Experimental Study On The Effects Of LA, Tau On Oxidative Injuries In Rats And The Effects Of RNAi On Bax, JNK On Apoptosis In 293 Cell Induced By Cadmium

ObjectiveCadmium (Cd) is an important industrial and environmental pollutant that poses a significant health risk to humans and animals. Depending on. the dose, route, and duration of exposure, Cd can cause damage to various organs including the lung, liver, bone, kidney and so on. Kidney is the major target organ of Cd - induced damage. The mechanism of damage induced by cadmium is very complex and infected by various facters. Oxidative stress and apoptosis may be the crucial mechanism which contributes to Cd toxicity reported by a lot of correlative studies.The aims of the present studies are as follows:1. To study the effects of LA,Tau on acute toxicity of cadmium in rats.2. To study the effects of LA and Tau on sub - chronic toxicity of cadmium in rats.3. To investigate the effects of JNK (c - Jun N - terminal Kinase)/c - jun transmit access and Bax gene in 293 cell apoptosis induced by cadmium with small interference RNA on JNK and Bax genes.Method1. Experimental study on the effects of alpha - lipoic acid and taurine on a-cute oxidative injurys induced by cadmium in vivo32 male and female Wistar rats, 150 ± 10 g of weight, were purchased from Laboratory Animal Center in China Medical University. The animals werehoused at 17 -23CC and 45 -55 percentage humidity. Rat chow was provided by Laboratory Animal Center. The animals were fed for five days before experiment, and then they were divided into four groups at random;the control and three experimental groups, 8 in each group. The first group was given subcutaneous injection of normal saline 5ml/kg. The second group received subcutaneous injection of CdCl2 35 |xrnol/kg. The third and the fourth groups were respectively injected alpha - lipoic acid 175 jxmol/kg and taurine 4mmol/kg in abdominal cavity, after two hours the rats were given subcutaneous injection of CdCl2 35ujnol/kg. After 24hours the rats were anesthetized with ethyl ether and then turned over and abdomen was opened, 5ml blood sample was collected from abdominal aorta. After the rats were killed, liver and renal were taken out. The activity of GPT and LDH in serum, the content of GSH, MDA, Cd and the activity of SOD, GSH - Px in the liver and renal cortex were determined.2. Experimental study on the effects of alpha - lipoic acid and taurine on sub - chronic toxicity of cadmium in vivo32 male and female Wistar rats, 150 i 10 g of weight, were purchased from Laboratory Animal Center in China Medical University. The animals were housed at 17 -23^ and 45 -55 percentage humidity. Rat chow was provided by Laboratory Animal Center. The animals were fed for five days before experiment, and then they were divided into four groups at random;the control and three experimental groups, 8 in each group. Control group was injected sc with 0.9 % NaCl;Cadmium chloride group, LA treated group and Tau treated groups were injected sc with 7jxmol CdCl2/kg, 5 times a week, for up to 6 weeks;Then LA treated group and Tau treated group were injected ip with 35|xmol/kg LA and 0. 8mmol/kg Tau respectively for 2 weeks, 5 times a week;Cadmium chloride group and control group were injected with 0. 9 % NaCl at the same time. At the end of the eighth week,the animals were placed in individual metabolic cages, and 24 hours urinary samples were collected. Then the animals were anesthetized with ethyl ether. After the animals were killed, liver and kidney cortex samples were collected. Cd, protein contents and ALP, NAG activities in urine were determined. Cd, MDA and GSH contents in liver and renal cortex as well as GSH - Px, SOD activities were measured.3. Study on the effects of JNK /c - jun transmit access and bax gene in 293 cell apoptosis induced by cadmium with small interference RNA on JNK and Bax genes.293 cell was routinely maintained in Dulbecco's modified Eagle medium (DMEM) containing 10% fetal bovine serum, 371 in a humidified atmosphere with 5% CO2 in air. When cells reached 60 -70% area of bottle, the cells were shifted one in two.3. 1 Expression of genes in 293 cell treated by cadmiumTwo groups were settled: the control and Cd treated group. When cells reached 60-70% area of bottle, cultures were refreshed, DMEM only for control and DMEM containing 30|xM cadmium chloride without fetal bovine serum for Cd treated group. After 4h, lOh and 48h refreshed, expression level of JNK, c -jun, Bax and the rates of apoptosis were measured respectively.3.2 The effect of Bax on apoptosis of 293 cell induced by cadmium with RNAi on BaxFive groups were settled: the control, Cd treated group, siRNA - Bax treated group, false siRNA treated group and transfection agent group. SiRNA - Bax (2nM) , false siRNA(2nM) and transfection agent(0.41(xg/ml) were tranfect-ed to cells in siRNA - Bax treated group, false siRNA treated group and transfection agent group respectively after the cultures were refreshed using DMEM containing 1 % fetal bovine serum without antibiotics. After 36h, cultures were refreshed again, DMEM for control and DMED containing 30u,M Cd for another four groups. Incubated with Cd for lOh and 48h, the expressions of bax and the rates of apoptosis were measured respectively.3. 3 The effect of JNK/c - jun on apoptosis of 293 cell induced by cadmium with RNAi on JNKFive groups were settled: the control, Cd treated group, siRNA - JNK treated group, false siRNA treated group and transfection agent group. siRNA -JNK (0. 8nM) , false siRNA (0. 8nM) and transfection agent (0. 16|xg/ml) were tranfected to cells in siRNA - JNK treated group, false siRNA treated group and transfection agent group respectively after the cultures were refreshed using DMEM containing 1 % fetal bovine serum without antibiotics. After 36h,cultures were refreshed again, DMEM for control and DMED containing 30jxM Cd for another four groups. Incubated with Cd for 4h, lOh and 48h, the expressions of JNK, c - jun, bax gene and the rates of apoptosis were measured respectively.3.4 Investigation of cell activity with MTT293 cells were incubated in 96 wells, seven groups were settles: the control , siRNA - Bax treated group, false siRNA on Bax treated group, transfection agent for Bax group, siRNA - JNK treated group, false siRNA on JNK treated group and transfection agent for JNK group, 8 wells for each group. Cell activity was determined after 36h tranfection with MTT.Result1. Experimental study on the effects of alpha — lipoic acid and taurine on a-cute oxidative injurys induced by cadmium in vivoCompared with the control group, the activity of GPT and LDH in serum, the content of Cd in tissues of Cd group increased evidently. The activity of SOD in tissues declined evidently. The concentration of GSH, MDA increased, and the activity of GSH - Px declined significantly in liver. After pretreated with LA or Tau, the activity of GPT and LDH in serum, the content of Cd in liver declined, and the activity of GSH -Px increased evidently. LA made the content of MDA in liver lower and the activity of SOD in both tissues higher evidently. Tau made the concentration of GSH in liver and the content of Cd in renal cortex lower significantly.2. Experimental study on the effects of alpha - lipoic acid and taurine on sub - chronic toxicity of cadmium in vivoCompared with the control group, the content of Cd in liver, kidney cortex and urine increased evidently with Cd group. The activity of ALP and NAG were obviously increased as well as the content of protein in urine. The contents of GSH in both tissues and MDA in kidney cortex were increased evidently. The activity of SOD in liver and activity of GSH -Px in both tissues were declined. Contrast with the group given Cd alone, the concentration of Cd in urine was de-clined in company with reducement of the activity of ALP and the content of u-rine protein in the group given LA. The contents of GSH and MDA in kidney cortex were declined. The activitys of GSH - Px in both tissues and SOD in liver were increased significantly.. After pretreated with Tau, the contents of Cd in liver and urine were declined. The content of GSH in kidney cortex was reduced in the meanwhile the activity of GSH - Px was increased significantly.3. Study on the effects of JNK /c - jun transmit access and bax gene in 293 cell apoptosis induced by cadmium with small interference RNA on JNK and Bax gene.Results were as follows:1. After treated with CdCl2 the expression levels of JNK, c - jun and Bax were higher than that of the control group also as the rate of apoptosis.2. The apoptosis rates of 293 cells treated with 30|xM CdCl2were higher than that of the control accompanied by increased expressions of bax. siRNA -Bax could inhibit those changes but false siRNA and transfection agent couldnt.3. The apoptosis rates of 293 cells treated with 30jxM CdCl2 were higher than that of the control group accompanied by increased gene expressions level of JNK, c - jun and Bax. siRNA - JNK could inhibit those changes except protein activity of c - jun but false siRNA and transfection agent couldnt.4. False siRNA and Sofast only had nothing effect on cell activity.Conclusion1. LA and Tau may prevent liver from damage induced by acute Cd exposure to some extension, which may be related to the light of oxidative stress.2. LA and Tau had some accelerating effect to healing of kidney after sub- chronic oxidative injury by Cd.3. The results were suggested that: JNK was a key role during cadmium induced 293 cells apoptosis, which bring its effect into play by phosphorylating c- jun;Bax has been shown to play some role in the apoptosis of 293 cell line induced by cadmium and regulated by JNK/c - jun to some extension.