Effects of AP214 treatment on liver damage in lethal CLP

We previously reported liver damage in mouse CLP model.10 The lethal CLP model showed

significant increases of aspartate aminotransferase (AST) and alanine aminotransferase (ALT)

at 18 hr after surgery compared with sham-operated animals [AST: vehicle 680.9 ± 92.5 U/l

(n = 14), sham 126.4 ± 22.3 U/l (n = 5), p<0.05; ALT: vehicle 314.3 ± 30.1 U/l (n = 14), sham

40.6 ± 3.8 U/l (n = 5), p<0.05]. AP214 treatment at the dose of 10 μg significantly attenuated

liver damage in lethal CLP (Figure 4A,B) and did not cause any change in sham-operated mice

(data not shown). Delayed 10 μg AP214 treatment that was started at 6 hr after surgery also

showed a protective effect against liver damage by lethal CLP (Figure 4C,D).

AP214 treatment improved hypotension in lethal CLP

Blood pressure and heart rate measurement was performed in conscious animals by

radiotelemetery. Injection of AP214 on normal CD-1 mice at the dose of 10 μg did not alter

blood pressure and heart rate compared with vehicle injection (Figure 5A,B). Sepsis induced

by lethal CLP surgery caused severe hypotension and decreases of heart rate. AP214 treatment

prevented the progression of severe hypotension and bradycardia [blood pressure at 18 hr:

sham 112.4 ± 2.8 mmHg (n = 3), CLP + vehicle 54.4 ± 4.7 mmHg (n = 5), CLP + AP214 75.7

± 6.4 mmHg (n = 5), p<0.05 (vs CLP + vehicle); heart rate: sham 459.4 ± 77.0 bpm (n = 3),

CLP + vehicle 197.6 ± 14.2 bpm (n = 5), CLP + AP214 459.4 ± 77.0 bpm (n = 5), p<0.05 (vs

CLP + vehicle)]. (Figure 5C,D).

Serum TNF-α and IL-10 levels in lethal CLP were suppressed by AP214

We evaluated serum TNF-α and IL-10 levels by ELISA 18 hr after surgery in lethal CLP

animals. Both serum TNF-α and IL-10 levels increased in CLP animals [TNF-α: Vehicle 572.5

± 65.5 pg/ml (n = 14), sham 55.3 ± 5.3 pg/ml (n = 5), p<0.05, IL-10: Vehicle 1365.2 ± 182.4

pg/ml (n = 14), sham 20.2 ± 9.3 pg/ml (n = 5), p<0.05]. Administration of AP214 reduced

serum TNF-α and IL-10 levels with bell-shaped dose-response curves (Figure 6A,B). AP214

had a maximum effect on sepsis-induced AKI at the dose of 10 μg and the same dose of 10

μg also had a maximum effect on reducing serum TNF-α and IL-10. Delayed AP214 treatment

(10 μg) also reduced serum TNF-α and IL-10 (Figure 6C,D).

Nuclear factor-κB activation in kidney and spleen was inhibited by AP214

We examined nuclear factor-κB (NF-κB) activation in kidney and spleen because α-MSH has

been reported to inhibit NF-κB activation induced by several inflammatory signals.27 NF-κB

activation in kidney and spleen at 18 hr after lethal CLP surgery was significantly increased

compared with sham-operated animals. AP214 treatment at the dose of 10 μg significantly

inhibited the increase of NF-κB activation both in kidney and spleen (Figure 7A,B). Delayed

AP214 treatment (10 μg) also decreased NF-κB activation (Figure 7C,D).

AP214 reduced splenocyte apoptosis in lethal CLP

Lymphocyte apoptosis is reported to contribute to sepsis severity.28 Therefore, we investigated

the effect of AP214 on splenocyte apoptosis. Splenocyte apoptosis was evaluated by

immunohistochemical analysis of activated caspase-3. In sham operated animals, activated

caspase-3 positive stained cells were rarely found in spleen (Figure 8A). CLP increased

activated caspase-3 positive stained cells profoundly in the white pulp of spleen at 18 hr after

surgery and AP214 treatment at the dose of 10 μg significantly reduced splenocyte apoptosis

(Figure 8B,C,D). Delayed AP214 treatment (10 μg) also reduced splenocyte apoptosis (Figure

8E). No apoptotic cell was detectable in the kidney as previously reported 10.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscript