| The treatment of ischemic stroke is a major problem in the current society,and morbidity of ischemic stroke in China is significantly higher than the world level,which seriously affects the lifetime and quality of life of our residents.Cerebral ischemia-reperfusion injury is one of the most critical factors that influence the prognosis of ischemic stroke,including a variety of pathophysiological mechanisms such as oxidative stress,apoptosis,neuroinflammation outbreak,lipid peroxidation and so on.Therefore,we believe that alleviating the severity of ischemia reperfusion injury can be regarded as an important aim to improve the prognosis of ischemic stroke.PI3K/Akt pathway can mediate cell survival,with the effect of anti-apoptosis and anti-oxidative stress.During the cerebral ischemia-reperfusion injury,PI3K/Akt pathway was found to be suppressed.Several investigations have revealed that both cerebral infarct volume and Longa neuroethology score of rats were significantly reduced when PI3K/Akt was activated.LY294002 is the specific inhibitor of PI3 K,and is used widely as the inhibitor of PI3K/Akt pathway.By using PI3 K specific inhibitor LY294002,with the methods of TUNEL apoptotic staining and western blot,we chose middle cerebral artery occlusion(MCAO)model to explore the role of PI3K/Akt pathway in cerebral ischemia-reperfusion injury;Besides,by using PCR Array,we detected genes expressions related to oxditive stress after MCAO and selected four genes(DHCR24,HO-1,Fth1,Nos2)connected with cerebral ischemia-reperfusion injury closely for the following part.In additoin to the widely study of Seladin-1 in lipid and Alzheimer’s Disease,recently,it has also been found to be associated with cerebral ischemia and has a protective role.By reading other researches,we found at DNA level,we would like to call it DHCR24,while at protein level both are correct.Known as an inducible antioxidase,HO-1 plays an antioxidant effect.Under physiological conditions,HO-1’s expression is rather low.After ischemic stress,the expression elevates quickly.Many studies reported that Seladin-1 and HO-1 could be regulated by PI3K/Akt pathway.H-Ferritin,encoded by Fth1,can combine with free iron and alleviate oxidative stress injury induced by free iron.Nos2 encode i NOS,which can produce NO causing nitrosative stress.What’s more,i NOS is also an important regulator in neuroinflammation.Tongxinluo(TXL),as a kind of compound Chinese medicine consisting of 12 drugs,could effectively promote blood circulation,regulate the meridians and relieve pain.Recent studies suggested that TXL has various effects including anti-apoptosis,anti-oxidative stress and anti-inflammation.Our study used the methods of TUNEL staining,rats’ neurobehavior score,HE staining to evaluate the protection of Tongxinluo on rats cerebral ischemia-reperfusion injury;By using PCR Array,we detected genes expressions related to oxditive stress after using TXL;By using western blot and immunohistochemical to explore the influence of TXL on the changes of p-Akt,Akt,Seladin-1,HO-1,H-Ferritin and i NOS’expression.Our study explored the anti-oxidative effects of TXL on cerebral ischemia-reperfusion injury in rats,in order to provide a theoretical foundation for its wider application in clinical treatment.Part Ⅰ The effect of PI3K/Akt pathway and the possible regulatory mechanism in cerebral ischemia reperfusion injuryObjective: To explore the effect of PI3K/Akt pathway and the possible regulatory mechanism in cerebral ischemia reperfusion injury.Methods: Healthy adult male SD rats were chosen.These SD rats were randomly divided into sham group,MCAO group,MCAO+LY294002 group,7 rats in each group.LY294002 is the specific inhibitor of PI3 K.The rat model of MCAO was established and ischemia was sustained for 90 min before the suture pulled out.Reperfusion was sustained for 24 h and then the rats were sacrificed.At 30 min before MCAO operation,a 10μl of LY294002 was infused intracerebroventricularly.TUNEL staining was evaluted as the apoptosis degree of neuron in the ischemic penumbra;Using PCR Array to work out the changes of expressions of the m RNA levels of genes related to oxditvie stress after MCAO,we selected the genes related to cerebral ischemia reperfusion injury closely and explored the expressions of the proteins encoded by these genes in the following part;Western blot method was used to detect the expressions of p-Akt,Akt,Seladin-1 and HO-1.Results: Comparing between the MCAO group and the MCAO+LY294002 group,the MCAO+LY294002 group apoptosis index was significantly elevated,P<0.001;According to PCR Array,we selected four genes related to cerebral ischemia reperfusion injury closely: DHCR24,HO-1,H-Ferritin and Nos2.The expressions of Nos2,HO-1,and H-Ferritin were up-regulated,the expression of DHCR24 was down-regulated;compared with the sham group,the expressions of p-Akt/Akt(P<0.01),Seladin-1(P<0.05)were inhibited in the MCAO group.However,the expression of HO-1 was elevated in the MCAO group(P < 0.001).Western blot shows that the expressions of p-Akt/Akt(P < 0.01),Seladin-1(P < 0.01)and HO-1(P < 0.001)were inhibited in MCAO+LY294002 group.Part Ⅱ Neuroprotective effect of TXL on focal cerebral ischemia reperfusion injury in ratsObjective: To explore the neuroprotective effect of TXL on focal cerebral ischemia reperfusion injury in ratsMethods: Healthy adult male SD rats were selected.These SD rats were randomly divided into sham group,MCAO group,MCAO+TXL group,7 rats in each group.The rat model of MCAO was established and ischemia was sustained for 90 min before the suture pulled out.Reperfusion was sustained for 24 h and then the rats were sacrificed.The rats in TXL group were orally administered TXL twice a day at 8:00 and 18:00 for three days before MCAO surgery until the animals were executed.The same volume of distilled water was given to rats in other groups.The evalution of rats’ behavior was conducted after MCAO operation 24 h.After executing the rats,the brain samples were taken.Using TTC staining to work out the changes of infarct volume;Using HE staining to observe the neuron damage;Using TUNEL to detect the apoptosis index;the above experimental operating procedures were conducted to evaluate the neuroprotective effect of TXL on focal cerebral ischemia-reperfusion injury in rats.Results: According to BIVP,MCAO+TXL group(22.49±1.93%)has a much smaller volume of infaction than MCAO group(40.32±1.99%),P<0.001.According to the morphology and apoptosis of neuron,the MCAO+TXL group has a better outcome(P<0.001);Compared with the MCAO group(2.67±0.516),TXL(1.67±0.817)improved the rats’ neurological function(P<0.05);Part Ⅲ The regulation on PI3K/Akt pathway by TXL and the protective effect of TXL in rat focal cerebral ischemia reperfusionObjective: To explore the regulation on PI3K/Akt pathway by TXL and the protective effect of TXL in rat focal cerebral ischemia reperfusion.Methods: Healthy adult male SD rats were chosen.These SD rats were randomly divided into sham group,MCAO group,MCAO+TXL group,7 rats in each group.The rat model of MCAO was established and ischemia was sustained for 90 min before the suture pulled out.Reperfusion was sustained for 24 h and then the rats were sacrificed.The rats in TXL group were orally administered TXL twice a day at 8:00 and 18:00 for three days before MCAO surgery until the animals were executed.The same volume of distilled water was given to rats in other groups.Using PCR Array to examin the effects of TXL on the m RNA levels of genes related to oxditvie stress;Using immunohistochemical to semiquantitative analysis the expressions of p-Akt,Akt,Seladin-1,HO-1;Using western blot to detect the expressions of p-Akt,Akt,Seladin-1,HO-1,H-Ferritin,i NOS.Results: According to PCR Array,the m RNA expressions of DHCR24,HO-1,Fth1 increased and Nos2 was inhibited in MCAO+TXL group;According to immunohistochemical and western blot,the expressions of p-Akt/Akt(P<0.01),Seladin-1(P<0.05),HO-1(P<0.001),H-Ferritin(P<0.05)were elevated and the expression of i NOS(P<0.05)was inhibited in the MCAO+TXL group compared with the MCAO group.Conclusion: 1.PI3K/Akt pathway plays a key role in the neuron apoptosis during the cerebral ischemia-reperfusion injury.2.PI3K/Akt pathway has a regulative role on Seladin-1 and HO-1 during the cerebral ischemia-reperfusion injury.3.TXL has a neuroprotective effect on rats with focal cerebral ischemia-reperfusion,which can significantly reduce infarct volume,inhibit the apoptosis of neurons and improve neurological function.4.TXL is capable of increasing the expressions of the Seladin-1 and HO-1 to play the role of anti-oxidative stress,probably by activating PI3K/Akt pathway.5.TXL can increase the expression of H-Ferritin and reduce the expression of i NOS,exerting the effect of anti-oxidative stress. |
PDF Full Text Request