| Objective:Ma Huang-Gui Zhi herb pair is a canonical herb pair used for mutual potentiation and mutual assistance to treat wind cold syndromes in China for many years.Due to the excitatory adverse effects of Ma Huang in the CNS(central nervous system),its medicinal use has been restricted.Previous studies have found Gui Zhi may exhibit a neuroprotective effect against Ma Huang-induced toxicity in CNS when used in combination,though the underlined mechanism is not clear.This study is to investigate the molecular mechanisms of ephedrine-induced neuronal hyperactivity,and neuroprotective effect of Guizhit against Ma Huang-induced neurotoxicity via the cAMP/PKA signaling pathway,explaining the scientific and reasonable compatibility of Ma Huang and Gui Zhi.Methods:(1)PC12/HT22 cells were treated with ephedrine of gradient concentration.After 24 h of incubation,the survival rate of PC 12/HT22 cells was detected by MTT method,and the changes of cell structure and morphology were observed in bright field.(2)PC12/HT22 cells were treated with ephedrine of appropriate concentration without affecting cell viability.The mRNA levels of Arc,c-fos,Nr4al and klf10 were detected by QPCR assay,and the genes with significant differences were selected as indicators of cell excitability in vitro,which were verified by Western blot.(3)PC12/HT22 cells were incubated with ephedrine and western blot was used to detect the effect on CREB phosphorylation.H-89(a PKA inhibitor)was used to elucidate the involvement of cAMP/PKA signaling pathway.After pretreated with H-89 1 h to inhibit cAMP/PKA pathway,PC12/HT22 cells were incubated with ephedrine for 24 h.CREB phosphorylation was detected by Western blot.The effect of ephedrine and H-89 on c-fos protein expression in PC12/HT22 cells were detected by western blot and immunofluorescence.(4)The rats were randomly divided into control,H-89,ephedrine and H-89+ephedrine groups.The rats of control group and H-89 group were stereo-located injected with solvent and H-89(5 μM)for 3 days respectively.And the two groups above were intragastrically administrated with normal saline after stereo-located injection.And the rats of ephedrine and H-89+ephedrine group were stereo-located injected with solvent and H-89(5 μM)for 3 days respectively and the rats in these two groups were intragastrically administrated with 48 mg/kg ephedrine after injection.The rats were detected by open field test and the elevated plus maze 1 h after intragastrical administration.Western blot was used to detect the phosphorylation of CREB in cortex and striatum.The expressions of c-fos in the cortex and the striatum were detected by Western blot and immunofluorescence.(5)The effect of cinnamaldehyde on ephedrine induced PC 12 cell toxicity was determined via the MTT assay.After treatment of cinnamaldehyde for 24 h,QPCR assay was used to detect the mRNA level of c-fos in PC 12 cells.And after treatment of cinnamaldehyde for 1 h,PC 12 cells were incubated with or without of 2 mM ephedrine,c-fos mRNA expression was detected.(6)HPLC methods was established for the determination of ephedrine in decoction.Different decotions were prepared and the concentrations of ephedrine in ephedrine solution/Ma Huang decoction/Ma Huang Gui Zhi decoction/Gui Zhi decoction were kept with the same.(7)The rats were randomly divided into control,ephedrine,Ma Huang,Ma Huang+Gui Zhi and Gui Zhi groups.The behavioral changes of rats in open field test and elevated plus maze test were observed after 1 h of intragastric administration of the indicated drug.The expression of c-fos in cortex and striatum of rats in every group was detected by Western blot and immunofluorescence.The concentrations of cAMP in the cortex and the striatum were measured by ELISA assay.And the phosphorylation levels of PKA and CREB were detected by Western blot.Results:(1)A certain doses of ephedrine was cytotoxic in PC12/HT22 cells.High dose of ephedrine not only reduced the cell survival rate of PC 12/HT22 cells,but also significantly changes the cellular morphology and structure.(2)Ephedrine treatment increased the mRNA expression of arc,c-fos and Nr4al in PC 12/HT22 cells.The expression of c-fos mRNA and protein showed a concentration dependent manner,which can be used as an indicator of ephedrine induced cellular excitability.(3)Ephedrine could increase the phosphorylation level of CREB,the downstream of cAMP/PKA pathway.There was significant difference at the dosage of 500 μM.The cAMP/PKA pathway inhibitor H-89(10 μM)could inhibit the increase of CREB phosphorylation level and the increase of c-fos expression caused by ephedrine.(4)The behavioral test showed that after 48 mg/kg ephedrine was given to rats for 1 h,the locomotor activity increased significantly,and the rats showed obvious anxiety and tension.After preventive injection of H-89,the excitatory behaviors of rats induced by ephedrine was significantly reduced.The results of western blot showed that ephedrine alone increased the expression of c-fos in the cortex and striatum.And the phosphorylation level of CREB was also increased,which was consistent with the experimental results in vitro.The expression of c-fos in the brain has not been changed when the rats were treated by H-89 alone.Compared with ephedrine alone,CREB phosphorylation and c-fos expression were significantly reduced in the pretreatment with H-89 group.The results of immunofluorescence staining of c-fos were consistent with those of Western blot.(5)The safe concentration range of cinnamaldehyde in PC 12 cells was small.And it could not reverse the cytotoxicity induced by ephedrine in vitro.However,cinnamaldehyde could reduce the mRNA level of c-fos and antagonize the increase of c-fos expression induced by ephedrine.(6)Through the investigation of specificity,stability and precision,a rapid and simple HPLC method for the determination of ephedrine in water decoction was established.(7)The results of behavioral tests showed that the compatibility of Ma Huang and Gui Zhi could inhibit the increase of spontaneous activity induced by Ma Huang in rats.And tension and anxiety caused by Ma Huang could be alleviated.Western blot and immunofluorescence results showed that Ma Huang could significantly increase the expression of c-fos in the cortex and the striatum.Compared to the ephedrine/Ma Huang group,expression of c-fos in the cortex and the striatum reduced in the herd pair group by Western blot and immunofluorescence.After compatibility with Gui Zhi,the increased concentrations of cAMP and the raised phosphorylation levels of PKA/CREB caused by Ma Huang could be significantly inhibited in the cortex and the striatum.Conclusion:(1)Ephedrine could increase the expression of c-fos via cAMP/PKA signaling pathway in vitro.(2)H-89 could inhibit ephedrine induce hyperactivity by blocking cAMP/PKA signaling pathway in vivo.(3)The compatibility of Ma Huang and Gui Zhi herb pair could reduce the excitatory toxicity in CNS induced by Ma Huang via inhibiting cAMP/PKA signaling pathway... |
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