Effect And Mechanism Of Acacetin And NLRC3 On Acute Lung Injury

Acute lung injury（ALI）refers to the injury of lung parenchyma cells caused by various direct or indirect causes,diffuse pulmonary edema,and subsequent ventilation insufficiency in clinical acute critical disease.Sepsis is one of the common risk factors for ALI.Cytokine storm and immunosuppression produced by the body are the two main pathophysiological features of sepsis.With the in-depth study of the mechanism of ALI,inhibition or reversal of sepsis has become the main purpose of prevention and treatment of ALI and its complications,but there is still a lack of specific treatment measures.Acacetin is a kind of natural flavonoid compound,which has a wide range of biological activities,such as antioxidant,anti-inflammatory,antibacterial,antiviral and tumor proliferation inhibition.Previous studies have shown that acacetin can inhibit the oxidative stress response induced by lipopolysaccharide（LPS）and inhibit the development of acute lung injury,but its mechanism remains unclear.Excessive inflammatory responses are associated with the dysfunctioning of immune cells,which are regulated jointly by the NOD-like receptor family（NLRs）and its downstream signaling pathways.NLRC3（NOD-like receptor family CARD domain containing-3）,as a member of NLRs family,was initially discovered and cloned in human Jurkat T lymphocytes.Cell localization studies showed that NLRC3 was a cytoplasmic protein.It is one of the important molecules that negatively regulate immunity and proliferation.Current studies have shown that NLRC3 is significantly down-regulated when LPS stimulates macrophages,and the nuclear factor kappa B（NF-κB）pathway closely related to inflammatory response is over-activated after NLRC3 is completely knocked out,leading to the production of a large number of inflammatory mediators.Therefore,increasing the expression of NLRC3 may help to inhibit inflammatory response,prevent and treat sepsis induced ALI,and improve the prognosis of patients with lung injury.In this study,the inhibitory effect of acacetin on sepsis induced ALI and its effect on NLRC3 at the global and cellular level were observed to explore the possible mechanism of acacetin in treating ALI.Furthermore,NLRC3 knockout mice were further used to observe the possible role of NLRC3 in ALI at the whole and cellular level,and to explore the related mechanism.This study is expected to provide new strategies and means for the prevention and treatment of ALI.Study on the prevention and treatment of ALI by acacetin and its mechanismObjectiveTo observe the inhibitory effect of acacetin on sepsis induced ALI and its effect on NLRC3 and NF-κB/NLRP3 signaling pathway at both global and cellular levels,and to explore the mechanism of acacetin in preventing ALI and inhibiting excessive inflammatory response.MethodsBy setting the drug concentration gradient,the optimal drug concentration was selected.Mice or RAW264.7 were divided into three groups:Control group（Control）,LPS group（LPS）,and administration group（LPS+A）.ALI model of mice was induced by intraperitoneal injection of LPS.Lung moisture-dry ratio and lung body ratio were calculated.HE staining of lung tissue sections was performed to observe pathological changes and calculate pathological scores.The expressions of inflammatory cytokines,NLRC3,Sirt1,NLRP3 and NF-κB pathway in lung tissues and cells were detected by real-time quantitative PCR and Western Blot.Results（1）Animal experiment results:acacetin can significantly reduce lung tissue damage caused by sepsis.LPS inhibited the expression of NLRC3 and Sirt1 in mouse lung tissue,and may activate the NF-κB signaling pathway by promoting the phosphorylation of p65and increase the expression of NLRP3.Acacetin can up-regulate the expression of NLRC3 and Sirt1,reduce the phosphorylation of p65,inhibit the activation of NF-κB signaling pathway,and down-regulate the expression of NLRP3.（2）Cell experiment results:LPS stimulated the expression of inflammatory cytokines in RAW264.7,and acacetin significantly reduced the expression of inflammatory cytokines.LPS inhibited the expression of NLRC3 and Sirt1 in RAW264.7,and increased the expression of NLRP3 downstream by activating the NF-κB signaling pathway by promoting the phosphorylation of p65.By up-regulating the expression of NLRC3 and Sirt1,acacetin inhibits the phosphorylation of p65 and the activation of NF-κB signaling pathway,and reduces the expression of NLRP3.ConclusionAcacetin inhibits NF-κB/NLRP3 in the prevention and treatment of ALI,and its mechanism may be realized by up-regulating the expression of NLRC3 and Sirt1.The role and mechanism of NLRC3 in ALIObjectiveTo observe the role of NLRC3 in sepsis induced ALI and its effect on NF-κB signaling pathway at both global and cellular levels,and to explore the mechanism of NLRC3 inhibiting the development of ALI.MethodsMice or primary cultured mouse BMDM were divided into four groups:normal Control group（Control）,knockout control group(NLRC3^-/-),LPS injection group（LPS）,knockout mouse injection group(LPS+NLRC3^-/-).ALI model was induced by intraperitoneal injection of LPS.Lung moisture-dry ratio and lung body ratio were calculated.HE staining of lung tissue sections was performed to observe pathological changes and calculate pathological scores.Lung tissue MPO activity detection;Immunofluorescence staining of lung tissue sections;Real-time quantitative PCR and Western Blot were used to detect the expression of cytokines,Sirt1,and NF-κB pathway in lung tissues and cells.The content of cytokines in the supernatant of cell culture was detected by ELISA.Results（1）Animal experiment results:acute lung injury model was successfully replicated,NLRC3^-/-mice had more obvious inflammatory symptoms and serious lung tissue injury.LPS increased the content of immune cells and inflammatory cytokines,inhibited the expression of NLRC3 and Sirt1,and increased phosphorylation of p65 promoted the activation of NF-κB signaling pathway.NLRC3^-/-mice stimulated by LPS not only significantly increased the content of inflammatory cytokines and increased the aggregation of macrophages,but also significantly decreased the expression of Sirt1 and increased phosphorylation of p65,resulting in excessive activation of NF-κB signaling pathway.（2）Cell experiment results:LPS stimulated the expression and secretion of inflammatory cytokines in BMDM,down-regulated the expressions of NLRC3 and Sirt1,and increased phosphorylation of p65 promoted the activation of NF-κB signaling pathway.NLRC3^-/-BMDM after LPS stimulation not only significantly increased the synthesis and secretion of inflammatory cytokines,but also decreased the synthesis and secretion of anti-inflammatory cytokines,significantly down-regulated the expression of Sirt1,and increased phosphorylation of p65,leading to excessive activation of NF-κB signaling pathway.ConclusionNLRC3 can resist ALI by down-regulating the production of inflammatory cytokines,which may be realized by inhibiting the abnormal activation of NF-κB signaling pathway.