The Effect Of Thymosin Beta4on Angiogenesis In Rat Cerebral Cortex After Focal Cerebral Ischemia/Reperfusion And Mechanism

Ischemic cerebrovascular disease has a higher mortality andpermanent disability on a worldwide scale. The most common cause ishypertension. As statistics suggesting that about80%patients survivedstroke would leave sequelae, which brought a heavy burden for the societyand family.Researchers still make efforts to reduce the sequelae after ischemiccerebrovascular disease. After focal cerebral ischemia/reperfusion injury,inflmmtory meditor release into surrounding tissue, nerves and vesselswere damaged. So fast and early arterial recanalization and angiogenesis isa key to reduce anoxic damage of brain and reduce sequelae.Revascularization has three models, vasculogenesis, angiogenesis andarterial formation. In the past, people think mechanism of revascularizationin individual after birth is vasculogenesis. But the EPCs(endothelialprogenitor cells，EPCs) extracted from the blood, which making us realizethat a new mechanism of revascularization is angiogenesis. Angiogenesis is that EPCs which come from bone marrow proliferation, chemotaxis anddifferentiation to form new vessels.Studies have shown that eNOS secretion increased in focal cerebralischemia/reperfusion cerebral cortex, which can promote EPCsmobilization, transfer and homing. The eNOS is upstream of PI3K/Aktpathway which affect eNOS transcription and translation.Electroacupuncture (EA) is extensive used as a " non-drug " treatment. Ourteam previous studies have suggested that EA can improve Aktphosphorylation and eNOS expression, and promote angiogenesis in thebounding of infarct zone after focal cerebral ischemia/reperfusion.Studies have shown that vascular density increased in the bounding ofinfarct zone after intraperitoneal injection Tβ4, but the mechanism isunclear. In vitro Tβ4promote EPCs migration and angiogenesis byPI3K/Akt/eNOS pathway. Our study focuses on the effect of EA on Tβ4expression and PI3K/Akt/eNOS pathway after focal cerebralischemia/reperfusion rats. Whether intraperitoneal injection Tβ4peptidecan promote angiogenesis in the bounding of infarct zone and neurologicalrehabilitation after focal cerebral ischemia/reperfusion.Objective (1) To explore the effect of EA on the expression of Tβ4peptide in the cerebral cortex after focal cerebral ischemia/reperfusion,observe the target and mechanisms of EA promoting angiogenesis afterfocal cerebral ischemia/reperfusion. (2) At the same time, intraperitoneal injection Tβ4peptide for focalcerebral ischemia/reperfusion to observe the effect of angiogenesis andexplore the mechanisms.Methods (1) The male SD (Sprague-Dawley) rats were randomly dividedinto sham group (S), model group (IR), Electroacupuncture group (EA).The focal ischemia/reperfusion rat model was established by filamentoccluding the right middle cerebral artery for2hours. Rats were dividedinto1、2、3and7day subgroups in IR and EA group. EA stimulation"Baihui" point (GV20) and "Siguan " point (" hegu " point LI4+"Taichong " point LR3), frequency selection2/20HZ,20min, electriccurrent intensity1mA, once a day, the longest continuous stimulation7days. Pathological changes were observed by HE staining. Tβ4and eNOSmRNA expression were detected by reverse transcription-polymerase chainreaction(RT-PCR) in S group, IR group and EA group. Tβ4, eNOS andCD31protein expression were checked by immunohistochemistry in Sgroup, IR group and EA group. Akt phosphorylation level was detected byWestern blot in S group, IR group and EA group. Longe5points systemwas choosed to evaluate neurological function score after focal cerebralischemia/reperfusion.(2) Rats were randomly divided into sham group(S), model group(IR), Tβ4group(IRT), Tβ4+LY294002group(IRTL),0.9%sodium chloride injectiongroup(IRN). The focal ischemia/reperfusion rat model was established by filament occluding the right middle cerebral artery for2hours. Rats weredivided into3and7day subgroups in IR、IRN and IRT group. The proteinexpression of eNOS、p-Akt and Akt were detected by western blotting.Immunohistochemical method was selected to detect the expression of Tβ4and CD31. The mRNA expression of eNOS、SDF-1and CXCR4weretested by fluorogenic quantitative PCR. Longa score was selected toevaluate neurobehavioral.Result(1)RT-PCR ResultCompare with the S group, IR1day Tβ4mRNA expression increasedsignally(P <0.01), got the peak at second day and decrease at the third day(P <0.01); Compare with IR group, EA1day and EA2day Tβ4mRNAexpression did not change significantly, but EA3day Tβ4mRNAexpression was significantly higher. Compare with the EA3day, EA7dayTβ4mRNA decreased, but still significantly higher than the IR group(P<0.05).Compare with the S group, IR1day eNOS mRNA expression wassignificantly increased, and reached the peak at3day, decreasing at7day(P<0.01); compare with IR group, EA can significantly increased eNOSexpression, especially at3day, decreasing at7day (P<0.01)(2) Wester blot ResultCompare with the S group, Akt phosphorylation levels increased significantly at3day but decreasing at7day(P<0.01);compare with IRgroup, EA stimulation "Baihui" point and "siguan" point can promote Aktphosphorylation level, EA1day Akt phosphorylation levels increasedsignificantly, got peak at EA3day, decreasing at7day(P<0.01).(3) Immunohistochemical resultsA few Tβ4positive cells were expression in cerebral cortex,hippocampus, surrounding infarct zone. Compare with the S group，Tβ4positive cells increased significantly in cerebral cortex, hippocampus andsurrounding infarct zone in IR group(P<0.01). Tβ4is expressed mainly inglial cells, nerve cells.Tβ4expression were similar in EA group and IRgroup, but the amount of Tβ4positive cells increased significantly.Compare with S group, Tβ4protein expression was significantly increasedin IR1day, got the peak at2day and declined at3day (P <0.01); Comparewith IR group, Tβ4protein expression did not change significantly at EA1day and EA2day(P>0.05), but EA3day increased significantly, reducingat EA7day. Tβ4protein expression at EA7day significantly higher thanthe IR7day(P<0.05).eNOS protein expressed in nerve cells, vascular endothelial cells,glial cells in IR and EA group. A frew eNOS protein expressed in S group.The eNOS positive cells mainly located in ischemic cerebral cortex,hippocampus, periventricular tissue surrounding the brain. EA group andthe IR group expression position is similar, but the number of eNOS positive cells increased significantly in EA group(P<0.05). Compare withthe S group, eNOS protein expression was significantly increased at1dayin IR group, and got peaked at3day, declined at7day (P<0.01); comparewith IR group, eNOS protein expression increased at3day and7daysignificantly in EA group (P<0.01).A frew CD31positive micrangium were expression in S group.Compare with the S group, CD31positive micrangium density increasedsignificantly at3day, and most highest at7day in IR group (P<0.05);Compare with the IR group, CD31positive micrangium density increasedsignificantly at3day, especially at7day in EA group (P<0.05).(4) HE stain resultsS group basically can not see red necrotic neurons, but the IR groupand EA group showed a lot of red neuronal necrosis. a little inflammatorycells in S group, but a large number of inflammatory cells in IR and EAgroup. Compare with the IR group, the number of red necrotic neuron werelower than EA group and the inflammatory cells were also lower.(5) Neurological scoreEA could promote neural functional recovery significantly. Comparewith the IR2hour, the neurological score reduced little by little at1day,2day,3day and7day. Compare with IR group, the changes of neurologicalscore at EA1day, EA2day, EA3day were not significantly (P>0.05), butruduced significantly at7day(P<0.05). 2.1Q-PCR resultCompare with IRN, eNOS mRNA expression was increasedsignificantly at IRT3day, reduced at7day(P<0.05); SDF-1, CXCR4andeNOS mRNA has a parallel trends, which increased significantly at IRT3day, reduced7day (P<0.05).2.2Western blot resultWester blot showed that p-Akt expression less in S group than othergroups. In IRN group Akt phosphorylation levels were significantly higherthan S group. Compared with the S group, p-Akt expression increasedsignificantly at IRN3day (P<0.05), reduced at7day(P<0.05). Comparewith IRN group, IRT3day Akt phosphorylation levels increasedsignificantly, but still remained at a high level at7day(P<0.05); However,after blocking the PI3K/Akt pathway, Akt phosphorylation levels weresignificantly decrease in IRTL group, compare with IRN and IRT group(P<0.01).The expression of eNOS has the same trend with p-Akt. Comparedwith IRN group, The expression of eNOS significantly increased at IRT3day (P<0.01); compared with the IRT group, p-Akt and eNOS expressionsignificantly decreased in IRTL group(P<0.01).2.3Immunohistochemical resultA few Tβ4expressed in S group, mainly located in the hippocampus.A mount of Tβ4expressed in IRN group, mainly located in glial cells and neurons in the hippocampus and the bounding of infarct. In IRT group Tβ4expression was significantly higher than the IR group (P<0.05).A frew CD31positive micrangium were expression in S group. CD31positive micrangium significantly increased in IR group. Compare withIRN3day, vascular density is significantly increased than IRN7day(P<0.05). Compare with IRN group, CD31positive micrangiumincreased significantly at IRT3day, especially at7day in IRT group(P<0.05); The CD31positive micrangium significantly reduced in IRTLgroup (P<0.01).2.4HE stain resultS group basically can not see red necrotic neurons, but the IRN groupand IRTL group showed a lot of red neuronal necrosis. A littleinflammatory cells in S group, but a large number of inflammatory cells inIRN and IRTL group. Compare with the IRN and IRTL group, the numberof red necrotic neuron were lower in IRT group and the inflammatory cellswere also lower(P<0.05).2.5Neurological scoreTβ4could promote neural functional recovery significantly. Comparewith the IRN2hour, the neurological score reduced little by little at3dayand7day in IRT group. Compare with IRN group, the changes ofneurological score at IRT3day were not significantly (P>0.05), butruduced significantly at7day(P<0.05). Conclusion （1）EA may promote the expression of endogenous Tβ4incerebral cortex after focal cerebral ischemia/reperfusion, promote theexpression of p-Akt and eNOS, promote angiogenesis and neurologicalrehabilitation.(2) Intraperitoneal injection exogenous Tβ4peptide for focal cerebralischemia/reperfusion rats, which may promote promote angiogenesis byup-regulation p-Akt and eNOS expression and promote neurologicalrehabilitation.