RNAiso Plus, PrimeScript^TM RT reagent kit with gDNA Eraser and SYBR^® Premix Ex Taq^TM II were purchased from Takara Biotech, Inc. Rabbit NPR-B polyclonal antibody, rabbit CNP polyclonal antibody, and rabbit beta-actin polyclonal antibody were purchased from Beijing Biosynthesis Biotechnology Co., Ltd. Peroxidase-conjugated affini-pure goat anti-rabbit IgG was purchased from Beijing ZSGB Biotech Co., Ltd. Other reagents were obtained from standard commercial suppliers.

Male Sprague Dawley rats (License No.: SCXK (LIAO) 2008-0002; certificate No. 0003496) of clean grade (200 ± 20 g) were purchased from the Experimental Animal Center of Dalian Medical University. Animals were housed at a rearing temperature of 23 ± 2 °C and relative humidity of 55% ± 2% for one week under standard conditions with a 12 h light/dark cycle (light: 7:00 am to 7:00 pm) before experiments.

A total of 45 rats were divided into five groups: N, M, D, Z, and G (primary control, depression model, low XYS dose, middle XYS dose, and high dose XYS groups, respectively) after one week of adaptive feed. The rats were housed singly, with the exception of the primary control group, which were housed three rats per cage. Primary controls were fed normally, ad libitum. Groups M, G, Z, and D were subject to classic chronic mild unpredictable stimulation each day. Groups D, Z, and G were perfused intragastrically with low, middle, and high dose of XYS, respectively, one hour before stimulation. Groups N and M were perfused intragastrically with normal saline at the same time. The experiment continued for 3 wk. The remaining rats received one of these chronic mild unpredictable irritations daily: bondage, swimming-induced fatigue, electrical stimulation, fasting, or concussion. For bondage, the rat was bound into a narrow tube for 5 h in order to allow only very limited activity space. For swimming-induced fatigue, the rat was placed into water (20 °C, 15 cm) and then removed after 20 min. For electrical stimulation, the rat was placed into a cage connected to an electronic stimulator, and then irritated with 1.5 mA, 45 V, for 4 s at intermittent intervals of 1 min for 30 min each day. Fasting was induced for 24 h once a week. Concussion consisted of placing the rat on a horizontal oscillator and then shocking it at a frequency of once every second for 30 min. XYS gavage doses were (gavage volume 2 mL): 7.65 g/kg per day for the low dose group, 15.3 g/kg per day for the medium dose group, and 30.6 g/kg per day for the high dose group. Primary controls were fed with 2 mL of saline. All experimental protocols were approved by the local Animal Care Committee and conformed to the Guide for the Care and Use of Laboratory Animals published by the Science and Technology Commission of the People’s Republic of China (STCC Publication No. 2, revised 1988).

Conditions such as mood, fur color, irritability, eye mucosal color, and feces color were examined every day.

The body weight of each rat was recorded before commencing the model, on the sixth day of modeling, on the 13^th day of modeling, and on the 21^st day of modeling.

Open-field test was performed before modeling, on the 16^th day of modeling, on the 13^th day of modeling, and on the 21^st day of modeling.

Briefly, rats were placed in the middle of an open field box and had their activity observed for 5 min. Outcome measures included: (1) The number of grids crossed (the number of times three or more paws crossing into adjacent grids); (2) Standing number (the number of times two forelimbs were lifted up from the bottom of the box to make the body upright); and (3) Grooming times (the number of times two forelimbs were used for grooming, scratching, or licking face and paws).

The rats were anesthetized by 10% chloral hydrate (350 mg/kg) by intraperitoneal injection. After sacrifice, the rectum of each rat was washed in ice-cold PBS and bisected longitudinally. The connective tissue was carefully removed from the rectum by mechanical scraping on ice. For RNA and protein extraction, sections of the rectum were flash-frozen in liquid nitrogen and stored at -80 °C. For immunohistochemistry, the rectum was fixed immediately in 4% paraformaldehyde solution at 4 °C for 12 h.

Western blotting analysis was performed to analyze protein expression in the rat rectum samples. Frozen rectum tissue was crushed in liquid nitrogen using a mortar and pestle. Each rectum sample was homogenized with lysis reagent (P0033; Beyotime Chemical Co., Jiangsu, China) according to the manufacturer’s instructions, with the protein concentration then being determined according to the Lowry method using bovine serum albumin as a standard. Total protein was separated on 10% polyacrylamide gel and then transferred onto polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA, United States). The membrane was blocked in 5% (w/v) skimmed milk powder with Tris Buffered Saline-Tween 20 (TBST) for 1 h at 37 °C and then incubated with the primary antibodies, rabbit polyclonal anti-rat NPR-B antibody (bs-2348R; Biosynthesis Biotechnology, Beijing, China; 1:500 dilution) and rabbit polyclonal anti-rat β-actin antibody (bs-0061R; Biosynthesis Biotechnology, Beijing, China; 1:1000 dilution) for 1 h at 37 °C. After incubation, the membrane was washed three times with TBST and then incubated with horseradish peroxidase (HRP)-linked secondary antibody, goat anti-rabbit IgG (ZB-2301; ZSGB-Biotechnology, Beijing, China; 1:5000 dilution) for NPR-B, and β-actin. Probed membrane was immersed for 1 min in enhanced chemiluminescence (ECL) detection reagent (Amersham Biosciences, Piscataway, NJ, United States). After development and image acquisition, the image from each western blotting was quantitatively analyzed using Quantity One software (Bio-Rad Laboratories, CA, United States) and normalized to their corresponding β-actin expression levels.

The localization and distribution of NPR-B and CNP in the rectum was investigated by immunohistochemistry. After being affused with 4% paraformaldehyde (4 °C, 12 h), rectum tissue samples were paraffin-embedded, cut into 5 μm-thick sections, and mounted serially on de-paraffinized and rehydrated glass slides. Sections were deparaffinized in three changes of xylene, hydrated in a graded ethanol series, and then washed in tap water. For antigen retrieval, sections were incubated in 10 mmol/L sodium citrate tissue antigen retrieval solution (pH = 6) that contained 0.05% Tween-20 at a high temperature and pressure for 2 min, and then cooled to room temperature. The sections were washed in distilled water, immersed in PBS, and then incubated with 0.3% H₂O₂ in distilled water for 20 min to block endogenous peroxides. After rinsing three times in PBS for 2 min each at room temperature, slides were incubated for 15 min at 37 °C with normal goat serum (Vector, Burlingame, CA, United States; 1:20) to block non-specific binding. The blocking serum was removed by gentle tapping, with the samples then being incubated in a humidified container with either rabbit polyclonal anti-NPR-B antibody (bs-2348R; Biosynthesis Biotechnology, Beijing, China; 1:75 dilution) or rabbit polyclonal anti-CNP antibody (bs-1000R; Biosynthesis Biotechnology, Beijing, China; 1:400 dilution) for 1 h at 37 °C, and washed with PBS 2 min × 3, followed by incubation in a humidified container with biotin-labeled goat anti-rabbit antibody (KIT-5030, Maixin Biotechnology, Fuzhou, China) for 15 min. Slides were washed with PBS 2 min × 3. The sections were stained with 3, 3’-diaminobenzidine tetrahydrochloride (DAB-0031; Maixin Biotechnology, Fuzhou, China) for approximately 2 min at room temperature and then washed with distilled water. The nuclei were stained with hematoxylin and examined using a LEICA DM 2500 light microscope (Leica Microsystem, ON, Canada). A minimum of six fields were examined for each rectum tissue, with representative tissue sections photographed with LEICA DFC450 (Leica Camera AG, ON, Canada). DAB signal examined as showing immunopositive staining was a “brown” label. The staining indices were compared among five groups and calculated from the staining intensity and percentage of cells staining per section, with three sections per group. Samples were scored from 0 to 4 according to the percentage of positive-stained cells of 0%, 1%-25%, 26%-50%, 51%-75%, and 76%-100%, respectively. Staining intensity was scored from 0 to 3 (0 = negative, 1 = weak, 2 = moderate, and 3 = intense). An histological score formula of ∑pi was used, where “i” represents the score of staining intensity and “p” represents the percentage score of the same staining intensity cells in total cells[25].

Real time fluorescence quantitative PCR (Real time-PCR) was performed to analyze the gene expression levels of NPR-B and CNP in the rat rectum samples. Total RNA was isolated from the rectum as recommended by the manufacturer of RNAiso Plus (9109, Takara Biotechnology Inc., Japan). RNA concentration was determined spectrophotometrically (Bio-photometer Eppendorf, Milan, Italy) at 260 nm and 280 nm. The ratio of readings at 260 nm and 280 nm (A260/A280) provided an estimate of the purity of RNA. The integrity and purity of total RNA was also detected by electrophoresis of samples on GelStar Stain agarose gels. Only samples that showed clear and distinct 28S and 18S ribosomal RNA bands and had spectrophotometric A260/A280 ratios of 1.9-2.1 were used. Reverse transcription was performed as recommended by the manufacturer of the PrimeScript^TM RT reagent kit with gDNA Eraser (RR047A; Takara Biotechnology Inc., Japan), which was incubated at 37 °C for 15 min, followed by incubation at 85 °C for 5 s. cDNA samples were used for analyzing specific cDNA of CNP and NPR-B. cDNA were examined by agarose gel electrophoresis to assure near equivalent levels of cDNA synthesis. Following DNase treatment, real-time PCR was performed in an optical 96-well plate using real-time monitoring of nucleic acid green dye fluorescence (SYBR Green I) with SYBR^® Premix Ex Taq^TM II (RR820A; Takara Biotechnology Inc., Japan) with the StepOnePlus^TM System (Applied Bio-systems). Two-step standard real-time PCR procedure: Stage 1: Reps 1, 95 °C, 30 s; Stage 2: Reps 40, 95 °C, 5 s; 60 °C, 30 s. Table 1 depicts the specific primers for rat β-actin, GAPDH, CNP, and NPR-B (Bioneer, Inc., South Korea) which were used. The genes of β-actin and GAPDH were used as housekeeping gene constitutively expressed to a constant amount in cells, and gene expression of CNP and NPR-B was normalized to β-actin or GAPDH. All samples were amplified in triplicate. Ct values were analyzed and the ΔΔCt value of amplification was calculated. The relative increase/decrease of mRNA in the target genes of the experimental groups were calculated using the model group as the calibrator.

Differences between more than two independent groups were analyzed by Fisher’s test after ANOVA[26]. Where required, the data was transformed by the appropriate method to obtain a normal distribution. All experiments were repeated at least three times. The results are expressed as mean ± SE and a P value < 0.05 was considered significant.

The method of chronic mild unpredictable irritations combined with solitary raising work together to make a depression model. The state, weight, and open-field testing of rats were observed to identify whether the depression model was successful.

Performance of normal rats: Good mood, agile activity, thick and shiny hair, pale pink ear mucosa, normal eating, normal drinking, and normal stool.

Performance of depressed rats: Exhausted demeanor, dull and brown hair, dark eyes with secretions, arched and huddled together, loose stool, weight loss, reduced numbers of crossed-grids, reduced instances of standing, and reduced grooming.

On the 21^st day, the average weight of the rats was 309 ± 1 g in primary control group (N), 254 ± 1 g in depression model group (M), 265 ± 1 g in low dose group (D), 281 ± 2 g in middle dose group (Z), and 296 ± 3 g in High dose group (G). The difference between the M and the N was significant (n = 9, ^bP < 0.01; Figure 1). The difference between the XYS groups and the depression model group was also significant (n = 9, ^bP < 0.01; Figure 1).

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Figure 1 Weight changes of the five groups of rats. Data are presented as mean ± SE. N: Primary control group; M: Depression model group; D; Low dose Xiaoyaosan (XYS) group; Z: Middle dose XYS group; G: High dose XYS group. On the 21^st day, the difference between the depression model group and the primary control group was significant (n = 9; ^bP < 0.01 vs M). The difference between the XYS groups and the depression model group was significant (n = 9; ^bP < 0.01 vs M).

On the 21^st day, the number of crossed-grids, instances of standing, and number of grooming times were compared. The differences between the depression and primary control groups was significant (n = 9, ^bP < 0.01; Figures 2). The differences between the XYS and depression groups were also significant (n = 9, ^bP < 0.01; Figures 2).

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Figure 2 Open-field test of the five groups of rats. A: Number of grids crossed: (instances that three or more paws crossed into adjacent grids); B: Standing number (number of times two forelimbs were lifted up from the bottom of box to make the body upright); C: Grooming times (number of times two forelimbs were used for grooming, scratching, or licking face and paws). Data are presented as mean ± SE. N: Primary control group; M: Depression model group; D: Low dose Xiaoyaosan (XYS) group; Z: Middle dose XYS group; G: High dose XYS group. On the 21^st day, the difference between the depression model group and the primary control group was significant (n = 9; ^bP < 0.01 vs M). The difference between the XYS groups and the depression model group was significant (n = 9; ^bP < 0.01 vs M).

Immunohistochemistry was used to detect the expression of CNP in the rectum of depressed rats. The doses of XYS administered were as follows: 7.65 g/kg per day for D, 15.3 g/kg per day for Z, and 30.6 g/kg per day for G. CNP immunopositive brown granules were detected in the mucosa, muscle, and serosa of the rats’ rectum. CNP positive-staining was evidenced in the nucleus and cytoplasm of smooth muscle cells, basal granular cells, and stromal cells. The results showed that smooth muscle cells (n = 9; Figure 3A) and basal granular cells (n = 9; Figure 3B) in the five groups all expressed CNP, but differentially. The staining index of CNP was 1.50 ± 0.224 in N, 13.00 ± 0.365 in M, 11.67 ± 0.211 in D, 7.50 ± 0.224 in Z, and 4.83 ± 0.307 in G. The expression of CNP in the depression model group (M) was significantly higher than that in the primary control group (N). The high and middle XYS dose groups (G and Z) were not significantly different compared with the primary control group (N), but significantly decreased (n = 9; ^bP < 0.01; Figure 3C) compared with the depression model group (M). The low XYS dose group (D) was not significant different compared with the depression model group (M).

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Figure 3 Immunohistochemical analysis of C-type natriuretic peptide in rat rectum. A: C-type natriuretic peptide (CNP) expression in rectum smooth muscle layer of rats (arrows); B: CNP expression in rectum mucosal/serosal layers of rats (arrows); C: Staining index of CNP expression in the rectum of the five groups. Data are presented as mean ± SE. Arrows denote areas of positivity. N: Primary control group M: Depression model group; D: Low dose Xiaoyaosan (XYS) group; Z: Middle dose XYS group; G: High dose XYS group. The difference between the depression model group and the primary control group was significant (n = 9; ^bP < 0.01 vs M). The high and middle XYS dose groups were significantly different compared with the depression model group (n = 9; ^bP < 0.01 vs M). Scale bar = 50 μm.

The mRNA expression levels of CNP in the various groups corroborated with the immunohistochemical data. The average ratios of CNP/β-actin between the five groups and the depression model group were 0.534 ± 0.013 in G, 0.785 ± 0.017 in Z, 0.967 ± 0.015 in D, 1.000 ± 0.025 in M, and 0.289 ± 0.017 in N (n = 9; ^bP < 0.01; Figure 4A). The average ratios of CNP/GAPDH between the five groups and the depression model group were 0.579 ± 0.072 in G, 0.801 ± 0.041 in Z, 0.984 ± 0.032 in D, 1.000 ± 0.031 in M, and 0.428 ± 0.022 in N (n = 9; ^bP < 0.01; Figure 4B). The expression of CNP in the depression model group (M) was significantly higher than that in the primary control group (N). The high and middle XYS dose groups (G and Z) were significantly decreased compared with the depression model group (M). The low XYS dose group (D) was not significant different compared with the depression model group (M).

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Figure 4 Real-time polymerase chain reaction analysis of C-type natriuretic peptide in rat rectum. A: C-type natriuretic peptide (CNP) mRNA transcript levels were normalized to β-actin. B: CNP mRNA transcript levels were normalized to GAPDH. Values of ΔΔCt are expressed as ratios of model group expression level. β-actin and GAPDH are used as the internal control. Bars represent data presented as mean ± SE. N: Primary control group; M: Depression model group; D: Low dose Xiaoyaosan (XYS) group; Z: Middle dose XYS group; G: High dose XYS group. The difference between the depression model group and the primary control group was significant (n = 9; ^bP < 0.01 vs M). The high and middle XYS dose groups were significantly different compared with the depression model group (n = 9; ^bP < 0.01 vs M).

NPR-B (molecular weight: 120 kDa) expression levels in the rectum of the rats were detected using the method of western blotting. The doses were as follows: 7.65 g/kg per day for the low dose group (D), 15.3 g/kg per day for the medium dose group (Z), and 30.6 g/kg per day for the high dose group (G). The ratio of NPR-B/β-actin was 0.552 ± 0.026 in N, 2.039 ± 0.047 in M, 1.873 ± 0.048 in D, 1.136 ± 0.030 in Z, and 0.814 ± 0.033 in G (n = 9; ^bP < 0.01; Figure 5B).

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Figure 5 Western blotting analysis of natriuretic peptide receptor B expression in rat rectum. A: Natriuretic peptide receptor B (NPR-B) and β-actin protein expression levels in rat rectum tissue. Assayed NPR-B protein or β-actin by immunoblot analysis. β-actin was used as a loading control. Lane N: Primary control group; Lane M: Depression model group; Lane D: Low Xiaoyaosan (XYS) dose group; Lane Z: Middle XYS dose group; Lane G: High XYS dose group. Protein bands have been altered via image processing software (Adobe Photoshop CS3) to get a cleaner background; B: Panel shows the expression of NPR-B and β-actin protein levels by relative optical density measurement in rat rectum. Data are presented as mean ± SE. The difference between the depression model group and the primary control group was significant (n = 9; ^bP < 0.01 vs M). The high and middle XYS dose groups were significantly different compared with the depression model group (n = 9; ^bP < 0.01 vs M). CNP: C-type natriuretic peptide.

Using immunohistochemistry evaluation, NPR-B immunopositive brown granules were expressed in the mucosa, muscle, and serosa of the rats’ rectum. NPR-B positive staining was located in the membrane of smooth muscle cells, basal granular cells, and stromal cells. The immunohistochemistry results showed that smooth muscle cells (n = 9; Figure 6A) and basal granular cells (n = 9; Figure 6B) in the five groups all expressed NPR-B, but differentially. The staining index of NPR-B was 1.33 ± 0.333 in N, 13.17 ± 0.307 in M, 11.50 ± 0.224 in D, 5.83 ± 0.307 in Z, and 4.00 ± 0.258 in G (n = 9, ^bP < 0.01; Figure 6C).

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Figure 6 Immunohistochemical analysis of natriuretic peptide receptor B in rat rectum. A: Natriuretic peptide receptor B (NPR-B) expression in rectum smooth muscle layer of rats; B: NPR-B expression in rectum mucosal/serosal layers of rats; C: Staining index of NPR-B expression in rat rectum of the five groups. Data are presented as mean ± SE. Arrows denote areas of positivity. N: Primary control group; M: Depression model group; D; Low dose Xiaoyaosan (XYS) group; Z: Middle dose XYS group; G: High dose XYS group. The difference between the depression model group and the primary control group was significant (n = 9; ^bP < 0.01 vs M). The high and middle XYS dose groups were significantly different compared with the depression model group (n = 9; ^bP < 0.01 vs M). Scale bar = 50 μm.

The mRNA expression level of NPR-B in the rectum of the rats corroborated with the immunohistochemistry result. The average ratio of NPR-B/β-actin between the five groups and the depression model group were 0.609 ± 0.017 in G, 0.817 ± 0.028 in Z, 0.974 ± 0.022 in D, 1.000 ± 0.030 in M, and 0.313 ± 0.020 in N (n = 9, ^bP < 0.01; Figure 7A). The average ratio of NPR-B/GAPDH between the five groups and the depression model group were 0.614 ± 0.051 in G, 0.862 ± 0.035 in Z, 0.969 ± 0.029 in D, 1.000 ± 0.039 in M, and 0.428 ± 0.015 in N (n = 9, ^bP < 0.01; Figure 7B).

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Figure 7 Real-time polymerase chain reaction analysis of natriuretic peptide receptor B in rat rectum. A: Natriuretic peptide receptor B (NPR-B) mRNA transcript levels were normalized to β-actin; B: NPR-B mRNA transcript levels were normalized to GAPDH. Values of ΔΔCt are expressed as ratios of model group expression level. β-actin and GAPDH are used as the internal control. Bars represent data presented as mean ± SE. N: Primary control group; M: Depression model group; D: Low dose Xiaoyaosan (XYS) group; Z: Middle dose XYS group; G: High dose XYS group. The difference between the depression model group and the primary control group was significant (n = 9; ^bP < 0.01 vs M). The high and middle XYS dose groups were significantly different compared with the depression model group (n = 9; ^bP < 0.01 vs M).

NPR-B mRNA data were consistent with NRP-B IHC and western blotting data. The expression of NPR-B in the depression model group (M) was significantly higher than that in the primary control group (N). The high and middle XYS dose groups (G and Z) were not significantly different compared with the primary control group (N), but significantly decreased compared with the depression model group (M). The low XYS dose group (D) was not significant different compared with the depression model group (M).

A common complication of depression is depressive digestive disorders. However, its pathogenesis is not yet clear. A recent study has indicated that C-type natriuretic peptide (CNP) is secreted from the alimentary canal and plays an inhibitory role in the contraction of smooth muscles. However, whether the expression of CNP is increased in the rectum and its effect on depressive digestive disorders has not been reported.

CNP is distributed in diverse cell types such as cardiac fibroblasts, chondrocytes, glial cells, macrophages, and endothelial cells. Enterochromaffin cells in gastrointestinal mucosa and smooth muscle cells also secrete CNP. However, the other functions of the CNP signal pathway in the gastrointestinal tract in terms of physiological and pathophysiological conditions still need to be explored. In the present study, the possibility as to whether the CNP/natriuretic peptide receptor B (NPR-B) signal pathway is involved in depressive digestive disorders was investigated in depressed rats.

Recent reports have highlighted the pathogenesis of depressive digestive disorders, with previous studies focusing on the relationship between the CNP/NPR-B pathway and gastric motility. This is the first study to report that the expression of CNP and NPR-B in rectum tissue is increased in depressed rats. This study suggests that the CNP/NPR-B signal pathway may be involved in depressive digestive disorders.

By understanding that the CNP/NPR-B signal pathway may be involved in depressive digestive disorders, this study may represent a future strategy for therapeutic or preventive intervention in the treatment of patients with depression.

Depression induces many significant changes in digestive disorders, absorption, endocrine, and immune function dysfunction. However, digestive disorders improve as a precondition to the recovery of absorption, endocrine, and immune function dysfunction. At the same time, digestive disorder diseases are often accompanied by poor mood. NPR-B is the natriuretic peptide receptor for ANP, BNP, and CNP. NPR-B has a considerably higher affinity for CNP than ANP or BNP.

An interesting article pointing to a novel mechanism that may explain key factors in the CNP signaling pathway in depressive digestive disorders. The results are logical, attractive, and congruent. In many ways, the work is interesting, quite novel, and probably worthy of publication.