Study On The Regulatory Mechanism Of Tibetan Medicine Baimai Therapy On Limb Recovery In Ischemic Stroke Rats

ObjectiveInternational general middle cerebral artery occlusion(MCAO)method was adopted to establish the model of focal cerebral ischemia rats.With Notch signaling pathway as the core,we explored curative effect mechanism of Tibetan Baimai therapy(BT)for the treatment of ischemic stroke(BT,in this research,is the therapy oral Ruyizhenbao pills combined with painting Baimai ointment on affected limb),using molecular biology techniques,such as PCR,Western blot detection related indicators,statistical methods and the Tibetan medicine theory.As well as we hope to provide the basis for clarifying adjustment mechanism of BT helping rat,with ischemic stroke,recover limb function.Methods(1)Model formation and grouping:Referencing line embolism method reported by Belayev and improving it,we established the right middle cerebral artery occlusion(MCAO)model.We did grade according to Longa scoring method after modeling,pointed out the rats of score of 0 and 4,chose the rats of score of 1-3 in this study,then applied random table divide each score of rats into six groups:Sham group(S),model group(M),positive control group(P),Ruyizhenbao pills group(R),Baimai ointment group(BO),Baimai therapy group(BT),each group is divided into two subgroups based on ischemia reperfusion time,namely,7th day group and 14th day group.Each day each group rats were gavaged every morning,and ointments were applied on the left limb.Positive group rats were gavaged simvastatin solution,groups of R and BT were gavaged Ruyizhenbao pills,the rest groups with physiological saline;groups of BO and BT apply Baimai ointment,the rest of the group are smearing vaseline.(2)Indicators detection:?Conventional detection:We observed and recorded fur status of each group rats every day.?The brain physiological pathology change:We observed the condition of inflammatory cells infiltration,neuronal nuclear pyknosis and softening oven in cortex,ischemic penumbra and the hippocampus by HE staining in microscope cortex;We observed nissl body changes of cortex neurons by Nissl's staining.?Behavioral detection:After operation 3,7 and 14 days,we did ethology score of each group rats,the concrete content includes:posture reflex test and limb placement test.?Nerve regeneration and angiogenesis:We used immunohistochemistry detect the expression of GAP-43 and VEGF1.?Notch signaling pathways:Using immunohistochemistry.Western Blot technology,Real-time PCR technology from the level of protein and gene,we detected the dynamic change of Hes1,and Jagged1.Results(1)The conventional detection:the differences of each group rats' fur emerged with the increase of ischemia reperfusion time,the differences emerged on 7th day and significantly on 14th day:BO group was similar to the sham,their colour is normal,the other groups appeared yellow status in different degree,model group was the wors t;(2)Behavior detection:The two kinds of behavior score both showed the limb function state of BT group rats were better than the model group with significant difference(P<0.05),compared with the model group in two time points of 7th and 14th day and two kinds of behavior score showed the limb function of BT group rats in 14th day better than 7th day.(3)The brain physiological and pathological effects:The condition of inflammatory cells infiltration,neuronal nuclear pyknosis and softening oven in cortex,ischemic penumbra and the hippocampus,by HE staining,showed that BT group was lower than model group.The difference between BT group and model group was further increased;Nissl's staining showed the number of nissl body of BT group was higher than model group on 7th and 14th day.(4)Nerve regeneration and angiogenesis:the results showed that the GAP-43 and VEGF1 protein expression of BT group rats was siguificantly higher thar that of model group(P<0.05)on 7th and 14th day,and two classes of protein expression on 14th day was slightly less than 7th day.(5)Notch signaling pathways:on 7th day,BT can inhibit Hest protein expression,promote the Jagged1 protein expression,and there was significant difference compared with the sham(P<0.05).On 14th day,the degree of BT group inhibiting Hes1 protein expression level was reduced,and there was no significant difference between the sham,at the same time BT promoted the Jaggedl protein expression,but the expression was less than that of 7th day significantly.ConclusionTibetan "white pulse therapy" could improve the rats' limb function after they were ischemia reperfusion.The mechanism is probably the treatment can promote the expression of GAP 43 and VEGF1,at the same time regulate Notch signaling pathway by inhibiting Hes1 protein expression,promote the Jagged 1 protein expression,to promote the regeneration of the nerves and blood vessels,to reduce the extent of the neurons damage after cerebral ischemia,thereby reducing cerebral infarction area,and then improving the ischemic rat limb function.Its mechanism need further discussion,also need more study.