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. 2012 Nov;42(11):1131-40.
doi: 10.1111/j.1872-034X.2012.01030.x. Epub 2012 May 14.

Effect of specific activation of γ-aminobutyric acid receptor in vivo on oxidative stress-induced damage after extended hepatectomy

Lindsay B Gardner  1 , Tomohide Hori, Feng Chen, Ann-Marie T Baine, Toshiyuki Hata, Shinji Uemoto, Justin H Nguyen
Affiliations

Effect of specific activation of γ-aminobutyric acid receptor in vivo on oxidative stress-induced damage after extended hepatectomy

Lindsay B Gardner et al. Hepatol Res. 2012 Nov.

Abstract

Aim: γ-Aminobutyric acid (GABA) is a multifunctional molecule with various physiological effects throughout the body. The regulation of GABA receptor (GABAR) plays a key role in reducing the damage mediated by oxidative stress (OS). Extended hepatectomy causes fatal OS-induced injury in the liver remnant. We aimed to investigate the effect of a GABAR agonist in extended hepatectomy.

Methods: Saline or a GABAR agonist (43.56 nmol/g bodyweight of muscimol) was administrated intravenously at 4 h preoperatively. C57BL/6 mice were divided into three groups: laparotomy only, 90% hepatectomy with saline and 90% hepatectomy with a GABAR agonist. Liver samples were obtained at 6 h after surgery.

Results: Survival curves were prolonged by the GABAR agonist. Histopathological findings and biochemical profiles showed that the GABAR agonist reduced liver damage. Immunohistological assessment demonstrated that the GABAR agonist prevented apoptotic induction. As shown by 4-hydroxynonenal, which reflects OS-induced damage, 90% hepatectomy caused OS and the GABAR agonist reduced OS. We measured ataxia-telangiectasia mutated kinase (ATM), H2AX, Akt and free radical scavenging enzymes because they may be affected by GABAR regulation, and found that Akt was greatly decreased after 90% hepatectomy, but it recovered with the GABAR agonist.

Conclusion: GABAR is activated by a specific agonist in the liver in vivo. This activation reduces OS-mediated damage after extended hepatectomy in vivo, and the mechanism via an Akt-dependent pathway may be a key.

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Conflict of interest statement

All authors have no financial conflict of interest.

Figures

Figure 1
Figure 1. Immunohistological assessment by TUNEL and caspase-3 staining
(A) Laparotomy with saline (TUNEL, x100). (B) 90% hepatectomy with saline (TUNEL, x100). TUNEL-positive nuclei were stained brown and negative nuclei were counterstained light blue. (C) 90% hepatectomy with GABAR agonist (TUNEL, x100). (D) Changes in the ratio of TUNEL-positive nuclei (†p <0.05). These results suggested that the GABAR agonist decreased the ratio of TUNEL-positive cells. (E) Laparotomy with saline (caspase-3, x100). (F) 90% hepatectomy with saline (caspase-3, x100). Caspase-3-positive nuclei were stained brown and negative nuclei were counterstained blue. (G) 90% hepatectomy with GABAR agonist (caspase-3, x100). (H) Changes in the ratio of caspase-3-positive nuclei (†p <0.05). These results indicated that the GABAR agonist decreased the ratio of caspase-3-positive cells. Abbreviations: cysteine aspartic acid protease, caspase-3; γ-aminobutyric acid receptor, GABAR; hepatic vein, HV; not significant (p ≥ 0.05), NS; portal vein, PV; terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling. TUNEL.
Figure 1
Figure 1. Immunohistological assessment by TUNEL and caspase-3 staining
(A) Laparotomy with saline (TUNEL, x100). (B) 90% hepatectomy with saline (TUNEL, x100). TUNEL-positive nuclei were stained brown and negative nuclei were counterstained light blue. (C) 90% hepatectomy with GABAR agonist (TUNEL, x100). (D) Changes in the ratio of TUNEL-positive nuclei (†p <0.05). These results suggested that the GABAR agonist decreased the ratio of TUNEL-positive cells. (E) Laparotomy with saline (caspase-3, x100). (F) 90% hepatectomy with saline (caspase-3, x100). Caspase-3-positive nuclei were stained brown and negative nuclei were counterstained blue. (G) 90% hepatectomy with GABAR agonist (caspase-3, x100). (H) Changes in the ratio of caspase-3-positive nuclei (†p <0.05). These results indicated that the GABAR agonist decreased the ratio of caspase-3-positive cells. Abbreviations: cysteine aspartic acid protease, caspase-3; γ-aminobutyric acid receptor, GABAR; hepatic vein, HV; not significant (p ≥ 0.05), NS; portal vein, PV; terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling. TUNEL.
Figure 1
Figure 1. Immunohistological assessment by TUNEL and caspase-3 staining
(A) Laparotomy with saline (TUNEL, x100). (B) 90% hepatectomy with saline (TUNEL, x100). TUNEL-positive nuclei were stained brown and negative nuclei were counterstained light blue. (C) 90% hepatectomy with GABAR agonist (TUNEL, x100). (D) Changes in the ratio of TUNEL-positive nuclei (†p <0.05). These results suggested that the GABAR agonist decreased the ratio of TUNEL-positive cells. (E) Laparotomy with saline (caspase-3, x100). (F) 90% hepatectomy with saline (caspase-3, x100). Caspase-3-positive nuclei were stained brown and negative nuclei were counterstained blue. (G) 90% hepatectomy with GABAR agonist (caspase-3, x100). (H) Changes in the ratio of caspase-3-positive nuclei (†p <0.05). These results indicated that the GABAR agonist decreased the ratio of caspase-3-positive cells. Abbreviations: cysteine aspartic acid protease, caspase-3; γ-aminobutyric acid receptor, GABAR; hepatic vein, HV; not significant (p ≥ 0.05), NS; portal vein, PV; terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling. TUNEL.
Figure 1
Figure 1. Immunohistological assessment by TUNEL and caspase-3 staining
(A) Laparotomy with saline (TUNEL, x100). (B) 90% hepatectomy with saline (TUNEL, x100). TUNEL-positive nuclei were stained brown and negative nuclei were counterstained light blue. (C) 90% hepatectomy with GABAR agonist (TUNEL, x100). (D) Changes in the ratio of TUNEL-positive nuclei (†p <0.05). These results suggested that the GABAR agonist decreased the ratio of TUNEL-positive cells. (E) Laparotomy with saline (caspase-3, x100). (F) 90% hepatectomy with saline (caspase-3, x100). Caspase-3-positive nuclei were stained brown and negative nuclei were counterstained blue. (G) 90% hepatectomy with GABAR agonist (caspase-3, x100). (H) Changes in the ratio of caspase-3-positive nuclei (†p <0.05). These results indicated that the GABAR agonist decreased the ratio of caspase-3-positive cells. Abbreviations: cysteine aspartic acid protease, caspase-3; γ-aminobutyric acid receptor, GABAR; hepatic vein, HV; not significant (p ≥ 0.05), NS; portal vein, PV; terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling. TUNEL.
Figure 1
Figure 1. Immunohistological assessment by TUNEL and caspase-3 staining
(A) Laparotomy with saline (TUNEL, x100). (B) 90% hepatectomy with saline (TUNEL, x100). TUNEL-positive nuclei were stained brown and negative nuclei were counterstained light blue. (C) 90% hepatectomy with GABAR agonist (TUNEL, x100). (D) Changes in the ratio of TUNEL-positive nuclei (†p <0.05). These results suggested that the GABAR agonist decreased the ratio of TUNEL-positive cells. (E) Laparotomy with saline (caspase-3, x100). (F) 90% hepatectomy with saline (caspase-3, x100). Caspase-3-positive nuclei were stained brown and negative nuclei were counterstained blue. (G) 90% hepatectomy with GABAR agonist (caspase-3, x100). (H) Changes in the ratio of caspase-3-positive nuclei (†p <0.05). These results indicated that the GABAR agonist decreased the ratio of caspase-3-positive cells. Abbreviations: cysteine aspartic acid protease, caspase-3; γ-aminobutyric acid receptor, GABAR; hepatic vein, HV; not significant (p ≥ 0.05), NS; portal vein, PV; terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling. TUNEL.
Figure 1
Figure 1. Immunohistological assessment by TUNEL and caspase-3 staining
(A) Laparotomy with saline (TUNEL, x100). (B) 90% hepatectomy with saline (TUNEL, x100). TUNEL-positive nuclei were stained brown and negative nuclei were counterstained light blue. (C) 90% hepatectomy with GABAR agonist (TUNEL, x100). (D) Changes in the ratio of TUNEL-positive nuclei (†p <0.05). These results suggested that the GABAR agonist decreased the ratio of TUNEL-positive cells. (E) Laparotomy with saline (caspase-3, x100). (F) 90% hepatectomy with saline (caspase-3, x100). Caspase-3-positive nuclei were stained brown and negative nuclei were counterstained blue. (G) 90% hepatectomy with GABAR agonist (caspase-3, x100). (H) Changes in the ratio of caspase-3-positive nuclei (†p <0.05). These results indicated that the GABAR agonist decreased the ratio of caspase-3-positive cells. Abbreviations: cysteine aspartic acid protease, caspase-3; γ-aminobutyric acid receptor, GABAR; hepatic vein, HV; not significant (p ≥ 0.05), NS; portal vein, PV; terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling. TUNEL.
Figure 1
Figure 1. Immunohistological assessment by TUNEL and caspase-3 staining
(A) Laparotomy with saline (TUNEL, x100). (B) 90% hepatectomy with saline (TUNEL, x100). TUNEL-positive nuclei were stained brown and negative nuclei were counterstained light blue. (C) 90% hepatectomy with GABAR agonist (TUNEL, x100). (D) Changes in the ratio of TUNEL-positive nuclei (†p <0.05). These results suggested that the GABAR agonist decreased the ratio of TUNEL-positive cells. (E) Laparotomy with saline (caspase-3, x100). (F) 90% hepatectomy with saline (caspase-3, x100). Caspase-3-positive nuclei were stained brown and negative nuclei were counterstained blue. (G) 90% hepatectomy with GABAR agonist (caspase-3, x100). (H) Changes in the ratio of caspase-3-positive nuclei (†p <0.05). These results indicated that the GABAR agonist decreased the ratio of caspase-3-positive cells. Abbreviations: cysteine aspartic acid protease, caspase-3; γ-aminobutyric acid receptor, GABAR; hepatic vein, HV; not significant (p ≥ 0.05), NS; portal vein, PV; terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling. TUNEL.
Figure 1
Figure 1. Immunohistological assessment by TUNEL and caspase-3 staining
(A) Laparotomy with saline (TUNEL, x100). (B) 90% hepatectomy with saline (TUNEL, x100). TUNEL-positive nuclei were stained brown and negative nuclei were counterstained light blue. (C) 90% hepatectomy with GABAR agonist (TUNEL, x100). (D) Changes in the ratio of TUNEL-positive nuclei (†p <0.05). These results suggested that the GABAR agonist decreased the ratio of TUNEL-positive cells. (E) Laparotomy with saline (caspase-3, x100). (F) 90% hepatectomy with saline (caspase-3, x100). Caspase-3-positive nuclei were stained brown and negative nuclei were counterstained blue. (G) 90% hepatectomy with GABAR agonist (caspase-3, x100). (H) Changes in the ratio of caspase-3-positive nuclei (†p <0.05). These results indicated that the GABAR agonist decreased the ratio of caspase-3-positive cells. Abbreviations: cysteine aspartic acid protease, caspase-3; γ-aminobutyric acid receptor, GABAR; hepatic vein, HV; not significant (p ≥ 0.05), NS; portal vein, PV; terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling. TUNEL.
Figure 2
Figure 2. Western blot analysis of HNE
(A) Changes in the intensity of 4-HNE and GAPDH. (B) Changes in quantitative HNE/GAPDH (†p <0.05). Ninety percent hepatectomy resulted in lipoperoxidation and the GABAR agonist reduced lipoperoxidation. Abbreviations: 4-hydroxynonenal, 4-HNE; γ-aminobutyric acid receptor, GABAR; glyceraldehyde-3-phosphate dehydrogenase, GAPDH.
Figure 2
Figure 2. Western blot analysis of HNE
(A) Changes in the intensity of 4-HNE and GAPDH. (B) Changes in quantitative HNE/GAPDH (†p <0.05). Ninety percent hepatectomy resulted in lipoperoxidation and the GABAR agonist reduced lipoperoxidation. Abbreviations: 4-hydroxynonenal, 4-HNE; γ-aminobutyric acid receptor, GABAR; glyceraldehyde-3-phosphate dehydrogenase, GAPDH.
Figure 3
Figure 3. Western blot analyses of ATM and H2AX
(A) Changes in the intensity of ATM, H2AX, Akt and GAPDH. (B) Changes in quantitative ATM/GAPDH (†p <0.05). Notably, there were no differences in ATM between 90% hepatectomy with saline and that with the GABAR agonist. (C) Changes in quantitative H2AX/GAPDH (†p <0.05). Notably, there were no differences in H2AX between 90% hepatectomy with saline and that with the GABAR agonist. Abbreviations: ataxia-telangiectasia mutated kinase, ATM; γ-aminobutyric acid receptor, GABAR; glyceraldehyde-3-phosphate dehydrogenase, GAPDH; not significant (p ≥ 0.05), NS.
Figure 3
Figure 3. Western blot analyses of ATM and H2AX
(A) Changes in the intensity of ATM, H2AX, Akt and GAPDH. (B) Changes in quantitative ATM/GAPDH (†p <0.05). Notably, there were no differences in ATM between 90% hepatectomy with saline and that with the GABAR agonist. (C) Changes in quantitative H2AX/GAPDH (†p <0.05). Notably, there were no differences in H2AX between 90% hepatectomy with saline and that with the GABAR agonist. Abbreviations: ataxia-telangiectasia mutated kinase, ATM; γ-aminobutyric acid receptor, GABAR; glyceraldehyde-3-phosphate dehydrogenase, GAPDH; not significant (p ≥ 0.05), NS.
Figure 3
Figure 3. Western blot analyses of ATM and H2AX
(A) Changes in the intensity of ATM, H2AX, Akt and GAPDH. (B) Changes in quantitative ATM/GAPDH (†p <0.05). Notably, there were no differences in ATM between 90% hepatectomy with saline and that with the GABAR agonist. (C) Changes in quantitative H2AX/GAPDH (†p <0.05). Notably, there were no differences in H2AX between 90% hepatectomy with saline and that with the GABAR agonist. Abbreviations: ataxia-telangiectasia mutated kinase, ATM; γ-aminobutyric acid receptor, GABAR; glyceraldehyde-3-phosphate dehydrogenase, GAPDH; not significant (p ≥ 0.05), NS.
Figure 4
Figure 4. Western blot analysis of Akt
(A) Changes in the intensity of Akt and GAPDH. (B) Changes in quantitative Akt/GAPDH (†p <0.05). Notably, there was a considerable difference in Akt between 90% hepatectomy with saline and that with the GABAR agonist. Abbreviations: γ-aminobutyric acid receptor, GABAR; glyceraldehyde-3-phosphate dehydrogenase, GAPDH.
Figure 4
Figure 4. Western blot analysis of Akt
(A) Changes in the intensity of Akt and GAPDH. (B) Changes in quantitative Akt/GAPDH (†p <0.05). Notably, there was a considerable difference in Akt between 90% hepatectomy with saline and that with the GABAR agonist. Abbreviations: γ-aminobutyric acid receptor, GABAR; glyceraldehyde-3-phosphate dehydrogenase, GAPDH.
Figure 5
Figure 5. Western blot analyses of SOD1, SOD2 and catalase
(A) Changes in the intensity of SOD1, SOD2, catalase and GAPDH. (B) Changes in quantitative SOD1/GAPDH (†p <0.05). These results showed that SOD1 was decreased with 90% hepatectomy with saline compared with laparotomy. Additionally, there was a large difference in SOD1 between 90% hepatectomy with saline and that with the GABAR agonist. (C) Changes in quantitative SOD2/GAPDH (†p <0.05). These results showed that SOD2 was decreased with 90% hepatectomy with saline compared with laparotomy. Additionally, there was no difference in SOD2 between 90% hepatectomy with saline and that with the GABAR agonist. (D) Changes in quantitative catalase/GAPDH. There were no differences in catalase, between laparotomy and 90% hepatectomy with saline, and between 90% hepatectomy with saline and that with the GABAR agonist, respectively. Abbreviations: γ-aminobutyric acid receptor, GABAR; glyceraldehyde-3-phosphate dehydrogenase, GAPDH; not significant (p ≥0.05), NS; superoxide dismutase, SOD.
Figure 5
Figure 5. Western blot analyses of SOD1, SOD2 and catalase
(A) Changes in the intensity of SOD1, SOD2, catalase and GAPDH. (B) Changes in quantitative SOD1/GAPDH (†p <0.05). These results showed that SOD1 was decreased with 90% hepatectomy with saline compared with laparotomy. Additionally, there was a large difference in SOD1 between 90% hepatectomy with saline and that with the GABAR agonist. (C) Changes in quantitative SOD2/GAPDH (†p <0.05). These results showed that SOD2 was decreased with 90% hepatectomy with saline compared with laparotomy. Additionally, there was no difference in SOD2 between 90% hepatectomy with saline and that with the GABAR agonist. (D) Changes in quantitative catalase/GAPDH. There were no differences in catalase, between laparotomy and 90% hepatectomy with saline, and between 90% hepatectomy with saline and that with the GABAR agonist, respectively. Abbreviations: γ-aminobutyric acid receptor, GABAR; glyceraldehyde-3-phosphate dehydrogenase, GAPDH; not significant (p ≥0.05), NS; superoxide dismutase, SOD.
Figure 5
Figure 5. Western blot analyses of SOD1, SOD2 and catalase
(A) Changes in the intensity of SOD1, SOD2, catalase and GAPDH. (B) Changes in quantitative SOD1/GAPDH (†p <0.05). These results showed that SOD1 was decreased with 90% hepatectomy with saline compared with laparotomy. Additionally, there was a large difference in SOD1 between 90% hepatectomy with saline and that with the GABAR agonist. (C) Changes in quantitative SOD2/GAPDH (†p <0.05). These results showed that SOD2 was decreased with 90% hepatectomy with saline compared with laparotomy. Additionally, there was no difference in SOD2 between 90% hepatectomy with saline and that with the GABAR agonist. (D) Changes in quantitative catalase/GAPDH. There were no differences in catalase, between laparotomy and 90% hepatectomy with saline, and between 90% hepatectomy with saline and that with the GABAR agonist, respectively. Abbreviations: γ-aminobutyric acid receptor, GABAR; glyceraldehyde-3-phosphate dehydrogenase, GAPDH; not significant (p ≥0.05), NS; superoxide dismutase, SOD.
Figure 5
Figure 5. Western blot analyses of SOD1, SOD2 and catalase
(A) Changes in the intensity of SOD1, SOD2, catalase and GAPDH. (B) Changes in quantitative SOD1/GAPDH (†p <0.05). These results showed that SOD1 was decreased with 90% hepatectomy with saline compared with laparotomy. Additionally, there was a large difference in SOD1 between 90% hepatectomy with saline and that with the GABAR agonist. (C) Changes in quantitative SOD2/GAPDH (†p <0.05). These results showed that SOD2 was decreased with 90% hepatectomy with saline compared with laparotomy. Additionally, there was no difference in SOD2 between 90% hepatectomy with saline and that with the GABAR agonist. (D) Changes in quantitative catalase/GAPDH. There were no differences in catalase, between laparotomy and 90% hepatectomy with saline, and between 90% hepatectomy with saline and that with the GABAR agonist, respectively. Abbreviations: γ-aminobutyric acid receptor, GABAR; glyceraldehyde-3-phosphate dehydrogenase, GAPDH; not significant (p ≥0.05), NS; superoxide dismutase, SOD.
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