Elucidating the Mechanism of the Liqi Yangyin Formula in Treating Depression-Constipation Comorbidity: An Integrative Approach Using Network Pharmacology and Experimental Validation methods
Aim. Evidence-backed execution summary for Elucidating the Mechanism of the Liqi Yangyin Formula in Treating Depression-Constipation Comorbidity: An Integrative Approach Using Network Pharmacology and Experimental Validation methods from Elucidating the Mechanism of the Liqi Yangyin Formula in Treating Depression-Constipation Comorbidity: An Integrative Approach Using Network Pharmacology and Experimental Validation.
Show snapshot details
On this page
This experiment, in seven questions
Jump straight to the part of the recipe you need. Data and provenance labels stay close to the action they support.
Shopping and prep list
What do I need before I start?
mouse
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
4. Materials and Methods
reagent used in the protocol.
- Use
- The crude materials for the LQYY formula were purchased from Jiangsu Huahong Pharmaceutical Technology Co., Ltd. (Zhenjiang, China), and marked with their respective batch numbers: Nanshashen (Cat: 240620); Maidong (Cat: 241203); Xuanshen (Cat: 240905); Shudihuang (Cat: 241012); Baizhu (Cat: 250331); Zhiqiao (Cat: 2...
4. Materials and Methods
reagent used in the protocol.
- Use
- For metabolite extraction, 200 µL of the filtrate was transferred to a 1.5 mL microcentrifuge tube and mixed with 200 µL of 70% methanol containing the internal standard. Samples were vortex-mixed for 15 min at 12,000 rpm at 4 °C and centrifuged for 3 min. The supernatant was subjected to UPLC-ESI-MS/...
4.2. Network Pharmacology
reagent used in the protocol.
- Use
- To ensure comprehensive and accurate coverage, all compounds detected by UPLC-MS/MS were screened in the Traditional Chinese Medicine Systems Pharmacology database (TCMSP; https://tcmsp-e.com/tcmsp.php (accessed on 5 June 2025)). Retention criteria were oral bioavailability (OB) ≥ 30%, drug-likeness (DL) Ͱ...
4.3. Molecular Docking
reagent used in the protocol.
- Use
- Molecular docking was used to validate compound-target interactions and estimate binding affinities. The six consensus hub proteins identified by CytoHubba were treated as receptor targets, and the ten highest-degree components from the component-target-pathway-disease network were selected a...
4.4.6. First Black Stool
reagent used in the protocol.
- Use
- A charcoal solution was prepared by dissolving gum acacia (1 g) in 80 mL distilled water with heating until clear, adding kaolin (5 g), boiling the mixture three times, cooling to room temperature, and adjusting to 100 mL with distilled water to obtain a stable suspending vehicle. An activated carbon (charcoal) susp...
4.4.9. Quantitative Real-Time PCR
reagent used in the protocol.
- Use
- Total RNA was isolated from PFC and colonic tissues using Trizol reagent (15596026CN, Invitrogen) and reverse-transcribed into cDNA using the ReverTra Ace qPCR RT kit (FSQ-301, ToYoBo). Quantitative PCR (qPCR) was performed on a QuantStudio™ 5 Real-Time PCR System using SYBR Green Realtime PCR Master Mix (QPK-...
4.4.10. TUNEL Staining
reagent used in the protocol.
- Use
- Paraffin-embedded PFC sections were deparaffinized in xylene, rehydrated through graded ethanol to water, and treated with Proteinase K. Sections were rinsed with PBS and gently air-dried. TUNEL reaction mixture (G1510, Servicebio, Wuhan, China) was applied per the manufacturer's instructions and incubated at...
4.4.11. Immunofluorescence Staining
reagent used in the protocol.
- Use
- Paraffin sections (4 µm) of PFC were deparaffinized in xylene, rehydrated with ethanol and water, and subjected to heat-induced antigen retrieval in citrate buffer (pH 6.0). After PBS washes, endogenous peroxidase activity was quenched with 3% H 2 O 2 for 15 min at room temperature, followed by additional PBS w...
4. Materials and Methods
For metabolite extraction, 200 µL of the filtrate was transferred to a 1.5 mL microcentrifuge tube and mixed with 200 µL of 70% methanol containing the internal standard. Samples were vortex-mixed for 15 min at 12,000 rpm at 4 °C and centrifuged for 3 min. The supernatant was subjected to UPLC-ESI-MS/...
- Use
- For metabolite extraction, 200 µL of the filtrate was transferred to a 1.5 mL microcentrifuge tube and mixed with 200 µL of 70% methanol containing the internal standard. Samples were vortex-mixed for 15 min at 12,000 rpm at 4 °C and centrifuged for 3 min. The supernatant was subjected to UPLC-ESI-MS/...
4.4.3. Open Field Test (OFT)
The OFT was used to evaluate anxiety- and locomotion-related behaviors. Mice were placed in a 45 cm × 45 cm × 45 cm arena for a 5 min acclimation, and then returned to the starting position for a 5 min recording session. Movements were tracked with an automated system (TopScan v3.0, Clever Sys Inc., Reston...
- Use
- The OFT was used to evaluate anxiety- and locomotion-related behaviors. Mice were placed in a 45 cm × 45 cm × 45 cm arena for a 5 min acclimation, and then returned to the starting position for a 5 min recording session. Movements were tracked with an automated system (TopScan v3.0, Clever Sys Inc., Reston...
4.4. Experimental Validation
After a 7-day acclimation, mice were randomized into five groups ( n = 8/group): Control, CUMS, LQYY, mosapride citrate (MC), and fluoxetine (FLX). Except for the Control group, all groups underwent the 6-week CUMS procedure and then received daily oral treatment for 4 weeks: LQYY (30 g·kg -1 ·day &#...
- Use
- After a 7-day acclimation, mice were randomized into five groups ( n = 8/group): Control, CUMS, LQYY, mosapride citrate (MC), and fluoxetine (FLX). Except for the Control group, all groups underwent the 6-week CUMS procedure and then received daily oral treatment for 4 weeks: LQYY (30 g·kg -1 ·day &#...
4.4.8. Histopathological Testing of PFC and Colon
PFC and colonic tissues were fixed in 4% paraformaldehyde for 48 h, embedded in paraffin, and sectioned at 4 µm. PFC and colonic tissue sections were stained via hematoxylin-eosin (H&E) to evaluate histological structure. Additionally, PFC tissue sections underwent Nissl staining to assess neuronal struct...
- Use
- PFC and colonic tissues were fixed in 4% paraformaldehyde for 48 h, embedded in paraffin, and sectioned at 4 µm. PFC and colonic tissue sections were stained via hematoxylin-eosin (H&E) to evaluate histological structure. Additionally, PFC tissue sections underwent Nissl staining to assess neuronal struct...
4.4.9. Quantitative Real-Time PCR
Total RNA was isolated from PFC and colonic tissues using Trizol reagent (15596026CN, Invitrogen) and reverse-transcribed into cDNA using the ReverTra Ace qPCR RT kit (FSQ-301, ToYoBo). Quantitative PCR (qPCR) was performed on a QuantStudio™ 5 Real-Time PCR System using SYBR Green Realtime PCR Master Mix (QPK-...
- Use
- Total RNA was isolated from PFC and colonic tissues using Trizol reagent (15596026CN, Invitrogen) and reverse-transcribed into cDNA using the ReverTra Ace qPCR RT kit (FSQ-301, ToYoBo). Quantitative PCR (qPCR) was performed on a QuantStudio™ 5 Real-Time PCR System using SYBR Green Realtime PCR Master Mix (QPK-...
4.4.10. TUNEL Staining
Paraffin-embedded PFC sections were deparaffinized in xylene, rehydrated through graded ethanol to water, and treated with Proteinase K. Sections were rinsed with PBS and gently air-dried. TUNEL reaction mixture (G1510, Servicebio, Wuhan, China) was applied per the manufacturer's instructions and incubated at...
- Use
- Paraffin-embedded PFC sections were deparaffinized in xylene, rehydrated through graded ethanol to water, and treated with Proteinase K. Sections were rinsed with PBS and gently air-dried. TUNEL reaction mixture (G1510, Servicebio, Wuhan, China) was applied per the manufacturer's instructions and incubated at...
4.4.11. Immunofluorescence Staining
Paraffin sections (4 µm) of PFC were deparaffinized in xylene, rehydrated with ethanol and water, and subjected to heat-induced antigen retrieval in citrate buffer (pH 6.0). After PBS washes, endogenous peroxidase activity was quenched with 3% H 2 O 2 for 15 min at room temperature, followed by additional PBS w...
- Use
- Paraffin sections (4 µm) of PFC were deparaffinized in xylene, rehydrated with ethanol and water, and subjected to heat-induced antigen retrieval in citrate buffer (pH 6.0). After PBS washes, endogenous peroxidase activity was quenched with 3% H 2 O 2 for 15 min at room temperature, followed by additional PBS w...
4.4.13. Statistical Analysis
Software used for acquisition, scoring, statistics, or reporting.
- Use
- Analyses were performed using IBM SPSS Statistics 25.0 and GraphPad Prism 9.5. Data are expressed as mean ± SD from at least three independent experiments. Group differences were assessed by one-way ANOVA followed by Tukey's multiple-comparisons test. When normality or homoscedasticity assumptions were vi...
Before you run
What should be confirmed before execution?
First confirmation
Equipment is listed but no product mappings are linked.
Confirm before execution
This page is backed by a publishable Replication Data Ledger package with zero critical source-verification issues.
Confirm before execution
Open the source paper before finalizing run-specific details.
Procurement checkpoint
Use source-stated vendors where present. Treat mapped products as sourcing options unless the page marks an exact source match.
Open quote workflowStep-by-step procedure
What do I do, in order?
2.8. LQYY Attenuates CUMS-Induced Depression-Like Behaviors and Pathological Damage of PFC in Mice
The CUMS model was used to evaluate LQYY after 6 weeks of stress exposure ( A). Compared with Controls, CUMS mice showed typical depression-like phenotypes: reduced body weight, lower sucrose preference, decreased total distance traveled and time in the center zone in the OFT (with thigmotactic trajectories), and increased immobility in the FST. Treatment with LQYY or FLX significantly mitigated these abnormalities ( B-G). Consistently, mice subjected to CUMS exhibited significant neuronal pathology in the PFC, characterized by disorganized neuronal soma arrangement, nuclear pyknosis, and deep nuclear staining, and Nissl staining revealed a marked reduction or loss of Nissl bodies in PFC neurons, whereas LQYY and FLX attenuated these histopathological changes ( H). These histopathological improvements provide direct visual evidence of LQYY's neuroprotective efficacy, effec...
4. Materials and Methods
The crude materials for the LQYY formula were purchased from Jiangsu Huahong Pharmaceutical Technology Co., Ltd. (Zhenjiang, China), and marked with their respective batch numbers: Nanshashen (Cat: 240620); Maidong (Cat: 241203); Xuanshen (Cat: 240905); Shudihuang (Cat: 241012); Baizhu (Cat: 250331); Zhiqiao (Cat: 241111); Houpo (Cat: 250205); Gualouren (Cat: 250217); Huomaren (Cat: 241209); Yuliren (Cat: 241218); Muxiang (Cat: 230710); and Xingren (Cat: 241115). The total crude drug weight (228 g) was soaked in water for 45 min and decocted twice, and the combined filtrates were concentrated to a final concentration of 3 g/mL.
4. Materials and Methods
For metabolite extraction, 200 µL of the filtrate was transferred to a 1.5 mL microcentrifuge tube and mixed with 200 µL of 70% methanol containing the internal standard. Samples were vortex-mixed for 15 min at 12,000 rpm at 4 °C and centrifuged for 3 min. The supernatant was subjected to UPLC-ESI-MS/MS. Chromatographic analysis was performed on a UPLC system coupled via electrospray ionization to a triple quadrupole/linear ion trap (QTRAP) mass spectrometer. Separation used an Agilent SB-C18 column (2.1 mm × 100 mm, 1.8 µm). Mobile phase A was water with 0.1% formic acid; mobile phase B was acetonitrile with 0.1% formic acid. The gradient program was as follows: 0.0 min, 95:5 (A:B); ramp to 5:95 by 12.0 min; and re-equilibrate to 95:5 by 14.0 min. Flow rate was 0.35 mL·min -1; injection volume was 2 µL. ESI source settings were as follows: te...
4.4.3. Open Field Test (OFT)
The OFT was used to evaluate anxiety- and locomotion-related behaviors. Mice were placed in a 45 cm × 45 cm × 45 cm arena for a 5 min acclimation, and then returned to the starting position for a 5 min recording session. Movements were tracked with an automated system (TopScan v3.0, Clever Sys Inc., Reston, VA, USA). Outcomes included time spent in the central zone and total distance traveled. The apparatus was cleaned with 75% ethanol between trials to eliminate olfactory cues.
4.4. Experimental Validation
A CUMS model was used as previously described [ ]. Mice were exposed daily, for six weeks, to a random pair of two distinct stressors selected from the following: 24 h water and food deprivation; 45° cage tilt (24 h); empty-cage exposure (24 h); horizontal shaking (10 min); swimming in 40 °C water (10 min); reversal of the light/dark cycle (24 h); tail pinch (5 min); wet bedding (24 h); and restraint (3 h).
4.4. Experimental Validation
After a 7-day acclimation, mice were randomized into five groups ( n = 8/group): Control, CUMS, LQYY, mosapride citrate (MC), and fluoxetine (FLX). Except for the Control group, all groups underwent the 6-week CUMS procedure and then received daily oral treatment for 4 weeks: LQYY (30 g·kg -1 ·day -1 ), MC (3 mg·kg -1 ·day -1; H19990313; Chengdu Kanghong Pharmaceutical Group Co., Ltd., Chengdu, China), or FLX (10 mg·kg -1 ·day -1; HJ20181215; Patheon France, Évreux, France). The dose of LQYY was determined by converting the clinical human dose to a mouse-equivalent dose based on body surface area, followed by confirmation in a pilot dose-finding experiment, and the doses of MC and FLX were selected based on studies and verified in our laboratory [ ]. The treatment period was set according to previous studies, whi...
4.4.4. Forced Swimming Test (FST)
The FST was performed as described previously [ ]. Each mouse was placed individually in a transparent cylinder (height 25 cm, diameter 10 cm) containing 20 cm of water maintained at 25 ± 1 °C. Behavior was recorded for 6 min in a quiet room. Post-test, the immobility time of each mouse during the final 4 min of swimming was measured using a stopwatch. Immobility is defined as the period during which mice cease active struggling and only make slight limb movements necessary to keep their heads above water.
4.4.5. Fecal Water Content
Fresh fecal pellets were collected into pre-weighed 2 mL tubes. Wet weight (W1) was determined as the weight of tube + wet feces minus the empty tube weight. Samples were dried at 100 °C for 3 h in a thermostatic oven, and dry weight (W2) was recorded as tube + dried feces minus the empty tube weight. Fecal water content (%) was calculated as follows: FWC = (W1 - W2)/W1 × 100.
Measurement outputs
What raw and processed outputs should exist?
The overlapping targets were submitted to STRING to construct a PPI network, which was imported into Cytoscape for visualization and analysis ( B). Hub genes were ranked in Cyto...
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
KEGG analysis identified the top 20 pathways ( B). Among these, HIF-1, FoxO, and JAK-STAT signaling pathways are closely linked to inflammatory responses and apoptosis. Notably,...
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
KEGG enrichment highlighted the JAK-STAT pathway as a key axis potentially mediating the effects of LQYY in depression with constipation. Consistently, molecular docking support...
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
Apoptosis contributes to neuronal injury and depression-like behaviors. Consistent with H&E findings of PFC pathology in CUMS mice, TUNEL staining and NeuN immunofluorescence re...
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
Analysis plan
How should the outputs become interpretable results?
Acquisition
Capture matched images from the relevant tissue region using the same acquisition settings across samples.
inferred from protocolPreprocessing / cleaning
The CUMS model was used to evaluate LQYY after 6 weeks of stress exposure ( A).
from paperScoring or quantification
Quantify the primary readouts for this experiment: The overlapping targets were submitted to STRING to construct a PPI network, which was imported into Cytoscape for visualization and analysis ( B). Hub genes were ranked in Cyto...; KEGG analysis identified the top 20 pathways ( B). Among these, HIF-1, FoxO, and JAK-STAT signaling pathways are closely linked to inflammatory responses and apoptosis. Notably,...; KEGG enrichment highlighted the JAK-STAT pathway as a key axis potentially mediating the effects of LQYY in depression with constipation. Consistently, molecular docking support...; Apoptosis contributes to neuronal injury and depression-like behaviors. Consistent with H&E findings of PFC pathology in CUMS mice, TUNEL staining and NeuN immunofluorescence re....
from paperNormalization
Normalize image-derived measurements against the matched acquisition or segmentation rules before comparing groups.
inferred from protocolStatistical comparison
The CUMS model was used to evaluate LQYY after 6 weeks of stress exposure ( A). Compared with Controls, CUMS mice showed typical depression-like phenotypes: reduced body weight,...; Analyses were performed using IBM SPSS Statistics 25.0 and GraphPad Prism 9.5. Data are expressed as mean ± SD from at least three independent experiments. Group difference...
from paperReporting output
Report representative outputs alongside summary comparisons for The overlapping targets were submitted to STRING to construct a PPI network, which was imported into Cytoscape for visualization and analysis ( B). Hub genes were ranked in Cyto..., KEGG analysis identified the top 20 pathways ( B). Among these, HIF-1, FoxO, and JAK-STAT signaling pathways are closely linked to inflammatory responses and apoptosis. Notably,..., KEGG enrichment highlighted the JAK-STAT pathway as a key axis potentially mediating the effects of LQYY in depression with constipation. Consistently, molecular docking support..., Apoptosis contributes to neuronal injury and depression-like behaviors. Consistent with H&E findings of PFC pathology in CUMS mice, TUNEL staining and NeuN immunofluorescence re....
inferred from protocolStructured statistical methods
The CUMS model was used to evaluate LQYY after 6 weeks of stress exposure ( A). Compared with Controls, CUMS mice showed typical depression-like phenotypes: reduced body weight,...; Analyses were performed using IBM SPSS Statistics 25.0 and GraphPad Prism 9.5. Data are expressed as mean ± SD from at least three independent experiments. Group difference...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (8)
The CUMS model was used to evaluate LQYY after 6 weeks of stress exposure ( A). Compared with Controls, CUMS mice showed typical depression-like phenotypes: reduced body weight, lower sucrose preference, decreased total distance traveled and time in the center zone in the OFT (with thigmotactic trajectories), and increased immobility in the FST. Treatment with LQYY or FLX significantly mitigated these abnormalities ( B-G). Consistently, mice subjected to CUMS exhibited significant neuronal pathology in the PFC, characterized by disorganized neuronal soma arrangement, nuclear pyknosis, and deep nuclear staining, and Nissl staining revealed a marked reduction or loss of Nissl bodies in PFC neurons, whereas LQYY and FLX attenuated these histopathological changes ( H). These histopathological improvements provide direct visual evidence of LQYY's neuroprotective efficacy, effectively bridging our molecular findings on apoptosis inhibition with the recovery of behavioral functions.
The crude materials for the LQYY formula were purchased from Jiangsu Huahong Pharmaceutical Technology Co., Ltd. (Zhenjiang, China), and marked with their respective batch numbers: Nanshashen (Cat: 240620); Maidong (Cat: 241203); Xuanshen (Cat: 240905); Shudihuang (Cat: 241012); Baizhu (Cat: 250331); Zhiqiao (Cat: 241111); Houpo (Cat: 250205); Gualouren (Cat: 250217); Huomaren (Cat: 241209); Yuliren (Cat: 241218); Muxiang (Cat: 230710); and Xingren (Cat: 241115). The total crude drug weight (228 g) was soaked in water for 45 min and decocted twice, and the combined filtrates were concentrated to a final concentration of 3 g/mL.
For metabolite extraction, 200 µL of the filtrate was transferred to a 1.5 mL microcentrifuge tube and mixed with 200 µL of 70% methanol containing the internal standard. Samples were vortex-mixed for 15 min at 12,000 rpm at 4 °C and centrifuged for 3 min. The supernatant was subjected to UPLC-ESI-MS/MS. Chromatographic analysis was performed on a UPLC system coupled via electrospray ionization to a triple quadrupole/linear ion trap (QTRAP) mass spectrometer. Separation used an Agilent SB-C18 column (2.1 mm × 100 mm, 1.8 µm). Mobile phase A was water with 0.1% formic acid; mobile phase B was acetonitrile with 0.1% formic acid. The gradient program was as follows: 0.0 min, 95:5 (A:B); ramp to 5:95 by 12.0 min; and re-equilibrate to 95:5 by 14.0 min. Flow rate was 0.35 mL·min -1; injection volume was 2 µL. ESI source settings were as follows: temperature, 500 °C; ion spray voltage, +5500 V (positive mode) and -4500 V (negative mode); and source gas 1, source gas 2, and curtain gas at 50, 60, and 25 psi, respectively. Multiple reaction monitoring (MRM) transitions were acquired with compound-specific declustering potentials (DPs) and c...
The OFT was used to evaluate anxiety- and locomotion-related behaviors. Mice were placed in a 45 cm × 45 cm × 45 cm arena for a 5 min acclimation, and then returned to the starting position for a 5 min recording session. Movements were tracked with an automated system (TopScan v3.0, Clever Sys Inc., Reston, VA, USA). Outcomes included time spent in the central zone and total distance traveled. The apparatus was cleaned with 75% ethanol between trials to eliminate olfactory cues.
A CUMS model was used as previously described [ ]. Mice were exposed daily, for six weeks, to a random pair of two distinct stressors selected from the following: 24 h water and food deprivation; 45° cage tilt (24 h); empty-cage exposure (24 h); horizontal shaking (10 min); swimming in 40 °C water (10 min); reversal of the light/dark cycle (24 h); tail pinch (5 min); wet bedding (24 h); and restraint (3 h).
After a 7-day acclimation, mice were randomized into five groups ( n = 8/group): Control, CUMS, LQYY, mosapride citrate (MC), and fluoxetine (FLX). Except for the Control group, all groups underwent the 6-week CUMS procedure and then received daily oral treatment for 4 weeks: LQYY (30 g·kg -1 ·day -1 ), MC (3 mg·kg -1 ·day -1; H19990313; Chengdu Kanghong Pharmaceutical Group Co., Ltd., Chengdu, China), or FLX (10 mg·kg -1 ·day -1; HJ20181215; Patheon France, Évreux, France). The dose of LQYY was determined by converting the clinical human dose to a mouse-equivalent dose based on body surface area, followed by confirmation in a pilot dose-finding experiment, and the doses of MC and FLX were selected based on studies and verified in our laboratory [ ]. The treatment period was set according to previous studies, which is sufficient to observe neuronal apoptosis and treatment effects [, ]. Body weight was recorded weekly. Following treatment, behavioral assessments were performed, including the sucrose preference test (SPT), open field test (OFT), and forced swim test (FST). Gastrointestinal outcomes included...
The FST was performed as described previously [ ]. Each mouse was placed individually in a transparent cylinder (height 25 cm, diameter 10 cm) containing 20 cm of water maintained at 25 ± 1 °C. Behavior was recorded for 6 min in a quiet room. Post-test, the immobility time of each mouse during the final 4 min of swimming was measured using a stopwatch. Immobility is defined as the period during which mice cease active struggling and only make slight limb movements necessary to keep their heads above water.
Fresh fecal pellets were collected into pre-weighed 2 mL tubes. Wet weight (W1) was determined as the weight of tube + wet feces minus the empty tube weight. Samples were dried at 100 °C for 3 h in a thermostatic oven, and dry weight (W2) was recorded as tube + dried feces minus the empty tube weight. Fecal water content (%) was calculated as follows: FWC = (W1 - W2)/W1 × 100.
Machine-readable layer
[
{
"@context": "https://schema.org",
"@type": "HowTo",
"name": "Elucidating the Mechanism of the Liqi Yangyin Formula in Treating Depression-Constipation Comorbidity: An Integrative Approach Using Network Pharmacology and Experimental Validation methods",
"description": "Evidence-backed execution summary for Elucidating the Mechanism of the Liqi Yangyin Formula in Treating Depression-Constipation Comorbidity: An Integrative Approach Using Network Pharmacology and Experimental Validation methods from Elucidating the Mechanism of the Liqi Yangyin Formula in Treating Depression-Constipation Comorbidity: An Integrative Approach Using Network Pharmacology and Experimental Validation.",
"totalTime": "PT24081M",
"step": [
{
"@type": "HowToStep",
"position": 1,
"name": "2.8. LQYY Attenuates CUMS-Induced Depression-Like Behaviors and Pathological Damage of PFC in Mice",
"text": "The CUMS model was used to evaluate LQYY after 6 weeks of stress exposure ( A). Compared with Controls, CUMS mice showed typical depression-like phenotypes: reduced body weight, lower sucrose preference, decreased total distance traveled and time in the center zone in the OFT (with thigmotactic trajectories), and increased immobility in the FST. Treatment with LQYY or FLX significantly mitigated these abnormalities ( B-G). Consistently, mice subjected to CUMS exhibited significant neuronal pathology in the PFC, characterized by disorganized neuronal soma arrangement, nuclear pyknosis, and deep nuclear staining, and Nissl staining revealed a marked reduction or loss of Nissl bodies in PFC neurons, whereas LQYY and FLX attenuated these histopathological changes ( H). These histopathological improvements provide direct visual evidence of LQYY's neuroprotective efficacy, effec..."
},
{
"@type": "HowToStep",
"position": 2,
"name": "4. Materials and Methods",
"text": "The crude materials for the LQYY formula were purchased from Jiangsu Huahong Pharmaceutical Technology Co., Ltd. (Zhenjiang, China), and marked with their respective batch numbers: Nanshashen (Cat: 240620); Maidong (Cat: 241203); Xuanshen (Cat: 240905); Shudihuang (Cat: 241012); Baizhu (Cat: 250331); Zhiqiao (Cat: 241111); Houpo (Cat: 250205); Gualouren (Cat: 250217); Huomaren (Cat: 241209); Yuliren (Cat: 241218); Muxiang (Cat: 230710); and Xingren (Cat: 241115). The total crude drug weight (228 g) was soaked in water for 45 min and decocted twice, and the combined filtrates were concentrated to a final concentration of 3 g/mL."
},
{
"@type": "HowToStep",
"position": 3,
"name": "4. Materials and Methods",
"text": "For metabolite extraction, 200 µL of the filtrate was transferred to a 1.5 mL microcentrifuge tube and mixed with 200 µL of 70% methanol containing the internal standard. Samples were vortex-mixed for 15 min at 12,000 rpm at 4 °C and centrifuged for 3 min. The supernatant was subjected to UPLC-ESI-MS/MS. Chromatographic analysis was performed on a UPLC system coupled via electrospray ionization to a triple quadrupole/linear ion trap (QTRAP) mass spectrometer. Separation used an Agilent SB-C18 column (2.1 mm × 100 mm, 1.8 µm). Mobile phase A was water with 0.1% formic acid; mobile phase B was acetonitrile with 0.1% formic acid. The gradient program was as follows: 0.0 min, 95:5 (A:B); ramp to 5:95 by 12.0 min; and re-equilibrate to 95:5 by 14.0 min. Flow rate was 0.35 mL·min -1; injection volume was 2 µL. ESI source settings were as follows: te..."
},
{
"@type": "HowToStep",
"position": 4,
"name": "4.4.3. Open Field Test (OFT)",
"text": "The OFT was used to evaluate anxiety- and locomotion-related behaviors. Mice were placed in a 45 cm × 45 cm × 45 cm arena for a 5 min acclimation, and then returned to the starting position for a 5 min recording session. Movements were tracked with an automated system (TopScan v3.0, Clever Sys Inc., Reston, VA, USA). Outcomes included time spent in the central zone and total distance traveled. The apparatus was cleaned with 75% ethanol between trials to eliminate olfactory cues."
},
{
"@type": "HowToStep",
"position": 5,
"name": "4.4. Experimental Validation",
"text": "A CUMS model was used as previously described [ ]. Mice were exposed daily, for six weeks, to a random pair of two distinct stressors selected from the following: 24 h water and food deprivation; 45° cage tilt (24 h); empty-cage exposure (24 h); horizontal shaking (10 min); swimming in 40 °C water (10 min); reversal of the light/dark cycle (24 h); tail pinch (5 min); wet bedding (24 h); and restraint (3 h)."
},
{
"@type": "HowToStep",
"position": 6,
"name": "4.4. Experimental Validation",
"text": "After a 7-day acclimation, mice were randomized into five groups ( n = 8/group): Control, CUMS, LQYY, mosapride citrate (MC), and fluoxetine (FLX). Except for the Control group, all groups underwent the 6-week CUMS procedure and then received daily oral treatment for 4 weeks: LQYY (30 g·kg -1 ·day -1 ), MC (3 mg·kg -1 ·day -1; H19990313; Chengdu Kanghong Pharmaceutical Group Co., Ltd., Chengdu, China), or FLX (10 mg·kg -1 ·day -1; HJ20181215; Patheon France, Évreux, France). The dose of LQYY was determined by converting the clinical human dose to a mouse-equivalent dose based on body surface area, followed by confirmation in a pilot dose-finding experiment, and the doses of MC and FLX were selected based on studies and verified in our laboratory [ ]. The treatment period was set according to previous studies, whi..."
},
{
"@type": "HowToStep",
"position": 7,
"name": "4.4.4. Forced Swimming Test (FST)",
"text": "The FST was performed as described previously [ ]. Each mouse was placed individually in a transparent cylinder (height 25 cm, diameter 10 cm) containing 20 cm of water maintained at 25 ± 1 °C. Behavior was recorded for 6 min in a quiet room. Post-test, the immobility time of each mouse during the final 4 min of swimming was measured using a stopwatch. Immobility is defined as the period during which mice cease active struggling and only make slight limb movements necessary to keep their heads above water."
},
{
"@type": "HowToStep",
"position": 8,
"name": "4.4.5. Fecal Water Content",
"text": "Fresh fecal pellets were collected into pre-weighed 2 mL tubes. Wet weight (W1) was determined as the weight of tube + wet feces minus the empty tube weight. Samples were dried at 100 °C for 3 h in a thermostatic oven, and dry weight (W2) was recorded as tube + dried feces minus the empty tube weight. Fecal water content (%) was calculated as follows: FWC = (W1 - W2)/W1 × 100."
}
],
"tool": [
{
"@type": "HowToTool",
"name": "4. Materials and Methods"
},
{
"@type": "HowToTool",
"name": "4.4.3. Open Field Test (OFT)"
},
{
"@type": "HowToTool",
"name": "4.4. Experimental Validation"
},
{
"@type": "HowToTool",
"name": "4.4.8. Histopathological Testing of PFC and Colon"
},
{
"@type": "HowToTool",
"name": "4.4.9. Quantitative Real-Time PCR"
},
{
"@type": "HowToTool",
"name": "4.4.10. TUNEL Staining"
},
{
"@type": "HowToTool",
"name": "4.4.11. Immunofluorescence Staining"
}
],
"supply": [
{
"@type": "HowToSupply",
"name": "4. Materials and Methods"
},
{
"@type": "HowToSupply",
"name": "4. Materials and Methods"
},
{
"@type": "HowToSupply",
"name": "4.2. Network Pharmacology"
},
{
"@type": "HowToSupply",
"name": "4.3. Molecular Docking"
},
{
"@type": "HowToSupply",
"name": "4.4.6. First Black Stool"
},
{
"@type": "HowToSupply",
"name": "4.4.9. Quantitative Real-Time PCR"
},
{
"@type": "HowToSupply",
"name": "4.4.10. TUNEL Staining"
},
{
"@type": "HowToSupply",
"name": "4.4.11. Immunofluorescence Staining"
}
],
"isBasedOn": {
"@type": "ScholarlyArticle",
"headline": "Elucidating the Mechanism of the Liqi Yangyin Formula in Treating Depression-Constipation Comorbidity: An Integrative Approach Using Network Pharmacology and Experimental Validation",
"datePublished": "2026",
"author": [
{
"@type": "Person",
"name": "Lianjie Xu"
},
{
"@type": "Person",
"name": "Shun Seng Ong"
},
{
"@type": "Person",
"name": "Xiaoyue Deng"
},
{
"@type": "Person",
"name": "Yunzhi Qian"
},
{
"@type": "Person",
"name": "Zhao Tang"
},
{
"@type": "Person",
"name": "Ming Li"
},
{
"@type": "Person",
"name": "Tianshu Xu"
}
],
"identifier": "10.3390/ph19010106"
}
},
{
"@context": "https://schema.org",
"@type": "BreadcrumbList",
"itemListElement": [
{
"@type": "ListItem",
"position": 1,
"name": "Experiments",
"item": "https://replicatescience.com/experiments"
},
{
"@type": "ListItem",
"position": 2,
"name": "Elucidating the Mechanism of the Liqi Yangyin Formula in Treating Depression-Constipation Comorbidity: An Integrative Approach Using Network Pharmacology and Experimental Validation methods",
"item": "https://replicatescience.com/experiments/elucidating-the-mechanism-of-the-liqi-yangyin-formula-in-treating-depression-8211-constipation-comorbidity-an-integrative-approach-using-network-pharmacology-and-experimental-valid/elucidating-the-mechanism-of-the-liqi-yangyin-formula-in-treating-depressioncons"
}
]
}
]