Research On The Antidepressant Effects And Mechanisms Of Cistanche Phenylethanol Glycosides in Mice

Abstract:

 

This study aims to investigate the therapeutic effects and mechanisms of Cistanche phenylethanol glycosides (CPhGs) on depression-like behavior induced by chronic unpredictable mild stress (CUMS) in mice. Mice were randomly divided into control group, model group, high dose CPhGs group (700 mg/kg), middle dose CPhGs group (350 mg/kg), low dose CPhGs group (175 mg/kg), and fluoxetine group (15 mg/kg). After four weeks of corresponding drug interventions, neurobehavioral tests were conducted; enzyme-linked immunosorbent assay was used to detect levels of neurotransmitters in serum, as well as the content of inflammatory factors and mediators in hippocampal tissue. Hematoxylin-eosin and Nissl staining were employed to observe pathological changes in hippocampal tissue. Immunohistochemistry and immunofluorescence methods were used to analyze the density of astrocytes and microglia in the hippocampal tissue of mice, as well as the expression levels of toll-like receptor 4/nuclear factor κB/Nod-like receptor protein 3 (TLR4/NF-κB/NLRP3). Results showed that CPhGs could improve depression-like behavior in mice, increase the preference for sweet water, and reduce immobility time, significantly enhancing the levels of serotonin and dopamine in mouse serum, while inhibiting the expression of inflammatory mediators and factors in the hippocampus. Immunohistochemical analysis indicated that CPhGs could increase the density of astrocytes in the hippocampus, reduce the activation of microglia, and inhibit the expression of NLRP3 inflammasome-related proteins. Immunofluorescence experiments demonstrated that the mechanism through which CPhGs reduce inflammasome formation is by decreasing the activation of the TLR4/NF-κB signaling pathway in hippocampal tissue. In summary, CPhGs exhibit significant antidepressant effects on CUMS-induced depression like behavior, potentially through the regulation of neurotransmitter levels and inhibition of neuroinflammatory responses, providing experimental evidence for its application in the treatment of depression.

Key words: Cistanche phenylethanol glycosides; depression; neuroinflammatory response; astrocytes; TLR4/NFκB/NLRP3 pathway

Herbal Cistanche Raw Materials 

 

Depression is a common and complex affective mental disorder characterized by symptoms such as low mood, anhedonia, slow thinking and physiological dysfunction. In severe cases, suicidal tendencies may even occur[1]. According to the "Report on the Nutrition and Chronic Disease Status of Chinese Residents (2020)", the prevalence of depression in my country reached 2.1%, and was listed by the World Health Organization as one of the diseases with the highest disability rates in the world[2]. Since the pathogenesis of depression is very complex, the specific mechanism remains unclear to date, which poses a huge challenge to its treatment. In recent years, domestic and foreign scholars have found that depression may be closely related to immune inflammatory activation. In the process of inflammatory response activation in the central nervous system, the activation of hippocampal inflammatory cells is its characteristic manifestation. Continuously activated hippocampal inflammatory cells induce the release of multiple inflammatory cytokines, forming an inflammatory cytokine "cascade effect", thereby participating in the pathogenesis and pathological process of mental illnesses such as depression[3]. Currently, the treatment of this disease mainly relies on Western medicine, such as fluoxetine, venlafaxine, duloxetine, etc. However, there are shortcomings such as low clinical cure rate, prominent residual symptoms, high recurrence rate, and easy to cause organ dysfunction, which limits its clinical application [4]. Traditional Chinese medicine has the advantages of more targets and fewer side effects in the treatment of emotional diseases, and has broad application prospects in the treatment of depression and its complications [5].

 

Cistanche deserticola is the dried fleshy stem with phosphorus leaves of Cistanche deserticola Y.C.Ma and/or Cistanche tubulosa (Schenk) Wight, both of which are plants of the Orobanchaceae family. It is a good food and medicine product. Modern pharmacological research has confirmed that Cistanche deserticola has a neuroprotective effect (it can inhibit Alzheimer's disease, Parkinson's disease, etc.). "Cistanche total glycosides" capsules, which are mainly composed of phenylethanol glycosides in Cistanche deserticola, are widely used in the treatment of neurological diseases [6]. Phenylethanol glycosides (CPhGs) are important active ingredients in Cistanche total glycosides. They have multiple biological functions such as anti-tumor, immunomodulation, and antioxidant, and show good application prospects [7]. Previous studies have shown that CPhGs can protect the effects of monoamines and relieve depressive symptoms by increasing the ceramide metabolic pathway in the substantia nigra and the content of dopamine (DA) in the striatum[8]; it can also adjust the depressive-like behavior of mice with perimenopausal syndrome by improving the level of monoamine neurotransmitters in the brain[9]. However, there have been no reports on its use in treating the chronic unpredictable mild stress (CUMS)-induced mouse depression model, and its mechanism of action remains to be elucidated. Based on previous research and combined with the current potential of CPhGs in anti-oxidative stress, regulating neuroinflammation and inhibiting neuronal apoptosis, this study used the CUMS-induced mouse depression model to study the antidepressant effect of CPhGs and the mechanism of action of the TLR4/NF-κB/NLRP3 pathway in improving neuroinflammation, providing experimental basis for further exploring the antidepressant effect of CPhGs, expanding its clinical application, and developing and utilizing it as a health food.

 

1 Materials and methods

1.1 Materials and instruments

CPhGs extract (phenylethanol glycosides in the extract ≥ 85%, including echinacoside ≥ 40%, verbascoside ≥ 16%; batch number: 20231221, Xinjiang Hotan Dichen Pharmaceutical Biological Co., Ltd.); fluoxetine hydrochloride capsules (fluoxetine, FLX) (batch number: 8533C, Patheon France Pharmaceutical Company, France); 5-hydroxytryptamine (5-HT), DA detection kit (batch number: 20240305, 20240305, Shanghai Future Industrial Co., Ltd.); inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (cyclooxygenase-2, COX-2), prostaglandin E2 (prostaglandin E2, PGE2), tumor necrosis factor alpha (tumor necrosis factor alpha, TNF-α), interleukin 1β (interleukin-1β, IL-1β), IL-18 (interleukin-18, IL-18), IL-6 (interleukin 6, IL-6), interferon-γ (interferon-γ, IFN-γ) enzyme-linked immunosorbent assay kits (Batch No.: ER050A332132, ER035PD09812, ER048H753146, ER10X6398452, ER051H648219, ER09X2865471, ER049H746823, ER07X1729784, Wuhan Elarite Biotechnology Co., Ltd.); ionized calcium-binding adapter molecule 1 (ionized calcium-binding adapter molecule 1, Iba-1) monoclonal antibody, glial fibrillary acidic protein (GFAP) monoclonal antibody, NOD Nod-like receptor protein 3 (NLRP3) monoclonal antibody (Batch No. GR3417890-15, GR3379124-30, GR3285067-28, Abcam, UK); Toll-like receptor 4 (TLR4) monoclonal antibody, phosphorylated nuclear factor-κB p65 (phospho-nuclear factor-κB p65, p-NF-κB p65) monoclonal antibody (Batch No. 10032512, 00128965, Wuhan Sanying Biotechnology Co., Ltd.); 4% paraformaldehyde, immunofluorescence staining kit (Batch No. 20240106, 20240315, Shanghai Bio-Technology Co., Ltd.); hematoxylin and eosin (HEXA) eosin, HE) staining kit, Nissl staining kit (batch number: 20240401, 20240402, Liaoning Meilun Biology); Immunohistochemistry universal kit (batch number: 43731A, Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.).
ME204E 1/10,000 electronic balance (Mettler Toledo, USA); Milli-Q laboratory pure water system (Merck, Germany); SpectraMax iD3 multifunctional microplate reader (Molecular Devices, USA); LEICADM 2000 fluorescence upright microscope (Leica, Germany); 63008 open field, 63010 elevated plus maze, 63022 forced swimming, DB001 Morris water maze, SMART 3.0 animal behavior video analysis system (Shenzhen Ruiwode Life Science Technology Co., Ltd.).

60 SPF C57BL/6 mice, weighing 18-22 g, were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. (Production License No. SCXK (Beijing) 2020-0033, Experimental Animal Use License No. SYXK (Beijing) 2021-0011).
The mice were kept in an SPF environment, with the ambient temperature controlled at (21 ± 2) °C, the humidity maintained at 60% ± 10%, and a 12 h light-dark cycle. The mice had free access to food and water, and the experiment began after 1 week of adaptive feeding. The experimental animals were kept at the Experimental Animal Center of Shaanxi University of Chinese Medicine. All mouse experimental procedures in this study have been approved by the Animal Experiment Ethics Committee of Shaanxi University of Chinese Medicine (SUCMDL20231015003).

 

1.2 Experimental methods

1.2.1 Grouping, modeling and drug administration

After 1 week of adaptive feeding of 7-week-old SPF C57BL/6 mice, non-elastic mice that met the requirements of subsequent experiments were screened out through the sugar water preference test and randomly divided into a control group (Con), a model group (Mod), a high dose CPhGs group (CPhGs-H; 700 mg/kg), a middle dose CPhGs group (CPhGs-M; 350 mg/kg), a low dose CPhGs group (CPhGsL; 175 mg/kg) and a fluoxetine group (FLX; 15 mg/kg), with 10 mice in each group. Except for Con, the mice in the other groups began to be subjected to CUMS stress for 4 weeks[10]. A total of 10 stressors were used, including restraint (1 h), shaking (1 h), tail suspension (1 h), 45-degree cage tilt (24 h), wet bedding (24 h), empty cage (24 h), night lighting (12 h), 41 ℃ hot water swimming for 5 min, 4 ℃ ice water swimming for 5 min, and tail clamping for 2 min. Two stimuli were randomly selected every day, and the same stimulus did not appear on two consecutive days. After 4 weeks of modeling, CPhGs-H, CPhGs-M, and CPhGs-L mice were given corresponding CPhGs doses by gavage according to body weight, and FLX mice were given fluoxetine by gavage (10 mg/kg). The dosage of CPhGs-M and FLX in mice was converted according to the equivalent coefficient of body surface area between adults and mice, and the fluctuation of 2 times was the low and high doses of CPhGs. Con and Mod mice were given an equal volume of deionized water by gavage. The mice were gavaged once a day for 4 weeks. Neurobehavioral tests were performed on the 8th week.

 

1.2.2 Neurobehavioral experiment

1.2.2.1 Sugar water preference experiment

Before the sucrose preference test (SPT), mice should be housed in a single cage and then adapted to sugar water for 3 days. On the first day, two bottles of 1% sucrose water were given for 24 hours, and on the second day, one bottle of sugar water and one bottle of deionized water were given for 24 hours. On the third day, the positions of sugar water and pure water were exchanged for 24 hours, and the experiment ended 24 hours later. On the fourth day, the mice were fasted and deprived of water for 24 hours. On the fifth day, one bottle of sugar water and one bottle of pure water were given after weighing, and the positions were exchanged after 12 hours. After 24 hours, the two bottles were taken out and weighed again, and the sugar water consumption rate was calculated (sugar water consumption rate = sugar water consumption/(sugar water consumption + water consumption) × 100%).

 

1.2.2.2 Open field test

The open field test (OFT) was conducted in a 50 cm×50 cm×50 cm cube open box. The center of the box was defined as a square with a side length of 25 cm. The mice were placed in the center of the box and allowed to adapt for 5 min. The movement trajectory of the mice was analyzed in the last 5 min. The evaluation indicators included the total movement distance of the mice in the open field, the activity time in the center of the open field, and the number of times they entered the center. 1.2.2.3 Tail suspension test The tail suspension test (TST) is to suspend the mouse by its tail so that its head is kept at a certain distance from the ground. The struggle of the mice within 6 min was recorded, and the immobility time of the mice in the last 4 min was analyzed.

 

1.2.2.4 Forced swimming test

The forced swimming test (FST) is to place mice in a transparent cylindrical barrel (15 cm diameter × 50 cm height) filled with (25 ± 2) ℃ water, with the water level reaching 20 cm. The struggling of mice in the water within 6 min was recorded, and the immobility time of mice in the last 4 min was analyzed.

 

1.2.3 Sample collection

At the end of the 8th week, mice were deeply anesthetized by intraperitoneal injection of sodium pentobarbital (3%, 10 mL/kg). After anesthesia, the anesthesia state was confirmed by observing slow breathing, muscle relaxation, and no response to surgical operation. Then, the blood of mice was collected by eye removal, and mice were killed by cervical dislocation. The heart was perfused with saline, and then the brain was quickly opened and the intact brain was removed. It was rinsed with pre-cooled saline, and half of it was fixed with 4% paraformaldehyde for 72 h; the hippocampal tissue of the other half was separated on ice and stored in liquid nitrogen for later use. Take 10 mg of mouse hippocampal tissue sample, add pre-cooled physiological saline at a mass-volume ratio of 1:9, and mechanically homogenize in an ice water bath. After homogenization, centrifuge at 4 000 r/min for 15 min (centrifugal radius 4.5 cm), collect the supernatant for detection.

 

1.2.4 Determination of neurotransmitter index

Take appropriate amount of mouse serum and hippocampal tissue homogenate supernatant, and detect the content of 5-HT and DA content according to the kit operation method.

 

1.2.5 Determination of hippocampal tissue biochemical index

Take the supernatant of hippocampal tissue homogenate, and use the BCA kit to determine the protein concentration. According to the kit instructions, the absorbance A of inflammatory mediators (iNOS, COX-2 and PGE2) and inflammatory factors (TNFα, IL-1β, IL-18, IL-6, IFN-γ) in the supernatant of hippocampal tissue homogenate is determined by an enzyme marker, and the content of each substance is calculated, and the data is recorded and statistically analyzed.

 

1.2.6 HE staining of hippocampal tissue

Whole brain tissue samples fixed for more than 72 h were taken and paraffin sections were made with a thickness of 4 μm. After dewaxing with xylene, the samples were hydrated with gradient ethanol (immersed in 100% ethanol I and II for 5 min, and in 95%, 90%, 80% and 70% ethanol for 2 min respectively), and washed three times with PBS, stained with eosin for 5 min, rinsed in tap water for 5 min, counterstained with hematoxylin for 1 min, differentiated with 1% hydrochloric acid ethanol for 2 s, and rinsed in tap water for 10 min. Dehydrated with gradient ethanol (80% ethanol for 2 min, 95% ethanol for 2 min, ethanol I and 100% ethanol II for 5 min each), transparentized with xylene, sealed with neutral gum and air-dried. The morphological changes of hippocampal tissue cells in each group of mice were observed under a microscope.

 

1.2.7 Nissl staining of hippocampal tissue

The paraffin sections prepared under "1.2.5" were dewaxed and hydrated, then stained with Nissl staining solution for 10 min, washed twice with distilled water for 3 min each time, dehydrated with gradient ethanol (80% ethanol for 2 min, 95% ethanol for 2 min, ethanol I and 100% ethanol II for 5 min each), transparentized with xylene, sealed with neutral gum and air-dried. The morphological changes of hippocampal tissue cells in each group of mice were observed under a microscope.

 

1.2.8 Immunohistochemistry of hippocampal tissue

The paraffin-embedded hippocampal tissue specimens were processed by immunohistochemistry (IHC) staining according to the instructions of the universal immunohistochemistry kit. After dewaxing, hydration, antigen repair, peroxidase blocking, and blocking, Iba-1 (1:400), GFAP (1:400), LRP31:300) and Caspsae1 (1:400) primary antibodies were added and incubated overnight at 4°C. After DAB color development, nuclear staining, dehydration and sealing, the positive expression of Iba1, GFAP, NLRP3 and Caspsae-1 was observed under a light microscope and the images were analyzed by Image J.

1.2.9 Immunofluorescence detection of hippocampal tissue

The paraffin-embedded hippocampal tissue specimens were processed by immunofluorescence staining according to the instructions of the immunofluorescence kit. The paraffin sections prepared under "1.2.5" were dewaxed, hydrated, antigen repaired, and blocked, and then TLR4 (1:400) and p-NF-κB p65 (1:200) primary antibodies were added to the sections and incubated overnight at 4 °C. The primary antibody was removed the next day, washed three times with PBS, and the secondary antibody (1:500) was added and incubated in the dark for 1 h. After washing three times, the sections were sealed with a fixative containing DAPI. The fluorescence signal of the hippocampal tissue area was observed by fluorescence microscopy and the images were analyzed by Image J.

 

1.3 Data processing

The experimental data were statistically analyzed using statistical software SPSS 26.0 and Graphpad Prism 9.0. The data were expressed as mean ± standard deviation (x ± s), and the analysis was performed using One-way ANOVA. The comparison between the two groups was performed using Dunnett's test. P<0.05 indicated that the difference was statistically significant.