Elaidic Acid Induces NLRP3 Inflammasome Activation In Rat Hepatic Kupffer Cells And SD Rat Liver

This work was funded by the National Key Research and Development Program of China(2019YFC1606200).Trans fatty acids(TFAs)are unsaturated fatty acids containing one or more non-conjugated double bonds in the trans configuration,with a high proportion of elaidic acid(EA).In food industry,in order to improve the stability and extend shelf life,TFAs are used in a wide range,such as fried and baked foods.Nutritional and epidemiological studies have shown that high intake of TFAs can cause a variety of adverse effects on human health,including inflammation,lipid metabolism,Alzheimer's disease,cardiovascular disease,diabetes and cancer.Several studies in recent years have shown that the inflammatory response is a causative factor in TFAs triggering several types of diseases,but the exact mechanism is not yet clear.Since inflammatory vesicles are involved in the body's defence response against a variety of pathogenic stimuli,abnormalities in NLRP3 inflammasome are responsible for the pathogenesis of several inflammatory diseases.Studying the effect of EA on NLRP3 inflammasome activation provides a theoretical basis for clarifying the pathogenesis of TFAs-related diseases.In addition,diet is an important factor in regulating the intestinal flora,but there are few studies on the changes of intestinal flora induced by TFAs in the organism.Exploring the effects of EA on the gut-liver axis will help to study the effects of TFAs on the liver and intestinal innate immune system,and investigate in depth with the mechanisms of EA-induced inflammatory responses in the organism.The main study contents and findings are as follows.(1)The cell model was established using rat hepatic Kupffer cells to explore the effects of different doses of EA(0,0.05,0.1 m M)on NLRP3 inflammasome in rat hepatic Kupffer cells.By detecting the m RNA and protein expression levels of NLRP3 inflammasome in rat hepatic Kupffer cells induced by EA,it was demonstrated that EA induced the activation of NLRP3 inflammasome in rat hepatic Kupffer cells.EA induced an increase in the release of ROS,a significant decrease in the activity of SOD and GSH,and a significant increase in the content of MDA in rat hepatic Kupffer cells,all of these proved that EA caused oxidative damage to cells.The expression results of ERS-related molecules at m RNA and protein levels demonstrated that EA induced ERS in rat hepatic Kupffer cells.Finally,ROS scavengers,ERS inhibitors and MAPK selective inhibitors were used to clarify the effects of OS,ERS and MAPK signaling pathway on EA-induced NLRP3inflammasome activation in rat hepatic Kupffer cells,demonstrating that EA activates NLRP3 inflammasome by MAPK signaling pathway through the regulation of OS and ERS in rat hepatic Kupffer cells,with massive release of inflammatory factors.(2)Immunofluorescence experiments revealed that EA induced a significant increase in the degree of endoplasmic reticulum-mitochondrial superposition in rat hepatic Kupffer cells,the co-localization of the endoplasmic reticulum membrane marker protein IP3R and the mitochondrial outer membrane marker protein VDAC1on the MAM was also significantly improved.Meanwhile,immunoprecipitation experiments demonstrated that the linkage of IP3R-Grp75-VDAC1 was enhanced.The above studies confirmed that EA induced the enhancement of MAM structure in rat hepatic Kupffer cells.EA induced a significant increase in both intracellular and mitochondrial Ca²⁺levels in rat hepatic Kupffer cells,while the use of IP3R inhibitor and MCU inhibitor inhibited intracellular and mitochondrial Ca²⁺release levels,respectively,as well as suppressed MCU protein expression levels,demonstrating that the excessive Ca²⁺released from endoplasmic reticulum of rat hepatic Kupffer cells induced by EA was released into the mitochondria and accumulated via the IP3R-Grp75-VDAC1-MCU based on MAM structure.Laser confocal microscopy results showed that EA induced a large release of mt ROS from rat hepatic Kupffer cells,while the IP3R inhibitor and MCU inhibitor inhibited the release of mt ROS induced by EA,demonstrating that Ca²⁺transfer at the MAM was closely related to mitochondrial damage.Subsequently,EA-induced increase in m PTP permeability in rat hepatic Kupffer cells eventually led to a significant decrease in mitochondrial membrane potential and ATP.The results of transmission electron microscopy results showed that the cells showed swelling of the inner mitochondrial cristae and mitochondrial vacuolization.In summary,EA induced mitochondrial Ca²⁺imbalance in rat hepatic Kupffer cells through the MAM structure,which ultimately led to mitochondrial dysfunction.(3)The effects of EA on inflammatory injury in the liver of SD rats were investigated by constructing an SD rat toxin model(0,50,100,150 mg/kg).EA induced a significant increase in the expression levels of m RNA and protein of NLRP3 inflammasome related molecules in the SD rat liver.It was determined that EA activated NLRP3 inflammasome in the liver of SD rats.EA induced an increase in ROS release,a decrease in SOD and GSH activities,and a significant increase in8-OHdG and MDA concentration in SD rat liver,which proved that EA induced OS injury in SD rat liver.The expression levels of ERS and MAPK signaling pathway related proteins were examined,and it was found that EA activated the ERS as well as MAPK signaling pathway in the liver of SD rats and promoted the inflammatory response,which was consistent with the results of cellular level research.EA induced the activation of NLRP3 inflammasome and the release of inflammatory factors in SD rat liver by regulating MAPK signaling pathway mediated by OS and ERS.The detection of ATP,mitochondria membrane potential and ultrastructural observations of rat liver showed that EA induced the significant decrease of mitochondrial membrane potential and ATP in SD rats.The ultrastructural characterization showed that EA induced the swelling and vacuolation of liver mitochondrial ridge,which eventually led to the damage of liver mitochondria.(4)In the SD rat toxicity model,the pathological histological results showed that EA induced obvious lesions in the intestinal structures of rats,the increased release of the intestinal inflammatory factor TNF-?,as well as a significant increase in serum,liver and intestinal LDH levels,further indicating that EA induced inflammatory damage in the liver and intestine of rats.The results of 16S r DNA high-throughput sequencing showed that the abundance of Proteobacteria induced by EA in high-dose EA group was significantly higher than control group in the phylum level,the abundance of Prevotella,Ruminococcaceae and Burkholderiaceae,which are related to obesity and inflammation,was significantly up-regulated.ELISA results indicated that EA induced a significant increase in LPS levels in rat serum,liver and intestine.The protein expression levels of intestinal tight junction proteins ZO-1,Occludin and Claudin-1 were found decreased significantly.Combined with the characterization of disrupted intestinal tight junctions by transmission electron microscopy,further indicated that EA led to an increase in the permeability of the intestinal barrier and disruption of the intestinal barrier in rats.The expression levels of key proteins of the TLR4/My D88/NF-?B signalling pathway in rat liver were significantly increased,and the LPS levels in serum,intestine and liver were significantly increased,further demonstrating that EA induced the release of LPS and pathogenic bacteria in rat intestine to activate the TLR4/My D88/NF-?B signalling pathway,thereby exacerbating liver inflammation and injury.In summary,EA can activate NLRP3 inflammasome by triggering OS and ERS,while EA modulated ROS release and ERS to induce massive Ca²⁺release into mitochondria through the MAM structure based on IP3R-Grp75-VDAC1-MCU,causing mitochondrial Ca²⁺overload and mitochondrial damage.EA can exacerbate liver inflammatory injury by inducing the proliferation of harmful bacteria in the rat intestine and the release of the endotoxin,which activated the TLR4/MyD88/NF-?B signalling pathway.