Cyanidin-3-O-Glucopyranoside And Liquiritin Reduce Toxic Effects On Human Neuroblastoma Cells Induced By Tri-Ortho-Cresyl Phosphate And Mechanism Discussion

Objective:To provide a new idea for the prevention and treatment of neurotoxicity caused by organic phosphate compounds,the regulatory effects and mechanisms of cyanidin-3-O-glucoside（C3G）and liquiritin（LIQ）in tri-ortho-cresyl phosphate（TOCP）-induced neurotoxicity have been investigated in vitro SK-N-SH cell experiments.Methods:1.The CCK-8 method was used to determine the cell activity of SK-N-SH cells after 48 h of exposure to a series of concentrations of TOCP（0.00 mmol/L、0.25 mmol/L、0.50 mmol/L、0.75 mmol/L and 1.00mmol/L）,and the cell activity of SK-N-SH cells after treatment with a series of concentrations of C3G or LIQ for 6 h,12 h,24 h and 48 h.2.SK-N-SH cells were pretreated with 100μmol/L C3G for 1 h and then treated with 0.75 mmol/L TOCP for 48 h.After cell viability was measured by CCK-8,the expression levels of endoplasmic reticulum calcium transport-related proteins（IP3R and Ry R）and endoplasmic reticulum stress-related proteins（CHOP and GRP78）were detected by western blot assay combined with indirect immunofluorescence.The activity of Ca²⁺-ATP ase was detected by phosphorus fixation colorimetric method.Cellular superoxide dismutase activity,malondialdehyde and glutathione contents were determined with microporous enzyme labeling method.Detecting the concentration of intracellular free calcium ions and the total active oxygen level by a spectrophotometer.Detection of mitochondrial membrane potential by fluorescence microscopy.3.SK-N-SH cells were pretreated with 75μmol/L LIQ for 1 h and then treated with 0.75 mmol/L TOCP for 48 h.The cell viability was determined by CCK-8 and the expression of TRPA1 m RNA was detected by RT-PCR.The expression levels of calcium translocation-related protein（TRPA1）and endoplasmic reticulum stress-related protein（CHOP and GRP78）were detected by western blot combined with indirect immunofluorescence.The activity of superoxide dismutase and the contents of malondialdehyde and glutathione in the cells were determined with microporous enzyme labeling method.Detecting the concentration of intracellular free calcium ions and the total active oxygen level by a spectrophotometer.Detection of mitochondrial membrane potential by fluorescence microscopy.Results:1.With the increase of TOCP exposure,the proliferation of SK-N-SH cells was inhibited and a dose-response dependent death occurred.SK-N-SH cell activity was not significantly inhibited with the increase in dose and exposure time of C3G or LIQ.2.Compared with the 0.75 mmol/L TOCP group,the survival rate of SK-N-SH cells in the 100μmol/L C3G+0.75 mmol/L TOCP group was increased,the concentration of free calcium ions in the cells was decreased,the protein expression levels of IP3R and Ry R,as well as CHOP and GRP78 were decreased,the Ca²⁺-ATPase activity and superoxide dismutase activity were increased,the content of glutathione was increased,the levels of malondialdehyde and total active oxygen were decreased,and the mitochondrial membrane potential was increased.3.Compared with the 0.75 mmol/L TOCP group,the survival rate of SK-N-SH cells in the 75μmol/L LIQ+0.75 mmol/L TOCP group was increased,the concentration of free calcium ions in the cells was decreased,the protein and m RNA levels of TRPA1 were decreased,the protein expression levels of CHOP and GRP78 were decreased,the activity of superoxide dismutase and the content of glutathione.Conclusion:1.100μmol/L C3G can protect 0.75 mmol/L TOCP-induced SK-N-SH cells by restoring Ca²⁺-ATPase activity,regulating IP3R and Ry R,and alleviating oxidative stress and endoplasmic reticulum stress.2.75μmol/L LIQ reduced extracellular calcium influx by inhibiting the opening of TRPA1 channel and protected 0.75 mmol/L TOCP-induced SK-N-SH cells by alleviating oxidative stress and endoplasmic reticulum stress.