Effect Of KTC On Antioxidant Enzymes In H₂O₂-induced Oxidative Damage Of MES23.5Nerve Cells

ObjectiveThis study was designed to discuss the antioxidative mechanism of kidney-tonifying compound(KTC) used to treat Parkinson Disease(PD).To investigate the effects of KTC on antioxidant enzymes expression after H2O2-induced oxidative damage of MES23.5dopaminergic cells. In order to provide a certain theoretical basis for the clinical application of KTC.MethodsMES23.5dopaminergic cells were divided into control group(fresh culture medium), H2O2group(H2O2culture medium), Ginkgo biloba extract (GE) group(H2O2and GE culture medium), KTC low dose group, KTC middle dose group, KTC high dose group(H2O2and different dose of KTC culture medium). MES23.5nerve cells at logarithmic phase were seeded in6/96-well plates coated by poly-L-lysine(PLL) for1d. The control group was treated with the fresh culture medium,other groups were treated with the culture medium containing150μmol/L H2O2for12h.Then the control group and H2O2group were treated with the fresh culture medium,GE group was treated with the culture medium containing12.5μg/ml GE, the groups of KTC were respectively treated with containing3.125,6.25,12.5μg/ml of KTC for24h.Cell viability of every group was measured by MTT. Cellular morphology of every group was observated by inverted phase contrast microscope.Cellular ultrastructure of every group was observated by transmission electron microscope. The activity of antioxidant enzymes of every group were measuredof by ELISA. Then HO-1,CAT,GPx-1protein were determined by Western blot. HO-1, CAT, GPx-1mRNA were determined by RT-PCR.Results1. Changes in cell viability1.1The effects of different concentrations of H2O2treatment on cell viability150-500μmol/L H2O2exposure induced a dose-and time-dependent reduction of cell viability. After12h exposure with150μmol/L H2O2, the viability decreased to52.81%; and at24h, the viability decreased to50.61%;48h post-treatment, the viability decreased to25.35%Therefore,150μmol/L H2O2for12h was used in the following experiments.1.2The effects of different concentrations of KTC treatment on cellular viability Treatment with KTC alone did not cause any cytotoxic effect on the cell viability up to the highest concentration (100μg/ml).1.3KTC ameliorated H2O2-induced loss of neuronal cell viabilityWhen the cells were treated with150μmol/L H2O2for12h, their viability decreased to46.76%. The cell viability was71.78%with12.5μg/ml GE treatment for24h. The H2O2-induced viability loss was attenuated by KTC in a concentration-dependent manner. Viability rate with3.125μg/ml,6.25μg/ml,12.5μg/ml of KTC treatment for24h,were95.86%,96.49%and99.04%, respectively.2. Changes in cell structure2.1Observation of cellular morphologyThe cells of control group showed the neurite of neuron. The number of neurons showed a significant reduction after H2O2treatment, and the cells exhibited round and degenerated morphology(shrinkage and had bubble on the neurite). However, the number of cells increased, and exhibited improved cellular morphology after treatment with KTC or GE, respectively.2.2Observation of cellular ultrastructureThe cells in control group were most nearly oval, and cytomembrane did not rupture. Their surface was characterized by many corrugations and microvilli-like structures. Nucleus was showed elliptic and in the center with a single nucleolus; and had normal euchromatin, scattered heterochromatin. Mitochondria showed rod-like or elliptic,scattered with control ridge.The cells treated with H2O2showed typical oxidative damage phenomena. The nucleus decreased in size and changed in shape, Their surface had fewer microvilli-like structures. mitochondria showed swelled with ridge fractured or disappeared, Rough endoplasmic reticulum had obvious degranulation.The cells treated with KTC ameliorated H2O2-induced oxidative damage phenomena, their surface had microvilli-like structures. mitochondria showed rod-like or elliptic,scattered with control ridge.Rough endoplasmic reticulum hadn’t obvious degranulation.3. Changes in antioxidant enzyme3.1Intracellular SOR,CAT,GPx-1,GSHCompared with the control group, H2O2treatment markedly downregulated the levels of HO-1,CAT,GPx-1,GSH and upregulated SOR of MES23.5cells in vitro. GE and KTC treatment upregulated the levels of HO-1,CAT,GPx-1,GSH and downregulated SOR. KTC-H caused a greater increase in the levels of HO-1,CAT,GPx-1,GSH and downregulated SOR.3.2The expression of HO-1,CAT,GPx-1proteinCompared with the control group, H2O2treatment markedly downregulated the expression levels of HO-1, CAT, GPx-1protein and upregulated the expressions of Caspase-3protein. GE and KTC treatment upregulated the expressions of HO-1, CAT, GPx-1protein,and decreased the expression level of Caspase-3protein. As expected, KTC-H caused a greater increase in the expression levels of HO-1, CAT, GPx-1protein. Expression of Caspase-3protein was also greater decreased in the KTC-H group.3.3The expression of HO-1,CAT,GPx-1mRNAAs shown in Fig.7,compared with the control group, H2O2treatment markedly downregulated the expression levels of HO-1,CAT,GPx-1mRNA of MES23.5cells in vitro. GE and KTC treatment upregulated the expressions of HO-1,CAT,GPx-1mRNA. KTC-H caused a greater increase in the expression levels of HO-1,CAT,GPx-1mRNA.Conclusion1.KTC can up-regulate antioxidant enzymes expression after oxidative stress damage of MES23.5nerve cells, playing a role of resistance to oxidative stress is one of the mechanisms to ameliorate the symptoms of Parkinson’s disease.2.This study provides a evidence for developing KTC as a therapeutic agent to ameliorates oxidative stress damage.