Protective effect and mechanism of Lycium barbarum L. polyphenol on cognitive impairment induced by ethanol in mice.

BACKGROUND: Chronic excessive ethanol consumption damages the central nervous system and causes neurobehavioral changes, such as cognitive impairment, which is related to oxidative stress and inhibition of neurogenesis in the hippocampus. It is known that promoting neurogenesis improves learning memory, anxiety and depression. Lycium barbarum L. polyphenol (LBP) is the main active ingredient of Lycium barbarum L., which has excellent neuroprotective effects. However, the effects and mechanisms of LBP on ethanol-induced cognitive impairment are unclear. PURPOSE: To assess the effects and mechanisms of LBP on ethanol-induced cognitive impairment in mice. METHODS: Eight-weeks-old adult C57BL/6J mice were allowed to drink ethanol (10%) to establish a model of ethanol-induced cognitive impairment. From the 29th day of LBP (25, 50, 100, 200, 400 mg/kg, intragastric administration), the locomotor activity, novel object recognition (NOR), Y maze and Morris water maze (MWM) were sequentially performed to investigate the effect of LBP on ethanol-induced cognitive impairment in mice. Next, enzyme-linked immunosorbent assay, immunofluorescence, and western blotting were used to study the underlying mechanism of LBP on ethanol-induced cognitive impairment. RESULTS: LBP significantly decreased the escape latency and increased the number of crossings of the original platform in MWM, increased the spontaneous alteration behavior in the Y maze, and increased the preference index in the NOR in ethanol-induced mice. Notably, LBP significantly promoted the proliferation of neural stem cells, neural progenitor cells and neuroblasts, and increased the proportion of activated NSCs in mice with ethanol-induced cognitive impairment. Similarly, LBP significantly increased the number of newborn immature neurons and mature neurons. Moreover, LBP increased the levels of nuclear factor erythroid2-related factor 2 (Nrf2) and the downstream heme oxygenase-1(HO-1) protein expression, which led to a decrease of oxidative stress levels. CONCLUSION: LBP significantly improves cognitive impairment in ethanol-induced mice, which is attributed to the promotion of hippocampal neurogenesis and reduction of oxidative stress.

Potential mechanism of action of Jing Fang Bai Du San in the treatment of COVID-19 using docking and network pharmacology.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), severely infects people and has rapidly spread worldwide. JingFangBaiDu San (JFBDS) has been used to treat prevalent epidemic pathogens, common cold, headache, cough due to lung-cold, and other symptoms; however, its treatment for COVID-19 is unknown. Molecular docking and network pharmacology were applied to obtain ingredient-protein structures and the herb-ingredient-disease target network model, respectively, to explore the potential mechanism of JFBDS in COVID-19 treatment. Network pharmacology analysis showed that acacetin, wogonin, and isorhamnetin were the main active ingredients of JFBDS, and EGFR, PIK3CA, LCK, MAPK1, MAPK3, MAPK8, STAT3, TNF, IL2, and RELA were speculated to be crucial therapeutic targets. Moreover, the Toll-like receptors, HIF-1, PIK3K/AKT, MAPK, NF-κB and NOD-like receptor signaling pathways were important for JFBDS in COVID-19 treatment. Molecular docking analysis indicated that ingredients of JFBDS could bind to angiotensin converting enzyme II, spike protein, and chymotrypsin like protease (3CLpro), which inhibits virus entry and replication in host cells. This study provides a new perspective for understanding potential therapeutic effects and mechanisms of JFBDS in COVID-19 and may facilitate its clinical application.

Neuroprotective effect of pseudoginsenoside-F11 on permanent cerebral ischemia in rats by regulating calpain activity and NR2A submit-mediated AKT-CREB signaling pathways.

BACKGROUND: N-methyl-d-aspartate receptors (NMDARs) have been demonstrated to play central roles in stroke pathology and recovery, including dual roles in promoting either neuronal survival or death with their different subtypes and locations. PURPOSE: We have previously demonstrated that pseudoginsenoside-F11 (PF11) can provide long-term neuroprotective effects on transient and permanent ischemic stroke-induced neuronal damage. However, it is still needed to clarify whether NMDAR-2A (NR2A)-mediated pro-survival signaling pathway is involved in the beneficial effect of PF11 on permanent ischemic stroke. MATERIAL AND METHODS: PF11 was administrated in permanent middle cerebral artery occlusion (pMCAO)-operated rats. The effect of PF11 on oxygen-glucose deprivation (OGD)-exposed primary cultured neurons were further evaluated. The regulatory effect of PF11 on NR2A expression and the activation of its downstream AKT-CREB pathway were detected by Western blotting and immunofluorescence in the presence or absence of a specific NR2A antagonist NVP-AAM077 (NVP) both in vivo and in vitro. RESULTS: PF11 dose- and time-dependently decreased calpain1 (CAPN1) activity and its specific breakdown product α-Fodrin expression, while the expression of Ca2+/calmodulin-dependent protein kinase II alpha (CaMKII-α) was significantly upregulated in the cortex and striatum of rats at 24 h after the onset of pMCAO operation. Moreover, PF11 prevented the downregulation of NR2A, p-AKT/AKT, and p-CREB/CREB in both in vivo and in vitro stroke models. Finally, the results indicated treatment with NVP can abolish the effects of PF11 on alleviating the ischemic injury and activating NR2A-mediated AKT-CREB signaling pathway. CONCLUSIONS: Our results demonstrate that PF11 can exert neuroprotective effects on ischemic stroke by inhibiting the activation of CAPN1 and subsequently enhancing the NR2A-medicated activation of AKT-CREB pathway, which provides a mechanistic link between the neuroprotective effect of PF11 against cerebral ischemia and NR2A-associated pro-survival signaling pathway.

Seed oil of Brucea javanica induces apoptosis through the PI3K/Akt signaling pathway in acute lymphocytic leukemia Jurkat cells.

Brucea javanica oil emulsion (BJOE) has been used to treat tumor in China for more than 40 years. However, its components and effectiveness in the treatment of acute lymphocytic leukemia (ALL) and its mechanism of anti-cancer activity remain unknown. In the current study, high-performance liquid chromatography-evaporative light scattering detector (HPLC-ELSD) was used to analyze the components of BJOE. Then, the anti-leukemia effects of BJOE were examined both in vitro and in vivo using ALL Jurkat cells and the p388 mouse leukemia transplant model, respectively. The primary ALL leukemia cells were also used to confirm the anti-leukemia effects of BJOE. The apoptotic-related results indicated that BJOE induced apoptosis in Jurkat cells and were suggestive of intrinsic apoptotic induction. Moreover, BJOE inhibited Akt (protein kinase B) activation and upregulated its downstream targets p53 and FoxO1 (forkhead box gene, group O-1) to initiate apoptosis. The activation of GSK3ß was also involved. Our findings demonstrate that BJOE has anti-leukemia effects on ALL cells and can induce apoptosis in Jurkat cells through the phosphoinositide3-kinase (PI3K) /Akt signaling pathway.

Assessment of reproductive toxicity and genotoxicity of Aconiti Lateralis Radix Praeparata and its processed products in male mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Aconiti Lateralis Radix Praeparata (Chinese name: Fuzi), the root of Aconitum carmichaelii Debx., is a representative medicine for restoring yang and rescuing patient from collapse. However, less studies had been reported on the reproductive toxicity and genotoxicity of Fuzi. According to the principle of reducing toxicity and preserving efficiency, only processed products of Fuzi are commonly applied in clinic, including Baifupian, Heishunpian and Danfupian. However, whether processing could alleviate the reproductive toxicity and genotoxicity of Fuzi had not been revealed. AIM OF THE STUDY: To assess the effect and possible mechanism of Fuzi and its processed products on reproductive toxicity and genotoxicity in male mice. MATERIALS AND METHODS: Aqueous extracts of Fuzi and its processed products (Baifupian, Heishunpian and Danfupian, 5.85 g/kg) were administrated by gavage once daily for fourteen consecutive days. The reproductive toxicity was evaluated by testis weight, testis ratio, testis histopathology, sperm count, sperm viability rate and sperm deformity rate. The genotoxicity was evaluated by comet assay and micronucleus test in sperm, peripheral blood cell and bone marrow cell. Possible mechanisms of attenuating toxicity by processing were analyzed by detecting the level of testosterone, superoxide dismutase (SOD), glutathione (GSH), malondialdehyde (MDA) and catalase (CAT). RESULTS: Fuzi significantly caused different degrees of reproductive toxicity and genotoxicity, specifically reducing the weight and testicular coefficient of testis, causing obvious pathological changes in testicular tissue, reducing sperm count and sperm viability rate, increasing sperm deformity rate and DNA damage in sperm/peripheral blood cells/bone marrow cells. Moreover, Fuzi decreased the level of testosterone, SOD, GSH and CAT, while increased the level of MDA in serum. Notably, the reproductive toxicity and genotoxicity induced by the processed products, especially Heishunpian and Danfupian, were significantly lowered compared to Fuzi. Processing could increase the level of testosterone, SOD, GSH, CAT and decrease the level of MDA compared to Fuzi. CONCLUSION: Fuzi and its processed products had reproductive toxicity and genotoxicity, but the toxicity of processed products was significantly weakened compared to Fuzi. The protective mechanism of processing to reduce the toxicity of Fuzi might be related to increasing the level of testosterone and decreasing oxidative stress.

Oxytocin signaling in the treatment of drug addiction: Therapeutic opportunities and challenges.

Drug addiction is one of the leading causes of mortality worldwide. Despite great advances were achieved in understanding the neurobiology of drug addiction, the therapeutic options are severely limited, with poor effectiveness and serious side effects. The neuropeptide oxytocin (OXT) is well known for its effects on uterine contraction, sexual/maternal behaviors, social affiliation, stress and learning/memory by interacting with the OXT receptor and other neuromodulators. Emerging evidence suggests that the acute or chronic exposure to drugs can affect the OXT system. Additionally, OXT administration can ameliorate a wide range of abused drug-induced neurobehavioral changes. Overall, OXT not only suppresses drug reward in the binge stage of drug addiction, but also reduces stress responses and social impairments during the withdrawal stage and, finally, prevents drug/cue/stress-induced reinstatement. More importantly, clinical studies have also shown that OXT can exert beneficial effects on reducing substance use disorders of a series of drugs, such as heroin, cocaine, alcohol, cannabis and nicotine. Thus, the present review focuses on the role of OXT in treating drug addiction, including the preclinical and clinical therapeutic potential of OXT and its analogs on the neurobiological perspectives of drugs, to provide a better insight of the efficacy of OXT as a clinical addiction therapeutic agent.

Network pharmacology reveals pharmacological effect and mechanism of Panax notoginseng (Burk.) F. H. Chen on reproductive and genetic toxicity in male mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Cisplatin (CP), one of the most commonly used antitumor drugs in clinic, could induce reproductive and genetic toxicity. Traditional Chinese medicine believed that this side effect might be caused by the deficiency of both qi and blood. Panax notoginseng (Burk.) F. H. Chen (PN) is a traditional precious Chinese medicine for nourishing blood and hemostasis, which had the synergistic antitumor and reducing toxicity effects. However, the protective effect and mechanism of PN on CP-induced reproductive and genetic toxicity were still unknown. AIM OF THE STUDY: This study was designed to illuminate the possible protective effect and mechanism of PN on CP-induced reproductive and genetic toxicity. MATERIALS AND METHODS: Network pharmacology was first applied to analyze the potential components and targets of PN against CP-induced reproductive and genetic toxicity. Then, the results of network pharmacology were validated in a mouse model of reproductive and genotoxicity induced by CP. Body weight, testis weight, epididymis weight, sperm count, sperm viability and sperm morphology were used to assess protective effects of PN on CP-induced reproductive toxicity. Tail moment in peripheral blood cells and micronucleus in bone marrow cells were used to assess protective effects of PN on CP-induced genetic toxicity. Finally, possible protective targets obtained from network pharmacology, including 8-hydroxy-2-deoxyguanosine (8-OHdG), malondialdehyde (MDA), total superoxide dismutase (T-SOD) and glutathione peroxidase (GSH-Px), were experimentally validated by ELISA. RESULTS: One hundred and nineteen components of PN and sixty-eight targets of reproductive/genetic toxicity were acquired and constituted as the component-target network. Network pharmacology analysis showed alleviating oxidative stress might play important role in therapeutic mechanism of PN. In verified experiments, PN significantly improved the decline of body weight, testis weight and epididymis weight, increased sperm count and viability, decreased abnormal sperm morphology rate induced by CP in mice. Moreover, PN also significantly decreased the tail moment in peripheral blood cells and micronucleus formation rate in bone marrow cells in CP-induced mice. Finally, not only the decrease of T-SOD and GSH-Px level but also the increase of 8-OHdG and MDA level in serum were restored under PN treatment. CONCLUSION: Current study found that PN could improve CP-induced reproductive and genetic toxicity, which were probably attributed to alleviating oxidative stress. This finding provided the new perspective for understanding the therapeutic effect of PN on CP-induced reproductive and genetic toxicity and facilitating the clinical use of PN.

Pseudoginsenoside-F11 attenuates cognitive dysfunction and tau phosphorylation in sporadic Alzheimer's disease rat model.

We previously reported that pseudoginsenoside-F11 (PF11), an ocotillol-type saponin, significantly ameliorated Alzheimer's disease (AD)-associated cognitive defects in APP/PS1 and SAMP8 mice by inhibiting Aß aggregation and tau hyperphosphorylation, suggesting a potential therapeutic effect of PF11 in the treatment of AD. In the present study we further evaluated the therapeutic effects of PF11 on relieving cognitive impairment in a rat model of sporadic AD (SAD). SAD was induced in rats by bilateral icv infusion of streptozotocin (STZ, 3 mg/kg). The rats were treated with PF11 (2, 4, 8 mg·kg-1·d-1, ig) or a positive control drug donepezil (5 mg·kg-1·d-1, ig) for 4 weeks. Their cognitive function was assessed in the nest building, Y-maze, and Morris water maze tests. We showed that STZ icv infusion significantly affected the cognitive function, tau phosphorylation, and insulin signaling pathway in the hippocampus. Furthermore, STZ icv infusion resulted in significant upregulation of the calpain I/cyclin-dependent protein kinase 5 (CDK5) signaling pathway in the hippocampus. Oral administration of PF11 dose-dependently ameliorated STZ-induced learning and memory defects. In addition, PF11 treatment markedly reduced the neuronal loss, protected the synapse structure, and modulated STZ-induced expression of tau phosphorylation by regulating the insulin signaling pathway and calpain I/CDK5 signaling pathway in the hippocampus. Donepezil treatment exerted similar beneficial effects in STZ-infused rats as the high dose of PF11 did. This study highlights the excellent therapeutic potential of PF11 in managing AD.

Gut microbiota and Chinese medicine syndrome: altered fecal microbiotas in spleen (Pi)-deficient patients.

OBJECTIVE: To investigate the fecal microbiota changes in patients with spleen (Pi)-deficiency (SPD), a common Chinese medicine syndrome with digestive and absorptive disturbances and to provide insight into the relationship between Chinese medicine syndrome and gut dysbiosis. METHODS: Fecal microbiotas from the stool samples of 53 SPD patients and 35 healthy subjects were analyzed via 16S rRNA gene polymerase chain reaction (PCR)-denaturation gradient gel electrophoresis (DGGE). SPD-related marker genes from 20 SPD patients and 20 healthy subjects were identified through gene sequencing, while some genes were quantified using quantitative PCR (qPCR). Discriminant analysis was conducted using SPSS software, and the canonical discriminant function formula for Pi-deficiency was established. RESULTS: Alterations in microbiota diversity and composition between the SPD and healthy groups were demonstrated via 16S rRNA gene PCR-DGGE combined with multivariate statistical analysis. Fecal microbiota changes were also observed among different SPD-subtype patients. Eight SPD-related markers were found, and putative species corresponding to these markers were identified through gene sequencing, which may have potential associations with the common digestive dysfunctions in SPD patients. qPCR methods were established for two of these markers, which were significantly altered in the SPD patients. The canonical discriminant function formula was calculated for SPD, and the validity rates of these markers were over 85%. CONCLUSION: Fecal microbiotas are altered in patients with SPD, which may provide insight for further studies on clinically diagnosing and treating SPD. The results may also provide data to gain a better understanding of Traditional Chinese Medicine syndrome and gut dysbiosis.

Antidepressant-like effects of Xiaochaihutang in perimenopausal mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Xiaochaihutang (XCHT) is a traditional Chinese medicine prescription for thousand years in China. Our previous researches show that XCHT has antidepressant-like effects in several depression models, but effect and mechanism of XCHT in perimenopausal depression are still vague. AIM OF THE STUDY: To reveal the antidepressant-like effect and mechanism of XCHT in perimenopausal mice. MATERIALS AND METHODS: Perimenopausal depression model is executed by ovariectomy combined with chronic unpredictable mild stress (OVX-CUMS). Tail suspension test (TST), forced swim test (FST), elevated plus-maze (EPM), novelty suppressed feeding (NSF) and locomotor activity are used to assess antidepressant-like effects of XCHT. The Level of estradiol (E2), follicle-stimulating hormone (FSH), gonadotrophin releasing hormone (GnRH), corticosterone (CORT), adrenocorticotrophic hormone (ACTH) and corticotropin releasing hormone (CRH) are evaluated by ELISA. Antidepressant mechanisms of XCHT in OVX-CUMS mice are analyzed by 5-hydroxytryptamine (5-HT), tryptophan hydroxylase 2 (TPH2) and estrogen receptor α and ß (ERα/ß). RESULTS: The results show that OVX-CUMS significantly increases the immobility time in TST and FST, increases latency to feed, decreases food consumption in NSF and both the time spend and number of entries in open arms in EPM. While, oral administration of XCHT can significantly normalize above depression-like behaviors in OVX-CUMS mice. Moreover, XCHT also remarkably normalized levels of 5-HT, 5-HIAA, E2, GnRH, CORT, ACTH and CRH in OVX-CUMS mice. Finally, the expression of ERß and TPH2 are decreased by OVX-CUMS in prefrontal cortex and hypothalamus, and XCHT can restore these decrease. CONCLUSION: Current findings suggest XCHT can alleviate perimenopausal depression-like behaviors, restore 5-HT and hormones in OVX-CUMS mice, which may be related to normalizing the functions of HPA/HPO axis and enhancing expression of ERß and TPH2 in prefrontal cortex and hypothalamus.

Protective effect of Liuwei Dihuang Pill on cisplatin-induced reproductive toxicity and genotoxicity in male mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Cisplatin (CP) is the classical chemotherapeutic drug for various cancer, but it also accompanies reproductive toxicity and genotoxicity. Liuwei Dihuang Pill (LW) is the traditional Chinese medicine prescription for treating Kidney-Yin deficiency syndrome, which has been reported to prevent and treat various diseases. However, the protective effect of LW on CP-induced reproductive toxicity and genotoxicity has not been reported. AIM OF THE STUDY: To investigate the potential protective effect and mechanism of LW on CP-induced reproductive toxicity and genotoxicity in male mice. MATERIALS AND METHODS: Mice were given LW (0.4, 1.2 and 3.6 g/kg) or Vitamin C (0.1 g/kg) once daily by oral gavage for thirteen consecutive days. Then, CP (3.00 mg/kg) was given intraperitoneal injection once daily for five consecutive days starting on the ninth day. The protective effects of LW against CP-induced reproductive toxicity and genotoxicity were evaluated by body weight, testis ratio, sperm count, sperm viability, sperm abnormal morphology type, micronuclei test, testicular histopathology, serum malondialdehyde (MDA), total superoxide dismutase (T-SOD), glutathione peroxidase (GSH-Px) and catalase (CAT) level. RESULTS: The results demonstrated that LW could significantly increase CP-induced the reduction of sperm count and sperm viability, then decrease abnormal sperm type rate and micronucleus rate. Moreover, LW also could improve testicular abnormal histopathologic morphology induced by CP exposure. Meanwhile, LW decreased serum MDA level and increased T-SOD, GSH-Px and CAT level compared to CP group. CONCLUSION: our findings show that LW has protective effects on CP-induced reproductive toxicity and genotoxicity. LW decreases serum MDA level and increases T-SOD, GSH-Px and CAT level, which indicates that antioxidant activity may be the potential mechanism of LW to resist reproductive toxicity and genotoxicity.

Evaluation and Identification of the Neuroprotective Compounds of Xiaoxuming Decoction by Machine Learning: A Novel Mode to Explore the Combination Rules in Traditional Chinese Medicine Prescription.

Xiaoxuming decoction (XXMD), a classic traditional Chinese medicine (TCM) prescription, has been used as a therapeutic in the treatment of stroke in clinical practice for over 1200 years. However, the pharmacological mechanisms of XXMD have not yet been elucidated. The purpose of this study was to develop neuroprotective models for identifying neuroprotective compounds in XXMD against hypoxia-induced and H2O2-induced brain cell damage. In this study, a phenotype-based classification method was designed by machine learning to identify neuroprotective compounds and to clarify the compatibility of XXMD components. Four different single classifiers (AB, kNN, CT, and RF) and molecular fingerprint descriptors were used to construct stacked naïve Bayesian models. Among them, the RF algorithm had a better performance with an average MCC value of 0.725±0.014 and 0.774±0.042 from 5-fold cross-validation and test set, respectively. The probability values calculated by four models were then integrated into a stacked Bayesian model. In total, two optimal models, s-NB-1-LPFP6 and s-NB-2-LPFP6, were obtained. The two validated optimal models revealed Matthews correlation coefficients (MCC) of 0.968 and 0.993 for 5-fold cross-validation and of 0.874 and 0.959 for the test set, respectively. Furthermore, the two models were used for virtual screening experiments to identify neuroprotective compounds in XXMD. Ten representative compounds with potential therapeutic effects against the two phenotypes were selected for further cell-based assays. Among the selected compounds, two compounds significantly inhibited H2O2-induced and Na2S2O4-induced neurotoxicity simultaneously. Together, our findings suggested that machine learning algorithms such as combination Bayesian models were feasible to predict neuroprotective compounds and to preliminarily demonstrate the pharmacological mechanisms of TCM.

Quantitative proteomics reveal antidepressant potential protein targets of xiaochaihutang in corticosterone induced model of depression.

ETHNOPHARMACOLOGICAL RELEVANCE: Xiaochaihutang (XCHT), one of famous Chinese herbal prescription for treating Shaoyang symptom, has been used successfully in depressive disorders for many years. Our laboratory has demonstrated that XCHT remarkably alleviated various depressive behaviors induced by several depressive animal models, but previous studies only focused on one or several protein targets, lacked dynamic change and interrelation of proteins. Therefore, potential protein targets and mechanisms are required further systematic investigation. AIM OF THE STUDY: To discover and assess the differentially expressed proteins (DEPs) of hippocampus after oral administration of XCHT in corticosterone (CORT) induced model of depression by using isobaric tags for relative and absolute quantification (iTRAQ) analysis. MATERIALS AND METHODS: The antidepressant effects of XCHT were assessed by two behavioral despair models (forced swimming test and tail suspension test) in CORT induced model of depression. The DEPs of hippocampus after XCHT treatment were investigated by iTRAQ analysis. Potential protein targets and mechanisms were assessed by gene ontology (GO), Kyoto encyclopedia of gene and genomes (KEGG) and protein-protein interaction (PPI) network. RESULTS: Our data demonstrated XCHT could significantly improve depressive behaviors. A total of 241 DEPs were identified after XCHT treatment, including 68 up regulation and 173 down regulation proteins. GO enrichment results indicated that XCHT mainly regulated intracellular structural proteins involved in cellular response to stress, transferase activity and steroid hormone. KEGG enrichment analysis showed that endocytosis might be the principal pathway of XCHT on depression. PPI analysis predicted cell division cycle and apoptosis regulator protein 1 (Ccar1) and Calretinin (Calb2) might play the central roles in XCHT's antidepressant network. CONCLUSION: Our results indicate that XCHT plays the important roles in antidepressant action by restoring DEPs, which results in the dysregulation of hippocampal neurogenesis, neurotransmitter and steroid hormone. The current results wish to provide novel perspectives for revealing the potential protein targets of XCHT on depression.

Activation of an AKT/FOXM1/STMN1 pathway drives resistance to tyrosine kinase inhibitors in lung cancer.

BACKGROUND: Tyrosine kinase inhibitors (TKIs) have demonstrated clinical benefits in the treatment of several tumour types. However, the emergence of TKI resistance restricts the therapeutic effect. This study uses non-small cell lung cancer (NSCLC) to explore the mechanisms contributing to TKI resistance in tumours. METHODS: Biological phenotypes and RNA microarray expression data were analysed in NSCLC cells with and without TKI pretreatment. Specific inhibitors and siRNAs were used to validate the direct involvement of an AKT/FOXM1/STMN1 pathway in TKI resistance. Patients' tissues were analysed to explore the clinical importance of FOXM1 and STMN1. RESULTS: In vitro and in vivo studies showed that TKIs induced the enrichment of cancer stem cells (CSC), promoted epithelial to mesenchymal transition (EMT), and conferred multidrug resistance on NSCLC cells in a cell type- and TKI class-dependent manner. Mechanistically, TKIs activated an AKT/FOXM1/STMN1 pathway. The crucial role of this pathway in TKI-induced enrichment of CSC and drug resistance was verified by silencing FOXM1 and STMN1 or blocking the AKT pathway. Additionally, overexpression of STMN1 was associated with upregulation of FOXM1 in advanced NSCLC patients, and STMN1/FOXM1 upregulation predicted a poor outcome. CONCLUSIONS: Our findings elucidate an additional common mechanism for TKI resistance and provide a promising therapeutic target for reversing TKI resistance in NSCLC.

Xiaochaihutang attenuates depressive/anxiety-like behaviors of social isolation-reared mice by regulating monoaminergic system, neurogenesis and BDNF expression.

ETHNOPHARMACOLOGICAL RELEVANCE: Xiaochaihutang (XCHT), as a classical herbal formula for the treatment of "Shaoyang syndrome" has been demonstrated to exert an antidepressant effect in multiple animal models of depression as shown in our previous studies. However, the effects of XCHT on social isolation (SI)-reared mice have not been investigated. This study aims to explore the effects of XCHT on depressive/anxiety-like behaviors of SI-reared mice, and its implicated mechanisms, including alterations in the monoaminergic system, neurogenesis and neurotrophin expression. MATERIALS AND METHODS: Male C57 BL/6J mice (aged 4 weeks after weaning) were reared isolatedly for 8 weeks and XCHT (0.8, 2.3, 7.0g/kg) were given by gavage once a day. Forced swimming test (FST), tail suspension test (TST), open field test (OFT), elevated-plus maze test (EPM) and intruder-induced aggression test were used to explore the effects of XCHT on depressive/anxiety-like behaviors of SI-reared mice after administration of XCHT for 6 weeks. HPLC-MS/MS was performed to quantify the levels of neurotransmitters in the hippocampus by in vivo microdialysis, while western immunoblotting was used to evaluate the action of XCHT on the synthesis, transport and degradation of monoamine neurotransmitters. Immunofluorescence was used to study the effects of XCHT on neurogenesis and neurotrophin expression, including Ki-67, DCX, BrdU and BDNF. RESULTS: Our results showed that administration of XCHT (0.8, 2.3 and 7.0g/kg) for 6 weeks significantly attenuated the increase in immobility time in TST and FST, improved the anxiety-like behaviors in OFT and EPM, and improved the aggressive behaviors of SI-reared mice. XCHT significantly elevated monoamine neurotransmitters levels and inhibited 5-HT turnover (5-HIAA/5-HT) in hippocampal microdialysates of SI-reared mice. In addition, we found XCHT enhanced monoamine neurotransmitter synthesis enzymes (TPH2 and TH) expressions, inhibited serotonin transporter (SERT) expression and decreased monoamine neurotransmitter degradation enzyme (MAOA) expression in the hippocampus of SI-reared mice for the first time. Moreover, XCHT significantly augmented hippocampal neurogenesis and BDNF expression in hippocampus of SI-reared mice. CONCLUSIONS: Our results showed for the first time that XCHT improved depressive/anxiety-like behaviors of SI-reared mice by regulating the monoaminergic system, neurogenesis and neurotrophin expression. The findings indicate that XCHT may have a therapeutic application for early-life stress model of depression and in turn provide further evidence supporting XCHT a novel potential antidepressant from a distinct perspective.

Development and validation of a UHPLC-MS/MS method for the simultaneous determination of five bioactive flavonoids in rat plasma and comparative pharmacokinetic study after oral administration of Xiaochaihu Tang and three compatibilities.

An accurate, rapid, and reliable ultra high performance liquid chromatography with tandem mass spectrometry method was developed and validated for the simultaneous determination of baicalin, wogonoside, baicalein, wogonin, and oroxylin A in rat plasma. Then, the stability of baicalin and baicalein in the preparation of plasma sample was systematically investigated. The Waters BEH C18 column was used with a gradient mobile phase system of acetonitrile and water containing 0.1% formic acid. The analytes were detected in the multiple reaction monitoring mode with positive electrospray ionization. 100 µL fresh plasma was added with 50 µL antioxidant reagent (1 mol/L HCl containing 0.5% Vitamin C), and liquid-liquid extraction with ethyl acetate was used to extract the analytes from plasma. Lower limits of quantification of baicalin, wogonoside, baicalein, wogonin, and oroxylin A were 21.9, 4.80, 1.20, 0.848, and 0.800 ng/mL, respectively. The mean extract recoveries of five flavonoids were 69.1∼89.2%, and the precision and accuracy were within the acceptable limits. This method was further successfully applied to the comparative pharmacokinetic study of these five flavonoids in rats after oral administration of Xiaochaihutang and three compatibilities. The obtained results may be helpful to reveal the mechanism of Xiaochaihutang formula compatibility.

Antidepressant-like effects of Xiaochaihutang in a neuroendocrine mouse model of anxiety/depression.

ETHNOPHARMACOLOGICAL RELEVANCE: Hyperactivity of hypothalamic-pituitary-adrenal (HPA) axis is often observed in the pathophysiology of depression. Antidepressant therapy can restore hippocampal neurogenesis to rescue the HPA axis regulation defects. Xiaochaihutang (XCHT), a famous Chinese herbal formula, has been used clinically in depressive disorders in China. Our previous studies have demonstrated XCHT improved depressive-like behaviors in chronic unpredictable mild stress rat, but the underlying mechanisms of XCHT on hippocampal neurogenesis and the HPA axis were still unclear. MATERIALS AND METHODS: We used chronic corticosterone (CORT)-induced mouse model of anxiety/depression to investigate antidepressant-like effects of XCHT by several physical and behavioral testing, including body weight, coat state, open field test, elevated plus maze, tail suspension test and forced swimming test. The integrity of negative feedback function on HPA axis was assessed by the dexamethasone (DEX) suppression test. In addition, Ki-67 and doublecortin (DCX) were performed to assess hippocampal cell proliferation and neurogenesis by immunohistochemistry. Chemical profile of active constituents in brain after oral administration of XCHT was revealed by ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). RESULTS: Our results showed that oral administration of XCHT (2.3, 7 and 21g/kg) for 30 days remarkably normalized chronic CORT-induced the slowness in weight gain, the deterioration in coat state, the escape behavior in open field test and elevated plus maze, and the increase of immobility time in tail suspension test and forced swimming test. Moreover, XCHT significantly reversed chronic CORT-induced the reduction of DEX-induced plasma corticosterone/c-Fos suppression and Ki-67/DCX positive cells. Finally, a total 13 potential active constituents in brain were identified by UPLC-MS/MS after oral administration of XCHT, including 10 prototype components and 3 metabolites. CONCLUSIONS: Our findings showed that XCHT could remarkably alleviate chronic CORT-induced anxiety/depression-like behaviors, which were probably attribute to promoting hippocampal neurogenesis and remodeling the integrity of the negative feedback loop on HPA axis. The constituents identified in brain might contribute to understanding the therapeutic basis of XCHT on depression.

Serum metabonomics study of anti-depressive effect of Xiao-Chai-Hu-Tang on rat model of chronic unpredictable mild stress.

Xiao-Chai-Hu-Tang (XCHT) has been proven to be effective for the clinical treatment of depression. However, the mechanisms of definite antidepressant-like effects and detailed metabolic biomarkers were still unclear in this prior study. Here, we have investigated the metabolic profiles and potential biomarkers in a chronic unpredictable mild stress model after treatment with XCHT. Metabonomics based on ultra-high performance liquid chromatography coupled with mass spectrometry was used to profile the metabolic fingerprints of serum obtained from a rat model with chronic unpredictable mild stress with and without XCHT treatment. The model rats showed a significant decrease in sucrose preference and food consumption, and these depression-like symptoms were significantly improved by XCHT. Through principal component analysis (PCA), nine potential biomarkers of tryptophan, uric acid, phenylalanine, cholic acid and lysophosphatidylcholine (C18:0 LPC, C16:0 LPC, C16:1 LPC, C18:1 LPC, C20:4 LPC) were characterized as potential biomarkers involved the pathogenesis of depression. The therapeutic effect of XCHT on depression may involve in amino acid metabolism, lipid metabolism, oxidative stress and inflammation response. The present investigation highlights that metabonomics is a valuable tool for studying the essence of depression as well as evaluating the efficacy of the corresponding drug treatment.

Study of Malformin C, a Fungal Source Cyclic Pentapeptide, as an Anti-Cancer Drug.

Malformin C, a fungal cyclic pentapeptide, has been claimed to have anti-cancer potential, but no in vivo study was available to substantiate this property. Therefore, we conducted in vitro and in vivo experiments to investigate its anti-cancer effects and toxicity. Our studies showed Malformin C inhibited Colon 38 and HCT 116 cell growth dose-dependently with an IC50 of 0.27±0.07µM and 0.18±0.023µM respectively. This inhibition was explicated by Malformin C's effect on G2/M arrest. Moreover, we observed up-regulated expression of phospho-histone H2A.X, p53, cleaved CASPASE 3 and LC3 after Malformin C treatment, while the apoptosis assay indicated an increased population of necrotic and late apoptotic cells. In vivo, the pathological study exhibited the acute toxicity of Malformin C at lethal dosage in BDF1 mice might be caused by an acute yet subtle inflammatory response, consistent with elevated IL-6 in the plasma cytokine assay. Further anti-tumor and toxicity experiments proved that 0.3mg/kg injected weekly was the best therapeutic dosage of Malformin C in Colon 38 xenografted BDF1 mice, whereas 0.1mg/kg every other day showed no effect with higher resistance, and 0.9mg/kg per week either led to fatal toxicity in seven-week old mice or displayed no advantage over 0.3mg/kg group in nine-week old mice. Overall, we conclude that Malformin C arrests Colon 38 cells in G2/M phase and induces multiple forms of cell death through necrosis, apoptosis and autophagy. Malformin C has potent cell growth inhibition activity, but the therapeutic index is too low to be an anti-cancer drug.