The Effect Of Curcumin On Stroke Prevention & The Effect Of Renal Dopamine Receptor In Hypertension

1. The effects of Curcumin on Stroke Prevention from Stroke-prone Spontaneously Hypertensive RatsStroke is an important cause of mortality and severe morbidity, with its accompanying economic and social burden worldwide. Prevention of stroke is a major public health priority in this present day and age.Curcumin is a safe, natural polyphenol compound isolated from the plant curcuma longa, which is a widely cultivated plant in the tropical regions of Asia. Cucurmin is well recognized as a dietary spice for a long time and exerts a wide spectrum of biological functions including anti-canceranticancer, antioxidation, anti-inflammatory, anti-platelet. Moreover, curcumin also ameliorates artery dysfunction associated with aging and reduces infarct size in cerebral ischemia rat model. The effects of curcumin on neuroprotection, antioxidant, anti-inflammatory and antilipemic, anti-platelet properties could make it suitable for stroke prevention.Previous studies suggested that the arterial vascular endothelium may play an essential role in the pathophysiologic process of cardiovascular diseases(CVD). Vascular endothelial dysfunction, as reflected by impaired endothelium-dependent dilation(EDD), is thought to increase the risk of CVD. Endothelium-derived NO is an important endogenous mediator of cerebral blood flow and cerebrovascular protection. Therefore, up-regulation of vascular endothelial NO production and amelioration of vascular dysfunction is a valid strategy to decrease the risk of stroke.In our previous experiment, camparing with the control, curcumin has been delayed and the survival time has been prolonged. However, the mechanism is dose not clear. According to the previous reports, the level of oxidative stress rised and the vascular function is impaired significantly among the stroke-prone spontaneously hypertensive rats(SHRsp), so we suggest that it may be related to curcumin reducing the oxidative stress level and protecting vascular endothelial cell and improving vascular dyfunction.UCP2 is a member of the mitochondrial uncoupling proteins, belonging to the mitochondrial inner anion carrier family. Evidence suggests that UCP2 is a negative regulator of ROS generation within the mitochondrion. UCP2 inhibition leads to a significant increase of oxidative stress in endothelial cells, whereas overexpression of UCP2 inhibits endothelial cells apoptosis and ameliorates vascular endothelial function by inhibiting ROS production. So we suggest that curcumins may play an important role in stroke prevention by upregulating the UCP2 expression to protect endothelial cells and improve the endothelial dysfunction.In order to confirm the hypothesis, we take the stroke-prone hypertension rats and human umbilical vein endothelial cells(HUVECS) as the research object during the experiments in vivo and in vitro, and study if and how curcumin prevents stroke.1.1 Methods1.1.1 The effect of curcumin on accurance time and death time of stroke.Twenty female stroke-prone spontaneously hypertensive rats, at 8 weeks of age, were randomly divided into 2 groups and observe the accurance time and the death time of stroke.1.1.2 Assessment of arteries relaxation in vitroThe rats were anesthetized three weeks later, after their blood collected, the rats were killed. The carotid arteries were carefully dissected with the fat and connective tissues removed for vascular function determination.1.1.3 Measurement of NO and ROS levelNO levels in the basilar artery wall and HUVECs were assessed by using DAF-2DA. The level of ROS in the basilar artery wall and HUVECs were analyzed by dihydroethidium(DHE) stain. Accumulation of nitrate and nitrite in the blood is recognized as the end products of NO metabolism. The level of nitrate and nitrite levels were measured by Nitrate reduction method.1.1.4 Measurement of plasma malondialdehyde, and superoxide dismutasePlasma malondialdehyde(MDA) concentration and superoxide dismutase(SOD) activity were assayed using a Lipid Peroxidation MDA Assay Kit and a Cu/Zn-SOD and Mn-SOD Assay Kit with WST-1.1.1.5 UCP2 expression in the carotid artery and HUVECsTo observe the effect of regulation curcumin on UCP2 expression in carotid arteries and HUVECs. UCP2 m RNA expression in the carotid artery by RT-PCR and protein expression in HUVCEs by immunoblotting.1.1.6 Cells autophagy and apotosis messuredIn order to detect the effects of curcumin on autophagy and apotosis from carotid arteries and HUVECs, LC-3 protein, a marker of autophagy were messured by immunoblotting. The percentages of apoptotic nuclei were calculated by determining the number of Hoechst-stained nuclei that were positive for TUNEL staining.1.2 Results1.2.1 General characteristicsTo investigate the effect of curcumin on general characteristics of SHRsp, We measured blood pressure and body weight at the 0, 2 and 4 weeks after curcumin in treatment. Results showed that curcumin treatment did not influence blood pressure, body weight and the ratio of brain weight(g)/body weight(kg) from saline group and treatment with curcumin SHRsp.1.2.2 Curcumin treatment delayed the onset of stroke and increased survival timeTo investigate whether curcumin treatment has a preventive effect on stroke, we observed the occurrence of stroke and death in all SHRsp with or without curcumin treatment. Compared to the saline group, curcumin delayed the onset of stroke, and made the stroke rats survive for a longer time.1.2.3 Curcumin treatment ameliorated arterial dysfunction in SHRspDue to the role of the artery in the stroke, we checked arterial function in the carotid arteries of SHRsp. Compared with saline SHRsp, administration of curcumin significantly enhanced the relaxation of carotid artery response to ACH and SNP, but did not change its response to PHE.1.2.4 Curcumin treatment increased the NO levels, but decreased ROS expressions in basilar arterial wall and plasma from SHRsp.Due to the role of ROS in the pathogenesis of stroke, we checked the ROS in the plasma and artery. The results showed that, compared with the saline group, curcumin increased plasma nitrate/nitrite levels and plasma SOD activity, an important antioxidant enzyme and decreased plasma MDA levels. We also observed ROS in the artery instead of MDA in the plasma, we found that curcumin decreased ROS levels in the basilar artery wall.1.2.5 Role of UCP2 on the curcumin protection in artery and HUVECs.To check whether UCP2 is involved in the anti-oxidative effect of curcumin, UCP2 expression was determined by RT-PCR. The results demonstrated that curcumin significantly increased UCP2 levels in the carotid artery from SHRsp. The effect of curcumin on UCP2 was also confirmed in the HUVECs, H2O2 was used to simulate oxidative stress in vitro. The results showed that H2O2 significantly decreased cell viability, which was reduced by curcumin in a concentration-dependent manner. consistent with the in-vivo study, curcumin inceased NO levels, decreased ROS levels in the HUVECs. Moreover, the UCP2 expression was increased, in the presence of UCP2 inhibitor, genipin(10 mol/L), the effects of curcumin on ROS and NO were blocked indicating that UCP2 is an important signaling factor in the antioxidant role of curcumin. we also determin UCP2 is invoved in the effects of curcumin on cells autophagy and apotosis.1.3 Conclusion1.3.1 Curcumin delayed the onset of stroke and increased the survival of stroke-prone spontaneously hypertensive rats(SHRsp)1.3.2 Curcumin improved the vascular dysfunction of stroke-prone spontaneously hypertensive rats(SHRsp).1.3.3 Curcumin enhanced the antioxidant ability of stroke-prone spontaneously hypertensive rats(SHRsp)1.3.4 Curcumin may play a role of antioxidant, anti-apotosis, protecting endothelial cells, increasing NO levels and increasing atouphagy of endothelial cells, improving the vascular dilatation function. These effects of curcumin may through upregulation of UCP2 expression.2. Machenism of dopamine D3 receptor increasing dopamine D4 receptor expression and function in Renal Proximal Tubule CellsDopamine regulates natriuresis through its binding with dopamine receptors in kidneys. Dopamine receptors belong to G-protein coupled receptor superfamily and can be classified into D1-like(D1 and D5) receptors and D2-like(D2, D3 and D4) receptors pharmacologically. Renal dopamine receptor takes care of more than 50% sodium excretion when sodium is replete.All subtypes of dopamine receptors, directly or indirectly, are involved into regulation of natriuresis and blood pressure.It has been demonstrated that some interactions can be found between different dopamine receptors. Our previous studies showed positive interactions between D3 and D1 receptors in renal proximal tubule(RPT) cells. The expression and function of each receptor is increased when the other receptor is stimulated. Similar effects are found between D3 and D4 receptors. Theses interactions can be found in RPT cells from WKY rats but not from SHR. However the machenism of these effects is not clear.2.1 Methods2.1.1 To further determine the mechanisms underlying the regulation of D3 receptor on D4 receptor expression, we checked D4 receptor protein degradation in the presence of 10 μg/ml cycloheximide(CHX), which inhibited the de-novo protein synthesis.2.1.2 To detect whether D3 receptors can directly interact with D4 receptors, we observed the colocalization of D3 and D4 receptors in RPT cells by using laser confocal microscopy and co-immunoprecipitation. To check the physiological significance of D3 and D4 receptor interaction,D4 receptor localization in the plasma membranes of both WKY and SHR RPT cells by using laser confocal microscopy.2.1.3 To study the signal of the regulation of D3 receptor on D4 receptor expression, WKY RPT cells were pretreated with PLC and PKC blokers, then D4 receptor expression were messured by immunoblotting.2.2 Results2.2.1 PD128907(10-7mol/L) decreased D4 receptor degradation in WKY RPT cells but not in SHR RPT cells.2.2.2 stimulation of D3 receptor with PD168907(10-7mol/L) for 30 minutes had no effect on D4 receptor localization in the plasma membranes of both WKY and SHR RPT cells, indicating the regulation of D3 and D4 receptor-mediated inhibitory effect on Na+-K+-ATPase activity in RPT cells is not accounted by receptor translocation, rather by receptor expression changed.2.2.3 The immunostaining showed that D3 and D4 receptors had abundant expression in RPT cells. D3 receptors colocalized with D4 receptors in WKY cells The co-localization was further confirmed by co-immunoprecipitation, D3 and D4 receptors co-immunoprecipitated in RPT cells from both WKY rats and SHRs, which was increased by PD128907 in WKY cells, not in SHR cells.2.2.4 Pretreatment with PLC blocker significantly inhibited PD128907 induced up-regulation of D4 receptors, we also checked the role of PKC in the regulation process, it resulted that PKC inhibitor exerted similar effects as PLC blocker.2.3 ConclusionThe post-translational mechanism is involved in the effect of dopamine D3 receptor increasing dopamine D4 receptor expression in Renal Proximal Tubule Cells.The regulation of dopamine D3 receptor and D4 receptor mediated inhibitory effect on Na+-K+-ATPase activity in RPT cells is not accounted by receptor translocation, rather by receptor expression changed.D3 receptor colocalizes and directly interacts with D4 receptor in RPT cells.PLC/PKC are involved in the regulation of PD128907 on D4 receptor expression in RPT cells from WKY rats.