| Hippocampal neurons/astrocytes in vitro co-culture system has been widely used to prevent the screening of natural active ingredients of neurodegenerative diseases and its mechanism,but few reports have been reported co-culture system as a experimental model for early screening of neurotoxicity.In view of this,neurotoxic acrylamide(ACR)as a positive drug,acted on co-cultured cells to establish early neurotoxicity in vitro evaluation method.On the basis of this,the method was used to evaluate the in vitro neurotoxicity of Areca catechu L.and Polygonum cuspidatum decoction.The main contents and results are as follows:1.The establishment of hippocampal neurons and astrocytes co-culture model in vitroTwo kinds of cell models were established by direct co-culture and transwell chamber non-contact co-culture of hippocampal neurons and astrocytes.It was found that in two co-culture methods astrocytes were able to promote the growth of neurons and promote the increase of the number of neurons,the number and length of neurite in mixed co-culture in different culture days were higher than transwell culture.The above results showed that mixed co-culture could better simulate the in vivo state,maintained the growth of neurite,mixed co-culture as the experimental model was used for follow-up experiments.2.The establishment of in vitro neurotoxicity early evaluation method(1)The co-cultured cells were treated with ACR(2~10 mmol/L)for 24 h,48 h and 72 h,which had general toxicity(P<0.01).Follow-up experiments were performed on ACR-treated co-cultured cells for 24 h at the concentration of 3,7.26 and 10 mmol/L.(2)ACR at the concentration of 7.26 and 10 mmol/L treatment increased LDH level,the increments were 1.19 and 2.53 folds compared with the control.The increments of Ca2+ were 0.5 and 0.71 folds compared with the control;the mitochondrial membrane potential in ACR group decreased,the decrements were from 0.26 to 0.56 folds compared with control(P<0.01).Indicating that ACR within the concentration range produced a general toxic effect in co-cultured cells.IV((3)After treatment with 7.26 and 10 mmol/L ACR,ACR caused the number of neurite decreased and the length of neurite shortened,the expression of synapsic plasticity related proteins Arc,SYN and ?-???-Tubulin were decreased,the above differences were statistically significant(P<0.05).Indicating that ACR caused synaptic plasticity changes,affected the release of neurotransmitters,and ultimately produced neurotoxicity.3.Betel nut decoction and Polygonum cuspidatum decoction early safety in vitro neurotoxicity evaluationTo evaluate the early safety in vitro neurotoxicity of betel nut and Polygonum cuspidatum.the raw materails were respectively decocted,concentrated at 45?,and freeze-dried to obtain dry powder.(1)Evaluation of neurotoxicity of betel nut decoctionThe co-cultured cells were treated with 25-1000 ?g/mL betel nut decoction for6-96 h,when 150 ?g/mL betel nut decoction treated with cells for 96 h,cell viability was 77 ± 4.5%(P<0.01);25-200 ?g/m L betel nut decoction treated with cells for 24 h,150 ?g/m L group increased LDH release,intracellular Ca2+ concentration,decreased mitochondrial membrane potential level(P<0.01).Indicating that the concentration of the 150 ?g/mL produced a general toxic effect.The expression of synaptic plasticity-related proteins Arc,SYN and?-???-Tubulin were not significantly different after treatment with 25-200 ?g/mL betel nut decoction for 1 h and 3 h(P>0.05,compared with control).The expression of SYN protein was significantly decreased at concentration of 50 ?g/mL betel nut decoction,the contents of NO and glutamate were increased and the content of ACh decreased,the above differences were statistically significant(P<0.05).It was shown that 50 ?g/mL betel nut decoction treated with co-cultured cells for 6 h have a neurotoxicity effect on co-cultured cells,SYN can be used as a sensitive protein for rapid detection betel nut decoction neurotoxicity.(2)Evaluation of neurotoxicity of Polygonum cuspidatum decoctionThe co-culture cells were treated with 25-1000 ?g/m L Polygonum cuspidatum decoction for 6-96 h,when Polygonum cuspidatum decoction at the concentration of300 ?g/mL treated with cells for 48 h,the cell viability was 79 ± 6.5%(P<0.01,);25-400 ?g/mL Polygonum cuspidatum decoction treated with cells for 24 h,300?g/mL group increased LDH release,intracellular Ca2+ concentration and decreased mitochondrial membrane potential(P<0.01).The above showed that the concentration of 300 ?g/mL produced a general toxic effect.The expression of synaptic plasticity-related Arc,SYN and ?-???-Tubulin were not significantly different after treatment with 50-400 ?g/mL Polygonum cuspidatum decoction for 1 h and 3 h(P>0.05).The expression of SYN protein was significantly decreased at concentration of 400 ?g/mL Polygonum cuspidatum decoction,the contents of NO and glutamate were increased and ACh decreased(P<0.01).It was shown that 400 ?g/mL group treated with co-cultured cells for 6 h have a neurotoxicity effect on co-cultured cells,Arc can be used as a sensitive protein for rapid detection Polygonum cuspidatum decoction neurotoxicity. |
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