Protection Effect And Mechanism Of Compound Isatis Root Polysaccharide On Lead Induced Injuries In NRK Cells

Lead toxicity to the kidney has raised the public awareness. At present, the mechanism of toxicity of the lead is not very clear. The effect of drugs that can reduce lead levels are not ideal. Exploring the drugs that are natural, have little risk and residue, can effectively prevent and treat animals’ lead toxicity will contribute to the prevention and treatment of lead toxicity with new ideas and approaches.Objective:In this study, we examined the protective effects of Compound Isatis Root Polysaccharide on lead induced renal tubular epithelial cell injury in rats with oxidation and the effects on mitochondria pathway of apoptosis. We studiedthe protective effects of Compound Isatis Root Polysaccharide on renal toxicity of lead and its regulation mechanism of apoptosis.Methods:(1) In vitro culture of rat renal tubular epithelial(NRK) cells, Compound Isatis Root Polysaccharide(0、0.5、1、2、5、10、20、50、100 μg/m L) and lead acetate(0、0.5、1、2、5、10、20、50 μmol/L) were added and incubated at 37 ℃ for 24 hours. MTT method was used to detect the effect of Compound Isatis Root Polysaccharide and lead acetate to the relative cell viability.(2) In accordance with the former result, 50 μmol/Llead acetate and Compound Isatis Root Polysaccharide(2 μg/m L、10 μg/m L、50 μg/m L) were used in the grouping trials. WST-1 and Chromogenic substrate method were used to determine the activity of superoxide dismutase(SOD) and catalase(CAT) in cells, respectively. TBA method was used to detect cell of MDA content. Ultraviolet colorimetry was used to detect cell total glutathione peroxidase(GSH-PX) activity; Fluorescent probe DCFH-DA method was used to detect the content of active oxygen(ROS) cells. Hoechst33258, Annexin V- FITC/PI double staining and flow cytometry instrument were used to detect the effect of Compound Isatis Root Polysaccharide on apoptosis inhibition.Western Blot was used to detect the protein content of Caspase-3, Caspase-9,Bax, Bcl-2, and the expression of P53.Results:(1) In this experiments, the increasing concentration of Compound Isatis Root Polysaccharide were used to determine the effect of Polysaccharide on NRK cells. The results showed that when the concentration of Compound Isatis Root Polysaccharide reached 50~100 μg/m L, it would promote the proliferation of NRK cells.(2) Lead acetate can inhibit the growth of NRK cells. When the concentrate of lead acetate exceeded 1μmol/L, it significantly inhibited on the growth of NRK cells and showed a dose-dependent manner. When the concentration of lead acetate exceeded 10μmol/L, the cell viability was reduced to less than 60%.(3) 50μg/m LCompound Isatis Root Polysaccharide can significantly reduce the inhibitory effect of lead acetate on the growth of NRK cells.(4) 2、10、50 μg/m LCompound isatis root polysaccharide can significantly increase the activity of SOD, GSH-Px, CAT in the cells and decrease the content of MDA. The Compound Isatis Root Polysaccharide had greater effect on GSH-Px and MDAcompared to the other.(5) When the concentration of Compound Isatis Root Polysaccharide was 2 μg/m L, it can significantly reduce the ROS content and apoptosis rate of NRK cells in a dose-dependent manner.(6) Compound isatis root polysaccharide can prevent the activation of Caspase-9 and Caspase-3 precursor protein, block the activation of the mitochondrial apoptosis pathway and prevent irreversible cell apoptosis by up-regulation of anti-apoptotic protein Bcl-2 and down-regulation of pro-apoptotic proteins P32 and Bax.Conclusion: Compound Isatis Root Polysaccharide can promoteproliferation of NRK cells and can effectively reduce the oxidative damage induced by lead on NRK cells. The mechanism may be related to the up-regulation of anti-apoptotic protein Bcl-2 and down-regulation of pro-apoptotic proteins P32 and Bax to prevent the cell apoptosis.