| Objective: The goal of this study was to define the role of mi R-146 a and TLR4-TRAF6-NF-κB signal pathway in intestinal response to damage using an ischemia- or ischemia-reperfusion induced injury model, which may help to provide theoretical basis for the early diagnosis of intestinal ischemia and the effective therapeutic strategies of intestinal ischemia-related injury. Methods: The cultured IEC-6 cells were used to establish models, ischemic groups with different ischemic times for 1h, 2h or 3h(I1h、I2h、I3h)respectively, ischemia-reperfusion group with ischemia solution for 1h and the reperfusion fluid for 1h(I1h R1h), while the control group was cultured in normal condition. Then, we regulated the expression of mi R-146 a in I1 h group and I1 h R1h group. In every group, detected the expression of mi R-146a、TLR4、TRAF6、RIP、XIAP m RNA by Real time quantitative fluorescence PCR test, the apoptosis of cells was detected by flow cytometry, and the Western blot was used to detect the protein expression of HIF-1α、TLR4、TRAF6、RIP、XIAP、SCOS-3、NF-κB and cleaved-Caspase-3. Statistical analysis was performed by using two-tailed Student t test or ANOVA for comparisons as appropriate using SPSS software(Version 13.0). In all cases, P< 0.05 was considered significant. Results:(1) Cells in the ischemia groups exhibited significantly higher expression of HIF-1α protein than that in the control group(P< 0.01), whereas the expression of HIF-1α protein was decreased quickly after reperfusion treatment.(2)The expression of mi R-146 a was significantly decreased in the plasma of patients with intestinal ischemia(P< 0.01).(3)The expression of mi R-146 a was significantly decreased in the I1 h group(P< 0.01) and showed a slowly recovery with prolonged ischemic time, but it always still below the control level. The expression of mi R-146 a in the I1 h R1h group was higher than those of I1 h group, but it was still significantly lower than control(P< 0.05).(4)The apoptosis index gradually increased with the prolonged ischemic time, but there was no difference between I2 h group and I3 h group. Compared with I1 h group, the apoptosis index in the I1 h R1h group was significantly increased(P< 0.05).(5)The expression of TLR4, TRAF6, RIP m RNA levels and TLR4, TRAF6, RIP, NF-κB, cleaved-Caspase-3 protein levels had the similar trends which were significantly increased in ischemia groups and ischemic-reperfusion group, especially in I1 h group(P< 0.01), which were always higher than normal levels(P<0.05), even though they all decreased on the basis of I1 h group with prolonged ischemic time or after reperfusion treatment. The expression of XIAP m RNA levels and SCOS-3, XIAP protein levels all significantly decreased in the I1 h group(P< 0.01), which also showed a slow recovery with prolonged ischemic time or after reperfusion treatment, but they were always lower than the control levels(P< 0.05).(6)After transfection with mimic or inhibitor of mi R-146 a, the expression of mi R-146 a was significantly up- or down-regulated in the control group(P<0.01), but there was no significant change in the apoptosis rate of cells.(7)After transfection with mimic of mi R-146 a in the I1 h group, the expression of mi R-146 a was significantly increased(P< 0.01), but there was no significant downward trend and statistically significant after transfection with inhibitor of mi R-146 a in the same group.(8)After transfection with mimic or inhibitor of mi R-146 a in the I1 h R1h group, the expression of mi R-146 a was significantly up- or down-regulated(P<0.01).(9)Compared with I1 h group or I1 h R1h group, the related groups which were transfeced with mi R-146 a mimic could significantly inhibit the upregulation of TLR4, TRAF6, RIP, NF-κB and cleaved-Caspase-3, whereas SOCS-3 and XIAP were increased, and the apoptosis index was decreased. The results showed that overexpression of mi R-146 a plays a protective role in cells injury caused by ischemia or ischmia/reperfusion.(10)Compared with I1 h R1h group, after transfection of mi R-146 a inhibitor could significantly promote the upregulation of TLR4, TRAF6, RIP, NF-κB and cleaved-Caspase-3, whereas SOCS-3 and XIAP were decreased, and the apoptosis index was increased(P< 0.05). These suggest that the inhibition of mi R-146 a may exacerbate cells injury. Conclusions:(1) mi R-146 a involved in intestinal ischemia and ischemia-reperfusion process, and plasma mi R-146 a is expected to become an early biomarker of clinical diagnosis of intestinal ischemia.(2) The signaling pathway of TLR4-TRAF6-NF-κB involved and induced cells damage in intestinal ischemia and ischemia-reperfusion process.(3) mi R-146 a may inhibit apoptosis of cells by negative regulating TLR4-TRAF6-NF-κB signaling pathway and thus play a protective role in intestinal ischemia and ischemia-reperfusion process.(4)Upregulating mi R-146 a or inhibiting activation of TLR4-TRAF6-NF-κB pathway is expected to become an effective target in the prevention and treatment of intestinal ischemia-related diseases. |
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