Molybdenum And Cadmium Combine To Induce Sheep Hepatocyte Inflammation By Targeting Mitochondria-related Endoplasmic Reticulum Membrane

To investigate whether induction of Mo and Cd co-exposure dysfunction of MAM and causes the production of NLRP3 inflammasome leading to the onset of inflammatory responses in sheep hepatocytes.A modified two-step collagenase perfusion method was used to extract primary sheep hepatocytes to establish a model of Mo,Cd and their combined exposure.The experiment was divided into four groups:control group,Mo group（600μM Mo）,Cd group（4μM Cd）,Mo combined group(600μM Mo+4μM Cd.The cells were collected after treatment for 24 h.The MAM structure,function and MAM-related factor levels,and production of NLRP3 inflammasome and inflammation levels were assessed by transmission electron microscopy observation,antioxidant index assay,flow cytometry,immunofluorescence observation,quantitative real-time fluorescence assay and protein immunoblotting assay.TThe results showed that compared with the control group,Mo,Cd and their combined exposure shortened the functional regions of MAM in hepatocytes,abnormal changes in the number of mitochondrial and endoplasmic reticulum,VDAC1 and IP3R,NLRP3 and ASC co-localized fluorescent spots,elevated LDH and NO levels in cell supernatants（P<0.05）,and significantly elevated intracellular and mitochondrial Ca²⁺levels（P<0.05）.In parallel,the campared with the control group,Mo,Cd and their combined exposure interference with the expression of MAM-related factors and upregulated the expression levels of NLRP3 inflammasome and inflammation-related factors（P<0.05）.In contrast,the use of 2-APB,an inhibitor of IP3R,resulted in a significant remission of these indicators.These data suggest that Mo and Cd co-exposure causes production of NLRP3 inflammasome by inducing MAM dysfunction,which drives inflammatory responses in sheep hepatocytes,while inflammation of sheep hepatocytes induced by Mo and Cd targeting MAM can be alleviated by inhibiting IP3R.