| ObjectiveAcute alcoholism refers to the excessive intake of ethanol at a time,so that the absorption of ethanol is greater than the metabolism,resulting in poisoning symptoms such as coma.According to the World Health Organization,the number of deaths caused by alcohol consumption(including those caused by alcoholism and drunk driving accidents)has reached 2.5 million worldwide each year,which has a serious impact on individuals,families and society.Therefore,it is of great and important practical significance to seek effective hangover drugs.Xingnaojing Injection(XNJI)is derived from the classic prescription AnGong NiuHuang Pill in Wen Bing Tiao Bian written by Wu Jutong.It is mainly composed of four traditional Chinese medicines,namely Moschus,Borneolum,Curcumae radix and Gradeniae fructus.It has the characteristics of exact curative effect and wide application,and is often used in clinical treatment of various coma syndromes,such as alcohol coma.Alcohol is mainly metabolized by the liver,in which acetaldehyde and reactive oxygen species can induce oxidative stress and cause liver damage.Alcohol has inhibitory effect to central nervous system.Ethanol absorbed into the blood can enter the brain through blood-brain barrier,causing dysfunction of the body,resulting in multiple dysfunctions such as memory cognition and motor coordination.In this study,an animal model of acute alcoholic coma was established by intraperitoneal injection of 34%ethanol,and three different doses of XNJI were administered to observe the activities of ethanol metabolic enzyme,oxidative stress reaction and pathological changes in rat liver.In addition,microdialysis experiments were conducted to observe the dynamic changes of adenosine(AD)and Orexin A in the inhibitory-excitatory neurotransmitters,and the mRNA expression levels of the receptors and proteins involved in the two.In this paper,microdialysis,molecular biology and other techniques were used to study the mechanism of XNJI's awakening and nerve action on alcoholic coma,laying a foundation for the development of more effective hangover drugs.Methods1.Effects of XNJI on ethanol metabolic enzymes and oxidative stress in the liver of ethanol-induced coma ratsIn this experiment,34%ethanol was used to establish a rat model of ethanol-induced coma,and the effect of XNJI on the duration of Loss of the righting reflex(LORR)in rats with ethanol-induced coma was studied.In order to investigate the effect of XNJI on ethanol metabolic enzymes and oxidative stress in the liver of ethanol-induced coma rat,the kits were used to determine the activities of Alcohol dehydrogenase(ADH),Aldehyde dehydrogenase(ALDH),Superoxide dismutase(SOD)and the content of Glutathione(GSH),Malondialdehyde(MDA)in rat liver.Hematoxylin-eosin staining(HE)was used to observe pathological changes in rat liver.2.Optimization of conditions for determination of adenosine in artificial cerebrospinal fluid by high-performance liquid chromatographyIn this experiment,high-performance liquid chromatography(HPLC)was used to determine the peak area and retention time of AD at different detection wavelengths and different mobile phase ratios.The HPLC chromatographic conditions of AD were optimized to maximize absorption.It provides a reliable method for the determination of AD in artificial cerebrospinal fluid.3.The effect of XNJI on the awakening nerve mechanism in ethanol-induced coma ratsThe microdialysis technology were used in this experiment to collect rat cerebrospinal fluid.The AD level in cerebrospinal fluid(CSF)was determined by HPLC combined with UV detector,and the orexin A level in CSF was determined by enzyme-linked immunosorbent assay(ELISA)to investigate the dynamic changes of AD and orexin A in the hypothalamus CSF in ethanol-induced coma rats.The mRNA expression levels of OX1R,AiR,ENT1 and ADK in the hypothalamus of rats were determined by fluorescence quantitative PCR(qPCR).Result1.Effect of XNJI on ethanol metabolic enzymes in liver of ethanol-induced coma ratsCompared to the control group,the activity of ADH and ALDH in the liver of the model group decreased significantly(P<0.01).LORR(2.92±0.24h)in the medium dose XNJI group was shorter than that in the low dose group(3.92 ±0.68h)and the high dose group(3.59 ± 1.16h),and was statistically significant(P<0.01)as compared to that in the model group(4.26±0.56h)while the other groups were not.The activity of ADH and ALDH in rat liver was increased in both the medium and high dose groups of XNJI(P<0.01),while the activity of ADH and ALDH was not increased in the low dose group of XNJI(P>0.05).2.Effect of XNJI on oxidative stress in liver of ethanol-induced coma ratsCompared to the control group,SOD activity in the liver of the model group was significantly decreased(P<0.01),while MDA content was significantly increased(P<0.01)and GSH content was not significantly changed(P>0.05).Compared to the model group,SOD activity was significantly increased(P<0.01)and MDA content was significantly decreased(P<0.01)in the low-dose XNJI group,while GSH content was not significantly increased(P>0.05).SOD activity and GSH content in XNJI medium dose group were increased(P<0.01),and MDA content was decreased(P<0.01).In the high-dose group of XNJI,GSH content was effectively increased and MDA content was decreased(P<0.01),but SOD activity was not significantly changed(P>0.05).XNJI can improve liver cell cytopathic,reduce hepatocyte edema and maintain liver cell morphology to be normal.3.Optimization of chromatographic conditions for the determination of AD by HPLCThe retention time of AD at the wavelength of 250-260 nm was about 5.9min,and the peak area of AD increased with the increase of wavelength.The ratio of methanol to 8 mmol/L NaH2PO4 had little effect on the peak area of AD,but had a significant effect on its retention time.As the proportion of methanol increases,the retention time of AD was shortened.The optimal HPLC condition for AD was mobile phase A:mobile phase B=20%methanol:8 mmol/L NaH2PO4,and the detection wavelength was 260nm.4.Effect of XNJI on AD and orexin A levels in CSF of ethanol-induced coma ratsCompared to the control group,adenosine levels in the model group were significantly higher from 135min to 270min(P<0.01).Compared to the model group,adenosine levels in the low-dose and high-dose XNJI groups showed a downward trend,but no statistical significance(P>0.05).Adenosine levels in the medium-dose XNJI group were all lower than those in the model group within 0 min to 270 min,and the adenosine levels in 135min to 270min were significantly lower than those in the model group(P<0.05).Compared to the control group,the level of orexin A in the model group did not change significantly(P>0.05).Compared to the model group,no significant changes were observed in the low,medium and high dose groups of XNJI(P>0.05).5.The effect of XNJI on the expression levels of OX1R,A1R,ENT1 and ADK mRNA in the lateral hypothalamus of ethanol-induced coma ratsCompared to the control group,the OX1R and ENT1 mRNA of the model group showed a significant decrease,and the A1R expression increased significantly(P<0.05),while there was no significant difference in ADK.Compared to the model group,the OX1R expression was significantly upregulated in the low,medium and high dose groups of XNJI(P<0.05),while the A1R expression was effectively downregulated(P<0.05).For ENT1,the expression was significantly decreased in the medium dose group of XNJI(P<0.05),but not in the low and high dose groups(P>0.05).However,for ADK,the expression in the high-dose group of XNJI was significantly upregulated(P<0.01),but not in the medium-dose and high-dose groups(P>0.05).ConclusionXNJI has awakening effect on ethanol-induced coma rat,and its mechanism may be related to excitatory neurotransmitter orexin A and inhibitory neurotransmitter adenosine and their related receptors OX1R,A1R.The regulation of proteins ENT1 and ADK mRNA expression is also related. |
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