Unraveling the role of Epac1-SOCS3 signaling in the development of neonatal-CRD-induced visceral hypersensitivity in rats

Abstract

Aims: Visceral hypersensitivity in irritable bowel syndrome (IBS) is widespread, but effective therapies for it remain elusive. As a canonical anti-inflammatory protein, suppressor of cytokine signaling 3 (SOCS3) reportedly relays exchange protein 1 directly activated by cAMP (Epac1) signaling and inhibits the intracellular response to inflammatory cytokines. Despite the inhibitory effect of SOCS3 on the pro-inflammatory response and neuroinflammation in PVN, the systematic investigation of Epac1-SOCS3 signaling involved in visceral hypersensitivity remains unknown. This study aimed to explore Epac1-SOCS3 signaling in the activity of hypothalamic paraventricular nucleus (PVN) corticotropin-releasing factor (CRF) neurons and visceral hypersensitivity in adult rats experiencing neonatal colorectal distension (CRD).

Methods: Rats were subjected to neonatal CRD to simulate visceral hypersensitivity to investigate the effect of Epac1-SOCS3 signaling on PVN CRF neurons. The expression and activity of Epac1 and SOCS3 in nociceptive hypersensitivity were determined by western blot, RT-PCR, immunofluorescence, radioimmunoassay, electrophysiology, and pharmacology.

Results: In neonatal-CRD-induced visceral hypersensitivity model, Epac1 and SOCS3 expressions were downregulated and IL-6 levels elevated in PVN. However, infusion of Epac agonist 8-pCPT in PVN reduced CRF neuronal firing rates, and overexpression of SOCS3 in PVN by AAV-SOCS3 inhibited the activation of PVN neurons, reduced visceral hypersensitivity, and precluded pain precipitation. Intervention with IL-6 neutralizing antibody also alleviated the visceral hypersensitivity. In naïve rats, Epac antagonist ESI-09 in PVN increased CRF neuronal firing. Consistently, genetic knockdown of Epac1 or SOCS3 in PVN potentiated the firing rate of CRF neurons, functionality of HPA axis, and sensitivity of visceral nociception. Moreover, pharmacological intervention with exogenous IL-6 into PVN simulated the visceral hypersensitivity.

Conclusions: Inactivation of Epac1-SOCS3 pathway contributed to the neuroinflammation accompanied by the sensitization of CRF neurons in PVN, precipitating visceral hypersensitivity and pain in rats experiencing neonatal CRD.

Keywords: Epac1; SOCS3; corticotrophin-releasing factor; neonatal colorectal distension; visceral hypersensitivity.

FIGURE 1

Downregulated Epac1‐mediated neonatal‐CRD‐induced visceral hypersensitivity. (A) Schematic description of time course of experiments. Neonatal CRD was performed on postnatal days 8, 10, and 12. Adult CRD was applied between weeks 8 and 10. At 120 min after adult CRD, all rats were sacrificed with brain samples isolated for RT‐PCR, Western blotting, ELISA, and immunofluorescent staining. (B) Neonatal‐ and dual‐CRD rats presented a significant decrease in pain threshold vs. control and adult‐CRD rats, respectively (n = 8). (C) Neonatal‐ and dual‐CRD rats displayed a significant increase in AWR score (n = 8). (D) Epac1 mRNA expression was significantly decreased in neonatal‐CRD and dual‐CRD groups vs. control group (n = 6). (E) Epac1 protein expression was significantly decreased in neonatal‐CRD and dual‐CRD groups vs. control group (n = 6). (F) Epac2 protein had no significant alterations in each group (n = 6). Data are presented as the mean ± SD. *p < 0.05, **p < 0.01 vs. indicated group

FIGURE 2

Activation of Epac1 in PVN alleviated the visceral hypersensitivity and decreased the excitability of PVN CRF neurons in neonatal‐CRD rats. (A) Epac agonist 8‐pCPT significantly elevated the visceral nociceptive threshold 15 min after administration and 120 min after adult‐CRD stimuli in neonatal‐CRD group (n = 6). (B) Epac agonist 8‐pCPT significantly decreased the AWR scores at 30, 40, and 50 mmHg 15 min after administration (n = 6). (C) Epac agonist 8‐pCPT significantly decreased the AWR scores at 30, 40, and 50 mmHg 120 min after adult‐CRD stimuli in neonatal‐CRD group (n = 6). (D) Electrophysiological recording of specific AAV labeled CRF neurons. (E) Schematic diagram of discharge of CRF neurons before and after 8‐pCPT administration. (F) In vitro electrophysiology revealed that 8‐pCPT perfusion inhibited the firing of CRF neurons in PVN from neonatal‐CRD group (n = 14 cells from 6 rats). Data are presented as the mean ± SD. *p < 0.05, **p < 0.01 vs. indicated group

FIGURE 3

Inhibition of Epac1 in PVN facilitated visceral hypersensitivity and increased the excitability of PVN CRF neurons in control rats. (A) Epac antagonist ESI‐09 reduced the nociceptive threshold 15 min after administration and 120 min after adult‐CRD stimuli in control group; however, Epac2 antagonist HJC0350 had no evident effect on the pain threshold in control group (n = 6). (B) ESI‐09 significantly elevated the AWR scores at 20 and 30 mmHg 15 min after administration in control group (n = 6). (C) ESI‐09 significantly elevated the AWR scores at 20, 30, 40, and 50 mmHg 120 min after adult‐CRD stimuli in control group (n = 6). (D) Schematic diagram of discharge of CRF neurons before and after ESI‐09 administration. (E) In vitro electrophysiology revealed that ESI‐09 perfusion increased the firing of CRF neurons in PVN from control group (n = 12 cells from 6 rats). (F) The nociceptive threshold significantly decreased by 36 h after Epac1‐siRNA injection into PVN in naïve rats (n = 6). (G) The AWR scores were significantly elevated at 30, 40, and 50 mmHg 36 h after Epac1‐siRNA injection into PVN in naïve rats (n = 6). (H) Immunofluorescent analyses showed the activated PVN neurons were labeled with c‐Fos 36 h after Epac1‐siRNA injection in naïve rats (n = 3–4 sections from 5 rats, scale bar = 100 μm). (I) Cell counts of c‐Fos‐positive neurons in PVN 36 h after Epac1‐siRNA injection (n = 6). (J) CRF mRNA in PVN from control group was significantly increased 36 h after Epac1‐siRNA, as well as (K) CRF protein expression. Meanwhile, (L) pituitary CRF protein was significantly elevated (n = 5). Data are presented as the mean ± SD. *p < 0.05, **p < 0.01 vs. indicated group

FIGURE 4

Downregulation of downstream effector SOCS3 participated in visceral hypersensitivity in neonatal‐CRD or dual‐CRD rats. (A–D) The expression of Rap1 mRNA, Rap1 protein, SOCS3 mRNA, and SOCS3 protein in PVN in each group (n = 6). (E) SOCS3 double‐labeled with CRF and GFAP, rather than Iba‐1 (Scale bar = 100 μm). (F) Co‐expression of CRF with SOCS3 in PVN in each group. (G) Statistical diagram for SOCS3 and CRF co‐labeling (n = 6, scale bar = 100 μm). (H) Schema of PVN, and immunofluorescent staining of SOCS3 in PVN. Data are presented as the mean ± SD. *p < 0.05, **p < 0.01 vs. indicated group

FIGURE 5

Activation of SOCS3 in PVN alleviated visceral hypersensitivity with the decreased excitability of PVN CRF neurons in neonatal‐CRD rats. (A) Typical immunofluorescent image of c‐Fos protein after AAV9‐SOCS3 intervention (Scale bar = 100 μm). (B) c‐Fos was significantly decreased in PVN in neonatal‐CRD group 21 days after AAV‐SOCS3 injection (n = 6). (C) Overexpressed SOCS3 protein was detected in PVN 21 days after AAV‐SOCS3 injection in neonatal‐CRD group (n = 6). (D) The nociceptive threshold elevated 21 days after AAV‐SOCS3 injection in neonatal‐CRD group (n = 6). (E) The AWR scores were decreased at 40 and 50 mmHg 21 days after AAV‐SOCS3 injection. Data are presented as the mean ± SD. **p < 0.01 compared with indicated group

FIGURE 6

Inhibition of SOCS3 in PVN facilitates visceral hypersensitivity with the increased excitability of PVN neurons in control rats. (A) The nociceptive threshold declined 36 h after SOCS3‐siRNA injection in naïve rats (n = 6). (B) The AWR scores were elevated at 20, 30, 40, and 50 mmHg 36 h after SOCS3‐siRNA injection in naïve rats (n = 6). (C) Immunofluorescence of c‐Fos and DAPI in PVN 36 h after SOCS3‐siRNA injection in naïve rats (n = 3–4 sections from 6 rats, scale bar = 100 μm). (D) Cell counts of c‐Fos‐positive neurons in PVN 36 h after SOCS3‐siRNA injection (n = 6). (E–G) CRF, ACTH, and CORT in peripheral plasma were significantly increased 36 h after intra‐PVN injection of SOCS3‐siRNA (n = 3). Data are presented as the mean ± SD. *p < 0.05, **p < 0.01 vs. indicated group

FIGURE 7

Involvement of IL‐6 in neonatal‐CRD‐induced chronic visceral hypersensitivity. (A) Western blotting indicated that IL‐6 protein expression was significantly upregulated in neonatal‐CRD, adult‐CRD, and dual‐CRD groups, respectively (n = 6). (B) ELISA indicated that IL‐6 protein expression was also significantly upregulated (n = 6). (C) The visceral nociceptive threshold decreased 120 min after intra‐PVN administration of exogenous IL‐6 in control group (n = 6). (D) The AWR scores were increased at 30, 40, and 50 mmHg 120 min after intra‐PVN administration of exogenous IL‐6 in control group (n = 6). (E) Nociceptive threshold increased 120 min after PVN administration of IL‐6 neutralizing antibody (NA) in neonatal‐CRD group (n = 6). (F) The AWR scores were decreased at 30, 40, and 50 mmHg 120 min after intra‐PVN administration of exogenous IL‐6 NA in neonatal‐CRD group (n = 6). Data are presented as the mean ± SD. *p < 0.05, **p < 0.01 vs. indicated group

FIGURE 8

Schema of the experiment. In neonatal‐CRD‐induced visceral hypersensitivity, both Epac1 and SOCS3 were downregulated, resulting in the disinhibition of IL‐6 receptor‐mediated signaling and amplification of IL‐6 pro‐inflammatory signals and further activation of CRF neurons in PVN and the HPA axis. Activation of either Epac1 or SOCS3, or antagonism on IL‐6, reversed the visceral nociceptive effects in the neonatal‐CRD group. The upward arrow represents upregulation and the downward arrow stands for downregulation