Study On Cellular And Molecular Mechanisms Of Brain And Kidney Injury Induced By Lead Acetate

BACKGROUNDLead,which is one of the most popular contaminations in the environment, is able to cumulate for a long time and damage various of tissue systems of human body, especially on the children. Recent study showed that Lead acquires neurotropism and cumulates in the nervous system, then further induces long and inconvertible damage. The kidney, as the main organ of distribution and excretion of Lead, is one of the target organs of Lead damage. Some researches demonstrated that there is no safe threshold in the Lead toxicity, that's to say, the toxicity exist when lead invades the human body. Although many countries'governments have reduced the Lead contaminations in the environment through many measures, chronic harm of Lead on the body is still one of the most essential healthy problems which people have to face nowadays.Blood brain barrier (BBB), which consists of the endothelial cells of brain blood capillaries, tight junction between endothelial cells, basement membrane and neuroglial cells(gelatinous membrane), is not only an important part of the central nervous system but also ensures internal environment of the nervous system to keep highly stable. Therefore, BBB supplies the base for various normal activities of central nervous system (CNS). The nephron is the structural and functional unit of the kidney, comprising renal corpuscle and an epithelial renal tubule. The renal corpuscle consists of blood capillaries and mesenteria which develop a significant function, while the renal tubule functions as reabsorption and excretion. The structural and functional changes of BBB or renal unit may lead to pathophysiological changes of many diseases.Nowadays many researches about Lead toxicity on the brain and kidney mainly focus on the animals models, epidemiological investigations, and some cells like hippocampal neurons and human kidney epithelial cell line(HK-2), however, studies on the relationship between Lead and mouse astrocytes(C8), cerebral capillary vessel endothelial cell and human glomerular mesangial cells are rare. Based on previous animal experience, we studied the effects of Lead toxicity on C8, human femoral artery endothelial cell (HFAEC), Human mesangial cells (HMC) and HK-2, and tried to explore the cellular and molecular mechanisms of Lead effecting on the brain and kidney. PartⅠEffects of Lead toxicity on astrocytes(As) and human femoral arterial endothelial cell(HFAEC)Objection To explore Lead acetate inducing apoptosis of mice astrocytes(C8) and human femoral arterial endothelial cell (HFAEC), and provide theoretical basis for the damage of Lead to blood-brain barrier and nephron.Methods1. C8 and HFAEC in logarithmic growth phase were treated to different concen- -tration Lead acetate(5,10,20,40μmol/L)for different time (6,12,24,48h). Mean- -while, the cell treated without Lead acetate was served as the control group, and the prorolifcations of the cells were detected by MTT.2. C8 and HFAEC were treated with 5,10,20μmol/L Lead acetate for 24h respect- -ively .the cell treated without Lead acetate was served as the control group, Cell morphology changes were observed by Giemsa and HE stain and cell ultrastruc- -tures were observed by transmission electron microscopy.3. LDH activity and MDA content of cellular supernatant of Lead acetate-treated groups (5, 10, 20μmol/L) for 24h and blank group of C8 and HFAEC were detected by spectrophotometer in order to investigate cell damage level.4. DNA damage of Lead acetate-treated grous (5, 10, 20μmol/L) and control group of C8 and HFAEC were detected by DNA ladder.5. The expression of c-jun,P53, Bax, Bcl-2 and Caspase-3 of Lead acetate-treated groups (5,10,20μmol/L) and control group of C8 and HFAEC were detected by cell immunohistochemistry.6. Caspase-3 mRNA level of Lead acetate-treated groups (5, 10, 20μmol/L) and control group of C8 and HFAEC were detected by RT-PCR .7. Caspase-3 protein level of Lead acetate-treated groups (5, 10, 20μmol/L) and control group of C8 and HFAEC were detected by Western blotting. 8. Cell apoptosis rates of Lead acetate-treated groups (5, 10, 20μmol/L) and control group of C8 and HFAEC were detected by PI-Hoechst33342 stain and flow cytometry.Results1. With different concentration of Lead acetate(5,10,20,40μmol/L)and different time (6,12,24,48h), the cell growth activity in treated groups reduced significantly compared to the control group (P<0.01), presenting a dose and time-dependent manner.2. C8 and HFAEC treated with the Lead acetate (5,10,20μmol/L)for 24h showed that cell density was lower, and synapses became shorter and thinner, and intercellular junction reduced compared to control group. Giemsa and HE stain showed that cell nucleolus and cytoplasm dense stained, pyknosis,nucleolus cracking, and part of nucleolus showed kindey and horseshoe type. Electron microscopy showed nuclear pyknosis, side shift, nucleus and cytoplasm space increased on exposed groups of Lead acetate, and with the increasing concentration of Lead acetate, the apoptotic bodies appeared.3. LDH and MDA content in supernatant of treated groups (5, 10, 20μmol/L) increased significantly compared to control group (P<0.01).4. The result of DNA ladder showed that, diffused bands appeared in treated groups (5, 10, 20μmol/L) compared to control group by agarose gel electrophoresis.5. Results of immunohistochemistry showed that the expression of c-jun, P53, Bax and Caspase-3 increased, meanwhile Bcl-2 decreased in treated groups (5, 10, 20μmol/L) compared to control group.6. The result of RT-PCR showed that Caspase-3 mRNA level increased in 5, 10, 20μmol/L toxicant groups compared to control group,and the expression of Capase-3 was positively correlated with Lead dose.)7. Western blotting suggested that Caspase-3 expression of C8 and HFAEC in the treated group was higher than that in the control group,and the expression of Capase-3 was positively correlated with Lead dose.8. PI-Hoechst33342 double staining and flow cytometry demonstrated that cell apoptosis rates of 5, 10, 20μmol/L toxicant groups increased significantly compared to control group(P<0.01)。 Conclusion Lead acetate was able to induce the formation of lipid peroxide and result in the damage of genome DNA of C8 and HFAEC, and then influence the expression of apoptosis-related factors, increase the ratio of Bax/Bcl-2, activate the high expression of Caspase-3, finally lead to the apoptosis of C8 and HFAEC and destroy the structure and function of brain blood barrier. PartⅡEffects of Lead toxicity on human mesangial cells and human renal tubular epithelial cellsObjetion To find the effects of Lead acetate induced apoptosis of human mesangial cells(HMC) and human renal tubular epithelial cells(HK-2), and provide theoretical basis for the damage of Lead to nephron. Methods The same to the PartⅠResults1. With different concentration of Lead acetate(5,10,20,40μmol/L)and different time (6,12,24,48h), the cell growth activity in toxicant groups reduced significantly compared to the control group (P<0.01), presenting a dose and time-dependent manner.2. Under inverted phase contrast microscope observing,the Lead acetate(5,10,20μmol/L)treated HMC and HK-2 for 24h showed that cell density was lower, synapses became shorter and thinner and intercellular junction reduced compared to control group. Giemsa and HE stain showed that cell nucleolus and cytoplasm dense stained, pyknosis, nucleolus cracking, and part of nucleolus showed kindey and horseshoe type. Electron microscopy showed nuclear pyknosis, side shift, nucleus and cytoplasm space increased on exposed groups of Lead acetate, and with the increasing concentration of Lead acetate, the apoptotic bodies appeared.3. LDH and MDA content in supernatant of treated groups (5, 10, 20μmol/L) increased significantly compared to control group (P<0.01).4. The result of DNA Ladder showed that, diffused bands appeared in treated groups(5,10,20μmol/L) compared to control group by agarose gel electrophoresis.5. Results of immunohistochemistry showed that the expression of c-jun, P53, Bax and Caspase-3 increased, meanwhile Bcl-2 decreased in treated groups(5,10,20μmol/L) compared to control group.6. The result of RT-PCR showed that Caspase-3 mRNA level increased in treated groups (5,10,20μmol/L) compared to control group,and the transcription of Capase-3 was positively correlated with Lead dose.7. Western blotting suggested that Caspase-3 expression of HMC and HK-2 in the treated group was higher than that in the control group,and the expression of Capase-3 was positively correlated with Lead dose.8. PI-Hoechst33342 double staining and flow cytometry demonstrated that cell apoptosis rates of treated groups (5, 10, 20μmol/L) increased significantly compared to control group(P<0.01).Conclusion Lead acetate was able to induce the formation of lipid peroxide and result in the damage of genome DNA of HMC and HK-2, and then influence the expression of apoptosis-related factors, increase the ratio of Bax/Bcl-2, activate the high expression of Caspase-3, finally lead to the apoptosis of HMC and HK-2 and damage the structure and function of the kidney.