| Objective: Cerebrovascular disease is a very serious health and social problem. Owing to the complex mechanisms and the lack of effective treatment, ischemic cerebrovascular disease is one of the most urgent problems at present.Focal cerebral ischemia, infarct secondary mechanism of neuronal death mediated by oxidative stress remains to be further studied. Autophagy under ischemia situation is a double sword: moderate autophagy can clear damaged organelles and abnormal folded proteins which is protective role, but incompletely or excessive autophagy may lead to cell death. Recently, more and more experimental evidence that oxidative stress can induce autophagy, and the application of antioxidants can inhibit autophagy and reduce cell death.Sal B is a water-soluble component extracted from Salvia, Sal B can protect myocardial ischemia by inhibiting apoptosis, and it is also reported that in the Sal B can inhibit autophagy to protect the fasted cardiomyocytes.This experiment is designed to study the mechanism of Sal B on focal cerebral ischemia.Methods: Male CD1 mice(weight 25-30g) were subjected to a modified Longa suture p MCAO procedure to establish mouse model on the right side. Experiment was divided into three parts:Experiment 1: neuroprotective effect of Sal B on acute focal cerebral ischemia in miceNeuroprotective effects of acute Sal B after focal cerebral ischemia clear. Mice were randomly divided into four groups: Sham group(Sham + saline), p MCAO group(p MCAO + saline), Sal B low-dose group(p MCAO + Salvianolic acid B 10 mg / kg, SAL-L), Sal B high-dose group(p MCAO + Salvianolic acid B 25 mg / kg, SAL-H). The mice were anesthetized in accordance with uniform standards. Right after p MCAO procedure, drug intervention group was injected intraperitoneally with Sal B solution, Sham group and p MCAO group was given saline. 24 h after surgery, mice were neurological scored(Longa law and Clark Law)(n=15). And brain water content was measured using wet and dry weight method, infarct volume was measured by TTC staining, BBB permeability was measured by Evans blue extravasation, Claudin5 expression was measured by western blot.The infarct border zone neuronal injury was mesuared by Nissl staining and electronic microscopy(ultrastructural changes including the formation of autophagy, apoptosis, microvascular injury and so on).Experiment 2: Expressing patterns of numerous factors in focal cerebral ischemiaSham group and p MCAO group were measued immunohistochemistry positive cells of LC3, beclin1, p62, bcl2, bax, cleaved Caspase-3. And PGC-1α-Sirt1 expression was measured by western blot in different sections(core, pericore, contralateral) of the coretex.Experiment 3: Exploring neuroprotective mechanism of Sal BFirst, according to the expression pattern illustrated by histochemisty result. LC3, beclin1, p62 protein expression and m RNA content was observed in the pericore section, while bcl2, bax and Caspase-3 were observe in the central area of infarction. RT-q PCR and western blot were measured in Sham group, p MCAO group, Sal B low-dose group and Sal B.Corresponding to different sectional expression patterns, apoptosis related signaling pathways(MAPKs) and energy metabolism pathways(PGC-1α-Sirt1) were detected by western blot and RT-q PCR. Determination of the amount of infarcted cortex generation of ROS are conducted by DHE staining, MDA level by kit and SOD activity by kit.In short, apoptosis, autophagy and oxidative stress are respectively investigated to explore Sal B neuroprotective mechanism in a sectional manner.Results:1 Sal B protected acute focal brain ischemic mice.Sal B improve behavioral score. When using Longa score and Clark score, compared with p MCAO group, Sal-L group and Sal-H group significantly improved neurological function, and the difference was statistically significant(P<0.01). Sal B reduced infarct volume. Infarct volume of Sal-L group and Sal-H group was significantly reduced, and the difference was statistically significant(Sal-L group and Sal-H group vs. p MCAO group:51.09%±12.65% and 34.31%±7.93% vs. 65.94%±2.96%, P<0.05).Sal B protective blood-brain barrier. Sal B could alleviate cerebral edema, Sal-L group and Sal-H group infarcted brain tissue water content was significantly reduced compared with p MCAO group. Evans blue extravasation result indicted Sal-H group can reduce permeability of BBB, this may owing to upregulated expression of Claudin5. Claudin5 protein expression was increased in Sal B intervened groups. Sal B could also protect blood-brain barrier ultrastructure.Sal B reduced brain cell damage. Sal B increased the number of surviving cells by Nissl staining. There are more viable cells in Sal-L group and Sal-H group compared with p MCAO group, and the difference was statistically significant. Sal B improve tissue ultrastructure. Autophagy and apoptosis can be detected after focal cerebral ischemia.2 Factor expression in different regions of cerebral ischemia differedImmunohistochemistry showed the expression pattern of apoptosis related factors: In p MCAO group Caspase-3, bax positive cells were detected mainly in core section. bcl2 expression were reduced in this secton, however, upregualted in the pericore section. In contralateral section, each factor expression(positive cell number) had no significant difference with sham group; Caspase-3 and bax expression was significantly increased and the difference was statistically significant(Core vs. Sham, P<0.05, Fig.6b, C, G), bcl2 expression was significantly reduced and the difference was statistically significant(Core vs. Sham, P<0.05, Fig.6b, K); In pericore group, Caspase-3 and bax expression has been raised and was statistically significant(P<0.05, Fig.6b, A, E) but the magnitude is much smaller than the center of cortical infarction, bcl2 upregulation and increased the number of positive cells, the difference was statistically significant( P<0.05, Fig.6b, I).In p MCAO group, almost no positive cell of autophagy related protein can be detected in core group which is a significant difference versus Sham group. In contralateral group, there was no significant change in the level of autophagy compared with the sham group. Pericore section’s expression of LC3, beclin1 and p62 was significantly increased.Immunofluorescence result revealed that autophagy related factor-beclin1 and apoptosis related factor caspase-3 had no co-mark.3 Exploring Sal B neuroprotective mechanismAntioxidant effects of Sal B were confirmd by DHE staining showing reduction of DHE flourscence(ROS amout) and a significant decrease in MDA content. Also, Sal B restored the activity of SOD.Anti-apoptotic effects of Sal B: western blot showed Cleaved Caspase-3 / GADPH and bax / bcl2 ratio was significantly increased compared with Sham group. Comparision of Sal-L group and Sal-H group with p MCAO group revealed that the ratio of Cleaved Caspase-3 / GADPH and bax / bcl2 significantly reduced. RT-q PCR results showed Sal-L group and Sal-H group’s Caspase-3 and bax gene expression was decreased and there was statistically significance.Sal B inhibited autophagy. Autophagy-related protein expression level indicators include LC3 II / GADPH, beclin1 / GADPH and p62 / GADPH, which is correspond to the level of gene expression including MAP1LC3 b / ACTB, BECN1 / ACTB and SQSTM1 / ACTB. Compared with Sham group, the expression of factors above were significantly increased in p MCAO group, consistent with the histochemistry results. Low doses of Sal B intervention can significantly reduce the expression of various indicators, slightly less than the effect of high doses.And the expression of m RNA of Sal-H and Sal-L groups also decreased in consistence with western blot results. Sal B downregulated p-p38 and p-erk1 / 2 expression in core section and raised Sirt1-PGC-1α pathway expression in pericore section.Conclusion: Sal B has cerebralprotective effect on acute focal cerebral ischemia in a mice model of stroke. It can improve the neurological deficit scores, reduce infarct volume, and it can protect BBB ultrastructure. Sal B can also increases the number of Nissl staining viable cells.Immunohistochemistry showed different distribution pattern of autophagy and apotosis related proteins. Autophagy-related proteins mainly up-regualted in pericore section(such as LC3 and beclin1), and apoptosis-related proteins the center of cortical infarction(such as cleaved Caspase-3 and bax raised, bcl2 downregulated). Sal B can inhibit excessive activation of autophagy in pericore section and aptotosis in core section. Sirt1-PGC-1α activation is also evident in pericore section, which may related to the alleviation of excessive activation of autopagy.The anti-apoptotic effect may be through downregulation of p-erk1 / 2 and p-p38 protein expression. |
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