Activation of α7nAChR Protects Against Gastric Inflammation and Dysmotility in Parkinson's Disease Rats

Abstract

The cholinergic anti-inflammatory pathway (CAIP) has been proposed to regulate gastrointestinal inflammation via acetylcholine released from the vagus nerve activating α7 nicotinic receptor (α7nAChR) on macrophages. Parkinson's disease (PD) patients and PD rats with substantia nigra (SN) lesions exhibit gastroparesis and a decayed vagal pathway. To investigate whether activating α7nAChR could ameliorate inflammation and gastric dysmotility in PD rats, ELISA, western blot analysis, and real-time PCR were used to detect gastric inflammation. In vitro and in vivo gastric motility was investigated. Proinflammatory mediator levels and macrophage numbers were increased in the gastric muscularis of PD rats. α7nAChR was located on the gastric muscular macrophages of PD rats. The α7nAChR agonists PNU-282987 and GTS-21 decreased nuclear factor κB (NF-κB) activation and monocyte chemotactic protein-1 mRNA expression in the ex vivo gastric muscularis of PD rats, and these effects were abolished by an α7nAChR antagonist. After treatment with PNU-282987 in vivo, the PD rats showed decreased NF-κB activation, inflammatory mediator production, and contractile protein expression and improved gastric motility. The present study reveals that α7nAChR is involved in the development of gastroparesis in PD rats and provides novel insight for the treatment of gastric dysmotility in PD patients.

Keywords: 6-hydroxydopamine; Parkinson’s disease; gastric inflammation; macrophage; α7nAChR.

FIGURE 1

Expression of tyrosine hydroxylase (TH) in the substantia nigra (SN) and choline acetyltransferase (ChAT) in the dorsal motor vagal nucleus (DMV) and gastric muscularis. (A) TH-IR neurons in the SN and ChAT-IR neurons in the DMV. n = 3 per group. (B) Representative western blots showing the expression of TH in the SN (left) and ChAT in the dorsal medulla (middle) and summary boxplot (right) showing significant reductions in TH expression in the SN and ChAT expression in the dorsal medulla in 6-OHDA rats. n = 8 per group. (C) Representative western blot (left) and summary boxplot (right) showing significant reductions in ChAT expression in the gastric muscularis of 6-OHDA rats. n = 8 per group. (D) ACh content in the muscularis of the gastric corpus. n = 7 per group. ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001 vs. the control group by unpaired Student’s t-test.

FIGURE 2

Gastric muscular inflammation in 6-OHDA rats. (A) The levels of TNF-α, IL-1β and IL-6 in the gastric mucosa (left) and muscularis (right) at 6 weeks after microinjection of 6-OHDA into the SN. n = 6 per group. (B) The architecture of the muscularis of the gastric corpus in control rats [(B), a] and 6-OHDA rats at 4 weeks [(B), b] and 6 weeks ([B), c]; [(B)a’–c’] are high-magnification images of the white framed regions in [(B), a–c], respectively. n = 3 per group. (C) Representative western blot (left) and boxplot (right) showing increased expression of α7nAChR in the gastric muscularis of 6-OHDA rats. GAPDH was analysed as a loading control. n = 8 per group. (D) The colocalization of α7nAChR with CD163 (left) or CD68 (right) in the gastric myenteric plexus in control and 6-OHDA rats. Green signal, CD163 or CD68 positive macrophages; red signal, α7nAChR positive cells. GC, gastric corpus; MGC, muscularis of the gastric corpus. n = 3 per group. ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001 vs. the control group by unpaired Student’s t-test. n = 3–8 per group.

FIGURE 3

Effects of activating α7nAChR on the production of NF-κB p65 and MCP-1 and the infiltration of macrophages. (A) The α7nAChR agonists PNU-282987 (PNU) and GTS-21 decreased the protein expression of phosphorylated nuclear NF-κB p65 subunit (p-p65) (left) and the mRNA expression of MCP-1 (right) in cultured gastric muscularis from 6-OHDA rats. Methyllycaconitine citrate (MLA), a selective α7nAChR antagonist, significantly blocked the effects of PNU and GTS-21 on the expression of p-p65 (left) and MCP-1 (right). ^* p < 0.05, ^** p < 0.01 compared with their respective controls or between the two groups indicated. n = 8–10 per experimental group. (B) The effects of early-PNU (left) and late-PNU (right) treatment on the mRNA levels of MCP-1, TNF-α, IL-1β and IL-6 in the muscularis of the gastric corpus. n = 6–7 per experimental group. (C) Early treatment group: typical immunohistochemical staining of CD163 and CD68 in rat myenteric plexus whole mounts from the control, 6-OHDA, and 6-OHDA + early-PNU (left) groups; and a summary boxplot (right) showing decreased numbers of CD163-and CD68-positive macrophages in 6-OHDA + early-PNU rats. n = 5 rats per group, 2 whole-mount preparations per rat and 2–3 visual fields per section. (D) Late treatment group: typical immunohistochemical staining of CD163 and CD68 in rat myenteric plexus whole mounts from the control, 6-OHDA, and 6-OHDA + late-PNU (left) groups and a summary boxplot (right) showing decreased numbers of CD163-and CD68-positive macrophages in 6-OHDA + late-PNU rats. MGC, muscularis of the gastric corpus; early-PNU, injection of PNU on day 1 after 6-OHDA/saline microinjection. Late PNU, injection of PNU-282987 beginning from day 28 after 6-OHDA/saline microinjection. n = 5 rats per experimental group, 2 whole-mount preparations per rat and 2–3 visual fields per section. ^** p < 0.01, ^*** p < 0.001 compared with control rats; ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 compared with 6-OHDA rats by Kruskal-Wallis test followed by Dunn’s multiple-comparisons test.

FIGURE 4

Effects of PNU-282987 (PNU) treatment on the expression of iNOS, COX-2 and smooth muscle contractile markers in the gastric muscularis of 6-OHDA rats. (A) Representative western blot (upper) and boxplot (lower) showing reduced nuclear p-p65 protein expression and increased cytosolic IκB protein expression in the gastric muscularis of 6-OHDA rats subjected to early-PNU (left) and late-PNU (right) treatment. Lamin B and Actin were used as the loading controls for nuclear protein and cytosolic protein, respectively. n = 7–8 per group. (B) Representative western blot (upper) and boxplot (lower) showing decreased expression of iNOS and COX-2 in the gastric muscularis of 6-OHDA rats subjected to early-PNU (left) and late-PNU (right) treatment. GAPDH was used as a loading control. n = 8 per experimental group. (C) Representative western blot and boxplot showing decreased α-smooth muscle actin (α-SMA) and myosin light chain 20 (MLC20) expression in the gastric muscularis of 6-OHDA rats. Early-PNU treatment significantly increased α-SMA and MLC20 expression without statistical significance. n = 7 per group. Early-PNU, injection of PNU on day 1 after 6-OHDA/saline microinjection; late-PNU, injection of PNU beginning from day 28 after 6-OHDA/saline microinjection. ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001 compared with control rats; ^# p < 0.01, ^## p < 0.01 compared with 6-OHDA rats. by one-way ANOVA followed by Turkey’s multiple comparisons test or Kruskal-Wallis test followed by Dunn’s multiple-comparisons test.

FIGURE 5

Effects of PNU-282987 (PNU) treatment on gastric motility in 6-OHDA rats. (A) Carbachol-induced contraction of gastric muscular strips (left, n = 8 animal per group, 1–2 muscular strip per animal) and the gastric emptying (GE) rate (right, n = 8 per experimental group) after a solid meal in 6-OHDA rats were improved after early-PNU treatment. (B) Representative images (left) and boxplot (right) showing that late-PNU treatment significantly increased the amplitude of gastric muscle contraction in 6-OHDA rats. n = 8 per experimental group. (C) Representative images (left) and a boxplot (right) showing that late-PNU treatment ameliorated the decreased GE rate after a barium meal in 6-OHDA rats. n = 6 per experimental group. (D) Morphometric analysis by electron microscopy revealed hypertrophy of smooth muscle cells, increased collagen deposition in the extracellular matrix (white arrows) and a decreased number of dense plaques (white triangles) in the gastric corpus muscularis in 6-OHDA rats (middle) compared with control rats (left); these ultrastructural features were greatly ameliorated by late-PNU treatment (right). n = 3 per group. Early-PNU, injection of PNU on day 1 after 6-OHDA/saline microinjection; late-PNU, injection of PNU beginning from day 28 after the 6-OHDA/saline microinjection. ^** p < 0.01, ^*** p < 0.001 compared with control rats. ^# p < 0.05 compared with 6-OHDA rats by one-way ANOVA followed by Turkey’s multiple comparisons test or Kruskal-Wallis test followed by Dunn’s multiple-comparisons test. n = 6–8 per group.

FIGURE 6

Expression of tyrosine hydroxylase (TH) in the substantia nigra (SN) and choline acetyltransferase (ChAT) in the dorsal motor vagal nucleus (DMV) and gastric muscularis in control, 6-OHDA and 6-OHDA rats treated with PNU-282987 (PNU). (A) Representative western blot (upper) and boxplot (lower) showing that early-PNU treatment failed to improve TH expression in the SN in 6-OHDA rats. n = 3 per group. (B) Representative western blot (upper) and boxplot (lower) showing that early-PNU treatment failed to improve ChAT expression in the dorsal medulla of 6-OHDA rats. n = 3 per group. (C) Representative western blot (upper) and boxplot (lower) showing that late-PNU treatment failed to improve TH expression in the SN in 6-OHDA rats. n = 3 per group. (D) Representative western blot (upper) and boxplot (lower) showing that late-PNU treatment failed to improve ChAT expression in the dorsal medulla of 6-OHDA rats. n = 3 per group. Early-PNU, injection of PNU on day 1 after 6-OHDA/saline microinjection; late-PNU, injection of PNU beginning from day 28 after 6-OHDA/saline microinjection. ^** p < 0.01, ^*** p < 0.001 compared with control rats; ns, not significant compared with 6-OHDA rats by one-way ANOVA followed by Turkey’s multiple comparisons test or or Kruskal-Wallis test followed by Dunn’s multiple-comparisons test. n = 8 per group.

FIGURE 7

Illustration of the working hypothesis of the role of the cholinergic anti-inflammatory pathway (CAIP) in control and 6-OHDA rats and the role of the α7nAChR in preventing inflammation in 6-OHDA rats. At steady state, the strong vagal tone determines the shift in the CAIP towards tolerance by causig the release of a sufficient amount of ACh, and muscular macrophages exhibit a resident phenotype. In 6-OHDA rats, lesions in the SN induce a decrease in vagal tone, and the CAIP shifts towards inflammation due to inadequate release of ACh, which induces an increased number of resident/proinflammatory macrophages and the subsequent production of inflammatory mediators. Stimulating α7nAChR on activated macrophages leads to the inhibition of inflammatory mediator production and restoration of intestinal immune homeostasis.