Xiao Yao San Improves the Anxiety-Like Behaviors of Rats Induced by Chronic Immobilization Stress: The Involvement of the JNK Signaling Pathway in the Hippocampus.

The current study evaluated the effects of Xiao Yao San (XYS) on anxiety-like behaviors and sought to determine whether the c-Jun N-terminal kinase (JNK) signaling pathway is involved. A total of 40 rats were divided into 5 groups (n=8): the control group (deionized water, per os (p.o.)), the model group (deionized water, p.o.), the SP600125 group (surgery), the per se group (surgery), and the XYS group (3.9 g/kg/d, p.o.). A 1% dimethyl sulfoxide (DMSO) citrate buffer solution (2 µL/ventricle/d) and SP600125 (10 µg/ventricle, 2 µL/ventricle/d) were separately and bilaterally injected into the rats of the two surgery groups via the ventricular system of the brain. All but the control group underwent 14 d of chronic immobilization stress (CIS; 3 h/d). On day 15, the body weights of all of the rats were measured; additionally, the rats were subjected to the elevated plus maze (EPM) and novelty suppressed feeding (NSF) tests. Finally, JNK signaling pathway indices, including phosphorylated JNK (P-JNK), JNK, phosphorylated c-Jun (P-c-Jun) and cytochrome C (Cyt-C), were examined. After modeling, the body weight and behavioral analyses of the model rats indicated that this modeling method induced anxiety-like behaviors. P-JNK, JNK, and P-c-Jun were altered in the hippocampus of the model rats. After 14 d of treatment with XYS and SP600125, rat body weight and behaviors as well as P-JNK, JNK, and P-c-Jun had changed. However, no significant difference in Cyt-C was found. XYS improves the anxiety-like behaviors induced by CIS, which might be related to the JNK signaling pathway in the hippocampus.

Relationship between Insulin Levels and Nonpsychotic Dementia: A Systematic Review and Meta-Analysis.

Objectives: To explore the relationship between insulin levels and nonpsychotic dementia. Methods: Six electronic databases (PubMed, Cochrane, SCI, CNKI, VIP, and Wanfang) were searched from January 1, 2007, to March 1, 2017. Experimental or observational studies that enrolled people with nonpsychotic dementia or abnormal insulin levels in which insulin levels or MMSE scores (events in nonpsychotic dementia) were the outcome measures. Random-effects models were chosen for this meta-analysis. Sample size, mean, s.d., and events were primarily used to generate effect sizes (with the PRIMA registration number CRD42017069860). Results: 50 articles met the final inclusion criteria. Insulin levels in cerebrospinal fluid were lower (Hedges' g = 1.196, 95% CI = 0.238 to 2.514, and P = 0.014), while the levels in peripheral blood were higher in nonpsychotic dementia patients (Hedges' g = 0.853 and 95% CI = 0.579 to 1.127), and MMSE scores were significantly lower in the high insulin group than in the healthy control group (Hedges' g = 0.334, 95% CI = 0.249 to 0.419, and P = 0.000). Conclusions: Our comprehensive results indicate that blood insulin levels may increase in patients with nonpsychotic dementia.

Norcantharidin Induces HL-60 Cells Apoptosis In Vitro.

Norcantharidin (NCTD) is the demethylated form of cantharidin, which is the active substance of mylabris, and is known to have anticancer potentials. The aim of this paper was to assess the apoptosis-inducing effect of NCTD on HL-60 cells. Methods. The effects of NCTD were detected by flow cytometer on the cell toxicity, cell cycle, and apoptosis of HL-60 cells cultured in vitro. Results. After 48-hour treatment with NCTD, the growth of HL-60 cells was inhibited significantly. The summit of apoptosis appeared after 24 hours. The percentage of the cells in G(1) phase decreased and then increased in S and G(2)+ M phase, while the S and G(2)+ M phases were blocked after treatment with 5, 10, and 50 µmol/L NCTD for 24 hours. Conclusions. NCTD can induce the apoptosis of HL-60 cells and inhibit the fissiparism, and the domino effect was obviously correlated with the time and dosage.

Saikosaponin D exerts antidepressant effect by regulating Homer1-mGluR5 and mTOR signaling in a rat model of chronic unpredictable mild stress.

BACKGROUND: Many studies about depression have focused on the dysfunctional synaptic signaling in the hippocampus that drives the pathophysiology of depression. Radix Bupleuri has been used in China for over 2000 years to regulate liver-qi. Extracted from Radix Bupleuri, Saikosaponin D (SSD) is a pharmacologically active substance that has antidepressant effects. However, its underlying mechanism remains unknown. MATERIALS AND METHODS: A chronic unpredictable mild stress (CUMS) paradigm was used as a rat model of depression. SD rats were randomly assigned to a normal control (NC) group or one exposed to a CUMS paradigm. Of the latter group, rats were assigned to four subgroups: no treatment (CUMS), fluoxetine-treated (FLU), high-dose and low-dose SSD-treated (SSDH and SSDL). SSD was orally administrated of 1.50 mg/kg and 0.75 mg/kg/days for three weeks in the SSDH and SSDL groups, respectively. Fluoxetine was administrated at a dose of 2.0 mg/kg/days. SSD's antidepressant effects were assessed using the open field test, forced swim test, and sucrose preference test. Glutamate levels were quantified by ELISA. Western blot and immunochemical analyses were conducted to quantify proteins in the Homer protein homolog 1 (Homer1)-metabotropic glutamate receptor 5 (mGluR5) and mammalian target of rapamycin (mTOR) pathways in the hippocampal CA1 region. To measure related gene expression, RT-qPCR was employed. RESULTS: CUMS-exposed rats treated with SSD exhibited increases in food intake, body weight, and improvements in the time spent in the central are and total distance traveled in the OFT, and less pronounced pleasure-deprivation behaviors. SSD also decreased glutamate levels in CA1. In CA1 region of CUMS-exposed rats, SSD treatment increased mGluR5 expression while decreasing Homer1 expression. SSD also increased expressions of postsynaptic density protein 95 (PSD95) and synapsin I (SYP), and the ratios of p-mTOR/mTOR, p-p70S6k/p70S6k, and p-4E-BP1/4E-BP1 in the CA1 region in CUMS-exposed rats. CONCLUSIONS: SSD treatment reduces glutamate levels in the CA1 region and promotes the expression of the synaptic proteins PSD-95 and SYP via the regulation of the Homer1-mGluR5 and downstream mTOR signaling pathways. These findings suggest that SSD could act as a natural neuroprotective agent in the prevention of depression.

Antidepressant Mechanism of Traditional Chinese Medicine Formula Xiaoyaosan in CUMS-Induced Depressed Mouse Model via RIPK1-RIPK3-MLKL Mediated Necroptosis Based on Network Pharmacology Analysis.

Background: Depression is a stress-related disorder that seriously threatens people's physical and mental health. Xiaoyaosan is a classical traditional Chinese medicine formula, which has been used to treat mental depression since ancient times. More and more notice has been given to the relationship between the occurrence of necroptosis and the pathogenesis of mental disorders. Objective: The purpose of present study is to explore the potential mechanism of Xiaoyaosan for the treatment of depression using network pharmacology and experimental research, and identify the potential targets of necroptosis underlying the antidepressant mechanism of Xiaoyaosan. Methods: The mice model of depression was induced by chronic unpredictable mild stress (CUMS) for 6 weeks. Adult C57BL/6 mice were randomly divided into five groups, including control group, chronic unpredictable mild stress group, Xiaoyaosan treatment group, necrostatin-1 (Nec-1) group and solvent group. Drug intervention performed from 4th to 6th week of modeling. The mice in Xiaoyaosan treatment group received Xiaoyaosan by intragastric administration (0.254 g/kg/d), and mice in CUMS group received 0.5 ml physiological saline. Meanwhile, the mice in Nec-1 group were injected intraperitoneally (i.p.) with Nec-1 (10 mg/kg/d), and the equivalent volume of DMSO/PBS (8.3%) was injected into solvent group mice. The behavior tests such as sucrose preference test, forced swimming test and novelty-suppressed feeding test were measured to evaluate depressive-like behaviors of model mice. Then, the active ingredients in Xiaoyaosan and the related targets of depression and necroptosis were compiled through appropriate databases, while the "botanical drugs-active ingredients-target genes" network was constructed by network pharmacology analysis. The expressions of RIPK1, RIPK3, MLKL, p-MLKL were detected as critical target genes of necroptosis and the potential therapeutic target compounds of Xiaoyaosan. Furthermore, the levels of neuroinflammation and microglial activation of hippocampus were measured by detecting the expressions of IL-1ß, Lipocalin-2 and IBA1, and the hematoxylin and eosin (H&E) stained was used to observe the morphology in hippocampus sections. Results: After 6-weeks of modeling, the behavioral data showed that mice in CUMS group and solvent group had obvious depressive-like behaviors, and the medication of Xiaoyaosan or Nec-1 could improve these behavioral changes. A total of 96 active ingredients in Xiaoyaosan which could regulate the 23 key target genes were selected from databases. Xiaoyaosan could alleviate the core target genes in necroptosis and improve the hippocampal function and neuroinflammation in depressed mice. Conclusion: The activation of necroptosis existed in the hippocampus of CUMS-induced mice, which was closely related to the pathogenesis of depression. The antidepressant mechanism of Xiaoyaosan included the regulation of multiple targets in necroptosis. It also suggested that necroptosis could be a new potential target for the treatment of depression.

Xiaoyaosan Alleviates Hippocampal Glutamate-Induced Toxicity in the CUMS Rats via NR2B and PI3K/Akt Signaling Pathway.

Purpose: It is revealed that Xiaoyaosan could reduce glutamate level in the hippocampus of depressed rats, whose metabolism leads to the pathophysiology of depression. However, the underlying mechanism remains unclear. This study aims to explore the effect of Xiaoyaosan on glutamate metabolism, and how to regulate the excitatory injury caused by glutamate. Methods: Rats were induced by chronic unpredictable mild stress, then divided into control, vehicle (distilled water), Xiaoyaosan, fluoxetine, vehicle (DMSO), Xiaoyaosan + Ly294002 and Ly294002 groups. Ly294002 was microinjected into the lateral ventricular catheterization at 5 mM. Xiaoyaosan (2.224 g/kg) and fluoxetine (2.0 mg/kg) were orally administered for three weeks. The open field test (OFT), forced swimming test (FST), and sucrose preference test (SPT) were used to assess depressive behavior. The glutamate and corticosterone (CORT) levels were detected by ELISA. Western blot, immunochemistry or immunofluorescence were used to detect the expressions of NR2B, MAP2, PI3K and P-AKT/Akt in the hippocampal CA1 region. The mRNA level of MAP2, NR2B and PI3K were detected by RT-qPCR. Results: Compared to the rats in control group, body weight and food intake of CUMS rats was decreased. CUMS rats also showed depression-like behavior as well as down regulate the NR2B and PI3K/Akt signaling pathway. Xiaoyaosan treatments could increase food intake and body weight as well as improved time spent in the central area, total distance traveled in the OFT. Xiaoyaosan could also decrease the immobility time as well as increase the sucrose preference in SPT. Moreover, xiaoyaosan decreased the level of glutamate in the hippocampal CA1 region and serum CORT in CUMS rats. Furthermore, xiaoyaosan improved the expression of MAP2 as well as increased the expression of NR2B, PI3K and the P-AKT/AKT ratio in the hippocampal CA1 region in the CUMS rats. Conclusion: Xiaoyaosan treatment can exert the antidepressant effect by rescuing hippocampal neurons loss induced by the glutamate-mediated excitotoxicity in CUMS rats. The underlying pathway maybe through NR2B and PI3K/Akt signaling pathways. These results may suggest the potential of Xiaoyaosan in preventing the development of depression.

Xiaoyaosan Produces Antidepressant Effects in Rats via the JNK Signaling Pathway.

BACKGROUND: Xiaoyaosan (XYS) has achieved definite curative effects in clinic. However, the mechanism is not clear. Previous studies of our team indicated XYS improved anxiety-like behaviors through inhibiting c-Jun N-terminal kinase (JNK) signaling pathway of hippocampus. OBJECTIVES: In the study, we explored whether the JNK signaling pathway is involved in the mechanism of XYS treating depression. METHOD: Forty-eight rats were divided randomly into 4 groups (n = 12): the control group (deionized water, p.o.), the model group (deionized water, p.o.), the fluoxetine group (2.08 mg/kg/day, p.o.), and the XYS group (3.9 g/kg/day, p.o.). All rats except for the control group were given continuous 21 days of chronic immobilization stress (CIS; 3 h/day). On day 29, the body weights and the behavioral tests, including the novelty suppressed feeding test, the open field test, and the elevated plus maze test, were measured. On day 30, all the rats were sacrificed, and three indices of the JNK signaling pathway were tested by Western blot. RESULTS: The body weight and behavioral tests of all groups indicated that 21 days of CIS induced depression-like behaviors. After 21 days of treatment with fluoxetine and XYS, changes were seen in body weight, behaviors, and JNK, phosphorylated JNK (P-JNK), and phosphorylated c-Jun (P-c-Jun) levels in the hippocampus. CONCLUSIONS: XYS ameliorated the depression-like behaviors, potentially through affecting the JNK signaling pathway in the hippocampus.

Xiaoyaosan exerts anxiolytic-like effects by down-regulating the TNF-α/JAK2-STAT3 pathway in the rat hippocampus.

Although the anxiolytic-like effects of Xiaoyaosan, a Chinese herbal formula, have been described in many previous studies, its underlying mechanism remains undefined. The cytokine tumour necrosis factor-α (TNF-α) and its closely associated janus kinase 2 (JAK2)-signal transducer and activator of transcription (STAT3) signalling pathway regulate the neuro-inflammatory response in the brain, thus participating in the development of anxiety. Our purpose was to investigate whether the anxiolytic-like effects of Xiaoyaosan are related to the TNF-α/JAK2-STAT3 pathway in the hippocampus. We examined the effects of Xiaoyaosan on behaviours exhibited in the elevated plus maze test, open field test and novelty-suppressed feeding test as well as hippocampal neuron damage and changes in the TNF-α/JAK2-STAT3 pathway in a rat model of chronic immobilization stress (CIS)-induced anxiety. Xiaoyaosan exerts anxiolytic-like effects on CIS-induced anxiety, with a significant alleviation of anxiety-like behaviours, an attenuation of hippocampal neuron damage, and a reversal of the activation of the TNF-α/JAK2-STAT3 pathway in the hippocampus that are similar to the effects of the JAK2 antagonist AG490. However, Xiaoyaosan and AG490 failed to effectively regulate apoptosis-related factors, including Bax and Caspase-3. These results suggest that Xiaoyaosan attenuates stress-induced anxiety behaviours by down-regulating the TNF-α/JAK2-STAT3 pathway in the rat hippocampus.

Effects of Xiaoyaosan on Stress-Induced Anxiety-Like Behavior in Rats: Involvement of CRF1 Receptor.

Background. Compared with antidepressant activity of Xiaoyaosan, the role of Xiaoyaosan in anxiety has been poorly studied. Objective. To observe the effects of Xiaoyaosan on anxiety-like behavior induced by chronic immobilization stress (CIS) and further explore whether these effects were related to CRF1R signaling. Methods. Adult male SD rats were randomly assigned to five groups (n = 12): the nonstressed control group, vehicle-treated (saline, p.o.) group, Xiaoyaosan-treated (3.854 g/kg, p.o.) group, vehicle-treated (surgery) group, and antalarmin-treated (surgery) group. Artificial cerebrospinal fluid (0.5 µL/side) or CRF1R antagonist antalarmin (125 ng/0.5 µL, 0.5 µL/side) was bilaterally administered into the basolateral amygdala in the surgery groups. Except for the nonstressed control group, the other four groups were exposed to CIS (14 days, 3 h/day) 30 minutes after treatment. On days 15 and 16, all animals were subjected to the elevated plus-maze (EPM) and novelty suppressed feeding (NSF) test. We then examined the expression of CRF1R, pCREB, and BDNF in the amygdala. Results. Chronic pretreatment with Xiaoyaosan or antalarmin significantly reversed elevated anxiety-like behavior and the upregulated level of CRF1R and BDNF in the amygdala of stressed rats. pCREB did not differ significantly among the groups. Conclusions. These results suggest that Xiaoyaosan exerts anxiolytic-like effects in behavioral tests and the effects may be related to CRF1R signaling in the amygdala.

Effect of xiaoyaosan decoction on learning and memory deficit in rats induced by chronic immobilization stress.

Xiaoyaosan (XYS) decoction is a famous prescription which can protect nervous system from stress and treat liver stagnation and spleen deficiency syndrome (LSSDS). In this experiment, we observed the effect of XYS decoction on chronic immobilization stress (CIS) induced learning and memory deficit in rats from behaviors and changes of proteins in hippocampus. We used XYS decoction to treat CIS induced learning and memory deficit in rats with rolipram as positive control, used change of body weight and behavioral tests to determine whether the rats have LSSDS and have learning and memory deficit or not. We used Western blotting to determine the content of postsynaptic density protein 95 (PSD-95) and synaptophysin (SYP) in hippocampus. Results showed that XYS could improve the situation of slow weight gain induced by CIS, improve the ability of learning and memory, reverse the symptom of liver stagnation and spleen deficiency syndrome (LSSDS) in rats, and increase the levels of PSD-95 and SYP on the hippocampal nerve synapses. These findings suggested that XYS decoction may be helpful in reversing CIS induced learning and memory deficit by increasing the levels of PSD-95 and SYP on the hippocampal nerve synapses and improving synaptic plasticity.

Metabonomics-based study of clinical urine samples in suboptimal health with different syndromes.

Objective. To explore the urinary biochemistry features of syndromes of traditional Chinese medicine (TCM) such as syndrome of stagnation of liver Qi, spleen deficiency, liver Qi stagnation, and spleen deficiency (LSSDS) in sub-optimal health status (SHS). Methods. 12 cases for each syndrome group in SHS were selected, 12 subjects were used as a normal control group, and (1)H NMR detection was, respectively, carried out, and the data was corrected by the orthogonal signal correction (OSC) and then adopted a partial least squares (PLS) method for discriminate analysis. Results. The OSC-PLS (ctr) analysis results of the nuclear overhauser enhancement spectroscopy (NOESY) detection indicated that the syndromes in SHS could be differentiated, and there were significant differences in the levels of metabolites of the urine samples of the four groups; the biomarkers of LSSDS in SHS were found out. The contents of citric acid (2.54 and 2.66), trimethylamineoxide (3.26), and hippuric acid (3.98, 7.54, 7.58, 7.62, 7.66, 7.82, and 7.86) in the urine samples of LSSDS group were lower than that of the normal control group. Conclusion. There are differences in the (1)H-NMR metabolic spectrum of the urine samples of the four groups, and the specific metabolic products of the LSSDS in SHS can be identified from metabonomics analysis.

Gene expression profile of the hippocampus of rats subjected to chronic immobilization stress.

OBJECTIVE: This study systematically investigated the effect of chronic stress on the hippocampus and its damage mechanism at the whole genome level. METHODS: The rat whole genome expression chips (Illumina) were used to detect gene expression differences in the hippocampus of rats subjected to chronic immobilization stress (daily immobilization stress for 3 h, for 7 or 21 days). The hippocampus gene expression profile was studied through gene ontology and signal pathway analyses using bioinformatics. A differentially expressed transcription regulation network was also established. Real-time quantitative polymerase chain reaction (RT-PCR) was used to verify the microarray results and determine expression of the Gabra1, Fadd, Crhr2, and Cdk6 genes in the hippocampal tissues. RESULTS: Compared to the control group, 602 differentially expressed genes were detected in the hippocampus of rats subjected to stress for 7 days, while 566 differentially expressed genes were expressed in the animals experiencing stress for 21 days. The stress significantly inhibited the primary immune system functions of the hippocampus in animals subjected to stress for both 7 and 21 days. Immobilization activated the extracellular matrix receptor interaction pathway after 7 day exposure to stress and the cytokine-cytokine receptor interaction pathway. The enhanced collagen synthesis capacity of the hippocampal tissue was the core molecular event of the stress regulation network in the 7-day group, while the inhibition of hippocampal cell growth was the core molecular event in the 21-day group. For the Gabra1, Fadd, Crhr2, and Cdk6 genes, RT-PCR results were nearly in line with gene chip assay results. CONCLUSION: During the 7-day and 21-day stress processes, the combined action of polygenic, multilevel, and multi-signal pathways leads to the disorder of the immunologic functions of the hippocampus, hippocampal apoptosis, and proliferation disequilibrium.