Neuroprotective Effects And Mechanisms Of Angelica Sinensis Polysaccharides Combined With Tetramethylpyrazine On Cerebral Ischemia/Reperfusion Injury In Rats

Objective To get Angelica Sinensis polysaccharide(ASP), and isolated acid or neutral, molecular weight less or more than20KDa fragments, then test the antioxidant activity of different fragments, choose the fragments with antioxidant activity, and following research the compatibility of ASP and tetramethylpyrazine (TMP) to protective PC12cells damaged by hydrogen peroxide. Then established a middle cerebral artery occlusion (MCAO)/reperfusion rat model, to study the protective effect of ASP-on ischemic reperfusion rat, explore the possible mechanisms, and provide a theoretical basis of the clinical application of herbal extracts.Methods ASP was obtained by boiled-alcohol precipitation method, and separated less or more than20KDa by dextran gel column, or acidic and neutral fragments by anion exchange column; cultured PC12cells and interfere with H2O2to produced cell model occured oxide stress injury, testing the antioxidant activit of different ASP fragments, and get the fragment through ultrafiltration separation; research the anti-oxidative effect of different concentratons of ASP-TMP on PC12cells;84rats were randomly divided into control group (A), model group (B),25:10group (C),25:20group (D),50:10group (E),50:20group (F), sham group (G); A group does not perform any surgical treatment, B to F rats were established MCAO reperfusion model, G group were operated same to B group except unplugged suture, then C to F group was intraperitoneally injected different proportions of ASP and TMP, A, B and G groups were ingected the same volume of saline, injection began immediately after reperfusion, once a day, a total of seven times; Neurological severity scores (NSS)of rats were evaluated at the points4h,24h,3d and7d after reperfusion. six rats of each group were perfused with saline, then their bain was removed to examined the antioxidant kinase(SOD, GSH-PX)activity and the lipid peroxide content (MDA) around the infarct cortex, then to detect BDNF, Bcl-2/Bax expression by Western blot method; the other six rats of each group were decapitated after fixed using paraformaldehyde, then cerebral cortex were taken to detect CD31, Bcl-2/Bax expression with immunohistochemistry. Results The yield of totle ASP was4.72%, the frament which have antioxidative effect is a frament with molecular weight less than20Kda, and its yield was0.89%, the ASP protective PC12cells by preventing the accumulation of reactive oxygen species (ROS) in the cells, prevent the decline of mitochondrial membrane potential(MMP), then protect PC12cells from apoptosis; TMP did not showned any protective effect on PC12cells injured by H2O2, nor when ASP were used in the same time; The NSS of each group at4h,24h have no significant difference (P>0.05), otherwise, the score of all the groups received drug intervention were less than model group at3d and7d points (P<0.05); but there is no significant difference between any two examining groups (P>0.05); Findings of antioxidant kinase showned, in examing groups, the activity of SOD, GSH-PX were higher than control group, then the MDA content were lessthan the control group (P<0.05), comparing the dataes between two examing groups, we found that the activity of oxidative stress come from ASP but not TMP; The results of immunohistochemical detection showed, compared with control group (group A), the CD31positive cell of MCAO rats (group B) around the ischemic infarct cortex increased (P<0.05), then compared with group B, CD31positive cells of group C increased again (P <0.05), TMP or ASP increased alone, CD31positive cells further increased (P<0.05), However, no significant differences between the25mg ASP+20mg TMP/kg group (group D) and50mg ASP+10mg TMP/kg (group E)(P>0.05), compared with the C,D and E groups,50mg ASP+20mg TMP/Kg group (group F) have maximum CD31positive cells (P<0.05); Brain-derived neurotrophic factor (BDNF) expression was detected by Western blot, compared with the control group, the BDNF levels of treatment groups increased, and there presence synergistic effects of ASP and TMP; By immunohistochemistry and Western blot method, Bcl-2/Bax expression were detained, the ratio of Bcl-2/Bax of treatments groups raised compared with the control group, ASP and TMP showned synergistic effects again.Conclusions The fragments of ASP with molecular weight<20Kda have activity against oxidative stress, but TMP showed no the same activity;The motor function of MCAO rat can be partially self-healing, ASP and TMP intervention promoted their recovery, but there were not obvious differences between different concentration of drug; ASP and TMP could promote MCAO rats angiogenesis in cortex around ischemic core, and them shownd a synergistic effect; ASP, TMP could promote the expression of BDNF in cortex around ischemic core, and has synergistic effects; ASP and TMP could prtect MCAO rats apoptosis in cortex around ischemic core in MCAO rats, and has synergistic effects.