The Role Of Dimethylarginine Dimethylaminohydrolase In Differentiation And Anti-apoptotic Effect Of Durg In PC12 Cells

It is well known that nitric oxide (NO) synthase (NOS)/NO play an important role in neuronal differentiation. dimethylarginine dimethylaminohydrolase (DDAH), which widely expresses in different cell and tissue including cardiovascular and neuronal system is responsible for the degradation of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of NOS. Various studies have reported that endogenous ADMA is a key factor contributing to endothelial dysfunction and is associated with development of some cardiovascular diseases. And the reduced DDAH activity is believed to be the main cause of elevated ADMA levels, suggesting that the DDAH/ADMA pathway may be a new target for pervention and treatment of cardiovascular diseases. Recently, studies showed that DDAH mRNA in rat brain was up-regulated in axotomized motoneurons and the reduction of DDAH activity and elevation of ADMA level were abserved in Hcy-treated neuronal cells, suggesting that DDAH may be involved in neuronal development and some neuronal disease.The thesis systematically investigated the role of DDAH in neuronal differentiation and apoptosis induced byβ-amyloid (Aβ) or cobalt chloride (CoCl₂), and explored the anti-apoptotic effects of 3,4,5,6-tetrahydroxyxanthone or all-trans retinoic acid (atRA) through improving the DDAH/ADMA pathway. The main research can be summarized as following.By gene silencing and overexpression technology, we systemically investigate the regulatory role of DDAH1 in nerver growth factor (NGF) -induced differentiation of PC12. During the exposure of NGF (50 ng/ml) on PC12 cells, DDAH1 mRNA and protein expression increased progressively with time. By contrast, the reduction in DDAH2 mRNA and protein levels were observed. The ADMA level and DDAH activity did not changed during NGF-induced differentiation of PC12 cells. The differentiation was significantly diminished in DDAH1-depleted PC12 cells treated with NGF, concomitantly with the reduction of MAP2 (neuronal-cell-specific marker) and nNOS mRNA expression. Overexpression of DDAH1 inhibited cell cycle progression and induced neuronal differentiation, and increased the nNOS mRNA expression.Xanthones are polyphenol compounds existing in many plants which have potent anti-inflammation and anti-oxidation effects. 3,4,5,6-tetrahydroxyxanthone is synthesized by the department of chemical pharmacy. The previous work of our lab has shown that 3,4,5,6-tetrahydroxyxanthone has protective effects on endothelium and myocardium by reduction of ROS and ADMA production and enhancement of DDAH activity. According to the oxidative stress is invovled in the apoptosis induced by Aβand the anti-apoptotic effect of 3,4,5,6-tetrahydroxyxanthone, we studied the relationship between anti-apoptotic effect of xanthone on Aβ_25-35-treated PC 12 cells and DDAH/ADMA pathway. Treatment with Aβ_25-35 (10μM) for 48 h induced apoptosis of PC12; After 3 hours of incubation with Aβ_25-35 (10μM), the intracellular ROS level and caspase-3 activity were increased; Pretreatment with PDTC (5μM) or DEVD-CHO (100μM) significantly attenuated the apoptosis induced by Aβ_25-35 (10μM); Pretreatment with xanthone (3, 10 or 30μM) significantly attenuated the apoptosis elicited by Aβ; Xanthone treatment significantly inhibited the elevated ROS level and caspase-3 activity induced by Aβ_25-35 (10μM for up to 48 hours). Incubation of PC12 with Aβ_25-35 for 6 hours led to decrease in the DDAH activity and increase in ADMA level; Preincubation of PC 12 with xanthone (3, 10 or 30μM) significantly inhibited the decreased DDAH activity and elevated ADMA induced by Aβ_25-35. ADMA induced apoptosis of PC12 in time and concentration-dependent way; After 3 hours of incubation with ADMA (10μM), the intracellular ROS level and caspase-3 activity were increased; Pretreatment with PDTC (5μM) or DEVD-CHO (100μM) significantly attenuated the apoptosis elicited by ADMA (10μM); PDTC or DEVD-CHO itself had no effect on cell apoptosis.AtRA has a wide range of biological processes, such as cell growth, differentiation and apoptosis. It was reported that atRA regulated gene transcription via binding with retinoic acid receptor (RAR) and retinoid X receptor (RXR). Recent study showed that atRA induced the expression of DDAH2 in endothelial cells. Therefore, in the present study we investigated the role of DDAH/ADMA pathway in CoCl₂-induced apoptosis of PC12 cells and studied the relationship between anti-apoptotic effect of atRA on CoCl₂-treated PC 12 cells and DDAH/ADMA pathway. Treatment with CoCl₂ (125μM) for 48 h induced apoptosis of PC12; After 3 hours of incubation with CoCl₂ (125μM), the intracellular ROS level and caspase-3 activity were increased; Pretreatment with PDTC (5μM) or DEVD-CHO (100μM) significantly attenuated the apoptosis induced by CoCl₂ (125μM); Pretreatment with atRA (0.1, 1 or 10μM) significantly attenuated the apoptosis elicited by CoCl₂; AtRA treatment significantly inhibited the elevated ROS level and caspase-3 activity induced by CoCl₂. Incubation of PC 12 with CoCl₂ for 6 hours led to decrease in the DDAH activity, and the decreased levels of DDAH2 mRNA and protein also reached significance after 8 hours and 12 hours of incubation, respectively. However, the level of ADMA in the medium was elevated after 6 hours of incubation with CoCl₂. Preincubation of PC12 with atRA (0.1, 1 or 10μM) significantly inhibited the reduction of DDAH activity and DDAH2 expression, and elevation of ADMA induced by CoCl₂. The inhibitory effects of atRA (0.1μM) on cell apoptosis were blocked in the cells depleted of DDAH2; The inhibitory effects of atRA (0.1μM) on ROS production, caspase-3 activity and ADMA accumulation also were attenuated after 12 hours of incubation with CoCl₂.