NPs/NPRs Signaling Pathways May Be Involved in Depression-Induced Loss of Gastric ICC by Decreasing the Production of mSCF

Abstract

It is well known that natriuretic peptides (NPs) are involved in the regulation of gastrointestinal motility. Interstitial cells of Cajal (ICC) are the pacemaker cells of gastrointestinal motility and gastrointestinal dyskinesia is one of the important digestive tract symptoms of depression. However, it is unclear whether they are involved in depression-induced loss of ICC. The aim of the present study was to investigate the relationship between the natriuretic peptide signaling pathway and depression-induced loss of gastric ICC in depressed rats. These results showed that the expression of c-kit and stem cell factor (SCF) in smooth muscle layers of stomach were down-regulated in depressed rats at the mRNA and protein levels. The expression of natriuretic peptide receptor (NPR)-A, B and C were up-regulated in the stomach of depressed rats at the mRNA and protein levels. NPR-A, B and C can significantly decrease the expression of SCF to treat cultured gastric smooth muscle cells (GSMCs) obtained from normal rats with different concentrations of C-type natriuretic peptide (CNP). Pretreatment of cultured GSMCs with 8-Brom-cGMP (8-Br-cGMP, a membrane permeable cGMP analog), cANF (a specific NPR-C agonist) and CNP (10-6 mol/L) demonstrated that 8-Br-cGMP had a similar effect as CNP, but treatment with cANF did not. The results of the methyl thiazolyl tetrazolium bromide (MTT) assay indicated that high concentrations of cANF (10-6 mol/L) restrained the proliferation of cultured GSMCs. Taken together, these results indicate that the up-regulation of the NPs/NPR-C and NPs/NPR-A, B/cGMP signaling pathways may be involved in depression-induced loss of gastric ICC.

Fig 1. Rats show depressive-like symptoms at the end of the experiment.

(A) Body weight. (B) Percentage of sucrose solution from the total liquid in sucrose preference test. The results of the percentage were 59.05±3.97 in group M and 76.73±3.60 in group N. (C) The rate of gastric residual. The results were 55.18±0.52 in M and 41.68±1.97 in N.

Fig 2. The result of open-field test.

(A) Crossed-grids times. At the end of the experiment, the average crossed-grids times were 10.33±1.27 in M and 49.70±1.12 in N. (B) Standing times. At the end of the experiment, the average standing times were 5.00±1.17 in M and 18.60±0.82 in N. (C) Grooming times. At the end of the experiment, the average grooming times were 2.00±0.37 in M and 7.70±0.56 in N.

Fig 3. Western blotting and real-time PCR analyses of SCF and c-kit in the stomachs of rats.

(A) The average ratio of c-kit/β-actin was 0.959±0.056 in N and 0.571±0.116 in M. (B) The average ratio of SCF/β-actin was 0.871±0.078 in N and 0.251±0.030 in M. The differences between M and N were significant (compared with normal, *P<0.05, Independent-Samples T-test, A and **P<0.01, Independent-Samples T-test, B). (C-D) The result of c-kit is 1.000±0.000 in N and 0.380±0.053 in M. The result of SCF is 1.000±0.000 in N and 0.308±0.069 in M. The differences between N and M were significant (**P<0.01, Independent-Samples T-test).

Fig 4. Western blotting and real-time PCR analyses of NPR-A, B and C in the stomachs of rats.

(A-C) The ratio of NPR-A/β-actin was 0.427±0.013 in N and 0.927±0.038 in M. The ratio of NPR-B/β-actin was 0.172±0.012 in N and 0.400±0.048 in M. The ratio of NPR-C/β-actin was 1.261±0.151 in N and 3.287±0.474 in M. The differences between M and N were significant (compared with normal, **P<0.01, Independent-Samples T-test, A-B; *P<0.05, Independent-Samples T-test, C). (D-F) The result of NPR-A is 1.000±0.000 in N and 2.019±0.005 in M. The result of NPR-B is 1.000±0.000 in N and 2.969±0.012 in M. The result of NPR-C is 1.000±0.000 in N and 1.936±0.012 in M. The differences between N and M were significant (**P<0.01, Independent-Samples T-test).

Fig 5. Western blotting analyses of SCF expression in cultured GSMCs with different pretreatment conditions.

(A) The ratio of SCF/β-actin was 0.460±0.051 in N, 0.448±0.038 in cANF(10⁻⁶ mol/L) group, 0.276±0.047 in 8-Br-cGMP (10⁻⁶ mol/L) group and 0.253±0.055 CNP (10⁻⁶ mol/L) group. The expression levels were significantly different in the 8-Br-cGMP (10⁻⁶ mol/L) group and CNP (10⁻⁶ mol/L) group (*P<0.05, one-way ANOVA), but not in the cANF(10⁻⁶ mol/L) group (P>0.05, one-way ANOVA) compared with N. (B) The ratio of SCF/β-actin was 0.429±0.022 in N, 0.362±0.037 in CNP (10⁻⁸ mol/L) group, 0.282±0.020 in CNP (10⁻⁷ mol/L) group, and 0.217±0.019 CNP (10⁻⁶ mol/L) group. These results showed that high concentrations of CNP (10⁻⁷ mol/L and 10⁻⁶ mol/L) could significantly decrease the expression of SCF (**P<0.01, one-way ANOVA).

Fig 6. Effects of cANF on A value of MTT of rat GSMCs.

cANF The average value was 0.599±0.016 in control group, 0.597±0.033 in 10⁻⁸ mol/L group, 0.459±0.024 in 10⁻⁷ mol/L group and 0.356±0.025 in 10⁻⁶ mol/L group.