Elucidating the Mechanism of the Liqi Yangyin Formula in Treating Depression-Constipation Comorbidity: An Integrative Approach Using Network Pharmacology and Experimental Validation methods
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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.
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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.
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