| Objective:To explore the correlation between the expression and secretion of inflammatory factors in the body of mice with sepsis,and further explore whether Blimp-1 can mediate monocyte/macrophage pyroptosis through NLRP3 inflammasome,which in turn affects the expression and secretion of inflammatory factors in the body of septic mice and the mechanism,and to provide a theoretical basis and a new target for the development of new drugs against and against sepsis.Part I: correlation between Blimp-1 expression and the expression and secretion of inflammatory factors in liver and lung tissues of septic mice.Methods:Firstly,the cecum ligation and puncture(CLP)method was used to replicate the mouse model of sepsis and its identification.C57BL/6 mice were randomly divided into 3 groups: blank control group(Control),sham-operated group(Sham),and sepsis disease animal model group(CLP).The survival curves were plotted for 5 consecutive days of observation.The expression of Blimp-1 in the liver and lung tissues of mice was measured by q RT-PCR and Western Blot.The expression of TNF-α,IL-1β and IL-6 in the homogenates of liver and lung tissues of each group of mice was examined by ELISA.The correlation between Blimp-1 and the expression and secretion of inflammatory factors in septic mice was investigated.Results:(1)The survival rate of mice at 120 h was found to be 100% in the Control and Sham groups;compared with the Control and Sham groups,the survival rate of mice in the CLP group was 65% at 48 h,and the survival rate of mice in the CLP group decreased to 25% at 72h(P < 0.05),and all mice in the CLP group died at 120 h.(2)The results of pathological examination of liver and lung tissues from mice in each group showed that the alveolar wall of lung tissue from mice in the Sham group consisted of a single layer of epithelium with a clear structure;The pulmonary interstitium,including connective tissue and blood vessels in the lung,was unremarkable,and no obvious inflammatory changes were observed.Compared with the Sham group,the CLP group showed alveolar wall thickening,with more red blood cells and inflammatory infiltration in the alveoli as well as interstitial edema;The lung tissue injury scores of septic mice in the CLP group were significantly higher than those in the Sham group(P < 0.001).The liver lobules of mice in Sham group were sharply demarcated and regularly arranged,and the hepatocytes and liver sinusoids were round and full;There were no significant abnormalities in the portal area between adjacent hepatic lobules;Cells showed no degeneration,edema.Compared with Sham group,CLP group showed more liver tissues with hepatocyte hydropathy,loose and pale cytoplasm staining,and a large number of inflammatory cells infiltration in the confluent zone.The liver injury score of septic mice in CLP group was significantly higher than that in Sham group(P < 0.001).(3)The results of q RT-PCR and Western Blot showed that the expression levels of m RNA and protein of Blimp-1 in septic mice in the CLP group increased significantly compared with those in the Sham and Control groups,and the expression of m RNA and protein of Blimp-1 started to increase after 24 h of modeling(P < 0.01).The expression levels of m RNA and protein of Blimp-1 continued to increase at 72h(higher than24 h,P < 0.001),and reached the highest level at 120h(higher than 72 h,P < 0.05).(4)The results of ELISA showed that the expression of TNF-α,IL-1β and IL-6 in liver and lung tissues of mice with sepsis was significantly higher in the CLP group compared with the Sham group(P < 0.001);the expression of TNF-α,IL-1β and IL-6 in the CLP group was consistently higher at 1d,3d and 5d(P <0.001).The expression of TNF-α,IL-1β and IL-6 in liver and lung tissues of septic mice in the CLP group was significantly higher in 3d compared with 1d(P < 0.01),and the levels of inflammatory factors were also significantly higher in 5d compared with 3d(P < 0.001).(5)Using the JASPAR database analysis,one to more than one Blimp-1 binding elements were found in the promoter region of the genes encoding the inflammatory factors TNF-a,IL-1β and IL-6 in sepsis,and five Blimp-1 binding sites were also found in the promoter region of NLRP3 gene,indicating that Blimp-1may regulate the inflammatory factors at the transcriptional level.The expression of TNF-a,IL-1β and IL-6was also found in the promoter region of NLRP3 gene.(6)The correlation analysis between the expression of Blimp-1 and the expression and secretion of inflammatory factors TNF-α,IL-1β and IL-6 in liver and lung tissues of each group of mice showed that the expression of Blimp-1 in liver tissues was significantly and positively correlated with the expression and secretion of TNF-α(r = 0.9809,p < 0.0001),and the expression of Blimp-1 was significantly and positively correlated with the expression and secretion of The expression of Blimp-1 was significantly and positively correlated with the expression and secretion of IL-1β(r = 0.9934,p < 0.0001)and Blimp-1 was also significantly and positively correlated with the expression and secretion of IL-6(r = 0.9403,p < 0.0001);the expression of Blimp-1 was also significantly and positively correlated with the expression and secretion of TNF-α,IL-1β and IL-6,respectively,in lung tissues(r = 0.9934,p < 0.0001).The expression of Blimp-1 in lung tissues was also significantly and positively correlated with the expression and secretion of TNF-α,IL-1β and IL-6(r = 0.8985,p < 0.0001),(r = 0.923,p < 0.0001)and(r = 0.9249,p < 0.0001),respectively.Part II: Blimp-1 mediates monocyte/macrophage scorching via NLRP3 inflammatory vesicles,which in turn regulates the expression and secretion of inflammatory factors in septic mice.Methods:In vivo animal experiment: the cecum ligation perforation(CLP)method was used to replicate the mouse model of sepsis and to identify it.The mice were divided into Sham group,CLP group,and CLP+TAK-242(TLR4 antagonist)group.The expression of inflammatory factors TNF-α,IL-1β and IL-6 in the liver and lung homogenates of mice in each group were detected by ELISA;the expression of TLR4,Blimp-1 and scorch death pathway(NLRP3,ASC,Caspase-1,GSDMD)proteins in liver and lung tissues of mice in each group were detected by Western Blot.The m RNA expression of TLR4 and NLRP3 in liver and lung tissues of each group of mice was detected by q RT-PCR;the expression of Blimp-1 in peripheral blood monocytes/macrophages of each group of mice was detected by flow cytometry.In vitro cell experiments: LPS-induced RAW 264.7 cells were used to establish a sepsis cell model and identification.They were divided into Control group,LPS group and LPS+TAK-242 group.The expression of TLR4 and Blimp-1 in each group was detected by immunofluorescence staining;the expression of inflammatory factors TNF-α,IL-1β and IL-6 in the culture supernatant of each group was detected by ELISA;the expression of TLR4,Blimp-1 and focal death pathway(NLRP3,ASC,Caspase-1,GSD)in each group was detected by Western Blot.The expression of TLR4,Blimp-1 and focal pathway(NLRP3,ASC,Caspase-1,GSDMD)proteins was detected by Western Blot.Blimp-1 gene was silenced in RAW 264.7 cells by si RNA interference technique,and the experiment was divided into si NC,LPS,si BLIMP1#1 and TAK-242 groups.The expression of inflammatory factors TNF-α,IL-6 and IL-1β in the supernatant of each group was measured by ELISA,and the expression of TLR4,Blimp-1 and focal death pathway(NLRP3,ASC,Caspase-1,GSDMD)proteins in each group was measured by Western Blot.Results:(1)In vivo animal experiments,the expressions of inflammatory factors TNF-α,IL-1β and IL-6 in liver and lung tissues of mice in each group were significantly higher in the CLP group compared with the Sham group(P < 0.001);The expression of TNF-α,IL-1β and IL-6 in the TAK-242 group was significantly lower than that in the CLP group(P < 0.001),but the expression levels of inflammatory factors in the TAK-242 group were still higher than those in the Sham group.(2)In vivo animal experiments,the expression of TLR4,Blimp-1 and scorch death pathway(NLRP3,ASC,Caspase-1,GSDMD)proteins in liver and lung tissues of mice in each group were significantly higher in the CLP group compared with the Sham group(P < 0.001),while the expression levels of TLR4,Blimp-1,NLRP3,Caspase-1,ASC and GSDMD were significantly lower in the CLP+TAK-242 group compared with the CLP group(P < 0.001).(3)In vivo animal experiments,the m RNA expression of TLR4 and NLRP3 in liver and lung tissues of mice in each group was significantly higher in the CLP group compared with the Sham group(P < 0.001),while the m RNA expression of TLR4 and NLRP3 in the CLP+TAK-242 group was significantly lower than that in the CLP group(P < 0.001).(4)In vivo animal experiments,the peak plots of Blimp-1 expression in peripheral blood monocytes/macrophages in each group of mice using flow cytometry showed that the protein expression of Blimp-1 was highest in the CLP group,followed by the TAK-242 group and lowest in the Sham group.The differences between the groups were statistically significant(P < 0.001).(5)In vitro cell experiments,the results of TLR4 and Blimp-1 protein expression in each group using immunofluorescence staining showed that TLR4 and Blimp-1 expression were significantly higher in the LPS group compared with the Control group(P < 0.001),and Blimp-1 and TLR4 expression were significantly lower in the TAK-242 group compared with the LPS group(P < 0.05).(6)In vitro cell experiments,the results of the expression of inflammatory factors TNF-α,IL-6 and IL-1β in cell culture supernatants of each group using ELISA showed that the expression of TNF-α,IL-1β and IL-6 was significantly higher in the LPS group compared with the Control group(P < 0.001),and the expression of TNF-α,IL-1β and IL-6 was significantly higher in the TAK-242 group compared with the LPS group(P < 0.001),but their expression was higher than that in the Control group,and the difference between the groups was statistically significant(P < 0.001).(7)In vitro cell experiments,the results of TLR4,Blimp-1 and scorch death pathway(NLRP3,ASC,Caspase-1,GSDMD)protein expression in each group were detected by Western Blot method,which showed that the expression of TLR4,Blimp-1 and NLRP3,Caspase-1,ASC,GSDMD in the LPS group was significantly lower than that in the Control group(P < 0.001),while TLR4,Blimp-1 and NLRP3,Caspase-1,ASC,and GSDMD were significantly lower in the LPS+TAK-242 group compared with the LPS group(P< 0.001).(8)In vitro cell assay,the results of the expression of inflammatory factors TNF-α,IL-6 and IL-1β in cell culture supernatants of each group using ELISA showed that the expression of pro-inflammatory factors TNF-α,IL-1β and IL-6 were significantly higher in the LPS,si Blimp-1#1 and TAK-242 RAW 264.7 cell culture supernatants compared with the si NC group(P < 0.001).The expression of pro-inflammatory factors TNF-α,IL-1β and IL-6 was significantly higher in the si Blimp-1#1 group compared with the LPS group(P< 0.001),while the expression of all three inflammatory factors was lower in the TAK-242 group compared with the si Blimp-1#1 group,lower than that in the LPS group and higher than that in the si NC group,with statistically significant differences between the groups.The differences were statistically significant(P <0.001).(9)The results of TLR4,Blimp-1 and scorch death pathway(NLRP3,ASC,Caspase-1,GSDMD)protein expression in each group by Western Blot showed that the expression of Blimp-1 protein was significantly higher in LPS,si BLIMP1#1 and TAK-242 groups compared with si NC group,while the expression of Blimp-1 protein was significantly higher in LPS(P < 0.001),si BLIMP1#1 and TAK-242 groups.The expression of TLR4 protein in the LPS and si BLIMP1#1 groups was higher than that in the TAK-242 group(P < 0.001),but there was no significant difference between the two groups.The expression of NLRP3,Caspase-1,ASC and GSDMD were significantly higher in the LPS,si BLIMP1#1 and TAK-242 groups compared with the si NC group(P < 0.001);the expression of scorch death-related proteins was the highest in the si BLIMP1#1 group,followed by the LPS group and the TAK-242 group,and the difference was statistically significant(P < 0.001).Conclusion.Part I:(1)The expression of Blimp-1 protein was upregulated in liver and lung tissues of septic mice,and the expression level was correlated with the expression and secretion of inflammatory factors TNF-α,IL-1β and IL-6,which showed a significant positive correlation.(2)One to more Blimp-1 binding sites exist in the promoter regions of inflammatory factors TNF-α,IL-1β and IL-6.Blimp-1 may act as a transcriptional repressor downstream of the TLR signaling pathway,regulating the expression of inflammatory factors TNF-α,IL-1β and IL-6 at the gene transcription level.Part II:(1)TLR4 inhibitor downregulated the expression of Blimp-1 protein in liver and lung tissues of septic mice,and Blimp-1 mediated monocyte/macrophage scorching through NLRP3 inflammatory vesicles,which in turn inhibited the expression and secretion of inflammatory factors TNF-α,IL-1β and IL-6.(2)After si RNA silencing of Blimp-1 gene,the expression of NLRP3,GSDMD,Caspase-1 and ASC,and the expression and secretion of inflammatory factors TNF-α,IL-1β and IL-6 were increased in mouse monocytes/macrophages.(3)Blimp-1 mediates NLRP3 inflammatory vesicles in septic mice,inhibits monocyte/macrophage scorch death,and thereby reduces the secretion of inflammatory factors TNF-α,IL-1β,and IL-6. |
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