| Part I The effect of hot partial hepatic ischemia-reperfusion injury on the expression of microRNA-155 in the liver of mice[Objective]To investigate the effect of hot partial hepatic ischemia-reperfusion injury (IRI) on the expression of microRNA-155 (miR-155) in the liver of mice[Methods]Forty wild type C57BL/6 mice (WT) were randomly divided into two groups to establish the model of hot partial hepatic ischemia-reperfusion injury. For the sham group, all the procedures were the same with I/R group except blockage of blood to the middle and left lobes. The mice in the surgical group were blocked the blood supplying to the middle and left lobes for an hour followed by the movement of the vascular clamp, and the blood began to supply to the lobes. The survival rate was estimated after reperfusion for 6 hours following one-hour ischemia. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were examined to assess hepatic function using plasma. HE was performed to estimate the degree of liver damage and make statistical analysis for the magnitude of the liver damage, according to Suzuki's standard. MiR-155 expression was determined by qPT-PCR with TaqMan probe to assess the difference of the level for miR-155 expression between the livers from both groups.[Results]After reperfusion for 6 hours following one-hour ischemia, the survival rate for the mice in both groups was more than 95% and it can meet the experimental requirements. Hepatic function was obviously damaged after hot partial hepatic ischemia-reperfusion injury. The level of ALT and AST in the I/R group was significantly elevated than that in the sham group (P<0.0001). Pathology showed that there was no damage in liver tissue structure, lobular central vein and hepatic cord and no liver congestion, no cell degeneration, necrosis and no inflammatory cell infiltration in the sham group. After reperfusion for 6 hours following one-hour ischemia, the pathology for the I/R group showed there was significant liver congestion, disorder arrangement of liver cell cord, varying degrees of edema and vacuolar degeneration and a lot of flaky necrosis, accompanied by inflammatory cells infiltration. Compared with the sham group, Suzuki's scores in the I/R group were significantly increased, the difference was statistically significant (P<0.0001). Compared with the sham group, the expression of miR-155 was significantly increased in liver tissue of the mice in I/R group after reperfusion, and there was a significant difference with a p value below 0.05.[Conclusion]We have successfully established the model of hot partial hepatic ischemia-reperfusion injury in mice and ischemia-reperfusion injury increased the expression of miR-155 in liver.Part ?miR-155 deficiency ameliorates liver ischemia-reperfusion injury in mice associated with Kupffer cells[Objective]To investigate whether the absence of miR-155 was through acting on Kupffer cells in the liver and then further improved warm ischemia-reperfusion injury.[Methods]Polymerase chain reaction (PCR) was performed to identify microRNA-155 knockout (miR-155-/-) mice. The mice were divided into four groups:(1) WT mice in the control group, WT mice were injected intravenously with the same amount of phosphate buffer with the volume of GdCl3 solution in the experimental group of mice 24 hours prior to the operation, (2) miR-155-/- mice in the control group, miR-155-/- mice were injected intravenously with the same amount of phosphate buffer with the volume of GdCl3 solution in the experimental group of mice 24 hours prior to the operation (3) WT mice in the experimental group, WT mice were injected intravenously with GdCl3 solution (10 mg/kg) 24 hours prior to the operation (4) miR-155-/- mice in the experimental group, miR-155-/-mice were injected intravenously with GdCl3 solution (10 mg/kg) 24 hours prior to the begin of the operation. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were examined to assess hepatic function using plasma. HE was performed to estimate the degree of liver damage and make statistical analysis for the magnitude of the liver damage, according to Suzuki's standard.[Results]Expectedly, the picture showed that there was 465bp-strip for WT mice and 600bp-strips for miR-155-/- mice. ALT and AST levels in WT mice and miR-155-/- mice injected with GdCl3 via the tail vein were significantly lower than those in the corresponding control group of mice and the difference was statistically significant (P <0.0001). Furthermore, the transaminase levels in WT mice and miR-155-/- mice injected with GdCl3 solution were not significantly different (P> 0.05). ALT and AST in the WT ice injected with phosphate buffer were significantly higher than those in miR-155-/- mice injected with phosphate buffer. Compared to the corresponding control groups, tissue damage of both experimental groups was significantly decreased shown in liver pathology sections. The histology of WT mice in the control group injected intraperitoneally phosphate buffer 24 hours before surgery and experienced liver ischemia reperfusion showed obvious liver congestion, disorder arrangement of liver cell cord, varying degrees of edema and vacuolar degeneration, necrosis and inflammatory cell infiltration. Compared with WT mice in control group, the liver injury of miR-155-/- mice in the control group significantly reduced and its Suzuki's scores were lower, and there was a significant difference with a p value below 0.05. The histology of WT mice and miR-155-/- mice in the experiment group showed that there were reduction of liver tissue structural damage, a little spotty necrosis, punctate cell degeneration and necrosis, clear lobular central vein, intact hepatic cord cell structure, weakened liver sinusoidal congestion, and reduced inflammatory cell infiltration.[Conclusion]The absence of miR-155 was through acting on Kupffer cells in the liver and then further improved warm ischemia-reperfusion injury.Part ? MiR-155 deficiency ameliorates liver ischemia-reperfusion injury by regulating polarization and phenotype of Kupffer cells in mice[Objective]To examine the relationship between the protective effect of the deficiency of miR-155 on ischemia-reperfusion injury and it's regulation on Kupffer cell polarization.[Methods]Liver hot part ischemia-reperfusion injury was performed in WT mice and miR-155"'' mice. After reperfusion for 6 hours following 1 hour-ischemia, we collected whole blood serum. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were examined to assess hepatic function using plasma. HE was performed to evaluate the degree of liver damage and make statistical analysis for the magnitude of the liver damage, according to Suzuki's standard. We detected liver cell apoptosis with TUNEL technique and the mRNA expression of inflammatory factors in liver tissue were detected by RT-PCR. Kupffer cells were harvested by density gradient centrifugation from the livers of mice. The phenotype and classification differences of Kupffer cells derived from WT mice and miR-155-/- mice challenged with hepatic ischemia-reperfusion injury were examined by flow cytometry. Peritoneal macrophages were extracted from WT mice and miR-155-/- mice and were purified by adherent culture. Then the purified cells were divided into four groups: A, WT peritoneal macrophages unstimulated group; B, miR-155-/- peritoneal macrophages unstimulated group; C, WT peritoneal macrophages with LPS (50ng/ml) stimulation 24 hours; D, miR-155-/- peritoneal macrophages with LPS (50ng/ml) stimulation 24 hours. The cells in each group were collected and the phenotype and polarization differences were detected by flow cytometry. ELISA assay was performed to examine the concentration of TNF-a and IL-10 in the culture supernatant of peritoneal macrophages. The impact of Kupffer/macrophage on hepatocytes apoptosis were detected by flow cytometry after co-culture withTranswell assays.[Results]ALT and AST levels in miR-155-/- mice were significantly lower than those in WT mice group. Liver histopathology showed that, compared with WT mice, the area of tissue necrosis in miR-155-/- mice was significantly reduced, and there was less congestion and structural damage in miR-155-/- mice. The congestion of liver tissue was significantly increased, liver cell cord was severely damaged, and more patchy necrosis areas appeared in WT mice group. In miR-155-/- mice group, Suzuki's scores were significantly lower, and there was a significant difference with a p value below 0.001. Compared with WT group, apoptosis liver cell significantly reduced in miR-155-/- mice group (P<0.05). Although the expression of inflammatory factors were significantly increased after ischemia-reperfusion injury in both group, there was lower level of TNF-a expression and higher level of IL-10 expression in miR-155-/- mice than those in WT mice group. Compared with WT group, the expression levels of CD80, CD86 and MHC-IIin Kupffer cells were much lower in miR-155-/- mice after ischemia-reperfusion injury and there was a significant difference with a p value below 0.05. In miR-155-/- mice group, M1 type (CD11c+CD206-) Kupffer cells were significantly reduced compared with WT group, and M2 type (CD11c-CD206+) Kupffer cells have increased significantly and there was a significant difference with a p value below 0.05. The purity of peritoneal macrophages isolated and purified was more than 90%, and it can meet experimental needs. Compared with WT mice macrophages, the expression of CD80, CD86 and MHC-II in peritoneal macrophages derived from miR-155-/- mice without LPS stimulation group (PBS incubation group) was significantly lower and the difference was statistically significant (CD80 P<0.01; CD86 P<0.05; MHC-IIP<0.05). Although the expression of CD80, CD86 and MHC-II in peritoneal macrophages derived from both WT mice and miR-155-/- mice was significantly increased after LPS stimulation, the expression level of CD80, CD86, CD40 and MHC-II in miR-155-/- mice group was lower than those in WT mice and the difference was significant (CD80 P<0.01; CD86 P<0.05; CD40 P<0.01; MHC-IIP<0.05).Compared with WT mice group, the percent of M1-type (CD11c+CD206-) macrophages was significantly lower but M2-type (CD11c-CD206+) macrophages was significantly higher without LPS stimulation in miR-155-/- mice group (P<0.05). After LPS stimulation, the percent of M1-type macrophage derived from both WT mice and miR-155-/- mice was increased, while the percent of M2-type cell were both decreased. However, compared with WT mice group, M1-type cells were significantly reduced and M2-type cells were significantly increased in miR-155-/- mice group, and there was a significant difference with a p value,M1 P<0.05, M2 P<0.01. After LPS stimulation, the concentrations of both TNF-a and IL-10 were increased significantly in the culture supernatant of macrophages derived from both WT mice group and miR-155-/- mice group. However, compared with WT mice group, the concentration of TNF-a was significantly lower in miR-155-/- mice group while IL-10 concentration was significantly higher and the difference was statistically significant (P values were both less than 0.05). Macrophages and Kupffer cells accelerated hepatocyte apoptosis. However, compared with WT mice group, apoptosis hepatocytes induced by Kupffer cells derived from miR-155-/- mice were significantly less and the difference was statistically significant (P<0.05).[Conclusion]MiR-155 absence regulated KCs shifting to anti-inflammatory M2 phenotype other than M1 phenotype, resulting in reducing TNF-? secretion, increasing IL-10 secretion and weakening hepatic inflammation, and further improved the liver ischemia-reperfusion injury. |
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