Mechanism Of NOD1 Expression And MAPK Signaling Pathway In Diabetic Guinea Pigs Infected With Legionella

Objective: Legionella is a gram-negative bacterium widely existing in nature and artificial water system.It is an intracellular pathogen that can cause two forms of Legionella disease,namely Legionella pneumonia and Pontiac fever.It can cause both outbreaks and sporadic cases.Legionella pneumonia accounts for approximately 2-7%of all community-acquired pneumonia and has a high mortality rate,which can be as high as 80% in immunosuppressed patients without proper treatment.The genus Legionella includes at least 58 species,among which legionella pneumophila is the most important pathogenic bacteria,and about 84% of legionella pneumophila serotype1 causes legionella pneumophila disease.At present,patients with diabetes complicated with Legionella pneumophila infection are mostly reported as clinical cases,and there is no relevant basic research on the mechanism of difference in disease severity between patients infected with Legionella pneumophila and normal immune hosts.In this study,a guinea pig model of diabetes mellitus complicated with Legionella pneumophila infection was established to study the expression level of intracellular receptor NOD1 and the activation of MAPK signaling pathway in macrophages,so as to further study the immune mechanism of legionella infection in the two groups of models.Methods: This study was divided into three parts.The first part studied the expression of intracellular receptor NOD1 and the activation level of MAPK signaling pathway in macrophages infected with Legionella pneumophila induced by PMA.Experimental cells were divided into two groups: normal macrophage infection time gradient group,including 6h group,12 h group,24 h group and 48 h group,and macrophage infection time gradient group under high glucose condition,including 6h group,12 h group and24 h group.Firstly,CCK8 method was used to measure the effect of different concentrations of glucose on cell survival rate to determine the glucose concentration used in the experiment.Western blot was used to detect the expression of NOD1 and the activation of MAPK signaling pathway.In addition,the supernatant of cell culture of each group was collected,and the expression levels of inflammatory factors TNF-αand IL-6 were detected by ELISA.The expression levels of intracellular receptor NOD1 and activation levels of MAPK signaling pathway in macrophages were compared under high glucose state,as well as the secretion of inflammatory factors in the two groups.In the second part,after adding NOD1 inhibitor,To verify that NOD1 receptor is the upstream regulatory molecule of MAPK signaling pathway in the immune response of macrophages infected with Legionella pneumophila,and to study the expression of intracellular receptor NOD1 and the activation level of MAPK pathway in macrophages infected with Legionella pneumophila diabetes mellitus.The macrophage model was constructed in the same way as the first part.The experimental cells were divided into eight groups: control group,simple inhibitor group,normal infection group,infection plus inhibitor group,high glucose group,high glucose plus inhibitor group,and high glucose infection group.High glucose-contaminated bacteria plus inhibitor group.Western blot was used to detect the expression of NOD1 and the activation of MAPK signaling pathway.The expression levels of inflammatory factors TNF-α and IL-6 in supernatant of cell culture from each group were detected by ELISA.The results verified that NOD1 inhibitor was effective,and the expression of NOD1 and MAPK signaling pathway was also inhibited,which further verified that NOD1 was the regulatory molecular protein of MAPK signaling pathway in the process of macrophage infection with Legionella.The third part studied the expression of intracellular receptor NOD1 and the activation of MAPK signaling pathway in diabetic guinea pigs and normal guinea pigs infected with LEGIonella bacteria induced by STZ.The experimental cells were divided into four groups: control group,legionella pneumophila infection group,diabetic guinea pig group,diabetic guinea pig Legionella infection group;Legionella pneumophila infection at 24 h,48h and 72 h at three test time points.Western blot was used to detect the expression of NOD1 and the activation of MAPK signaling pathway at different time points in the two groups.By HE staining and immunohistochemistry of lung tissues at different time points of the two groups of guinea pigs,the inflammatory cell infiltration and lung tissue structural changes as well as the positive expression of NOD1 protein in the lung tissues of guinea pigs were observed.The expression levels of TNF-α and IL-6 were detected by real-time PCR.Results: In the first part,the activation of intracellular receptor NOD1 and signaling pathway MAPK in macrophages after legionella infection under normal and high glucose conditions was compared.The cell viability of U937 cells was detected by CCK8 method.The survival rates of U937 cells were 83.6%,71.6%,89.3% and 80.0%after treatment with glucose concentration of 25 m M for 6 h,12 h and 24 h.After comprehensively considering cell concentration and human blood glucose,25 m M glucose concentration was selected as the representative condition of high glucose concentration for further experiments.The cells were divided into control group and high glucose group.Human histiocytic lymphoma cell U937 was induced as macrophages by PMA.After the macrophages were infected with Legionella pneumophila type 1,the expression level of intracellular receptor NOD1 was detected by Western blot.The expression level of NOD1 peaked 12 hours after normal macrophages were infected.After 24 h,the expression of NOD1 decreased gradually,and the downstream signaling pathway MAPK family proteins were activated,among which the activation of p38 and erk1/2 peaked at 12 h after infection.In the high glucose infection group,the expression level of NOD1 peaked 6 h after macrophage infection,and began to decrease 12 h after infection.The expression of P38,ERK1/2 and JNK proteins increased after the infection of U937 cells by Legionella under high glucose condition,and reached the peak at 6 h after infection.These results suggest that macrophages infected with Legionella pneumophila can activate P38,ERK1/2 and JNK signaling pathways in MAPK under high glucose state.The levels of inflammatory cytokines IL-6 and TNF-α in normal macrophages were higher than those in high glucose group at 6h,12 h and 24 h.TNF-α and IL-6 secreted by macrophages in normal infection group peaked at 24 h and decreased at 48 h.The inflammatory mediators TNF-α and IL-6 reached the peak at 12 h and began to decrease at 24 h in the hyperglycemic macrophage-infected group.Infection increased the secretion of inflammatory mediators by macrophages,and the decrease time of the secretion of inflammatory mediators was earlier in the high glucose infection group.At each infection point,the secretion of inflammatory factors TNF-α and IL-6 decreased in the diabetic infection group compared with the normal infection group.In the second part,NOD1 inhibitors were added to further explore the relationship between NOD1 and MAPK protein family in the process of legionella pneumophila infection of macrophages.CCK8 assay was used to detect cell viability.The survival rates of NOD1 inhibitor(ML130)treated with 25 u M for 6 h,12 h,24 h and 48 h were93.6%,91.1%,95.1% and 93.5%,respectively.According to the experimental results.Finally,the concentration of NOD1 inhibitor was selected to be 25 u M for further experiment.The cells were pretreated with NOD1 inhibitor,and the expression level of intracellular protein NOD1 and the phosphorylation level of MAPK were detected by Western Blot.Compared with the control group,the expression level of NOD1 decreased in the group with NOD1 inhibitor alone,and the expression level of NOD1 decreased in the group infected with Legionella bacteria with inhibitor compared with the group infected with normal Legionella bacteria.Meanwhile,phosphorylation levels of downstream P38,JNK and ERK1/2 signaling pathways were also decreased.These results indicate that Legionella infection can increase the expression of NOD1,and NOD1 is the upstream signaling molecule of MAPK pathway.In the high glucose contaminated group,macrophages were incubated with NOD1 specific inhibitors for 2hours,and Legionella bacteria infected macrophages at time points.Western Blot was used to detect the expression level of intracellular protein NOD1 and the phosphorylation level of MAPK.The expression level of NOD1 decreased in the hyperglycemia infected GROUP compared with the hyperglycemia infected group.At the same time,the phosphorylation level of downstream MAPK signaling pathway in Lp group with hyperglycemia infection also decreased.These results indicate that macrophage infection with Lp can increase the expression of NOD1 in hyperglycemia,and NOD1 is the upstream signaling molecule of MAPK pathway.The levels of TNF-α and IL-6 in the cell culture medium of the L P infection group with inhibitor were significantly lower than that of the LP infection group,and there was no statistically significant difference between the addition of inhibitor alone and the blank control group.Activation of NOD1 receptor can promote the release of inflammatory factors by macrophages.In the third part,diabetic guinea pigs were modeled by intraperitoneal injection of STZ,and the control group was injected with the same dose of citric acid buffer.Glucose tolerance test was performed 2 weeks later,and the blood glucose was higher than11.1mmol/L after 2 hours of glucose feeding.HE staining of lung tissue in L P infected group showed progressively aggravated injury,72 h lung tissue structure disorder,alveolar and alveolar wall structure unclear,extensive and numerous inflammatory cells mainly neutrophils exudate.In the diabetic group,HE staining of lung tissue showed normal structure,and a small amount of neutrophil infiltration was observed in alveolar wall 24 h after infection.HE staining of the lung tissue of guinea pigs with diabetes mellitus combined with Lp infection showed that the injury was aggravated,and the lung tissue structure was widely disorganized at 72 h,a large number of alveolar cavities were filled with inflammatory exudate,the alveolar wall was extensively destroyed,and the number of alveoli was reduced.Immunohistochemical results showed that NOD1 expression increased at all time points after infection with Legionella pneumophila in normal guinea pigs,and the difference was statistically significant.At each time point of infection,the expression of NOD1 in diabetic group with Legionella infection was lower than that in normal guinea pig group,and the difference was statistically significant.Western blot results showed that The expression of NOD1 was up-regulated by Legionella infection.The comparison of NOD1 expression levels at each infection time point showed that at 24 h and 72 h of infection,NOD1 expression in the diabetic infection group was lower than that in the normal infection group,indicating that the expression of NOD1 was inhibited by diabetes.In the diabetic guinea pig infected group,the expression was increased at 48 h compared with 24 h,the expression peak was at 48 h,and the expression decreased at 72 h.The phosphorylation level of downstream MAPK signaling pathway in diabetic infected group was lower than that in normal infected guinea pigs,and the activation time was shorter.These results suggest that the expression of NOD1,the immune-associated pathogen recognition receptor,is inhibited and the immune response of MAPK signaling pathway is weakened after diabetic guinea pigs are infected with Legionella.Conclusions: 1.Infection of Macrophages by Legionella pneumophila can up-regulate the expression of intracellular receptor NOD1 and activate MAPK signaling pathway.After macrophages in high glucose group were infected with Legionella bacteria,the activation duration of NOD1 receptor was shorter and the secretion of inflammatory factors was less.2.In the immune response of macrophages infected with Legionella,NOD1 is the upstream regulatory molecule of MAPK signaling pathway,and the expression of NOD1 is related to the inflammatory mediators secreted by macrophages.3.After diabetic guinea pigs were infected with Legionella,the expression of NOD1 receptor in lung tissues was inhibited,the activation of P38,ERK1/2 and JNK proteins in MAPK signaling pathway was weakened,and the production of inflammatory factors in lung tissues of diabetic guinea pigs was reduced.