Study On Mechanisms Of Cerebral Ischemical Reperfusion Injury And The Preventive And Theapeutic Effect Of The Conjugate Of Superoxide Dismutase And Low Molecular Weight Heparin

IntroductionTumor necrosis factor-α (TNFα) is a cytokine generated by macrophage,monocyte and glia cell. TNFa has many kinds of biological effects,it is an important excitatory transmitter in nerves-endocrine-immunological regulation system.It expresses abnormally and plays many important roles in many pathology courses.TNFα participates in inflammation and cell death course after cerebral ischemia.Its overexpression could intensify cerebral injury by inducing and promoting inflammation,cytotoxicity,blood clotting and many apoptosis pathways.Its overexpression not only could induce apoptosis and active glia cells,but also could induce overexpression of c-Jun and active c-jun N-terminal kinases (JNK).C-Jun is one of immediate early genes(IEGs).Cerebral ischemia reperfssion could induce c-Jun quickly and temporal expression in brain. C-Jun participates in many effective enzymatic transcription courses of signal transmission system.It could regulate other many kinds of genic express and target proteins synthesis.C-jun N-terminal kinase(JNK)belongs to one of the superfamilyof mitogen activated protein kinase(MAPK).It is distributed intracytoplasm generally. It is a gen of protien kinases containing a -amino- P -hydroxy-propionic acid / threonine residue. It is an important signal transducting system.lt can transfer ecto-spike to cell nucleus.The overexpress of c-jun and JNK activation can lead to neurons apoptosis. The phosphorylation of c-jun would increase activity of c-jun activation of genetic transcription.JNKcould induce overexpress and phosphorylation of c-jun,up-regulate express of TNFa, mediate activation of caspases and lead to cell apoptosis.Cerebral ischemia could induce overexpress of c-jun and activation of JNK.Neurocyte apoptosis was decreased obviously and cerebral ischemic injuries were palliated significantly by inhibition of c-jun overexpress and JNK activation.There are not yet reports on the effects of overexpression of murine TNFo gene on glia and infarct volume, and the dynamic expression of TNFa and dynamic effects on microglia activation of murine TNFa transgenic rats with cerebral ischemia,and there is not yet the study on apoptosis, microglia activation, expression of c-JUf^ p-JUN and p-JNK of murine TNFa transgenic rats with cerebral ischemia now.Free radical excess generation and leukocyte mediated inflammatory reaction are the important factors to result in cerebral ischemic reperfusion injury. The research on mechanisms of action of nitrogen monoxidum (NO) ,glutathion peroxidase(GSH-PX) andmyeloperoxidase(MPO) in cerebral ischemic reperfusino injury is a hot spot in recent years.Some researches have found that the roles of NO have dualism in cerebral ischemic injury possiblly. it could dilate blood vessel,sustain cerebral blood flow, prevent platelet and leukocyte aggregation and adhere, down regulation NMDA acceptor, and it may have neuroprotective effect.lt could participate excitability toxic effect,mediate cortex and hippocampus neuron aminoglutaminic acid(Glu) neurotoxicity, aggravate neuron injury after cerebral ischemia. GSH-PX is one of important antioxidases related to free radical metabolism,it can catch and clean up organism excess superoxide anion and hydrogen peroxide, block and prevent a series of free radical reaction due to superoxide anion, maintain oxadation and dynamic equilibrium,protect cellularmembrane to exempt oxidization and lipid peroxidation injury.A lot of findings manifestate that activity of SOD and GSH -PX lower significantly in ischemic region after cerebral ischemic reperfusion, the level of GSH -PX can reflect cerebral ischemia graveness degree. GSH-PX activity can mitigate cerebral ischemic reperfusion injury,this is one of mechanisms to educe neuroprotective effect.MPO is typical desmoenzymes in heterophil granulocyte and histoleucocyte.Its activity has become one of indicatrixes to assess leukocytic infiltrate and nerve injury degree after cerebral ischemia.Superoxide dismutase(SOD) is one of natural biocatalyst barrier against free radical injury, its main biological function is that O2"is catalyzed to become H2O2 and O2 by dismutation reaction, so it has obvious neuroprotective effect,but natural SOD has limitations in clinical application,for exzample, plasma half-life is short,it could not permeate blood-brain barrier of normal brain tissue,cellular permeability is very low,and it is very easy to be degradated by intaorgan enzymes. The researches have found in recent years that it could have different neuroprotection effect after SOD is modified by different modifiers .Our recent recearch has confirmed that extraorgan stability of LMWH-SOD (LMWH modificated enzyme) was increased obviousely ,intraorgan antigenicity of LMWH-SOD was decreasedsignificantly,plasma half-life of LMWH-SOD was lengthened significantly , and LMWH-SOD is much easier to pass blood brain barrier than SOD,LMWH-SOD could lessen cerebral edema,reduce lipid peroxidation level,, inhibite cellular adhesion molecule express and apoptosis, and reduce TNF-a and IL-ip express significantly after global brain ischemia reperfusion, so it has obvious brain protection.There are not yet reports on effects of of NS,LMWH,SOD,LMWH+SOD and LMWH-SOD on express of TNF-a,c-Jun and NF-kBP65 and on the changes of serum NO,GSH-PX and MPO levels after global brain ischemia reperfusion. ObjectivesTo observe protein expression of TNFo and activation of microglia, astrocyte and oligodendrocyte.To investigate the effects of overexpression of TNFq on neurologic impairment scales and volume of cerebral infarct.To explore the effects ofoverexpression of murine tumor necrosis factor-a(TNFa) gene of Sprague-Dawley(SD) transgenic rats on glia and infarct volume in focal cerebral ischemia.To observe the dynamic expression of tumor necrosis factor (TNFa) and dynamic effects on microglia activation of murine TNFa transgenic rats with cerebral ischemia.To explore the effects of over expression of tumor necrosis factor-a(TNF-a) in cerebral ischemia tissue on apoptosis,microglia activation, expression of c-JUN,phospho-c-jun (p-JUN) and phospho-c-jun N-terminal kinase (p-JNK).To observe the effects of LMWH-SOD on astrocyte activation of IL-6 transgenic mice.To observe the effects of NS,LMWH,SOD,LMWH+SOD and LMWH-SOD on express of TNF-a,c-Jun and NF-kBP65 after global brain ischemia reperfusion.To observe the effects of NS,LMWH,SOD,LMWH+SOD and LMWH-SOD on the changes of serum NO,GSH-PX and MPO levels after global brain ischemia reperfusion. MethodsProtein expression of TNFa and activation of microglia, astrocyte and oligodendrocyte were examined by immunohistochemistry of TNFa, interginaM (0X42) , glial fibrillary acidic protein (GFAP) and myelin.Using suture occlusion technique,the right middle cerebral artery in rats was occluded.SD rats and murine TNFa transgenic rats were subjected to 1 hour of the right middle cerebral artery occlusion.Seventy-two hours after reperfusion,neuronal deficit scores were calculated,and infarct volume was calculated by 2,3,5-triphenyltetrazolium chloride monohydrate (TTC) staining.Using suture occlusion technique,the right middle cerebral artery in rats was occluded.Protein expression of TNFa and activation of microglia were examined by immunohistochemistry of TNFa and interginaM (0X42) . The dynamic expression of TNFa and dynamic effects on microglia activation were observed in murine TNFa transgenic rats subjected to 1 hour occlusion and 3 hours,! 2hours,24hours,72hoursand 7 day s reperfusion.Using suture occlusion technique,the right middle cerebral artery in Sprague-Dawley rats and murine TNFa transgenic rats was occluded. Protein expression of TNFa and activation of microglia were examined by imrnunohistochemistry of TNFa and interginaM ( 0X42 ) in the cerebral non-ischemia tissue of Sprague-Dawley rats and murine TNFa transgenic rats.Apoptosis, activation of microglia, protein expression of c-JUN, p-JUN and p-JNK were examined by immunohistochemistry of terminal deoxynucleotidyl transferase(TdT)-mediate biotinylated UTP nick end labeling (TUNEL)technique, 0X42, c-JUN, p-JUN and p-JNK in the cerebral ischemia tissue of Sprague-Dawley rats and murine TNFa transgenic rats subjected to 1 hour occlusion and 24hours reperfusion.Using suture occlusion technique,the right middle cerebral artery in IL-6 transgenic mice was occluded. By immunohistochemistry of glial fibrillary acidic protein (GFAP) ,to investigate the effects of LMWH-SOD on astrocyte .Using global brain ischemia reperfusion of gerbil model,to investigate the effects of NS,LMWH,SOD,LMWH+SOD and LMWH-SOD on express of TNF-a,c-Jun and NF-kBP65 by immunohistochemistry of TNFa,c-JUN and NF- k BP65,.Using global brain ischemia reperfusion of gerbil model,the serum levels of N0,MP0 and GSH-PX were evaluated by biocatalyst indication meter and spectrophotometric method. To observe the effects of NS,LMWH,SOD, LMWH+SOD and LMWH-SOD on the changes of serum NO,GSH-PX and MPO levels after global brain ischemia reperfusion. ResultsTNFa overexpression and hypertrophic and proliferative changes of microglia, astrocyte and oligodendrocyte in the non-ischemia brain tissue of murine TNFa transgenic rats were observed. Neuronal deficit scores and infarct volume of murine TNFa transgenic rats for seventy-two hours reperfusion after 1 hour of occlusion were higher than those of SD rats .TNFa overexpression at 3 hours after reperfusion in the ischemic brain tissue ofmurine TNFa transgenic rats was observed and reached the peak.At 12hours,24hours,72hours and 7 days reperfusion, TNFa overexpression decreased gradually. Microglia activation at 3 hours after reperfusion in the ischemic brain tissue of murine TNFa transgenic rats was observed and reached the peak.At 12hours,24hours,72hours and 7 days reperfusion, microglia activation decreased gradually.TNFa expression and hypertrophic and proliferative changes of microglia in the cerebral non-ischemia tissue of murine TNFa transgenic rats were observed. An increase in the level of apoptosis, microglia activation, c-JUN, p-JUN and p-JNK overexpression found in the murine TNFa transgenic rats subjected to 1 hour occlusion and 24hours reperfusion were higher than those in the Sprague-Dawley rats.The cell population of astrocyte was more,but cell body was fairly smaller, and branches were fewer,shorter and thinner in IL-6 transgenic mice cerebral non-ischemic tissue.The quantity of astrocyte in NS group was fewer than sham group.The quantity of astrocyte in LMWH-SOD group was more than that in sham group and NS group.And cell body became bigger,and branches became longer and thicker,astrocyte was observed to offer the changes of hypertrophy and accrementition.There was no express of TNFa and c-jun in non-ischemic cerebral tissue, only a little NF- k BP65 express.There was much express of TNFa,c-JUN,NF- k BP65 in NS group. There was express of TNFa,c-JUN,NF- k BP65 in LMWH group,but less than that in NS group. There was some express of TNFa,c-JUN,NF- k BP65 in SOD group,but less than that in NS group. There was some express of TNFa,c-JUN,NF- k BP65 in LMWH+SOD group,but less than that in NS group,LMWH group and SOD group. There were fewer cells to express TNFa,c-JUN and NF- k BP65 in LMWH-SOD group,but fewer than those in NS group,LMWH group, SOD group and LMWH+SOD group.After global brain ischemia reperfusion of gerbil , serum NO and MPO levels would be increased in NS group significantly, and serum NO and MPO levels were lower in LMWH group , SOD group, LMWH+SOD group and LMWH-SOD groupthan that in NS group. But serum NO and MPO levels were higher in LMWH group , SOD group, LMWH+SOD group than that in LMWH-SOD group. After global brain ischemia reperfusion of gerbil , serum GSH-PX level would be decreased in NS group significantly, and serum GSH-PX level was higher in LMWH group , SOD group, LMWH+SOD group and LMWH-SOD group than that in NS group. But serum GSH-PX level was lower in LMWH group , SOD group, LMWH+SOD group than that in LMWH-SOD group. ConclusionOverexpression of TNFa gen could activate microglia, astrocyte and oligodendrocyte in the non-ischemic brain tissue,and increase neuronal deficit scores and infarct volume significantly. Overexpression of TNFa can aggravate cerebral ischemic injury.The dynamic expression of TNFa and dynamic changes of microglia activation of murine TNFa transgenic rats with cerebral ischemia was same,this suggests that the microglia activation is strongly related with overexpression of TNFa .The mechanisms of overexpression of TNFa gen effects on cerebral ischemia injury are probably microglia activation, c-JUN, p-JUN and p-JNK overexpression.After the treatment of LMWH-SOD,at earliest stage of cerebral ischemic injury,astrocyte was observed to offer the changes of hypertrophy and accrementition.It is one mechanism to protect brain.TNF-a,c-Jun and NF-kBP65 were observed to be inhibited significantly in LWMH-SOD group at the stage of cerebral ischemic reperfusion, The effect of LWMH-SOD group surpassed that of SOD group,LMWH group and combined SOD with LMWH group in preventive and therapeutic effects of cerebral ischemic reperfusion injury significantly.The global brain ischemia model of gerbil was done well, LMWH-SOD group would decrease serum NO and MPO levels, and increase serum GSH-PX level significantly. All effects of group treated with LWMH-SOD are better than those of LMWH group,SOD group and combined LMWH wiyh SOD group.So LMWH-SOD is hopeful to be exploitated as a new drug for the treatment of cerebralischemic reperfusion. SignificanceTNFq overexpression could intensify cerebral ischemic injuries,its mechanism is related with the activation of glia cells,The methods of TNFa overexpress inhibition and regulation would be a new device for prevention and cure cerebral ischemic injuries and regulation microglia activation.For prevention and cure cerebral ischemic injuries after cerebral ischemia,besides TNFa overexpress inhibition and rivalry the effect of TNFa,the catastaltic of overexpress and phosphorylation of c-Jun and JNK activation would be applicated as a new means,too.After super-early cerebral ischemic reperfusion , LWMH-SOD could protect and active astrocyte,inhibit express of TNF-a,c-Jun and NF-icBP65,decrease serum NO and MPOlevels obviously,increase serum GSH-PX level.AH effects of group treated with LWMH-SOD are better than those of LMWH group,SOD group and combined LMWH with SOD group.So LMWH-SOD is hopeful to be exploitated as a new drug for the treatment of cerebral ischemic reperfusion.