Effect And Mechanism Of ZS And XH In AGNH Improving Brain Injury

Effect and mechanism of ZS and XH in AGNH improving brain injuryAngongniuhuang (AGNH) is a traditional medicine used in emergency of infectious diseases. It has efficacies of relieving fever, detoxicating, relieving convulsion and waking up a patient from unconsciousness. Its indications are infectious disease with hyperpyrexia, coma, facial distortion, blood-stroke, muscle and vessel locking, phlegmatic epilepsy. It is used in conscious disturbance and coma come from all kinds of reasons in clinical. But because of Zhusha (ZS) and Xionghuang (XH) which contain HgS and As2S2, and the toxicity and side effects, such as nephronia and hypersensitive reactions induced by AGNH, were reported for the past few years, the safety of AGNH is called in question. So the removing and remaining of ZS and XH in the prescription is the investigation focus. So much work on the pharmacologic actions of ZS and XH in AGNH was done.The study indicated that AGNH could protect brain cell obviously, and put forward that protection on brain was one of the mechanism of its Xinnao Kaiqiao. In some effects, AGNH were better than AGNH without ZS and XH (QZX-AGNH). AGNH could increase c-fos expression in cortex, directly activate cortical neurons, but the activation of QZX-AGNH was weak. All the data indicated that ZS and XH are maybe the important material foundation of AGNH in improving brain injury. More study on the effects of ZS and XH in improving brain injury may provide experiment bases for its existing in AGNH.In the dissertation, brain injury rat model caused by LPS injected through caudal vein and by middle cerebral artery occlusion were employed respectively to investigate the effect of AGNH on electocortocogram (ECoG). By the means of radio ligand binding technique, immunohistochemistry and biochemistry, molecular biology techniques, with electronmicroscope, the effect and the mechanisms of AGNH and QZX-AGNH in improving brain injury, and the significance of ZS and XH in prescription were studied.1 Effect of AGNH and QZX-AGNH on EEG in brain injured ratsEEG as a kind of spontaneous electric activity is an objective index of brain function changing, which is closely correlated to cerebral cortex activity level. And EEG is closely correlated to conscious state changing. When conscious state improving, the EEG was activated. EEG activation is the EEG changing from sleep to waking up, the beta (14-30Hz) increase is the characteristic of EEG activation, which indicates behavior arousal. The delta (0-3Hz) increase is the typical feature of all brain injury with conscious disturbance. So increasing the beta-waves and decreasing delta-waves were may be the important indications of AGNH improving brain injury. In this part, with LPS brain injury model, cerebral ischemia model by middle cerebral artery occlusion, effects of AGNH and QZX-AGNH on the power and relative power of the beta- and delta-wave of EEG in brain injury were studied.1.1 Effect on EEG of cerebral ischemia ratsNormal rats implanted cortical electrode in advance were divided into 6 groups: sham, vehicle, AGNH (0.4g/kg, 0.2g/kg), QZX-AGNH (0.32g/kg, 0.16g/kg), after three times administration, MCAO model was made,and then EEG was traced immediately. The results were that MCAO could decrease the power of data- and beta-waves, and the relative power of beta-waves, but raise the relative power of data-waves. AGNH could reverse the changing of data- and beta-waves induced by MCAO, and QZX-AGNH had no effect on beta-waves, AGNH and QZX-AGNH could inhabit the relative power of data-waves increasing.1.2 Effect on EEG of brain injured rats induced by LPSNormal rats with implanting cortical electrode in advance were divided into 9 groups: control, model, AGNH (0.4g/kg, 0.2g/kg), QZX-AGNH (0.32g/kg, 0.16g/kg), ZS+XH (0.08g/kg), XH (0.04g/kg) and ZS (0.04g/kg). After three administrations, brain injured rat model was made by injecting LPS 16mg/kg through caudal vein, and then tracing EEG in awake rats at 1h, 2h, 4h, 6h after LPS injection. The results indicated that the power and relative power ofβdecreased persistently, and reached the lowest point at 6h, the relative power of delta-wave raised at 4h, 6h. AGNH could increase the power and the relative power of beta-waves, and inhabit the relative power of delta-waves. QZX-AGNH could inhabit the relative power of delta-waves, and increased the relative power of beta-waves at high dose. The effects of ZS and XH were same as AGNH on beta-waves.Summary: LPS and cerebral ischemia could increase the relative power of delta, and decrease the power and the relative power of beta. AGNH could increase the power and relative power of beta, and the action was not observed in QZX-AGNH (P<0.05), ZS and XH had the same effect with AGNH on beta. So ZS and XH are important in improving EEG changing in AGNH in brain injuried.2 Effect of AGNH and QZX-AGNH on the acetylcholine system of cortex and brainstem Normal rats were divided into 7 groups: control, model, AGNH (0.4g/kg), QZX-AGNH (0.32g/kg), ZS+XH (0.08g/kg), XH (0.04g/kg) and ZS (0.04g/kg). After administrating for three days, brain injured rat model by LPS was made at an hour after the third administration. With biochemistry methods, radio ligand binding technique, immunohistochemistry and molecular biology techniques, the activity of choline acetyl transferase (ChAT), cholinesterase (ChE), ChATmRNA expression, content of acetylcholine (Ach), affinity of M-R, content of G-protein were detected.2.1 Effect on the content of acetylcholine in cortex and brainstemWith hydroxylamine chromatometry, the contents of Ach in cortex and brainstem were detected. The results indicated that the Ach content of cortex did not change 6h after LPS injection, and that of brainstem decreased. AGNH could obviously increase the Ach level of brainstem, but QZX-AGNH, ZS and XH had no effects on it.2.2 Effect on the activity of ChAT and ChE in cortex and brainstemLPS could increase the ChAT content of cortex and brainstem, and had no influence on ChE. AGNH could reverse the increasing of ChAT in cortex and brainstem caused by LPS injection, QZX-AGNH could not inhabit the increasing of ChAT content in cortex and brainstem, comparing with AGNH,P<0.05. ZS+XH and XH had the same effects. QZX-AGNH could also obviously increase ChE activities of cortex and brainstem.2.3 Effect on the expression of ChAT and ChATmRNA in cortexThe third pyramidal cells of cortex, CA1 and CA3 region were the main observation fields in this experiment. The results were that LPS could increase ChAT expression in three regions. AGNH, ZS+XH, XH could decrease the expression of ChAT in all regions. But QZX-AGNH and ZS could further increase ChAT expression, especially in CA3 region, comparing with AGNH, P<0.05. ChAT mRNA expressions of cortex were all same in different groups.2.4 Effect on the affinity of M-receptor in cortex and brainstemBy radio ligand binding technique, the affinities of M-receptor (M-R) in cortex and brainstem were detected. The results indicates that the affinity of M-R in cortex decreased obviously and the affinity of M-R in brainstem increased. AGNH could increase the affinity of M-R in cortex, but QZX-AGNH had no effect (P<0.05). ZS+XH could increase the affinity of M-R in cortex as well as AGNH.2.5 Effect on the content of G-protein in cortex and brainstemG-protein is the door that information getting into cells from extro-cellular. It is also the important components of M-R coupled signal transduction. The main kinds of G-protein are Gsαand Giα, its level and the ratio of Gsα/Giαare very important for keeping and controlling normal condition of cell in physiology as well as in pathologic state. The results were that LPS could decrease Gsαand increase Giα, make the ratio of Gsα/Giαdecreasing. AGNH could obviously increase Gsα, raise the ratio of Gsα/Giα, QZX-AGNH could decrease Giα, then increase Gsα/Giαratio. ZS+XH, XH had the same effect with AGNH. Summary: LPS could decrease Ach content in brainstem, increase ChAT content in cortex and brainstem, cut down the affinity of M-R and Gsα/Giαratio, cause acetylcholine system injury of brain. AGNH could increase Ach content, decrease ChAT in cortex and brainstem, raise the affinity of M-R and Gsα/Giα, improved acetylcholine system function. QZX-AGNH had no effect on ChAT, Ach contents, the affinity of M-R, comparing with AGNH, P<0.05. ZS+XH, XH had the same effect with AGNH. So ZS, XH are important in AGNH improving acetylcholine system injury of brain.3 Effect of AGNH and QZX-AGNH on the monoamine system of cortex and brainstem Noradrenaline (NE), Dopamine (DA), and 5-hydroxytryptamine (5-HT) are important in keeping the excitability of central nervous system. With high-performance liquid chromatography with electrochemical detector, the contents of monoamine transmitters and the metabolites, including NE, DA, 5-HT and dihydroxy-phenyl acetic acid (DOPAC), homovanillic acid (HVA), 3-methoxy-4- hydroxyl-phenylglycol (MHPG), 5-hydroxyindoleacetic acid (5-HIAA), were detected. With biochemistry methods, immunohistochemistry and molecular biology techniques, the activity of monoamine oxidase (MAO), expression of Tyrosine Hydroxylase (TH) and THmRNA were measured.3.1 Effect on the monoamine transmitters and its metabolite of cortex and brainstemAfter LPS injection, the contents of NE, 5-HT, 5-HIAA in cortex and brainstem increased, and the E, DOPAC contents in cortex decreased, without DA changing. In cortex, AGNH could decrease NE, 5-HIAA (P<0.05) contents, increase DOPAC (P<0.05) and E contents, but QZX-AGNH could further increase NE contents, and comparing with AGNH and model, P<0.05. ZS and XH could also decrease the content of NE.In brainstem, AGNH also decreased the content of NE, QZX-AGNH did not inhabit NE increasing. ZS and XH could decrease NE content.3.2 Effect on tyrosine hydroxylase and monoamine oxidase of cortex and brainstem The expressions of TH and THmRNA in cortex in different group were all the same. The activity of MAO in cortex decreased and that in brainstem increased slightly after LPS injection. AGNH and QZX-AGNH could recovery the changing of MAO in cortex and brainstem.Summary: After LPS injection, the contents of NE, 5-HT, 5-HIAA increased in cortex and brainstem(P<0.05), the activity of MAO in cortex decreased and that in brainstem increased. AGNH could inhabit the increasing of NE in cortex and brainstem (P<0.05), improve the metabolism of 5-HT (P<0.05), and reverse the activity changing of MAO caused by LPS. QZX-AGNH could not decrease the NE (comparing with AGNH, P<0.05), it also can increase the content of DA in cortex. ZS and XH could decrease NE increase in cortex and brainstem. This indicated that ZS and XH produced a marked effect in improving monoamine transmitter disorder of brain injury in AGNH4 Effect of AGNH and QZX-AGNH on morphous of pyramidal neurons and synapse ultramicrostructure in cortexNeuron is the functional unit of central nervous system, normal morphous of neurons is the foundation of brain function. LPS and ischemia can damage neurons and cause the function depletion. Synapse as the connection of neurons is the key position for neuron to accomplish physiologic function, and is important for keeping brain function. With Haematoxylin-eosin staining (HE), the morphous of pyramidal cell and the synapse structure in third layer of cortex is observed, the texture parameters of synapse were calculated.4.1 Effect on morphous of pyramidal neurons in the third layer of cortex After LPS injection, obvious tissue hydroncus occurred, and many nerve cell bodies deflated with karyopycnosis, part of them became triangulum, chromatospherite of many pyramidal neurons disappeared. AGNH could improve pyramidal cell deteriorating, abate karyopycnosis, and many neurons had clear chromatospherite, tissue hydroncus was lightened. The effect of AZX-AGNH was weak than AGNH in the above aspect, ZS+XH could also improve deteriorating and karyopycnosis. Cinnabaris and realgar also showed some effects.4.2 Effect on the synapses ultramicrostructure in the third layer of cortexAfter LPS injection, the numbers of synapses in cortex decreased, the ultramicrostructure was destroyed, synaptic cleft was unclear. Packs of vesicles gathered near presynaptic membrane, and post-synaptic density (PSD) decreased. AGNH could improve synapse injury caused by LPS, improve vesicles gathering, PSD was kept well, some large laugh synapse could be seen under electronmicroscope. In the QZX-AGNH group, the synapse structure was kept well, the number of synapse increased, and PSD were protect to some extent, but QZX-AGNH could not improve synaptic cleft changing, especially vesicles gathering was not improved. The common features of ZS+XH, XH and ZS were that the structure of synapse was clear, vesicles were less than normal group, and larger synapses could be viewedQuantitative analysis indicated that synapse number, thickness of PSD, length of PSD decreased obviously with synaptic cleft stegnosis after LPS injection. AGNH could improve the above indexes. QZX-AGNH could increase the number of synapse and protect PSD in some degree, the effect of QZX-AGNH on synaptic cleft and PSD were different from AGNH (P<0.05), ZS+XH could increase the number of synapses, and increase synapse cleft and protect PSD as same as AGNH.Summary: AGNH could improve pyramidal cell injury, synapse structure destruction and vesicles gathering near presynaptic membrane cause by LPS, with obviously protection on PSD. QZX-AGNH could increase the number of synapse, and protect PSD in some degree, but could not improve synapse cleft, compare with AGNH, P<0.05, especially vesicles gathering were not improved. ZS+XH could increase PSD and synapse cleft stegnosis as same as AGNH, vesicles gathering was inhabited obviously. So AGNG was superior to QZX-AGNH in protecting neurons and synapses structure, in the improving of vesicles gathering of AGNH, ZS and XH produced a marked effect.5 Effect of AGNH and QZX-AGNH on ATP and synapsinⅠin cortex and brainstemReleasing of transmitters is the common link of central neurotransmitter actions, and maybe improving transmitter releasing is the important action of AGNH in improving brain injury. Many factors influence the course, synapsinⅠinfluences the dynamics of transmitter releasing, Na+-K+-ATP, Ca2+-ATP can keep resting membrane potential (RMP) and steady state of Ca2+ in lower concentration in neurons. The study mainly focus on the effect of AGNH and QZX-AGNH on ATP and synapsinⅠin cortex and brainstem after LPS injection. 5.1 Effect on expression of synapsinⅠand SynapsinⅠmRNA in cortexExpression of synapsinⅠdecreased in DG region of hippocampus 6h after LPS injection. AGNH could increase the expression of synapsinⅠ, QZX-AGNH could not, comparing with AGNH, P<0.05. ZS+XH, ZS could also increase synapsinⅠexpression, compared ZS with AGNH, P<0.05. ZS could also increase synapsinⅠmRNA expression.5.2 Effect on activity ATPase in cortex and brainstemAfter LPS injection, the activities of Na+-K+-ATP and Ca2+-ATP decreased slightly. AGNH could increase the activities of Na+-K+-ATP in cortex and brainstem, and QZX-AGNH and XH could only increase the activities of Na+-K+-ATP in brainstem.Summary: AGNH could increase the activity of Na+-K+-ATP in cortex and brainstem which inhabited by LPS, increase the expression of synapsinⅠ. QZX-AGNH could raise the activity of Na+-K+-ATP in brainstem, without improving on synapsinⅠexpression, comparing with AGNH, P<0.05. XH could increase the activity of Na+-K+-ATP in brainstem and ZS increase synapsinⅠexpression in cortex. ZS and XH made its role respectively in improving ATP activity and synapsinⅠexpression of AGNH.Conclusion: The power and relative power of beta-waves of EEG decreased in brain injured rats caused by ischemia and LPS injection, the relative power of delta-waves increased. Disorder of Ach and monoamine transmitter, injury of synapse structure was maybe the base of the brain injury. AGNH could increase the power and relative power of beta-wave, inhabit delta relative power and make EEG activation. It raised Ach content, inhabited ChAT, increased the affinity of M-R, increased Gsαand made Gsα/Giαup-regulation, improved the function of Ach. It inhabited the increase of NE, improved 5-HT metabolism and improved monoamine transmitter function, protected pyramidal cell and synapse structure. So through above mechanism, AGNH could increase the function of Ach and monoamine transmitter, inhabit brain injury, then made EEG activation, improve the function of brain, and promoted consciousness recovery. QZX-AGNH was different from AGNH in the following effects: EEG activation, ChAT content, the affinity of M-R, the contents of NE and DA, synapse cleft, PSD, synapsinⅠexpression. ZS and XH were same as AGNH in above effects. So, ZS and XH are important in the promoting consciousness recovery of AGNH.Innovation:In this experiment, through observation of EEG activation of AGNH and QZX-AGNH in different brain injured rats, the effect of AGNH in improving brain function were reported, and ZS, XH make an important role in the effect.By studying on the mechanism of AGNH improving brain injury, it is prove that ZS and XH are correlated to Ach and monoamine transmitters improving of AGNH in brain injury. [Key words] Angongniuhuang; Zhusha; Xionghuang; EEG; Ach; monoamine transmitters; brain injury...