01 and P < 0.001) TNF-α, IL-6, and IL-12p40 levels (pg/mL) in PACAP27/PACAP38 groups (Fig. 5A-C: TNF-α: 1,074.1 ± 33.8/1,117.8 ± 22.6; IL-6: 1,690.9 ± 174.9/1,986.4 ± 97.6; and IL-12p40: 4,805.1 ± 271.0/5,347.1 ± 168.1), compared with PACAP cultures only. Moreover, IL-10 levels decreased (P < 0.001) in BMM cultures supplemented with PACAP plus H-89 (Fig. 5D: 833.2 ± 124.9/981.1 ± 126.8), compared with PACAP alone. To analyze the immunomodulatory click here function of cAMP-PKA signaling

in hepatocytes, we designed primary hepatocyte culture systems to mimic liver IR-mediated hepatocellular damage in vivo. Because necrosis and apoptosis are essential in the mechanism of liver IRI, we used H2O2 to mimic in vivo ROS-triggered hepatocyte necrosis or TNF-α/ActD to induce apoptosis. Native mouse hepatocytes were cultured in the presence of PACAP, with H-89 (PKA antagonist) or DMSO (control). The addition of PACAP27/PACAP38 consistently suppressed hepatocyte death, assessed by fluorescence-activated cell-sorting (FACS)-assisted frequency (%) of Annexin V+7-AAD+ cells (Fig.

6A: H2O2: 3.3 ± 2.6/3.4 ± 2.8 versus 13.8 ± 3.6; TNF-α+ActD: 4.8 ± 2.3/3.1 ± 2.5 versus http://www.selleckchem.com/products/GDC-0449.html 15.6 ± 2.5; P < 0.001), diminished caspase-3 activity (pmol/min/5 × 10 E4 cells) (Fig. 6B: H2O2: 0.09 ± 0.07/0.09 ± 0.07 versus 0.29 ± 0.17; TNF-α+ActD: 0.58 ± 0.13/0.58 ± 0.13 versus 1.91 ± 0.32; P < 0.001), LDH release (%) (Fig. 6C: H2O2: 10.39 ± 2.29/10.36 ± 2.28 versus 19.19 ± 5.26; TNF-α+ActD: 15.58 ± 4.23/15.54 ± 4.22 versus 37.62 ± 9.58; P < 0.01), and ALT release (%) (Fig. 6D: H2O: 10.98 ± 2.06/11.06 ± 2.03 versus 22.58 ± 4.58; TNF-α+ActD: 13.97 ± 3.80/14.10 ± 3.75 versus 36.36 ± 8.58; P < 0.01), as compared to controls. In contrast, PKA inhibition enhanced hepatocyte

death (Fig. 6A: H2O2: 10.1 ± 3.1/11.2 ± 3.2; TNF-α+ActD: 13.4 ± 2.7/13.3 ± 2.8) and MCE公司 caspase-3 activity (Fig. 6B: H2O2: 0.27 ± 0.17/0.26 ± 0.16; TNF-α+ActD: 1.85 ± 0.31/1.74 ± 0.30). In addition, PKA inhibition increased LDH (Fig. 6C: H2O2: 18.63 ± 5.03/18.45 ± 5.03; TNF-α+ActD: 36.22 ± 9.24/35.88 ± 9.22), and ALT (Fig. 6D: H2O2: 21.97 ± 4.63/22.20 ± 4.57; TNF-α+ActD: 35.15 ± 8.49/35.52 ± 8.39) release in hepatocyte cultures. Although PACAP neuropeptide regulates macrophage cytokine programs and stimulates hepatocyte glucose production,22 its role in innate immunity-driven liver inflammation and IR hepatocellular injury have not been explored. Here, we show that (1) PACAP and its intrinsic receptors were induced in mouse livers subjected to warm IR, (2) PACAP deficiency exacerbated liver damage, implying that PACAP is essential for liver homeostasis, (3) exogenous PACAP protected livers against IRI by inhibiting macrophage function and improving hepatocyte survival, and (4) PACAP-mediated regulatory/cytoprotective function was cAMP-PKA dependent.