The Effects And Mechanism Of Safflower Yellow Pigment On Hypoxic-ischemic Brain Damage Which Induced By Controlled Hypotension

BackgroundControlled hypotension (Controlled Hypotention, CH) is a clinically anesthetic technique which widely used based on the surgery s condition and needs.The purpose is to reduce bleeding,improve the surgical field environment, reduce blood transfusions,increases the security of surgery.However,some patients may often be heart rate, blood pressure,respiratory depression and other symptoms during the surgery due to their own conditions,anesthesia drugs, the surgery and other stimuli.It is often difficult to ensure the supply to vital organs blood perfusion and oxygen during controlled hypotension and it is easy to induce the hypoxic-ischemic damage on tissue and organ.But compared to with other organs,the brain tissue will be more susceptible to damage because of their strong metabolic and the poor hypoxia tolerance.So the biggest concern during controlled hypotension is cerebral insufficiency and brain damage caused by cerebral hypoxia and reperfusion injury after re-pressed.Hypoxic-ischemic brain injury include the ischemia primary brain injury and the reperfusion of secondary brain injury.The root treatment of cerebral ischemia is to eliminate the cause of ischemia,restore the blood perfusion as soon as possible and short the time of tissue ischemia. However,the secondary injury after reperfusion is inevitable, which directly determines the prognosis of brain injury. The secondary brain injury,that is cerebral ischemia-reperfusion injury (CIRI) refers to before the cerebral ischemic tissue occurred cell necrosis and after recover the blood perfusion of ischemic tissue,its function is not only failed to restore but also appears to further aggravated phenomenon on the basis of the original injury.CIRI is a complex pathophysiological process, its pathogenesis involves many aspects, including the toxicity of excitatory amino acids, calcium overload,the metabolism of mitochondrial energy and acidosis, free radicals and lipid peroxidation, nitric oxide,inflammatory cytokines and apoptosis.Thus,the key to the treatment of cerebral ischemia and reperfusion injury is to protect the structure and function of brain cells, neurons avoid stress injury and apoptosis.Apoptosis is a active death controlled by gene,also known as programmed cell death (PCD).It can be roughly divided into three stages from receiving the apoptosis inducing factor to apoptosis, that is:the phase of apoptotic signal transduction,the activation of apoptotic gene and the implementation phase, impact any one of these stages will be interrupted apoptosis.Mitochondria are the cellular energy metabolism center,also the central part of connecting the cell stress response and neuronal apoptosis execution.It is the most sensitive to a variety of injuries and therefore it is the subcellular targets ischemic lesions, which directly induce apoptosis while dysfunction.Nitric oxide synthase (nNOS) is a calcium-dependent enzyme, present in the nerve cells,it is less active under the normal conditions,and result in physiological doses of NO to transfer the signal.Studies have shown that the hypoxic-ischemic brain injury can activate the nNOS to produce excessive NO and induced Glutamate receptor 6 (GluR6) nitrosylation, thereby activating GluR6 · PSD95 · MLK3 signal pattern and c-Jun N-terminal kinase (JNK) signaling pathway and apoptosis signal-regulated kinase 1 (apoptosis signal-regulating kinase 1, ASK1).The ASK1 is always a neurotransmitter of cells death which respond to various stimuli and the JNK pathway is also considered to play an important role in cerebral ischemia reperfusion injury, especially the process of programmed cell death.Hypoxia-inducible factor -1a (Hypoxia-inducible factor-la, HIF-1a),as a hypoxia-sensitive transcription factor,it is upregulated in ischemic stroke with its downstream genes.Safflower as a traditional Chinese medicine, is a class Asteraceae annual herb Its warm spicy, owned by two darling.it has been widely used in the treatment of cardiovascular disease and other organ systems in recent years.Safflower yellow pigment (safflor yellow pigment, SYP), also known as safflower Flavin, is one of the main active ingredient of Chinese medicine safflower.it is extracted from safflower and refined to intravenous formulation recently.Modern pharmacology Pharmacodynamic studies have found that the Safflower yellow pigment has a variety of pharmacologic-al effect,including microcirculation, expansion of coronary, blood pressure, anti-oxidation, immune suppression,protecting cardiac and so on.In recent years studies have reported safflower and its extract enhanced the antioxidant capacity of spinal cord tissue mainly by reducing the superoxide dismutase (MDA) concentration, increased the malondialdehyde (SOD) activity,thus reducing the schemia-reperfusion injury of the heart, brain, muscle, lung and other tissues and organs.Our previous findings on spinal cord ischemia-reperfusion also showed safflower injection could well reduce the spinal cord ischemia-reperfusion injury and could reduce the apopto -sis spinal cord anterior horn neurons.Chen Duo Bao, Zhang Ji Qing and other scholars also confirmed SYP had a protective effect in the study of ischemia-reper-fusion injury,and also thought it may be related to inhibition of brain Ca2+ and excitatory amino acids and thereby inhibiting apoptosis.Howeve,Seldom literature has been reported the protective effects and mechanism of safflower yellow pigment on hypoxic-ischemic brain damage which induced by lower levels of controlled hypotension in rabbits.ObjectiveTo establish the animal model of simple induced hypotension by sodium nitroprusside on rabbits to investigate the effects of safflower yellow pigment on the degree of mitochondrial ultrastructure after a hypoxic-ischemic brain injury and the expression of ischemic injury associated proteins (Caspase-3, iNOS and HIF-1α),to study the protective effect and possible mechanisms of SYP on hippocampal neurons under the lower hypotension and provide a theoretical basis for clinical research.Methods1. Animals and groups:40 healthy New Zealand white rabbits (male and female, weighing 1.8-2.2Kg, provided by the Experimental Animal Center of Southern Medical University) were randomly divided into 5 groups,8 of each:Normal group (N), Controlled hypotension group (ie CH group, the group of mean arterial pressure [MAP] down to 45% of the underlying value),SYP preconditioning group (SYP1 group,which giving ntravenous SYP,2ml/kg 30min before induced hypotension,the SYP formulated with saline containing SYP 1.6mg/ml),SYP post-treatment group (SYP2 group,giving intravenous injection SYP,2ml/kg at the time of 30min after the controlled hypotension beginning,the SYP formulated with saline containing SYP 1.6mg/ml),SYP pretreatment+post-treatment group (SYP1+SYP2 group, giving intravenous SYP,2ml/kg,before the start of 30min and 30min after the start of controlled hypotension, the SYP formulated with saline containing SYP 1.6mg/ml).2. Model Making:This study follows our previous experiments that using the New Zealand white rabbits to make the induced hypotension model,concrete steps are as follows:Anesthesia the fasting rabbits which forbidden to drink water and food 12h through intraperitoneal injection with chloral hydrate.fixed the head and limbs of rabbit to the rabbit stage,Shave the rabbit hair in the front of the neck, disinfection,and then tracheotomy endotracheal tube, ventilator animals pick mechanical ventilation (tidal volume 21ml, breathing 51bpm, FiO21.0).Isolated on the right internal jugular vein catheterization, and then to extend the pipe passage infusion infusion. Shearing at the four weeks left groin,disinfection, separate the femoral artery at the groin by lower edge of measured and catheterization,then connect the load converter passage to row mean arterial pressure measurement.End, sterile wet gauze to cover the wound, infusion the right amount of Ringer’s solution,supply the loss liquid of fasting total about 15ml/kg,let the Rabbit table flat on 39.5℃ thermostat blanket insulation, Until blood pressure is stable 15min later,recording the time of basic mean arterial pressure (MAP), heart rate (HR) and central venous pressure (CVP).Normal group use continuous infusion with 5% glucose injection at 4ml/(kg.h),Buck and treatment groups were started pumping by sodium nitroprusside which formulated with 5% glucose solution (400ug/ ml),dropped to the target blood pressure at 10-15 minute,interrupted adjusting the pump speed to maintain the target blood pressure 1h. According to the above grouping scheme, giving the safflower yellow pigment at corresponding time,on the basis of CVP to adjust the amount of fluid in all animals during the buck (including the amount of antihypertensive drugs liquid),to minimize the impact of fluid volume on the volume,after the completion of the buck, each experimental animal use Ringer’s solution at 4ml/(kg·h) pump speed,and reperfusion 2 h then death.Take the hippocampal CA1 neuronal tissue of animals to examine pathological,ang use the transmission electron microscopy to observe the change of ultrastructural alterations,TUNEL staining was used to observe the apoptotic cells case of brain tissue samples,Use immunohistochemistry and Western blot analysis to detect the expression of nNOS, HIF-1α,as well as Caspase-3 protein in the hippocampal tissue.Results1.All animal models were produced successfully, accidental death due to anesthesia or surgery did not appear, Normal mortality rate was 0,;CH group died six:two were died at the time of 45 min after controlled hypotension,and other four rabbits were died after the multi-pressure start,whose mortality rate is 75%; SYP1, SYP2, SYP1+SYP2 groups were respectively one,two,one death,which gradually died at the time after recovery pressure 1h, the mortality rate were lower than the CH group, the difference was statistically significant (P<0.05).2. Under TEM to observe the ultrastructural of hippocampal CA1 and Flameng Rating:N group:cell structure was intact,organelles clearly visible nucleolus chromatin uniform, no karyopycnosis fragmentation or dissolution;CH group showed ultrastructural damage is more serious, showing nuclear fragmentation, dissolution, and the cell structure is substantially destroyed; the injury of group SYP1, group SYP2 and group SYP1+SYP2 have been reduced compare to the CH group,no significant nuclear fragmentation and dissolution of intercellular and the connections between cells are tight;The mitochondrial Flameng scores in CH group were higher (P<0.05) than other groups, the remaining difference between the groups was not statistically significant (P> 0.05).3.TUNEL staining of apoptotic cells and calculate apoptotic index:N cells was normal, showing a very small amount of TUNEL-positive staining cells;CH group, the number of positive cells increased significantly;while the TUNEL-positive staining cells in Safflower yellow pigment treated groups (SYP1 group, SYP2 group, SYP1+SYP2 group) were significantly reduced than CH group (P<0.05);But the apoptotic cells of group SYP2 were more than the other two groups (SYP1 group, SYP1+SYP2 group), the difference between them were not statistically significant (P> 0.05).4.1mmunohistochemistry and Western blot to analysis the expression of protein nNOS, HIF-laand Caspase-3:Compared with the N group,the expression of nNOS, HIF-la, and Caspase-3 were significant increase in CH group;the safflower yellow pigment treated groups (SYP1 group, SYP2 group, SYP1+SYP2 group) were decreased than the CH group;compare to the other two groups,the protein content was increased in group SYP2,the difference was not statistically significant (P> 0.05). Conclusions1.Morphological and other studies confirmed that safflower yellow pigment can protect the cerebral ischemia-reperfusion injury which induced by controlled hypotension in rabbits, may reduce the mortality to some extent;2.In the cerebral ischemia-reperfusion injury, protective effect of safflower yellow pigment may be related to reducing the content of HIF-1α and nNOS;3.It may be that Safflower yellow pigment reduced apoptosis by inhibiting the expression of Caspase-3 protein, and thereby inhibiting cerebral ischemia and reperfusion injury in neurons;4.Different administration time of safflower yellow have a protective effect in hypoxic brain injury, there is no significant time-effect relationship between them.