| Objective: Legionella is a gram-negative bacterium that exists widely in nature.It can grow and multiply in natural environment,artificial environment water source and moist soil.Legionella is an opportunistic pathogen.Diabetic patients have an increased risk of pulmonary infection with Legionella,a low disease control rate,and a high case fatality rate.At present,most of the studies on diabetes with Legionella are clinical case reports and clinical retrospective studies,but there is a lack of research on the specific mechanism of diabetes with Legionella.Hypoxia-inducible factor 1α(HIF-1α)is a transcription factor activated in the state of tissue hypoxia.Previous studies have shown that HIF-1α has a regulatory effect on the inflammatory response caused by pathogen infection.This study will establish a model of diabetes complicated with Legionella pneumonia to study its pathological changes and the regulatory role of HIF-1α in it.Methods: This study is divided into three parts.The first part studies the expression changes of HIF-1a and related pathway proteins after lung infection of L.pneumophila type 1 in diabetic guinea pigs.The experimental animals were divided into four groups:normal control group,diabetes control group,Legionella infection group,and diabetes combined with Legionella infection group.Diabetic guinea pigs are established by the destruction of islet cells by the combined application of yohimbine and streptozotocin.After the diabetes model was successfully established,the guinea pigs in the infection group were inoculated with Legionella pneumophila(Lp)bacterial suspension through tracheal intubation to establish the Legionella pneumonia model.The proliferation of Lp in lung tissue was confirmed by Giemsa staining and in situ immunofluorescence detection of Legionella,and the inflammatory infiltration of lung tissue was confirmed by HE staining.Afterwards,Western blot and immunohistochemistry of lung tissue homogenate were performed to confirm the changes of HIF-1α,i NOS,NF-κb and SIRT6 protein expression.The second part applied FG4592 to increase HIF-1a expression on lung Legionella infection after infection of diabetic guinea pigs.Experimental animals were divided into diabetes group control group,diabetes combined with Legionella infection group,FG4592 treated diabetes group,FG4592 treated diabetes combined with Legionella infection group.The establishment method of diabetic guinea pigs was the same as that of the first part.The FG4592 group was intraperitoneally injected with a dose of 10 mg/kg every other day one week before infection,covering the whole infection process.The proliferation of Lp in lung tissue was confirmed by Giemsa staining and in situ immunofluorescence detection of Legionella,and the inflammatory infiltration of lung tissue was confirmed by HE staining.Afterwards,Western blot and immunohistochemistry of lung tissue homogenate were performed to confirm the changes of HIF-1α,i NOS,NF-κb and SIRT6 protein expression.Clearly increase the expression of HIF-1α on Lp proliferation in lung tissue,inflammatory infiltration of lung tissue and the regulation of related proteins.The third part is the effect and mechanism of hypoxia-inducible factor on the differentiation of human macrophages induced by Legionella infection under high glucose conditions.Using PMA to induce THP-1 to differentiate into macrophages,after differentiation,normal medium,high glucose medium and FG4592 containing 5n M concentration were treated for 24 hours,respectively,normal culture group,high glucose culture group and FG4592 treated high glucose culture Group.After stimulation,the Lpcontaining medium with MOI=1 was used to co-culture Lp with macrophages for 2 hours to remove extracellular bacteria.The expression of-1α protein,ROS,mitochondrial membrane potential and the level of lactate in the cell supernatant clearly indicated the regulatory effect of increased HIF-1α expression on Lp-infected macrophages under high glucose environment.Results:1.Part 1: The expression changes of HIF-1a and related pathway proteins in diabetic guinea pigs complicated with pulmonary infection with Legionella pneumophila type 1.HE staining indicated that diabetes aggravated the progression and inflammatory infiltration after pulmonary Lp infection.Giemsa-stained lung tissue Lp in situ fluorescence hybridization detection showed that compared with the non-diabetic group,the proliferation of Legionella in the lungs of diabetic guinea pigs was significantly increased after 48 hours of infection with Legionella.Both Werstern blot and immunohistochemistry showed that there was no clear HIF-1a protein expression in the uninfected guinea pig lung tissue of diabetic and non-diabetic groups.After infection,HIF-1α was significantly expressed in the post-infection inflammatory infiltration area,and the expression level increased with the aggravation of inflammatory infiltration.The expression of HIF-1α in the diabetic group after infection was significantly lower than that in the non-diabetic group.The expression of i NOS in the non-diabetic group was significantly higher than that in the diabetic group at 24 hours after infection,and began to decrease at 48 hours.The i NOS expression level in the diabetic group reached a peak at 48 hours and decreased rapidly at 72 hours.The protein expression level of NF-κb,the level of NF-κb in the diabetic group increased significantly under non-infection conditions,and the protein expression level increased after infection,and the protein level in the two groups decreased at 72 hours,and the level in the diabetic group was significantly higher than that in the non-diabetic group.increase.SIRT6 protein levels were slightly elevated in the lung tissue of diabetic uninfected guinea pigs,but not statistically significant.The expression was significantly increased after Legionella infection,and the protein expression level of the diabetic group was significantly lower than that of the non-diabetic group at the 72-hour time point.2.The second part: the effect of applying FG4592 to increase the expression of HIF-1a after infection of diabetic guinea pigs on Legionella infection in the lungs.The results of Western blot and immunohistochemistry showed that the application of FG4592 increased the expression of HIF-1α after infection in diabetic guinea pigs.HE staining showed that the inflammation in the diabetic combined infection group was significantly reduced after the increase of HIF-1α.Giemsa and in situ fluorescence hybridization detection showed that Lp proliferation in lung tissue was significantly inhibited compared with diabetes group.The Western blot and immunohistochemical results of i NOS,NF-κb and SIRT6 proteins showed that the expression of i NOS in the FG4592 treatment group was significantly lower than that in the diabetes group,but 24 hours after infection,the i NOS level in the FG4592 treatment group increased rapidly compared with the diabetes group.However,the protein expression level dropped sharply at 48 hours,and the protein expression level of diabetic histones could still be maintained.The expression level of NF-κb in the lung tissue of diabetic guinea pigs was significantly higher than that in the FG4592-treated group.The protein expression of the FG4592 group was higher than that of the diabetic group at 24 hours after infection.There was no significant difference between the two groups at 48 hours,but the protein expression levels of the two groups at72 hours.The levels of SIRT6 in the diabetes group were significantly lower than those in the FG4592 treatment group.Compared with the diabetes group,the SIRT6 level in the FG4592 treatment group was slightly lower than that in the diabetes group,but there was no significant difference in immunohistochemical statistics.The SIRT6 protein expression in the FG4592 treatment group after infection The increase was obvious,but it was lower than that of the diabetes group at 24 hours,and the protein levels of the two groups were significantly decreased at 72 hours,and the FG4592 treatment group was higher than the diabetes group.3.The effect and mechanism of hypoxia-inducible factor on the differentiation of human macrophages induced by Legionella infection under high glucose conditions.Cell lysis was performed on the cells after co-culture,and there was no significant difference in the proliferation level of Lp in the cells of each group at 24 hours.The proliferation of Lp in the cells of the high-glucose culture group at 48 hours and 72 hours was significantly higher than that of the normal culture group and FG4592 treatment.sugar culture group.Twenty-four hours after the cells were infected with Lp,the expression of HIF-1α protein in the normal culture group was higher than that in the high-glucose culture group,and no significant difference was observed at 48 and 72 hours.The high-glucose cultured cells treated with FG4592 drug showed a higher level of HIF-1α when uninfected.The expression of HIF-1α was higher than that in the high-glucose culture group.There was no significant difference in the expression of HIF-1α protein at 24 and 48 hours after infection.At 72 hours,the FG4592 treatment group increased significantly in the high-glucose culture group.The results of ROS detection showed that there was no significant difference in the expression of ROS between the three groups at 24 hours after infection,and the expression level of ROS increased significantly at 48 hours.In the culture group,the ROS expression in the 72-hour high glucose treatment group was also higher than that in the normal culture group and the FG4592 treatment group.The detection of mitochondrial membrane potential showed that in the absence of Lp infection,the mitochondrial membrane potential level of the high-glucose medium-treated group was lower than that of the normal culture group,and the mitochondrial membrane potential level of the highglucose medium-treated group recovered after treatment with FG4592.There was no significant decrease in the membrane potential of the three groups 24 hours after infection,and the trend of the membrane potential of the three groups was similar to that of the uninfected group.48 hours after infection,the level of membrane potential decreased significantly,and the expression level in the high glucose treatment group was significantly lower than that in the normal culture group,and the level of mitochondrial membrane potential recovered after treatment with FG4592.Membrane potential levels of the three groups decreased further in the 72-hour group.Conclusion: 1.Diabetes increased lung damage after Legionella infection,increased inflammatory infiltration,increased bacterial replication,inhibited the expression of HIF-1α after pulmonary infection with Legionella,and increased the expression of i NOS,NF-κb,and SIRT6 in non-infected states,And it affects the degradation of i NOS,NF-κb and the maintenance of SIRT6 level in the late stage of infection.2.Application of FG4592 to increase the expression of HIF-1α after Legionella infection in diabetic guinea pigs can reduce the replication and inflammatory infiltration of Legionella in lung tissue.Increase HIF-1α in the early stage of infection by promoting the expression of i NOS and NF-κb,and reducing the inhibitory effect of SIRT6 on HIF-1α,so as to inhibit the proliferation of Legionella in lung tissue,and in the late stage of infection by inhibiting the expression of i NOS and NF-κb.Expression,increased expression of SIRT6,alleviated excessive inflammatory response.3.High glucose conditions inhibited the expression of HIF-1α in macrophages infected with Legionella,increased the proliferation of Legionella in cells,and increased the level of HIF-1α could inhibit the replication of Legionella in cells.Increasing HIF-1α expression can reduce the increased cellular ROS level under high glucose conditions,reduce the inflammatory damage of cells after infection,and can reduce the damage to mitochondrial membrane potential caused by high glucose,improve the respiratory function of cells after infection,and reduce the incidence of high glucose conditions after infection.The level of glycolysis in cells inhibits the promoting effect of high glucose on intracellular Legionella replication. |
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