Study On Leptin Receptor Express After Ischemic Reperfusion Damage In The Cortex And Changes Of ERK2,STAT3,NOS In Rat

ObjectiveBrain tissue will react rapidly as changes of function,metabolism and gene express et al to compensate when body meet with ischemic reperfusion damage. In aspect of function, when transient ischemia occur, brain cells will lose its function transiently which called transient ischemic attack. At the same time, brain cell metabolism will change obviously, too. Expresses of enzyme related to glycolysis such as PGK - 1 , GAPDH, LDH - A,pyruvate kinase increase, the energy supply of brain cells change from aerobic metabolism to anaerobic glycolysis. Furthermore, the production of protective protein in brain increase, too. The upper changes may have important significance to brain cell protection and injury repair. The reaction of ischemia reperfusion injury has complex molecular basis just as other physiologic and pathologic phenomena, and the main aspect is change of gene express. Some studies indicate that brain ischemic reperfusion injury can induce express of a series of early genes immediately. Most of these genes belong to oncogen family such as c - fos,c - jun,c - myc,jun - B and fos- B et al, coding many transcription factors. These transcription factors regulate many gene expresses of lower level so brain cells can modulate metabolism,function and repair after ischemic reperfusion injury. Second, the expresses of anti- oxygenic enzyme (SOD,NOS et al) ,hot shock protein gene( HSP70,HSP25 et al) , glycolysis enzyme gene(PGK - 1,GAPDH,LDH - A,pyruvate kinase et al) , calcium management gene(Ca²⁺ -ATP enzyme,hypo - calcium protein etal) , hypo - oxygen induce factor - 1 ( HIF - 1) gene and growth factor gene ( VEGF^FGF - l^TGF - |3| et al) increase after transient ischemic reperfusion injury in brain. The changes of gene express may have important significance in the protection of brain ischemia and ischemic reperfusion injury, so it may provide new method to prevent and cure brain ischemic reperfusion injury to explore such changes.Obese (ob) gene was successfully cloned in 1994. Its express production leptin was found soon. Leptin is an acute reactor in the course of inflammation, pro - inflammatory factor can influence its synthesis. Cellular study and animal experiment have proved that leptin can inhibit inflammation as a factor of inflammatory factor network. Since they lack leptin in obese rat, the time of thrombosis and vascular occlusion will prolong markedly after arterial wall damage;and the extent of thrombosis damage will lighten obviously if they are treated with leptin before damage. This may relate to the interaction of leptin and platelet and leptin receptors in the vascular endothelial cells. Studied by Dicou found that leptin could protect nerve by reducing toxic function of NMDA to cortical neuron in rats, it functions mainly by AG490 which is an inhibitory factor of JAK2. TNF - aand IL — 6 can reduce protective function of leptin to cerebral neuron by binding with STAT site so as to form leptin resistance.Mitogen - activated protein kinase ( MAPK) is a kind of serine/threonine protein kinase found in the study of.phosphorylation product of growth factor receptor at the end of eighty years. It can be activated by cytokinchormone^neu-rotransmitter. It can play an important role in cell differentiation ^developments apoptosisN malignant transformation and function in cellular multiplication. In mammal, MAPK at least has three wide — ranging sub - family series;C - Jun amino acid terminal kinase (Jun N - terminal kinase, JNK ) ^P38 MAPKs and extra cellular regulated protein kinase ( ERK). ERKs consist of P44 ERK, ^P42 ERK^PlKKERKs and P60 ERK7 ,they all can be activated by bi - phosphoryla-tion of serine/threonine protein kinase, and ERK, ^ ERK2 are the most widely studied ERK family members. ERKt ^ERKj can be activated by MAPK enzyme ( MEKj and MEKj) , active MEK - ERK1/2 signals can induce the formation of ERK monomer and dimmer, which enter nucleus by passive diffusion and activetransmission to regulate nuclear protein by binding with nuclear substrate. ERK activity can be regulated by the balance between kinase which can be activated and in - activated phosphatase. The signal pathway of these kinases is highly conservative, they transmit extra - cellular stimulus to nucleus by cascade signals through three key kinases and they regulate cellular function by phosphoryl-ating transcriptive factors. Different kinases act on different substrates to produce corresponding effect. JAK/STATs cascade is activated by others cytokine, super -family kinase series which regulate cell growth and differentiation, they are formed of Janus kinase (JAK) ^ signal transmitters and STAT, the former consists of JAK, >JAK2 NJAK3 and TYK2;the latter consists of STAT^STAT^ STAT3NSTAT4^STAT5ANSTAT5B^STAT6 Two kinase families in JAK/STAT pathway are both protein kinase with tyrosine residuals. Cytokine and growth factor bind with receptor to form dimmers which phosphorylate JAK tyrosine residuals, then activated JAK will have binding site for STAT to phosphorylate tyrosine residuals to form homogenous or heterogeneous dimmers with STAT, the dimmers transform from cytoplasm to nucleus to bind with related DNA sequences in order to take part in gene transcription and regulate protein synthesis.NOS has three types at the basis of its tissue sources and induction character, that is neural NOS(nNOS) .endothelial NOS(eNOS) ^inducible NOS( iN-OS) , the former two are called CNOS, mainly distribute in vascular endothelial cells, nerve cells and platelets, their catalytic activity depend on intracellular ca2* concentration, NO is released by pulsate formation to take part in normal physiological exercise such as maintaining vascular moderate relaxing state, inhibiting platelet congregation, regulating white blood cells chemotaxis and adhesion , regulating local organ blood volume, inhibiting interstitial cells in vascular smooth muscle, it plays an important role in neurotransmitter transferring^cellular apoptosis^study and memory. In pathologic condition, nNOS and eNOS are activated continuously when intracellular Ca2+ concentration increases to produce a lot of NO which has toxic effect. iNOS orient in immune cells ^glial cells N neuron and vascular smooth muscle cells. In normal physiological condition, iNOS genes can only express when cells are stimulated by endotoxin and cytokines to produce lots of NO. In recent years, the function of NO in ischemic cerebralvascular diseases and its mechanism are our studying emphasis, and the certain conclusion is that NO has bilateral functions as cerebral protection and nerve toxicity.This experiment studies leptin receptor express in cerebral cortex and its express after ischemic reperfusion injury, and we explore the express of STAT3 ^ ERK2>,NOS in signal transmission pathway after ischemic reperfusion injury.Methods1. Focal ischemic reperfusion injury model;We make further improvement at the basis of Zea Longa method. Anesthesia through abdominal cavity, cutting cervical skin, separating common carotid artery (CCA) , external carotid artery ( EC A) >, internal carotid artery (ICA) ^ligating CCA and EC A from site near the heart. We put a spare line from the dividing site of CCA to the dividing site of middle cerebral artery, the mean length of line entered is 18. 5 ±0. 5 mm (from the dividing site of ECA and ICA) , the end of the line is left to the skin, The reperfusion occurred after an hour of occlusion . The control group was the same in the other steps except the length of line entered with 9mm. The studying model standard: animals turn right when they climbed after awake, the right anterior limbs contract internal and bending.2. Wax specimen preparation;Injecting more anesthetic drugs to rats after 24/72 hours from ischemic reperfusion injury, cut the brain immediately after pouring NS, 4% poly - methanal. Fixed 48 hours in 4% poly - methanal, dehydration in alcohol, enveloped in wax, coronary cutting continuously, the thickness is 7jxm.3. Immunohistochemical specimen;Staining based on SABC method, to keep activity\of endogenous peroxidase by 0.3% H2O2 ,Hot repair 15 minutes at 92t by PH =6.0 buffering liquid, washing three times with PBS. Incubating 1 hour(18 -20t) of 1. 5% sealed serum, Abandon but not wash, add anti -leptin ob - R rat serum( 1:100) to incubate 36 hours at 4t, then washing with PBS and add goat IgC which is marked to incubate 3 hours in room temperature. A and B complex incubated 2 hours in room temperature, colour showed byDAB, common dehydration, hyaline, neutral gum sealed, observed under microscope.4. Electroscope^immuno -electroscope specimen;Following upper steps except electroscope specimen, fixed 1 hour in 1% OsO4, washing by PBS buffering liquid, dehydration by alcoholNpyruvate, enveloped by Epon 812, continuously cutting by LKB - 2088 machine, stain by uranium and lead, observation under H - 600 electroscope.5. Semi - quantitative RT - PCR;First extracting cerebral cortical RNA, then synthesize cDNA, last multiplication by PCR. ERK2 primer sequence as, sense;5GCA GGT GTT CGA GGG 3', antisense: 5'GTG CAG A AC ATT AGC TGA AT 3', multiplication segments 394bp. STAT3 primer sequence as, sense;5'TGG GTC TGG CTA GAC AAT 3', antisense;5'CGT TGG TGT CAC ACA GAT 3', multiplication segments 465bp. (3 -actin primer sequence as, sense;5 'GCC AAC CGT GAA AAG ATG 3', antisense: 5'CCA GGA TAG AGC CAC CAA T 3', multiplication segments 700bp. Putting CDNA lOui to 0. 2 ml Ep-pendorf tube, adding 5XPCR buffer 10ui^ sterilized water 28. 75jaU TaKaRa TaqHS 0.25uJ^upper floating matter 0. 5jxl> lower floating matter 0. 5|xl separately. Pre - degeneration 2 minutes at 94X., degeneration 30 seconds at 94X,, annealing 30 seconds at 57t, prolonging 1 minute at 12X., circulating 35 times and then prolonging 5 minutes at 72^.6. Western blot: Injecting more anesthetic drugs to rats after 24 hours from ischemic reperfusion injury, getting brain and separate cortical brain tissue of parietal lobe, keep in the liquid nitrogen. Adding lml splitting buffering fluid to 200mg cortical brain tissue of rat, taking 25ui to measure protein content. Measure the concentration of protein by phenol reagent method, electrophoresis, blot . Pre - stain protein to ensure it transformed to membrane. Incubating with rabbit anti - iNOS^ nNOS^ eNOS ( 1:300 ) antibody the whole night at 4^. Washing with TIBS fluid twice, adding goat anti - rabbit IgG (1:2000) marked by alkaline phosphatase to keep 2 hours in room temperature, washing by TIBS twice. Putting filter membrane to developer 15 -30 minutes, taking out it when it is clear, washing by distilled water. Taking out the filter membrane to make it dry. Scanning by computer, analysis and manage.7. Statistical analysis;Results are presented as means SE. Paired and Stu-dence s t - tests were used as appropriate to evaluate the statistical significance of differences between two groups means , and ANOVA was used for multiple groups. Values of p <0.05 were considered to indicate statistical significance.Results1. Expression of leptin receptor on cerebral cortex: It can express on pyramidal cells of cerebral cortex Nvascular endothelium cells ? vein plexus. The reactive product with osmic acid on immuno - electroscope showed brown, it appeared on nuclear membrane intermittently and scattered/concentrated in cytoplasm, and brown particles can also be seen at the edge of mitochondrion.2. Ischemic reperfusion of 24 hours group ob - R expression decreased compared with control group ( p < 0.05);and ischemic reperfusion of 72 hours group ob - R expression decreased compared with control group( p <0. 001);72 hours group ob - R expression decreased compared with 24 hours group ( p <0.05) .3. Result of HE stain in cerebral cortical nerve cells;In control group, the shape of cerebral cortical cells is normal, without cerebral edema. In ischemic reperfusion of 24 hours group, the volume of most of these cells are small and appear as triangle, nucleus concentrate and stained darkly, the structure disappeared, the gap around vessel is widen, appeared as light cerebral edema. In ischemic reperfusion of 72 hours group, cellular necrosis and eeddema are more obvious, loss of nerve cells, pan - shaped bubble changes around cells, structure appears loose, the surrounding edema is more obviously.4. Electroscope result of cerebral cortical nerve cells;In control group, nuclear membrane is complete, nucleolus is clear, cellular apparatus structure is clear, endoplasmic reticulum is abundant. In ischemic of 24 hours group, cellular membrane is not so regular, it shrinks slightly, cytoplasm concentrate obviously, but the nucleus concentration and nuclear membrane changes are not obvious, small amount of chromatin gather. In ischemic of 72 hours group, degeneration and necrosis of nerve cells can be seen which mainly appeared as cellular dissolution, mitochondrion swollen, cristae break, cytoplasm bubble, eel-lular dissolution and darkly stained, nuclear membrane shrunk, chromatin appeared compact mass and gathered in the peripheral area, nucleus concentrate.5. Semi - quantitative RT - PCR: In ischemia reperfusion of 12^24^72 hours, ERK2 expression increased in cerebral cortex, it began to increase at 12th hour, and reached the peak at 24 hour, but began to decrease at 72" hour. In ischemia reperfusion of 12^24^72 hours, STAT3 expression increased in cerebral cortex, it began to increaseat 12th hour, and reached the peak at 24th hour, but began to decrease at 72 nd hour.6. Western blot: In ischemia reperfusion of 12^24^72 hours group iNOS expression increased obviously compared with control group. It began to increase at 12 hour, and the expression increased gradually as the ischemic reperfusion time prolonging;ischemia reperfusion of 12^24 hours group, nNOS expression increased obviously compared with control group, yet there was no increase in 72 hour group, and the expression was the most obvious at 12' hour, it began to decrease at 24' hour;ischemia reperfusion of 12^24^72 hours group, eNOS expression increased obviously compared with control group, especially in the 12 hours group. The expression of iNOS increased gradually from 12 to 72 hours, and the express of nNOS decreased gradually from 12 to 72 hours;the expression of eNOS decreased gradually from 12 to 72 hours.Conclusions1. Leptin receptor can express in pyramidal cells of cerebral cortex N vascular endothelium cells^vein plexus;2. The expression of leptin receptor in cerebral cortical neuron decreased after ischemic reperfusion injury;3. Ob - R expression decreased obviously compared ischemic of 72 hours group with that of 24 hours, the neuron damage is more severe in 72 hours group;so it indicates the decrease of leptin receptor after ischemic reperfusion injury is parallel to the extent of neuron damage;4. In ischemia reperfusion of 12^24^72 hours, ERK2 expression increased in cerebral cortex, it began to increase at 12th hour, and reached the peak at24th hour, but began to decrease at 72nd hour;5. In ischemia of 12 ,,24 ,,72 hours, STAT3 expression increased in cerebral cortex, it began to increase at 12th hour, and reached the peak at 24th hour, but began to decrease at 72nd hour;6. In ischemia of 12 ,,24 ,,72 hours group iNOS expression increased obviously compared with control group. It began to increase at 12th hour, and the expression increased gradually as the ischemic reperfusion time prolonging;ischemia reperfusion of 12,,24 hours group, nNOS expression increased obviously compared with control group, yet there was no increase in 72 hour group, and the expression was the most obvious at 12th hour, it began to decrease at 24' hour;ischemia reperfusion of 12 ^24 ,,72 hours group, eNOS expression increased obviously compared with control group, especially in the 12 hours group.