| Objective:1. To observe the inhibitory effect of MAG on neurites outgrowth, Its role in enhancing Rho A and activating ROCK phosphorylation.2. To observe the stimulative role of Bo NT/A HC on neuritogenesis in cultured neuro-2a cells, and explore the related intracellular mechanisms as well.3. To investigate whether the Bo NT/A HC is able to antagonize MAG in neuritogenesis.Methods:1. The expression of Ng R in neuro-2a cells, one of the specific receptors for MAG,was detected by immunofluorescence in order to decide if the exogenous MAG can get endocytozed. With the Ng R expression, the intracellular localization of MAG was identified by immunofluorescent staining. Meanwhile, the neurites outgrowth of neuro-2a cells at different time points(24 h, 48 h and 72 h) of different doses of MAG treatment was evaluated by measuring the length of neurites and the percentage of cells with neurites.Based on the above, the minimal dose of MAG with obvious effect on neurites outgrowth was chosed and added into cell cultures. The activity of Rho A was assayed by ELISA and the phosphorylation of ROCK by SDS-PAGE/western-blot(WB) after the different period of MAG treatment were assessed based on whole cell protein.2. Neuro-2a cells were treated with different doses of BoNT/A HC first, and then the cells were harvested at 24 h, 48 h and 72 h of Bo NT/A HC exposure. The neurites length and the percentage of cells with neurites to total cells on image were measured and calculated based on bIII-tubulin immunofluorescent staining. Depending on the results, the most efficient dose of Bo NT/A HC was chosen added into cell cultures. Harvesting cells at different time points. Thephosphorylation of ERK1/2 and Akt were detected by SDS-PAGE/ western-blot(WB) and immunofluorescent staining.3. Based on the above, choose 1 nmol/L Bo NT/A HC and 20 nmol/L MAG as theexperimental concentration, then add into cell culture as the following three different ways:1) add Bo NT/A HC 1 h ahead of MAG; 2) Add MAG 1h ahead of Bo NT/A HC; 3) add Bo NT/A HC and MAG at the same time. Cells were collected at 24 h, 48 h, 72 h for immunofluorescence staining to detect the neurites length and the percentage of cells with neurites. Meanwhile, the phophorylation of ROCK, ERK1/2 and Akt were also investigated by Western-blot after 10 min, 15 min, 1 h and 2 h of the above treatments.Results:1. The results in this study displayed that: 1) Immunofluorescence showed that neuro-2a cell expressed Ng R; 2) MAG strong positive staining was observed in the cytoplasm of neuro-2a cells after 6h of exogenous giving of MAG, which hinted that exogenous MAG could be endocytozed by its binding to Ng R; 3) Administration of exogenous MAG inhibited the neurites growth of neuro-2a cells, which was evidenced by assessing the neurites length and the percentage of cells with neurites. The results showed the average length of neurites was significantly shorter than those of the control group(P<0.05), and the percentage of the cells with neurites to all of the cells on the same image was less than that in control group(P<0.05); 4) Rho A activity was increase when adding20 nmol/L MAG into cell culture for 5 min, 10 min, 20 min, the difference of Rho A between MAG treatment and control group was statistically significant(P<0.05). The phosphorylation of ROCK was significant increased compared with control group(P<0.05).2. Both the length of neurites and the percentage of cells with neurites were increased after Bo NT/A HC treatment with different concentration(0.1 nmol/L, 1 nmol/L and 10nmol/L). The difference between Bo NT/A HC and control group was statistical significant(P<0.05), especially in the group with 1 nmol/L of Bo NT/A HC treatment. The obvious increase of p-ERK1/2 was seen from 60 min~5 h when adding 1 nmol/L Bo NT/A HC into cell culture for 15 min,30 min,60 min,90 min,2 h,3 h,4 h,5 h(P < 0.05), for 15 min,30 min,60 min,120 min to detect phosphorylation of Akt respectively. The increase of p-Akt appeared at the 15 min and 60 min of Bo NT/A HC treatment(P< 0.05).3. After adding 1 nmol/L Bo NT/A HC and 20 nmol/L MAG into cell cultures as the above three different ways, the length of neurites and the percentage of cells with neurites didn’t show statical difference at the 24 h,48 h and 72 h of treatments(P>0.05). The phosphorylation of ERK1/2, Akt and ROCK had no significant difference as well(P >0.05).Conclusion:1. Neuro-2a cells expressed Ng R. The binding of exogenous MAG and Ng R inhibit neurites outgrowth, activated intracellular signaling pathways, including the increase of Rho A activity and the phosphorylation of ROCK.2. Mini-dose of Bo NT/A HC is able to stimulate neurites outgrowth of neuro-2a cells.The intracellular signal proteins related to nerve regeneration(ERK1/2, Akt) were also activated by Bo NT/A HC, which might be one of the mechanisms of Bo NT/A HC stimulating neuritogenesis.3. The results from the intervention of Bo NT/A HC and MAG suggested that Bo NT/A HC antagonized the inhibitory role of MAG in neuritogenesis. |
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