High Glucose And Lipopolysaccharide Induce Activation Of NLRP3Inflammasome Through ROS/TXNIP Pathway In Rat Mesangial Cells

Objective: Diabetic nephropathy (DN) is one of thespecific microvascular complications of diabetes and the leading cause ofend-stage renal disease in the western. The onset of diabetic nephropathyis insidious, and the mechanism of it is unclear. Now it is considered thatinflammatory immune response is the central link in the development ofdiabetic nephropathy. The innate immune system builds up the body‘sfirst line. It initiates immune response and responses to themicroorganism or physicochemical damage in environment, to maintainbody homeostasis by pattern recognition receptors recognizing andbinding pathogen associated molecular patterns (such as virus, pathogenof the bacterial) and damage associated molecular patterns (such ashyperglycemia, hyperlipidemia). Nucleotide binding and oligomerizationdomain-like receptor family pyrin domain-containing3(NLRP3), amember of the innate immune family, is indicated as an importantcomponent of the inflammatory immune response. It is not only the"sensor", but also a "regulator" of inflammation response. The NLRP3inflammasome can identify exogenous or endogenous danger signals,lead to the activation of caspase-1and then active cytokines interleukin(IL)-1beta, IL-18, IL-33, and trigger the inflammatory cascade reaction.Recent studies have shown that NLRP3inflammasome plays animportant role in the pathogenesis of metabolic diseases. However, itsrole and mechanism in diabetic nephropathy is still unclear. This studyobserves the expression of NLRP3inflammasome signaling stimulated byhigh glucose, lipopolysaccharide (LPS) and reactive oxygen species(ROS) inhibitor NAC in glomerular mesangial cells, aiming to explorethe control effects and related mechanism of the NLRP3inflammasomesignaling pathway in diabetic nephropathy. Methods:1. Rat glomerular mesangial cells cultured with high glucose and LPS as stimulating factor,ROS inhibitor NAC as the intervention factors, were divided into6groups:①normal control group(group NC):5.6mmol/L glucose;②different concentrations of high glucose group (group HG):10,20and30mmol/L glucose;③Osmotic pressure group (group OP):5.6mmol/Lglucose+24.4mmol/l mannitol;④The NAC intervention in high glucosegroup (group HG+NAC):10μmol/l NAC added in the best effect of highglucose medium;⑤different concentrations of LPS group(group LPS):1,5and10ug/L LPS;⑥The NAC intervention in LPS group(groupLPS+NAC):10μmol/L NAC added in the best effect of LPS medium.The each group was induced12,24and48h, then detected the expressionof NLRP3, Caspase-1, IL-1β, thioredoxin-interacting protein (TXNIP)protein and mRNA by Western blot and RT-PCR.2.Establishing andgrouping of DM model：20healthy male wistar rats randomly dividedto two group：10diabetes mellitus (DC) and10normal control (NC).DC rats are once peritoneally injected with STZ (60mg/kg) while normalcontrol rats are injected with isovolumetric citral damping fluid. After6weeks and8weeks, rats’24h urine protein ration and blood fastingglucose were detected. Kidneys were embedded with paraffin and TXNIPand IL-1β protein expression of renal tissue were detected byimmunohistochemisty. Result: In vitro, compared with normal group, theexpression of TXNIP, NLRP3, Caspase-1, IL-1β was upregulated in highglucose and LPS group in a dose-dependent manner (P<0.05). Comparedwith group HG3and LPS3, the expression of TXNIP, NLRP3, Caspase-1,IL-1β was decreased by NAC intervention (P<0.05). In vivo, an increasedtendency of body weight with diabetes rats was delayed compared withnormal group (P<0.05). However, diabetes rats’ blood fasting glucoseand24h urine protein ration were higher than control rats (P<0.05). There was no obvious difference between8weeks and6weeks in rats (P>0.05).In immunohistochemisty result, TXNIP or IL-1β expression was notfound in NC group. Compared with NC group, the expression of TXNIPand IL-1β were increased. The expression of TXNIP or IL-1β in8weekswas stronger than that in6weeks. Conclusion: Our data support that highglucose and LPS may activate the ROS/TXNIP/NLRP3/IL-1βinflammasome signaling pathway and mediate inflammation in diabeticnephropathy.