| Objective:Rhubarb is a blind application of a wide range of traditional Chinese medicine, one of the province four rare herbs, with a variety of active ingredients and a wide range of pharmacological effects, have been applied to the clinical variety of critical illness life-saving treatment. The rhubarb pharmacological active ingredient of anthraquinones, including emodin, ether, chrysophanol, rhein, aloe emodin, rhubarb glycosides such as. The plateau region is cold, hypoxia, dry, high ultraviolet radiation, can easily result in high altitude hypoxic brain damage. Combined with the Northwest economy, transportation, backward, and local health resources are limited, treatment difficulties; the characteristics of high-altitude hypoxic injury where the development of a new plateau hypoxia brain injury and brain edema is more efficient and effective drug has important practical significance and significant social and economic benefits. Studies have shown that relieve the nerve cells again ischemia emodin (emodin) interleukin-1β (IL-1β), tumor necrosis factor-alpha (TNF-a) expression[1]; intercellular adhesion molecule-1in (ICAM-1) decreased by inhibiting of the cysteine protease-3(caspase-3) activation and activity, inhibit the adhesion of leukocytes on the vascular endothelial reduce the inflammatory response, so that the narrowing of intracerebral hemorrhage surrounding tissue degeneration and necrosis area, to reduce the role of neuronal apoptosis and neuronal cell protection [2]. Chrysophanol can enhance blood glutathione peroxidase (GSH-PX), and superoxide dismutase (SOD), vitality, anti-ischemic injury[3]; can reduce the mouse brain of nitric oxide (NO) content and nitric oxide synthase (of nitric oxide, synthase NOS) activity, and significantly improved the scopolamine induced spatial learning and memory impairment[4]; can significantly reduce cerebral ischemia and reperfusion in mice brain H2O2content, and markedly increase cerebral ischemia and reperfusion in mice brain through catalase (catalase, CAT), vitality [5]. Multi-target therapeutic effect of rhubarb extract plays an important role in the protection of nerve cells after traumatic brain injury, nerve cell damage caused by a variety of trauma from the multi-factor, multi-link, multi-channel play a protective role, particularly suitable for the treatment of neurosurgery diseases [6]. Therefore, the study emodin and chrysophanol, have broad prospects for development of anti-nervous system damage in Chinese medicine, new drugs and compound drugs. The topics to be adopted rhubarb extract emodin the chrysophanol, the system of the cerebral protective effects on nerve cells in the hypoxia model, to provide a theoretical basis for the further development made reagents for clinical Methods:PC12cells are rat adrenal medulla chromaffin cell tumor differentiation of strains, with the neuron characteristics, widely used in neural physiological and pharmacological studies [7]. This paper simulates the nerve cell damage model of the pathological process of PC12cells hypoxia induced nerve cell damage model; to observe emodin and chrysophanol on the protective effect of hypoxia in PC12cells. MTT assay PC12cell proliferation activity changes and observed by light microscopy in PC12cells morphological changes, fluorescence microscopy oil immersion lens to observe the morphological changes; determination of the content of the supernatant of lactate dehydrogenase (LDH) and superoxide dismutase (SOD); the expression of BCL-2and c-fos immunofluorescence and by RT-PCR, analysis of the expression of nNOS, iNOS, p38MAPK, and Caspase-3mRNA’s.Results:(1) tetrazolium salt (the multiply-table tournament, MTT) and determination of cell viability results:emodin experimental group:control group, cell viability increased with time and increased vitality; cell viability of the model group with the prolongation of hypoxia time significantly decreased cell survival (model group, the OD/control group, the OD×100%) decline; cells after hypoxia emodin group activity enhanced compared with the model group, cell viability (emodin group OD/control group OD×100%) increase. There are significant differences between the two. The emodin enhanced cell viability, has a protective effect on hypoxic cells. Emodin monomethyl ether the5μg/ml group in decline in cell viability and survival of hypoxic late larger flavin ether1μg/ml group; the chrysophanol experimental group:control group, cell viability increased with time and increased vitality; cell viability of the model group with prolonged hypoxia significantly decreased cell survival (model group OD/control group, down from the OD×100%); chrysophanol group hypoxic cell vitality compared with model group, enhanced cell survival (of chrysophanol group OD/control group OD×100%) increase. There are significant differences between the two. Chrysophanol can enhance cell viability, and has a protective effect on hypoxic cells. Chrysophanol1μg/ml in an increase in cell viability and survival of hypoxic medium-term larger yellow phenol5μg/ml(2) lactate dehydrogenase (LDH) and superoxide dismutase (SOD), determination of results:in PC12cells after hypoxia, LDH was significantly increased, the emodin group and chrysophanol group compared with model group LDH was significantly reduced; in PC12cells after hypoxia SOD significantly reduced, emodin group and the the chrysophanol group compared with model group SOD was significantly higher.the emodin ether5μg/ml and emodin of1μg/m was no significant difference; the chrysophanol5μg/ml and Chrysophanol,1μg/m and no significant difference(3) B-cell lymphoma/leukemia-2(Bcl-2) and c-fos immunofluorescence results: Emodin groups:after hypoxia at all time points both Bel-2expression. Control group maintained at about30%; model group in lh positive rate of33.33%,2h positive rate was30.87%,6h positive rate of37.69%, the12h positive rate was43.33%, the24h positive rate was53.33%; emodin1ug/ml1h positive rate was32%,2h positive rate was40.87%,6h positive rate of57.69%,12h positive rate of53.33%, the24h positive rate was63.33%, emodin5ug/ml1h positive rate was38.70%,2h positive rate was35.20%,6h positive rate of33.26%,12h positive rate of45.73%, the24h positive rate was64.35%.Chrysophanol group:hypoxia at various time points are the Bcl-2protein expression. Control group the positive rate was12.94%,17.78%positive rate of model group,1h,2h positive rate was12.63%,6h positive rate of18.89%,12h positive rate of25.00%, the24h positive rate was23.93%; chrysophanol1ug/ml1h the positive rate was19.02%,2h positive rate was29.17%,6h positive rate was43.45%,12h positive rate was57.14%, the24h positive rate was49.16%; the chrysophano;5ug/ml1h positive rate was17.39%,2h-positive rate26.67%,6h positive rate was39.52%,12h positive rate was39.29%,24h-positive rate of45.45%.Emodin groups:hypoxia at various time points are c-fos protein expression. Positive control group was13.99%shown in Figure (2).36.19%positive rate of model group,1h,2h positive rate was40.48%, the6h positive rate was45.83%,12h positive rate was63.89%, the the24h positive rate of73.61%shown in Figure (3); emodin1μg/ml1h the positive rate was34.09%,2h positive rate was35.26%,6h positive rate was17.13%,12h positive rate was14.85%,24h-positive rate of46.10%shown in Figure (4); emodin5μg/ml1h positive rate2h-positive rate was25.20%14.54%, the6h positive rate was14.81%, the12h positive rate was16.24%, the24h positive rate was46.51%;Chrysophanol group:each time point after hypoxia are the c-fos expression. Control group positive rate of29.44%,41.11%positive rate of model group,1h,2h positive rate was47.94%, the the6h positive rate of51.85%the12h positive rate of56.70%, the24h positive rate was65.00%, the rhubarb phenol1μg/ml1h positive rate32.22%,2h positive rate was29.17%,6h positive rate of15.63%,12h positive rate of29.81%, the24h positive rate was41.67%, the chrysophanol5ug/ml1h positive rate was34.72%,2h positive rate26.67%the6h positive rate of22.07%, the12h positive rate was21.14%, the24h positive rate was37.90%.(3) reverse transcription-polymerase chain reaction (the reverse transcription polymerase chain reaction, by RT-PCR) results:all groups extracted total RNA value (the A260/A280) in the range of1.05to4.228, indicating that total RNA was no degradation The purity test requirements; of GAPDH reference gene dissolution curve of normal distribution, no degradation of the measured total RNA. Real Time-PCR results showed that: of nNOS in iNOS in gene expression between emodin Group:hypoxic different periods:(1) control group of nNOS in visible trace, iNOS mRNA expression;(2) model group of nNOS in hypoxia lh and iNOS mRNA expression increased more significantly, the model group and control group and emodin group significant difference (p<0.05).(3) model group, hypoxia2h nNOS, iNOS mRNA were higher than that nNOSmRNA model group and control group, and emodin group significant difference (p <0.05), model group, the most obvious expression of nNOS mRNA.(4) model group, hypoxia6h, nNOS mRNA expression started to decline, of iNOS mRNA expression was significantly increased, the model group and control group and emodin group have significant difference (p <0.05);(5) hypoxia12h to the model group and iNOS in mRNA were higher and emodin group, the comparison between the two significant differences (p<0.05), but the expression of nNOS mRNA decreased significantly compared with the control group and emodin ether group showed no significant difference (p>0.05);(6) model group, hypoxia for24h, the expression of iNOS decrease the model group and emodin of group were higher than the two more significant difference (p<0.05). But model group emodin of group no significant difference (p>0.05). nNOS mRNA expression of no significant change, and normal group emodin of group no significant difference (p>0.05); hypoxia in different periods between the groups of p38MAPK and Caspase-3mRNA gene expression:(1) control group showed trace expression of p38MAPK and Caspase-3mRNA;(2) hypoxia lh group of Caspase expression-3mRNA, increase the amount of difference between model group and normal group and emodin group was statistically significant (p>0.05).(3) hypoxia for2h of p38MAPK model group were higher, p38MAPKmRNA model group and control group, and emodin group difference was statistically significant (p<0.05).(4) hypoxia for6h, expression of p38MAPK and Caspase-3mRNA continue to increase, model group and control group and emodin group difference was statistically significant (p<0.05);(5) hypoxia12h of p38MAPK expression significantly the amount of Caspase-3mRNA model group were higher than, and emodin group, the difference being statistically significant (p<0.05);(6) hypoxia for24h, p38MAPK and caspase-3mRNA the expression of the model group were higher, the difference was statistically significant (p<0.01) and Caspase-3mRNA model group emodin of group difference was not statistically significant (p>0.05);The chrysophanol group:hypoxia in different periods of gene expression:(1) control group were seen of nNOS in the expression of iNOS in mRNA in trace;(2) of nNOS in hypoxia lh group, iNOS mRNA expression increased, the model group and control group and rhubarb phenol group difference was statistically significant (p<0.05).(3) hypoxia for2h of nNOS in iNOS mRNA model group were higher than that of nNOS in mRNA in model group and control group and chrysophanol group difference was statistically significant (p<0.05), model group, the nNOS mRNA to express the most obvious.(4) hypoxia for6h, nNOS expression levels began to decline significantly expressed, iNOSmRN A expression of the model group and control group and chrysophanol group difference was statistically significant (p<0.05);(5) lack of oxygen for12h, iNOS mRNA in model group was higher in model group and control group and chrysophanol group difference was statistically significant (p<0.05), but significantly decreased nNOS mRNA expression, with the control group and rhubarb phenol group was no significant difference (p>0.05);(6) hypoxia for24h, iNOS mRNA in model group were higher, the difference was statistically significant (p<0.01). model group and chrysophanol group difference (p>0.05) of nNOS in mRNA expression showed no significant change compared with the control group and chrysophanol group was no significant difference (p>0.05); hypoxia in different periods groups p38MAPK and Caspase-3mRNA expression:(1) control group were seen p38MAPK and caspase-3mRNA in micro-expression;(2) model group increased p38MAPK and caspase-3mRNA expression in hypoxic1h is not obvious, the model group control group and chrysophanol group showed no significant difference (p>0.05).(3) hypoxia for2h p38MAPK and Caspase-3mRNA in model group were higher than that of p38MAPK model group and control group and the group of chrysophanol significant difference (p<0.05).(4) hypoxia for6h, p38MAPK and caspase-3mRNA expression continue to increase, model group and control group and chrysophanol group there was significant difference (p<0.05);(5) hypoxia12h of p38MAPK expression level of the the Caspa se-3mRNA in model group were higher and chrysophanol group, there was significant difference (p<0.05);(6) hypoxia for24h, p38the MAPK expression of model group were higher than and chrysophanol group, There was a significant difference (p<0.01), the expression of Caspase-3mRNA in model group were higher, more significant differences (p<0.01). The model group and chrysophanol group no significant difference (p>0.05).Conclusion:The model of this lack of oxygen can cause the phenomenon of apoptosis in PC12cells. the emodin and chrysophanol can reduce PC12cells injured by hypoxia has a protective effect on neurons. |
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