Effect And Mechanism Of Salvianolic Acid A On Cerebral Microvascular Embolism In Rats

In the sixth nationwide population census of china, aging of population sound the alarm for people. Human have already and will suffer more from cerebrovascular disease and dementia. Lacunar infarction was a kind of cerebral microvascular disease, constituted approximately 25% of first in-a-lifetime ischemic stroke. It is one of the most common type of stoke. Patients with lacunar infarction do not usually present with neuropsychological abnormalities or cognitive impairment during the acute phase of stroke, but easy to recurrence, and repeated relapse could lead to dementia. Current treatments only can slow or delay progression of symptoms. So research of prevention and cure strategies are of the utmost importance for lacunar infarction.Salvianolic acid A (Sal A) is the major water-soluble active constituent of traditional Chinese herb, salvia miltiorrhiza. Sal A has many pharmacological effects, such as stronganti-oxidation, protective effect against brain and heart injury, therapeutic effects on hepatic injury, hepatic fibrosis and anti-tumor. Previous study reported the Sal A ameliorated the cerebral ischemia/reperfusion injury, improved the cognitive impairment induced by ischemia/reperfusion, and exerted antiamnesic effect on diazepam-induced anterograde amnesia in mice. The thromboembolic model is of great interest to stroke researchers because of its close resemblance to human ischemic stroke. In this project, according to the previous research cerebral microvascular embolic model induced by injection of emboli was made and the effect and mechanisms of Sal A on it was investigated.Chapter I Effect of different size of exogenous emboli on the cerebral injury and cognitive impairment in ratsThe cerebral microvascular embolic disease was induced by injection of three different size of emboli through the right internal carotid artery of a rat. This study was to present the effect of different size of homologues exogenous emboli on the cerebral injury and cognitive function in rats.1. The rCBF was significantly decreased fewer than 30% at once after three sizes of emboli (150μm-178μm,74μm-124μm and 48μm-74μm) injection. Then, the rCBF has been increasing at various degrees. At 1 h, the rCBF was up to 37.7%,40.2% and 60.2%, respectively. Injection of different size of blood emboli in rats also significantly increased the neurological deficit scores to 2.55,1.75 and 1.67 at 24 h after emboli injection, respectively. These results suggested that the bigger of the emboli injected, the more decrease of regional cerebral blood flow (rCBF) and more increase of neurological deficit scores induced. Basically, the decrease of rCBF and increase of nerurological deficit scores had positive correlation to the size of blood emboli. HE staining revealed that HE staining revealed that The injured areas of CA1 and CA2-3 in rats of group S were larger and more uniform than those of group L and M that were relative smaller and had more disparities, all the brain cortexes in the three model groups were damaged severely.2. In Morris water maze test we found that rats of group S and L reached the platform with longer latency, adopted a spatial strategy different from those of group normal (group N) and normal saline (group NS). Moreover, they took more time and swam longer in the goal quadrant although they swam almost the same total distance as the rats of group N and NS in 120 seconds. The rats injected with emboli of 74μm-124μm in diameter (group M) also had cognitive impairment but with no significant difference compared to NS group. It is related to the larger and more uniform injured area of hippocampus especially CA1 in rats.Combined the result of 24 h and 2 months, we can concluded that the emboli of 48μm-74μm could lead to a larger and more uniform injured area in hippocampus CA1 in rats, and cause significantly cognitive impairment. So dose the emboli of 150μm-178μm. The microvasculature is less than 100μm diameter. Therefore, we choose the emboli of 48μm-74μm to induce cerebral microvascular embolic disease.ChapterⅡProtective effect of Sal A on cerebral microvascular embolic model induced by injection of microemboliEffect of Sal A was investigated on cerebral microvascular embolic disease was induced by microemboli of 48μm-74μm.1. In the cerebral microvascular embolic model, pretreatment of Sal A could significantly decrease the increase of neurological deficit scores and cerebral MDA level, and at the same time increase the level of GSH-Px, NO and tNOS. And at last the injured blood-brain barrier (BBB) and neuron was recovered in some degree. The results were also showed that the ability of scavenging free radical of Sal A can compared to the free radical scavenger-edaravone.2. Hemodynamics assay showed that pretreatment of SAA could significantly decrease the whole viscosity, plasma viscosity and erythrocytes aggregation, as well as inhibit platelet aggregation induced by adenosine diphosphate (ADP). However, the effect of Sal A on APTT, PT, FIB and TT was poor. The results were also showed that the ability of inhibiting platelet aggregation of Sal A can compared to aspirin.ChapterⅢEffect of Sal A on the ability of learning and memory of rat with cerebral microvascular embolic disease induced by injection of microemboliMorris water maze was employed to investigate the effect of Sal A on the ability of learning and memory of rats with cerebral microvascular embolic model. Changes of cholinergic system, nerve growth factor and soluble epoxide hydrolase were observed by immunohistochemistry after treated with Sal A. The results showed that:1. Treated with Sal A for 2 months could significantly decrease the escape latency, increase the number of crossings over the former platform location and the distance in goal quadrant of rats in probe trial performance at 2 months after injection of microemboli. Therefore, administration of Sal A could improve the ability of learning and memory of rats with cerebral microvascular embolic model for 2 months.2. Treated with Sal A for 2 months could significantly decrease the cerebral tissue's ROS level of rats with cerebral microvascular embolic model.3. Results of MRI and HE staining showed that treated with Sal A for 5 months could significantly decrease the brain infarction of rats with cerebral microvascular embolic model.4. In vitro, results showed that Sal A was a weak inhibitor of acetylcholine esterase (AChE) and has slightly higher inhibited activity on butyrocholinesterase (BuChE). In vivo results showed that treated with Sal A for 2 months could significantly decrease the serum and cerebral tissue's AChE level of rats with cerebral microvascular embolic model.5. Compared to rats of group normal, the expression of choline acetyl transferase (ChAT) in CA1 of hippocampus has no changes in rats with cerebral microvascular embolic model. However, after treated with Sal A for 2 months the expression of ChAT was significantly increased in CA1 of hippocampus.6. Compared to rats of group normal, the expression of brain derived neurophic factor (BDNF) and vascular endothelial growth factor (VEGF) in CA1 of hippocampus were significantly decrease in rats with cerebral microvascular embolic model. However, after treated with Sal A for 2 months the expression of BDNF and VEGF was markedly increased in CA1 of hippocampus.7. In vitro, Sal A is a better inhibitor of soluble epoxide hydrolase (sEH). IC50 is 7.78μg/ml. In vivo, Sal A could markedly decrease the increase of sEH in CA1 of hippocampus in rats of group model.In summary, Administrated Sal A for 2 or 5 months could decrease the brain infarction, the brain tissue ROS level, recovered the injured brain tissue, and improve the learning and memory capability of rats with cerebral microvascular embolic model. Improving the function of cholinergic system, enhancing the expression of BDNF and VEGF and decreasing the activity of sEH may be all involved in the protective effect of Sal A.Chapter IV Protective effect of Sal A on SH-SY5Y and mechanism researchSal A showed obvious protective effect on the brain tissue in rat with cerebral microvascular embolic model. In the chapter, effect of Sal A on SH-SY5Y and related mechanism was observed.1. In the correction experiment, Sal A, as a strong reductant, at the concentration of 10-5 M could lead to abnormal increase of OD value when determined by MTS; and our results also suggested that 10-5 M Sal A could decrease the viability of SH-SY5Y. Therefore,10-6 M-10-9 M Sal A was chose for cell level experiment.2. Sal A showed obvious protective effect on the cell viability of SH-SY5Y and rat primary cultured cortical and hippocampus neurons subjected to H2O2 for 4 h.10-8 M Sal A showed the best effect, while 10-6 M Sal A showed lowest.3. Sal A showed obvious protective effect on the cell viability of SH-SY5Y subjected to H2O2 for 24 h. Sal A could increase the ATP content and mitochondrial membrane potential, diminish apoptosis and promote cell survival in SH-SY5Y subjected to 200μM H2O2 for 24 h. And 10-8 M Sal A also showed the best effect, while 10-6 M Sal A showed lowest.4. Results of Western Blot suggested that the AMPK was activated subjected to 200μM H2O2 for 5 min, and pretreated with Sal A could inhibit the AMPK activation.10-9 M Sal A showed the best inhibited effect.5. Results of Western Blot suggested that the Akt was activated subjected to 200μM H2O2 for 5 min, and pretreated with Sal A could inhibit the Akt activation.10-9 M Sal A also showed the best inhibited effect.6. Results of Western Blot suggested that the ERK1/2 was inhibited subjected to 200μM H2O2 for 5 min, and pretreated with Sal A could significantly activate the ERK1/2.10-8 M Sal A showed the best effect.In summary, Sal A possessed protective effect by increasing ATP content, mitochondrial membrane potential, diminishing cell apoptosis and promoting cell survival in SH-SY5Y subjected toμM H2O2. It may be related to the inhibition of Sal A on AMPK and Akt, and the activation of ERK1/2.Gather up the content of second, third and forth chapters, we can conclude that Sal A mainly prevents and treats cerebral microvascular embolic model as follows:1. Antioxidation ability to enhance and restore the body oxidant/antioxidant balance in steady-state system2. Regulating the function of cholinergic system3. Regulating signal pathway of energy metabolism4. Improving the hemodynamics, resisting platelet aggregation5. Inhibition of sEH6. Increase the expression of nerve growth factor.