of different kinds of neuropathic pains as an adjuvant to conventional medicines.

Involvement of miR-144-3p in Cbs+/-mouse hepatocyte autophagy induced by high-methionine diet / 中国组织工程研究

BACKGROUND:High-methionine diet can cause liver injury in Cbs+/-mice,and hyperhomocystinemia is related to the occurrence and progression of various liver-related diseases,such as hepatic steatosis,autoimmune hepatitis,and alcoholic fatty liver disease.MicroRNAs(miRNAs)are involved in various cellular processes including cell survival,differentiation and autophagy,which are of great significance. OBJECTIVE:To investigate the critical role of miR-144-3p on Cbs+/-mouse hepatocyte autophagy induced by high methionine die. METHODS:(1)Ten male cystathione-β-synthase normal(Cbs+/+)mice and another 10 male mice with single gene knockout(Cbs+/-)of similar body mass,4 weeks of age,were fed a high-methionine diet and executed after 12 weeks to take liver tissue.(2)Human hepatocytes(HL-7702)were cultured in vitro and divided into control[0 μmol/L homocysteine(Hcy)],Hcy(100 μmol/L Hcy),mimic-NC(transfected with mimic-NC),mimic-NC + Hcy(mimic-NC transfecton+100 μmol/L Hcy),miR-144-3p mimic(transfected with miR-144-3p mimic),and miR-144-3p mimic + Hcy(miR-144-3p mimic transfection+100 μ mol/L Hcy),inhibitor-NC(transfected with inhibitor-NC),inhibitor-NC + Hcy(inhibitor-NC transfection + 100 μmol/L Hcy),miR-144-3p inhibitor(transfected with miR-144-3p inhibitor),and miR-144-3p inhibitor + Hcy(miR-144-3p inhibitor transfection + 100 μmol/L Hcy).Quantitative real-time PCR was used to detect the expression of miR-144-3p in liver tissue and hepatocytes.After transfection of miR-144-3p mimic or inhibitor,quantitative real-time PCR and western blot were used to detect the transfection efficiency of miR-144-3p and its effect on the expression of autophagy-related proteins LC3B and p62.The levels of alanine transferase and aspartate aminotransferase in hepatocyte supernatants were determined by enzyme linked immunosorbent assay.The correlation between the expression of miR-144-3 in hepatocyte and the levels of alanine transferase and aspartate aminotransferase in hepatocyte supernatants were analyzed by Pearson correlation analysis. RESULTS AND CONCLUSION:Compared with the Cbs+/+ group and control group,the expression of miR-144-3p in the liver tissue of the Cbs+/-group and in hepatocytes of the Hcy group was decreased(P<0.01).The expression of LC3B-Ⅱ/Ⅰ was decreased in hepatocyte after transfection of miR-144-3p mimic,while the protein expression of p62 was increased(P<0.01).The opposite results were obtained after transfection of miR-144-3p inhibitor(P<0.01).Compared with the mimic-NC group,the levels of alanine transferase and aspartate aminotransferase were decreased in the miR-144-3p mimic group(P<0.01),while the opposite results were obtained in the inhibitor-NC group(P<0.01).The expression of miR-144-3p in hepatocytes was negatively correlated with the levels of alanine transferase(P<0.01,r=-0.887 6)and aspartate aminotransferase(P<0.01,r=-0.829 9)in the supernatant of hepatocytes.To conclude,Hcy promotes hepatocyte autophagy by inhibiting the expression of miR-144-3p,which subsequently aggravates liver injury.

Hypoxic postconditioning protects myocardium by regulating autophagy in aging cardiomyocytes through piRNA-005854 / 中国组织工程研究

BACKGROUND:Ischemic postconditioning is one of the effective ways to reduce ischemia-reperfusion injury and has been more and more widely used in clinical practice in recent years,but its specific molecular mechanism has yet to be studied. OBJECTIVE:To investigate the role and mechanism of piRNA-005854 in the aging cardiomyocytes caused by hypoxic postconditioning. METHODS:In vitro,cardiomyocytes were administered 8 mg/mL D-galactose for 9 days to induce their aging.β-Galactosidase staining was used to observe the aging of cardiomyocytes.Senescent cells were treated with hypoxia/reoxygenation and hypoxic postconditioning.ELISA was utilized to detect changes in myocardial injury markers creatine kinase isoenzyme MB and lactate dehydrogenase levels.Western blot assay was applied to detect the expression changes of autophagy-related proteins LC3II,p62,ULK1 and phosphorylated ULK1 in aging cardiomyocytes.qRT-PCR was employed to determine the expression level of piRNA-005854.piRNA-005854 inhibitor and piRNA-005854 mimics were transferred into aging cardiomyocytes and followed with hypoxic postconditioning.Western blot assay was used to examine the expression of LC3II,p62,ULK1 and phosphorylated ULK1. RESULTS AND CONCLUSION:(1)D-galactose induced obvious senescence of cardiomyocytes 9 days later.(2)Compared with the normoxia group,creatine kinase isoenzyme MB and lactate dehydrogenase levels increased in the hypoxia/reoxygenation group(P<0.01);LC3 II/I expression was increased;p62 expression was decreased;ULK1 phosphorylation level was increased,and piRNA-005854 expression was increased(P<0.01).(3)Compared with the hypoxia/reoxygenation group,creatine kinase isoenzyme MB and lactate dehydrogenase levels significantly reduced in the hypoxic postconditioning group(P<0.01);LC3 II/I expression significantly decreased(P<0.05);p62 expression increased(P<0.01);ULK1 phosphorylation level decreased(P<0.05),and piRNA-005854 expression decreased(P<0.01).(4)After transfection of piRNA-005854 inhibitor,LC3II/I expression was decreased(P<0.01);the expression of p62 was increased significantly(P<0.05);the phosphorylation level of ULK1 was decreased significantly(P<0.01).After transfection of piRNA-005854 mimics,LC3II/I expression was increased significantly;the expression of p62 was decreased,and the phosphorylation level of ULK1 was increased significantly(P<0.01).(5)The results show that piRNA-005854-mediated reduction of ULK1-dependent autophagy level is a possible mechanism that hypoxic postconditioning exerts its protective effect on aging cardiomyocytes.

Effect of cyclic RNA hsa-circ-0001360 on homocysteine-induced apoptosis of human umbilical vein endothelial cells / 中国组织工程研究

BACKGROUND:Increased homocysteine level induces apoptosis of human umbilical vein endothelial cells,but the mechanism remains unclear. OBJECTIVE:To investigate the role of hsa-circ-0001360 in human umbilical vein endothelial cell apoptosis induced by homocysteine. METHODS:In vitro cultured human umbilical vein endothelial cells were divided into control group,homocysteine group,interference control group,interference control + homocysteine group,hsa-circ-0001360 interference group,hsa-circ-0001360 + homocysteine interference group,overexpression control group,overexpression control + homocysteine group,hsa-circ-0001360 overexpression group and hsa-circ-0001360 + homocysteine overexpression group.All groups were treated with 100 μmol/L homocysteine.After 72 hours of intervention,the expressions of apoptosis-related proteins Bax,Bcl-2,and Caspase-3 were detected by western blot assay.The apoptotic rate was detected by flow cytometry.Quantitative real-time PCR was used to detect the expression of hsa-circ-0001360. RESULTS AND CONCLUSION:(1)Compared with the control group,the expression of Caspase-3 and Bax was significantly increased(P<0.01),and the expression of Bcl-2 was significantly decreased(P<0.01),and the apoptotic rate was significantly increased(P<0.01)in the homocysteine group.(2)Compared with control group,the expression of hsa-circ-0001360 was significantly increased in the homocysteine group(P<0.01).(3)The expression of hsa-circ-0001360 was significantly higher in the cytoplasm than that in the nucleus(P<0.01).(4)Compared with the interference control C group and interference control + homocysteine group,the expressions of Caspase-3 and Bax were significantly decreased(P<0.01),while the expression of Bcl-2 was significantly increased(P<0.01);the apoptotic rate was significantly decreased(P<0.01)in sh-hsa-circ-0001360 interference group and sh-hsa-circ-0001360 + homocysteine interference group.(5)Compared with overexpression control group and overexpression control + homocysteine group,the expressions of Caspase-3 and Bax were significantly increased(P<0.01),while the expression of Bcl-2 was significantly decreased(P<0.01);the apoptotic rate was significantly increased(P<0.01)in the hsa-circ-0001360 overexpression group and the hsa-circ-0001360 + homocysteine overexpression group.(6)In conclusion,hsa-circ-0001360 can promote the apoptosis of human umbilical vein endothelial cells induced by homocysteine.

Effect of EZH2 on Hcy-induced cholesterol accumulation of foam cells / 实用医学杂志

Objective To explore the effect of EZH2 on Hcy-induced cholesterol accumulation of foam cells. Methods THP-1 foam cells were divided into control group ,100 μmol/L Hcy group and folic acid group. Lipid droplet in foam cells was tested by Oil red O. TC and TG contents in cells were determined by enzymic meth od. H3K27me3 level and EZH2 protein expression were detected by Western-blot. EZH2 mRNA expression was assayed by q-PCR. H3K27me3 level and TC and TG contents were examined followed by overexpression or knock- down of EZH2. Results After administration of Hcy,TC and TG contents in foam cells were increased (P <0.05). H3K27me3 level and EZH2 expression were also increased(P<0.05). Overexpression of EZH2 caused the expansion of H3K27me3 level,and the TC and TC contents were also increased(P<0.05). Conclusion Regula-tion of H3K27me3 by EZH2 might be involved in Hcy-induced accumulation of cholesterol in foam cells.

Study on the Diastolic Effect and Mechanism of Hui Medicine Hexin Oil Solution on Isolated Thoracic Aor-tic Vascular Rings of Rats / 中国药房

OBJECTIVE:To study the diastolic effect and mechanism of Hui medicine Hexin oil solution on isolated thoracic aortic vascular rings of rats,and provide reference for its treatment for cardiovascular diseases. METHODS:Thoracic aortic vascu-lar rings of rats were taken and then soaked in Kelvin's nutrient solution(K-H). Using 1×10-6 mol/L norepinephrine(PE)or 60 mmol/L potassium chloride (KCl) for inducing the contraction of vascular rings,biological signal acquisition and analysis system was used to determine the diastolic effect and mechanism of Hexin oil solution with concentrations of 0.0204,0.0408,0.0612, 0.0816,0.1020 mg/mL on vascular rings,and diastolic rate was calculated. After culturing vascular rings by 0.1 mmol/L nitric ox-ide synthase inhibitor L-nitro-arginine methyl ester (L-NAME),cyclooxygenase inhibitor indomethacin (INDO),and potassium ion channel blocker glibenclamide(Gli)for 20 min,the diastolic effects of above-mentioned 5 mass concentrations of Hexin oil so-lution on the contraction of vascular rings pre-contracted by PE were determined,and diastolic rate was calculated. The test was based on K-H solution as blank control. RESULTS:Compared with blank control,Hexin oil solution with concentration of 0.0204-0.1020 mg/mL had obvious diastolic effect on the contraction of vascular rings induced by PE and KCl (P<0.05 or P<0.01), showing concentration-dependent relationship. INDO pre-treatment can relieve the diastolic effect of Hexin oil solution on vascular rings pre-contracted by PE;and compared with blank control group,the diastolic rate had no statistical significance (P>0.05). While the pre-treatment of Gli,L-NAME did not affect the diastolic effect of Hexin oil solution on vascular rings pre-contracted by PE;and compared with blank control group,diastolic rate was obviously increased(P<0.05 or P<0.01). CONCLUSIONS:Hex-in oil solution can concentration-dependently conduct the relaxation of thoracic aortic vascular rings pre-contracted by PE,KCl. The mechanism may be associated with activation of cyclooxygenase pathway.

Effect of EZH2 on Hcy-induced cholesterol accumulation of foam cells / 实用医学杂志

Objective To explore the effect of EZH2 on Hcy-induced cholesterol accumulation of foam cells. Methods THP-1 foam cells were divided into control group ,100 μmol/L Hcy group and folic acid group. Lipid droplet in foam cells was tested by Oil red O. TC and TG contents in cells were determined by enzymic meth od. H3K27me3 level and EZH2 protein expression were detected by Western-blot. EZH2 mRNA expression was assayed by q-PCR. H3K27me3 level and TC and TG contents were examined followed by overexpression or knock- down of EZH2. Results After administration of Hcy,TC and TG contents in foam cells were increased (P <0.05). H3K27me3 level and EZH2 expression were also increased(P<0.05). Overexpression of EZH2 caused the expansion of H3K27me3 level,and the TC and TC contents were also increased(P<0.05). Conclusion Regula-tion of H3K27me3 by EZH2 might be involved in Hcy-induced accumulation of cholesterol in foam cells.

Study on the Diastolic Effect and Mechanism of Hui Medicine Hexin Oil Solution on Isolated Thoracic Aor-tic Vascular Rings of Rats / 中国药房

OBJECTIVE:To study the diastolic effect and mechanism of Hui medicine Hexin oil solution on isolated thoracic aortic vascular rings of rats,and provide reference for its treatment for cardiovascular diseases. METHODS:Thoracic aortic vascu-lar rings of rats were taken and then soaked in Kelvin's nutrient solution(K-H). Using 1×10-6 mol/L norepinephrine(PE)or 60 mmol/L potassium chloride (KCl) for inducing the contraction of vascular rings,biological signal acquisition and analysis system was used to determine the diastolic effect and mechanism of Hexin oil solution with concentrations of 0.0204,0.0408,0.0612, 0.0816,0.1020 mg/mL on vascular rings,and diastolic rate was calculated. After culturing vascular rings by 0.1 mmol/L nitric ox-ide synthase inhibitor L-nitro-arginine methyl ester (L-NAME),cyclooxygenase inhibitor indomethacin (INDO),and potassium ion channel blocker glibenclamide(Gli)for 20 min,the diastolic effects of above-mentioned 5 mass concentrations of Hexin oil so-lution on the contraction of vascular rings pre-contracted by PE were determined,and diastolic rate was calculated. The test was based on K-H solution as blank control. RESULTS:Compared with blank control,Hexin oil solution with concentration of 0.0204-0.1020 mg/mL had obvious diastolic effect on the contraction of vascular rings induced by PE and KCl (P<0.05 or P<0.01), showing concentration-dependent relationship. INDO pre-treatment can relieve the diastolic effect of Hexin oil solution on vascular rings pre-contracted by PE;and compared with blank control group,the diastolic rate had no statistical significance (P>0.05). While the pre-treatment of Gli,L-NAME did not affect the diastolic effect of Hexin oil solution on vascular rings pre-contracted by PE;and compared with blank control group,diastolic rate was obviously increased(P<0.05 or P<0.01). CONCLUSIONS:Hex-in oil solution can concentration-dependently conduct the relaxation of thoracic aortic vascular rings pre-contracted by PE,KCl. The mechanism may be associated with activation of cyclooxygenase pathway.

Protective effects of Lycium barbarum polysaccharide on neonatal rat primary cultured hippocampal neurons injured by oxygen-glucose deprivation and reperfusion.

This study investigated the protective effects of Lycium barbarum polysaccharide (LBP) on alleviating injury from oxygen-glucose deprivation/reperfusion (OGD/RP) in primary cultured rat hippocampal neurons. Cultured hippocampal neurons were exposed to oxygen-glucose deprivation (OGD) for 2 h followed by a 24 h re-oxygenation. The MTT assay and the lactate dehydrogenase (LDH) release were used to determine the neuron viability. Superoxide dismutase (SOD), Glutathione peroxidase (GSH-PX), malondialdehyde (MDA) were determined by spectrophotometry using commercial kits. Mitochondrial membrane potential (MMP) and the intracellular free calcium concentration ([Ca(2+)](i)) in hippocampal neurons were measured using the confocal laser scanning microscope (CLSM). Treatment with LBP (10-40 mg/l) significantly attenuated neuronal damage and inhibited LDH release in a dose-dependent manner. Furthermore, LBP enhanced activities of SOD and GSH-PX but it decreased their MDA content, inhibited [Ca(2+)](i) elevation and decrease of MMP in ischemia-reperfusion treated hippocampal neurons. These findings suggested that LBP may be a potential neuroprotective agent for cerebral ischemia-reperfusion injury.