# Helicid Reverses the Effect of OverexpressingNCALD, Which Blocks the sGC/cGMP/PKG Signaling Pathway in the CUMS-Induced Rat Model

Authors: Xiao-Tong Zhang; Yuan Zhang; Yuan-Xiang Zhang; Zhen-Yi Jiang; Hui Yang; Lan Jiang; Bin Yang; Jiu-Cui Tong
Journal: Journal of Healthcare Engineering
 (2021)

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## Abstract

Background. Increasing evidence has shown that apoptosis in the hippocampus is closely related to depressive-like behavior. We previously reported that helicid had good antidepressant activities, which manifested as the alleviation of depression-like behaviors and the reversal of the high expression of neurocalcin delta (NCALD) in chronic unpredictable mild stress (CUMS) rats. The aim of this study was, therefore, to characterize the antidepressant-like effects and underlying mechanism of helicid on CUMS rats by silencing NCALD and using rescue experiments. Methods. We developed the CUMS rat model using CUMS stimulation from week 0 to week 6. The rats were treated with helicid, or NCALD silenced, then we overexpressed NCALD using adeno-associated virus. We also measured the protein levels of sGCα1, sGCβ1, PKG1/2, and cleaved caspase-3 in hippocampal tissues using western blotting and measured cGMP using an ELISA. Results. Treating CUMS rats by silencing NCALD or by the administration of helicid improved the depressive-like behavior. The levels of proteins, including sGC, PKG, cleaved caspase-3, and cGMP, in hippocampus all decreased. NCALD overexpression reversed these decreases and reversed the alleviation of depression-like behaviors in CUMS rats. Limitation. We only detected the antidepressant effects of helicid in the hippocampus; therefore, other parts of brain should also be studied. Conclusions. Inhibition of NCALD, as well as helicid administration, alleviated antidepressant-like behavior by regulating the expressions of apoptotic cytokines and the sGC/cGMP/PKG signaling pathway. Overexpressing NCALD reversed the amelioration effects of silenced NCALD and helicid administration.


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## Body

## 1. Introduction

As a concerning disorder, depression decreased the quality of human life for its symptoms such as depressed mood and anhedonia. The World Health Organization ranks depression as a major cause of disabilities (accounting for 7.5% of all persons with disabilities in 2015). Depression is also the main cause of suicide-related deaths, with nearly 800,000 suicide deaths each year [1]. The pathogenesis of depression is currently not clear, but the monoamine neurotransmitter hypothesis, neuroendocrine hypothesis, neurotransmitter receptor hypothesis, cytokine hypothesis, neurotrophic hypothesis, and epigenetic hypothesis have all tried to explain the pathogenesis of this disorder. It has also been reported that neuronal apoptosis/death in the brain may play a decisive part in major depressive disorder (MDD) [2]. Previous researches have pointed out that mice with chronic unpredictable mild stress (CUMS)-induced depression showed decreased pleasure, and many of pyramidal cells in the hippocampus decreased, while immobility time and the number of apoptotic cells increased [3]. The reduction in hippocampal volume may be the most common neuroimaging finding associated with MDD [4]. Hippocampal apoptosis leads to depression. It has been reported that apoptosis is expressed more in the dentate gyrus of maternal rats with repeated separation from their pups, with accompanying damage to memory functions and depression-like behaviors [5].Neurocalcin delta (NCALD) is a brain-enriched neuronal calcium sensor, abundantly expressed in the central nervous system and the brain, testis, ovary, and small intestine [6–8]. Several studies have reported that helicid plays a protective role in the cellular inflammatory response by regulating NCALD function [9, 10]. Moreover, NCALD is involved in cyclic guanosine phosphate (cGMP) signaling by regulating the activity of guanylyl cyclase (GC) [11]. Soluble GC can be activated by nitric oxide to release the second messenger, cyclic guanosine monophosphate (cGMP), then cGMP further activates cGMP-dependent protein kinase (PKG) to regulate many physiological functions [12]. Although it is known that sGC and its related cytokines are associated with depression [13], it is not clear whether and how NCALD regulates the sGC/cGMP/PKG signaling pathway.Helicid (Figure1), which is an active component of Helicia nilgirica, is extensively used in the clinic to relieve headache dizziness and sleep disorders and is an auxiliary treatment for primary headaches as well as for relief from depression [14]. Our reports have shown that helicid alleviates depressive behaviors and inflammation, then to reverse the high expression of NCALD through IKK/IkappaBalpha/NF-kappaB pathway in CUMS rats. [15, 16]; however, there is no definitive evidence showing how helicid mediates the apoptosis relative to sGC/cGMP/PKG signaling pathway by regulating NCALD.Figure 1
Structural formula of helicid (4-formylphenyl-β-D-allopyranoside). MW: 284.2 g/mol, SID: 126,631,785 (CID: 4447573800), melting point: 199–200°C.Based on the above theories and previous experimental results, we silenced and overexpressedNCALD to identify the specific mechanisms involving helicid regulation of the sGC/cGMP/PKG signaling pathway.


## 2. Materials and Methods

### 2.1. Animals

Male Sprague-Dawley rats (170–210 g) were from the Qinglong Mountain Animal Breeding Farm (Nanjing, China). The rats were first adapted in cages for a week before the beginning of experiments, including a 12/12 h light/dark cycle (8 AM/8 PM) and a temperature of 23 ± 2°C. We provided them with standard food and water. The procedures for care and use of animals were approved by Wannan Medical College Animal Care Committee.


### 2.2. Drugs and Treatments

The experimental design and specific routes of administration are shown in Figure2. The dose of helicid is 32 mg/kg/day. This dose is an effective dose which has verified in our previous studies [10, 15–17].Figure 2
Timeline of the study. The establishment of CUMS rat model and drug treatment.Helicid (purity ≥98%) was purchased from Tokyo Chemical Industry (Tokyo, Japan). Saline was purchased from Anhui Fengyuan Pharmaceutical (Anhui, China). Adeno-associated virus (AAV), AAV-siNCALD, and AAV-NCALD were purchased from Hanbio Biotechnology (Hanbio, Anhui, China). The sequences of NCALD siRNA are shown in Table 1. As the most preferred option, we selected siRNA-NCALD-003 for RT-PCR.Table 1
The sequences of siRNA-NCALD to be selected.
Primer (5′-3′)siRNA-NCALD-001ATGGAGATGGGACGATAGAsiRNA-NCALD-002ACATCAGCAAGGCAGAATsiRNA-NCALD-003ACACCAACAGAGATGGAAAAfter establishing the CUMS model, 90 rats were randomly assigned to the CUMS (n = 70) and control (n = 20) groups. We divided the experimental protocol to first characterize the effect of NCALD on the CUMS rat model, and then to determine whether helicid played a role in its antidepressant effect through NCALD using rescue experiments. The grouping of experiment 1 rats was as follows (n = 10 per group): control + saline (CON + SAL); CUMS + saline (CUMS + SAL); CUMS + AAV; and CUMS + AAV-siNCALD. The grouping of experiment 2 was as follows (n = 10 per group): control + saline (CON + SAL); CUMS + saline (CUMS + SAL); CUMS + helicid + saline (CUMS + helicid + SAL); CUMS + helicid + AAV; and CUMS + helicid + AAV-NCALD.


### 2.3. Chronic Unpredictable Mild Stress Procedure

CUMS is a mature depression model, which has been studied in the past few years [18]. We subjected the experimental rat group to CUMS stimulation for 12 weeks, which involved (1) light/dark changes, (2) a polluted environment (250 mL water mixed with sawdust cushion), (3) a tail pinch for 1 min, (4) immersion in frigid water (4°C for 5 min), (5) water or food deprivation for 24 h), (6) a tilted cage (45° for 24 h), and (7) a tail suspension for 1 min. In the CUMS procedure, we randomly selected a stimulus each day to ensure that each stimulus was used, and the same stimulus was used at least 1 day apart to ensure unpredictability. The control rat group received no stress stimuli.


### 2.4. Lateral Ventricle Injection

At the seventh week after stimulation, the rats were anesthetized with 1% sodium pentobarbital (50 mg/kg). AAV-NCALD, AAV-siNCALD, or saline was then injected into the right lateral ventricle of rats using a brain stereotaxic apparatus. The specific parameters were as follows: the coordinate of the right midline was 1.5 mm, the caudal was 1.1 mm, and the depth was 4.5 mm. The concentration of AAV was 1 × 1012 copies/mL and the volume was 10 μL. The rat wounds were disinfected and sutured after injection.


### 2.5. Behavioral Testing

#### 2.5.1. The Sucrose Preference Test (SPT) and Body Weight (BW) Measurements

The SPT is a common method for detecting anhedonia. Briefly, rats were given two bottles of sucrose solution and free contact for 12 h to adapt. The bottles were replaced by a bottle of preweighed sucrose solution and a bottle of preweighed pure water. To ensure the randomness of the experiment, the order of the two bottles was constantly changed. Finally, solution consumption in the two bottles was recorded and converted to the sucrose preference ratio (SPR) and calculated using the formula: sucrose intake (g) × 100%/[sucrose intake(g) + water intake (g)].The rats were then weighed once per week.


#### 2.5.2. The Open Field Test (OFT)

The open field test is one of the most popular ethological tests to assess anxiety-like behavior in rodents [19, 20]. The open field apparatus (120 × 120 × 60 cm) consisted of a black metallic enclosure with an open floor. The camera above the apparatus recorded all activities of the rats and recorded them in real time using advanced motion recognition software. The behavior of the rats was automatically recorded for 5 min and analyzed by the software. Total distance, average speed, and motion duration indicated spontaneous activity, and regional crossing indicated the willingness to explore. Before the start of each experiment, the device was cleaned with 10% ethanol and water to remove olfactory cues.


#### 2.5.3. The Forced Swimming Test (FST)

The rats were place in a cylindrical Plexiglas swimming device (25 cm × 65 cm) with a water depth of 35 cm. The water temperature was controlled between 23 and 25°C. On the first day of the experiment, each rat swam for 15 min, after which they were dried and put in a cage. The rats were then tested 24 h later, when they were continuously observed in the water for 5 min to determine the accumulated immobility times in the water, including stoppage of struggling, floating upright, and only occasional body movements to keep the head floating on the surface of the water.


### 2.6. Sample Collection

On the tenth and twelfth weeks of the experiment, the rats were anesthetized with 1% sodium pentobarbital (50 mg/kg) and then decapitated execution in the light environment. The blood collection method is decapitation. After high-speed centrifugation (3,000 × g for 10 min), the upper serum was collected. The hippocampus of the rat was quickly removed and placed in liquid nitrogen for 10 min for rapid freezing, and then stored at −80°C.


### 2.7. ELISA

The ELISA kit (Suzhou Calvin Biotechnology, Jiangsu, China) was used to detected the levels of cGMP. The antigen was diluted to 5μg/mL and coated overnight at room temperature, 4°C, or 37°C with 50 μL antigen per well. The coating buffer was then discarded and the plate was washed with 1 × PBST washing solution (pH 7.2–7.4) three times, with each time being 3–5 min. A total of 250 μL of 1% bovine serum albumin blocking buffer was then added to each well, which was sealed and incubated at 35 ± 2°C for 2 h. The wells were then washed with 1 × PBST three times, for 3–5 min each time. The hippocampal supernatants were diluted with a 1 × PBST buffer at 1 : 1,000, with 100 μL added per well. The negative control was incubated at 37°C for 1.5 h. The 1 × PBST buffer diluted to 1 : 1000 was used to add 50 μL of enzyme-labeled goat anti-mouse horseradish peroxidase-labeled IgG to each well, followed by incubation at 37°C for 1 h. Substrate solution was added to each well, then incubated for 10 min at 37°C with light excluded. After color development, 50 μL of termination solution was added to each well to terminate the reaction. The OD value at 450 nm was read by a microplate reader.


### 2.8. Western Blotting (WB)

Nuclear and cytoplasmic protein extraction kits were used to extract nuclear and cytoplasmic proteins, respectively. The sample was then incubated in boiling water for 5 min. The protein concentration was determined using a BCA kit, and then protein samples were added to the SDS-PAGE gel wells and electrophoresed using a 10% separation gel and 5% stacking gel. The initial voltage during electrophoresis was 90 volts, and the adjusted voltage was 110 volts after the sample entered the separation gel. The protein was then transferred to a polyvinylidene difluoride membrane using 110 volts and a constant current for 75 min. Blocking solution (TBST containing 5% skim milk) was then added to the membrane at room temperature for 2 h, and the blocking solution was discarded, followed by the addition of the following diluted antibodies: anti-NCALD (1 : 1,000) (Cell Signaling Technology, Danvers, MA, USA), anti-PKG1/2 (1 : 1,000) (BioVision, Milpitas, CA, USA), anti-sGCα1 (1 : 1,000) (Cayman Chemical, Ann Arbor, MI, USA), anti-sGCβ1 (1 : 1,000) (Cayman Chemical), and anti-cleaved caspase-3 (1 : 1,000) (Cell Signaling Technology). After overnight incubation at 4°C, the membrane was rinsed three times with TBST, for 10 min each time. The corresponding goat anti-rabbit (1 : 3,000; Abcam, Cambridge, MA, USA) antibody was then added for 2 h, then washed with TBST at room temperature three times, with 10 min each time. After being treated with the ECL chemiluminescent agent, the membranes were exposed to film, exposed, developed, fixed, and washed, and the results were scanned and recorded.


### 2.9. Statistical Analysis

Data are expressed as and analyzed by one-way analysis of variance followed by Turkey’s test.P<0.05 was assumed to be statistically significant. The data were analyzed and plotted using Prism 7.0 software, ImageJ software, and SPSS statistical software.


## 2.1. Animals

Male Sprague-Dawley rats (170–210 g) were from the Qinglong Mountain Animal Breeding Farm (Nanjing, China). The rats were first adapted in cages for a week before the beginning of experiments, including a 12/12 h light/dark cycle (8 AM/8 PM) and a temperature of 23 ± 2°C. We provided them with standard food and water. The procedures for care and use of animals were approved by Wannan Medical College Animal Care Committee.


## 2.2. Drugs and Treatments

The experimental design and specific routes of administration are shown in Figure2. The dose of helicid is 32 mg/kg/day. This dose is an effective dose which has verified in our previous studies [10, 15–17].Figure 2
Timeline of the study. The establishment of CUMS rat model and drug treatment.Helicid (purity ≥98%) was purchased from Tokyo Chemical Industry (Tokyo, Japan). Saline was purchased from Anhui Fengyuan Pharmaceutical (Anhui, China). Adeno-associated virus (AAV), AAV-siNCALD, and AAV-NCALD were purchased from Hanbio Biotechnology (Hanbio, Anhui, China). The sequences of NCALD siRNA are shown in Table 1. As the most preferred option, we selected siRNA-NCALD-003 for RT-PCR.Table 1
The sequences of siRNA-NCALD to be selected.
Primer (5′-3′)siRNA-NCALD-001ATGGAGATGGGACGATAGAsiRNA-NCALD-002ACATCAGCAAGGCAGAATsiRNA-NCALD-003ACACCAACAGAGATGGAAAAfter establishing the CUMS model, 90 rats were randomly assigned to the CUMS (n = 70) and control (n = 20) groups. We divided the experimental protocol to first characterize the effect of NCALD on the CUMS rat model, and then to determine whether helicid played a role in its antidepressant effect through NCALD using rescue experiments. The grouping of experiment 1 rats was as follows (n = 10 per group): control + saline (CON + SAL); CUMS + saline (CUMS + SAL); CUMS + AAV; and CUMS + AAV-siNCALD. The grouping of experiment 2 was as follows (n = 10 per group): control + saline (CON + SAL); CUMS + saline (CUMS + SAL); CUMS + helicid + saline (CUMS + helicid + SAL); CUMS + helicid + AAV; and CUMS + helicid + AAV-NCALD.


## 2.3. Chronic Unpredictable Mild Stress Procedure

CUMS is a mature depression model, which has been studied in the past few years [18]. We subjected the experimental rat group to CUMS stimulation for 12 weeks, which involved (1) light/dark changes, (2) a polluted environment (250 mL water mixed with sawdust cushion), (3) a tail pinch for 1 min, (4) immersion in frigid water (4°C for 5 min), (5) water or food deprivation for 24 h), (6) a tilted cage (45° for 24 h), and (7) a tail suspension for 1 min. In the CUMS procedure, we randomly selected a stimulus each day to ensure that each stimulus was used, and the same stimulus was used at least 1 day apart to ensure unpredictability. The control rat group received no stress stimuli.


## 2.4. Lateral Ventricle Injection

At the seventh week after stimulation, the rats were anesthetized with 1% sodium pentobarbital (50 mg/kg). AAV-NCALD, AAV-siNCALD, or saline was then injected into the right lateral ventricle of rats using a brain stereotaxic apparatus. The specific parameters were as follows: the coordinate of the right midline was 1.5 mm, the caudal was 1.1 mm, and the depth was 4.5 mm. The concentration of AAV was 1 × 1012 copies/mL and the volume was 10 μL. The rat wounds were disinfected and sutured after injection.


## 2.5. Behavioral Testing

### 2.5.1. The Sucrose Preference Test (SPT) and Body Weight (BW) Measurements

The SPT is a common method for detecting anhedonia. Briefly, rats were given two bottles of sucrose solution and free contact for 12 h to adapt. The bottles were replaced by a bottle of preweighed sucrose solution and a bottle of preweighed pure water. To ensure the randomness of the experiment, the order of the two bottles was constantly changed. Finally, solution consumption in the two bottles was recorded and converted to the sucrose preference ratio (SPR) and calculated using the formula: sucrose intake (g) × 100%/[sucrose intake(g) + water intake (g)].The rats were then weighed once per week.


### 2.5.2. The Open Field Test (OFT)

The open field test is one of the most popular ethological tests to assess anxiety-like behavior in rodents [19, 20]. The open field apparatus (120 × 120 × 60 cm) consisted of a black metallic enclosure with an open floor. The camera above the apparatus recorded all activities of the rats and recorded them in real time using advanced motion recognition software. The behavior of the rats was automatically recorded for 5 min and analyzed by the software. Total distance, average speed, and motion duration indicated spontaneous activity, and regional crossing indicated the willingness to explore. Before the start of each experiment, the device was cleaned with 10% ethanol and water to remove olfactory cues.


### 2.5.3. The Forced Swimming Test (FST)

The rats were place in a cylindrical Plexiglas swimming device (25 cm × 65 cm) with a water depth of 35 cm. The water temperature was controlled between 23 and 25°C. On the first day of the experiment, each rat swam for 15 min, after which they were dried and put in a cage. The rats were then tested 24 h later, when they were continuously observed in the water for 5 min to determine the accumulated immobility times in the water, including stoppage of struggling, floating upright, and only occasional body movements to keep the head floating on the surface of the water.


## 2.5.1. The Sucrose Preference Test (SPT) and Body Weight (BW) Measurements

The SPT is a common method for detecting anhedonia. Briefly, rats were given two bottles of sucrose solution and free contact for 12 h to adapt. The bottles were replaced by a bottle of preweighed sucrose solution and a bottle of preweighed pure water. To ensure the randomness of the experiment, the order of the two bottles was constantly changed. Finally, solution consumption in the two bottles was recorded and converted to the sucrose preference ratio (SPR) and calculated using the formula: sucrose intake (g) × 100%/[sucrose intake(g) + water intake (g)].The rats were then weighed once per week.


## 2.5.2. The Open Field Test (OFT)

The open field test is one of the most popular ethological tests to assess anxiety-like behavior in rodents [19, 20]. The open field apparatus (120 × 120 × 60 cm) consisted of a black metallic enclosure with an open floor. The camera above the apparatus recorded all activities of the rats and recorded them in real time using advanced motion recognition software. The behavior of the rats was automatically recorded for 5 min and analyzed by the software. Total distance, average speed, and motion duration indicated spontaneous activity, and regional crossing indicated the willingness to explore. Before the start of each experiment, the device was cleaned with 10% ethanol and water to remove olfactory cues.


## 2.5.3. The Forced Swimming Test (FST)

The rats were place in a cylindrical Plexiglas swimming device (25 cm × 65 cm) with a water depth of 35 cm. The water temperature was controlled between 23 and 25°C. On the first day of the experiment, each rat swam for 15 min, after which they were dried and put in a cage. The rats were then tested 24 h later, when they were continuously observed in the water for 5 min to determine the accumulated immobility times in the water, including stoppage of struggling, floating upright, and only occasional body movements to keep the head floating on the surface of the water.


## 2.6. Sample Collection

On the tenth and twelfth weeks of the experiment, the rats were anesthetized with 1% sodium pentobarbital (50 mg/kg) and then decapitated execution in the light environment. The blood collection method is decapitation. After high-speed centrifugation (3,000 × g for 10 min), the upper serum was collected. The hippocampus of the rat was quickly removed and placed in liquid nitrogen for 10 min for rapid freezing, and then stored at −80°C.


## 2.7. ELISA

The ELISA kit (Suzhou Calvin Biotechnology, Jiangsu, China) was used to detected the levels of cGMP. The antigen was diluted to 5μg/mL and coated overnight at room temperature, 4°C, or 37°C with 50 μL antigen per well. The coating buffer was then discarded and the plate was washed with 1 × PBST washing solution (pH 7.2–7.4) three times, with each time being 3–5 min. A total of 250 μL of 1% bovine serum albumin blocking buffer was then added to each well, which was sealed and incubated at 35 ± 2°C for 2 h. The wells were then washed with 1 × PBST three times, for 3–5 min each time. The hippocampal supernatants were diluted with a 1 × PBST buffer at 1 : 1,000, with 100 μL added per well. The negative control was incubated at 37°C for 1.5 h. The 1 × PBST buffer diluted to 1 : 1000 was used to add 50 μL of enzyme-labeled goat anti-mouse horseradish peroxidase-labeled IgG to each well, followed by incubation at 37°C for 1 h. Substrate solution was added to each well, then incubated for 10 min at 37°C with light excluded. After color development, 50 μL of termination solution was added to each well to terminate the reaction. The OD value at 450 nm was read by a microplate reader.


## 2.8. Western Blotting (WB)

Nuclear and cytoplasmic protein extraction kits were used to extract nuclear and cytoplasmic proteins, respectively. The sample was then incubated in boiling water for 5 min. The protein concentration was determined using a BCA kit, and then protein samples were added to the SDS-PAGE gel wells and electrophoresed using a 10% separation gel and 5% stacking gel. The initial voltage during electrophoresis was 90 volts, and the adjusted voltage was 110 volts after the sample entered the separation gel. The protein was then transferred to a polyvinylidene difluoride membrane using 110 volts and a constant current for 75 min. Blocking solution (TBST containing 5% skim milk) was then added to the membrane at room temperature for 2 h, and the blocking solution was discarded, followed by the addition of the following diluted antibodies: anti-NCALD (1 : 1,000) (Cell Signaling Technology, Danvers, MA, USA), anti-PKG1/2 (1 : 1,000) (BioVision, Milpitas, CA, USA), anti-sGCα1 (1 : 1,000) (Cayman Chemical, Ann Arbor, MI, USA), anti-sGCβ1 (1 : 1,000) (Cayman Chemical), and anti-cleaved caspase-3 (1 : 1,000) (Cell Signaling Technology). After overnight incubation at 4°C, the membrane was rinsed three times with TBST, for 10 min each time. The corresponding goat anti-rabbit (1 : 3,000; Abcam, Cambridge, MA, USA) antibody was then added for 2 h, then washed with TBST at room temperature three times, with 10 min each time. After being treated with the ECL chemiluminescent agent, the membranes were exposed to film, exposed, developed, fixed, and washed, and the results were scanned and recorded.


## 2.9. Statistical Analysis

Data are expressed as and analyzed by one-way analysis of variance followed by Turkey’s test.P<0.05 was assumed to be statistically significant. The data were analyzed and plotted using Prism 7.0 software, ImageJ software, and SPSS statistical software.


## 3. Results

### 3.1. Establishment of the CUMS Model

#### 3.1.1. The Effect of Stimulation on BW

BW loss is a significant indicator of depression [21]. Figure 3(a) shows that the BW of rats in the control and CUMS groups showed different trends during the whole CUMS stimulation. The BW of rats at week 0 did not significantly differ from it of week 1 (P>0.05). However, the slow weight increase of rats in the CUMS group was lower than that in the control group during the same period (P<0.05) from the second week. In the third week, the gaps in growth weights between the two groups further increased (P<0.01). At week 4 to week 6, the differences of weight growths were maximized (P<0.001).Figure 3
The effect of 6 week’s CUMS stimulation on body weight (a), immobility time using the forced swimming test (b), sucrose preference ratio. (c), the indicators of open field test (d) (D1–D3). The results are expressed as the mean ± SEM;n = 20 control, n = 70 CUMS rats. P∗<0.05; ∗∗P<0.01, ∗∗∗P<0.001 versus the control. ns means no statistical differences.


#### 3.1.2. The Effect of Stimulation on Immobility times Using the FST

The FST is an effective method to evaluate depressive-like behavior in rodents [22]. We used the immobility time as an index to reflect the degree of despair of CUMS rats. Figure 3(b) shows that the rats tend to be more immobile when given CUMS stimulation (P<0.001).


#### 3.1.3. The Effect of Stimulation Using the SPT

As a core symptom of depression, anhedonia is characterized by the inability of feeling happiness from milieu exterieur [23]. We used the FST to measure this symptom. It was presented in Figure 3(c) that the control group is similar to the CUMS group in anhedonia during the baseline period, while this indicator of CUMS rats significantly reduced in week 6 (P<0.001).


#### 3.1.4. The Effect of Stimulation on Locomotor Activities Using the OFT

The OFT is a commonly used ethological test, which detects the locomotor activity and exploratory behavior of rats. The total distance, number of rearing, and zone crossing were regarded as indicators to evaluate the locomotor activities of rats in OFT. We found that the indicators above of CUMS rats were all decreased than those of the control group (P<0.001, P<0.05) (Figure 3(d)D1–D3).Together, we were informed from these results that we successfully developed the CUMS rat model.


### 3.2. The Effect of AAV-siNCALD on CUMS Rat Behavior

#### 3.2.1. The Effect of AAV-siNCALD on BW

In general, the body weight of the four groups of rats gradually increased during the entire period of experiment 1. Figure4(a) shows that the weight of rats of both the CUMS + SAL and CUMS + AAV groups were significantly decreased, when compared with the CON + SAL group (P<0.001). However, after NCALD-silenced treatment, the weight of rats was significantly higher than that in the CUMS group and lower than that in the CON + SAL group at week 9 (P<0.01).Figure 4
The effect of AAV-siNCALD on body weight (a), immobility times using the forced swimming test (b), sucrose preference ratio (c), the indicators of open field test (d) (D1–D3). The results are expressed as the mean ± SEM; n = 10. ∗∗∗P<0.001, CUMS + SAL group vs. the CON + SAL group; P#<0.05; P##<0.01, the CUMS + SAL group vs. the CUMS + AAV-siNCALD group. Among all indicators, there is no significant difference between the CON + SAL group and the CUMS + AAV-siNCALD group.


#### 3.2.2. The Effect of AAV-siNCALD on Immobility Time Using the FST

Regarding the FST (Figure4(b)), we found that the rat immobility times for the CUMS + SAL and CUMS + AAV groups were longer than that of the CON + SAL group (P<0.001), and the immobility time subsequently decreased after NCALD silencing (P<0.01).


#### 3.2.3. The Effect of AAV-siNCALD Using the SPT

As demonstrated in Figure4(c), the SPR significantly reduced after CUMS stimulation (P<0.001), but recover to a certain extent after silencing NCALD (P<0.05).


#### 3.2.4. The Effect of AAV-siNCALD on Locomotor Activities Using the OFT

By detecting the indicators mentioned in Section 3.1.4, we found that the indicators all decreased in both the CUMS + SAL and CUMS + AAV groups, when compared with the CON + SAL group (P<0.001), which was enhanced by NCALD silencing (P<0.05) (Figure 4(d)D1–D3).To avoid the influence of saline and AAV, we also tested the CUMS + SAL and CUMS + AAV groups. During the entire period of experiment 1, there was no statistical difference between the two groups (P>0.05).


### 3.3. The Effect of Helicid and Rescue Experiments on CUMS Rat Behavior

#### 3.3.1. The Effect of Helicid andNCALD-Overexpression Treatment on Body Weight

In general, the body weight of the five groups of rats gradually increased during the entire period of experiment 2. Figure5(a) shows that the weight of the CON + SAL group was significantly greater than that for the CUMS + SAL group (P<0.0001). After helicid administration, the body weight of the rats increased when compared with the CUMS group at week 9 (P<0.05) and the weight gap further widened at week 12 (P<0.001). However, treatment of the NCALD-overexpressed rats with helicid flattened the weight gain curve, which was lower than the CUMS + helicid + SAL group at week 11 (P<0.05).Figure 5
The effect of helicid andNCALD overexpression on body weight (a), immobility times using the forced swimming test (b), sucrose preference test (c), the indicators of open field test (d) (D1–D3). The results are expressed as the mean ± SEM; n = 10. ∗∗∗P<0.001; the CUMS + SAL group vs. the CON + SAL group; P###<0.001, the CUMS + helicid + AAV-NCALD group vs. the CUMS + helicid + SAL group; P&<0.05; P&&<0.01; P&&&<0.001; the CUMS + SAL group vs. the CUMS + helicid + SAL group.


#### 3.3.2. The Effect of Helicid andNCALD-Overexpression Treatment on Immobility times Using the FST

Figure5(b) shows that the immobility time of the CUMS + SAL group was significantly greater than that of the CON + SAL group (P<0.001). After helicid administration, the immobility time of rats decreased (P<0.01). Based on the results of helicid administration, we also simultaneously overexpressed NCALD, which showed an increased immobility time for the rats (P<0.05).


#### 3.3.3. The Effect of Helicid andNCALD-Overexpression Treatment Using the SPT

By calculating the sucrose preference, we found that the CON + SAL group preferred sucrose more than the CUMS + SAL group (P<0.001) (Figure 5(c)). The SPR of rats in the CUMS + helicid + SAL group increased after treating with helicid (P<0.001). However, when we overexpressed NCALD, the SPR of rats was lower than that of the CUMS + helicid + SAL group (P<0.005).


#### 3.3.4. The Effect of Helicid andNCALD-Overexpression Treatment on Total Distance, Number of Rearings, and Number of Zone Crossings Using the OFT

Regarding the OFT, the total distance, number of rearings, and number of zone crossings for the CUMS + SAL group were all decreased, when compared with the CON + SAL group (P<0.001). However, the results were altered after helicid administration, showing an increase of the above-listed parameters (P<0.001). After overexpressing NCALD, there was a statistical difference between the CUMS + helicid + SAL and CUMS + helicid + AAV-NCALD groups (P<0.05).In order to avoid the influence of saline and AAV, we tested the CUMS + helicid + SAL and CUMS + helicid + AAV groups. During the entire period of experiment 2, the difference between the two groups was negligible (P>0.05).


### 3.4. Caspase-3 and the sGC/cGMP/PKG Signaling Pathway Changes in the Hippocampus of CUMS Rats

#### 3.4.1. The Effects of AAV-siNCALD on Apoptosis, Cytokines, and the sGC/cGMP/PKG Signaling Pathway in the Hippocampus of CUMS Rats

Numerous antidepressants inhibit hippocampal nerve apoptosis [24, 25]. For this reason, we measured the expressions of cleaved caspase-3 in the hippocampus using WB. Figure 6(b)B2 shows that there was an enhancement of cleaved caspase-3 expression in the CUMS + SAL group, when compared with the CON + SAL group (P<0.001), while after silencing NCALD, a decrease of cleaved caspase-3 expression was found in the CUMS + AAV-siNCALD group (P<0.01).Figure 6
The effect of AAV-siNCALD on the regulation of the sGC/cGMP/PKG signaling pathway and apoptosis cytokine caspase-3 using western blotting and an ELISA. (A1, B1, C1) Representative NCALD, sGCα1, sGCβ1, PKG1/2, cleaved-caspase-3, and β-actin bands. The relative protein levels of NCALD (A2), sGCα1 (C2), sGCβ1 (C3), PKG1/2 (C4), and cleaved-caspase-3 (B2), n = 3. ∗∗P<0.01; ∗∗∗P<0.001 the CUMS + SAL group vs. the CON + SAL group; P#<0.05; P##<0.01, the CUMS + SAL group vs. the CUMS + AAV-siNCALD group. (D1) The level of cGMP in the hippocampus, n = 3. ∗∗∗P<0.001, the CUMS + SAL group vs. the CON + SAL group; P##<0.01, the CUMS + SAL group vs. the CUMS + AA V-siNCALD group. The results are expressed as the mean ± SEM.To verify whether silenced virus affected normal functioning, WB was used to detect the protein level ofNCALD. Figure 6(a)A2 shows that NCALD expression was significantly higher in the CUMS + SAL group, when compared with the CON + SAL group (P<0.01). After injecting AAV-siNCALD, the NCALD expression decreased in the CUMS + AAV-siNCALD group (P<0.05). These results confirmed that the silenced virus worked as expected. WB and ELISA were, respectively, used to examine the expressions of sGCα, sGCβ, PKG, and cGMP in the hippocampus. Compared with the CON + SAL group, the protein levels of sGCα, sGCβ, PKG, and cGMP significantly increased (P<0.001) (Figures 6(c)C2–C4, 6(d)D1), which later declined after AAV-siNCALD administration (P<0.01).To determine the influence of saline and AAV, we tested the CUMS + SAL and CUMS + AAV groups. During the entire period of experiment 1, the statistical differences between the two groups were negligible (P>0.05).


#### 3.4.2. The Effect of Helicid and Rescue Experiments on Apoptosis, Cytokines, And the sGC/cGMP/PKG Signaling Pathway in the Hippocampus of CUMS Rats

As previously mentioned, we detected the expression of apoptosis cytokine cleaved caspase-3 using WB. Figure7(b)B2 shows that the expression of cleaved caspase-3 significantly increased in the CUMS + SAL group when compared with the CON + SAL group (P<0.01). This increase subsequently fell back to a certain extent after treating with helicid (P<0.05), but was increased in the CUMS + helicid + AAV-NCALD group (P<0.05).Figure 7
The effect of helicid and rescue experiment on the regulation on the sGC/cGMP/PKG signaling pathway and apoptosis cytokine caspase-3 using western blotting and an ELISA. (A1, B1, C1) Representative NCALD, sGCα1, sGCβ1, PKG1/2, cleaved-caspase-3, and β-actin bands. The relative protein levels of NCALD (A2), sGCα (C2), sGCβ1 (C3), PKG1/2 (C4), and cleaved-caspase-3 (B2), n = 3. ∗∗P<0.001; ∗∗∗P<0.001, the CUMS + SAL group vs. the CON + SAL group; P#<0.05; P##<0.01; P###<0.001, the CUMS + helicid + AAV-NCALD group vs. the CUMS + helicid + SAL group; P&<0.05; P&&<0.01; P&&&<0.001; the CUMS + SAL group vs. the CUMS + helicid + SAL group. (G) The level of cGMP in the hippocampus, n = 3. ∗∗∗P<0.001, the CUMS + SAL group vs. the CON + SAL group; P###<0.001, the CUMS + helicid + AAV-NCALD group vs. the CUMS + helicid + SAL group; P&&&<0.001; the CUMS + SAL group vs. the CUMS + helicid + SAL group. The results are expressed as the mean ± SEM.Before we initiated experiment 2, we examined the expressions of NCALD in the five groups to ensure that the silenced virus was functioning normally. Figure7(a) A2 shows that NCALD expression was significantly enhanced in the CUMS + SAL group (P<0.001). The levels then decreased after treating with helicid (P<0.01). In rescue experiments, and based on the results of the helicid treatments, we injected AAV-NCALD into the brains and found that NCALD expression increased (P<0.05), which showed that the virus functioned properly. We then detected the expressions of sGCα, sGCβ, PKG, and cleaved caspase-3 in the hippocampus by WB and the expression of cGMP in the hippocampus using an ELISA. The expressions of sGCα, sGCβ, PKG, cGMP, and cleaved caspase-3 all increased after CUMS stimulation in the CUMS + SAL group (P<0.001) (Figures 7(c)C2–C4, 7(d)D1), which significantly decreased after treating with helicid (P<0.001). The expressions of sGCα, sGCβ, PKG, cGMP, and cleaved caspase-3 increased again when we overexpressed NCALD (P<0.01).To determine the influence of saline and AAV, we tested the CUMS + helicid + SAL and CUMS + helicid + AAV groups. During the entire period of experiment 2, the statistical difference was negligible (P>0.05).


## 3.1. Establishment of the CUMS Model

### 3.1.1. The Effect of Stimulation on BW

BW loss is a significant indicator of depression [21]. Figure 3(a) shows that the BW of rats in the control and CUMS groups showed different trends during the whole CUMS stimulation. The BW of rats at week 0 did not significantly differ from it of week 1 (P>0.05). However, the slow weight increase of rats in the CUMS group was lower than that in the control group during the same period (P<0.05) from the second week. In the third week, the gaps in growth weights between the two groups further increased (P<0.01). At week 4 to week 6, the differences of weight growths were maximized (P<0.001).Figure 3
The effect of 6 week’s CUMS stimulation on body weight (a), immobility time using the forced swimming test (b), sucrose preference ratio. (c), the indicators of open field test (d) (D1–D3). The results are expressed as the mean ± SEM;n = 20 control, n = 70 CUMS rats. P∗<0.05; ∗∗P<0.01, ∗∗∗P<0.001 versus the control. ns means no statistical differences.


### 3.1.2. The Effect of Stimulation on Immobility times Using the FST

The FST is an effective method to evaluate depressive-like behavior in rodents [22]. We used the immobility time as an index to reflect the degree of despair of CUMS rats. Figure 3(b) shows that the rats tend to be more immobile when given CUMS stimulation (P<0.001).


### 3.1.3. The Effect of Stimulation Using the SPT

As a core symptom of depression, anhedonia is characterized by the inability of feeling happiness from milieu exterieur [23]. We used the FST to measure this symptom. It was presented in Figure 3(c) that the control group is similar to the CUMS group in anhedonia during the baseline period, while this indicator of CUMS rats significantly reduced in week 6 (P<0.001).


### 3.1.4. The Effect of Stimulation on Locomotor Activities Using the OFT

The OFT is a commonly used ethological test, which detects the locomotor activity and exploratory behavior of rats. The total distance, number of rearing, and zone crossing were regarded as indicators to evaluate the locomotor activities of rats in OFT. We found that the indicators above of CUMS rats were all decreased than those of the control group (P<0.001, P<0.05) (Figure 3(d)D1–D3).Together, we were informed from these results that we successfully developed the CUMS rat model.


## 3.1.1. The Effect of Stimulation on BW

BW loss is a significant indicator of depression [21]. Figure 3(a) shows that the BW of rats in the control and CUMS groups showed different trends during the whole CUMS stimulation. The BW of rats at week 0 did not significantly differ from it of week 1 (P>0.05). However, the slow weight increase of rats in the CUMS group was lower than that in the control group during the same period (P<0.05) from the second week. In the third week, the gaps in growth weights between the two groups further increased (P<0.01). At week 4 to week 6, the differences of weight growths were maximized (P<0.001).Figure 3
The effect of 6 week’s CUMS stimulation on body weight (a), immobility time using the forced swimming test (b), sucrose preference ratio. (c), the indicators of open field test (d) (D1–D3). The results are expressed as the mean ± SEM;n = 20 control, n = 70 CUMS rats. P∗<0.05; ∗∗P<0.01, ∗∗∗P<0.001 versus the control. ns means no statistical differences.


## 3.1.2. The Effect of Stimulation on Immobility times Using the FST

The FST is an effective method to evaluate depressive-like behavior in rodents [22]. We used the immobility time as an index to reflect the degree of despair of CUMS rats. Figure 3(b) shows that the rats tend to be more immobile when given CUMS stimulation (P<0.001).


## 3.1.3. The Effect of Stimulation Using the SPT

As a core symptom of depression, anhedonia is characterized by the inability of feeling happiness from milieu exterieur [23]. We used the FST to measure this symptom. It was presented in Figure 3(c) that the control group is similar to the CUMS group in anhedonia during the baseline period, while this indicator of CUMS rats significantly reduced in week 6 (P<0.001).


## 3.1.4. The Effect of Stimulation on Locomotor Activities Using the OFT

The OFT is a commonly used ethological test, which detects the locomotor activity and exploratory behavior of rats. The total distance, number of rearing, and zone crossing were regarded as indicators to evaluate the locomotor activities of rats in OFT. We found that the indicators above of CUMS rats were all decreased than those of the control group (P<0.001, P<0.05) (Figure 3(d)D1–D3).Together, we were informed from these results that we successfully developed the CUMS rat model.


## 3.2. The Effect of AAV-siNCALD on CUMS Rat Behavior

### 3.2.1. The Effect of AAV-siNCALD on BW

In general, the body weight of the four groups of rats gradually increased during the entire period of experiment 1. Figure4(a) shows that the weight of rats of both the CUMS + SAL and CUMS + AAV groups were significantly decreased, when compared with the CON + SAL group (P<0.001). However, after NCALD-silenced treatment, the weight of rats was significantly higher than that in the CUMS group and lower than that in the CON + SAL group at week 9 (P<0.01).Figure 4
The effect of AAV-siNCALD on body weight (a), immobility times using the forced swimming test (b), sucrose preference ratio (c), the indicators of open field test (d) (D1–D3). The results are expressed as the mean ± SEM; n = 10. ∗∗∗P<0.001, CUMS + SAL group vs. the CON + SAL group; P#<0.05; P##<0.01, the CUMS + SAL group vs. the CUMS + AAV-siNCALD group. Among all indicators, there is no significant difference between the CON + SAL group and the CUMS + AAV-siNCALD group.


### 3.2.2. The Effect of AAV-siNCALD on Immobility Time Using the FST

Regarding the FST (Figure4(b)), we found that the rat immobility times for the CUMS + SAL and CUMS + AAV groups were longer than that of the CON + SAL group (P<0.001), and the immobility time subsequently decreased after NCALD silencing (P<0.01).


### 3.2.3. The Effect of AAV-siNCALD Using the SPT

As demonstrated in Figure4(c), the SPR significantly reduced after CUMS stimulation (P<0.001), but recover to a certain extent after silencing NCALD (P<0.05).


### 3.2.4. The Effect of AAV-siNCALD on Locomotor Activities Using the OFT

By detecting the indicators mentioned in Section 3.1.4, we found that the indicators all decreased in both the CUMS + SAL and CUMS + AAV groups, when compared with the CON + SAL group (P<0.001), which was enhanced by NCALD silencing (P<0.05) (Figure 4(d)D1–D3).To avoid the influence of saline and AAV, we also tested the CUMS + SAL and CUMS + AAV groups. During the entire period of experiment 1, there was no statistical difference between the two groups (P>0.05).


## 3.2.1. The Effect of AAV-siNCALD on BW

In general, the body weight of the four groups of rats gradually increased during the entire period of experiment 1. Figure4(a) shows that the weight of rats of both the CUMS + SAL and CUMS + AAV groups were significantly decreased, when compared with the CON + SAL group (P<0.001). However, after NCALD-silenced treatment, the weight of rats was significantly higher than that in the CUMS group and lower than that in the CON + SAL group at week 9 (P<0.01).Figure 4
The effect of AAV-siNCALD on body weight (a), immobility times using the forced swimming test (b), sucrose preference ratio (c), the indicators of open field test (d) (D1–D3). The results are expressed as the mean ± SEM; n = 10. ∗∗∗P<0.001, CUMS + SAL group vs. the CON + SAL group; P#<0.05; P##<0.01, the CUMS + SAL group vs. the CUMS + AAV-siNCALD group. Among all indicators, there is no significant difference between the CON + SAL group and the CUMS + AAV-siNCALD group.


## 3.2.2. The Effect of AAV-siNCALD on Immobility Time Using the FST

Regarding the FST (Figure4(b)), we found that the rat immobility times for the CUMS + SAL and CUMS + AAV groups were longer than that of the CON + SAL group (P<0.001), and the immobility time subsequently decreased after NCALD silencing (P<0.01).


## 3.2.3. The Effect of AAV-siNCALD Using the SPT

As demonstrated in Figure4(c), the SPR significantly reduced after CUMS stimulation (P<0.001), but recover to a certain extent after silencing NCALD (P<0.05).


## 3.2.4. The Effect of AAV-siNCALD on Locomotor Activities Using the OFT

By detecting the indicators mentioned in Section 3.1.4, we found that the indicators all decreased in both the CUMS + SAL and CUMS + AAV groups, when compared with the CON + SAL group (P<0.001), which was enhanced by NCALD silencing (P<0.05) (Figure 4(d)D1–D3).To avoid the influence of saline and AAV, we also tested the CUMS + SAL and CUMS + AAV groups. During the entire period of experiment 1, there was no statistical difference between the two groups (P>0.05).


## 3.3. The Effect of Helicid and Rescue Experiments on CUMS Rat Behavior

### 3.3.1. The Effect of Helicid andNCALD-Overexpression Treatment on Body Weight

In general, the body weight of the five groups of rats gradually increased during the entire period of experiment 2. Figure5(a) shows that the weight of the CON + SAL group was significantly greater than that for the CUMS + SAL group (P<0.0001). After helicid administration, the body weight of the rats increased when compared with the CUMS group at week 9 (P<0.05) and the weight gap further widened at week 12 (P<0.001). However, treatment of the NCALD-overexpressed rats with helicid flattened the weight gain curve, which was lower than the CUMS + helicid + SAL group at week 11 (P<0.05).Figure 5
The effect of helicid andNCALD overexpression on body weight (a), immobility times using the forced swimming test (b), sucrose preference test (c), the indicators of open field test (d) (D1–D3). The results are expressed as the mean ± SEM; n = 10. ∗∗∗P<0.001; the CUMS + SAL group vs. the CON + SAL group; P###<0.001, the CUMS + helicid + AAV-NCALD group vs. the CUMS + helicid + SAL group; P&<0.05; P&&<0.01; P&&&<0.001; the CUMS + SAL group vs. the CUMS + helicid + SAL group.


### 3.3.2. The Effect of Helicid andNCALD-Overexpression Treatment on Immobility times Using the FST

Figure5(b) shows that the immobility time of the CUMS + SAL group was significantly greater than that of the CON + SAL group (P<0.001). After helicid administration, the immobility time of rats decreased (P<0.01). Based on the results of helicid administration, we also simultaneously overexpressed NCALD, which showed an increased immobility time for the rats (P<0.05).


### 3.3.3. The Effect of Helicid andNCALD-Overexpression Treatment Using the SPT

By calculating the sucrose preference, we found that the CON + SAL group preferred sucrose more than the CUMS + SAL group (P<0.001) (Figure 5(c)). The SPR of rats in the CUMS + helicid + SAL group increased after treating with helicid (P<0.001). However, when we overexpressed NCALD, the SPR of rats was lower than that of the CUMS + helicid + SAL group (P<0.005).


### 3.3.4. The Effect of Helicid andNCALD-Overexpression Treatment on Total Distance, Number of Rearings, and Number of Zone Crossings Using the OFT

Regarding the OFT, the total distance, number of rearings, and number of zone crossings for the CUMS + SAL group were all decreased, when compared with the CON + SAL group (P<0.001). However, the results were altered after helicid administration, showing an increase of the above-listed parameters (P<0.001). After overexpressing NCALD, there was a statistical difference between the CUMS + helicid + SAL and CUMS + helicid + AAV-NCALD groups (P<0.05).In order to avoid the influence of saline and AAV, we tested the CUMS + helicid + SAL and CUMS + helicid + AAV groups. During the entire period of experiment 2, the difference between the two groups was negligible (P>0.05).


## 3.3.1. The Effect of Helicid andNCALD-Overexpression Treatment on Body Weight

In general, the body weight of the five groups of rats gradually increased during the entire period of experiment 2. Figure5(a) shows that the weight of the CON + SAL group was significantly greater than that for the CUMS + SAL group (P<0.0001). After helicid administration, the body weight of the rats increased when compared with the CUMS group at week 9 (P<0.05) and the weight gap further widened at week 12 (P<0.001). However, treatment of the NCALD-overexpressed rats with helicid flattened the weight gain curve, which was lower than the CUMS + helicid + SAL group at week 11 (P<0.05).Figure 5
The effect of helicid andNCALD overexpression on body weight (a), immobility times using the forced swimming test (b), sucrose preference test (c), the indicators of open field test (d) (D1–D3). The results are expressed as the mean ± SEM; n = 10. ∗∗∗P<0.001; the CUMS + SAL group vs. the CON + SAL group; P###<0.001, the CUMS + helicid + AAV-NCALD group vs. the CUMS + helicid + SAL group; P&<0.05; P&&<0.01; P&&&<0.001; the CUMS + SAL group vs. the CUMS + helicid + SAL group.


## 3.3.2. The Effect of Helicid andNCALD-Overexpression Treatment on Immobility times Using the FST

Figure5(b) shows that the immobility time of the CUMS + SAL group was significantly greater than that of the CON + SAL group (P<0.001). After helicid administration, the immobility time of rats decreased (P<0.01). Based on the results of helicid administration, we also simultaneously overexpressed NCALD, which showed an increased immobility time for the rats (P<0.05).


## 3.3.3. The Effect of Helicid andNCALD-Overexpression Treatment Using the SPT

By calculating the sucrose preference, we found that the CON + SAL group preferred sucrose more than the CUMS + SAL group (P<0.001) (Figure 5(c)). The SPR of rats in the CUMS + helicid + SAL group increased after treating with helicid (P<0.001). However, when we overexpressed NCALD, the SPR of rats was lower than that of the CUMS + helicid + SAL group (P<0.005).


## 3.3.4. The Effect of Helicid andNCALD-Overexpression Treatment on Total Distance, Number of Rearings, and Number of Zone Crossings Using the OFT

Regarding the OFT, the total distance, number of rearings, and number of zone crossings for the CUMS + SAL group were all decreased, when compared with the CON + SAL group (P<0.001). However, the results were altered after helicid administration, showing an increase of the above-listed parameters (P<0.001). After overexpressing NCALD, there was a statistical difference between the CUMS + helicid + SAL and CUMS + helicid + AAV-NCALD groups (P<0.05).In order to avoid the influence of saline and AAV, we tested the CUMS + helicid + SAL and CUMS + helicid + AAV groups. During the entire period of experiment 2, the difference between the two groups was negligible (P>0.05).


## 3.4. Caspase-3 and the sGC/cGMP/PKG Signaling Pathway Changes in the Hippocampus of CUMS Rats

### 3.4.1. The Effects of AAV-siNCALD on Apoptosis, Cytokines, and the sGC/cGMP/PKG Signaling Pathway in the Hippocampus of CUMS Rats

Numerous antidepressants inhibit hippocampal nerve apoptosis [24, 25]. For this reason, we measured the expressions of cleaved caspase-3 in the hippocampus using WB. Figure 6(b)B2 shows that there was an enhancement of cleaved caspase-3 expression in the CUMS + SAL group, when compared with the CON + SAL group (P<0.001), while after silencing NCALD, a decrease of cleaved caspase-3 expression was found in the CUMS + AAV-siNCALD group (P<0.01).Figure 6
The effect of AAV-siNCALD on the regulation of the sGC/cGMP/PKG signaling pathway and apoptosis cytokine caspase-3 using western blotting and an ELISA. (A1, B1, C1) Representative NCALD, sGCα1, sGCβ1, PKG1/2, cleaved-caspase-3, and β-actin bands. The relative protein levels of NCALD (A2), sGCα1 (C2), sGCβ1 (C3), PKG1/2 (C4), and cleaved-caspase-3 (B2), n = 3. ∗∗P<0.01; ∗∗∗P<0.001 the CUMS + SAL group vs. the CON + SAL group; P#<0.05; P##<0.01, the CUMS + SAL group vs. the CUMS + AAV-siNCALD group. (D1) The level of cGMP in the hippocampus, n = 3. ∗∗∗P<0.001, the CUMS + SAL group vs. the CON + SAL group; P##<0.01, the CUMS + SAL group vs. the CUMS + AA V-siNCALD group. The results are expressed as the mean ± SEM.To verify whether silenced virus affected normal functioning, WB was used to detect the protein level ofNCALD. Figure 6(a)A2 shows that NCALD expression was significantly higher in the CUMS + SAL group, when compared with the CON + SAL group (P<0.01). After injecting AAV-siNCALD, the NCALD expression decreased in the CUMS + AAV-siNCALD group (P<0.05). These results confirmed that the silenced virus worked as expected. WB and ELISA were, respectively, used to examine the expressions of sGCα, sGCβ, PKG, and cGMP in the hippocampus. Compared with the CON + SAL group, the protein levels of sGCα, sGCβ, PKG, and cGMP significantly increased (P<0.001) (Figures 6(c)C2–C4, 6(d)D1), which later declined after AAV-siNCALD administration (P<0.01).To determine the influence of saline and AAV, we tested the CUMS + SAL and CUMS + AAV groups. During the entire period of experiment 1, the statistical differences between the two groups were negligible (P>0.05).


### 3.4.2. The Effect of Helicid and Rescue Experiments on Apoptosis, Cytokines, And the sGC/cGMP/PKG Signaling Pathway in the Hippocampus of CUMS Rats

As previously mentioned, we detected the expression of apoptosis cytokine cleaved caspase-3 using WB. Figure7(b)B2 shows that the expression of cleaved caspase-3 significantly increased in the CUMS + SAL group when compared with the CON + SAL group (P<0.01). This increase subsequently fell back to a certain extent after treating with helicid (P<0.05), but was increased in the CUMS + helicid + AAV-NCALD group (P<0.05).Figure 7
The effect of helicid and rescue experiment on the regulation on the sGC/cGMP/PKG signaling pathway and apoptosis cytokine caspase-3 using western blotting and an ELISA. (A1, B1, C1) Representative NCALD, sGCα1, sGCβ1, PKG1/2, cleaved-caspase-3, and β-actin bands. The relative protein levels of NCALD (A2), sGCα (C2), sGCβ1 (C3), PKG1/2 (C4), and cleaved-caspase-3 (B2), n = 3. ∗∗P<0.001; ∗∗∗P<0.001, the CUMS + SAL group vs. the CON + SAL group; P#<0.05; P##<0.01; P###<0.001, the CUMS + helicid + AAV-NCALD group vs. the CUMS + helicid + SAL group; P&<0.05; P&&<0.01; P&&&<0.001; the CUMS + SAL group vs. the CUMS + helicid + SAL group. (G) The level of cGMP in the hippocampus, n = 3. ∗∗∗P<0.001, the CUMS + SAL group vs. the CON + SAL group; P###<0.001, the CUMS + helicid + AAV-NCALD group vs. the CUMS + helicid + SAL group; P&&&<0.001; the CUMS + SAL group vs. the CUMS + helicid + SAL group. The results are expressed as the mean ± SEM.Before we initiated experiment 2, we examined the expressions of NCALD in the five groups to ensure that the silenced virus was functioning normally. Figure7(a) A2 shows that NCALD expression was significantly enhanced in the CUMS + SAL group (P<0.001). The levels then decreased after treating with helicid (P<0.01). In rescue experiments, and based on the results of the helicid treatments, we injected AAV-NCALD into the brains and found that NCALD expression increased (P<0.05), which showed that the virus functioned properly. We then detected the expressions of sGCα, sGCβ, PKG, and cleaved caspase-3 in the hippocampus by WB and the expression of cGMP in the hippocampus using an ELISA. The expressions of sGCα, sGCβ, PKG, cGMP, and cleaved caspase-3 all increased after CUMS stimulation in the CUMS + SAL group (P<0.001) (Figures 7(c)C2–C4, 7(d)D1), which significantly decreased after treating with helicid (P<0.001). The expressions of sGCα, sGCβ, PKG, cGMP, and cleaved caspase-3 increased again when we overexpressed NCALD (P<0.01).To determine the influence of saline and AAV, we tested the CUMS + helicid + SAL and CUMS + helicid + AAV groups. During the entire period of experiment 2, the statistical difference was negligible (P>0.05).


## 3.4.1. The Effects of AAV-siNCALD on Apoptosis, Cytokines, and the sGC/cGMP/PKG Signaling Pathway in the Hippocampus of CUMS Rats

Numerous antidepressants inhibit hippocampal nerve apoptosis [24, 25]. For this reason, we measured the expressions of cleaved caspase-3 in the hippocampus using WB. Figure 6(b)B2 shows that there was an enhancement of cleaved caspase-3 expression in the CUMS + SAL group, when compared with the CON + SAL group (P<0.001), while after silencing NCALD, a decrease of cleaved caspase-3 expression was found in the CUMS + AAV-siNCALD group (P<0.01).Figure 6
The effect of AAV-siNCALD on the regulation of the sGC/cGMP/PKG signaling pathway and apoptosis cytokine caspase-3 using western blotting and an ELISA. (A1, B1, C1) Representative NCALD, sGCα1, sGCβ1, PKG1/2, cleaved-caspase-3, and β-actin bands. The relative protein levels of NCALD (A2), sGCα1 (C2), sGCβ1 (C3), PKG1/2 (C4), and cleaved-caspase-3 (B2), n = 3. ∗∗P<0.01; ∗∗∗P<0.001 the CUMS + SAL group vs. the CON + SAL group; P#<0.05; P##<0.01, the CUMS + SAL group vs. the CUMS + AAV-siNCALD group. (D1) The level of cGMP in the hippocampus, n = 3. ∗∗∗P<0.001, the CUMS + SAL group vs. the CON + SAL group; P##<0.01, the CUMS + SAL group vs. the CUMS + AA V-siNCALD group. The results are expressed as the mean ± SEM.To verify whether silenced virus affected normal functioning, WB was used to detect the protein level ofNCALD. Figure 6(a)A2 shows that NCALD expression was significantly higher in the CUMS + SAL group, when compared with the CON + SAL group (P<0.01). After injecting AAV-siNCALD, the NCALD expression decreased in the CUMS + AAV-siNCALD group (P<0.05). These results confirmed that the silenced virus worked as expected. WB and ELISA were, respectively, used to examine the expressions of sGCα, sGCβ, PKG, and cGMP in the hippocampus. Compared with the CON + SAL group, the protein levels of sGCα, sGCβ, PKG, and cGMP significantly increased (P<0.001) (Figures 6(c)C2–C4, 6(d)D1), which later declined after AAV-siNCALD administration (P<0.01).To determine the influence of saline and AAV, we tested the CUMS + SAL and CUMS + AAV groups. During the entire period of experiment 1, the statistical differences between the two groups were negligible (P>0.05).


## 3.4.2. The Effect of Helicid and Rescue Experiments on Apoptosis, Cytokines, And the sGC/cGMP/PKG Signaling Pathway in the Hippocampus of CUMS Rats

As previously mentioned, we detected the expression of apoptosis cytokine cleaved caspase-3 using WB. Figure7(b)B2 shows that the expression of cleaved caspase-3 significantly increased in the CUMS + SAL group when compared with the CON + SAL group (P<0.01). This increase subsequently fell back to a certain extent after treating with helicid (P<0.05), but was increased in the CUMS + helicid + AAV-NCALD group (P<0.05).Figure 7
The effect of helicid and rescue experiment on the regulation on the sGC/cGMP/PKG signaling pathway and apoptosis cytokine caspase-3 using western blotting and an ELISA. (A1, B1, C1) Representative NCALD, sGCα1, sGCβ1, PKG1/2, cleaved-caspase-3, and β-actin bands. The relative protein levels of NCALD (A2), sGCα (C2), sGCβ1 (C3), PKG1/2 (C4), and cleaved-caspase-3 (B2), n = 3. ∗∗P<0.001; ∗∗∗P<0.001, the CUMS + SAL group vs. the CON + SAL group; P#<0.05; P##<0.01; P###<0.001, the CUMS + helicid + AAV-NCALD group vs. the CUMS + helicid + SAL group; P&<0.05; P&&<0.01; P&&&<0.001; the CUMS + SAL group vs. the CUMS + helicid + SAL group. (G) The level of cGMP in the hippocampus, n = 3. ∗∗∗P<0.001, the CUMS + SAL group vs. the CON + SAL group; P###<0.001, the CUMS + helicid + AAV-NCALD group vs. the CUMS + helicid + SAL group; P&&&<0.001; the CUMS + SAL group vs. the CUMS + helicid + SAL group. The results are expressed as the mean ± SEM.Before we initiated experiment 2, we examined the expressions of NCALD in the five groups to ensure that the silenced virus was functioning normally. Figure7(a) A2 shows that NCALD expression was significantly enhanced in the CUMS + SAL group (P<0.001). The levels then decreased after treating with helicid (P<0.01). In rescue experiments, and based on the results of the helicid treatments, we injected AAV-NCALD into the brains and found that NCALD expression increased (P<0.05), which showed that the virus functioned properly. We then detected the expressions of sGCα, sGCβ, PKG, and cleaved caspase-3 in the hippocampus by WB and the expression of cGMP in the hippocampus using an ELISA. The expressions of sGCα, sGCβ, PKG, cGMP, and cleaved caspase-3 all increased after CUMS stimulation in the CUMS + SAL group (P<0.001) (Figures 7(c)C2–C4, 7(d)D1), which significantly decreased after treating with helicid (P<0.001). The expressions of sGCα, sGCβ, PKG, cGMP, and cleaved caspase-3 increased again when we overexpressed NCALD (P<0.01).To determine the influence of saline and AAV, we tested the CUMS + helicid + SAL and CUMS + helicid + AAV groups. During the entire period of experiment 2, the statistical difference was negligible (P>0.05).


## 4. Discussion

As a nonenveloped virus, adeno-associated virus (AAV) found in more than 50 years was one of the safest gene therapies in clinic [26]. To date, there have been a large number of preclinical data that indicate that AAV is viable as a carrier to treat central nervous system (CNS) diseases. Also, there have been many experiments that confirm that AAV can successfully achieve overexpression and silencing of genes in depression [27–29]. For these reasons, we selected AAV to achieve overexpression and silencing of NCALD. Further, to clarify the role played by NCALD in depression.CUMS is presently the most common, effective, and reliable depression model in animals. It is also closely related to the anhedonic behavior of animals [30]. It is also worth noting that CUMS overcomes possible stress adaptability issues, when compared with other depression models [31]. Helicid is an herbal medicine with a variety of pharmacological effects and biological activities, which has been shown to have an antidepressant effect. We previously showed that helicid administration relieved the depression-like behavior of CUMS rats [15, 17]. NCALD is a member of the neurotroponin family and was highly expressed in CUMS rats according to the results of WB. We therefore concluded that silencing NCALD significantly increased the body weight, sucrose preference using the SPT, immobility time using the FST, as well as number of rearings and other indicators using the OPT, which was equivalent to the antidepressant effect of helicid. After we overexpressed NCALD after helicid administration, the depression-like behaviors of CUMS rats tended to become more serious.Apoptosis is regulated by caspases, which are members of the cysteine protease family, with caspase-3 being the main inducer of apoptosis [32]. Accumulating evidence has shown that chronic stress can lead to depression-like behavior and neuronal reduction by activating inflammation and apoptosis. Antidepressant therapy may therefore help to inhibit cell apoptosis and may have neuroprotective effects [33]. In the present study, we used caspase-3 as an indicator of apoptosis. The results showed that the upregulation of caspase-3 in CUMS rats was moderated by silencing of NCALD. We also found that helicid administration alleviated the upregulation of caspase in CUMS rats, while the effect was reversed by overexpressing NCALD.The sGC is a mammalian nitric oxide (NO) sensor. Because NO integrates with sGC heme, its guanylate cyclase activity was used to produce cGMP, thereby regulating various cell signaling functions. The present study confirmed that NO decreased apoptosis through the sGC/cGMP signaling pathway, providing a new perspective for this pathway in the treatment of neurodegenerative diseases [34]. WB showed that the content of sGC protein in the hippocampus of CUMS rats increased, as well as the contents of cGMP detected by an ELISA, indicating that the sGC/cGMP signaling pathway was activated in CUMS rats. Silencing of NCALD expression and helicid administration inhibited the expression of this signal pathway, while the overexpression of NCALD promoted it, which was reversed by helicid administration. All these results suggested that helicid relieved depressive symptoms and that of NCALD might be involved in regulating the sGC/cGMP-related pathway. The cGMP-dependent PKG is expressed more abundantly in intestinal mucosa and the brain and is involved in a variety of pathological and physiological processes. Numerous reports have shown that the upregulation of PKG induced the apoptosis of cancer cells and suppressed its proliferation and metastasis [35]. We hypothesized that there was an association between the sGC/cGMP/PKG signaling pathway and hippocampal neuronal apoptosis in patients with depression. Our study showed that the levels of PKG protein in the hippocampus of CUMS-stimulated and NCALD-overexpressed rats increased, while the results were altered after we treated them with helicid and silenced NCALD.Most importantly, we showed that helicid administration exerted its antidepressant effect throughNCALD in CUMS rats by inhibiting hippocampal apoptosis and alleviating the activation of the sGC/cGMP/PKG pathway. As a natural antidepressant, helicid is not widely used in clinics, but it has less side effects and a wider range of treatments. The results of this study therefore provided important information for the possible clinical applications of helicid and NCALD.



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*Source: 7168397-2021-12-11.xml*