Effect Of Cerebral Ischemia/Reperfusion Injury On Hydroxysafflor Yellow A Penetrating Across The Blood-Brain Barrier

ObjectiveIschemic cerebrovascular diseases (ICVD) is an usually pathologic process and influence human health and life badly. As we know, today the therapy of thrombolysis and brain protection are still two major treatments for ICVD. Up to now, traditional Chinese medicines have been paid more and more attention because of the good curative effects in the treatment of the ICVD. The flower of the safflower plant, Carthamus tinctorius L, has been used extensively in traditional Chinese medicine for treatment of cerebrovascular and cardiovascular diseases. Safflower yellow pigments which water-soluted are responsible for the therapeutic effects. HSYA, the main chemical component of the safflower yellow pigments, has been demonstrated to antagonize platelet activating factor receptor binding and has inhibitory effects on both thrombosis formation and platelet aggregation. Wei and colleagues indicated that HSYA might exert neuroprotection against cerebral ischemia/reperfusion injury and the mechanism is related to the antioxidant action of HSYA. It is also reported that HSYA showed a neuroprotective action on the cortex mitochondrial injuries induced by cerebral ischemia and glutamate-mediated neuron injury.The blood-brain barrier (BBB) serves as an dynamic obstacle which restricts the transport of most substances from the blood to brain parenchyma. The BBB is made of a complex vasculature lined by brain capillary endothelial cells (BCECs) and supported by tight junctions, pericytes, astrocytic end-feet and extracellular matrix. BCECs that have few fenestrations, cytoplasmic vesicles, and more enzymes are the specialized system of the BBB. Therefore the BBB restricts the delivery of macromolecule and hydrophilic agents from the systemic circulation to the central nervous system in order to maintain homeostasis. BBB is prone to structure weakness and function instability after exposed to ischemic injury, a number of molecules are implicated in the mechanistic breakdown and increased permeability of the BBB including calcium ions, free radical, nitric oxide, inflammatory cytokines, matrix metalloproteinase and leucocytes released following cerebral ischemia/reperfusion injury (CIRI).In order to exert their therapeutic effect, the compounds for the treatment of stroke should adequately cross the BBB and reach effective concentration in the brain. The State Food and Drug Administration of China recently approved HSYA for clinical trials of treating patients with cerebral ischemia as HSYA has showed protective action on cerebral ischemia in preclinical studies. However, it remains unclear whether HSYA can penetrate across the BBB and the effects of CIRI on the penetration of HSYA in BBB. In this study, the ability of the penetration of HSYA in BBB, the effect of CIRI on the penetration of HSYA in BBB and the mechanism was investigated.MethodsMiddle cerebral artery occlusion was used to induce transient focal cerebral ischemia and reperfusion 3 h or 24 h. The changes in permeability of the BBB were investigated using the Evans Blue (EB) content of brain tissue. The concentration of HSYA was determined by reversed phase high performance liquid chromatography and the protein was assayed by Bradford assay in homogenate of brain. Brain tissue concentrations of HSYA were expressed as the ratio of the HSYA concentration to protein content in brain tissue. The expression of matrix metalloproteinase-9 (MMP-9) was measured by euzymelinked immunosorbent assay. Results(1) The brains of the sham group showed no grossly visible areas of EB extravasation. The EB content of brain obviously increased in the ipsilateral cerebrum compared with sham group after cerebral ischemia reperfusion 3 h, subsequently it continued increasing on reperfusion 24 h.(2) Lower concentration of hydroxysafflor yellow A could be detected in control group after 10 to 60 min administration. There were no significant difference between the sham groups and the control group in HSYA concentration in brain tissue. The HSYA concentration in ischemic hemisphere of ischemia/reperfusion rats significantly increased, while did not change in non-ischemic hemisphere compared with sham group.(3) MMP-9 were not detected in the brain of sham group and sham (HSYA) group. The level of MMP-9 expression in ischemia/reperfusion and ischemia/reperfusion (HSYA) groups obviously increased compared with sham group and sham (HSYA) group respectively. There was no significant difference between ischemia/reperfusion and ischemia/reperfusion (HSYA) group.ConclusionBBB permeability increased which encountered CIRI. HSYA can penetrate the BBB to reach the brain tissue with low penetration in physiologic condition, and the BBB penetration of HSYA is increased obviously after CIRI, paralled with BBB permeability. Increased BBB penetration of HSYA which encountered cerebral ischemia may have relation with its level of MMP-9 expression.