The Study Of Revival Effect And Mechanism Of Xingnaojing Injection In Alcoholic Coma Animal Models

ObjectiveAlcohol abuse is increasingly serious in the worldwide, alcohol coma is severe phase of acute alcoholism, promoting the patient to revive as soon as possible is the key of treatment. Glutamate (Glu) and Gama-aminobutyric acid (GABA) is the most important pair of exciting and inhibited neurotransmitters, theirs function relationship accordance with traditional Chinese medicine theory of "Ying Yang", ethanol makes the "Ying and Yang unbalance" mainly by injuring the neural exciting and inhibited functions in acute alcoholism. This project uses technologies of microdialysis, electrophysiology and molecular biology, to select emergency proprietary Chinese medicine-Xingnaojing Injection (XNJI) which is definite effect, wide application and originate from classic famous formulae Angong Niuhuang Pill, to research the promoting wakefulness mechanism of XNJI in treating alcohol coma model whether involved with reinforcing the exciting neural functions of Glu and confront inhibited neural function of GABA; to achieve the effect of resuscitation by balancing excited and inhibited neural functions in central nervous system. Giving clarification the neural mechanisms of alcohol coma and promoting wakefulness of XNJI, lay a foundation for research and development more effective promoting wakefulness drugs.Methods1. To establish quality control standards of XNJI by GC and HPLCDetermining the content of muscone, borneol, isoborneol and camphor, the main chemical constituent of XNJI by GC, determining the content of Curcuminoid and jiasminoidin by HPLC-UV.To Determin and compare sample from different manufacturers and different batch, establish quality control standards of XNJI.2. To study the effect of XNJI on alcohol metabolism and wake-promoting in alcoholic coma mouseBuilding alcoholic coma model, study the effect of XNJI on LORR of alcoholic coma mouse; Determining activity of ADH and ALDH by relevant kit; Determining ethanol concenteation in serum by GC, discuss he effect of XNJI on alcohol metabolism and wake-promoting in alcoholic coma mouse.3. Effect of different dose XNJI on EEG of alcoholic coma rat Recording EEG by electrophysiology, the ratio of δ wave was seen as digree and prognosis, to study effect of different dose XNJI on EEG of alcoholic coma rat.4. Effect of XNJI on content of Glu, GAB A and expression of revelent receptor NR2A^ NR2B and GABAAα2 in LHACollecting cerebrospinal fluid (CSF) by microdialysis, and determining content of Glu and GABA by HPLC-FLU; Determining expression of NR2A、NR2B and GABAAα2 in LHA by Real-Time PCR, to study effect of XNJI on content of Glu, GABA and expression of revelent receptor NR2A、NR2B and GABAAα2 in LHA.Results1. Establishment and validation of GC and HPLCExperiments established methods to determine content of muscone, borneol, isoborneol and camphor of XNJI by GC and to determine content of Curcuminoid and jiasminoidin by HPLC, analize samples from different manufacturers. Results we got presented that borneol, camphor and muscone of No.1 manufacturer is higher than No.2 and No.3, and the isoborneol can’t be detect in XNJI of No.2 and No.3, Curcuminoid and jiasminoidin of No.l and No.3 manufacturer is higher than No.2, in conclusion, we chose XNJI of No.1 for the later pharmacological effect experiments.2. Effect of XNJI on alcohol metabolism and wake-promoting in alcoholic coma mouseCompared to the control group, the activity of ADH and ALDH significantly reduced. Compared with the model group, XNJI 1.36g/kg,0.68g/kg,0.34g/kg group can significantly decreased LORR of alcoholic coma mouse (P<0.01), XNJI 1.36 g/kg,0.68 g/kg,0.34 g/kg group can significantly increased activity of alcoholic coma mouse s ADH(P<0.01) andXNJI 1.36 mg/kg group can significantly increased activity of alcoholic coma mouse s ALDH(P<0.05),XNJI 1.36 g/kg,0.68 g/kg,0.34 g/kg group can significantly decreased concentration of ethol in serum of alcoholic coma mouse(P<0.01).3. Effect of XNJI in alcoholic coma ratsCompared to the control group, the model group significantly increased the ratio of 8 wave(P<0.05).Compared to the model group, XNJI 0.96 g/kg,0.48 g/kg group significantly decreased the ratio of6 wave(P<0.05).4. Effect of XNJI on Glutamate and GABA in LHA of alcoholic coma ratCompared to the control group, the model group apparently decreased extracellular glutamate levels during 30 to 240 minute period. The injection of 1.36 mg/kg XNJI significantly increased extracellular glutamate levels during the 90 to 240 minute period compare with model group. The injection of 0.68 mg/kg XNJI significantly added extracellular glutamate levels during the 90,180 and 210 minute period. However, the injection of 0.34 mg/kg XNJI only increased extracellular glutamate levels during 210 minute period.Compared to the control group, the the model group significantly increased extracellular GABA levels during 30 to 240 minute period. The injection of 1.36 mg/kg XNJI significantly decreased extracellular GABA levels during the 60 to 240 minute period compare with model group. The injection of 0.68 mg/kg XNJI significantly decreased extracellular GABA levels during the 90 to 240 minute period. Moreover, the injection of 0.34 mg/kg XNJI also decreased extracellular GABA levels during the 90,180210 and 240 minute period.5. Effect of XNJI on expression of NR2A, NR2B and GABAA in LHACompared to the control group, the model group appeared a significant decrease in NR2Aand NR2BmRNAs express(P<0.01), and a significant increase in GABAα2 mRNAs express(P<0.01). When compared with the model group, group treated with XNJI (0.34, 0.68 and 1.36 mg/kg) induced a significant increase in NR2A and NR2B mRNAs (P<0.01) and decrease in GABAA mRNA (P<0.05).ConclusionXNJI can promote the level of consciousness in alcoholic coma model, whose mechanism may be related to its function of regulating exciting and inhibited neurotransmitters-Glutamate (Glu) and Gama-aminobutyric acid (GABA), and the expression of it s related recepter NR2A,NR2B an GABAA.