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Anti-hypoxic Effects and Action Mechanism of Rutin

Author: YaoJuan
Tutor: JiaZhengPing; MaHuiPing
School: Lanzhou University
Course: Pharmacology
Keywords: rutin hypoxia in PC12cells H9c2cells hypoxia-inducible factor nitricoxide
CLC: R285
Type: Master's thesis
Year: 2012
Downloads: 129
Quote: 0
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Abstract


Objective:1.Establish PC12cells hypoxia model, observe the injury in PC12cells of the hypoxic environment, screening the optimal concentration of rutin in hypoxic PC12cells, study the protective molecular mechanism of rutin in PC12cells by the hypoxic injury.2.Establish H9c2cells hypoxia model, observe the injury in H9c2cells of the hypoxic environment, screening the optimal concentration of rutin in hypoxic H9c2cells, research the protective mechanisms of rutin in H9c2cells by hypoxic injury.3.Observe the influence of rutin on hypoxic H9c2cells eNOS-NO-sGC-cGMP signaling pathway, investigate the possible mechanisms of rutin on activation of NO signaling pathway to protect H9c2cells.Method:1.Cultivate PC12cells in anoxic environment, screening the best concentration of rutin in hypoxic PC12cells, observe the cell morphology, determin the cell viability before and after hypoxia, assay LDH contents and the cell cycle, use Real-time PCR analysis HIF-la and Caspase-3mRNA expression levels, extract total protein and use Western-blot detect HIF-la proteins expression.2.Cultivate H9c2cells in anoxic environment, screening the best concentration of rutin in anoxic H9c2cells, observe the cell morphology, determin the cell viability, assay LDH contents, detect the HIF-la, VEGF, HO-1and Caspase-3mRNA expression levels by Real-time PCR.3.Use L-NAME, Carboxy-PTIO and ODQ blocked eNOS, NO and sGC, observe the change of eNOS,NO, sGC and cGMP in anoxic H9c2cells of rutin, detect LDH,CK,ATP contents,use Real-time PCR detect HIF-la,VEGF,HO-1, Caspase-3,Bcl-2,Mb and Tnn mRNA expression levels.Result:1.Hypoxic environment significantly inhibited the PC12cells activity, compared with normal control group, cultured PC12cells in hypoxic environment for48h, the cells activity significantly inhibited. The rutins final concentration in1×10-5mol·L-1 could significantly improve the anoxic cells activity.In the hypoxic PC12cells, the rutins concentration in1×-5mol·L-1can effectively reduce the content of the mediums LDH, compared with the normal group, oxygen-poor can significantly reduced PC12cells G1phase proportion after hypoxia for48h, PC12cells S-phase fraction increased. Compared with the hypoxia model group, the1×10-5mol-L"1rutin increased the proportion of PC12cells in Gi phase, decreased S phase cells after hypoxia for48h.Rutin can increased hypoxic PC12cells HIF-la mRNA expression, cultured PC12cells in hypoxia inveronment for12h,24h,48h, compared with hypoxia model group, rutin can significantly increase HIF-la mRNA; rutin can significantly reduce the expression of Caspase-3mRNA expression levels.Culture PC12cells48h in hypoxic environment, HIF-la proteins expression increased, compared with the hypoxia model group, rutin group’s expression is higher than the hypoxia model group.2.Hypoxic environment significantly inhibited the H9c2cells activity after anoxia for48h. The1×10-5mol·L-1rutin can significantly improve the H9c2cells vitality compared with hypoxia model group.Compared with the hypoxia model group, the1×10-5mol·L-1rutin can significantly reduced H9c2cells LDH contents in cells medium.Rutin has the effect to increase the expression levels of HIF-la、 VEGF and HO-1mRNA in hypoxic H9c2cells, reduced the expression levels of Caspase-3mRNA in the hypoxic environment.3.Hypoxic environment can significantly increased H9c2cells eNOS activity in12h compared with the normal control group, the contents of NO in H9c2cells was significantly increased after hypoxia for12h.Compared with the control group,the rutin can improve the hypoxic H9c2cells contents of eNOS、the eNOS proteins expression and the contents of NO, increased the gene expression levels of sGC, increased the contents of cGMP in blocked group.Compared with the hypoxia model group, rutin has the effect on reduce the contents of LDH and CK in hypoxic H9c2cells culture medium, reduce the content of ATP in H9c2cells.Rutin can increase HIF-la, VEGF and HO-1mRNA expression levels, reduce the expression levels of Caspase-3mRNA, has no significant effect on Bcl-2mRNA expression levels, rutin also can increase the expression levels of Mb and Tnn mRNA in hypoxic H9c2cells. Conclusion:1.Rutin has significant anti-hypoxia injury effect on PC12cells, the possible mechanism is related to increase the PC12cells activity, protect the integrity of the cell membrane, affecting the cell cycle, anti-apoptosis, increased the gene expression levels of HIF-la in hypoxic PC12cells.2.Rutin has significant anti-hypoxia injury effect on H9c2cells, the possible mechanism correlated to improve cells activity, protect the integrity of cell embranes, anti-apoptosis and increase the gene expression levels of HIF-la、VEGF and HO-1in hypoxic H9c2cells.3.Rutin has the obvious protective effect on hypoxic H9c2cells, the possible mechanism is related to activation the eNOS-NO-sGC-cGMP signaling pathway, then develop the anti-hypoxia effect.

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