Burn-Induced Apoptosis in the Livers of Aged Mice Is Associated With Caspase Cleavage of Bcl-xL

Abstract

Introduction: Older adult burn victims have poorer outcomes than younger burn victims. The liver is critical for the recovery of patients with burns. Postburn hepatic apoptosis in young individuals compromises liver integrity; however, this pathway has not yet been studied in older individuals. Because aged animals with burns suffer significant liver damage, we hypothesized that apoptosis is altered in these animals and may affect liver function. Understanding postburn hepatic apoptosis and its effects on liver function in aged animals may help improve outcomes in older patients.

Methods: We compared the protein and gene expression levels in young and aged mice after a 15% total-body-surface-area burn. Liver and serum samples were collected at different time points after injury.

Results: Caspase-9 expression in liver tissue was downregulated by 47% in young animals and upregulated by 62% in aged animals 9 h postburn (P < 0.05). The livers of aged mice showed a Bcl-extra-large (Bcl-xL) transcription increase only after 6 h; however, the livers of young mice exhibited 4.3-fold, 14.4-fold, and 7.8-fold Bcl-xL transcription increases at 3, 6, and 9 h postburn, respectively (P < 0.05). The livers of young mice showed no changes in Caspase-9, Caspase-3, or Bcl-xL protein levels during the early postburn period. In contrast, the livers of aged mice contained cleaved caspase-9, reduced full-length caspase-3, and an accumulation of ΔN-Bcl-x at 6 and 9 h postburn (P < 0.05). p21 expression decreased in aged mice; however, it was significantly increased in the liver tissue of young mice postburn (P < 0.05). Serum amyloid A1 and serum amyloid A2 serum protein levels were 5.2- and 3.1-fold higher in young mice than in aged mice, respectively, at 6 and 9 h postburn (P < 0.05).

Conclusions: Livers of aged mice exhibited different apoptotic processes compared to those of young mice early after burn injury. Collectively, burn-induced liver apoptosis in aged mice compromises hepatic serum protein production.

Keywords: Aging; Apoptosis; Bcl-extra-large; Burn; Liver; Trauma.

Figure 1.. Opposing effects of Caspase-9 transcription in the livers of young and aged mice after burn injury.

Whole livers were analyzed using qPCR. (A) Caspase-8 (B) Caspase-9 Results were determined using the ΔΔCt algorithm with GAPDH as an internal control. Data are presented as boxplots. n = 10 per group; *p<0.05, ****p<0.0001

Figure 2.. Expression of Bcl-2 family members in livers from mice post-burn.

Whole livers were analyzed using qPCR. (A) Bcl-2 (B) Bcl-xL (C) Bak (D) Bax Results were determined using the ΔΔCt algorithm with GAPDH as an internal control. Data are presented as boxplots. n = 10 per group; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001

Figure 3.. Apoptosis is not detected 3 h post-burn in livers from young or aged mice.

Western blot analysis was performed on liver lysates using antibodies specific to caspase-9 (C9), caspase-3 (C3), or Bcl-xL. Cleavage Caspase 9= c-C9, Cleavage Caspase 3= c-C3. GAPDH was used as a loading control. Densitometry was performed using GAPDH normalization to each one of the target proteins. Data are presented as boxplots. Representative images are shown, n= 10

Figure 4.. Apoptosis is detected in the livers of aged mice 6 and 9 h post-burn.

Liver lysates were probed at the indicated time points with antibodies specific to caspase-9 (C9), caspase-3 (C3), or Bcl-xL. GAPDH was used as a loading control. (A) Active Caspase-9 6 h post-burn, (B) Active Caspase-9 9 h post-burn, (C) Caspase-3 6 h post-burn, (D) Caspase-3 9 h post-burn, (E) ΔN-Bcl-xL 6 h post-burn, and (F) ΔN-Bcl-xL 9 h post-burn. Protein not detected (N.D.). Cleavage Caspase 9= c-C9, Cleavage Caspase 3= c-C3. Densitometry was performed using GAPDH normalization to each one of the target proteins. Data are presented as boxplots. Representative images are shown, n= 10 per group *p<0.05

Figure 5.. The livers of young and aged mice exhibit differential expression of p21 protein.

Liver lysates were analyzed at 6 and 9 h post-burn for p21 using western blotting. GAPDH was used as a loading control. Densitometry was performed using GAPDH normalization to each one of the target proteins. Data are presented as boxplots. Representative images are shown, n= 10 per group *p<0.05 compared to all other groups. p<0.05 compared to aged sham and young burn groups.

Figure 6.. Induction of Serum SAA in the Serum is reduced in aged mice after burn injury.

Serum SAA protein was measured using ELISA. SAA protein quantities are presented as ng/mL of Serum. Data are presented as boxplots. SAA fold change compared to the age-matched sham group. n = 10 per group; *p<0.05, ***p<0.001, ****p<0.0001