| Background and objectives:Flos Genkwa,the dried flower bud from Daphne genkwa Sieb.et Zucc.(Thymelaeaceae).it is warm and toxic,which tastes bitter and acrid,as a traditional Chinese medicine for expelling water retention with drastic purgative.However,it leads to many adverse reactions,such as dizziness,nausea and vomiting,aching limbs,dry mouth,abdominal pain,diarrhea and so on.In the preliminary study of our lab,a prolonged exposure assay system of C.elegans to perform the full in vivo toxicity assessment of raw-processed Flos Genkwa was employed.Our data showed that Flos Genkwa exposure could induce the toxicity on locomotion behavior.In order to further understand the toxic material basis of Flos Genkwa,Genkwanin was selected as a major component and an active ingredient of Flos Genkwa to explore its toxicity and mechanisms,and provide the basis for clinical application and safety of Flos Genkwa.Methods and results:1.Using C.elegans to study the toxicity of Genkwanin1.1 Using C.elegans to study the neurotoxicity(locomotion behavior)of Genkwanin1.1.1 Effects of Genkwanin on lifespan of C.elegansWild-type C.elegans were treated with Genkwanin 0.1,1,10 and 100 μmol·L-1 for 72 h.Observe the effects on lifespan of C.elegans.The results showed that,compared with the normal control,there were no significant changes in lifespan and half lethal time.1.1.2 Effects of Genkwanin on head thrash and body bend of C.elegansWild-type C.elegans were treated with Genkwanin 0.1,1,10 and 100 μmol·L-1 for 24,48 and 72 h,respectively.Observe the effects on head thrash and body bend of C.elegans.The results showed that,compared with the normal control,the Genkwanin groups(1,10 and 100μmol·L-1)showed significant decreases in head thrash frequency and body bend frequency.1.1.3 Effects of Genkwanin on defecation and pumping behavior of C.elegansWild-type C.elegans were treated with Genkwanin 0.1,1,10 and 100 μmol·L-1 for 24,48 and 72 h,respectively.Observe the effects on mean defecation cycle length and mean pumping frequency of C.elegans.The results showed that,compared with the normal control,after treated with Genkwanin for 24 h,there were no significant changes on mean defecation cycle length and mean pumping frequency of C.elegans.After treated with Genkwanin for 48 h,a significant increase of mean defecation cycle length and a decrease of mean pumping frequency were observed in the 100 μmol·L-1 group.After treated with Genkwanin for 72 h,a significant increase of mean defecation cycle length and a decrease of mean pumping frequency were observed in all Genkwanin group(0.1,1,10 and 100 μmol·L-1).1.2 Using C.elegans to study the neurotoxicity(learning behavior)of GenkwaninWild-type C.elegans were treated with Genkwanin 0.1,1,10 and 100 μmol·L-1 for 24,48 and 72 h,respectively.Observe the effects on chemotaxis towards NaCl of C.elegans.The results showed that,compared with the normal control,after treated with Genkwanin for 24 and 48 h,a significant difference of chemotaxis index was observed in the 100 μmol·L-1 group for C.elegans kept without NaCl(—NaCl—OP50)for 3 h;there were no significant changes on chemotaxis index of C.elegans kept with NaCl(+NaCl—OP50)for 3 h.After treated with Genkwanin for 72 h,significant decreases of chemotaxis index were observed in the 10 and 100 μmol·L-1 groups for C.elegans kept without NaCl(—NaCl—OP50)for 3 h;a significant change on chemotaxis index of 100 μmol·L-1 group for C.elegans kept with NaCl(+NaCl—OP50)for 3 h.1.3 Using C.elegans to study the reproduction of GenkwaninWild-type C.elegans were treated with Genkwanin 0.1,1,10 and 100 μmol·L-1 for 72 h.Observe the brood size of C.elegans.The results showed that,compared with the normal control,there were no significant changes on brood size of C.elegans.2.Using C.elegans to study the neurotoxic mechanisms of Genkwanin2.1 Effects of Genkwanin on five neurotransmitter systems of C.elegansTransgenic C.elegans were treated with Genkwanin 0.1,1,10 and 100μmol·L-1 for 24,48 and 72 h,respectively.Observe the changes of the fluorescence intensity and the number of neurons in the neurotransmitter system of C.elegans.The results showed that,compared with the normal control,the Genkwanin groups showed significant decreases in the relative fluorescence intensity and the number of GABAergic neurons.In contrast,cholinergic,dopaminergic,serotonergic and glutamatergic neurons did not exhibit obvious structural alterations in nematodes treated with Genkwanin 0.1,1,10 and 100 μmol·L-1 for 72 h.2.2 Effects of Genkwanin on GABAergic related genes of C.elegansWild-type C.elegans were treated with Genkwanin 0.1,1,10 and 100 μmol·L-1 for 72 h.The expression levels of gene unc-25,unc-30,unc-46,unc-47,unc-49 and exp-1 in GABA ergic neurotransmitter systems of C.elegans were detected by using the method of RT-qPCR.The results showed that,compared with the normal control,the Genkwanin groups showed significant decreases on the expression levels of related genes of GABAergic neurotransmitter system.2.3 Effects of Genkwanin on ASE sensory neuron of C.elegansTransgenic C.elegans were treated with Genkwanin 0.1,1,10 and 100 μmol·L-1 for 72 h.Observe the changes of the fluorescence intensity of ASEL and ASER neurons of C.elegans.The results showed that,compared with the normal control,the Genkwanin 100 μmol·L-1 groups showed significant decreases in the relative fluorescence intensity of ASEL and ASER neurons of C.elegans.2.4 Effects of Genkwanin on learning behavior related genes of C.elegansThree mutant C.elegans that knockout daf-2,age-1 and akt-1,respectively,were treated with Genkwanin 100 μmol·L-1 for 72 h.Observe the effects on chemotaxis towards NaCl of C.elegans.The results showed that,compared with the normal control,there were no significant changes on chemotaxis index of mutant C.elegans.Conclusions:1.Genkwanin has certain toxicity on C.elegans and the toxicity manifests itself mainly throughinhibiting the locomotion behavior,defecation and pumping behavior and learning behavior of C.elegans.The locomotion behavior,defecation and pumping behavior and learning behavior of C.elegans are mainly controlled by the nervous system.The results suggest that Genkwanin may have inhibitory effect on nervous system of nematodes.2.Genkwanin can lead to significant decreases in the relative fluorescence intensity and the number of GABAergic neurons.Moreover,the expression levels of related genes of GABAergic neurotransmitter system are generally decreased.These suggest that the underlying mechanism may be related to the impairment of GABAergic neurons.3.Genkwanin can lead to significant decreases in the relative fluorescence intensity of ASEL and ASER neurons of C.elegans,and it may influence learning behavior of C.elegans through daf-2,age-1 and akt-1 genes. |
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