The Effect Of Endotoxemia On Scavenger Receptor CD163 In Acute-on-chronic Liver Failure

【Objective】Investigated into clinical expression and significance of scavenger receptor CD163, which only expressed on monocyte-macrophage cell lines, and serum soluble CD 163 in acute-on-chronic liver failure (ACLF).Further, to investigate into the endotoxemia influencing on immune regulatory of CD 163 and into the protection mechanisms of increased CD 163 induced by cortex in rats with liver failure.【Methods】1.Samples from serum, from fresh blood, from tissue samples of liver transplantation and from clinical data in ACLF were collected.2.Serum sCD163 was detected by Elisa and the correction was evaluated between sCD163 and clinical data.3.Serum LPS was assayed by TAL.4.FACS analyzed the expression of CD163 and HLA-DR in peripheral blood monocytes.5.The transcription of CD 163 and TLR4 in peripheral blood monocyte was detected by real-time PCR.6.LPS stimulated peripheral blood monocytes in patients with ACLF in vitro. sCD163, IL-10 and TNF-αwere detection by Elisa.7.We detected CD163-positive Kupffer cells in patients liver tissue with ACLF by HE staining and immunohistochemistry.8.Kupffer cells induced by glucocorticoid were observed in rat liver by HE staining and immunohistochemical staining.9.Serum alanine aminotransferase and bilirubin were measured by automatic biochemical analysis instrument in rats with liver failure and in rats with the intervention after liver failure.10.Serum sCD163 by Elisa was determined in rats with liver failure and with glucocorticoid administration.11.The pathological changes were observed by HE staining in rats liver tissue with liver failure and with the intervention group. 12.Western blotting analysis the expression of CD163,HO-1 in the model group with liver failure, administration group after modeling at the same time,4h administration group, 8h administration group.【Results】1.sCD163 levels in ACLF patients were increased compared with liver cirrhosis, chronic hepatitis B, acute hepatitis group and the control group (P<0.05).2.The Pearson correlation analysis, serum sCD163 in ACLF patients in admission were positively related to the clinical total bilirubin and international normalized ratio, (P <0.05).3.In ACLF patients with infection or complications such as hepatic encephalopathy, or hepatorenal syndrome, serum sCD163 was significantly higher than non-infected group and that no complication group, (P<0.05).4.In the dead group of ACLF patients, sCD163 levels were higher than that in the survival group both on admission and at discharge (P<0.05),when the death group exacerbations, sCD163 tended to increase in the death at discharge compared to on admission,whereas, sCD163 tended to decrease in the survival group.5.In ACLF group the incidence of endotoxemia and endotoxin levels were significantly higher than liver cirrhosis group, chronic hepatitis B group, acute hepatitis group and normal group, (P<0.05).6.Endotoxemia was correlation with serum sCD163 in ACLF group, r=0.56.7.In ACLF death group, TNF-αfrom supernatant was significantly less than that in survival group when peripheral blood monocytes cells were stimulated by LPS in vitro (P <0.05).Intetestingly, IL-10 and sCD163 levels were significantly higher than that in both the normal group and the survival group (P<0.05).8.In ACLF patients who died, CD 163 MFI was less than that both in the survival group and in the normal group, (P<0.05);Expression of HLA-DR molecules of peripheral blood monocytes in the death group was the lowest, that was second in the survival group, at last the normal group (P<0.05).9.In ACLF patients,CD 163 mRNA of peripheral blood monocytes was up-regulation and was higher than that in chronic severe group and in the normal group, (P<0.05);TLR4 mRNA of peripheral blood monocytes was also up-regulation in ACLF patients. But the levels were less than that in chronic severe group and higher than that in the normal group and in mild to moderate chronic group (P<0.05).10.Liver tissues from transplantation in ACLF by HE staining showed that hepatocytes in regeneration nodules were extensively ballooning degeneration and necrosis, bile thrombus with bright yellow can be seen in sinusoidal. Necrosis with different degrees from the interface hyperplasia and fibrosis in lobular bile duct proliferation were also observed; the macrophages were more common.11.In ACLF liver tissue by immunohistochemical staining, triangular, slender, or irregularly shaped cells which were brown were more common in hepatic sinusoid, that is labeled CD163-positive Kupffer cells, but, in normal tissues Kupffer cells dyed brown were less.12.Normal rats group induced by dexamethasone compared with that were not induced after 6h:immunohistochemical staining of liver tissue showed that in the group induced by dexamethasone, CD 163-positive Kupffer cells were significantly higher than that in the normal group.13.In liver failure model,serum sCD163 levels was less than that in the treatment group, sCD163 in the immediate administration group after modeling was higher than that in administration group at 4h and at 8h administration (P<0.05).14.For administration group after modeling, the expression of CD163 and HO-1 in liver failure was higher than that in the model, (P<0.05);for the immediate administration group after modeling, the expression of CD 163,HO-1 were the lowest, was the second for the group at 4h administration, was at last for the group at 8h administration, (P<0.05).【Conclusion】1.Serum sCD163 in ACLF patients was significantly higher than liver cirrhosis group, chronic hepatitis B group, acute hepatitis group and healthy controls, suggesting that sCD163 is sensitive serum marker proteins;the level of sCD163 increased in patients with peritonitis, suggesting that the level of serum sCD163 were response to inflammation and were the clinical indicators of infection; To a certain extent, sCD163 was related to TBIL and INR, suggesting that the severity of patients with ACLF.2.In ACLF death patients, serum sCD163 levels were significantly higher than that in the survival group. In death group after treatment sCD163 remained at a high level, but, in the survival group after treatment sCD163 showed a decline, which suggested that sCD163 levels and change trends after treatment were related to treatment and prognosis, to some extent.3.In ACLF patients, sCD163 and LPS levels increased, accordingly, the expression also increases of TLR4 which was knowned as LPS recognition receptors. Pearson test showed the levels of sCD163 correlated with LPS,the correlation coefficient r=0.56, suggesting that increased sCD163 in ACLF patients were induced by endotoxemia in vivo.4.In ACLF patients, CD 163 mRNA in peripheral blood monocytes increased, however, the expression of CD 163 was decreased, which suggesting that serum sCD163 may be partly from CD 163 protein hydrolysis in the peripheral blood monocytes.5.LPS stimulating peripheral blood momocytes in vitro isolated from ACLF patients showed that the secretion of TNF-αin patients died was less than that in the normal group. However, secretion levels of sCD163,IL-10 increased; HLA-DR expression in peripheral blood monocytes from ACLF patients was reduced. Compared with the survival group and the normal group, down-regulation of HLA-DR in the death group were more significant, while, up-regulation of serum sCD163 were more significant, suggesting that on the basis of chronic liver disease, pathologically long-term stimulation by endotoxin can result in LPS tolerance in ACLF, which indicated immune disorders from inflammation reaction to anti-inflammatory response in patients. Very high sCD163 marked immune tolerance, after tolerance of macrophages the immune paralyzed, and increased risk of subsequent infection, and therefore high sCD163 had higher risk of death and poor prognosis.6.Our results confirmed that corticosteroids can induce the expression of CD 163 in Kupffer cells in vivo,immediate administration after modeling and that at 4h in treatment group has a protective effect on rats with acute liver failure, however, significantly increased serum sCD163,suggesting that glucocorticoid in the early can induce acute anti-inflammatory protein CD 163 to protect liver cells against inflammatory factor damage. The study provided with the new immunological basis for the clinical application with glucocorticoid to control endotoxemia.