Role Of Angiotensin ⅡInduced Neural Cell Senescence And Drug Intervention

Objective: Vascular dementia (VD) is an age-related disease characterized by progressive irreversible decline of intelligence-related functions. It is not only related to the vascular diseases, but also closely linked to abnormal death of neural cells and aging of central nervous system. Angiotensin Ⅱ (Ang II) is a core part of the renin-angiotensin system (RAS). Research has showed that the cerebral RAS can affect normal neuronal plasticity, learning, memory and other cognitive functions, which indicates that cerebral RAS is involved in the process of brain aging. Aging is a progressive recession process of structure and function of normal cells, which is also consistent with the progressive irreversible changes of dementia. Hereby, we hypothesize that Ang II may be induce neural cell senescence and then result in the occurrence of VD. Therefore, we investigated the role of Ang II-induced neural cell senescence in cultured PC12cells and rat hippocampal neurons in vitro.Methods:1. PC12cells were cultured in vitro. Cell viability was detected by MTT under different concentrations of Ang II to make sure of the best inducing condition. The action of irbesartan, PD123319and captopril on cell viability was also detected after Ang Ⅱ inducement. Senescence-associated β-galactosidase (SA-β-gal) staining was used to observe cell senescence, enzyme linked immunosorbent assay (ELISA) was used to examine the activity of telomerase reverse transcriptase (TERT), and Western blot was used to detect the expression of TERT, P53and P21in PC12cells.2. The primary neonatal rat hippocampal neurons were cultured using newborn rats within24h. SA-β-gal staining was used to detect the cell senescence induced by Ang Ⅱ in different concentration and different time to determine the best inducing condition. To further explore the mechanisms, the effects of AT1receptor blocker irbesartan and AT2receptor blocker PD123319on Ang Ⅱ-induced cell senescence were examined. The expression of cell cycle-related protein P53and P21were detected by Western blot assay.Results:1. Ang Ⅱ (1-1000μmol/L,48h) inhibited cell proliferation in a dose-dependent manner. Ang II in the concentration of10μmol/L could induce cell senescence after incubation for48h. Irbesartan, PD123319and captopril had no poisonous effect to PC12cells respectively. Both irbesartan and captopril improved the cell senescence induced by Ang II, while PD123319had no effect on it. After incubation with Ang II, both the activity and expression of TERT were decreased and the expression of P53and P21was increased. Both irbesartan and captopril increased the activity of TERT and expression of TERT, P53and P21, while PD123319had no effect on it.2. Ang II in the concentration of10μmol/L could induce cell senescence in the primary rat hippocampal neurons after incubation for48h. Irbesartan can inhibit cell senescence induced by Ang II and reduce the expression of P53and P21, whereas PD123319had no effect on both.Conclusion:Ang II in the concentration of10μmol/L could induce cell senescence after incubation for48h in both PC12cells and primary neonatal rat hippocampal neurons. Ang II induces neural cell senescence via AT] receptor, not AT2receptor. Ang Ⅱ-induced neural cell senescence is closely related to depression of TERT and overexpression of P53and P21. Objective:The constant generation and accumulation of free radicals in cells would damage the structure and function of proteins and nucleic acids, and thus lead to cell senescence or aging of the body, which is called the free radical theory of aging. Reactive oxygen species (ROS) are oxygen free radicals which can produce oxidative stress-induced damage to biological macromolecules and then affect the process of cell senescence. Hydrogen peroxide (H2O2), which can generate a large number of free radicals, is often used as a cell oxidative stress revulsant. Studies have confirmed that H2O2can induce cell senescence in the vascular endothelial cells, but the action of H2O2on neural cell senescence is rarely reported. Scutellarin, the main active ingredient of breviscapus, is widely used in the treatment of cardiovascular and cerebrovascular diseases. Recent studies have found that scutellarin is an antioxidant which has the capacity to reduce the accumulation of ROS. Therefore, we cultured PC12cells in vitro to study the mechanism of H2O2-induced cell senescence and to further explore the protective effects of scutellarin on PC12cell senescence.Methods:SA-β-gal staining was used to observe the cell senescence after incubation with different concentration of H2O2and different induction time of H2O2in PC12cells. SA-β-gal staining was also used to observe the effect of different concentrations of scutellarin on H2O2-induced PC12cell senescence. ELISA was applied to test the activity of telomerase reverse transcriptase (TERT). Real-time PCR was used to detect the mRNA expression of TERT, P53, P27and P21. Western blot was used to examine the protein expression of TERT, P53, P27and P21.Results:After incubation with10μmol/L H2O2for24h, cell pyknosis was found in PC12cells. With the increase of induction time, cell rupture and necrosis happened. After incubation with1μmol/L H2O2for48h,(β-gal positive cells (blue, aging cells) were significantly increased. Scutellarin in the concentration of1,10and100μmol/L relieved cell senescence in different degrees, while the action of10μmol/L scutellarin was most significantly (P<0.01). Both the activity and expression of TERT decreased significantly (P<0.05, P<0.05) and the expression of P53, P21and P27increased significantly (P<0.05, P<0.05, P<0.05) after H2O2induction. After preincutation of scutellarin, both the activity and expression of TERT were upregulated (P<0.05, P <0.05) and the expression of P53, P21and P27were downregulated (P<0.05, P<0.05,P<0.05)Conclusion:H2O2in the concentration of1μmol/L induces cell senescence after incubation for48h in PC12cells. The H2O2-induced cell senescence is related to the depression of TERT and the increment of P53, P27and P21. Scutellarin protects PC12cells from H2O2-induced cell senescence through up-regulating TERT and down-regulating P53, P27and P21.