Mechanism Of Vitamin D Binding Protein In Sepsis Secondary To Multiple Organ Dysfunction

Objective:The aim of this study was to assess the value of vitamin D binding protein (VDBP) and vitamin D metabolites in the prognosis of sepsis, and verificate the mechanism of VDBP in sepsis secondary to multiple organ function.Methods:(1) 78 patients that meet the sepsis diagnostic criteria were enrolled within the first 24h after onset of sepsis from 2013 to 2015 in department of respiratory care unit (RICU), emergency department care unit (EICU) and surgical intensive care unit (SICU) of Chinese PLA General Hospital. The application of ELISA were used to detect serum of VDBP,25 (OH) D3,1-25 (OH) D3 and IL-6 in 78 cases of patients with sepsis and 50 cases of healthy control group. The levels of VDBP and vitamin D related metabolites were compared between different prognosis of patients with sepsis. Correlation analysis was used to evaluate the relationship among VDBP, vitamin D metabolites and other clinical indicators. (2) Cecal ligation and puncture(CLP) was induced in mice to construct sepsis model. Serum, urine, liver, lung, kidney and spleen were collected on 1 d,3d,5d and 7d after operation. ELISA were used to detect the level of VDBP in serum and urine. HE staining and immunohistochemical methods were used to describe organ distribution of VDBP in sepsis. Real-time fluorescence quantitative PCR (qRT-PCR) and Western-blot methods were used to assess the correlation between apoptosis and expression of VDBP in liver. (3) By using qRT-PCR and Western blot analysis, we compared the correlation among synthesis of VDBP, apoptosis and proliferation in HepG2 induced with different concentration of LPS (Ong/ml, lOng/ml, 100 ng/ml, 1ug/ml and 10ug/ml) and time (Oh,6h,12h,24h and 48h). Examine the apoptosis in VDBP knocked down/stably overexpressing RAW264.7 macrophage cell lines induced by LPS. Western-blot and qRT-PCR were used to investigate the related pathway of VDBP in regulating LPS induced macrophage apoptosis.Results:(1) Age, blood urea nitrogen, APACHE Ⅱ score and SOFA score in death group were significantly higher than the survival group. Serum levels of 25 (OH) D3, 1,25 (OH) 2D3 and VDBP in death group were significantly lower than the survival group. Age is the independent risk factors of death in patients with sepsis, and 25 (OH) D3 is an independent protective factor. The correlation among VDBP, liver function (ALB, TP) and blood clotting index (PA, FIB) were positive(P<0.05). (2) VDBP levels in CLP operated mice exhibited significant continuous reduction with the lowest concentration at 5 days, which displayed a negtive relationship between serum VDBP and strength and duration of organ lesion. The immunohistochemistry stain of VDBP is seen in blood, airway secretions and on macrophages and granuloma in CLP mice and at low density in the control group. In CLP mice, VDBP synthesis ability of liver was negtively associated with injury and apoptosis of hepatocytes. (3) The synthesis of VDBP and proliferation in HepG2 liver cell line decreased, while the apoptosis increased with rising concentration and induction time of LPS. LPS induced apoptosis rate was significantly increased in VDBP stably overexpressing RAW264.7 compared to VDBP knocked down RAW264.7, with related to Caspase (Caspase3 and caspase8, caspase9, caspase-12) pathway, MAPK (p-p38, p-JNK and p-ERK) pathway and NF kappa B (P-F-kappa B) pathway.Conclusion:(1) 25 (OH) D3,1-25 (OH)2D3 and VDBP are warning of prognostic for sepsis.25 (OH) D3 is an independent protective factor. VDBP can early predict the disfunction of liver and microcirculation in sepsis. (2) The levels of VDBP in serum and urine can indirectly reflect synthesis disorders of liver, and the damage degree of lung, spleen and other tissues in sepsis. (3) LPS could impaire the ability to synthesize VDBP of liver cell by affecting the classical apoptotic pathways. VDBP can mediate LPS stimulated macrophage apoptosis through Caspase pathway, MAPK pathway and NF-κ B pathway.