Studies On The Epidemiological Characteristics Of Paraquat Poisoning And The Protection Of Resveratrol Against Paraquat-induced Lung Damage

With the extensive application of herbicides, paraquat poisoning is becoming increasingly popular and has become the second common pesticide poisoning following acute organophosphate poisoning. In recent years, the absolute number of paraquat-poisoning death was the first one in pesticide poisoning. Paraquat (PQ), the chemical name is 1,1'-dimethyl-4,4'-bipyridine dioxide dimethyl, is the widely used heterocyclic organic contact herbicide in the world, and it has strong toxicity to human body. Paraquat can be absorbed into the body through the skin, respiratory and gastrointestinal tract. The oral absorption rate of paraquat is 5% to 15%, and the plasma concentration can reach a peak concentration at 1-4 h after absorption. In the body paraquat may cause the damage of lung, kidney, liver and pancreas while the characteristic change is lung injury. The early signs are injury of epithelial cell, alveolar edema, hemorrhage and inflammatory cell infiltration. Alveolar and pulmonary interstitial fibrosis occurs in the advanced stage which can lead to acute respiratory distress, and that is the main cause of death in patients of paraquat poisoning. The main mechanism of lung injury caused by paraquat is that the paraquat is uptaked and transported to the lungs by alveolar epithelial cells through the polyamine uptake system, and then forms a redox cycle which can generate the reactive oxygen species including cation, superoxide anion, hydrogen peroxide and hydroxyl radical, which cause the oxidative damage of the lung. Because of the high mortality of paraquat poisoning and the shortage of effective antidote, the scholars do a series of related research with a variety of antioxidants drugs, such as EDTA, Edaravone, Salvia, Hypericum perforatum extract, ginkgo biloba extract, etc, to look for the effective drugs for paraquat poisoning. But it is difficult and the mortality rate is still very high. So how to relieve the damage induced by paraquat poisoning and improve the situation of the patients are the important task for our scientific workers.Resveratrol (Res) is widely distributed in plants. It has been found in 72 species of plants in 21 families and 31 genera, such as grape in vitaceae, peanuts and chamaecrista in leguminosae, veratrum in liliaceae, etc. Resveratrol is a plant phytoalexin caused by plants to resist the stimulus such as UV, fungus, virus infection or mechanical damage. The study results showed that resveratrol has many important functions such as anti-tumor, anti-inflammatory, antibacterial, antioxidant, anti-free radicals. It can protect liver, nervous, cardiovascular system, and has regulated immunity, affect bone metabolism and antagonize the effects of endothelin functions. In recent years, researchers attach importance to the antioxidant activity of resveratrol, which is stronger than vitamin E and vitamin C. They found out that resveratrol's strong activity of anti-free radical, especially hydroxyl radical, can protect cells and DNA from damage of various chemicals.The RLE-6TN cell line is the alveolar typeⅡepithelial cells of rats. Alveolar epithelial cells, especilly the alveolar typeⅡepithelial cells, play important roles in the development process of lung injury caused by paraquat. Alveolar typeⅡepithelial cells are the stem cells of alveolar epithelial cells, and they have variety of functions which can proliferate to new alveolar typeⅡepithelial cells, differentiate into other cells, such as typeⅠepithelial cells, and synthesize and secrete surfactant. They also can transfport the liquid in the lungs and increase immune function. In normal circumstances, alveolar typeⅡepithelial converse into typeⅠepithelial cells to repair the lung injury, but when alveolar typeⅡepithelial cells has serious injury even apoptosis, the repair processes will be blocked and cause more severe lung injury. In the paper we investigated the clinical literatures about paraquat poisoning in Chinese Mainland from 1991 to 2008, and analyzed the epidemiological characteristics of paraquat poisoning in order to find out the situation of paraqut poisoning and clinical treatment in China. Then we studied the damage of paraquat on RLE-6TN cells and the protection of resveratrol on RLE-6TN cells'oxidative damage caused by paraquat and tried to explore whether the antioxidant resveratrol can protect the cells from the damage. We hope our study can help people to enchance the understanding on paraquat poisoning and its treatment in the future.1 Materials and Methods1.1 Literature analysis on paraquat poisoning in China (1991-2008)The paraquat poisoning literature which published in the medical journals from the year 1991 to 2008 were searched from China Journal full-text Database by the keyword "paraquat poisoning". After classification and checking, the papers which are not related to medical cases or not contain complete information and or duplicate were excluded, and then 369 papers were adopted. Then the datas were collected and the general situation, regional distribution, poisoning reasons, exposure route, treatment and the prognosis of paraquat poisoning were analyzed.1.2 Establishment of the cell model of paraquat poisoningThe cell proliferation and cytotoxicity of RLE-6TN cells after paraquat poisoning were detected by MTT method.1×105 cells per mL were inoculated in 96-well plate. After 24h adherent culture, paraquat which final concentration were 200μmol/L,400μmol/L,600μmol/L,800μmol/L,1000μmol/L,2000μmol/L,4000μmol/L was added into the 96-well plate, and equivalent medium was added in the control group. After 20 h's culture,20μL MTT was added into each hole. After 4h's culture, the supernatant was thrown away, and 150μL DMSO was added into each hole. Then the 96-well plate was shoken 10mins in order to made the blue crystals disloved. After that, the 96-well plate was put in the microplate reader and detected at the wavelength of 570 nm. The cell activity which is represented by the absorbance of each drug group's compared with that of control group's was obtained and the best suitable concentration of paraquat injury was selected.1.3 MTT Detecttion of the cell activity after the cells treated with resveratrol and paraquat1×105 cells per mL were inoculated in 96-well plate. After 24 h adherent culture, resveratrol which final concentration were 0μmol/L,20μmol/L,40μmol/L,60μmol/L,80μmol/L,100μmol/L,120μmol/L was added in the 96-well plate. After 20 h's culture, paraquat which final concentration was 1000μmol/L was added in, and equivalent medium was added in the control group. After 20 h's culture,20μL MTT was added into each well. After 4h culture, the supernatant was threw away, and 150μL DMSO was added into each well, the 96-well plate was shook 10mins to make the blue crystals disloved. After that, the 96-well plate was put in the microplate reader and detected at the wavelength of 570 nm. The cell activity is represented by each drug group's absorbance compared to that of control group.1.4 Detection of the activity of cells' superoxide dismutase (SOD)Cells were divided into 4 groups, control group, paraquat group of 1000μmol/L final concentration, and two resveratrol groups which final concentration were 60μmol/L and 80μmol/L, respectively. After treated with drugs, the activity of cells' superoxide dismutase was detected following the operating instructions of Superoxide Dismutase Kit.1.5 Detection of the activity of cells' lactate dehydrogenase (LDH)Cells were divided into 4 groups, control group, paraquat group of 1000μmol/L final concentration, and two resveratrol groups which final concentration were 60μmol/L and 80μmol/L, respectively. After treated with drugs, the activity of cells' lactate dehydrogenase was detected following the operating instructions of Lactate Dehydrogenase Kit.1.6 Apoptosis examination by flow cytometry (FCM)Cells were divided into 4 groups, control group, paraquat group of 1000μmol/L final concentration, and two resveratrol groups which final concentration were 60μmol/L and 80μmol/L, respectively. After treated with drugs, the apoptosis was detected following the operating instructions of Annexin V-FIFC Apoptosis Detection Kit.1.7 Detection of the DNA damage of RLE-6TN cells caused by paraquat and the protection of resveratrolCells were divided into 4 groups, control group, paraquat group of 1000μmol/L final concentration, and two resveratrol groups which final concentration were 60μmol/L and 80μmol/L, respectively. After treated with drugs, the cells were detected by single-celled electrophoresis. The cellular DNA damage was determined and the DNA damage was classified according to the ratio of the tested cells in the total cells.1.8 Statistical analysisSPSS 13.0 version was used to statistical analysis. The cells' activity which was treated with paraquat, the cells' activity which was treated with resveratrol, the activity of cells' superoxide dismutase and the activity of cells' lactate dehydrogenase were analyzed by One-Way ANOVA and Multiple comparisons, then the data were presented as mean±standard deviation (x±s) and P<0.05 was statistically significant. The DNA damage of each groups analyzed by Chi-square test and P<0.05 was statistically significant.2 Results2.1 The situation of paraquat poisoning in China (1991-2008)The number of paraquat poisoning and deaths has increased markedly in China from 1991 to 2008, especially in Henan, Sichuan, Shandong, Hebei provinces. Most cases were young adults and two-thirds of cases were women. The main cause of poisoning was the suicide with oral intake of paraquat. The total mortality rate of reported paraquat poisoning was nearly 50%. Clinical treatment of paraquat poisoning was mainly to reduce the absorption of paraquat, promote its excretion from body and antagonize paraquat's toxic action. Early application of a variety of blood purification therapy may be effective.2.2 The changes of cell activity which treated with paraquatParaquat damaged cells and decreased the cell activity declined. The reduction of cell activity was significant concentration-dependent. The differences between the paraquat groups over 200μmol/L doses and the control group were statistically significant (P<0.05). The cells activity of the 1000μmol/L paraquat group was 57.17%, which was close to LC50. So the concentration of 1000μmol/L paraquat was chosen for paraquat damaged cell model.2.3 The changes of cell activity which treated with resveratrol and paraquatThe first treatment of resveratrol increased the cell activity. The cell activity in the different concentration resveratrol-treated groups were higher than that of the paraquat group, especially in 60μmol/L and 80μmol/L resveratrol-treated group (P<0.05).2.4 The changes of superoxide dismutase activity in cell culture fluidParaquat decreased the activity of superoxide dismutase in cell culture fluid.The activity of superoxide dismutase in each group was lower than the control group (P<0.05). But if it was treated with resveratrol (60μmol/L or 80μmol/L), the activity of superoxide dismutase in cell culture fluid increased (P<0.05).2.5 The changes of lactate dehydrogenase activity in cell culture fluidParaquat increased the lactate dehydrogenase activity in cell culture fluid, each group's lactate dehydrogenase activity was higher than the control group (P<0.05). But treated with resveratrol firstly declined the lactate dehydrogenase activity in cell culture fluid, especially the 60μmol/L and the 80μmol/L resveratrol-treated group (P<0.05).2.6 ApoptosisParaquat caused the apoptosis of RLE-6TN cells. The apoptosis rate of each paraquat treated group's was higher than the control group. But treated with resveratrol firstly lessened the apoptosis, the apoptosis rate of the 60μmol/L group and the 80μmol/L groups was lower than the paraquat group.2.7 The DNA damage of RLE-6TN cells by paraquat and the protection of resveratrolParaquat damaged the cellular DNA, each group's cells tailing rate was higher than the control group. But treated with resveratrol firstly eased the cellular DNA damage caused by paraquat, the cells tailing rate of the 60μmol/L group and the 80μmol/L group was lower than the paraquat group (P<0.05). 3 Conclusion3.1 Paraquat poisoning, especially caused by the suicide with paraquat, is still an important medical and social problem in China. The number of poisoning has increased markedly year by year, the total mortality rate of poisoning was nearly 50%, but the effective treatment has not found.3.2 The 1000μmol/L concentration of paraquat can cause severe oxidative damage of RLE-6TN cells which was showed by the reduction of superoxide dismutase activity and increasement of lactate dehydrogenase activity, and the inducement of the apoptosis and the damage of cells' DNA.3.3 Resveratrol can antagonize the oxidative damage of RLE-6TN cells caused by paraquat, protect the cells from DNA damage and apoptosis.