Deletion of IKK2 in hepatocytes does not sensitize these cells to TNF-induced apoptosis but protects from ischemia/reperfusion injury (original) (raw)
Generation of conditional Ikk2 and Nemo knockout mice.
Constitutive knockout mice for Ikk2 and Nemo die during embryogenesis from TNF-α–induced hepatic apoptosis (16–19). To study the function of these respective genes in the adult mouse liver, we generated conditional knockout mice. In the Ikk2 flox mouse, exons 6 and 7 are flanked by loxP sites (_Ikk2_f/f). Deletion of the respective exons introduces premature termination codons, producing an Ikk2 null allele (24). The _Ikk_2 flox mouse (24) (_Ikk_2f/f) was crossed with the Alfp-cre transgenic mouse (25), which shows hepatocyte-specific expression of Cre recombinase, to generate mice with _Ikk_2 ablated solely in hepatocytes (_Ikk_2Δhepa) but not in nonparenchymal liver cells. _Ikk_2Δhepa mice reached adulthood without any evidence of hepatic defects. In 8-week-old mice, efficiency of _Ikk_2 deletion in the liver was approximately 75% to 80% (Figure 1A, lane 5), reflecting the expected ratio of parenchymal to nonparenchymal liver cells. At the RNA (Figure 1B) and protein (Figure 1C) levels, almost no IKK2 signal was detectable. To verify specificity of the IKK2 knockout, we performed an immunohistochemical analysis of IKK2 expression. As seen in Figure 1D, IKK2 is expressed in all liver cells in Ikk2f/f mice, but its expression is restricted to nonparenchymal liver cells, e.g., bile duct cells, in Ikk2_Δ_hepa mice.
Conditional knockout of Ikk2 and Nemo in the mouse liver. (A) Deletion in the mouse liver is shown at the DNA level by Southern blot using genomic live DNA from mice with genetic status for the floxed (f) allele and positive (+) or negative (–) cre status as indicated. (B) Deletion in cre-positive floxed Ikk2 mice (Ikk2Δhepa) was verified in comparison to cre-negative control mice (Ikk2f/f) at the RNA level by semiquantitative RT-PCR using 1 μg of liver RNA and primers for IKK2 and GAPDH. (C) Western blot analysis with antibodies against IKK2 or α-tubulin (for loading control) from 100 μg of whole liver protein extracts from Ikk2Δhepa and Ikk2f/f mice. (D) Immunohistochemical staining of IKK2 on liver slides from Ikk2Δhepa and Ikk2f/f mice, showing that in Ikk2Δhepa mice, IKK2 expression is restricted to nonparenchymal liver cells such as bile duct cells (arrows). Original magnification, ×40. (E) For inducible knockout of NEMO, poly I/C was injected into both cre-positive (NemoΔ) mice and cre-negative control mice (Nemof/f). Efficiency of deletion in the mouse liver is shown at the RNA level by RT-PCR. (F) Western blot analysis with antibodies against NEMO and α-tubulin in NemoΔ and Nemof/f mice. (G) Inducible deletion of Ikk2. Deletion was induced by injection with poly I/C into Mx-cre–positive (Ikk2Δ) mice and Mx-cre–negative control mice (Ikk2f/f). Efficiency of the deletion in the mouse liver is shown at the RNA level by RT-PCR. (H) Western blot for IKK2 or α-tubulin from Ikk2Δ and Ikk2f/f mice.
To compare the effects of a lack of functional IKK2 with the effects of a Nemo knockout in the liver, we crossed the _Nemo_f/f mouse (26) with an Mx-cre mouse (27), in which the Cre-recombinase was induced by injection with poly-deoxyinosine-deoxycytodine (poly I/C) (_Nemo_Δ mice), which resulted in a knockout in parenchymal and nonparenchymal liver cells, and also other organs. Four days after induction, almost no NEMO could be detected in the livers at the RNA (Figure 1E) and protein (Figure 1F) levels. Whereas _Ikk2_Δhepa mice represent a developmental conditional knockout system, _Nemo_Δ mice are inducible conditional knockout mice. To exclude the possibility that differences in the phenotype between _Ikk_2Δhepa and _Nemo_Δ mice were caused by the induction with poly I/C, we also generated inducible _Ikk_2f/f_-Mx_-cre mice (_Ikk_2Δ), which, after induction, showed an efficient knockout of Ikk2 in the liver at the RNA (Figure 1G) and protein (Figure 1H) levels.
IKK2 in TNF-α–induced apoptosis in the liver
NEMO, but not IKK2, is essential for preventing TNF- α–induced apoptosis in the liver. TNF-α is a cytokine involved in mediating liver failure. It does not induce apoptosis unless cellular transcription is blocked. Previous experiments suggested that induction of antiapoptotic NF-κB target genes is critical to protecting hepatocytes from TNF-α–dependent apoptosis (20). Mouse embryonic fibroblast cells from Ikk2 and _Nemo_-deficient mice showed increased sensitivity towards TNF-α–induced apoptosis (16, 19). To test the function of IKK2 and NEMO in this context in the adult mouse liver, we injected TNF-α into _Ikk_2Δhepa mice, _Ikk2_Δ mice, and _Nemo_Δ mice or the respective _cre_-negative litter mates (_Ikk_2f/f and _Nemo_f/f). _Ikk_2Δhepa mice as well as the control _Ikk_2f/f mice did not show any increase in alanine aminotransferases (ALTs) as a marker of hepatocyte damage (Figure 2A) nor did they display a change in survival compared to control animals during the first 24 hours after TNF-α injection. Similarly, _Ikk_2Δ mice did not display any increase in ALT levels after TNF-α injection (Figure 2A). Likewise, after injection of bacterial LPS — which is a potent inducer of internal TNF-α — into _Ikk_2Δhepa and _Ikk_2Δ mice, no change in aminotransferases or survival compared to controls was noted (data not shown). When LPS injection was combined with administration of D-galactosamine (GalN), which sensitizes hepatocytes toward TNF/LPS-induced liver injury (20), no significant difference in the course of aminotransferases could be detected between these 2 groups (Figure 2A, top right panel). In contrast, while _Nemo_f/f mice showed no significant ALT increase after TNF-α, _Nemo_Δ mice displayed a massive increase in ALT levels and died between 2 and 3 hours after TNF-α stimulation (Figure 2A).
Nemo, but not Ikk2, deletion results in apoptotic cell death in the liver upon TNF-α stimulation. (A) Ikk2Δhepa, Ikk2Δ, or NemoΔ mice and their respective control litter mates (Ikk2f/f or Nemof/f) were injected intravenously with 6 μg/kg of recombinant TNF-α or intraperitoneally with LPS (100 μg/kg) and GalN (800 mg/kg) (right top panel). Liver injury was measured by determining ALT levels. Values are mean ± SD for independent animals (n = 6). Single asterisks indicate statistical significance with P < 0.01 versus Nemof/f control mice. All NemoΔ mice died between 2 and 3 hours after TNF-α stimulation from hepatic failure. (B) TUNEL staining of liver slides before and 2 hours after TNF-α stimulation showing clear positive staining in NemoΔ, but not in Ikk2Δhepa mice. Results are representative of those obtained in mice (n = 6). Original magnification, × 40. (C) Detection of caspase 3–like activity by an enzymatic, fluorometric assay from whole liver protein lysates at different time points after TNF-α stimulation in Ikk2Δhepa, NemoΔ, and control littermates. Values are mean ± SD for independent animals (n = 6). (D) ALT levels as markers for liver injury at different time points following ConA injection into Ikk2Δhepa and Ikk2f/f mice. Values are mean ± SD for independent animals (n = 6). (E) ALT levels following Fas-stimulating Jo-2 injection into Ikk2Δhepa and Ikk2f/f mice. Values are mean ± SD for independent animals (n = 6). U/l, units per liter; AFC, 7-amino-trifluoromethyl coumarin.
Histological analysis revealed no difference in the amount of TUNEL-positive cells between _Ikk_2f/f, _Ikk_2Δhepa, and _Nemo_f/f mice before and 2 hours after TNF-α administration whereas TUNEL-positive cells dramatically increased in _Nemo_Δ mice (Figure 2B). Moreover, almost no difference in caspase-3 activity was detected between _Ikk_2f/f and _Ikk_2Δhepa animals during the first 6 hours after TNF-α administration (Figure 2C). In _Nemo_f/f mice, caspase activity remained at baseline level whereas _Nemo_Δ mice responded with a strong increase in caspase-3–like activity 2 hours after TNF-α. Thus, absence of functional IKK2 in the adult mouse liver does not sensitize this organ to TNF-α–induced apoptosis, but lack of the NEMO subunit leads to massive hepatic apoptosis and liver failure upon TNF-α stimulation.
Concanavalin A (ConA) administration, which causes indiscriminate activation of T cells in the liver, results in acute hepatitis in rodents, which is accompanied by massive hepatocyte apoptosis (28). ConA stimulation in mice leads to TNF-α–dependent activation of NF-κB (29). Therefore, we tested to see whether IKK2 deficiency might influence susceptibility to ConA-induced liver damage. As shown in Figure 2D, _Ikk2_Δmice and _Ikk2_f/f mice did not differ in liver injury following ConA administration, which underlines the fact that IKK2-dependent signals are not essential for prevention of T cell–driven liver apoptosis. To evaluate a possible difference in Fas-mediated liver failure between these two groups, we injected the anti-Fas Jo-2 antibody into _Ikk2_Δhepa mice and _Ikk2_f/f mice (Figure 2E). Again, no significant difference in liver injury was seen between both groups, suggesting no major influence of the IKK2 subunit on Fas signaling in the liver.
TNF- α dependent NF-κB activation in the liver can occur independently of IKK2 but is dependent on functional NEMO. Given the dramatic difference in the sensitivity toward TNF-α–induced apoptosis, we wondered if this phenotype correlates with the activation of NF-κB in this model. We used nuclear protein extracts from whole mouse livers after TNF-α stimulation and performed gel-retardation assays. As shown in Figure 3A, _Ikk_2Δhepa mice exhibited no difference in the intensity and timing of NF-κB DNA binding compared with that shown in controls by gel-retardation assays, which was also the case in _Ikk_2Δ mice. In contrast, NF-κB activation was almost completely inhibited in the _Nemo_Δ mice. I-κBα degradation after TNF-α stimulation measured by Western blot analysis was clearly detected in both _Ikk_2Δ and _Ikk_2f/f mice 10 minutes after TNF-α whereas in _Nemo_Δ mice, it was almost completely blocked (Figure 3B).
NF-κB activation in the liver upon TNF-α stimulation is blocked in mice lacking Nemo but not Ikk2. (A) Liver nuclear protein extracts (5 μg) from the indicated mice and time points after TNF-α stimulation were subjected to a gel-retardation assay with an NF-κB consensus probe. In lanes 10/22/34 and 11/23/35, antibodies for the NF-κB subunits p50 or p65 were added as indicated as supershift control. The figure depicts results from 3 different assays. (B) I-κBα degradation in the different mouse groups was assessed by Western blot analysis with 50 μg of whole cell liver protein extracts before and 10 minutes after TNF-α stimulation using an antibody against I-κBα or α-tubulin (as loading control). (C) I-κBα phosphorylation was detemined after TNF-α stimulation by subjecting 50 μg of proteins to a Western blot analysis with an antibody detecting I-κBα phosphorylated at Ser32. (D) Evaluation of IKK activity. Proteins (300 μg) from mice stimulated with TNF-α were IP with a Nemo-antibody and subjected to a kinase assay using a truncated glutathione-S-transferase–I-κBα(1–54) protein as substrate. (E) Gel-retardation assay with an NF-κB consensus site using 5 μg of nuclear protein extracts from primary hepatocyte cultures. Results are representative of those obtained in mice (n = 4). (F) JNK activity was measured by Western blot using protein from mice stimulated with TNF, LPS, and ConA as indicated. Antibodies detecting c-Jun phosphorylated at Ser63 or JNK phosphorylated at Thr183/Tyr185 as well as nonphosphorylated JNK1 and α-tubulin as loading control were used.
We next examined the kinase activity of the IKK complex in _Ikk_2Δhepa and _Ikk_2f/f mice using a truncated I-κB protein as substrate (Figure 3C). In line with previous in vitro results (5, 11–13, 30), kinase activity in mice lacking IKK2 was weaker than in wild-type mice. Nevertheless, when we analyzed the phosphorylation status of internal I-κBα, no difference between the 2 groups could be detected (Figure 3D). These results were confirmed in hepatocyte cultures to exclude the possibility that the observed NF-κB activation in livers from _Ikk2_-deficient mice was occurring solely in cells that might have incompletely knocked out the respective gene. Primary hepatocytes were cultured from _Ikk_2Δ and _Nemo_Δ mice as well as the _cre_-negative control animals, and NF-κB activation was measured by gel-retardation assay (Figure 3E). Again, no difference in NF-κB activation was noted between hepatocytes from _Ikk2_Δ and _Ikk2_f/f control mice whereas NF-κB was blocked in primary hepatocytes from _Nemo_Δ mice. Therefore, although IKK activity is attenuated in the absence of IKK2, it is sufficient to fully activate NF-κB in response to TNF-α whereas this is not the case in the absence of NEMO. Moreover, the ability of the IKK complex to activate NF-κB appears to correlate strictly with its antiapoptotic role in TNF-α signaling.
JNK is activated upon cellular stress, including TNF-α–signaling (20). We tested to see whether JNK activation is preserved in Ikk2 knockout mice. As shown in Figure 3F, both p46- and p54-JNK phosphorylation, which are essential steps for JNK activation, and c-jun phosphorylation were almost equally induced after TNF stimulation in both groups. The same result was seen after LPS and ConA administration with a slightly enhanced signal in _Ikk2_Δhepa mice. Therefore, crosstalk between the NF-κB and JNK pathway does not strictly depend on IKK2.
A dominant-negative IKK2 mutant can block NF-κB activation in Ikk2Δ hepa mice. Our finding that NF-κB can be activated independently of IKK2 is surprising with regard to previous results in hepatocyte cultures (31). In this study, an adenoviral vector expressing a dominant-negative form of IKK2 with a mutation in the kinase domain but not a dominant-negative IKK1 adenovirus completely blocked TNF-α–dependent NF-κB activation, which suggests that this process is mediated primarily via IKK2. To examine this more closely, we first characterized the binding of the different IKK subunits in _Ikk2_Δhepa and _Ikk2_f/f mice. Protein extracts IP with a NEMO antibody showed that IKK1 could still be detected in the complex. As expected, no IKK2 was detectable in the IKK complex in _Ikk_2Δ mice (Figure 4A). IP with an IKK1 antibody showed equal amounts of IKK1 in the IKK complex in both groups (Figure 4B).
A dominant-negative IKK2 form can block NF-κB activation after TNF-α stimulation. (A) IP in 300 μg of whole cell liver protein extracts from Ikk2f/f and Ikk2Δhepa mice was performed with a polyclonal antibody against NEMO, followed by Western blot analysis with a monoclonal antibody against IKK1 or IKK2 as indicated. Association between NEMO and IKK1 was assessed before as well as 30 and 60 minutes after stimulation with TNF-α. (B) Western blot of protein extracts that were IP with anti-IKK1 and blotted with a monoclonal antibody against IKK1 or IKK2 as indicated. (C) Primary hepatocyte cultures were prepared from livers of Ikk2f/f and Ikk2Δhepa mice and either treated with PBS alone, infected with LacZAdv, or infected with dnIKK2Adv in a viral dose of 20 PFUs for 12 hours. Protein extracts (200μg) were subjected to IP with a NEMO antibody, followed by Western blot with an IKK2 antibody. (D) Primary hepatocytes from Ikk2f/f and Ikk2Δhepa mice were infected with LacZAdv or dnIKK2Adv in a viral dose of 20 PFUs for 12 hours and treated with TNF-α (30 ng/ml). Protein extracts were subjected to an NF-κB gel-retardation assay. (E) Protein extracts (100 μg) from primary hepatocytes infected with LacZAdv or dnIKK2Adv were IP with anti-Nemo and blotted with a monoclonal antibody against IKK1.
To test whether expression of dominant-negative IKK2 would eliminate the remaining NF-κB activation in hepatocytes lacking IKK2, we infected hepatocyte cultures from _Ikk_2Δ and _Ikk_2f/f mice with a dominant-negative IKK2 adenovirus (dnIKK2Adv) with a mutation in the kinase domain at position 44 (31). As a control, we used an adenovirus expressing the LacZ protein (LacZAdv). When dnIKK2Adv was added to cells from _Ikk_2f/f and _Ikk_2Δ mice, a strong IKK2 signal was seen in both groups after co-IP with an anti-NEMO antibody. This signal was detected slightly above the wild-type signal due to a flag tag (Figure 4C, Lanes 3 and 6). Thus, the externally expressed IKK2dn-mutant form was integrated into the IKK complex. We then stimulated these hepatocyte cultures with recombinant TNF-α and measured NF-κB activation in a gel-retardation assay. NF-κB DNA binding was induced 60 minutes after TNF-α stimulation in both groups when cells were treated with the control virus (Figure 4D). In contrast, NF-κB activation was completely blocked in cells from _Ikk_2f/f and _Ikk_2Δ mice infected with dnIKK2Adv. To test whether IKK1 is still recruited to the IKK complex in the presence of the dominant-negative IKK2 virus, we performed another Nemo/IKK1 co-IP on wild-type extracts from hepatocytes infected with the control virus and dnIKK2Adv. As clearly demonstrated in Figure 4E, no IKK1 signal is detected when the mutant IKK2 protein is overexpressed. These experiments provide a mechanistic explanation for the different phenotypes seen with the dnIKK2Adv and the Ikk2 knockout animals. The IKK1/Nemo complex is apparently sufficient to fully activate NF-κB in the absence of IKK2. In contrast, overexpression of the mutant IKK2 form prevents recruitment of IKK1 to the protein complex, which results in a nonfunctional IKK complex.
IKK2 in hepatic I/R injury
IKK2-dependent signaling in hepatocytes mediates liver damage in an I/R model. Previously we examined the role of IKK2 and NF-κB in the TNF-α pathway. NF-κB is also activated in other experimental models of liver injury. We evaluated the relevance of IKK2-dependent signaling in the liver after I/R. The degree of hepatic injury after 60 minutes I/R was measured as an increase in aspartate aminotransferases (ASTs) and ALT (Figure 5A). Unexpectedly, _Ikk_2Δhepa animals showed significantly lower levels of both AST and ALT at 3 and 6 hours after I/R than _Ikk_2f/f mice. On histological analysis, _Ikk_2f/f mice clearly displayed necrotic cell damage 6 hours and 24 hours after reperfusion, which was significantly reduced at both time points in _Ikk_2Δhepa animals (Figure 5B). In both groups, no signs of apoptosis — measured by TUNEL test and caspase-3–like activation — could be detected (data not shown).
Ikk2 deletion in hepatocytes protects against liver injury and inflammation after hepatic I/R. (A) Ikk2Δhepa and Ikk2f/f control mice underwent a procedure of partial hepatic ischemia lasting for 60 minutes, which was followed by reperfusion. Serum AST and ALT levels were measured at the indicated time points before the procedure and after reperfusion as markers for liver injury. Values are mean ± SD for independent animals (n = 8). Asterisks indicate statistical significance: *P < 0.02 versus Ikk2f/f control mice; **P < 0.05 versus Ikk2f/f control mice. (B) H&E staining of liver slides from Ikk2f/f and Ikk2Δhepa mice at 6 hours and 24 hours after reperfusion. N, necrotic area (arrows indicate margins of necrotic areas; P, portal vein; C, central vein. Results are representative of those obtained in 8 mice. Original magnification, ×20. The area of necrotic parenchymal surface was measured and quantified (right panel). Values are mean ± SD for independent animals (n = 4). Hatch mark indicates statistical significance: P < 0.01 versus Ikk2f/f control mice. (C) Quantification of PMN leukocytes per high power field (×40) at different time points after reperfusion. Values are mean ± SD for independent animals (n = 4). Double hatch marks indicate statistical significance: P < 0.001 versus Ikk2f/f control mice.
Inflammation and infiltration with polymorphonuclear (PMN) leukocytes is another process that occurs during liver injury after I/R (32). We found that infiltration with PMN leukocytes after reperfusion was significantly reduced in _Ikk_2Δhepa mice compared with _Ikk_2f/f control mice (Figure 5C). Therefore, IKK2-dependent signaling does play a role in hepatic I/R injury. In fact, in contrast to other models of liver injury where IKK-dependent signaling plays a protective role, IKK2 mediates necrosis and inflammation after I/R, and hepatocyte-specific knockout of the _Ikk_2 gene is protective in this context.
Activation of NF-κB DNA binding and induction of NF-κB target genes after I/R is dependent on functional IKK2. Recent studies have suggested that during I/R injury, NF-κB is activated independently of the IKK complex by alternative I-κBα tyrosine phosphorylation by Src family kinases (21, 33). _Ikk2_Δhepa mice are protected from liver damage after I/R compared with wild-type mice. We evaluated how the observed phenotype correlated with NF-κB activation in the liver. In _Ikk_2f/f mice, NF-κB DNA-binding was detected already at 30 minutes and peaked at 60 minutes after reperfusion (Figure 6A). In contrast, NF-κB activation was almost completely blocked in _Ikk_2Δhepa mice.
Activation of NF-κB and induction of NF-κB target genes after I/R is dependent on functional IKK2. (A) Nuclear protein extracts (5 μg) from livers of Ikk2f/f and Ikk2Δhepa mice at different time points after I/R were subjected to a gel-retardation assay with an NF-κB consensus oligonucleotide. In lanes 9 and 10, antibodies for the NF-κB subunits p50 or p65 were added as supershift control. (B) Immunohistochemical staining for iNOS at 6 hours after I/R in Ikk2f/f and Ikk2Δhepa mice. Original magnification, ×40. Results are representative of those obtained in mice (n = 4). (C) Immunohistochemical staining for TNF-α at 6 hours after I/R. Original magnification, ×40. The number of TNF-α–positive cells was quantified (right panel). Values are mean ± SD for independent animals (n = 4). The asterisk indicates statistical significance: P < 0.01 versus Ikk2f/f control mice. (D) Kupffer cells were isolated from livers of Ikk2f/f and Ikk2Δhepa mice and stained with an antibody against the F4/80 antigen, which is a macrophage-specific marker, to verify the specificity of the preparation procedure. Equal expression of IKK2 was verified by staining with a polyclonal antibody against IKK2. Cells were stimulated with 1 μg/ml LPS for 1 hour and TNF-α expression examined by immunohistochemical staining to prove that Kupffer cells in the livers of Ikk2Δhepa mice are functionally active. Original magnification, ×400. For negative control, no primary antibody was added.
iNOS is a known NF-κB target gene that regulates NO metabolism and plays a role in liver injury after I/R (2, 32). In contrast to _Ikk_2f/f mice, no iNOS-positive cells were detected in Ikk 2Δhepa mice 6 hours after reperfusion (Figure 6B). TNF-α, another NF-κB target, has been shown to be a key cytokine in I/R injury (34). On histological analysis, we found that TNF-α expression in liver cells was significantly reduced in _Ikk_2Δhepa mice (Figure 6C).
Kupffer cells play a central role in mediating liver injury after I/R. To ensure that these cells are functionally active in _Ikk2_Δhepa mice, Kupffer cells were isolated from _Ikk_2f/f and _Ikk_2Δhepa mice (Figure 6D). Cells were stained with an antibody against the F4/80 antigen, which is a macrophage-specific marker, to verify the specificity of the preparation procedure. As expected, IKK2 expression was not abolished in _Ikk2_Δhepa mice, underlining the restriction of the knockout to hepatocytes. Moreover, TNF-α expression in response to LPS stimulation was comparable in both _Ikk_2Δhepa and control mice, indicating that the observed phenotype was not caused by a possible Ikk2 knockout in Kupffer cells.
Pharmacological inhibition of IKK2 as a new therapeutic approach for prevention of liver injury after I/R. Hepatic I/R injury is a major problem and determines to a high degree the morbidity and mortality associated with liver surgery under vascular exclusion (Pringle maneuver), transplantation surgery, or hemorrhagic shock. Therefore, it is important to discover new ways for pharmacological intervention to improve the clinical outcome in these settings. AS602868 is a new, reversible, and ATP-competitive inhibitor of IKK2 (35). Wild-type mice received an oral administration of AS602868 20 hours and 2 hours before intervention whereas in the control group, mice received only the vehicle substance. Correlating with the results in _Ikk_2Δhepa mice, pharmacological inhibition of IKK2 in the absence of liver injury or after pure TNF-α stimulation had no effect on survival or the course of aminotransferases, as depicted in Figure 7A for ALT levels. Moreover, NF-κB activation after pharmacological inhibition of IKK2 was not impaired after TNF-α stimulation, again supporting the results found in the conditional Ikk2 knockout mice (Figure 7B). In the I/R model, mice treated with the IKK2 inhibitor showed a dramatic decrease in AST and ALT levels compared with the control group 6 hours after reperfusion (Figure 7C), which was even more pronounced than seen before in the _Ikk_2Δhepa animals. Histological analysis revealed a clear attenuation of necrosis and neutrophilic infiltration after AS602868 treatment (Figure 7, D and E). These results indicate a potential benefit of pharmacological IKK2 inhibition in the prevention of liver injury after I/R.
The pharmacological IKK2 inhibitor AS602868 protects mice from liver injury due to hepatic I/R. (A) C57BL6 mice received an oral administration of 150 μl of the IKK2 inhibitor AS602868 (10 μg/g body weight) or the vehicle substance (control) at 20 hours and 2 hours before stimulation with 6 μg/kg recombinant TNF-α. Evaluation of serum ALT levels at the indicated time points after TNF-α stimulation showing no significant difference between treated and untreated animals. (B) NF-κB EMSA using 5 μg of nuclear protein extracts from animals treated with AS602868 or control. (C) Serum AST and ALT levels were measured at the indicated time points after reperfusion in mice that were pretreated with AS602868 or vehicle substance (control) and had undergone partial I/R. Values are mean ± SD for independent animals (n = 4). The asterisk indicates statistical significance with P < 0.05 versus control mice. (D) H&E staining of liver slides at 6 hours after reperfusion from control mice and mice treated with AS602868. Original magnification, ×20. The area of necrotic parenchymal surface was measured and quantified (right panel). Values are mean ± SD for independent animals (n = 4). The double asterisk indicates statistical significance with P < 0.01 versus control mice. (E) Quantification of PMN leukocytes per high power field (×40) at 6 hours after reperfusion. Values are mean ± SD for independent animals (n = 4). The hatch mark indicates statistical significance with P < 0.001 versus control mice.