Study On The Mechanism Of Lead Exposure To RAW264.7 Cells

Immune system can regulate various physiological processes of the human body,improve immunity,enhance immune function,is the key to prevent diseases and restore health.Macrophage is one of the important cells in the body to play the immune function effect.It is widely distributed in the blood and tissues of the whole body and involved in various physiological,immune and morphogenetic processes.Studies have reported that lead can suppress immune system function.Although lead does not cause significant damage to major immune cells at low concentrations in the environment,nor does it lead to immune cell defects identified by routine tests,it can adversely affect immune cell regulation and function.The oxidative stress,inflammatory damage,apoptosis and autophagy concentration changes of macrophages to lead at the same time point have not been clarified.1.Study on oxidative stress mechanism of RAW264.7 cells induced by lead exposureThe effects of different concentrations of lead(0,3.125,6.25,12.5,25,50,100,200 μM)on cell viability were detected by MTT.The intracellular ROS content was detected by H2 DCFDA fluorescent probe method.The level of MDA was detected by TBA method.SOD activity was determined by hydroxylamine method.The activities of GSH-Px and CAT were determined by spectrophotometry.The expressions of Keap1,Nrf2 and HO-1 proteins as well as Keap1,Nrf2 cytoplasmic and nuclear proteins were detected by Western blot.The results showed that cell viability decreased after exposure to 25,50,100,200 μM lead for 24 h.ROS levels were increased after exposure to 25,50,100 and 200 μM lead.The content of MDA in 200 μM Pb-exposed cells was increased.The activity of SOD in 100 and 200 μM Pb-exposed cells decreased.CAT activity decreased in 50,100 and 200 μM Pbexposed cells.The expression of Nrf2 protein was increased in 50,100 and 200 μM Pbexposed cells.The expressions of Keap1 and HO-1 proteins were increased in 25,50,100 and 200 μM Pb-exposed cells.The expression of Keap1 protein was increased in 50,100 and 200 μM Pb-exposed cytoplasm.The expression of Nrf2 protein was increased in 100 μM Pbexposed cytoplasm.Keap1 protein expression in 50,100,200 μM Pb-exposed nucleus decreased;The expression of Nrf2 protein in 25,50,100 and 200 μM Pb-exposed nuclei increased.2.Study on the mechanism of lead exposure to inflammatory damage of RAW264.7 cellsThe content of NO in cells was detected by Griess method.The secretion levels of TNF-α and IL-6 in the supernatant were detected by ELISA.Western blot was used to detect the expression levels of i NOS,NLRP3,TXNIP and IL-1β,and MAPK and NF-κB signaling pathway related proteins.The results showed that there was NO significant difference in NO secretion and i NOS expression in 25,50,100,200 μM Pb-exposed cells.The levels of TNF-α in 25,50,100,and 200 μM Pb-exposed cells were increased.The level of IL-6 increased in 25 μM Pb-exposed cells.NLRP3 protein expression was decreased in 25,100 and 200 μM Pb-exposed cells.TXNIP expression was increased in lead exposed cells at 25,50,100 and 200 μM.There was no significant difference in IL-1β and p-ERK protein expression.The expression of p-JNK protein decreased in 25,50,100 and 200 μM Pb-exposed cells.The expression of p-p38 protein increased in 25 μM Pb-exposed cells,but decreased in 50,100 and 200 μM Pb-exposed cells.The expression of p-IKBα protein was decreased in 50,100 and 200 μM Pb-exposed cells.The expression of p-p65 protein in 25,50,100,200 μM Pb-exposed cells was increased;p65 protein expression was decreased in 25,50 and 100 μM Pb-exposed cells,but increased in 200 μM Pb exposed cells;p65 protein expression was increased in 25,50,100,and 200 μM Pb-exposed nuclei.3.Study on apoptosis and autophagy mechanism of RAW264.7 cells induced by lead exposureCytophagocytosis was determined by neutral erythrophagocytosis.The mitochondrial apoptosis was detected by JC-1 fluorescence probe.The changes of Cytochrome C,Caspase-3,Bax,Bcl-2,Bcl-2/Bax,and autophagy factors ATG5,ULKI,Beclin1,LC3 B were detected by Western blot.The results showed that the phagocytosis decreased in 100 and 200 μM Pb-exposure groups.The red-to-green fluorescence ratio decreased with the increase in Pb concentration.The expression of Cytochrome C protein increased in 100 μM Pb-exposed cells while decreasing in 25 and 200 μM cells.The expression of Caspase-3 protein was increased in 25,50,100,and 200 μM Pb-exposed cells.Bax expression was increased in 50,100,and 200 μM Pb-exposed cells.Bcl-2 protein expression was decreased in 100 and 200 μM Pb-exposed cells.The expression of Bcl-2/Bax ratio protein increased in 25 μM Pb-exposed cells but decreased in 100 and 200 μM Pb-exposed cells.Beclin1 expression was increased in 25,50,and 100 μM Pb-exposed cells.ATG5 and ULK1 protein expression was increased in 25,50,100,and 200 μM Pb cells.LC3 I protein expression decreased in 25 and 200 μM Pb-exposed cells,while histone expression increased in 100 μM Pb-exposed cells.LC3 II protein expression was increased in 50 and 100 μM Pb-exposed cells.LC3II/I protein expression was increased in 25,50,100 and 200 μM Pb-exposed cells.