Anti-inflammatory Effects Of Dopamine In Lipopolysaccharide(LPS)-stimulated RAW264.7 Cells Via Inhibiting NLRP3 Inflammasome Activation

Objective:Sepsis called a systemic inflammatory response syndrome was the common complication of trauma,major surgery and burns and also the leading cause of deaths in intensive care patients.LPS,the main pathogen causing sepsis was the main pathogenic component of Gram-negative bacteria.LPS is an effective activator of macrophages triggering a series of pathophysiological reactions.Macrophages as the first line of defense to recognize external stimuli and resist external reactions plays a key role in the inflammatory response by regulating cell activity and releasing various inflammatory mediators within and outside.Among the inflammatory bodies currently,the most popular form of NOD-like receptor protein 3(NLRP3)inflammasome is an important component of innate immunity,and also serves as the core of inflammatory response,providing theoretical basis for various inflammatory diseases.As a neurotransmitter,dopamine(DA)may effectively control the inflammatory response of the disease through anti-inflammatory effects and improve the prognosis of patients.This study was designed to investigate the mechanism of dopamine in LPS-induced RAW264.7 macrophages.Methods:RAW264.7 cells were kept in Dulbecco’s Modified Eagle Medium(DMEM)complemented with 10%fetal bovine serum(FBS)at 37℃ in a humidified 5%CO2 atmosphere.For all experiments,cells were seeded at 3×105cells/ml inoculated in 6-well plates or 5×103cells per well in 96-well plates,and treated with 1μg/ml LPS for 6 hr.Protocol Ⅰ Cell treatments were performed as follows:(1)control cells;(2)LPS(1μg/ml)treated cells;(3)LPS+10-3M DA treatment;(4)LPS+10-4M DA treatment;(5)LPS+10-5M DA treatment.In DA and LPS treated groups,cells were exposed to DA for 2 hr prior to the addition of 1μg/ml LPS.CCK8 assays were used for the assessment of cell viability and Quantitative real-time PCR(qRT-PCR)mRNA of IL-1β,IL-6,TNF-a and iNOS and protein expression levels of IL-1β,IL-6,iNOS,NLRP3 and Casapse-1,respectively.Protocol Ⅱ Cells were randomly divided into five groups to interpret the role of NLRP3 in the anti-inflammatory effects of DA:(1)control cells;(2)LPS(1μg/ml)treated cells;(3)LPS+ DA(10-3M)treatment;(4)LPS+MCC950(NLPR3 inhibitor,30μM)treatment;(5)LPS+DA(10-3M)+MCC950(NLPR3 inhibitor,30μM)treatment.Cells were treated with DA and MCC950 for 2 hr prior to the addition of 1μg/ml LPS.NLRP3,Casapse-1,IL-1β,IL-6,and iNOS were detected by Western blot analysis.Results:1.CCK8:To investigate DA induced cytotoxicity,RAW264.7 cells were treated with various concentrations of DA(10-3,10-4 and 10-5M)in the presence and absence of LPS for 6hr.CCk8 results revealed that there was no significance of the viability of RAW264.7 cells between each group(p>0.05).2.The results revealed that DA reduced the mRNA and protein expression of TNF-αIL-6 and IL-1β in RAW 264.7 cells stimulated with LPS.DA also inhibited the mRNA and protein expression levels of iNOS,and downregulated NLRP3 and Caspase-1 expression.In the presence of DA,the increased levels of these inflammatory mediators were inhibited in a concentration dependent manner,excluding iNOS mRNA following 10-5M DA treatment(p<0.05).3.Compared with LPS stimulation group,both NLRP3 inhibitor group and dopamine group showed inhibition of inflammatory response by down-regulating the expression of inflammatory factors(IL-6,IL-1β)and iNOS,and inhibiting the expression of NLRP3/caspase-1 signaling pathway(p<0.05);The effect made a contrast to that of DA administration:Dopamine combined with NLRP3 inhibitor(MCC950)group showed inhibition of inflammatory factors(IL-6,IL-10)and iNOS expression,and also inhibited the expression of NLRP3 and caspas-1 signaling pathways,and the inhibition is more obvious.The administration of DA and NLRP3 inhibitor,MCC950 played a synergistic role in suppressing NLRP3 activity(p<0.05).Conclusion:1.Dopamine does not inhibit cell viability in the inflammatory response of LPS-induced RAW264.7 cells in a certain concentration range.2.Dopamine inhibits the expression of inflammatory factors in LPS-induced murine macrophages,presenting a concentration-dependent manner.3.Dopamine inhibits NLRP3 inflammasome activation in LPS-induced inflammatory responses.The administration of DA and NLRP3 inhibitor,MCC950 played a synergistic role in suppressing NLRP3 activity(p<0.05).