The Effects And Oxidative Stress Mechanisms Of Cadmium Exposure On Hepatic Stellate Cell Activation

Cadmium?Cd?,a toxic heavy metal,posing a huge threat to human health.Acute high dose Cd exposure results in multiple organs damage,while for prolonged low dose Cd exposure,previous studies had confirmed its carcinogenic potential.When the Cd contaminated food orally ingested and absorbed,it firstly reached to liver through the portal system.Meanwhile,Cd has a long biological half-life between10-30 years,and repeats the process of accumulation.Therefore liver is one of the main target organs of Cd toxicity.In liver pathological changes of patients with“itai-itai”disease,the incidence of hepatic fibrosis was significantly higher than that of the control group.Liver fibrosis may be a specific change caused by persistent Cd exposure.Epidemiology has shown that progression of chronic liver disease may be associated with high Cd exposure.In vivo experiments,long-term Cd exposure can lead to hepatic interstitial fibrosis.However,the mechanisms of Cd exposure in the occurrence and progression of hepatic fibrosis are still unclear.Hepatic stellate cell?HSC?is a crucial mediator in the process of liver fibrosis.Following liver injury,the quiescent HSC activated and transformed into myofibroblast-like phenotype to secrete the alpha smooth muscle actin??SMA?and type?collagen to facilitate the liver fibrosis,and the perpetuation of its activation is related to the progress of liver fibrosis.Therefore,this research focused on Cd induced HSC activation and perpetuation and possible oxidative stress mechanisms by using in vivo and vitro experiments,and further to reveal the effects of long-term Cd exposure on the progression of hepatic fibrosis,and provide evidence for understanding the long-term effects of cadmium exposure.Methods:1.Cd exposure rats experiments,the rats were randomly divided into 4 groups:control group,GSH treated,Cd treated and Cd+GSH treated.Liver function index was detected by automatic biochemical analyzer,H&E staining was used to detect the pathological changes of liver tissue,Masson staining was used to detect the collagen fibers,immunohistochemistry staining was used to detect the expression of?SMA.Transmission electron microscopy was used to observe the changes of ultrastructure in hepatocytes and HSC.GSH,Vc,SOD,CAT and GSH-Px levels were detected by spectrophotometer or microplate reader.Western blotting was used to detect the specific markers of HSC activation and the expression of related protein in NF-?B and MAPK signaling pathways.2.HSC activation in vitro,the L02 exposed to Cd and the supernatant as conditioned medium?L02-CM?,?Cd-L02-CM?and?Cd+GSH-L02-CM?,were used to treat the LX2 cells,the expression of?SMA in LX-2 was detected.3.Mass cytometry to detect Cd signal and viability in LX-2 cells with different concentrations of Cd;Multiple low-dose Cd intervention?0.5?M,2?M?on HSC-T6cell phenotype and functions,CCK8 was used to measure the cell viability,flow cytometry was used to detect the apoptosis rate and the ROS levels,wound-healing test and transwell test to detect the migration ability,oil red O staining was used to detect the contents of lipid in HSC-T6,The level of ROS was also detected by fluorescence microscope,western blotting was used to detect specific markers of activated HSC and proteins in Nrf-2 and NF-?B signaling pathways.Results:1.The oxidative stress mechanism on Cd induced HSC activation.?1?Cd exposure and liver injury.Cd intervention can increase serum AST,GGT and XOD?P<0.05?,and given GSH can reduce the above liver damage index?P<0.05?.HE staining showed pathological changes such as inflammatory cell infiltration and hepatocyte necrosis in the Cd intervention group.Under the transmission electron microscope,the mitochondrial membrane and inner structure were partially destroyed.?2?Cd exposure and HSC activation.Cd exposure significantly increased the protein levels of?SMA?P<0.05?,and the immunohistochemical staining demonstrated that the?SMA positive cells?activated HSC?have more distribution out of the area of blood vessels.The lipid drops disappeared,and the collagen fibers surrounding the HSC,also confirmed the activation staus under the electron microscopy.However,Cd+GSH group decreased the expression of?SMA protein?P<0.05?,and the lipid drops in the HSC indicates a quiescent status.?3?Cd exposure and the oxidative status in liver.Cd exposure can increase the contens of small molecule antioxidants such as GSH and vitamin C?Vc??P<0.05?compared to the control group.The antioxidant enzyme activities such as superoxide dismutase?SOD?,catalase?CAT?and glutathione peroxidase?gsh-px?were inhibited?P<0.05?after Cd exposure,while given exogenous GSH attenuated these Cd induced alterations?P<0.05?.?4?The effects of Cd exposure on the NF-?B and MAPK signal pathway.Cd exposure can increase the protein levels of p-p65 and p-IkB?,as well as the p-JNK?p<0.05?compared to control group,while given GSH can significantly increase the p-p65 protein and reverse the levels of p-JNK?P<0.05?.?5?HSC activation in vitro,GSH intervention reduced Cd content and ROS production in L02 cells,and the expression of?SMA increased in LX-2 after Cd-L02-CM intervention.2.Effect of Cd intervention on HSC phenotype and function.?1?Cd uptake and viability of LX-2 cells.LX-2 cells were treated with different concentrations of Cd for 1h,the contents of ¹¹⁴Cd was 77.1?median intensity Value?higher in the 5?M Cd group than the others.And the proportion of ¹¹⁴Cd positive cells was 98.88%.However,the percentage of ¹⁹⁵pt strong positive cells?indicated death cells?is 35.5%.CCK8 tests also showed a significant inhibition of LX-2 cell viability after 5?M Cd exposure?P<0.05?.?2?Effects of multiple low-dose Cd interventions on the function and phenotype of HSC-T6 cells.There is no significantly difference on the proportion of apoptotic cells between the Cd treated group and the control group in the HSC-T6 cell line.The ability of proliferation migration was enhanced after 0.5?M Cd exposure.The oil red staining in HSC-T6 cells showed more lipid droplets in control cells than the Cd exposure group.The expression levels of fibrotic related protein?SMA in the Cd group was significantly increased?P<0.05?;After multiple low dose Cd interventions the ability of Cd uptake in HSC-T6 decreased when given a new Cd exposure,and the levels of p-P65 and HO-1 were increased compared to control group.?3?Low-dose Cd exposure protects against high-dose Cd toxicity.The 2?M+30?M Cd intervention group can resist high Cd toxicity to a certain extent,and its cell viability is higher than other groups.The ROS positive peaks were observed in the 0+30?M and 0.5+30?M Cd intervention groups.Conclusions:?1?Cd could activate HSC by decreasing the activity of antioxidant enzymes,up-regulating p-JNK,promoting ROS production and inducing oxidative injury.However,administration of GSH reduced intracellular Cd uptake,restored antioxidant enzyme activity to some extents,and might to up-regulate the p-P65 and inhibit p-JNK protein expression,reduce the oxidative injury and finally attenuate the HSC activation.?2?Cd could affect the phenotype and function of HSC-T6 cells.After multiple low doses Cd intervention,the expression of HSC activated markers increased and the proliferation and migration of HSC-T6 also enhanced.The phenotype and its protect role against to high-dose Cd toxicity may relate to the up-regulation of p-P65 and HO-1.?3?Mass cytometry could be a new and effective tool for studying Cd toxicity in single cell.