Free Fatty Acids Induce Activation Of ROS-NLRP3 Inflammasome Signaling In Rat Mesangial Cells

Objective: Diabetic nephropathy(DN) is the serious microvascular complications of diabetes, which has became the leading cause of end-stage renal disease in the western countries. It is considered that inflammation mediated by the innate immune is the significant link in the development of diabetic nephropathy in recent years’ studies. The innate immune system is the first line defense against pathogens, and it is usually involved in the initiation and propagation of inflammation, and activated by pattern recognition receptors to distinguish ‘self’ and ‘non-self’ antigens. It initiates the inflammatory reaction and responses to the infection and tissue damage in the environment by pattern recognition receptors which can recognize danger signals through damage-associated molecular patterns and pathogen-associated molecular patterns.NLRP3 inflammasome is a member of the innate immune system, it can be activated by endogenous and exogenous danger signals, then triggers the innate immune system, results in the activation of Caspase-1 and promotes the mature and secretion of pro-inflammatory cytokines IL-1β,IL-18 and IL-33,finally amplifies the pro-inflammatory response.Mounting evidences indicate that the NLRP3 inflammasome plays a role in a variety of inflammatory diseases, and pro-inflammatory cytokines play an important regulating role in the inflammatory process. Elevated plasma free fatty acids can lead to lipotoxicity, and the lipotoxicity by free fatty acids can stimulate tissue to release inflammatory factors, with which can cause metabolic organs in a chronic low-grade inflammatory state.However, whether free fatty acids could activate NLRP3 inflammasome and participate in the kidney inflammation in glomerular mesangial cells is unknown. Therefore, this study observes the expression of NLRP3 inflammasome signaling pathway which induced by palmitic acid and reactive oxygen species(ROS) inhibitors NAC in glomerular mesangial cells, aiming to find a new idea of therapeutic targets in the NLRP3 inflammasome signaling pathway. Methods:1.Rat glomerular mesangial cells, cultured with palmitic acid as stimulating factor and ROS inhibitor NAC as the intervention factor, were divided into 3 groups: ①normal control group(NC group):5.6mmol/L glucose; ②different concentrations gradient of high palmitic acid groups(HPA group):HPA1 group: culture medium containing 0.05 mmol/L palmitic acid; HPA2 group: culture medium containing 0.1 mmol/L palmitic acid; HPA3 group: culture medium containing 0.15 mmol/L palmitic acid; ③The NAC intervention in high palmitic acid group(NAC+HPA group):10μmol/L NAC added in advance in the best effect of 0.15 mmol/L palmitic acid medium to block the production of reactive oxygen species for observing the role of reactive oxygen species in the activation of NLRP3 signaling induced by palmitic acid. The each group was induced for 6,9 and 12 h, then detected the expression of NLRP3,Caspase-1,IL-1β protein and m RNA by Western-blot and RT-PCR. Results: Compared with normal control group, the protein and m RNA expression of NLRP3,Caspase-1 and IL-1β were up-regulated after palmitic acid treatment, in a dose- and time- dependent manner(P<0.05).However,compared with group HPA3, the protein and m RNA expression of NLRP3,Caspase-1 and IL-1β were obviously decreased by NAC intervention(P<0.05).Conclusion: The results indicate that palmitic acid could induces the activation of NLRP3 inflammasome signaling via oxidative stress machanisms in mesangial cells and this may be one of renal inflammation mechanisms induced by free fatty acids.