ABSTRACT
Objective:
To observe the effect of astragaloside Ⅳ on lysophosphatidic
acid(LPA)- induced
neurite retraction of N1E-115
cells and its potential mechanism.
Methods:
N1E-115
cells were divided into blank group, model group, the low, medium and high
dose groups of astragaloside Ⅳ. The blank group and model group was not intervened by astragaloside; while the low, medium and high
dose groups were treated with 20,40 and 80 μg/ml astragaloside Ⅳ for 24 h. Each group was cultured with
serum-free medium for 12 h. The model group and astragaloside Ⅳ groups were intervened by 40 μmol/L LPA for 10 min. Each group was observed and photographed with the inverted microscope, and the number of
neurites in N1E-115
cells was counted by Image J
software. The
fluorescence expression of recombinant
ras homolog
gene family member A (RhoA), rho associated coiledcoil
protein kinase 2 (ROCK2), phospho-rho associated coiledcoil
protein kinase 2 (p-ROCK2) and phospho-
myosin light chain 2 (p-MLC2)
proteins was detected by
immunohistochemistry. Real-
time fluorescent quantitative
polymerase chain reaction was used to detect the
mRNA expression levels of RhoA and ROCK2 ; the
protein expression levels of RhoA, ROCK2, p-MLC2 and
myosin light chain 2 (MLC2) were detected by
Western blotting.
Results:
Compared with 20 μg/ml astragaloside Ⅳ group, the inhibition rate of
neurite retraction in 40 and 80 μg/ml astragalosideⅣ groups increased ( P<0.05). Compared with model group, the average
fluorescence intensity of RhoA, p-ROCK2, p-MLC2 in 20, 40, 80 μg/ml astragaloside Ⅳ groups and the ROCK2 average
fluorescence intensity in 40 μg/ml astragaloside Ⅳ group were decreased ( P<0.05, P<0.01); the expression of RhoA
mRNA (0.89±0.09, 0.41±0.01, 0.09±0.03 vs. 1.50±0.01) and ROCK2
mRNA (0.89±0.09, 0.14±0.01, 0.20±0.01 vs. 1.62±0.17) decreased in 20, 40, 80 μg/ml astragaloside Ⅳ groups ( P<0.05, P<0.01); the ROCK2
protein (0.75±0.06, 0.57±0.02, 0.66±0.01 vs. 1.08±0.02), p-MLC2
protein (1.72±0.03, 1.40±0.04, 1.29±0.03 vs. 2.19±0.11), MLC2
protein (1.13±0.02, 0.68±0.03, 0.75±0.03 vs. 1.60±0.03) in 20, 40, 80 μg/ml astragaloside Ⅳ groups and the
RhoA protein (0.35±0.01, 0.40±0.03 vs. 0.57±0.08) in 20, 40 μg/ml astragaloside Ⅳ groups were decreased ( P<0.05, P<0.01).
Conclusion:
Astragaloside Ⅳ can prevent LPA-induced
neurite retraction and promote damaged
nerve regeneration. The mechanism may down-regulae the
protein expression levels of RhoA, ROCK2, p-ROCK2, p-MLC2 and MLC2 in RhoA-ROCK2 signaling pathway, and inhibite nerve
growth cone collapse.