The Study Of Blocking Tlr/Myd88/Nf-kb Signaling Pathway On Renal Ischemia Reperfusion Injury In Mice

PartⅠT-cell-specific intervention outreached the positive effects of systemic NF-κB blockade in attenuate renal ischemia reperfusion injury in miceObjective To investigate the effect of systemic NF-κB Inhibitor-ursolic acid on renal ischemia reperfusion injury and the mechanism in mice and compared the systemic and T-cell-specific inhibition of the NF-κB pathway on renal IRI in mice.Methods Mice were administrated ursolic acid (10mg/Kg) dissolved in carboxymethyl cellulose (CMC) intragastrically 48h,24h and 6h prior to renal ischemia. Mice intragastic administration with CMC served as controls. Renal IRI was induced in mice by clamping the left renal pedicle for 60min, while the right kidney was being removed. The protective effects of UA were evaluated by renal function, histological examination, and overall survival after lethal IRI. NF-κB expression was detected with TransFactor Colorimetric Kits and ROS Production was measure withDCFH-DA fluorescent probe; serum were collected 24h for detection of inflammatory factor such as IL-1β, IL-6 and TNF-α. MPO expression was measured by immunohistochemistry and TUNEL assays were used to evaluate the apoptosis of renal cells.Results The Ursolic Acid treated group and IkBΔN-Tg group showed mitigatory renal IRI (BUN 34.09±4.716 12.79±2.194 vs 72.63±9.216, p<0.01 p<0.01; sCr 60.67±12.61 27.33±6.31 vs 150.3±14.5, p<0.01 p<0.01); higher survival rate (62.5% and 75% vs 12.5%), less pathologic changes, lower levels of NF-κB translocation in vivo (0.18±0.05 0.14±0.02 vs 0.36±0.06,p=0.0035 p=0.0005) and the lower expression of downstream factors (IL-1β1.60±0.15 1.37±0.15 vs 3.80±0.40, p=0.0009, p=0.0006; IL-6 1.37±0.15 0.72±0.2vs3.57±0.65,p=0.0004, p=0.0002; TNF-α1.70±0.100.97±0.58 vs 2.40±0.26, p=0.0128, p=0.0008), less production of ROS (4.6±0.36,4.6±0.50 vs 10.07±0.9, p=0.0006, p=0.0008) in kidneys compared with the CMC group.Conclusion Systemic NF-κB Inhibitor-ursolic acid can protect kidneys from IRI through inhibiting NF-κB translocation, the expression of downstream factors (IL-1β, IL-6 and TNF-a) and the production of ROS in kidneys. T-cell-specific intervention outreached the positive effects of systemic NF-κB blockade in renal ischemia reperfusion injury. PartⅡCo-Blocking TLR/MYD88 signaling pathway and CD154-CD40T-Cell Co-stimulation Pathway Leads Profound Protection against Renal Ischemia-Reperfusion Injury in MiceObjective To inverstigated the effect of co-blocking TLR/MYD88 signaling pathway and CD154-CD40T co-stimulation pathway on renal ischemia reperfusion injury and relative mechanism in mice.Methods 84 male BALB/C mice were divided into 6 groups:the IRI group, MR1 group, TJ-2 group, MYD88KO group and the TJ-2+MR1 group,6 mice for each group (other 8 mice for each group for observation of survival rate). Renal IRI was induced by clamping the left renal pedicle for 80min, and some of the kidneys were removed immediately as others collected 24h,3d, and 7d later for detection of NF-κB expression with EMSA and ROS production with DCFH-DA fluorescent probe; serum were collected 24h for detection of inflammatory factor such as IL-1β, IL-6, TNF-a and IL-10. The therapeutic effects were evaluated in renal function after 24h,3d and 7d of reperfusion, also with histologic examination. MPO expression was measured by immunohistochemistry and TUNEL assays were used to evaluate the apoptosis of renal cells.Results The mean serum Cr and BUN levels of the IRI control mice were 190.6±12.4μmol/L and 56.7±8.3 mmol/L, TJ-2 and TJ-2+MR1 group significantly improved renal function after IRI, the mean serum Cr and BUN levels were 102.3±21.6μmol/L,51.2±8.9μmol/L and 47.2±9.5 mmol/L,20.4±5.2 mmol/L respectly(p< 0.05, p< 0.01 and p< 0.05, p< 0.05 vs IRI control group). Compared with the IRI control group, the survival rate of TJ-2 and TJ-2+MR1 group were greatly elevated from 12.5% to 57.1% and 100%. Consequently, the production of ROS were significantly reduced (5.16±1.24.17±0.81 vs 10.3±2.06, p=0.0205, p=0.0087), with the activation of NF-κB. Correspondently, Tissue damage caused by IRI was remarkedly reduced with less necrosis and hemorrhage as a result of TJ-2 and MR1 treatment. In addition, MPO expression was extremely diminished in treated mice compared with control kidneys, and apoptosis was also greatly decreased.Conclusion Blocking TLR/MYD88 signaling pathway can attenuate renal ischemia reperfusion injury, and Co-blocking TLR/MYD88 and CD154-CD40 patway showed stronger protective effect. The mainly mechanism mainly due to the inhibitory activation of NF-κB, lower procuction of ROS and the hypoexpression of inflammatory factor such as IL-1β, IL-6, TNF-αbut hypsi-expression of IL-10.