Screening the components in multi-biological samples and the comparative pharmacokinetic study in healthy and depression model rats of Suan-Zao-Ren decoction combined with a network pharmacology.

ETHNOPHARMACOLOGICAL RELEVANCE: Suanzaoren Decoction (SZRD) is a classic traditional Chinese prescription, which has been commonly used for treating insomnia, depression and other nerve system diseases for a long time. AIM OF THIS STUDY: The present study aimed to explore the metabolic profiles in multi-biological samples and pharmacokinetic mechanism between healthy and depression model rats combined with a network pharmacology approach after administration of SZRD. MATERIALS AND METHODS: In our study, an ultra-high performance liquid chromatography (UPLC)-Q-Exactive Orbitrap Mass Spectrometry method was firstly used to study the prototype components and metabolites of SZRD in plasma, brain, urine, and feces between healthy and depressed rats. The possible metabolic pathways were also speculated. Then a network pharmacological study was conducted on the components in the plasma of model rats. According to the above components screened by network pharmacology and the other reported representative active components, the comparative pharmacokinetic study was established for the simultaneous determination of mangiferin, spinosin, ferulic acid, liquiritin, formononetin. magnoflorine and isoliquiritin between healthy and depression model rats. Finally, molecular docking was used to validate the binding affinity between key potential targets and active components in pharmacokinetics. RESULTS: A total of 115 components were identified in healthy rats, and 101 components were identified in model rats. The prototype components and metabolites in plasma, brain, urine, and feces were also distinguished. The main metabolic pathways included phase I and phase II metabolic reactions, such as dehydrogenation, oxidation, hydroxylation, gluconaldehyde conjugation, glutathione conjugation and so on. These results provided a basis for the further study of antidepressive pharmacokinetic and pharmacological action in SZRD. Then, according to the degree value of network pharmacological study, it was predicted that 10 components and 10 core targets, which involved in the critical pathways such as neuroactive ligand-receptor interaction, cyclic adenosine monophosphate (cAMP) signaling pathway, serotonergic synapse, phosphatidylinositol-3 kinase (PI3K)-Akt signaling pathway, etc. Finally, the established pharmacokinetic method was successfully applied to compare the pharmacokinetic behavior of these 7 active components in plasma of healthy and depressed rats after oral administration of SZRD. It showed that except magnoflorine, the pharmacokinetic parameters of each component were different between healthy and depressed rats. Molecular docking analysis also indicated that the active compounds in pharmacokinetics could bind tightly to the key targets of network pharmacological study. CONCLUSION: This study may provide important information for studying the action mechanism of SZRD in treating depression.

A network pharmacology approach to investigate dehydrocostus lactone inhibits the proliferation and epithelial-mesenchymal transition of human gastric cancer cells via regulating the PI3K/Akt and extracellular signal-regulated kinases/mitogen-activated protein kinase signalling pathways.

OBJECTIVES: Dehydrocostus lactone (DHE), a sesquiterpene lactone, has been proven the significant inhibition of multiple cancer cells. However, there are limited reports on the activity of DHE in gastric cancer (GC). In this research, Network pharmacology predicted the anti-GC mechanism of DHE, and the prediction was verified by in-vitro experiments. METHODS: Network pharmacology confirmed the major effect signalling pathway of DHE in treating GC. Cell viability assay, colony formation assay, wound healing assay, cell migration and invasion assay, apoptosis assay, western blot and real-time quantitative polymerase chain reaction verified the mechanism of DHE in GC cell lines. KEY FINDINGS: The results showed that DHE inhibited the growth and metastasis of MGC803 and AGS GC cells. Mechanistically, the analysis results indicated that DHE significantly induced the apoptosis process by suppressing the PI3K/protein kinase B (Akt) signalling pathway, and inhibited epithelial-mesenchymal transition by suppressing the extracellular signal-regulated kinases (ERK)/MAPK signalling pathway. The Akt activator (SC79) inhibited DHE induced apoptosis, and DHE had similar effects with the ERK inhibitor (FR180204). CONCLUSIONS: All results suggested that DHE was a potential natural chemotherapeutic drug in GC treatment.

Biomarkers based on multiplatform comprehensive analysis: A systematic analysis of Geng-Nian-Shu in perimenopausal syndrome.

Although Geng-Nian-Shu has been shown to be clinically effective in perimenopausal syndrome, its active components and mechanism have not yet been elucidated. To demonstrate the mechanism-based biomarkers of Geng-Nian-Shu in treating perimenopausal syndrome, a total of 135 chemical constituents including 52 prototype blood constituents were identified via high-performance liquid chromatography-quadrupole-time of flight/mass spectrometry. Then, network pharmacology showed significant enrichment for the PhosphoInositide-3 Kinase/Akt pathway, suggesting that it may be the main regulatory pathway for the Geng-Nian-Shu treatment of the perimenopausal syndrome. Subsequently, multivariate analysis was performed between the Geng-Nian-Shu sham-treated and Geng-Nian-Shu ovariectomy-treated groups and further screened out 18 prototype blood constituents by correlation analysis with plasma estrogen levels to identify potential biomarkers associated with Geng-Nian-Shu treat the ovariectomy-induced perimenopausal syndrome. Finally, the results of pharmacological experimental verification and Pearson correlation analysis indicated that catalpol, ligustilide, paeoniflorin, and gallic acid were selected as biomarkers of Geng-Nian-Shu which were strongly and positively correlated with PhosphoInositide-3 Kinase/Akt signaling pathway. In this study, based on high-performance liquid chromatography-quadrupole-time of flight/mass spectrometry combined with pharmacodynamics, network pharmacology, pharmacology, and other disciplines, we explored the effects and mechanisms of Geng-Nian-Shu in the treatment of perimenopausal syndrome at multiple levels. Using multiplatform technology to investigate the role of Geng-Nian-Shu represents a new strategy for the selection and verification of biomarkers of Geng-Nian-Shu and provides a basis for further development and utilization of Geng-Nian-Shu.

Wei-Tong-Xin exerts anti-inflammatory effects through TLR4-mediated macrophages M1/M2 polarization and affects GLP-1 secretion.

OBJECTIVES: The present study was undertaken to explore the effects and mechanisms of Wei-Tong-Xin (WTX) in inhibiting lipopolysaccharide (LPS)-induced inflammatory response of macrophages, in turn, to study the influences on GLP-1 secretion of GLUTag cells. METHODS: We first evaluated the activation of Raw 264.7 cells and measured the intracellular ROS, CD86 and CD206 levels by flow cytometry. The expressions of proteins were detected by western blot and immunofluorescence. GLP-1 levels were detected by ELISA kits. TLR4 siRNA was used to investigate the role of TLR4 in the regulation of macrophage polarization by WTX. KEY FINDINGS: The results showed that WTX inhibited LPS-induced polarization of macrophages toward the M1 phenotype, but promoted the M2 phenotype. Meanwhile, WTX inhibited the TLR4/MyD88 pathway. The polarization of M1 phenotype promoted GLP-1 secretion by GLUTag cells, which was inhibited by WTX. The results of siRNA showed that WTX exhibited anti-inflammatory effects through targeting TLR4. CONCLUSIONS: Overall, WTX inhibited polarization of macrophages towards M1 phenotype but promoted the amounts of M2 phenotype, further the macrophages regulated by WTX alleviated GLP-1 content secreted by GLUTag cells. The aforementioned results were produced by WTX-mediated TLR4.

Alpinia oxyphylla Miq. volatile oil ameliorates depressive behaviors and inhibits neuroinflammation in CUMS-exposed mice by inhibiting the TLR4-medicated MyD88/NF-κB signaling pathway.

This study aimed to explore the antidepressant effect and underlying mechanism of the Alpinia oxyphylla Miq. volatile oil (AOVO) in mice exposed to chronic unpredictable mild stress (CUMS). C57BL/6 mice were grouped and administered with different dosages of AOVO (0.25, 0.50, 1.00, or 2.00 mL/kg body weight, i.g.), TAK242 (a TLR4 inhibitor, 0.75 mg/kg body weight, i.p.), or TAK242 (0.75 mg/kg body weight, i.p.) + AOVO (0.50 mL/kg body weight, i.g.) for 21 days. Depression-like symptoms in the mice were then evaluated through their body weight gain (BW), the open field test (OFT), the sucrose preference test (SPT), the novelty-suppressed feeding test (NSFT), and forced swimming test (FST). The concentrations of interleukin-1ß (IL-1ß), interleukin-6 (IL-6), tumor necrosis factor α (TNF-α), and 5-hydroxytyrptamine (5-HT) in the mice were determined using ELISA kits. Hematoxylin and eosin (HE) dying were performed for histopathological examination. The expression of inflammatory proteins was assessed through western blotting (WB) and immunofluorescence staining. AOVO was found to improve the behavioral indexes of CUMS-exposed mice behavioral and synergize TAK242 to mitigate both their depressive symptoms and neuroinflammation. Moreover, AOVO was found to inhibit the hippocampal damage, decrease inflammatory cytokines (Reduced IL-1ß, IL-6, and TNF-α by 19.97 %, 22.87 %, and 24.13 %, respectively), and downregulate the expression of TLR4/MyD88/NF-κB signaling pathway-related proteins in the hippocampus of CUMS-exposed mice (Reduced TLR4, MyD88, and NF-κB by 46.14 %, 42.48 %, and 38.08 %, respectively). These findings demonstrate that AOVO can ameliorate depressive behaviors and mitigate neuroinflammation in the CUMS-exposed mice via suppressing the TLR4-medicated MyD88/NF-κB signaling pathway.

A network pharmacology approach and experimental validation to investigate the anticancer mechanism and potential active targets of ethanol extract of Wei-Tong-Xin against colorectal cancer through induction of apoptosis via PI3K/AKT signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Wei-Tong-Xin (WTX), derives from the Chinese herbal decoction (CHD) of Wan-Ying-Yuan in ancient China, has been shown to be effective therapeutic herbal decoction for treating gastrointestinal diseases. Present studies have demonstrated that WTX had potential to alleviate the symptoms of gastrointestinal inflammation, gastric ulcer and improve gastric motility. AIM OF THE STUDY: The study primarily focused on exploring the therapeutic effect and possible pharmacological mechanism of WTX on colorectal cancer (CRC) based on network pharmacology, in vitro and in vivo experiments. MATERIALS AND METHODS: Firstly, colorectal cancer and WTX associated with targets were searched from GeneCards database and TCM Systems Pharmacology Database and Analysis Platform (TCMSP) respectively. The protein-protein interaction (PPI) network also was constructed to screening key targets. In addition, the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were applied to predict the underlying biological function and mechanism involving in the anti-colorectal cancer effect of WTX. Next, CCK-8, colony formation and transwell assays were performed to verify the influence of proliferation and metastasizing ability of HCT116 cells after treated with WTX. Cell cycle, apoptosis and reactive oxygen species (ROS) were analysis by flow cytometry. Hoechst 33258 staining was conducted to observe nuclear morphology changes. Protein expression of apoptosis and PI3K/AKT signaling as well as mRNA expression of ferroptosis and apoptosis were determined by Western Blotting and RT-qPCR. The effects of WTX and LY294002 combination on the PI3K/Akt/mTOR signaling pathway were measured by Western Blotting. Finally, the xenograft tumor mouse model was established by subcutaneous injection of CT26 cells to measure tumors volume and weight. Hematoxylin and eosin (HE) staining and immunohistochemical analysis were used to observe the pathological changes and the protein expression in tumor tissues. RESULTS: There were 286 potential treatment targets from 130 bioactive compounds in WTX, 1349 CRC-related targets were identified. Eleven core targets (TP53, AKT1, STAT3, JUN, TNF, HSP90AA1, IL-6, MAPK3, CASP3, EGFR, MYC) were found by PPI network analysis constructed of 142 common targets. The results of KEGG enrichment displayed PI3K/AKT signaling pathway as core pathway. After the treatment of WTX, the inhibitory of viability, metastases and cell cycle arrest at G2/M phase were observed in HCT116 cells. Moreover, WTX induced an increase in the expression of apoptosis proteins (Bak, cytochrome c, cleaved caspase-9/caspase-9 and cleaved caspase-3/caspase-3) and the levels of ROS and MDA, a decrease in the expression of PI3K/AKT signaling related proteins (PI3K, p-PI3K, p-AKT/AKT and p-mTOR/mTOR) and the level of SOD. WTX treatment significantly reduced the tumor weight, increased cleaved caspase-3 positive area and decreased that of ki67 in xenograft mouse model. CONCLUSION: Through a network pharmacology approach and in vitro experiments, we predicted and verified the effect of WTX on colorectal cancer cells mainly depended on the regulation of intrinsic apoptosis via PI3K/AKT signaling pathway, and further animal experiments proved that WTX has a good anti-colon cancer effect in vivo.

Effects of Alpinae Oxyphyllae Fructus on microglial polarization in a LPS-induced BV2 cells model of neuroinflammation via TREM2.

ETHNOPHARMACOLOGICAL RELEVANCE: As one of the important traditional Chinese medicines, Alpinia oxyphylla could warm and tonify the kidney and spleen. It has been used as anti-salivation, anti-diarrhea in various diseases. In recent years, many studies have reported the significant effect of Alpinia oxyphylla on improving cognitive ability, anti oxidative stress and protecting neurons. AIMS OF THE STUDY: In this paper, we studied whether AE and its main active components could improve M1 and M2 polarization, inhibit neuroinflammation through triggering receptor expressed on myeloid cells 2 (TREM2), and exert anti-inflammatory effects. MATERIALS AND METHODS: In this paper, the concentrations of inflammatory cytokines such as NO, TNF-α, IL-10 were assessed using detection kits respectively. Arg-1 and Iba-1, as polarized markers of M1 and M2, were detected by Immunofluorescence staining. CD86 and CD206 were tested by flow cytometry as surface markers of M1 and M2. Furthermore, RT-PCR was performed to determinate TNF-α, IL-10, Arg-1, and Iba-1. Western blot was used to test the activation of PI3K/AKT/GSK3ß and BDNF/TrkB/TLR4 signaling pathways. TREM2 siRNA treatment further verified the action target of Chrysin, the main active ingredient of Alpinia oxyphylla. Molecular docking study was performed to investigate the binding mode between Chrysin and the human TREM2. RESULTS: We found that AE could promote the phenotypic transformation of microglia from M1 to M2, and similar effects of Chrysin were observed. Furthermore, downregulation of TREM2 blocked the anti-neuroinflammation of Chrysin, and inhibited the shift of M1 phenotype to M2 phenotype. Additionally, TREM2-siRNA suppressed the effects of Chrysin on PI3K/AKT/GSK3ß and BDNF/TrkB/TLR4 signaling pathways. CONCLUSIONS: Our findings indicated that AE could improve the polarization response of microglia. TREM2 plays a vital role in the microglial repolarization effects of Chrysin through PI3K/AKT/GSK3ß and BDNF/TrkB/TLR4 signaling pathways regulated by neuroinflammation.

Combination of Alpinia Oxyphylla Fructus and Schisandra Chinensis Fructus ameliorates aluminum-induced Alzheimer's disease via reducing BACE1 expression.

Being the most common form of dementia, Alzheimer's disease (AD) has a series of modifiable risk factors, including metal ions represented by aluminium. Aluminium (Al) exhibits its neurotoxic effects, especially mainly by affecting amyloid-ß protein (Aß) aggregation and Tau hyperphosphorylation. As reported in our previous study, the combination of Alpinia Oxyphylla Fructus and Schisandra Chinensis Fructus (AS) had a neuroprotective effect. This study aimed to evaluate the anti-AD effect of AS and the mechanism by which AS reduces the neurotoxic effect of Al. Firstly, we used aluminium-maltol (Al(mal)3) to construct a mouse model of AD and performed oral administration of AS, followed by behavioral experiments, and we collected the mouse brain for immunohistochemistry analysis. In vivo results showed that AS significantly improved Al-induced cognitive decline in mice, and reduced the levels of Aß1-42 and P-Tau in the brain, which further proved the anti-AD effect of AS. Then, in order to explore the mechanism by which AS reduced Aß1-42, Al-induced PC12 cells were used for the in vitro experiments. Compared with other ratios, the ratio of Alpinia Oxyphylla Fructus: Schisandra Chinensis Fructus (AO:SC) = 1:2 could better improve the cell viability and reduce the Aß1-42 level. According to western blot and quantitative real-time polymerase chain reaction (qPCR) results, AS ameliorated the pathological process by downregulating the expression of ß-secretase (BACE1), rather than by reducing the expression of amyloid precursor protein (APP) or Tau. These results suggest that AS ameliorated Al-induced AD by affecting the expression of BACE1 and reducing the level of Aß1-42, thereby exerting a neuroprotective effect. Combined with previous studies, this study shows that AS has potential for further research and development in AD treatment.

An Investigation of the Anti-Depressive Properties of Phenylpropanoids and Flavonoids in Hemerocallis citrina Baroni.

The World Health Organization predicts that over the next several years, depression will become the most important mental health issue globally. Growing evidence shows that the flower buds of Hemerocallis citrina Baroni (H. citrina) possess antidepressant properties. In the search for new anti-depression drugs, a total of 15 phenylpropanoids and 22 flavonoids were isolated and identified based on spectral data (1D and 2D NMR, HR-ESI-MS, UV) from H. citrina. Among them, compound 8 was a novel compound, while compounds 1-4, 6, 9, 10, 15, 17, 24-26, 28, and 37 were isolated for the first time from Hemerocallis genus. To study the antidepressant activity of phenylpropanoids and flavonoids fractions from H. citrina, macroporous resin was used to enrich them under the guidance of UV characteristics. UHPLC-MS/MS was applied to identify the constituents of the enriched fractions. According to behavioral tests and biochemical analyses, it showed that phenylpropanoid and flavonoid fractions from H. citrina can improve the depressive-like mental state of chronic unpredictable mild stress (CUMS) rats. This might be accomplished by controlling the amounts of the inflammatory proteins IL-6, IL-1ß, and TNF-α in the hippocampus as well as corticosterone in the serum. Thus, the monomer compounds were tested for their anti-neuroinflammatory activity and their structure-activity relationship was discussed in further detail.

Salidroside Inhibits Ischemia/Reperfusion-Induced Myocardial Apoptosis by Targeting Mir-378a-3p Via the Igf1r/Pi3k/Akt Signaling Pathway.

BACKGROUND: The present study aimed to investigate the protective effects and mechanism of salidroside (SAL) on hypoxia/reoxygenation (H/R)-induced cardiomyocyte apoptosis and myocardial ischemia/reperfusion (I/R) injury. METHODS: We set up an H/R H9c2 cell model in vitro and an I/R rat model in vivo. Cell viability, apoptosis and histopathologic evaluation were conducted. RESULTS: The cell viability of H/R-induced cardiomyocytes was increased by pretreatment of SAL, whereas the release of lactate dehydrogenase, reactive oxygen species production, and apoptosis were decreased accompanied with reduced Cleaved-caspase-3 and Bax, and increased Bcl-2 expressions. The SAL restored mitochondrial membrane potential both in vitro and in vivo, and improved electrocardiographic abnormality, and attenuated myocardial apoptosis and injury in I/R-induced rats. The transfection of miR-378a-3p inhibitor counteracted the effects of SAL-induced increase of cell viability and decrease of cell apoptosis and mitochondrial membrane potential. SAL reduced the expression of insulin-like growth factor 1 receptor (IGF1R), and increased the expressions of PI3K and Akt, however, these alterations were blocked by miR-378a-3p inhibitor. CONCLUSIONS: miR-378a-3p might participate in the protective effect of SAL in I/R-induced myocardial apoptosis via the IGF1R/PI3K/AKT signaling pathway.

Schisandra chinensis (Turcz.) Baill. essential oil exhibits antidepressant-like effects and against brain oxidative stress through Nrf2/HO-1 pathway activation.

The present study aimed to evaluate the antidepressant-like effect of essential oils from Schisandra chinensis (Turcz.) Baill. (SEO) and its possible mechanisms of SEO. The behavioral despair mouse model in vivo and H2O2-induced PC12 cells model in vitro were employed. And the potential effective components were identified by the spectrum-effect relationships analysis. SEO significantly decreased the immobility time in the forced swimming test and tail suspension test, which indicated a promising antidepressant-like effect of SEO in depressed mice. The decreased levels of SOD, GSH, and CAT, and increased levels of MDA were significantly reversed by SEO treatment, which showed good antioxidant activities both in vitro and in vivo. Besides, SEO significantly promoted the nuclear translocation of Nrf2 and the expression of HO-1 in depressed mice and H2O2-induced PC12 cells. The histopathological examination results showed a potential neuronal protective effect of SEO in the hippocampus and cortex. Furthermore, the upregulation of PI3K/AKT/GSK3ß signaling was observed after SEO treatment in the H2O2-induced PC12 cells. Additionally, based on the spectrum-effect relationship analysis, 9 peaks were identified as positively correlated with the antioxidant activity of SEO. These results suggested that SEO promoted Nrf2/HO-1 pathway to improve the oxidative stress status and exerted the antidepressant-like effects.

Uncovering the pharmacological mechanism of Wei-Tong-Xin against gastric ulcer based on network pharmacology combined with in vivo experiment validation.

ETHNOPHARMACOLOGICAL RELEVANCE: The prescription of Wei-Tong-Xin (WTX) is improved based on the prescription "Wanyingyuan", a famous decoction documented in the book of Huatuozhongzangjing in the Han dynasty. Many years of clinical verification have demonstrated that WTX can be used to treat gastrointestinal diseases, especially gastric ulcer (GU). However, the potential pharmacological mechanism is undefined. AIM OF THE STUDY: This research was conducted to explore the pharmacological mechanisms under the consideration of the therapeutical effect of WTX against GU by combining the network pharmacology strategy and in-vivo verified experiments. MATERIALS AND METHODS: A prediction network describing the relationship between WTX and GU was established based on information collected from multiple databases. Then, the intersecting protein-protein interaction (PPI) network of the drug-disease overlapping gene targets was constructed, and several key targets related to both WTX and GU were obtained. Besides, the Gene Ontology (GO) biological enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed to investigate the key target genes and pathways of WTX against GU. Then, the candidate targets and signaling pathways of network pharmacology were validated in a rat model of GU induced by indomethacin following the results and available proof. RESULTS: There are 243 targets obtained from the 65 active ingredients in WTX, and 1362 disease targets related to GU were identified. Then, 6 key targets were determined with the PPI interaction network, which was structured from 126 overlapping gene targets. GO and KEGG analyses revealed that the phosphoinositide-3-kinase/protein kinase B (PI3K/AKT) signaling pathway might play a crucial role in the therapeutic mechanism of GU. In vivo verified experiments, WTX significantly reduced the ulcer area and improved the histopathological appearance of gastric tissues. Moreover, down-regulated the protein levels of IL6, TNF-α, and Caspase 3 in the gastric tissues while up-regulating the expression of p-PI3K, p-AKT, p-P53, and VEGFA compared to the model group. CONCLUSION: WTX, an ancient traditional Chinese medicine (TCM) compound prescription, may affect the inflammatory response and apoptosis process by regulating PI3K/AKT signaling pathway and related gene targets. Therefore, it is an effective drug candidate for the modern treatment of GU.

Silibinin improves L-cell mass and function through an estrogen receptor-mediated antioxidative mechanism.

BACKGROUND: Silibinin, a major component of milk thistle extract silymarin, promotes hypoglycemia by activating estrogen receptor (ER) α and ß-mediated pathways in pancreatic ß-cells. Glucagon-like peptide-1 (GLP-1) is the enteroendocrine peptide produced in L-cells, and it controls glucose homeostasis through multiple pathways. The effect of silibinin on L-cell mass and function is still unknown. PURPOSE: The protective effect of silibinin on palmitate (PA)-treated intestinal L-cell line GLUTag cells and the SHRSP•Z-Leprfa/Izm-Dmcr (SP•ZF) diabetic rat model was investigated in current study. METHODS: After pre-incubation with 50 µM silibinin for 4 h, GLUTag cells were treated with 0.125 mM PA. MTT, Annexin V/PI apoptosis, Hoechst 33342 staining, western blot, DCFH-DA, GLP-1 ELISA, qRT-PCR and immunofluorescence analyses were undertaken to determine ER-dependent protection of silibinin against PA-induced cellular damage. The differential protein expression of GLUTag cells under different treatments was examined by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). The SP•ZF diabetic rat model was chosen for in vivo study. After 4 weeks of gastric gavage with 100 or 300 mg kg-1 of silibinin, the physiological indexes of the rats were measured. Cells expressing GLP-1, 8­hydroxy-2'-deoxyguanosine (8-OHdG), ERα, and/or ERß in duodenum tissues were detected by immunofluorescence. RESULTS: The current study showed that the GLUTag cells preincubated with silibinin activated the transcription factor nuclear erythroid-2 like factor-2 (Nrf2)-antioxidant pathway, reduced reactive oxygen species (ROS) generation, and improved cell survival and GLP-1 content, while the antioxidative effect of silibinin was blocked by the selective ERα antagonist MPP or ERß antagonist PHTPP in GLUTag cells. Our proteomics data further revealed that ERα or ß inactivation reduced glutathione peroxide and proteins associated with endocytosis and reproduction, thus at least partially reversing the protective effect of silibinin. SP•ZF rats received silibinin treatment showed increased serum GLP-1 content and improved glucose homeostasis. Furthermore, silibinin upregulated ERα and ß levels and reduced the level of 8-OHdG in GLP-1-positive cells. CONCLUSIONS: Our study showed that silibinin improved L-cell mass and function through an ER-mediated antioxidant pathway, and the proteomics analysis revealed for the first time the differential regulation of proteins by PA and silibinin in GLUTag cells.

Alpinae Oxyphyllae Fructus alleviated LPS-induced cognitive impairments via PI3K/AKT/NF-κB signaling pathway.

Herein, we aim to investigate the effect of Alpinae Oxyphyllae Fructus (AOF) on cognitive impairments and neuroinflammation in a lipopolysaccharide (LPS)-induced models of AD. Mice were injected intracerebroventricularly with LPS, and then administrated AOF using a gavage for 6 weeks. Spatial working memory was assessed using the Y-maze and Morris water maze test, whereas the levels of PI3K, AKT, p-AKT, p-GSK3ß, GSK3ß, NF-κB, IL-1ß, IL-6, and TNF-α were evaluated using western blot and ELISA assay. Our data showed that AOF was able to significantly alleviate the memory decline in LPS-induced AD mice. Moreover, AOF was able to protect neurons through the PI3K/AKT signaling pathway and significantly decrease NF-κB, IL-6, IL-1ß, and TNF-α levels in the hippocampal and cortex tissues, which were reversed through the use of LY294002. Additionally, we discovered that AOF could significantly decrease the high expression of cytokines as well as the expression and translocation of NF-κB induced by LPS in PC12 cells. These results demonstrate the anti-neuroinflammatory effect of AOF in both cell and animal models of AD, thereby slowing down the process and development of the disease.

The flavonoids of okra insulates against oxidative stress, neuroinflammation and restores BDNF levels in Aß1-42 induced mouse model of Alzheimer's disease.

Okra (Abelmoschus esculentus [L.] Moench.) has been used as a natural drug in East or West Africa for many centuries, as well as consumed in most areas of the world as a tropical vegetable. The study aimed to evaluate whether the flavonoids of okra fruit (FOF) administration influence Aß1-42-induced learning and memory impairment, and explore the underlying mechanisms. The Y-maze task and the Morris water maze test were used for evaluating cognition processes. The levels of tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), superoxide dismutase (SOD), total antioxidant capacity (T-AOC), and glutathione peroxidase (GSH-Px) were detected by ELISA kits. The expressions of nuclear factor kappa-light chain-enhancer of activated B (NF-κB), brain-derived neurotrophic factor (BDNF), cAMP-response element-binding protein (CREB), extracellular signal-regulated kinase (ERK), phosphatidylinositol 3 kinase (PI3K), protein kinase B (AKT), glycogen synthase kinase-3ß (GSK-3ß) were studied by western blot. Histopathological changes were observed by H.E. straining. The results showed that intracerebroventricular injection of Aß1-42 was effective in producing memory deficits in mice. Besides, Aß1-42 exposure could significantly increase the levels of NF-κB, TNF-α, IL-1ß, and decreased T-AOC, the activities of SOD and GSH-Px in the hippocampus and cortex. Furthermore, the level of BDNF was also reduced, accompanied by down-regulated CREB/ERK and PI3K/AKT/GSK-3ß signaling pathways in the hippocampus and cortex. Nevertheless, chronic administration of FOF (100 or 300 mg/kg, i.g.) significantly prevented Aß1-42-induced behavioral and biochemical alterations. It also suggested that FOF could improve the cognitive deficits in AD-like model mice, which might be mediated by regulation of BDNF levels in cortex and hippocampus and up-regulating of CREB/ERK and PI3K/AKT/GSK3ß pathways, as well as alleviation of oxidative stress and neuroinflammation.

Nootkatone improves anxiety- and depression-like behavior by targeting hyperammonemia-induced oxidative stress in D-galactosamine model of liver injury.

Acute or chronic liver injury is closely related to hyperammonemia, which will result in oxidative stress and damage to nerve cells, and these factors are vital to the development of anxiety and depression. In this study, the effect of Nootkatone (NKT) on the anxiety- and depression-like behavioral changes in mice induced by liver injury was investigated. Liver injury was induced by D-galactosamine (D-GalN; 350 mg/kg) three times a week for 4 weeks. NKT (5 mg/kg or 10 mg/kg) was given as co-treatment daily for 4 weeks. NKT (5 mg/kg) co-treatment remarkably ameliorates D-GalN-induced anxiety- and depression-like behaviors as evident from the results of sucrose preference test, forced swimming test, tail suspension test, and novelty suppressed feeding test. Results showed that NKT could induce an elevation in serum alanine transaminase and aspartate transaminase level, alleviate the oxidative stress induced by hyperammonemia through activating Keap1/Nrf2/HO-1 antioxidant pathways, decrease the expression of inducible nitric oxide synthase and NOX2 in hippocampus and prefrontal cortex, enhance the vitality of superoxide dismutase, catalase, and glutathione levels in serum, liver, and brain, and significantly reduce the generation of malondialdehyde. At the same time, NKT also reduces the level of ammonia in serum and brain and upgrades the activity of glutamine synthetase in the hippocampus and prefrontal cortex. Taken together, the present results suggested that NKT has a significant antidepressant effect through modulation of oxidative stress induced by D-GalN administration.

Spinosin protects N2a cells from H2 O2 -induced neurotoxicity through inactivation of p38MAPK.

OBJECTIVES: Previous studies have suggested that spinosin (SPI) exerted neuroprotective effects through inhibition of oxidative damage, but the underlying mechanisms are still unclear. Herein, the mechanisms underlying the protective effects of SPI against oxidative stress induced by hydrogen peroxide (H2 O2 ) were examined in neuro-2a (N2a) mouse neuroblastoma cells. METHODS: N2a cells were pretreated with H2 O2 for 2 h, followed by a 24-h incubation with SPI. Intracellular reactive oxygen species (ROS) production was analysed by flow cytometry. Levels of Aß1-42 production were determined by ELISA assay. Levels of expression of c-Jun N-terminal kinase (JNK), p-JNK, extracellular signal-regulated kinase (ERK), p-ERK, p38 mitogen-activated protein kinase (p38MAPK), p-p38MAPK, p-Tau (Ser199), p-Tau (Ser202), p-Tau (Ser396), synaptophysin (SYP) and postsynaptic scaffold postsynaptic density-95 (PSD-95) were detected by Western blot analysis. KEY FINDINGS: Our results showed that H2 O2 treatment enhanced intracellular ROS production in N2a cells. SPI prevented H2 O2 -induced oxidative damage via inhibiting Aß1-42 production, decreasing Tau phosphorylation and improving synaptic structural plasticity. Notably, H2 O2 -increased p38MAPK activation was attenuated by SPI administration, and p38MAPK inhibitor BIRB796 markedly reduced H2 O2 -induced oxidative damage in N2a cells. CONCLUSIONS: Our findings suggest that SPI protects N2a cells from H2 O2 -induced oxidative damage through inactivation of p38MAPK.

Silychristin A activates Nrf2-HO-1/SOD2 pathway to reduce apoptosis and improve GLP-1 production through upregulation of estrogen receptor α in GLUTag cells.

Glucagon-like peptide-1 (GLP-1), a glucagon-like peptide secreted mainly from intestinal L cells, possesses the functions of promoting synthesis and secretion of insulin in pancreatic ß-cells, and maintaining glucose homeostasis in an insulin-independent manner. Silychristin A, a major flavonolignan from silymarin, was reported to protect pancreatic ß-cells from oxidative damage in streptozotocin (STZ)-induced diabetic rats. However, the role of silychristin A in the protection of intestinal L-cells is still unknown. Our current study demonstrated that palmitate (PA) inhibited protein expression of NF-E2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1) and superoxide dismutase 2 (SOD2), and subsequently increased reactive oxygen species level to induce apoptosis and decrease GLP-1 content in intestinal L-cell line GLUTag cells. Pre-incubation of silychristin A effectively reversed PA-inactivated Nrf2-HO-1/SOD2 antioxidative pathway accompanied with decreased apoptosis level and increased GLP-1 level in GLUTag cells. As a potential target of silychristin A, estrogen receptor α was shown to be downregulated by PA stimulation, and the expression of which was improved by silychristin A in a concentration-dependent manner. Further study revealed that the treatment of estrogen receptor α antagonist MPP induced apoptosis and blocked the stimulation of GLP-1 production by silychristin A through the activation of Nrf2-HO-1/SOD2 pathway in GLUTag cells. Taken together, our study found silychristin A activated estrogen receptor α-dependent Nrf2-HO-1/SOD2 pathway to decrease apoptosis and upregulate GLP-1 production in GLUTag cells.

Polysaccharide from Schisandra chinensis acts via LRP-1 to reverse microglia activation through suppression of the NF-κB and MAPK signaling.

ETHNOPHARMACOLOGICAL RELEVANCE: Schisandra chinensis (Turcz.) Baill (S. Chinensis), a traditional Chinese medicine frequently used in the traditional treatment of dementia, its polysaccharide component has been widely reported. AIM OF THE STUDY: In this paper, we studied whether SCP2-1, a natural product of homogeneous polysaccharide from S. Chinensis, could improve M1 and M2 polarization and inhibit neuroinflammation through lipoprotein receptor-related protein-1 (LRP-1), and futher exerted anti-inflammatory and neuroprotective effects. MATERIALS AND METHODS: SCP2-1 was obtained from crude polysaccharide of S. Chinensis, BV2 microglia cells and mice stimulated by LPS were served to detect the positive role of SCP2-1 in M1/M2 polarization. The concentration of cytokine expression, IL-1ß, TNF-α, IL-12 and IL-6 for M1 polarization and TGF-ß, IL-10, IL-4 and Arg-1 for M2 polarization, in the BV2 and hippocampus were tested by ELISA kits. CD86 and CD206, as surface markers of M1 and M2, were tested by flow cytometry. We examined the expression of LRP-1 in BV2 cells and mouse hippocampus. The addition of siRNA for LRP-1 demonstrated the important role of LRP-1 in the neuroprotection of SCP2-1. Western blot was used to detect the activation of various mitogen-activated protein kinase (MAPKs) pathway, i.e. the phosphorylation of JNK and ERK proteins, and nuclear translocation of nuclear factor κB (NF-κB). H.E. staining was used to observe Histopathological changes. RESULTS: SCP2-1 could reverse M1/M2 polarization in vitro culture and suppressed M1 polarization in the hippocampus of mice stimulated with LPS. After LPS stimulation, poor levels of LRP-1, hyperactivation of the JNK and NF-κB was appeared, which could improve by SCP2-1. The addition of siRNA for LRP-1 suppressed the protection of SCP2-1 in BV2 microglial cells. More importantly, SCP2-1 could improve LPS-induced cognitive dysfunction in mice in Y-maze and NOR test. CONCLUSIONS: SCP2-1 could improve M1/M2 polarization, especially inhibit M1 polarization, and ameliorate the cognition of mice in Y-maze and NOR test. SCP2-1 play a neuroprotective role through LRP-1 to reverse activation of microglia via suppressing the overactive NF-κB and JNK pathway.

Alpinia oxyphylla-Schisandra chinensis Herb Pair Alleviates Amyloid-ß Induced Cognitive Deficits via PI3K/Akt/Gsk-3ß/CREB Pathway.

Alzheimer's disease (AD), one of the most common neurodegenerative diseases, threatens people's health. Based on the theory of traditional Chinese medicine (TCM) efficacy and treatment theory, we first proposed the Alpinia oxyphylla-Schisandra chinensis herb pair (ASHP) for finding a candidate of AD treatment. This study aimed at exploring the effects of ASHP on improving the cognitive function and neurodegeneration, and revealing the possible mechanism. In this study, an amyloid-ß (Aß) induced AD model was established in mice via intracerebroventricular injection. The Y-maze test and Morris water maze test were carried out to observe the behavioral change of mice, which showed that ASHP significantly ameliorated cognitive impairment. In addition, ASHP reduced amyloid-ß deposition and downregulated the hyperphosphorylation of tau via immunofluorescence assay and western blot analysis, respectively. Subsequently we focused on the PI3K/Akt pathway that is a classical pathway related to nervous system diseases. It also noticeably ASHP improved the histopathological changes in the hippocampus and cortex. Moreover, it was found that ASHP could upregulate the PI3K/Akt/Gsk-3ß/CREB signaling pathway in N2a-SwedAPP cells. Taken together, it suggests that ASHP might reverse cognitive deficits and neurodegeneration via PI3K/Akt/Gsk-3ß/CREB pathway.