The Effect Of Astragaloside Ⅳ On Isoflurane-induced Neuro-apoptosis And Clinical Research Of Isoflurane Anesthesia On Cognitive Function

Part one:Astragaloside Ⅳ protects new born rats from anesthesia-induced neuro-apoptosis in the developing brainAnesthetics may damage brain during the synaptogeneic phase of neurodevelopment due to their property at the N-methul-D-aspartic acid(NMDA)andγ-aminoburyric acid(GABA)A receptors[1,2,3].The peak vulnerability window to the neurotoxicity is during synaptogenesis phase,also called brain growth spurt period[4,5].Such damage may affect the development of the neural network,and the effect can last to adult age for the behavior and recognition ability[6,7].These findings raised the concerns about the neurotoxicity of general anesthetics,particularly the drugs used in pediatric anesthesia,where human fetal brain may be damaged during the procedure[8].Neuron apoptosis is the main reason for the neurotoxicity,and the mechanisms of anesthesia-induced apoptosis have been well studied[5,9].Activation of pro-apoptotic proteins such as Bax,may lead to mitochondira membrane breakage and activation of caspases,which execute cell apoptosis.On the other hand,generation of free radicals such as reactive oxygen species(ROS)and reactive nitrogen species(RNS)leads to lipid peroxidation,which could cause brain damage.Such free radicals and damages may also elicit inflammatory responses,which further amplify the apoptotic response.By contrast,antioxidant enzymes such as superoxide dismutase(SOD),can scavenge excessive free radicals and alleviate their detrimental effect.Astragalosie Ⅳ(AS Ⅳ)is a saponin purified from a traditional Chinese herbal medicine component Astragalus membraneaceus(Fisch)Bunge[10,11].It showed antioxidant activity and anti-apoptosis function in various types of cells and tissues[12,13,14,15,16].For example,it ameliorates renal fibrosis by inhibiting MAPK activation and attenuating unilateral ureteral obstruction and TGF-β-induced renal tubular cell apoptosis[16,17].On the other hand,it can protect neuron cells from 1-methyl-4-phenylpyridnium ion-induced neurotoxicity by preventing ROS production and inhibition of Bax-mediated pro-apoptotic pathway[18].AS Ⅳ also reduced amyloid betal-42-induced neurotoxicity by inhibiting the mitochrondrial permeability transition pore opening[19].However,its role in anesthesia-induced neuro-apoptosis and related neurotoxicity has not been investigated.To determine if AS Ⅳ can protect developing neuron from anesthesia-induced apoptosis,we investigated the effect of AS Ⅳ pre-treatment on isoflurane-induced neuron apoptosis in hippocampus tissues of new born rats.The oxidative stress-related enzyme activities in hippocampus tissues and serum were quantified,and serum levels of pro-inflammatory cytokines were determined by ELISA.Protein expression levels of NF-κB,caspase-3,BCL-2,phosphorylated JNK,ERK,and AKT were detected by Western blot assay.Our study found out that AS Ⅳ had a protective effect in preventing isoflurane-induced neuro-degeneration,and identified potential mechanism for such protective effect.Overall,our study provides new experimental evidence for AS Ⅳ as a potential means for reducing anesthesia-associated neurotoxicity.Purpose:In the present study,we determined whether Astragaloside Ⅳ pre-treatment can reduce isoflurane exposure-induced neuro-apoptosis in rats.Methods:The rats(P5)were randomly divided into 5 groups with 6 rats pergroup:Group Ⅰ:Isoflurane;Groups II-Ⅳ:isoflurane with AS Ⅳ pre-treatment(10 mg/kg,40 mg/kg,100 mg/kg);and sham group without anesthesia.The rats were given AS Ⅳ pre-treatment for 3 days with different dosages(10,40,100 mg/kg),then exposed to anesthesia.The sham group was pre-treated with ethanol in 0.9%Saline vehicle.The oxidative stress-related enzyme activities in hippocampus tissues and serum were quantified,and serum levels of pro-inflammatory cytokines were determined by ELISA.Protein expression levels of NF-κB,caspase-3,BCL-2,phosphorylated JNK,ERK,and AKT were detected by Western blot assay.One-way ANOVA was used for analysis of difference between groups,and P<0.05 was considered statistically significant.Results:Isoflurane-induced neuron apoptosis in hippocampus CA1 regionWe first checked tissue damage in hippocampus area in isofluorane-exposed rats.There was almost no dead cells in hippocampus CA1 neuron in sham group rats,but the number of dead cells showing shrunken cytoplasm and degenerated nuclei significantly increased in tissues from isoflurane-treated group.Such results suggested that isoflurane exposure induced neuron cell death.By contrast,AS Ⅳ treatment significantly reduced isoflurane-induced neuron cell death.Such results imply that AS Ⅳ exerts neuroprotective effects on isoflurane-induced neuron cell damage in the CA1 region of hippocampus in rats.To confirm whether the cell death identified in the H&E stained tissues were apoptotic cells,we further eaxmined cell apoptosis in these tissues by TUNEL assay.In Sham group,the neuron in hippocampus CA1 zone was rarely stained positive in TUNEL assay.Isoflurane exposure significant increased the apoptotic neuron numbers(P<0.01 compared to Sham group).This indicates that the neurodegenerative damage caused by isoluorane was mainly from hippocampus neuron apoptosis.By contrast,AS Ⅳ treatment(both 40 and 100 mg/kg dosage groups)significantly reduced isoflurane-induced neuron cell apoptosis(P<0.01).SOD,iNOS,MDA,NO measurement in hippocampus tissue and rat serumTo test the levels of isoflurane-induced oxidative stress in different groups,we isolated rat serum and hippocampus tissues and measured the enzyme activities of some oxidative stress-related antioxidant enzymes such as Superoxide Dismutase(SOD),inducible nitric oxide synthase(iNOS),lipid peroxide marker Malondialdehyde(MDA),and nitric oxide(NO).In Sham group,MDA levels were relatively low in hippocampus CA1 zone and rat serum.Isoflurane exposure significant inhibited the enzyme activities of SOD levels,but increased total iNOS and MDA and NO levels,which may contribute to the neuron apoptosis.By contrast,AS Ⅳ treatment significantly inhibited isoflurane-induced MDA and iNOS and NO production and ameliorated the suppression of enzyme activity SOD production by isoflurane.Pro-inflammatory cytokine measurement in the serum and culture supernatantTo test the levels of isoflurane-induced pro-inflammatory responses in different groups,we isolated rat serum and measured levels of the major pro-inflammatory cytokines such as TNF-a,IL-6,and IL-1β.In Sham group,these cytokine levels were relatively low in rat serum.Isoflurane exposure significant increased the levels of all the three pro-inflammatory cytokines.By contrast,AS Ⅳ treatment(both low and high dosage groups)significantly reduced isoflurane-induced pro-inflammatory cytokine release(P<0.01).Such results suggested that isoflurane exposure induced systemic inflammatory responses in neonatal rats,and AS Ⅳ alleviated such inflammation.Inflammatory and anti-apoptotic signaling pathway analysisThe pro-inflammatory cytokine responses and reactive nitrogen species production are mainly regulated by NF-κB pathway.Thus,we measured the NF-κB activity in rat hippocampus tissues by Western blot.In protein samples from sham group,NF-κB protein was mainly in cytoplasm and low in nucleus,suggesting low NF-κB activity.Isoflurane exposure markedly increased nuclear level of NF-κB protein,indicating NF-κB activation in hippocampus tissue.By contrast,ASⅣ treatment(both low and high dosage groups)reduced isoflurane-induced NF-κB nuclear localization,indicating suppressed NF-κB activation.We further measured apoptosis-related protein expression levels in different groups.In protein samples from sham group,the pro-apoptotic marker protein,Caspase-3,was relatively low,and the anti-apoptotic marker protein,BCL-2,was abundantly expressed.Isoflurane exposure markedly increased caspase-3 protein levels and decreased BCL-2 protein levels.By contrast,AS Ⅳ treatment(both low and high dosage groups)markedly inhibited isoflurane-induced caspase-3 up-regulation and BCL-2 downregulation,suggesting that AS Ⅳ suppressed isoflurane-induced hippocampus apoptosis.Similarly,isoflurane exposure induced GSK-3P and decreased Klotho and phosphorylated Akt protein levels,but AS Ⅳ pre-treatment(both low and high dosage groups)markedly inhibited isoflurane-induced changes in these proteins.MAPK signaling pathways are intricately involved in apoptotic responses[20].We observed activation of JNK and ERK by isoflurane exposure(indicated by increased phosphorylated protein level);Furthermore,AS Ⅳ treatment(both low and high dosage groups)markedly inhibited isoflurane-induced MAPK activation.Conclusion:Overall,our study demonstrated that Astragaloside Ⅳ could protect developing brain from anesthesia-induced neuro-apoptosis through anti-oxidant and anti-inflammatory activities.Part two:Influence of cognitive function after isoflurane anesthesia on pediatric laparoscopic inguinalObjective To compare the cognitive function after isoflurane anesthesia on pediatric laparoscopic inguinal.Methods Select 60 cases which underwent laparoscopic inguinal repair surgery in our hospital from January 2013 to January 2015,the children were randomly divided into isoflurane group、sevoflurane group and control group 30 cases.isoflurane group and sevoflurane group were observed and recorded postoperative recovery time,postoperative adverse reactions,heart rate at different times during surgery,mean arterial pressure and all three groups postoperative WISC-Ⅳ score also be observed before operation and 3、7 day after operation.Results The heart rate at start of surgery and surgery 15min were lower than sevoflurane group,the difference was statistically significant,P<0.05,but the average arterial pressure a was no difference,P>0.05.The overall adverse reactions of sevoflurane group lower than the isoflurane group,the difference was statistically significant,P<0.05.The postoperative recovery time of sevoflurane group shorter than the isoflurane group,the difference was significant,P<0.05.POCD was confirmed in 4/26(15.0%)in isoflurane group compared with 3/25(12.0%)in sevoflurane group 3 days after surgery.Isoflurane group were significantly lower from patients in their scores of information,vocabulary and digit spandigit span at later testing sessions than the controls 3 days after surgery.Sevoflurane group were significantly lower in their scores of vocabulary and digit spandigit span at later testing sessions compared with the controls 3 days after surgery.There were no signigicantly defferences at testing sessions compared with-the controls 7 days after surgery both sevoflurane group and sevoflurane group.Conclusion Both isoflurane and sevoflurane may contribute to the postoperative cognitive dysfunction in pediatric laparoscopic inguinal,the time of the recovery of influence is three days.isoflurane and sevoflurane have no difference with the postoperative cognitive dysfunction.Anaesthesia only used isoflurane and sevoflurane was safe and effective in pediatric laparoscopic inguinal.