The Effect Of Salvia Miltiorrhiza Polyphenols Salt On Cerebral Ischemia/reperfusion Oxidative Injury And The Underlying Mechanisms

Background and purposeRestoration of blood flow following ischemic stroke plays a critical role in tissue repair and functional recovery. However, after a period of ischemia, reperfusion may exacerbate the injury initially caused by ischemia, producing a so-called "cerebral ischemia/reperfusion (I/R) injury". Multiple pathological processes are involved in ischemic neuronal damage, oxidative stress is attracted special attention.Salvia miltiorrhiza polyphenols salt (SMPS) are water-soluble extracts from Salvia miltiorrhiza, a traditional Chinese medicine known as Danshen, and magnesium lithospermate B (MLB) is the major ingredient of the extracts. It has been reported that MLB can prevent cerebral I/R injury, which is involved in its antioxidative activity. However, the exact mechanisms remain largely unknown.Potential sources of ROS in the body are produced by a variety of enzymes. Among them, NADPH oxidases (NOX) are considered as a major source of ROS production in the cardiovascular system. Our previous work have demonstrated that NOX2and NOX4, the subtypes of NADPH oxidases, are involved in mediation of cerebral I/R oxidative injury. It is not known, however, whether the protective effect of MLB on cerebral I/R oxidative injury is related to the inhibition of NOX.Based on the literatures as well as our preliminary data, this study is performed to explore the effect of SMPS and its major component MLB on cerebral I/R oxidative injury, and the underlying mechanisms.MethodsAnimal experiment, I/R model was established by occlusion of the middle cerebral artery (MCAO) in rats for2h followed by reperfusion for24h. For the study of the dose-effect relationship of SMPS, the male SD rats (250-300g) were randomly divided into8groups (n=8-14):the control group, the sham group, the I/R group, the I/R+SMPS (2mg/kg) group, I/R+SMPS (10mg/kg) group, I/R+SMPS (25mg/kg) group, I/R+SMPS (50mg/kg) group and the I/R+vehicle group. The infarct volume and neurological behavior was assessed by TTC staining and Bederson neuroscore, respectively. For the mechanisms exploration, the male SD rats were randomly divided into8groups (n=17-27):the control group, the sham group, the I/R group, the I/R+SMPS group, the I/R+MLB group, the I/R+edaravone group and the I/R+vehicle group. The infarct volume, neurological behavior, histopathological change and cellular apoptosis were assessed by TTC staining, Bederson neuroscore, HE staining and TUNEL assay, respectively. Expression of NOX2and NOX4were measured by Real-Time PCR and Western Blotting. The level of H2O2was determined by spectrophotometry kits. In the cell experiments, oxygen/glucose deprivation and reoxygenation model (OGD/R) was established by deprive the oxygen and glucose in cells for3h followed by reoxygenation for24h. The NG108-15cells were randomly divided into6groups:the control group, the OGD/R group, the OGD/R+MLB group, the OGD/R+edaravone group, the OGD/R+VAS2870group and the OGD/R+vehicle group. The cellular apoptosis were assessed by Hoechst stainning. The release of LDH in culture, the activity of NOX and the level of H2O2in the cell were determined by spectrophotometry kits.Results(1) Compared with control group, rats in the cerebral I/R group significantly displayed high mortality rate, defect in neurological behavior and cerebral infarction, these effects were attenuated by SMPS treatment at dose of lOmg/kg or above.(2) Compared with control group, rats in the cerebral I/R group showed vacuole in brain tissue, apoptosis and necrocytosis in neurocyte, pyknosis and lysis in cell, nucleolus vanishment, increase in gap and disarray in cell alinement. These phenomena were significant reversed by SMPS treatment.(3) Compared with control group, rats in the cerebral I/R group showed higher apoptosis ratio and Caspase-3activity in brain tissue. SMPS treatment significantly blocked the effects of cerebral I/R on apoptosis and Caspase-3activity.(4) Compared with control group, the mRNA and protein expressions of NOX2and NOX4were upregulated in the cerebral I/R group accompanied by increase in H2O2production, which was inhibited by SMPS.(5) Compared with control group, the apoptosis in nerve cells and the release of LDH was increased in the OGD/R group, which was attenuated in the presence of MLB.(6) Compared with control group, NOX activity and H2O2production in the OGD/R group was increased, which was reversed by MLB treatment.ConclusionsThe SMPS is able to protect brain from ischemia/reperfusion oxidative injury, which is related to the inhibition NOX2and NOX4expression and the reduction in oxidative stress.