Baicalin Protect Micovasular Endothelial Cell From Apoptosis And Necrosis Induced By Oxygen-Glucose Deprivation

With the aging process accelerated, the risk of prevalence of senile dementia rise sharply in our life. Because of an large population in our country, the senile dementia has become an important public health issue. Senile dementia is mainly divided into three types:one is Alzheimer’s diseases which is abbreviated to AD and Vascular dementia which is abbreviated to VD and mixed dementia. Nowdays, the number of patients with senile dementia grows rapidly and worldwidely. Vascular dementia is about 20% of the total dementia, and in Asia the number of vascular dementia patients out numbered the Alzheimer’s disease patients. The 5 years survival life of patients who suffer from vascular dementia the one of is much lower than patients who suffer from the Alzheimer disease. At the same time, with the aging process accelerated, cardiovascular and cerebrovascular disease have become the first risk factor to the public health issue, Thus it is very important to carry out studies on vascular dementia. For quit a long time, plenty of scientists have been engeged in the exploration of the mechanism of the disease to establish treatments and preventions. Although gained some progress, it is still lack of effective medications or measures for the control of the disease.Radix scutellariae is an ancient traditional Chinese medicine, applied for detoxification and stanching bleeding. Modern pharmacology studies show that radix scutellariae involves functions of antibacterial, antiviral, anti-inflammatory, antioxidative and other pharmacological effects. Baicalin, a flavonoids extracted from scutellaria root, is one of the most effective components. Several studies show that baicalin has antioxidative and anti-inflammatory effects, which can improve myocardial ischemia damage.ObjectiveTo simulate pathological damage of cerebral ischemia anoxic, model of OGD is established by depriving sugar and oxygen from mice microvascular endothelial cells (bEnd.3 cell). This study aimed to expound the protective effects of baicalin on bEnd.3 cell against apoptosis and necrosis casued by OGD.Methods1. The cultures were fist wash 2 times with PBS. Cover the cells with appropriate amount of serum and glucose-free DMEM. The cultures were immediately transferred to chamber filled with atmosphere air for 30 min and were put into the incubator.2. Evaluate the effects of baicalin on OGD induced cell damages by MTT and LDH assay.3. Examine the effect of baicalin on microvasular endothelial cell treated with OGD by the FCM.4. The impact of baicalin on OGD induced bEnd.3 cell morphology change observed by the Transmission electron microscope and Hoechst 33258/PI double staining.5. The baicalin with antioxidative effect on OGD induced cells damage by ROS kit.Results1. Incuded by OGD for 2,4 and 6 h, the bEnd.3 cells survival percentage respectively (x±s):62.31±2.91,48.32±0.49、36.95±6.46. Compared to the control group, the cell survival rate of the group of model is lower (p<0.05).2. The protection of Baicalin on OGD induced bEnd.3 cell damageThe effect of baicalin on OGD-induced bEnd.3 cell damage during the 2, 4 and 6 h. At three time points, compered to controlgroup, cells survival ratio of OGD group was lower (p<0.05). Compared to OGD group, the groups of baicalin (100 μM and 200 μM), obvious improved cell proliferation (p<0.05). Compared to OGD control group, LDH supernatant fluid content increased in OGD group (p<0.05), baicalin groups decreased, the LDH leakage as compared to OGD group (p<0.05). This effect of baicalin was up to the most significant level at 6 h (p<0.05).3. Antiapoptotic effect of baicalin:compared to control group, the ratio of Annexin V (+/-)/PI (+) of OGD group was significantly increased, which indicated advaned apoptosis and necrosis(p<0.05). On the contrary, Annexin V(+/-)/PI (+) ratio in baicalin group was significantly decreased, has which indicated the reduced apoptosis and necrosis (p<0.05).4. The Hoechst 33258/PI double staining results:compared to control group, OGD-treated cekks showed intense fluorescence of these two dyes which suggested that the necrosis and advanced apoptosis were increased by OGD for 6 h in bEnd.3 cells. Results showed that baicalin eliminated cell necrosis and advanced apoptosis remarkedly.5. The transmission electron microscopy result showed that compared to control group cell wlar vacuoles gradually increase in OGD group, apoptosis was observed in cells treated by OGD for 2 and 4 h. After treatment of OGD for 6 h, the cells showed that cytoplasm, nucler fragmentation and bursting of cell membranes. OGD baicalin treated cells induced by OGD for 6 h showed integrate cellular morphology suggesting that baicalin attenuated cell necrosis.6. The intracellular ROS content of OGD at 6 h, compared to contol group, ROS level significantly increased in OGD group, while the group of baicalin reduced the genertion of ROS induced by OGD treatment.Conclusions1. OGD led to severe and time-dependent damage in bEnd.3 cells, indicating by dramatically declined cell proliferation ratio. Thus the stability of this model is considered to meet the requirements of experiment.2. Baicalin significantly reduced OGD induced damage. According to LDH assay, the protective effect of baicalin reached the most significant level at 6 h of OGD treatment.3. Baicalin remarkedly decreased cellular apoptosis and necrosis, which induced by OGD.4. Baicalin maintained the stability of celluler physiological structure which was broke down under OGD condition.5. Baicalin eliminated ROS, suggesting an antioxidative effect.Baicalin reduced OGD-induced generation of ROS in bEnd.3 cells, and thus attenuated cell damage. Meanwhile, baicalin maintained the stability of cellular physiological structure, such as reserving the disrupted membrane, integrating the fragmented nuclear and relieving the swollen organells. Results above suggested that baicalin protected cells against apoptosis and necrosis. Based on the following experiment, we considened the mechanism of its effects as ROS scaveuging. In addition, the survival of the cells need energy supply. Under the condition of OGD, cells are in short of energy. Besides, apoptotic cascade reactions require a lot of energy. Combined OGD all the results above, it is concluded that the protective effects of baicalin on OGD-induced damages might involved key protein in necrosis and metabolism, which needs further investigation.