Protective Effects Of Gross Saponins Of Tribulus Terrestris On Cerebral Ischemia-reperfusion Damage

Cerebrovascular disease is very common and frequent, which severely threatens human health. Recently, cerebrovascular disease has become one of the three leading cause of death. Ischemic cerebrovascular disease has become the focus on preventing and treating cerebrovascular disease because of high mobidity, mortality and Mutilation rate. Thrombolysis is the main therapy method, but there are some dangers and restrictions for reperfusion damage. Consequently, to investigate the pathophysiology mecha- nism of the cerebral ischemia-reperfusion damage (CIRD) and develop the protective drugs for the CIRD have the important theoretical significance and clinical application value.Tribulus terrestris refers to the dry, ripe fruit of the natural herb caltrop. The main chemical compounds contained in Tribulus terrestris are saponins, ketone, alkaloid, et al. The gross saponins of Tribulus terrestris (GSTT) is one of the active principles which were extracted from the whole herb of caltrop, mainly includes furostanol and spirostanol. It is yellow powder and dissolvable in water. Research materials showed that GSTT has the effects of lowering blood pressure, diastolling blood vessel and inhibiting atherosclerosis occurrence and development. It has also anti-aging property and invigorating sexual function. Capsuled praeparatum of GSTT has been utilized for the diseases of cordis and cerebral vascular system generally in clinical treatment with curative effect obviously. We previously reported that GSTT has the effects of anti-thrombotic formation, ameliorating the blood viscosity, anti-myocardial ischemia and cerebral ischemia and protecting brain after intracerebral hemorrhage. However, how GSTT protects nerve cells against damage during CIRD and what is the mechanism need further investigate.To research the protective effect of GSTT in CIRD and its possible mechanism on the level of body and cell through setting up nerve cells ischemia-reperfusion model in vivo and in vitro, using the technique of pathology, biochemistry and molecular biology. These can provide theoretical basis for better, safer using and targeting application of this therapy in clinical treatment.Experiment in vivo: through setting up cerebral ischemia-reperfusion model in rats by blocking middle cerebral artery (MCA), the effects of GSTT on score of neurological deficit and infarction area in brain tissue were observed. The effect of GSTT on the pathomorphology of brain tissue with HE staining was observed. The effect of GSTT on ATPase activity in brain tissue was determined. The results of the experiment in vivo showed that after CIR, model rats had obvious behavior deficits, such as flexing or adducting the left limb, rotating to the left side when crawling, or even no crawling. GSTT (30,10mg/kg) reduced neurological deficit score obviously (P<0.001,P<0.05) and improved behavior deficits in MCAO rats. The brain tissue supplied by MCA becomes an infarction area after blocking it. GSTT (30,10mg/kg) could reduce infarction area (P<0.001,P<0.05) and relieve the further damage in brain tissue. CIRD leads to the changes of nerve cell morphology, including cell shrinking, endochylema stained deeply, nucleus contracted, or cell swelling and cellular structure unclear. GSTT (30,10,3mg/kg) could relieve brain tissue damage and protect cell normal morphology. The study of ATPase activity in brain tissue showed that GSTT could up-regulate ATPase activity (P<0.01,P<0.05), balance the ion concentration between cell exterior and interior, and protect the integrity of cell.Experiment in vitro: cultured PC12 cells, induced PC12 cells apoptosis model by H2O2. The morphology changes of cell were observed through invert microscope. The morphology changes of nucleus were observed through Fluophot. DNA ladder band was observed through agarose gel electro-phoresis. SOD activity and MDA content in medium were detected. The mitochondrial activity was analyzed using MTT assay. The cell apoptosis rate and mitochondrial membrane potential were detected by flow cytometry. PC12 cells with H2O2-induced oxidative-stress damage were observed that refractive index lowered, cell swelled and adherence cell quantity reduced, nucleus was stained deeply and concentrated, typical DNA ladder bands was observed. The results of the experiment showed that GSTT could protect the morphological integrity of PC12 cells induced by H2O2, reduce the quantity of nuclei stained deeply and dense, and there were no typical DNA ladder bands. Meanwhile, GSTT could increase SOD activity (P<0.001,P<0.01,P<0.05), lower MDA content (P<0.001, P<0.01) in medium , inhibit dose-dependently lipid peroxidation and reduce free radicals on nerve cells damage. Different GSTT dose groups could inhibit H2O2-induced apoptosis in PC12 cells more or less, and lower the rate of apoptosis significantly (P<0.001,P<0.01,P<0.05). GSTT (30,10mg/L) could maintain mitochondrial activity (P<0.001,P<0.01). GSTT (30,10,3mg/L) increased△Ψm (P<0.001,P<0.05) and positively correlated with apoptosis rate. The experiment showed that GSTT could inhibit the occurrence of apoptosis by protecting the integrity of mitochondria, maintaining energy metabolism, and improve environment inside the cell.Based on the above experiments in vivo and in vitro, it has been proved that GSTT restored ATPase activity of nerve cells, maintained the structure integrity of nerve cells, and reduced the infarction area after CIR and ischemic neurological dysfunction. Meanwhile, GSTT increased the activity of SOD and reduced the content of MDA under oxidative stress, reduced lipid peroxide- tion damage, stabilized△Ψm, maintained mitochondrial activity, improve cellular energy metabolism, and inhibited cell apoptosis.