Interleukin-6 mediates angiotensinogen gene expression during liver regeneration

Abstract

Background: Angiotensinogen is the precursor of angiotensin II, which is associated with ischemia-reperfusion injury. Angiotensin II reduces liver regeneration after hepatectomy and causes dysfunction and failure of reduced-size liver transplants. However, the regulation of angiotensinogen during liver regeneration is still unclear.

Aims: To investigate the regulation of angiotensinogen during liver regeneration for preventing angiotensin II-related ischemia-reperfusion injury during liver regeneration.

Methods: A mouse in vitro partial hepatectomy animal model was used to evaluate the expression of interleukin-6 (IL-6) and angiotensinogen during liver regeneration. Serum IL-6 and angiotensinogen were detected by enzyme immunoassay (EIA). Angiotensinogen mRNA was detected by RT-PCR. Tissue levels of angiotensinogen protein were detected by Western blot analysis. Primary cultures of mouse hepatocytes were used to investigate IL-6-induced angiotensinogen. Chemical inhibitors were used to perturb signal transduction pathways. Synthetic double-stranded oligodeoxynucleotides (ODNs) were used as 'decoy' cis-elements to investigate transcription. Ki 67 staining and quantification were used to verify liver regeneration.

Results: In the in vivo model, the levels of serum IL-6 and angiotensinogen correlated. In the in vitro model, IL-6 transcriptionally regulated angiotensinogen expression. Additionally, IL-6 mediated angiotensinogen expression through the Janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3) and JAK/p38 signaling. Decoy ODN analyses revealed that STAT3 and nuclear factor-kB (NF-kB) also played critical roles in the transcriptional regulation of angiotensinogen by IL-6. IL-6-mediated signaling, JAK2, STAT3 and p38 inhibitors reduced angiotensinogen expression in the partially hepatectomized mice.

Conclusion: During liver regeneration, IL-6-enhanced angiotensinogen expression is dependent on the JAK/STAT3 and JAK/p38/NF-kB signaling pathways. Interruption of the molecular mechanisms of angiotensinogen regulation may be applied as the basis of therapeutic strategies for preventing angiotensin II-related ischemia-reperfusion injury during liver regeneration.

Figure 1. Serum IL-6 and angiotensinogen levels, angiotenisogen mRNA and protein expression in remnant livers after 70% partial hepatectomy in mice.

(A) Serum angiotensinogen levels detected by enzyme immunoassay (n = 5/group). (B) Angiotensinogen mRNA detected by reverse transcription-polymerase chain reaction quantified by calculating the ratios of angiotensinogen/GAPDH. (C) Angiotensinogen protein expression detected by Western blot and quantified by calculating the ratios of angiotensinogen/ß-actin. The ratio in lane 1 is defined as 1. Comparison is between the time 0 group and specified time periods. *P<0.05, n = 5.

Figure 2. IL-6 induced angiotensinogen expression in primary cultures of mouse hepatocytes.

(A) Time course of IL-6-induced (10 ng/ml) angiotensinogen protein expression as detected by enzyme immunoassay (n = 3/group). (B) Dose response of IL-6-induced angiotensinogen protein expression after 24 hours of culture, as detected by enzyme immunoassay. Comparisons are between the indicated groups. *P<0.05, n = 3.

Figure 3. Signal transduction pathways involved in IL-6-induced angiotensinogen expression in primary cultures of mouse hepatocytes.

(A) Inhibition effects of chemical inhibitors 1 to 6 on IL-6-activated signaling mediators detected by Western blotting and quantified by calculating the ratios of phosphorylated/non-phosphorylated protein forms. The ratio in lane 1 is defined as 1. Comparison is between lanes 2 and 3 in each group. *P<0.05, n = 3. (B) The effects of different chemical inhibitors on IL-6-induced angiotensinogen protein expression as detected by enzyme immunoassay. Comparison is between the indicated groups. *P<0.05, n = 3.

Figure 4. Transcription factors and their upstream signal mediators involved in IL-6-induced angiotensinogen expression in primary cultures of mouse hepatocytes.

(A) Inhibition effects of different transcription factor oligodeoxynucleotide (ODN) decoys on IL-6-induced angiotensinogen protein expression as detected by enzyme immunoassay. Comparisons are between the indicated groups. *P<0.05, n = 3. (B) The effects of S31–201 and SB203580 on IL-6-induced STAT3 or NF-kB p65 subunit nuclear translocation were determined by Western blotting and quantified by calculating the ratio of n (nuclear)/c (cytosolic) proteins. The ratio in lane 1 is defined as 1. Comparisons are between lanes 2 and 3 or 4 in each group. *P<0.05, n = 3. (C) Effects of AG490 on IL-6-induced phospho-p38 (p-p38) and phospho-STAT3 (p-STAT3) were determined by Western blotting and quantified by calculating the ratio of phosphorylated/non-phosphorylated proteins. The ratio in lane 1 is defined as 1. Comparisons are between lanes 2 and 3 in each group. *P<0.05, n = 3.

Figure 5. Effect of IL-6-related signaling inhibition on angiotensinogen protein expression and liver regeneration in remnant livers after 70% partial hepatectomy in mice.

Mice were pretreated with chemical inhibitors of JAK2 (AG490, 10 mg/kg subcutaneously), p38 (intraperitoneal SB203580, 15 mg/kg), and STAT3 (intraperitoneal 5,15-DPP, 15 mg/kg) for 4 hours prior to partial hepatectomy. (A) Serum angiotensinogen levels of mice pretreated with different chemical inhibitors (AG490, SB203580, and 5,15-DPP) as detected by enzyme immunoassay. (B) Changes in the ratio of remnant to original liver weight after 70% partial hepatectomy. Remnant liver weight was estimated retrospectively from the excised liver weight after 70% PH. Data are presented as mean ± S.D., and comparisons were made between groups as indicated. *P<0.05. (C) Ki-67 staining of regenerated liver tissue sections of the indicated group. Magnification, 400x. (D) Quantification of Ki-67 staining. Data presented here are the quantification of Ki-67-positive nuclei per high-power field. Data are presented as mean percentage of positive nuclei ± S.D., and comparisons were made between groups as indicated. *P<0.05.