| Part I Effect of 3PO on Sepsis-induced Acute Lung Injury Objective:To expored the effect of 3PO on sepsis-induced acute lung injury Method:The C57BL/6 mice were randomly divided into three groups(10rats/group): control group,CLP group,and 3PO+CLP group.Control group(sham group): The caecal row of mice except for ligation and puncture was treated equally and returned to the abdominal cavity.CLP group: Follow the complete modelling procedure.In the 3PO+CLP group,mice were infused with 50 uL of DMSO containing 0.07 mg/g 3PO at 0 and 6 hours after CLP.The survival of the mice was recorded every 12 hours after 5 days of CLP in each group.The sham group was observed and recorded for the survival status of the mice.All the mice were euthanized 5 days later.HE staining was performed on the lungs of the mice to assess the degree of lung injury.The ratio of dry to wet weight of the lungs was measured.The bronchoalveolar lavage fluid was collected for detection of inflammatory factors,inflammatory cell exudation,lactate levels,and blood tests were collected to detect levels of inflammatory factors.Results:1.Establishment of sepsis-associated acute lung injury model: Clinical characteristics of typical sepsis such as apathy,shortness of breath,chills,standing hair,systemic weakness,and decreased or lost abilities after establishing CLP model by cecal ligation in mice After 24 hours,lung tissues were taken for HE staining to determine whether the mouse CLP model was successful.The results showed that in the sham operation group,the structure of the lung was clear,the epithelial cells were arranged neatly,and the alveolar space was clean and uniform.In the CLP group,however,the structure of the lung tissue was significantly damaged,and congestion,edema,and inflammatory cell infiltration were observed.2.Effect of 3PO on lung inflammation in sepsis mice: pathological results of mice in the Control group showed clear and complete structure of alveoli,normal alveolar septa,no obvious infiltration of inflammatory cells in the alveolar and lung interstitium,interstitial capillary The blood vessels showed no congestion and congestion;in the CLP group,the alveolar expansion was uneven,the alveolar wall was generally thickened,some alveolar wall structure was unclear,and the capillaries were dilated and congestion.There were more inflammations in the interstitial lung and alveolar space.Infiltration of cells,a large number of red blood cells can be seen at the same time;After 3PO treatment in CLP+3PO mice,the degree of lung tissue pathological damage was significantly lower than that of CLP group,alveolar dilation was evenly hooked,local alveolar wall thickened,and interstitial localized slight inflammation.Cell infiltration.3.The effect of 3PO on pulmonary edema in sepsis mice: Compared with control group,W/D increased after CLP,the difference was statistically significant(P<0.05,n=10).Compared with the CLP group,W/D decreased after 3PO intervention(P<0.05,n=10).4.3PO inhibits the degree of inflammatory cell exudation and glycolysis in the alveoli of mice with sepsis: compared with the sham group,the total cell number,the number of neutrophils,and the lactate level in the alveolar lavage fluid of the CLP group Significantly increased,P<0.01,n=10,the difference was statistically significant;and 3PO+CLP group compared with CLP group,total cell number,neutrophil count,and lactate in bronchoalveolar lavage fluid after intraperitoneal injection of 3PO.The average level was improved,P<0.05,n=10,and the difference was statistically significant.This result suggests that 3PO can inhibit the exudation and anaerobic glycolysis of inflammatory cells in the alveoli of sepsis mice.5.3PO can reduce the inflammatory response in the lung tissue of sepsis mice: Compared with the sham group,the levels of TNF-α,IL-6 and IL-1β in the bronchoalveolar lavage fluid of the CLP group increased significantly.P<0.01,n=10,the difference was statistically significant;and after intraperitoneal injection of 3PO,TNF-α,IL-6,and IL-1β in the bronchoalveolar lavage fluid of the 3PO+CLP group were improved compared to the CLP group.P<0.05,n=10,the difference was statistically significant.This result suggests that 3PO can inhibit the degree of inflammation in alveoli in sepsis mice.6.3PO can reduce the systemic inflammatory response in sepsis mice: the same trend as in bronchoalveolar lavage fluid.Compared with the sham operation group,serum levels of TNF-α,IL-6,and IL-1β in the CLP group have Significantly increased,P<0.01,n=10,the difference was statistically significant;and 3PO + CLP group serum TNF-α,IL-6,IL-1β improved after intraperitoneal injection of 3PO compared to CLP group,P <0.05,n = 10,the difference was statistically significant.This result suggests that 3PO can not only inhibit the degree of inflammatory reaction in the alveoli of sepsis mice,but also inhibit the degree of systemic inflammatory response in mice.7.3PO improved the survival rate of sepsis mice: compared with sham control group,the survival rate of mice in CLP group decreased significantly(P<0.01);compared with CLP group,the survival rate of mice in CLP+3PO group Increased(p<0.01).The results suggest that 3PO can improve the survival rate of sepsis mice.Conclusions:In this phase of the experiment,a mouse sepsis model was successfully established to simulate the pathological process of sepsis-associated acute lung injury,resulting in severe lung injury and decreased survival rate;3PO can inhibit lung pathological changes and inhibit alveolar Lactic acid levels reduce the levels of inflammatory cytokines TNF-α,IL-6 and IL-1β in the lung,reduce the levels of systemic inflammatory cytokines TNF-α,IL-6,and IL-1β,decrease the exudation of inflammatory cells in the lung tissue,and protect the lungs.Tissue cells improve mouse survival.Our study revealed the role of 3PO in promoting inflammation resolution and provided new strategies and therapeutic targets for the treatment of sepsis-induced acute lung injury.Part II 3PO inhibits glycolysis and inflammatory cytokine production sepsis-associated lung epithelial cells Objective:To study the effect of 3PO on glycolysis and inflammatory cytokine release in sepsis-associated lung epithelial cells Method:The human lung epithelial A549 cells were divided into three groups: Control,LPS,and LPS+3PO.The LPS group was stimulated with LPS(100μg/ml)for a specified period of time(0,2,and 4 hours)when the cell density was increased to 80%.A549 cells;LPS+3PO groups were treated with 3PO(20μmol/L)for 30 min before LPS stimulation,and then A549 cells were stimulated with LPS(100μg/ml)for a specified period of time(0,2,and 4 hours).Twenty-four hours after the administration of LPS,the glucose and lactic acid in the three cell culture media,the LDH activity in the cell lysate and the expression levels of TNF-α,IL-6 and IL-1β in the culture medium were detected.Results:1.3PO inhibited lipopolysaccharide-induced glycolysis: With the increase of treatment time,LPS group’s glucose consumption,lactate dehydrogenase(LDH)and lactic acid levels increased significantly,and at the same time point,LPS+3PO group The glucose consumption,lactate dehydrogenase(LDH)and lactic acid levels were significantly inhibited and the difference was statistically significant.2.3PO can inhibit LPS-induced release of inflammatory cytokines: With the increase of treatment time,the levels of TNF-α,IL-6 and IL-1β in LPS group increased significantly,and at the same time point,LPS+ The levels of TNF-α,IL-6 and IL-1β in the 3PO group were significantly inhibited,and the difference was statistically significant.Conclusions:By culturing human lung epithelial cell A549,LPS was added to simulate the sepsis environment,resulting in a significant increase in extracellular glucose consumption and lactate levels and intracellular lactate dehydrogenase(LDH)activity.The inflammatory cytokines TNF-α,IL-The expression of 6 and IL-1β was significantly increased,while the glycolysis and inflammatory cytokine release of the 3PO-treated cell group was decreased relative to the LPS group.It was verified at the cellular level that 3PO can suppress sepsis-induced glycolysis and inflammatory cytokine release.Part III 3PO reduces apoptosis in acute lung injury by inhibiting ROS production Objective:To investigate the effect of 3PO on sepsis-associated lung epithelial cell apoptosis and its mechanism Method:The A549 cells were seeded on a six-well plate and divided into seven groups: Control,LPS,LPS+3PO,Lactate,Lactate+NAC,HCL,and HCL+NAC.When the cell growth density reached 80%,the LPS group was stimulated with LPS(100μg/ml)for a specified period of time(0,2,4 hours);the LPS+3PO group was treated with 3PO(20μmol/L)for 30 min before LPS stimulation.Then,A549 cells were stimulated with LPS(100μg/ml)for a specified period of time(0,2,and 4 hours);A549 cells were directly stimulated with Lactate and HCL for a specified period of time(0,2,and 4 hours)in the Lactate and HCL groups.In the Lactate+NAC group and the HCL+NAC group,cells were treated with NAC 30 minutes before adding Lactate and HCL.After the treatment,apoptosis was collected and measured for reactive oxygen species.Results:1.3PO reduced LPS-induced ROS generation in A549 cells: ROS production was significantly increased in LPS-induced A549 cells,and inhibition of glycolysis by 3PO could significantly inhibit LPS-induced ROS production in A549 cells;whereas glycolysis product lactate was used.The ROS production in A549 cells can also be induced;more interesting is the generation of ROS in A549 cells that can also be induced by decreasing the pH of the medium with HCL.Combined with the previous results,it was suggested that LPS induces aerobic glycolysis of A549 cells and produces a large amount of lactate that lowers the PH and induces ROS production.2.3PO reduced the apoptosis of A549 cells induced by LPS: In LPS-induced A549 cells,apoptosis was reduced after pretreatment of ROS with the inhibitor NAC.At the same time,after inhibiting glycolysis with 3PO,LPS-induced apoptosis of A549 cells decreased;glycolytic products lactate and HCL can increase A549 cell apoptosis,its role can be inhibited by NAC.It is suggested that LPS can induce cell apoptosis by promoting the aerobic glycolysis of A549 cells,producing a large amount of lactate to reduce the PH,and then increasing the production of ROS;and 3PO can reduce the cell by inhibiting the production of glycolysis and glycolysis products and reducing the ROS.Apoptosis.3.3PO inhibited the apoptosis of alveolar cells in sepsis mice: Compared with the control group,the proportion of apoptosis in the CLP group was significantly higher,but the percentage of apoptosis in the 3PO group after CLP injection was significantly improved,*P<0.05.n=10,the difference was statistically significant.Conclusions:LPS(100ug/ml),lactate(5mmol/L)and HCl had similar effects on apoptosis,and LPS and lactic acid caused a significant increase in ROS.NAC(10mmol/L)significantly inhibited the increase of ROS induced by LPS and lactate.And apoptosis;3PO treatment can inhibit LPS-induced increase in ROS and apoptosis.It is suggested that 3PO can improve LPS-induced apoptosis by inhibiting ROS production in cells. |
