The Role Of Hypoxia Inducible Factor-1α In Klebsiella Pneumoniae Pneumonia In Diabetic Mice And Its Related Mechanisms

Objective:Clinical studies indicated that diabetes is an independent risk factor for Gram-negative bacilli infection.At the same time,the pneumonia patients complicated with diabetes always perform much more severe clinical manifestations,prolonged hospitalization duration and higher hospitalization costs.However,the reasons and mechanisms of the aggravation are still unclear.Previous investigations have confirmed that a hypoxic microenvironment can appear in the infected area when pulmonary infection occurs.Hypoxia inducible factor（HIF-1α）is a hypoxia response element,which can be activated when the oxygen concentration in cell or tissue decreased,and followed by the activation of the transcription and expression of a series of downstream molecules.In this study,we observed the changes of HIF-1αin Klebsiella pneumoniae（K.P）pneumonia in diabetic mice and explored its mechanism,so as to clarify the role of HIF-1αin Klebsiella pneumoniae pneumonia of diabetic mice.Methods:Part One:The infection characteristics of diabetes mellitus complicated with Klebsiella pneumoniae infection and the expression of HIF-1α.All the 180 SPF 8-week-old male C57/bl6 mice were randomly divided into:control group,diabetic non-infected group,non-diabetic infected group and diabetic infected group.Diabetic murine model was established by intraperitoneal injection of streptozotocin（STZ）150mg/kg in diabetic non-infected group and diabetic infected group.The model of pneumonia was established by using noninvasive tracheal intubation with 20μl K.P suspension.The control group and the non-infected group were inoculated with the same amount of sterile PBS.At 0h（before inoculation）,2h,8h,12h,24h,2 days,4 days,5 days,7 days and after inoculation,the infection and inflammatory reaction were evaluated by the general state of mice,animal behavior,survival rate,lung tissue weight,lung tissue pathology,bacterial load of lung tissue and spleen tissue,serum cytokines and alveolar lavage fluid（BALF）cytokines.HIF-1αexpression in lung tissue was detected by Western blot and immunohistochemistry.The above indexes were compared among the four groups.Mice in each group for 24 hours,the time of the most severe inflammatory reaction in lung,were selected.The proportion of macrophages in BALF was detected by flow cytometry.Part Two:Role of HIF-1αin diabetic mice with Klebsiella pneumoniae infection and its possible mechanism.In order to clarify the role of HIF-1α,we selected dimethyloxalylglycine（DMOG）,an inhibitor of HIF-1αhydrolase commonly used in animal experiments,to clarify the effect of inhibiting HIF-1αhydrolase on infection.Considering that a kind of clinical drug,HIF hydrolase inhibitor,rosalrestat（FG4592）,has been on the market,a separate group of animals was set up to observe the effect of FG4592 on pneumonia.All the 270 SPF8-week-old male C57/bl6 mice were randomly divided into:control group,diabetic non-infected group,non-diabetic infected group,diabetic infected group,diabetic-DMOG infected group and diabetic-FG4592 infected group.One week before infection,DMOG（30mg/kg）and FG4592（10mg/kg）were injected intraperitoneally every two days in diabetic-DMOG infected group and diabetic-FG4592 infected group until the samples were taken.The other models were established as before.the infection and inflammatory reaction were evaluated by the general state of mice,animal behavior,survival rate,lung tissue weight,lung tissue pathology,bacterial load of lung tissue and spleen tissue,serum cytokines and alveolar lavage fluid（BALF）cytokines.HIF-1αexpression in lung tissue was detected by Western blot and immunohistochemistry.The above indexes were compared among the mentioned groups.Part Three:Explore the effect of up-regulated HIF-1αon macrophage function in high glucose and hypoxia environment.Mouse monocyte macrophages（RAW264.7）were selected to detect their K.P clearance ability.In order to measure the effect of high glucose on scavenging ability,two glucose concentrations were set which are normal glucose（5.5m M）and high glucose（30m M）,and DMOG（200μM）and FG4592（50μM）were added respectively.24 hours later,K.P was added to co-culture with cells（100:1）.The experiment was divided into 5.5m M-K.P group,30m M-K.P group,30m M-K.P-DMOG and 30m M-K.P-FG4592 group.In order to measure the effect of high glucose and hypoxia on HIF-1αlevel of RAW264.7cells,hypoxia was used to stimulate RAW264.7 cells（37℃、5%CO₂、1%O₂,6h）,and DMOG（200μM）and FG4592（50μM）were added.To mimic pneumonia in vitro,lipopolysaccharide（LPS）was added.The experiment was divided into:normoxic normal glucose group（N5）,hypoxic normal glucose group（H5）,normoxic high glucose group（N30）,hypoxic high glucose group（H30）,hypoxic high glucose DMOG group（H30-DMOG）,hypoxic high glucose FG4592 group（H30-FG4592）,hypoxic normal glucose LPS group（H5-LPS）,hypoxic high glucose LPS group（H30-LPS）.HIF-1αlevel was detected by Western blot.To measure the effect of high glucose on the proliferation of RAW264.7 cells in vitro,two glucose concentrations were set（5.5m M and 30m M）and LPS was added to mimic pneumonia.The experiment was divided into normal glucose group（N5）,high glucose group（N30）,normal glucose LPS group（N5-LPS）,high glucose LPS group（N30-LPS）and high glucose DMOG-LPS group（N30-DMOG-LPS）.Cell proliferation was detected by 5-bromodeoxyuridine（Brd U）labeling.Results:Part One:Infection characteristics and expression of HIF-1αin diabetic mice with Klebsiella pneumoniae pneumonia.1.1 General status and survival rate:compared with the diabetic non-infected group,the activity,appetite and weight of the diabetic infected group were significantly reduced（P<0.05）.Only the diabetic infection group had purulent secretions in the cornea and nasal discharge,while the other three groups had no such changes.Death occurred within 2hours in the diabetic infected group and within 12 hours in the non-diabetic infected group.At the end of the observation period,the survival rate of diabetic infection group（67%）was significantly lower than that of non-diabetic infected group（89%,P<0.05）.There was no death in diabetes non-infected group and control group.1.2 Pathological findings of lung tissue:Compared with the control group,the weight of lung tissue in non-infected group was lighter（P<0.05）.Compared with the non-diabetic infected group,the lung tissue in the diabetic infected group showed more serious enlargement,larger consolidation range and heavier weight（P<0.01）.1.3 HE staining of paraffin section:Compared with non-diabetic infected group,diabetic infected group showed more serious capillary dilation,inflammatory cell infiltration and destruction of alveolar unit structure under light microscope.The score of inflammatory cell infiltration showed that both diabetic infection group and non-diabetic infected group had rapid inflammatory infiltration after infection,and the inflammatory reaction in diabetic infected group was more severe from about 12 hours to 4 days after infection（P<0.05）.1.4 BALF cytokines:compared with the control group,tumor necrosis factor-α（TNF-α）and interleukin-6（IL-6）were increased in the diabetic non-infected group（P<0.01）.Compared with non-diabetic infected group,TNF-αand IL-6 in diabetic infected group were increased（P<0.01）.1.5 Serum cytokines:Compared with the control group,the levels of TNF-αand IL-6were increased in the diabetic non-infected group（P<0.01）.Compared with non-diabetic infected group,TNF-αand IL-6 in diabetic infection group were significantly increased（P<0.01）.1.6 Serum procalcitonin（PCT）:Compared with the control group,the PCT in diabetic non-infected group was higher（P<0.01）.Compared with non-diabetic infected group,PCT in diabetic infected group was significantly higher（P<0.01）.1.7 Colony count of lung and spleen:Compared with non-diabetic infected group,the bacterial load in lung tissue of diabetic infection group was higher（P<0.01）.K.P was detected only in spleen of diabetic infected group 4 days after infection（P<0.01）.1.8 HIF-1αlevel in lung tissue:Compared with the normal control group,the level of HIF in the non-infected group of diabetes was decreased,but the difference was not statistically significant.Compared with the normal control group and the non-diabetic infection group,the level of HIF-1αin the diabetic and non-diabetic infected groups were significantly increased,but the increase degree in the diabetic infected group was significantly lower than that in the non-diabetic infected group（P<0.01）.1.9 Compared with the normal control group,the proportion of macrophages in BALF of diabetic infection group and diabetic infection group was higher（P<0.01）,and they were the main cells in BALF（over 70%）.Part Two:Role of HIF-1αin diabetic mice with Klebsiella pneumoniae infection and its possible mechanism.2.1 General status and survival rate:Compared with diabetic infected group,diabetic-DMOG infected group and diabetic-FG4592 infected group showed improved general condition,increased activity,decreased purulent secretion and improved survival rate（P<0.05）.2.2 Pathological findings of lung tissue:Compared with diabetic infected group,diabetic-DMOG infected group and diabetic-FG4592 infected group showed reduced hyperemia and edema in lung tissue,less consolidation and less lung weight（P<0.05）.2.3 HE staining of paraffin section:Compared with diabetic infected group,the degree of inflammatory cell infiltration in lung tissue of diabetic-DMOG infected group and diabetic-FG4592 infected group were significantly reduced（P<0.05）.2.4 Serum and BALF cytokines:Compared with diabetic infected group,the levels of inflammatory factors in serum and BALF in diabetic-DMOG infected group and diabetic-FG4592 infected group were significantly lower（P<0.05）.2.5 Colony count of lung and spleen:Compared with diabetic infected group,diabetic-DMOG infected group and diabetic-FG4592 infected group had lower bacterial load（P<0.05）.2.6 HIF-1αlevel in lung tissue:Compared with diabetic infected group,HIF in lung tissue of diabetic-DMOG infected group and diabetic-FG4592 infected group was activated.Part Three:Explore the effect of up-regulated HIF-1αon macrophage function in high glucose and hypoxia environment.3.1 Under high glucose condition,HIF-1αof RAW264.7 cells stimulated by hypoxia could not be activated normally.The survival number of K.P co-cultured with RAW264.7cells was significantly higher than that under normal glucose condition（P<0.01）.3.2 Under the condition of high glucose,the HIF-1αlevel of RAW264.7 cells inhibited by high glucose could be activated by DMOG and FG4592,and the survival number of K.P co-cultured with RAW264.7 cells decreased significantly（P<0.05）.3.3 Under the condition of high glucose,the proliferation of RAW264.7 cells was inhibited by LPS stimulation,and increased by dmog treatment（P<0.01）.Conclusion:1.Diabetes can aggravate the degree of Klebsiella pneumoniae infection in mice.The main manifestations were low survival rate,high bacterial load in lung tissue and spleen tissue,and high level of inflammatory factors in serum alveolar lavage fluid.The expression of HIF-1αwas inhibited in diabetic mice with Klebsiella pneumoniae pneumonia.2.Overexpression of HIF-1αby DMOG and FG4592 can reduce the infection condition of diabetic mice with Klebsiella pneumoniae,improve the survival rate,accelerate the bacterial clearance,and reduce the inflammatory reaction.3.HIF-1αcan improve the response of RAW264.7 cells to infection by enhancing the ability of scavenging Klebsiella pneumoniae and restoring the proliferation function activated by LPS.