To Explore The Mechanism Of Astragaloside Ⅳ Against Neurite Protrusion Injury Based On RhoA/ROCK/LIMK1/Cofilin Pathway

Objective To observe the effects of Astragaloside Ⅳ（AS-Ⅳ）on nerve protrusion injury,and explore the mechanism of Astragaloside Ⅳ inhibiting nerve protrusion injury based on Rho A/ROCK/LIMK1/Cofilin pathway.Methods Mouse neuroblastoma cells（N1E-115）were selected as the study object,and the toxicity of astragaloside Ⅳ was detected by CCK-8 method.The suitable injury concentration of Lysophosphatidic acid was selected by circular cell counting method.The experiment was divided into blank control group,model group（LPA group）and astragaloside Ⅳ low-medium-high concentration groups（20,40,80 μg/m L+LPA）.After cultured in serum-containing medium for 24 h,the nerve processes were induced by serum-free culture for 24 h.Each group containing astragaloside Ⅳ was replaced with different concentrations of drug containing medium for 24 h,respectively.40 μM Lysophosphatidic acid was added to model group and astragaloside Ⅳ low-medium high-concentration group for 20-30 min,resulting in nerve processes damage.The effect of astragaloside Ⅳ on nerve protrusion injury was analyzed by protuberance counting method.The m RNA contents of Rho A,ROCK2,LIMK1,Cofilin and MLC2 in each group were detected by real-time quantitative RT-PCR.Immunofluorescence technology and Western Blot were used to detect the protein expressions of Rho A,ROCK2,P-ROCK2,LIMK1,P-LIMK1,Cofilin,P-cofilin,MLC2 and P-MLC2 in N1E-115 cells in each group.Results 1.CCK-8assay showed that the survival rate of 120 μg/m L AS-Ⅳ was significantly lower than that of 0μg/m L and other concentrations of AS-Ⅳ.2.Circular cell count found that the concentration was the ratio of round cells in the 1-160 μM Lysophosphatidic acid group was higher than that in the 0 concentration group,and the rate of round cells was relatively high when the concentration was 40 μM.3.The process count analysis showed that the cell process ratio of model group was very low,the process ratio of astragaloside Ⅳ groups was significantly higher than that of model group,and the nerve process rate of 40 and 80 μg/m L astragaloside Ⅳ groups was higher than that of20 μg/m L.4.PCR results showed that compared with model group,Rho A m RNA expression in N1E-115 neurons treated with different doses was lower than that in model group;The expression level of ROCK2 m RNA was low in 40 and 80 μg/m L AS-Ⅳ group.The expression level of Cofilin m RNA was low in 20 and 40 μg/m L AS-Ⅳ group.LIMK1 and MLC2 m RNA expression showed no difference.Compared with blank group,Rho A and ROCK2 m RNA expression in each dose group was lower than that in blank group.5.Immunofluorescence results: Compared with the model group,Rho A,ROCK2,P-ROCK2,P-LIMK1,Cofilin,P-Cofilin in and P-MLC2 protein fluorescence intensity in 20,40 and 80 μg/m L AS-Ⅳ groups were all weakened,while LIMK1 and MLC2 showed no difference in fluorescence intensity.Compared with blank group,the fluorescence intensity of P-LIMK1,P-ROCK2,Cofilin,P-Cofilin in and P-MLC2 proteins in Lysophosphatidic acid group was stronger,while the fluorescence intensity of P-LIMK1 and P-MLC2 proteins in 80μg/m L AS-Ⅳ group was weaker.The fluorescence intensity of 80 μg/m L p-LIMK1 and Cofilin was weaker than that of other drug groups.6.Western blot results:Compared with the model group,the protein expression levels of Rho A,P-ROCK2 and P-MLC2 in 20,40 and 80 μg/m L AS-Ⅳ groups were significantly decreased,and the protein expression levels of Cofilin in 20 and 40 μg/m L AS-Ⅳ groups were significantly decreased.The protein expressions of P-Cofilin in and P-LIMK1 decreased in 40 and 80 μg/m L AS-Ⅳ group,ROCK2 protein expression decreased in80 μg/m L AS-Ⅳ group,and LIMK1 and MLC2 protein expression showed no difference.Compared with blank group,the protein expression levels of Rho A,Cofilin and P-cofilin in Lysophosphatidic acid group were significantly increased,while the protein expression levels of P-ROCK2 in 20,40 and 80 μg/m L AS-Ⅳ groups were decreased.The expression levels of ROCK2 and P-LIMK1 were lower at80 μg/m L compared with other drug groups.Conclusions 1.Lysophosphatidic acid can cause damage and retraction of neuronal processes,and significantly up-regulate the expression of related proteins in Rho A/ROCK/LIMK1 /Cofilin pathway.2.Astragaloside Ⅳ can effectively inhibit nerve protuberance damage and has protective effect on nerve protuberance.3.Astragaloside Ⅳ may reduce the expression of Rho A,ROCK2 and Cofilin m RNA,Decreased Rho A,ROCK2,P-ROCK2,P-LIMK1,Cofilin,p-Cofilin and P-MLC2 protein expressions in Rho A/ROCK/LIMK1/Cofilin pathway.It can down-regulate the activity levels of P-rock2,P-LIMk1,P-Cofilin and P-MLC2 to play an anti-neurite injury,which can be considered as neuroprotective drugs.