| So far a variety of theories about the pathogenic mechanism of carbon monoxide poisoning have been put forward, such as the blood anoxic theory, the free radical damage theory, the cell apoptosis theory, the blood vessels factors theory, immune injury theory et al, each of which may explain the brain damage caused by carbon monoxide reasonably in different ways. However, none of them can illustrate the exact mechanism thoroughly. Recently, more and more research shows that the neural cell apoptosis plays an important role in the pathological process of brain damage caused by carbon monoxide. Thus, research about the mechanism of neural cell apoptosis after carbon monoxide poisoning will provide the basic theory for the future clinical treatment.Xingnaojing (XNJ) Injection refined from Angongniuhuang Pill, which has the essential component including musk, borneol, gardenia and turmeric et al, has a significant effect on a variety of emergencies on central nervous system. The study suggests that XNJ Injection could inhibit neural cell apoptosis caused by traumatic brain injury apparent, as well as hypoxia and ischemia-reperfus ion. In our study, the effect of XNJ Injection on neural cell apoptosis caused by carbon monoxide releasing molecules and the possible mechanism were investigated.1. Model building of neural injury and preliminary study on XNJ Injection in protecting brainThe aim of this part is to build the model of neural cell injury caused by carbon monoxide releasing molecules and to discuss the protection effect of XNJ Injection. MTT test is carried out to determine the suitable concentration of carbon monoxide releasing molecules in inducing neural cell injury, and then to observe the protection effect of XNJ Injection. The study shows that neural cell induced by carbon monoxide releasing molecules-CORM-24h,8h,12h later, the survival rates of each group is not significantly impacted. However, compared to the control group, the survival rates of each group of CORM-2with different concentrations have declined after injury lasting for24h. The higher concentration of CORM-2is used, the lower cell survival rates are seen. In view of the number of living cells by MTT, concentration of CORM-2with200μmol·L-1is selected in the following experiments. Observation suggests that survival rates of each XNJ group have risen compared to model group after incubation for20h with XNJ Injection of different doses. Compared with model group, XNJ middle-dose group (10mL·L-1) and high-dose group (20mL·L-1) shows significant statistical difference (P<0.01). These results suggest XNJ Injection has a certain effect on protecting neural cell injury induced by CORM-2.2. Detection on cell apoptosis by Annexin V-PIThis part aims at observing on the rate of neuronal apoptosis induced by CORM-2and finding intervention effect on neuronal apoptosis of XNJ Injection with different doses. Annexin V-PI method is applied to detect apoptosis rate of early and late stage induced by CORM-2and the intervention effect of XNJ Injection. The study indicates that100umol·L-1,200μmol·L-1,400μamol·L-1and800μmol·L-CORM-2group could induce neuronal apoptosis. With the concentration of CORM-2increasing, the overall rates of neuronal apoptosis also increase. XNJ middle-dose group (10mL·L-1) and high-dose group (20mL·L-1) can lower neuronal apoptosis of early stage, which shows significantly different compared to model group (P<0.01), but the effect of these two groups is not obvious in the neuronal apoptosis of late stage. Compared with the model group, the difference is not statistically significant (P>0.05). The results suggest XNJ Injection could intervene neuronal apoptosis induced by CORM-2.3. Neuronal apoptosis induced by CORM-2and the mechanism of intervention effect of XNJ InjectionThis part of study is to investigate the mechanism of neuronal apoptosis induced by CORM-2and to explore the the mechanism of intervention effect of XNJ Injection. Western Blot method is used to observe whether caspase-dependent pathway play a role in the process of neuronal apoptosis induced by CORM-2and to study whether XNJ Injection inhibit neuronal apoptosis through caspase-dependent pathway. The research finds that compared to the control group,100μmol·L-1,200μmol·L-1,400umol·L-1and800μmol·L-1CORM-2group could increase the expression of caspase-3, caspase-9and Cyt C, all of which are key proteins in caspase-dependent mitochondrial pathway. Along with the rising concentration of CORM-2, the expression of caspase-3, caspase-9and of Cyt C is all on a rising trend. However, caspase-8and caspase-12, the two key proteins in caspase-dependent death receptor pathway and endoplasmic reticulum respectively, is not expressed, which indicates that CORM-2maybe induce neuronal apoptosis by activating caspase-dependent mitochondrial pathway. XNJ Injection with doses of10mL·L-1and20mL·L-1could decrease the expression of caspase-3, caspase-9and Cyt C significantly, and as the dose increasing, the expression of all these indexes gradually reduce, which shows XNJ Injection maybe intervene neuronal apoptosis induced by CORM-2through mitochondrial pathway.To sum up, XNJ Injection has a certain neuroprotective effect, which could inhibit the neuronal apoptosis induced by CORM-2, and it maybe intervene neuronal apoptosis by Caspase-dependent mitochondrial pathway. |
PDF Full Text Request