The Neuroprotective Effects And Mechanisms Of ZGF-2-130on Ischemic Neuronal Death

High incidence of cerebrovascular disease seriously harm human health. Stroke, the most common life-threatening disease of the nervous system, is currently the second largest cause of death and the first cause of disability. Cerebral ischemic injury-induced neuronal death is the main reason leading to loss of neurological function. The mechanism of ischemic brain damage has not been clarified and there is a lack of clinically effective therapies. While pharmacological therapy to reduce ischemic damage is being pursued, prevention and rehabilitation are still the only strategies.The brain damage produced by cerebral ischemia maturates over a period of several hours or days. Especially in global ischemia, a delayed hippocampal damage is observed3days or so after the insult in CA1pyramidal neurons, suggesting that mechanisms that develop slowly after ischemia have a role in ischemic cell death, including apoptosis, excitotoxicity and inflammation. The continued ischaemic injury to brain cells results in to a complex inflammatory cascade. It is characterized by infiltration of leukocytes mainly polymorphonuclear (PMN) cells, monocytes/macrophages lymphocytes, the activation of microglia and activated astrocytes release proinflammtory cytokines and chemokines. In cerebral ischaemia, microglia, resident brain macrophages become activated and release detrimental neurotoxic mediators like proinflammtory cytokines, superoxide, NO, TNF-a and proteases. Which contribute to the progression of ischaemic damage. At present, Inflammation is considered a major component in disseminating the detrimental effects of cerebral ischaemia. Therefore, antiinflammation can prove to be valuable drugs in attenuating CNS damage following ischaemia.Microglial cells are the resident macrophages of the brain and play a critical role as resident immunocompetent and phagocytic cells in the CNS. Excessive activation of microglia can cause too much pathological damage of CNS. Appropriate inhibiting its activation or against microglia which produces neurotoxicity molecules, can reduce further cytotoxic effects and pathological damage. But necessary intervention should be performed in early stage of microglia activation, namely the organization is in a reversible phase damage. According to this stage, exploration to the antiinflammatory drugs are already showing the preliminary results, Such as, acetylcholine receptor agonists, glucocorticoid, IL-1antagonists, NO inhibitors, Chinese traditional medicines, and immunity inhibitors, mitogen-activated inhibitors, antibiotics. Minozac(Mzc) is a bioavailable, brain-penetrant, small molecule experimental therapeutic. The mechanism of Mzc action is selective reduction of excessive proinflammatory cytokine production by activated glia back towards basal levels, and improves synaptic dysfunction and neurobehavioral impairment in mouse model of A lzheimer disease (AD). ZGF-2-130is the derivate of Mzc. Base on the above the results, we employed rat model of global ischemia to study whether ZGF-2-130has neuroprotective effects on delayed neuronal death in cerebral ischemia rat and its mechanism. Transient cerebral ischemia was performed by occluding4major arteries suppllying the brain i.e., the bolateral vertibral and common carotid arteries, modifying the original method by Pulsinelli on adult male wistar rats(body weight230±20g). The results showed that pretreatment of ZGF-2-130with low, middle and high dose (lmg/kg/time,5mg/kg/time,12.5mg/kg/time,2times/day for7days)2hours before ischemia are neuroprotective against ischemic stroke. The number of survival neurons in hippocampal CA1area was46.22±5.29cell/mm in the group of ZGF-2-130with1mg/kg/time, higher than that in saline treated group(19.40±2.43cell/mm)(P<0.05), there was significant difference compared with sham(277.57±5.48)(P<0.01). The number of survival neurons in hippocampal CA1area was109.09±10.99cell/mm and97.43±8.70cell/mm in the group of ZGF-2-130with5mg/kg/time and12.5mg/kg/time, the rate of the number for the sham was39.30%and35.10%, compared with saline treated groups, there was significant difference(P<0.01). The above results showed that ZGF-2-130could protect the neurons from death.In order to determine the effect of ZGF-2-130on microglia inflammatory reaction induced by reperfusion after ischemia, we observed the intensity of microglia(OX-42positive cells) in the CA1pyramidal cell layer with same drug administration at2h before15-minute global ischemia. In sham operated rats, the reaction intensity and number of microglia in the CA1pyramidal cell layer was very weak, whereas the intensity and number of microglia in CA1was highest in ischemia group. ZGF-2-130with different dose applied to ischemic rats can inhibit the activation of microglia in different degree.In order to further study whether ZGF-2-130has a direct antiinflammtory effects on astrocytes, according to the above dose and time of administration, we observed the intensity and number of astrocyte(GFAP positive cells) in the CA1pyramidal cell layer. The results showed that the saline-treated group had a2.72time increase compared to sham group(P<0.05). The results showed that the number of astrocyte in CA1applied with pretreatment of ZGF-2-130(1mg/kg/time,5mg/kg/time,12.5mg/kg/time,2times/day for7days)2hours before ischemia were53.63±3.54cell/mm2,54.60±2.74cell/mm2and55.80±2.88cell/mm2. There were significant difference between saline-treated rats, drug-treated rats and sham(P<0.01). But no difference was found between drug-treated rats and saline-treated rats. So drug administration before and after ischemia did not affect the intensity and number of astrocyte in the CA1.At the same time, in order to testify the function of left neurons or not, we adopted Morris water maze to detect the effect of ZGF-2-130on spatial learning and memory in rats after ischemia/reperfusion. The experiments were performed on the9th days. Results showed that compared to the control ischemic group, those treated with5mg/kg of ZGF-2-1302h before ischemia/reperfusion had the trend of an improved spatial learning and memory (P=0.07).However this above prevention is not suitable on clinical therapy, it is not humanistic to apply ZGF-2-130on healthy people before they are subject to stroke. Therefore we observed ZGF-2-130’s neuroprotective effects at different time after ischemia against reperfusion. ZGF-2-130-posttreated rats(5mg/kg, i.p. twice one day for7days) at30min,6h after ischemia had24.40%(67.06±6.75cell/mm),16.81%(46.21±4.72cell/mm) of the neuron profiles left in the CA1pyramidal cell layer respectively. The neuroprotection was statistically significant in every animal group (P<0.01), and the effect of ZGF-2-130is dose-dependent. Treatment of the same dose of saline did not provide any protection. Therefore, the earlier posttreatment of ZGF-2-130after ischemia, the better neuroprotection.Whereas7days after ischemia, the number of microglia was increased to13.33 times compared to sham goup (23.57±1.47cell/mm2). ZGF-2-130-posttreated rats at30min,6h after ischemia had4.96times (197.25±7.91cell/mm2),3.33times(236.51±7.71cell/mm2) of the microglia profiles left in the CA1pyramidal cell layer respectively. Treatment of the same dose of saline did not provide any anti-inflammation effects. Therefore, the earlier posttreatment of ZGF-2-130after ischemia, the better anti-inflammation effects. The anti-inflammation effects of ZGF-2-130are still statistically significant even applied6h after ischemia. Based on the above results, ZGF-2-130could inhibit the increased number of microglia induced by reperfusion after ischemia.In order to further study whether posttreatment of ZGF-2-130has a direct antiinflammtory effects on astrocytes, according to the above dose and time of administration, we observed the intensity and number of astrocyte(GFAP positive cells) in the CA1pyramidal cell layer. The results showed that the saline-treated group had a2.3time increase compared to sham group (P<0.01). The results showed that the number of astrocyte in CA1applied with posttreatment of ZGF-2-130at30min and6h were61.63±4.31cell/mm2,60.83±2.55cell/mm2. There were significant difference between saline-treated rats, drug-treated rats and sham(P<0.01). But no difference was found between drug-treated rats and saline-treated rats. So drug administration after ischemia did not affect the intensity and number of astrocyte in the CA1. Furthermore, in order to testify whether the ZGF-2130affected the expression of specific marker GFAP for astrocyte in CA1. Western bloting showed that the expression of GFAP in saline-treated rats was greatly enhanced, the increase was significant compared with sham group (P<0.01).30min and6h after ischemia, the rate of expression of GFAP was1.69time,1.72time compared to sham. There were significant between the drug treatment and sham (P<0.01). But no significant differences were found between drug treatment and saline-treated group (P>0.05). ZGF-2-130had no effect on the expression of GFAP. Taken together, ZGF-2-130could not obviously inhibit the inflammatory reaction of astrocyte induced by reperfusion after ischemia in hippocampal CA1area.In order to discuss whether ZGF-2-130affects NOS activity after ischemia, we observed the number of NADPH diaphorase positive cells in hippocampal CA1area. Treatment with5mg/kg dose of ZGF-2-130administered i.p. twice daily at30min(96.61±5.25cell/mm) and6h(131.10±7.43cell/mm) after ischemia/reperfusion could markedly reduce the number of NADPH diaphorase positive cells compared to saline treated group(149.33±5.31cell/mm)(P<0.05). There were also significant difference between drug-treated group and sham(50.71±2.14cell/mm)(P<0.01). It is thus clear that the earlier posttreatment of ZGF-2-130after ischemia, the more obvious decrease in NADPH diaphorase positive cells, which resulted in the greater inhibit of NOS activity.In order to further study the mechanisms of ZGF-2-130inhibiting the activity of microglia. We tested whether ZGF-2-130affected the expression of iNOS mRNA. RT-PCR results showed that3days after ischemia expression of iNOS mRNA were attenuated by39.4%in the hippocampus of ZGF-2-130-treated rats.In order to test the results in vivo, we also observed the effect of ZGF-2-130on the activation of in vitro BV2cells and production of inflammatory cytokines, BV2stimulated by LPS was established as the cell model of inflammation. We first observed the effect of ZGF-2-130on cell viability of BV2cells. The results showed that ZGF-2-130at0-20nM had no effect on the BV2cells. But ZGF-2-130at200nM could result in the remarkedly toxicity(P<0.01). At the same time, the soma of BV2showed pectination or rhabditiform, and cells extended the neurite. But LPS could stimulated the BV2and made the soma of cells expanded, showed spherical and neurite shrinked; MTT result demonstrated that ZGF-2-130(0-20nM) also did not affected the activation of BV2induced by LPS(P>0.05). ELISA and RT-PCR showed that ZGF-2-130could decreased the expression of LPS-induced IL-1β and TNF-α protein and mRNA (P<0.05).Many reports have found that the signal transduction mediated by MAPK has a relationship with the generation of proinflammatory factors. To further study the mechanism of ZGF-2-130inhibiting the production of inflammatory cytokines, we observed whether ZGF-2-130influenced the activities of p38、ERK and JNK via western blotting in cultured BV2cells. Results demonstrated that15min after LPS stimulation, the phosphorylation of p38were significantly enhanced, maximum at30min after LPS stimulation, and were restored to normal1h after LPS. The phosphorylation of ERK and JNK remained increased throughout0-15min after LPS. Western Blot showed that the phosphorylation of p38and ERK kinase can be decreased by treatment with ZGF-2-130(0.2-20nM) at15min after LPS. These results suggest that the inhibition of ZGF-2-130on inflammatory cytokines might be due to a decrease in the activity of p38and ERK kinase.In conclusion, ZGF-2-130could increase the number of neurons left in the CA1pyramidal cell layer after ischemia. The mechanism may decrease the activity of microglia in the hippocampal CA1and generation of inflammatory cytokines via inhibiting the activity of p38MAPK and ERK.