Study On The Role And Mechanism Of 1,25（OH）₂D₃ In Improving Alveolar Pro-coagulation And Fibrinolytic Inhibition In Acute Respiratory Distress Syndrome

Objective:The excessive inhibition of fibrinolysis and enhanced pro-coagulation within the alveoli,leading to massive fibrin deposition in the alveolar space,is a critical pathogenic factor in acute respiratory distress syndrome（ARDS）.However,the mechanisms involved in this process remain unclear,and effective treatment options are lacking.Previous research suggests that low levels of vitamin D are associated with the occurrence and progression of ARDS,making it a high-risk factor for development.Therefore,the primary objectives of this study are as follows:1.To investigate the correlation between vitamin D levels in the peripheral blood of ARDS patients and the degree of fibrinolysis inhibition and coagulation promotion within the alveoli.2.To explore the impact of vitamin D deficiency on coagulation promotion and fibrinolysis inhibition within the alveoli of ARDS patients.3.To explore the molecular mechanism by which 1,25（OH）₂D₃influences coagulation promotion and excessive fibrinolysis inhibition within the alveoli of ARDS patients,with a specific focus on the NF-κB p65 signaling pathway.Methods:1.Correlation between serum vitamin D levels and alveolar pro-coagulation as well as fibrinolytic inhibition in patients with ARDS:Clinical data were collected from ARDS patients who were hospitalized for treatment in the intensive care unit of Guizhou Medical University Affiliated Hospital between July 2019 and February 2023.Additionally,clinical data from individuals who visited the physical examination center of the same hospital for routine physical examinations and had similar basic characteristics as the ARDS patients were also collected.ELISA was employed to measure the levels of tissue factor（TF）and plasminogen activator inhibitor-1（PAI-1）in the bronchoalveolar lavage fluid（BALF）of ARDS patients,while the serum vitamin D levels in the subjects were measured using electrochemiluminescence immunoassay.Logistic regression analysis was conducted to explore the correlation between serum vitamin D levels and prognosis.Moreover,a multifactorial linear model was employed to adjust for confounding factors such as age and gender,and to examine the trends in serum vitamin D levels,TF,and PAI-1.All survey data were double-entered and analyzed using the R language for statistical analysis.2.Impact of 1,25（OH）₂D₃on alveolar pro-coagulation and fibrinolytic inhibition in ARDS mice through ameliorating vitamin D deficiency:Sixty C57BL/6 mice were randomly divided into five groups:a control group,a group receiving a normal diet with LPS stimulation（LPS group）,a group receiving a vitamin D-deficient diet（VD-group）,a group receiving a vitamin D-deficient diet with LPS stimulation（VD-+LPS group）,and a group receiving a vitamin D-deficient diet with LPS stimulation and 1,25（OH）₂D₃supplementation（VD-+LPS+1,25（OH）₂D₃group）.Each group consisted of 12 mice,with six mice undergoing alveolar lavage and the remaining six used for other measurements.ARDS was induced by intratracheal inhalation of LPS（200μg/50μL）.The control group and LPS group were fed a normal diet and exposed to normal light before the experiment.On the other hand,the VD-and VD-+LPS groups were fed a vitamin D-deficient diet and exposed to UV-free yellow light for six weeks before LPS intratracheal inhalation.The VD-+LPS+1,25（OH）₂D₃group followed a similar diet and light exposure for six weeks.Additionally,they received daily gavage of1,25（OH）₂D₃（2.5μg/kg）for two weeks before LPS intratracheal inhalation and were fed a normal diet and exposed to normal light.The experiment endpoint was set at eight hours after LPS administration.Serum 25（OH）D₃levels were measured using enzyme-linked immunosorbent assay（ELISA）,while lung tissue pathological changes were observed using hematoxylin-eosin（HE）staining and scored using the Smith score.The lung tissue wet/dry（W/D）ratio was calculated to assess the degree of pulmonary edema.Real-time fluorescence quantitative polymerase chain reaction（RT-q PCR）was employed to detect TF,PAI-1,p65,and VDR m RNA levels in lung tissue.Western blotting（WB）was used to detect TF,PAI-1,p65,p-p65,and VDR protein expression levels.The levels of TF and PAI-1 in bronchoalveolar lavage fluid（BALF）were measured using ELISA,while the expression of p65 and VDR in lung tissue was assessed through immunohistochemistry.3.Elucidating the molecular mechanism of 1,25（OH）₂D₃in alleviating LPS-induced expression of pro-coagulant and fibrinolytic inhibitory-related factors in type II alveolar epithelial cells:To determine the appropriate dose of1,25（OH）₂D₃for the experiment,the CCK-8 assay was conducted.The findings revealed that a dose of 0.01μM was optimal for pre-treating type II alveolar epithelial cells in rats with 1,25（OH）₂D₃for one hour,followed by 24-hour LPS treatment.To investigate whether 1,25（OH）₂D₃influences the expression of TF and PAI-1 in type II alveolar epithelial cells through the VDR/NF-κB p65 signaling pathway,VDR-specific si RNA was transfected into the cells to silence the VDR gene.Additionally,stable cell lines were established by overexpressing/silencing NF-κB p65 using a lentiviral vector.The same treatment regimen involving 1,25（OH）₂D₃and LPS was administered,and RT-q PCR was employed to detect the m RNA expression levels of TF,PAI-1,p65,and VDR.WB analysis was conducted to measure the protein expression levels of TF,PAI-1,p65,p-p65,and VDR in RLE-6TN cells.Furthermore,the expression level of nuclear p65 was determined through immunofluorescence staining.Results:1.Correlation between serum vitamin D levels and alveolar pro-coagulation as well as fibrinolytic inhibition in patients with ARDS:After adjusting for age,gender,and BMI,it was observed that the serum 25（OH）D levels in ARDS patients were significantly lower than those of the general population（p<0.001）.Among ARDS patients,applying the F-test correction for age and gender factors,17 factors were found to be associated with serum 25（OH）D levels.Except for TF and PAI-1concentrations in BALF,all other continuous variables exhibited an upward trend with increasing serum 25（OH）D levels.Upon further adjustment for age,gender,BMI,and significant factors,it was discovered that serum 25（OH）D levels were associated with prognosis.Higher serum 25（OH）D levels were associated with a lower risk of death.Specifically,with every 1 unit increase in 25（OH）D level,the risk of non-prescription discharge or death decreased by 2%.After controlling for age and gender,the beta coefficients for TF and PAI-1 in lung lavage fluid were determined as-0.0709（p<0.001）and-0.0795（p<0.001）,respectively.This indicates that for each unit increase in serum 25（OH）D level,the content of TF and PAI-1 decreased by0.0709 and 0.0795 units,respectively.2.Impact of 1,25（OH）₂D₃on alveolar pro-coagulation and fibrinolytic inhibition in ARDS mice through ameliorating vitamin D deficiency:Compared to the Control group,the VD-group exhibited a significant decrease in serum 25（OH）D₃levels,confirming the successful replication of the vitamin D deficiency animal model.The LPS group,VD-+LPS group,and VD-+LPS+1,25（OH）₂D₃group demonstrated significant increases in various parameters when compared to the Control and VD-groups.These parameters include lung tissue Smith pathology score,lung tissue wet/dry（W/D）ratio,lung tissue TF m RNA and protein expression,lung tissue PAI-1m RNA and protein expression,as well as TF and PAI-1 secretion in bronchoalveolar lavage fluid（BALF）.Furthermore,when comparing the VD-+LPS group to the LPS group,there were significant increases in the lung tissue Smith pathology score,lung tissue W/D ratio,lung tissue TF m RNA and protein expression,lung tissue PAI-1m RNA and protein expression,as well as BALF TF and PAI-1 secretion.However,these parameters showed significant decreases in the VD-+LPS+1,25（OH）₂D₃group compared to the VD-+LPS group.The LPS group and VD-+LPS group displayed significant increases in lung tissue p65 m RNA and protein expression,as well as p-p65 protein expression,and an increase in p65-positive staining compared to the Control group.When comparing the VD-+LPS group to the LPS group,there were significant increases in lung tissue p65 m RNA and protein expression,p-p65 protein expression,p65-positive staining,and VDR m RNA and protein expression.Conversely,VDR-positive staining decreased in the VD-+LPS group.However,in the VD-+LPS+1,25（OH）₂D₃group compared to the VD-+LPS group,there were significant decreases in lung tissue p65 m RNA and protein expression,p-p65 protein expression,p65-positive staining,as well as VDR m RNA and protein expression.Notably,VDR-positive staining increased in the VD-+LPS+1,25（OH）₂D₃group compared to the VD-+LPS group.3.Elucidating the molecular mechanism of 1,25（OH）₂D₃in alleviating LPS-induced expression of pro-coagulant and fibrinolytic inhibitory-related factors in type II alveolar epithelial cells:Pre-incubation with 1,25（OH）₂D₃demonstrated a significant reduction in both m RNA and protein levels of tissue factor（TF）and plasminogen activator inhibitor-1（PAI-1）when RLE-6TN cells were exposed to lipopolysaccharide（LPS）stimulation.Additionally,this pre-incubation upregulated the expression of the vitamin D receptor（VDR）and suppressed the NF-κB p65 signal,leading to a decrease in p65 expression,including phosphorylated p65 and nuclear p65.Moreover,the study observed that the transfection of VDR gene-specific si RNA to silence VDR signaling in RLE-6TN cells,followed by LPS stimulation,resulted in a significant increase in TF,PAI-1,p65 m RNA,and protein expression levels,as well as an elevation in nuclear p65 expression.Notably,these effects were not counteracted by 1,25（OH）₂D₃treatment.Conversely,in p65^+/+cells,LPS stimulation induced a notable upregulation of TF and PAI-1 m RNA and protein expression levels,which were effectively reversed by treatment with 1,25（OH）₂D₃.In contrast,p65^-/-cells did not exhibit any changes under LPS stimulation.Taken together,these findings suggest that 1,25（OH）₂D₃exhibits the ability to enhance the expression of coagulation and fibrinolysis inhibitors in type II alveolar epithelial cells through the upregulation of VDR expression and inhibition of the NF-κB p65 signal.Conclusions:1.Insufficient levels of vitamin D in patients with ARDS are linked to poor prognosis.2.Vitamin D deficiency exacerbates LPS-induced ARDS by promoting pulmonary coagulation and impeding fibrinolysis.3.1,25（OH）₂D₃mitigates augmented pro-coagulation and excessive fibrinolytic inhibition in ARDS by suppressing the NF-κB signaling pathway.