Curcumin Alleviates Acute Lung Injury Through Regulating SIRT1-NLRP3 Signaling Mediated Pyroptosis

The protective effects and mechanism of Curcumin in alleviating lung injury in ALI mice[Background/Objective]Acute lung injury(ALI)is an acute respiratory disease characterized by progressive dyspnea and refractory hypoxemia.Its pathogenesis is complex,with limited treatment options and poor clinical prognosis.In recent years,it has been found that the activation of NLRP3 inflammasome-mediated pyroptosis and the resulting inflammatory damage play an essential role in the pathogenesis of various respiratory diseases(such as asthma,pulmonary hypertension,and ALI).Hence,inhibiting the activation of NLRP3 inflammasome arid pyroptosis and blocking the inflammatory damage signaling pathway mediated by it may be the fundamental way to treat ALI.Curcumin,a natural yellow polyphenolic compound extracted from the rhizomes of Zingiberaceae,has attracted the attention of researchers because of its potent anti-inflammatory,anti-oxidative,anti-apoptotic,and other medicinal properties.To date,it has been found that curcumin can effectively inhibit the activation of NLRP3 inflammasome-mediated pyroptosis and prevent the release of inflammatory mediators.The role of inflammatory injury in lung tissue.However,the protective mechanism of curcumin in sepsis-induced ALI remains unclear.So far,we have constructed ALI mice model using cecal ligation and puncture(CLP)and used curcumin to intervene to explore the protective effect and mechanism of curcumin on ALI.[Methods]The C57BL/6 mice were gruoped by randomly digital table and given related intervention measures.The specific groups are as follows:①Acute lung injury model group(CLP group):Cecal ligation and puncture established a sepsis-sensitive lung injury model.②Sham operation group(Sham group):After the mice were anesthetized,an incision was made in the midline of the abdomen.Different doses of curcumin protection group(CLP+100/200-Cur group):mice were given different doses(100mg/kg/d or 200mg/kg/d)of curcumin,and CLP was performed after the last gavage.Dexamethasone protection group(CLP+5-DXM group):mice were intraperitoneally injected with 5 mg/kg dexamethasone at 0,2,and 12 hours after CLP.SIRT1 inhibitor intervention group(CLP+EX527 group):mice were given an intraperitoneal injection of SIRT1 inhibitor EX527 at a dose of 5 mg/kg/d once every two days before CLP for a total of 3 administrations,and the last injection of CLP was performed 24 hours after the end of the procedure.Subsequently,calculate the survival rate and observe the differences in the pathological changes of the lung tissue per group;measuring the wet to dry ratio(W/D)and bronchoalveolar lavage(Fluid,BALF)protein content,cell classification,and proportion;detection of myeloperoxidase(MPO)activity in mouse lung tissue;detection of proinflammatory cytokine CC chemokine 7 in serum of mice in each group by ELISA(chemokine(C-C motif)ligand 7,CCL7),tumor necrosis factor α(TNF-α),interleukin 6(interleukin 6,IL-6)expression levels;Western blot detection of SIRT1,NF in mouse lung tissue-Expression levels of κB,p-NF-κB and pyroptosis-related proteins(NLRP3,ASC,Caspase-1,Cleave-caspase-1,GSDMD,and GSDMD-N).[Results]Compared with the Sham group,the histopathology of the lungs of the mice in the CLP group showed that the typical structure of the alveoli and interstitium was severely damaged,and the alveolar septum was significantly widened.The inflammatory cells infiltrated and around the alveolar cavity.The wet-dry ratio increased,the MPO activity in the lung tissue increased,the inflammatory cell aggregation in the BALF of the mice and the expression of pro-inflammatory factors in the serum rise,and the SIRT1 protein in the lung tissue declined.Still,the NF-κB protein and the pyroptosis-related protein increased significantly,and the mouse survival ratio gradually dropped.When given different doses(100 mg/kg/d or 200 mg/kg/d)of curcumin for intervention,in contrast to the CLP group,the histopathological lung damage of the curcumin intervention group was sharply reduced,and the mortality dropped dramatically.Meanwhile,the protein content in BALF and the wet-dry ratio of lung tissue in the curcumin intervention group steadily declined;also,the MPO activity in lung tissue dropped,and the aggregation of inflammatory cells in BALF and the expression of pro-inflammatory factors in serum was decreased considerably.The content of SIRT1 protein in lung tissue was increased,accompanied by a fall trend in NF-κB and pyroptosis-related proteins.Whereas,after the intervention of SIRT1 protein inhibitor EX527,in contrast with the CLP+200-Cur group mice,the degree of histopathological lung damage in the CLP+200-Cur+EX527 group was marked aggravated,and the inflammatory cells in the BALF of the mice were concentrated.The expression of pro-inflammatory factors in serum rise,and SIRT1 protein in lung tissue declined,but NF-κB and pyroptosis-related proteins were steeply increased.[Conclusions]It is proven that CLP-mediated lung inflammation in ALI mice was evident.Given different doses(100 mg/kg/d or 200 mg/kg/d)of curcumin for interference,curcumin showed a markable inhibitory effect on the pyroptosis of lung tissue cells in ALI mice,hence,indicating the protective effect by reducing lung tissue inflammation.While after the intervention via giving the SIRT1 protein inhibitor EX527,the protective effect of curcumin on lung tissue inflammatory injury in ALI mice was significantly weakened.The mechanism of curcumin inhibits NLRP3 inflammasome-mediated lung macrophage pyroptosis via up-regulated expression of SIRT1 protein[Background/Objective]Macrophages are critical immune cells in the lungs,which have potent roles in initiating,regulating,and terminating inflammation.Macrophages play an essential role in the resistant stability of the lungs.Recherches have found that uncontrolled inflammatory responses in the pathogenesis of ALI are thought to be related to the functional status of macrophages,which mediates other immune cells and induce inflammatory damage through the release of chemotactic/inflammatory cytokines,ultimately contributing profound impacts to the development and prognosis of ALI(Acute lung injury).With the deepening of research,it has been found that macrophage pyroptosis and its mediated inflammatory injury also play an essential role in the pathogenesis of ALI.The results of our animal experiments suggest that curcumin can up-regulate the expression level of SIRT1 protein and inhibit the activation of NLRP3 inflammasome-mediated pyroptosis,thereby reducing pulmonary inflammatory injury in CLP mice.To this end,we will further clarify the mechanism by which curcumin up-regulates the expression of SIRT1 protein and inhibits the NLRP3 inflammasome-mediated pyroptosis of macrophages(RAW264.7)through cell experiments.[Methods]Firstly,the effect of the intervention of different curcumin concentrations on RAW264.7 cell viability was detected by the MTT method.The optimal curcumin concentration(20,40,80 μM)used in the experiment was screened.Then the effect of different concentrations of LPS intervention on RAW264.7 cell viability was detected by the MTT method,and the optimal LPS intervention concentration(1 μg/mL LPS)was screened.Then,the experimental grouping is carried out,and the specific set is as follows:①Control group:RAW264.7 cells were cultured generally without any intervention;②LPS+ATP group:RAW264.7 cells were treated with LPS(1 μg/mL)for 24 hours,and then ATP(5 mM)was added for 30 minutes;③20/40/80-Cur+(LPS+ATP)group:RAW264.7 cells were pretreated with 20,40,and 80 μM curcumin for 2 hours,then treated with LPS(1 μg/mL)for 24 hours.After that,ATP(5 mM)was added for 30 minutes;④10-EX527+80Cur group:RAW264.7 cells were pretreated with 80μM curcumin and 10 μM EX527 for 2 hours;⑤10-EX527+80Cur+(LPS+ATP)group:RAW264.7 cells were pretreated with 80 μM curcumin and EX527 for 2 hours and then treated with LPS(1μg/mL)for 24 hours.After that,ATP(5 mM)was added for 30 min.The final statistical observation indicators:MTT detected the changes in cell viability in each group;Hoechst33342/PI double staining method to detect the pyroptosis rate of cells in each group;ELISA to detect inflammatory factors(CCL7,TNF-α,IL-6 in the supernatant of each group))expression;immunofluorescence was used to detect the expression of Sirtl and NLRP3 proteins in each group of cells;Western blot was used to detect the expression of SIRT1,NF-κB,p-NF-κB and pyroptosis-related proteins(NLRP3,ASC,Caspase-1,Cleave-caspase-1,IL-1β,Cleaved IL-1β,GSDMD,and GSDMD-N).[Results]As the SIRT1 inhibitor was administered,EX527 down-regulated the expression of SIRT1 protein;meanwhile,EX527 diminished the effect of curcumin on LPS+ATP-mediated up-regulation of SIRT1 in macrophages,as well as down-regulation of NLRP3 protein and pyroptosis-related proteins.Therefore,EX527 weakened the protective effect of curcumin on LPS+ATP-mediated reduction of macrophage activity,the inhibitory effect of curcumin on macrophage pyroptosis,and the down-regulation of pro-inflammatory cytokine expression levels.[Conclusion]Curcumin reduces the inflammatory damage of macrophages by inhibiting the decrease of macrophage activity mediated by LPS+ATP,and curcumin has an inhibitory effect on macrophage pyroptosis.EX527 down-regulated the expression level of SIRT1 protein and faded the inhibitory effect of curcumin on macrophage pyroptosis and the protective result of inflammatory injury.