| Objective: This study aimed to observe the effects of hypnotic active components(HCCs)and isopimpinellin(IS)in Cnidii Fructuson the apoptosis of PHNs and secretion of neurotransmitters in the in vitro cultured PHNs,as well as the effects on the expression of neurotransmitter related receptor genes and circadian clock genes,and to explore the hypnotic mechanism of HCCs and IS.Methods1.Culture and identification of PHNs.The PHNs of Neonatal Sprague-Dawleyrats were cultured in vitro and the cell activity was detected by Cell Counting Kit-8(CCK-8)assay.The cell growth state was observed by an inverted microscope,and subsequent experiments were conducted when reaching the optimal state of cell growth according to the cell activity curve.The purity of PHNs was identified by immunohistochemistry of neuron-specific enolase.2.Screening of optimal fdosing concentrationsof HCCs and IS.The optimal concentrations of HCCs and IS were selected,and the dose interval was set at 1:10 and 1:2,respectively.CCK-8 assay was used to detect the survival rate of PHNs at 24 h and 48 h.3.Cell grouping and administrations.PHNs were randomized into eight groups,blank control group,diazepam positive group(25?g·m L-1),low-dose HCCs(25?g·m L-1),moderate-dose HCCs(50?g·m L-1),high-dose HCCs(100?g·m L-1),low-dose IS(5?g·m L-1),moderate-dose IS(10?g·m L-1),and high-dose IS(20?g·m L-1)groups.4.Indices detections.The apoptosis rate of PHNs was analyzed by flow cytometry(FCM)at 24 h and 48 h after administration.The levels of ?-aminobutyric acid(GABA),5-hydroxytryptamine(5-HT),norepinephrine(NA)and other neurotransmitters in the supernatant of PHNs were determined by enzyme-linked immunosorbent assay(ELISA).The expressions of GABA and 5-HT associated receptor genes and m RNAs of circadian clockgenes including Clock,Bmal1,Cry1,Per1 and Per2 in PHNs were detected by real-time fluorescence quantitative polymerase chain reaction(RT-PCR).Results1.Culture and identification of PHNs.On the 7~8th day,the PHNs grew in good condition with high activity,and the purity was above 90%.2.Survival rate of PHNs at 24 h or 48 h after administration with HCCs and IS,and the optimal dosing concentrations.The survival rates at 24 h or 48 h after administrationin the groups with 25?g·m L-1,50?g·m L-1 or 100?g·m L-1 HCCs were significantly higher than thosein the blank control group.The survival rates at 24 h or 48 h after administration in the group with 200 ?g·m L-1 HCCs were significantly lower than those in the blank control group.The survival rates at 24 h after administration in the groups with 10?g·m L-1 or20?g·m L-1 IS were significantly higher than that in the blank control group,while that in the group with 40 ?g·m L-1 IS was significantly lower than that in the blank control group.The survival rates at 48 h after administration in the groups with 5?g·m L-1,10 ?g·m L-1 or20?g·m L-1IS were significantly higher than those in the blank control group,while that in the group with 40?g·m L-1 IS was significantly lower than that in the blank control group.Therefore,HCCs ranging from 25?g·m L-1 to 100?g·m L-1 and IS ranging from 5?g·m L-1 to20?g·m L-1 in concentration reached the optimal survival rate,respectively.So as to HCCs,25?g·m L-1,50?g·m L-1,and 100?g·m L-1 were set as the low,moderate,and high concentrations respectively for further experiments,and as to IS,5?g·m L-1,10?g·m L-1,and20?g·m L-1 were set low,moderate,and high concentrations,respectively.3.Effects of HCCs and IS on the early-phase apoptosis rate of PHNs.After 24-hour administration of HCCs and IS on PHNs,the early-phase apoptosis rates of PHNs in the diazepam positive group,and the moderate-dose HCCs,low-dose HCCs,moderate-dose IS,and high-dose ISgroups were significantly lower than that in the blank control group.After48-hour administration,the early-phase apoptosis rates of PHNs in the diazepam positive group,the moderate-dose HCCs and high-dose IS groups were significantly lower than that in the blank control group,while that in high-dose HCCs group was significantly higher than that in the blank control group.4.Effects of HCCs and IS on the neurotransmitter content of PHNs.After 24-hour administration of HCCs and IS on PHNs,GABA contentsinthe diazepam positive group,the moderate-dose HCCs,high-dose HCCs,and high-dose IS groups were significantly higher than that in the blank control group;5-HT contentsin the high-dose HCCs and moderate-dose HCCs groups were significantly higher than that in the blank control group;there was no obvious changes in NA content in all groups.After 48-hour administration,the GABA contentsin the diazepam positive group,the high-dose HCCs,moderate-dose HCCs,high-dose IS and moderate-dose IS groups were higher than that in the blank control group;and the 5-HT contentsin the high-dose HCCs,moderate-dose HCCs and low-dose HCCs groups were significantly higher than that in the blank control group;the NA contents in the diazepam positive group,high-dose HCCs,moderate-dose HCCs,low-dose HCCs,and high-dose IS groups were significantly higher than that in the blank control group.5.Effects of HCCs and IS on the expressions of GABA and 5-HT related receptor genes in PHNs.After 24-hour administration of HCCs and IS on PHNs,the expression levels of Gabra1,Gabbr1,5-HT1A(A),5-HT1A(B),5-HT1 B and 5-HT1 D m RNAs in the diazepam positive group,the high-dose HCCs and moderate-dose HCCs groups were significantly higher than those in the blank control group;the expression levels of 5-HT1A(C)m RNA in the diazepam positive group,the high-dose HCCs,moderate-dose HCCs and low-dose HCCs groups were significantly higher than that in the blank control group;the expression levels of Gabrb5 m RNA in the diazepam positive group,the high-dose HCCs and high-dose IS groups were significantly higher than that in the blank control group;the expression level of Gabrb2 m RNA in high-dose HCCs group was significantly higher than that in the blank control group,while that in the low-dose HCCs group was significantly higher than that in the blank control group;the expression levels of Gabra1,5-HT1A(A),5-HT1A(B),5-HT1 B and5-HT1 D m RNAs in the high-dose IS and moderate-dose groups were higher than those in the blank control group;the expression levels of Gabbr1 and 5-HT1A(C)m RNAs in the diazepam positive group,the high-dose IS,moderate-dose IS and low-dose IS groups were significantly higher than those in the blank control group.6.Effects of HCCs and IS on expression of circadian clock genes in PHNs.The expression levels of Bmal1 m RNA in the diazepam positive group and the high-dose HCCs group were significantly lower than that in the blank control group.The expression level of Clock m RNA in the high-dose IS group was significantly lower than that in the blank control group.The expression levels of Cry1,Per1 and Per2 m RNAs in the diazepam positive group,the high-dose HCCs,moderate-dose HCCs,high-dose IS and moderate-dose IS groups were significantly higher than those in the blank control group.Conclusion:Hypnotic function of HCC?IS can decrease the apoptosis rate of PHNs,promote the secretion of GABA,5-HT,NA,up-regulate the m RNA expression of GABA and 5-HT related genes including Gabra1,Gabra5,Gabbr1,Gabrb2,5-HT1A(A),5-HT1A(B),5-HT1A(C),5-HT1 B,and 5-HT1 D,and the m RNA expression of circadian negative feedback regulation genes including Cry1,Per1 and Per2,and down-regulate the m RNA expression of circadian positive feedback regulation genes including Bmal1 and Clock,which enhances the secretion of inhibitory neurotransmitter and its receptor gene expression,and thus regulates the hypnotic effect of the clock genes. |
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