Study On The Anti-inflammatory Mechanism Of Rimulus Cinnamomi Extracts With Anti-inflammatory Effects Via NLRP3 Inflammasome Pathway

Objective: The study was designed according to the “cell/tissue with inflammatory,inflammatory cytokines,and signal transduction” research idea.The aim of this study was to evaluate the protective effects of Ramulus Cinnamomi extractions on lipopolysaccharide(LPS)-induced endotoxin poisoning mice and clarify the underlying mechanisms.Methods: The Ramulus Cinnamomi essential oils(RCEO)and cinnamaldehyde anti-inflammatory effects via NLRP3 inflammasome were studied using in vitro and in vivo experiments.THP-1 macrophage-like cells were used in vitro experiments.The effects of RCEO and cinnamaldehyde on the NLRP3 inflammasome signaling pathway were evaluated using different NLRP3 inflammasome activators.The levels of ASC,Caspase-1(p20),pro-IL-1?,and IL-1? in the supernatant were determined by ELISA.The expressions of NLRP3,ASC,Pro-Caspase-1,and Pro-IL-1? protein in cell lysate were detected by Western Blotting.In addition,The Cathepsin B,NLRP3,and ASC protein expressions were also detected by immunofluorescence method.The expressions of NLRP3,ASC,Caspase-1,IL-1?,IL-1?,and IL-6 mRNA in the cell lysate were detected by RT-PCR.ROS contents were measured using fluorescence microscopy.The endotoxin poisoning C57BL/6J mice were induced by LPS.The effects of RCEO and cinnamaldehyde on the general state and body weight of mice were observed and determined.The WBC and PLT in whole blood were measured by automatic blood cell counter.The organ indexes were determined,and the proinflammatory cytokines(IL-1?,IL-18,IL-5,IL-10,TNF-?,and IFN-?)and chemokines(MCP-1,MIP-1?,MIP-1?,M-CSF,GM-CSF,and G-CSF)in serum were measured by ELISA and luminex 200 liquid protein chip detection system.Further,the histological observation of lung tissue and determination of lung NO by Griess method were performed.In addition,the expressions of NLRP3,ASC,Caspase-1,IL-1?,IL-18,iNOS,p65,IFN-?,IFN-?,and STAT1 mRNA in lung tissue were detected by RT-PCR.The expressions of NLRP3,ASC,Pro-Caspase-1,Caspase-1(p20),Caspase-12,IL-1?,COP-1,and POP-1 protein were detected by Western Blotting.The expressions of P2X7 R and Cathepsin B protein in lung were detected by immunohistochemistry.The anti-inflammatory effects and underlying mechanisms of Ramulus Cinnamomi ethyl acetate extraction(EAE)and cinnamic acid were evaluated by the endotoxin poisoning KM mice.The WBC and PLT counts in whole blood were determined by automatic blood cell counter.The organ indexes,lung W/D ratio,BUN,CRE,ALT,AST,and SOD in serum,MDA in liver were determined.The levels of IL-1?,IL-18,IFN-?,and TNF-? in serum were measured by enzyme-linked immunosorbent assay(ELISA).Pathological observation of lung,spleen,liver and kidney was performed.In addition,the expressions of NLRP3,ASC,Caspase-1 and IL-1? mRNA in lung tissue were detected by RT-PCR.The expressions of NLRP3,ASC,Pro-Caspase-1,p20,and Pro-IL-1? protein in lung tissue were determined by Western Blotting.Results: using ATP and nigericin as the NLRP3 activators,the RCEO and cinnamaldehyde could down-regulate the expressions of Pro-Caspase-1,p20,Pro-IL-1?,and IL-1? protein,as well as the expressions of NLRP3,ASC,Caspase-1,and IL-1? mRNA in THP-1 macrophage-like cells.RCEO decreased the secretion of Pro-IL-1? in supernatant and cell lysates(P<0.05),inhibiting the expressions of NLRP3 and IL-1? mRNA in cell lysates(P<0.05).Cinnamaldehyde decreased the level of Caspase-1(p20)in supernatant and inhibited the expressions of NLRP3,ASC and IL-1? mRNA in cell lysates(P<0.05).Furthermore,RCEO and cinnamaldehyde could decrease the ROS levels in cells and relative co-localization area of NLRP3 and ASC protein detected by immunofluorescence,as well as the expression of Cathepsin B protein(P<0.05).RCEO and cinnamonaldehyde improved the mental state and activity of endotoxin poisoning C57BL/6J mice,decreasing the levels of IL-1?,IL-18,IL-5,IFN-?,MIP-1?,and M-CSF in serum,reducing the number of neutrophils and NO content in lung,down-regulating the NLRP3,p20,Pro-IL-1?,and P2X7 R protein in lung,as well as the expression of NLRP3,IL-1?,iNOS,and IFN-? mRNA(P<0.05).In addition,RCEO decreased the levels of MCP-1 in serum and STAT1 mRNA in lung,and cinnamaldehyde decreased the expression of Cathepsin B protein and increased the COP-1 protein in lung(P<0.05).EAE and cinnamic acid could increase the thymus indexes in endotoxin poisoning mice,decreasing the levels of BUN,IL-18 and TNF-? in serum,downregulating the expressions of NLRP3 and p20 protein,as well as the expressions of Caspase-1,and IL-1? mRNA in lung(P<0.05).Further,the lung and spleen histopathological changes induced by LPS were improved,the number of neutrophils in lung tissues were reduced,and the thymus indexes were increased(P<0.05).In addition,EAE could decrease the levels of serum CRE and MDA in liver,enhancing the SOD activity in liver and IFN-? levels in serum,down-regulating the expressions of NLRP3 mRNA and Pro-IL-1? protein in lung(P<0.05).Cinnamic acid decreased lung W/D ratio and IL-1? levels in serum(P<0.05).Conclusion: The EAE,RCEO,cinnamic acid,and cinnamaldehyde had protective effects on endotoxin poisoning mice via their' anti-inflammatory activities,and the mechanism was closely related to inhibit the activation of NLRP3 inflammasome.The results in the study provided a better scientific basis for the clinical application of Ramulus Cinnamomi to the treatment of "exterior syndromes",and enrich the modern scientific research on the theory of “divergence and relieving the exterior”.