Long non­coding RNA BLACAT2/miR­378a­3p/YY1 feedback loop promotes the proliferation, migration and invasion of uterine corpus endometrial carcinoma.

Uterine corpus endometrial carcinoma (UCEC) is a common gynecological malignancy with high rates of mortality and morbidity. The expression of long non­coding RNA bladder cancer­associated transcript 2 (BLACAT2) has been previously found to be aberrantly upregulated in UCEC. However, the regulatory consequences of this in UCEC progression remain poorly understood. In the present study, human UCEC cell lines AN3CA and HEC­1­A were infected with lentiviruses to overexpress BLACAT2 (Lv­BLACAT2) or knock down BLACAT2 using short hairpin RNA (Lv­shBLACAT2). BLACAT2 overexpression was found to promote the G1/S transition of cell cycle progression and UCEC cell proliferation. In addition, BLACAT2 overexpression was observed to facilitate UCEC cell migration and invasion. By contrast, BLACAT2 knockdown resulted in inhibitory effects in UCEC cell physiology. BLACAT2 overexpression also contributed to the activation of the MEK/ERK pathway. Subsequently, BLACAT2 was demonstrated to bind to microRNA (miR)­378a­3p according to dual­luciferase assays, where it appeared to function as a sponge of miR­378a­3p in 293T cells. miR­378a­3p overexpression was found to suppress UCEC cell proliferation, invasion, and ERK activation. Lentivirus­mediated knockdown of its target, the transcription factor Yin Yang­1 (YY1), was observed to reverse the oncogenic effects of BLACAT2 overexpression. Furthermore, YY1 was found to bind to the promoter of BLACAT2, suggesting that YY1 can regulate BLACAT2 expression. To conclude, results from the present study suggest that BLACAT2, miR­378a­3p and YY1 can form a feedback loop instead of an unidirectional axis, which can in turn regulate UCEC tumorigenesis through the MEK/ERK pathway. The present study furthered the understanding of UCEC tumorigenesis and may provide novel therapeutic targets for UCEC treatment.

Immunosuppressive effect of Columbianadin on maturation, migration, allogenic T cell stimulation and phagocytosis capacity of TNF-α induced dendritic cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Angelicae pubescentis radix (APR) has a long history in the treatment of rheumatoid arthritis (RA) in China. It has the effects of dispelling wind to eliminate dampness, removing arthralgia and stopping pain in the Chinese Pharmacopeia, but its mechanisms was unclear. Columbianadin (CBN) was one of the main bioactive compounds of APR, and has many pharmacological effects. But the immunosuppressive effect of CBN on DCs and the potential mechanism needed to be explored. AIM OF THE STUDY: The study was aimed to clarify the immunosuppressive effect of CBN on maturation, migration, allogenic T cell stimulation and phagocytosis capacity of TNF-α induced DCs. MATERIALS AND METHODS: Bone marrow-derived DCs were obtained and cultured from C57BL/6 mice in accordance with protocol. The phenotypic study (CD11c, CD40, CD80, CD86 and MHC Ⅱ) were measured by flow cytometry. FITC-dextran were uptaked by DCs and the change of endocytosis activity were mediated by acquired mannose receptor. Transwell chambers were used to detect the migration ability of DCs. Mixed leukocyte reaction (MLR) assay was used to detect the allostimulatory ability of CBN on TNF-α stimulated DCs. The secretion of cytokines and chemokines was measured by ELISA Kit. TLRs gene and MAPKs/NF-κB protein expression were checked by qRT-PCR and Western blot. RESULTS: CBN inhibited the maturation of TNF-α-induced DCs while maintaining phagocytosis capabilities. Additionally, CBN inhibited the migration of TNF-α stimulated DCs, which related to reduce the production of chemokines (MCP-1, MIP-1α). Notably, CBN could suppress the proliferation of CD4+T cells by inhibiting DCs maturation, and decrease the proinflammatory cytokines IL-6 production. Furthermore, CBN inhibited mRNA expression of TLR2, TLR7 and TLR9 in TNF-α-activated DCs. Meanwhile, the phosphorylation of p38, JNK1/2 and NF-κB protein were significantly inhibited in CBN treated DCs. CONCLUSIONS: These findings provided novel insights into the pharmacological activity of CBN. They also indicated that inhibition DCs maturation owning to the immunosuppressive effect of CBN. CBN was expected as a potential immunosuppressant and TLRs/MAPKs/NF-κB pathway may be an important mechanism for CBN's immunosuppressive activity.

In-vitro antiviral action of Eupatorium perfoliatum against dengue virus infection: Modulation of mTOR signaling and autophagy.

ETHNOPHARMACOLOGICAL RELEVANCE: Dengue virus (DENV) is a re-emerging mosquito-borne flavivirus that has recently engendered large epidemics around the world. Consequently antivirals with effective anti-DENV therapeutic activity are urgently required. In the 18th century, Europeans, as well as native inhabitants of North America, were known to adapt the medicinal property of the common perennial plant Eupatorium perfoliatum L. to treat fever and infections. Previous studies have shown that Eupatorium perfoliatum L. possesses anti-inflammatory, anti-oxidative, anti-plasmodial, anti-bacterial and antiviral activities. However, to the best of our knowledge, no anti-DENV activity of E. perfoliatum L. has been investigated at the molecular level so far. AIM OF STUDY: Here, for the first time we have attempted to study the action of E. perfoliatum extract and its few bioactive components i.e., quercetin, caffeic acid and eupafolin against wild primary clinical isolate of DENV-2 infection in an in vitro model. MATERIALS AND METHODS: The presence of the bioactive components in the E. perfoliatum extract, were analyzed by HPLC- DAD. Then, CC50 as well as IC50 values of the extract and its bioactive components were measured against DENV in HepG2 cell line. After that, the antiviral activity was studied by Time of addition assay using qRT-PCR. Further, the downstream signalling action of E. perfoliatum extract, was studied by Human phosphorylation MAPK antibody array, followed by immunofluorescence microscopy. Moreover, a molecular docking analysis was done to study the binding affinity of bioactive components of E. perfoliatum extract with TIM-1 transmembrane receptor protein, which is known for viral internalization. RESULT: We found that E. perfoliatum extract has marked antiviral activity during pre-treatment against DENV infection in HepG2 cell line. The extract also significantly reduced the DENV induced autophagy in HepG2 cell line as detected by LC3 II localization. The presence of different bioactive compounds in E. perfoliatum extract were confirmed by HPLC-DAD. In the bioactive components, in parallel to earlier studies, quercetin showed the most significant preventive action against DENV infection. Further, in molecular docking analysis also, quercetin showed the strongest binding affinity towards DENV membrane receptor TIM-1 protein. CONCLUSION: Our findings suggests that E. perfoliatum extract has significant potential to be an anti-DENV therapeutic agent. Moreover, among the bioactive components, quercetin may have a prophylaxis role in executing the antiviral activity of E. perfoliatum extract against DENV infection.

Qihuzha granule attenuated LPS-induced acute spleen injury in mice via Src/MAPK/Stat3 signal pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Qihuzha granule (QHZG), is one of traditional Chinese patent medicines composed of eleven edible medicinal plant, which has been used in the clinic for the treatment of indigestion and anorexia in children caused by deficiency of the spleen and stomach. Yet it is noteworthy that QHZG has therapeutic effect on recurrent respiratory tract infection (RRTI) in children. However, its potential molecular mechanisms remained unclear. AIM OF THE STUDY: The aim of this study was to investigate the therapeutic effect and potential mechanism of QHZG on lipopolysaccharide (LPS) induced acute spleen injury. MATERIALS AND METHODS: The acute spleen injury model was induced by intraperitoneal injection of LPS (10 mg/kg) and safe doses of QHZG was administered by gavage once a day for 23 days before LPS treatment. Serum inflammatory cytokines including interleukin-2 (IL-2), IL-1ß, IFN-γ, and tumor necrosis factor-α (TNF-α) were tested by ELISA. Related protein levels were detected by Western blotting. Hematoxylin-eosin (HE) staining was employed to observe the histological alterations. The distribution of macrophages and neutrophils in the mouse spleen was examined by immunofluorescence analysis. RESULTS: QHZG pretreatment significantly abolished the increased secretion of cytokines such as interleukin-2 (IL-2), IL-1ß, IFN-γ, and tumor necrosis factor-α (TNF-α), which were attributable to LPS treatment. Immunofluorescence staining and Histological analysis of spleen tissue revealed the protective effect of QHZG against LPS-induced acute spleen injury in mice. Further study indicated that pretreatment with QHZG significantly inhibited LPS-induced phosphorylation of Src. Accordingly, the increased phosphorylation of Src downstream components (JNK, ERK, P38 and STAT3) induced by LPS was remarkably diminished by QHZG, suggesting the involvement of Src/MAPK/STAT3 pathway in the inhibitory effects of QHZG on spleen injury in mice. CONCLUSION: Our study demonstrated that QHZG protected mice from LPS-induced acute spleen injury via inhibition of Src/MAPK/Stat3 signal pathway. These results suggested that QHZG might serve as a new drug for the treatment of LPS-stimulated spleen injury.

Neuroprotective effects of aucubin on hydrogen peroxide-induced toxicity in human neuroblastoma SH-SY5Y cells via the Nrf2/HO-1 pathway.

BACKGROUND: When redox balance is lost in the brain, oxidative stress can cause serious damage that leads to neuronal loss, in congruence with neurodegenerative diseases. Aucubin (AU) is an iridoid glycoside and that is one of the active constituents of Eucommia ulmoides, has many pharmacological effects such as anti-inflammation, anti-liver fibrosis, and anti-atherosclerosis. PURPOSE: The present study aimed to evaluate the inhibitory effects of AU on cell oxidative stress against hydrogen peroxide (H2O2)-induced injury in SH-SY5Y cells in vitro. METHODS: SH-SY5Y cells were simultaneously treated with AU and H2O2 for 24 h. Cell viability was measured by CCK-8. Additionally, mitochondrial membrane depolarization, reactive oxygen species (ROS) generation, and cell apoptosis were measured by flow cytometry. RESULTS: The results showed that AU can significantly increase the H2O2-induced cell viability and the mitochondrial membrane potential, decrease the ROS generation, malondialdehyde (MDA), and increase glutathione (GSH) contents and the superoxide dismutase (SOD) activity. We also found that H2O2 stimulated the production of nitric oxide (NO), which could be reduced by treatment with AU through inhibiting the inducible nitric oxide synthase (iNOS) protein expression. In H2O2-induced SH-SY5Y cells, the levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1ß (IL-1ß) content and cell apoptosis were significantly reduced by AU treatment through nuclear factor E2-related factor 2/hemo oxygenase-1 (Nrf2/HO-1) activation, inhibiting the expression of p-NF-κB/NF-κB and down-regulating MAPK and Bcl-2/Bax pathways. CONCLUSION: These results indicate that AU can reduce inflammation and oxidative stress through the NF-κB, Nrf2/HO-1, and MAPK pathways.

Sanweidoukou decoction, a Chinese herbal formula, ameliorates ß-amyloid protein-induced neuronal insult via modulating MAPK/NF-κB signaling pathways: Studies in vivo and in vitro.

ETHNOPHARMACOLOGICAL RELEVANCE: The traditional Chinese medicine Sanweidoukou decoction (DK-3) was a classical formula for the treatment of nervous system diseases, recorded in the Chinese medical classic Sibu Yidian. AIM OF THE STUDY: The present study is aim to investigate the neuroprotective effects of DK-3 on ß-amyloid (Aß) protein -induced AD-like pathologies and underlying molecular mechanisms both in vitro and in vivo studies. MATERIALS AND METHODS: Hydrolysates of DK-3 were analyzed by LC-ESI-MS/MS. In vitro, MTT was utilized to examine effects of DK-3 on Aß25-35-induced cytotoxicity in PC12 cells. In vivo, male Sprague-Dawley rats were administered with Aß25-35 to induce AD-like pathologies and behavioral evaluations were conducted via Morris water maze (MWM) test. Histopathological changes were observed by Hematoxylin-eosin (HE) straining. Immunohistochemistry (IHC) was used to detect the tau hyperphosphorylation at Thr181 site. The expression levels of tau hyperphosphorylation, inflammation-related cytokines such as COX-2, iNOS, TNF-α, IL-1ß, IL-6, the phosphorylated state of various mitogen-activated protein kinase (MAPK) signaling molecules (p38 MAPK, ERK, and JNK) and activation of nuclear factor κB (NF-κB) in vitro and in vivo were assessed via Western blot. RESULTS: In vitro, DK-3 dose-dependently increased cell viability of PC12 cells induced by Aß25-35. In vivo, DK-3 improved learning and memory abilities of Aß25-35-induced AD-like rats. Moreover, DK-3 reversed hyperphosphorylation of tau and reduced the production of inflammation-related cytokines through significantly inhibited MAPK and NF-κB signaling pathways both in vitro and in vivo studies. CONCLUSION: The present study suggested that the traditional Chinese medicine DK-3 may play a role in preventing and treating AD by reducing the hyperphosphorylation of tau protein and the expressions of inflammation-related cytokines via modulating the MAPK/NF-κB signaling pathways.

Asparanin A inhibits cell migration and invasion in human endometrial cancer via Ras/ERK/MAPK pathway.

Asparanin A (AA), a natural compound present in vegetables and medicinal herbs like Asparagus officinalis L., has been investigated extensively for its pharmacological attributes. So far, the effect of AA on endometrial cancer (EC) cell migration and invasion has not been explored. Herein, we elucidated the anti-metastasis mechanism of AA on Ishikawa cells based on miRNA-seq and mRNA-seq integrated analyses. AA treatment led to altered miRNAs expression in Ishikawa cells and inhibited the cell wound healing, cell migration and invasion. Gene Ontology and KEGG enrichment analyses showed that the target genes of different expression miRNAs were significantly enriched in Ras, Rap1 and MAPK signaling pathways. Further verification of these changes via qRT-PCR and Western blot assays in vitro and in vivo demonstrated that AA could suppress human EC cell migration and invasion through Ras/ERK/MAPK pathway. Furthermore, top two miRNAs (miR-6236-p5 and miR-12136_R+8) and top three target genes (KITLG, PDGFD, and NRAS) were identified as functional hub miRNAs and genes through miRNA-target gene network analysis. Our data presented a holistic approach to comprehend the anti-metastatic role of AA in EC after in vitro and in vivo analyses.

Qizhen capsule inhibits colorectal cancer by inducing NAG-1/GDF15 expression that mediated via MAPK/ERK activation.

ETHNOPHARMACOLOGICAL RELEVANCE: Qizhen capsule (QZC) is a traditional Chinese medicine (TCM) preparation that has been widely used in clinical practice and exerts promising therapeutic effects against breast, lung, and gastric cancers. However, studies have not reported whether QZC inhibits colorectal cancer (CRC) development and progression. Meanwhile, the underlying molecular mechanisms of its anticancer activity have not been studied. AIM OF THE STUDY: To investigate the anticancer effects of QZC on CRC and the possible underlying molecular mechanisms of QZC in vitro and in vivo. MATERIALS AND METHODS: The MTT assay and flow cytometry were used to determine the viability and apoptosis of HCT116 and HT-29 cancer cells. A xenograft nude mouse model was used to study the antitumor effects of QZC in vivo. Western blotting was performed to determine the expression of key proteins responsible for the molecular mechanisms elicited by QZC. Immunofluorescence staining was performed to detect the expression of nonsteroidal anti-inflammatory drug (NSAID)-activated gene-1 or growth differentiation factor-15 (NAG-1/GDF15). Small interfering RNAs (siRNAs) were used to silence NAG-1/GDF15 in cells. RESULTS: In this study, QZC significantly reduced the viability of HCT116 and HT-29 cells and induced apoptosis in dose- and time-dependent manners, but displayed much less toxicity toward normal cells. QZC-induced apoptosis in HCT116 cells was accompanied by the deregulation of the expression of the Bcl-2, Bax, PARP, caspase-3, and caspase-9 proteins. Furthermore, QZC induced NAG-1/GDF15 expression in HCT116 cells, while silencing of NAG-1/GDF15 attenuated QZC-induced apoptosis and cell death. Next, QZC increased the phosphorylation of mTOR, AMPK, p38, and MAPK/ERK in HCT116 cells. We then demonstrated that QZC-induced apoptosis and NAG-1/GDF15 upregulation were mediated by MAPK/ERK activation. Moreover, QZC significantly inhibited HCT116 xenograft tumor growth in nude mice, which was accompanied by NAG/GDF15 upregulation and MAPK/ERK activation. QZC also prevented 5-FU-induced weight loss or cachexia in tumor-bearing mice. The expression of Ki67 and PCNA was suppressed, while cleaved caspase-3 level and TUNEL staining were increased in the tumor sections from QZC-treated mice compared to the control. CONCLUSION: QZC is a novel anticancer agent for CRC that targets NAG-1/GDF15 via the MAPK/ERK signaling pathway.

Fermented ginseng attenuates lipopolysaccharide-induced inflammatory responses by activating the TLR4/MAPK signaling pathway and remediating gut barrier.

Generally, ginsenosides have the physiological effect of an anti-inflammatory immunity. After fermentation, the types of ginsenosides in ginseng change, and their physiological activity becomes a concern. L. plantarum KP-4 screened from Korean kimchi were used to ferment ginseng, and the changes of ginsenosides were observed. C57BL/6N mice were treated using fermented ginseng (390 mg kg-1 day-1), which was mixed with normal food, and an inflammatory mice model was established by the intraperitoneal injection of lipopolysaccharide (LPS) (2.5 mg per kg body weight) four weeks later. The liver index, pathological index, biochemical index, and inflammatory signaling pathway were determined. The results demonstrated that L. plantarum KP-4 fermentation increased the content of minor ginsenosides in ginseng and decreased the content of major ginsenosides. Fermented ginseng significantly reduced LPS-induced increases in ALT, AST, and pro-inflammatory cytokines IL-6, TNF-α, and IL-1ß in mice. Supplementation with fermented ginseng significantly ameliorated LPS-induced overexpression of Toll-like receptor 4 (TLR4), caspase3, phosphorylation p38 mitogen-activated protein kinase (p38MAPK), and phosphorylation extracellular signal-regulated kinase (ERK) compared with the control group. Moreover, fermented ginseng significantly increased the expression of claudin 1, the intestinal tight junction protein, caused by LPS. In conclusion, fermented ginseng alleviates LPS-induced inflammation through the TLR4/MAPK signaling pathway and increased intestinal barrier function in mice.

The intranasal administration of Carthamus tinctorius L. extract/phospholipid complex in the treatment of cerebral infarction via the TNF-α/MAPK pathway.

Carthamus tinctorius L.(Safflower), a herbal formula from Traditional Chinese Medicine (TCM), has been widely used for the treatment of cardio-cerebrovascular diseases, particularly cerebral infarction (CI) or cerebral ischemia-reperfusion injury. However, we know very little about the specific mechanisms associated with the therapeutic effect of Safflower on CI. In this study, we used a network pharmacology-based approach, together with rat model of CI, to gain more insight into of such mechanisms. Our analysis showed that Safflower contains 52 active compounds that target 247 genes, which were also cross-referenced with 299 genes associated with CI. Consequently, we identified 52 target genes in Safflower that were associated with CI. These 52 target genes were analyzed by gene ontology (GO) enrichment analysis, leading to the identification of 1491 biological process items, 90 molecular function items and 19 cell assembly items. Eighty-nine pathways were generated by KEGG enrichment (P < 0.05). Next, we investigated the effect of the extract of safflower (ES) and Safflower extract phospholipid complex (ESPC), delivered via the nasal route, on an animal model of the middle cerebral artery occlusion (MCAO). Our data confirmed that Safflower was able to treat CI by the regulating the TNF-α/MAPK pathway via CASP3. The therapeutic effect of ES and ESPC on CI acts by improving the circulation of blood in the central nervous system, reducing the inflammatory reaction, inhibiting apoptosis, and by protecting brain nerve cells from injury.

Efficacy of sodium butyrate in alleviating mammary oxidative stress induced by sub-acute ruminal acidosis in lactating goats.

Sub-acute ruminal acidosis (SARA) [1] is one of the most common problems of dairy animals causing great economical loss due to decreased milk production. Here we determined the antioxidant effect of sodium butyrate (NaB) [2] in experimentally induced SARA and its effects on mammary epithelial tissues of goat. Goats (n = 12) were equally divided into two groups: high-concentrate (HC) as control group fed with HC diet (concentrate: forage = 6:4) whereas HC + NaB as treatment group fed HC diet with NaB at 1% by weight for 24 weeks. Mammary epithelial tissue samples were analyzed for the expression of genes and proteins responsible for oxidative stress as well as biochemical markers of antioxidant activity in the form of Reactive Oxygen Species (ROS). The total antioxidant capacity (T-AOC) of antioxidant enzymes was also calculated. Butyrate induced antioxidant effect by increasing mRNA and protein abundance of antioxidants in mammary gland of HC + NaB group compared to HC group. Likewise, the total antioxidant capacity (T-AOC) was significantly increased and Malondialdehyde (MDA) concentration was decreased in HC + NaB group compared to HC group. It is concluded that oxidative stress in mammary gland of goats induced by high concentrate diet was alleviated by NaB supplementation.

Adventitious root cultures of Oplopanax elatus inhibit LPS-induced inflammation via suppressing MAPK and NF-κB signaling pathways.

Bioreactor-cultured adventitious roots (ARs) of the endangered medicinal plant Oplopanax elatus Nakai is a novel alternative plant material. To utilize ARs in the product production, the present study investigated the anti-inflammatory effect of O. elatus ARs. In the in vivo experiment, lipopolysaccharide (LPS)-induced acute lung injury disease model was established and several inflammatory indexes were determined. For the LPS-stimulated mice, after pretreatment of AR crude extract (200 mg/kg), cell infiltration in lungs was decreased, the production of proinflammatory mediators, including nitric oxide (NO), tumor necrosis factor (TNF)-α, and interleukin (IL)-6, and 1ß in the bronchoalveolar lavage fluid was evidently reduced, which indicated that O. elatus ARs had an anti-inflammatory effect. In the in vitro experiment, ethyl acetate (EtOAc) fractions (12.5, 25, and 50 µg/mL) were used to treat LPS-induced peritoneal macrophages (PMs) of mice. The production of NO, prostaglandin E2, TNF-α, IL-6, and IL-1ß in LPS-stimulated PMs was obviously inhibited (p < 0.05) after pretreatment with EtOAc fractions, and the expression of the inducible nitric oxide synthase and cyclooxygenase were also suppressed. To clarify the anti-inflammatory mechanism, effects of EtOAc fraction on changes of proteins related to the pathways of mitogen-activated protein kinases (MAPKs) and nuclear factor-kappa B (NF-κB) were investigated. The phosphorylation of extracellular regulated protein kinases, c-jun n-terminal kinase, and p38 MAPK in LPS-induced PMs was inhibited after pretreatment of EtOAc fractions. In addition, EtOAc fractions enhanced inhibitor of nuclear factor-kappa B-α expression and decreased nuclear translocation of p65 NF-κB. Thus, EtOAc from O. elatus ARs is involved in regulating MAKP and NF-κB signaling pathways to inhibit LPS-induced inflammation.

The dietary freeze-dried fruit powder of Actinidia arguta ameliorates dextran sulphate sodium-induced ulcerative colitis in mice by inhibiting the activation of MAPKs.

In this study, we aimed at investigating the antiinflammatory activity of the freeze-dried fruit powder of Actinidia arguta (FAA) on dextran sulphate sodium (DSS)-induced ulcerative colitis (UC) in mice and the effect of its extract on lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. For pharmacodynamic studies, the oral administration of FAA (300 or 600 mg kg-1) could decrease the disease activity index (DAI), reduce the incidence of colon and spleen edemas (caused by inflammation), and alleviate the pathological changes in UC. For research involving biochemical indicators, FAA could decrease the expression of inflammatory markers (such as myeloperoxidase (MPO)) and attenuate the oxidative stress levels. ELISA results revealed that the expressions of proinflammatory cytokines (IL-1ß, IL-6, and TNF-α) were downregulated by FAA. Furthermore, the expression levels of the inflammation-induced activation of p38, JNK, and ERK were decreased by FAA. Hence, it was concluded that FAA could alleviate the UC symptoms in mice and the inflammatory response of macrophages via the MAPK signal pathway. Overall, FAA might have the potential to treat UC when used as a dietary supplement.

Lycium barbarum polysaccharides extend the mean lifespan of Drosophila melanogaster.

The fruits of Lycium barbarum are considered medicinal foods with high nutritional value and bioactivity. In this study, we aimed to evaluate the effect of a crude L. barbarum polysaccharide (LBP) and two derived fractions, LBP-1 and LBP-2, on the lifespan of Drosophila melanogaster (fruit fly). The average lifespan of fruit flies was extended by supplementing their diet with either of the three LBP preparations. In vivo analysis of antioxidant activities detected increased superoxide dismutase (SOD) and catalase (CAT) activities and decreased malondialdehyde (MDA) levels. Dietary LBP supplements significantly reduced the mortality rate of fruit flies induced by paraquat and hydrogen peroxide. Importantly, the strongest anti-aging activity was exhibited by the LBP-2 fraction, containing arabinogalactan with a molecular weight of 9 × 104 Da. Further studies showed that the anti-aging activity of LBP was, at least in part, mediated by an age-related signaling pathway (MAPK, TOR, S6K) and the expression of longevity genes (Hep, MTH, and Rpn11).

Bee Pollen Extracts Modulate Serum Metabolism in Lipopolysaccharide-Induced Acute Lung Injury Mice with Anti-Inflammatory Effects.

Bee pollen (BP) collected from different floras possesses various potential bioactivities, but the mechanism-related research on anti-inflammatory effects is limited. Here, three types of BP originating from Camellia sinensis L. (BP-Cs), Nelumbo nucifera Gaertn. (BP-Nn), and Brassica campestris L. (BP-Bc) were assessed using molecular and metabolomics methods to determine their anti-inflammatory effects. The differences in polyphenolic abundance of three types of BP extracts were determined by HPLC-DAD/Q-TOF-MS. In vitro anti-inflammatory effects of three BP extracts were evaluated in a lipopolysaccharide (LPS)-induced RAW 264.7 cells model. BP-Cs extract with the most abundant polyphenols was found to be the most effective in reducing inflammation by downregulating inflammatory-related genes expression and blocking the activation of MAPK and NF-κB signaling pathways. Polyphenol-rich BP-Cs was further evaluated for their in vivo anti-inflammatory effect in a LPS-induced acute lung injury mouse model. An UPLC-Q-TOF/MS-based metabolomics approach was applied to analyze metabolite changes in mouse serum. Weshowed that the pretreated BP-Cs extract alleviated inflammation and regulated glycerophospholipid metabolism significantly. Our findings provide a foundation for developing and justifying BP as a potential anti-inflammatory ingredient in functional foods or nutraceutical formulations.

Neferine suppresses osteoclast differentiation through suppressing NF-κB signal pathway but not MAPKs and promote osteogenesis.

Osteoporosis is an ageing disease characterized by elevated osteoclastic bone resorption resulting in bone loss, decrease bone strength, and elevated incidence of fractures. Neferine, a natural compound isolated from the traditional Chinese medicine Nelumbo nucifera (Lotus), has been reported exhibit anti-inflammatory, antioxidant, and anticancer properties. However, its effect on bone remains to be determined. Here we showed that Neferine inhibits RANKL-induced osteoclast formation in a dose- and time-dependent manner. Furthermore, Neferine also demonstrated antiresorptive properties by effectively ameliorating the bone resorptive activity of mature osteoclasts. Mechanistically, Neferine suppressed RANKL-induced activation of NF-κB signaling pathway. This in turn hindered the induction and activation of NFATc1 resulting in downregulation of osteoclast marker genes closely related to differentiation, fusion as well as bone resorption. Interestingly, we found Neferine enhanced the differentiation and bone mineralization activity of MC3T3-E1 preosteoblast cells. Finally, mice treated with Neferine was protected against ovariectomy (OVX)-induced bone loss. The Neferine treatment improved bone volume following ovariectomy and also exhibited less TRAP-positive osteoclasts on bone surface. Collectively our data provide promising evidence that Neferine could be a potential therapeutic application for against osteolytic bone conditions such as osteoporosis.

Activity and molecular targets of pioglitazone via blockade of proliferation, invasiveness and bioenergetics in human NSCLC.

BACKGROUND: Pioglitazone, a synthetic peroxisome proliferator activated receptor (PPAR-γ) ligand, is known as an antidiabetic drug included in the thiazolidinediones (TZDs) class. It regulates the lipid and glucose cell metabolism and recently a role in the inhibition of numerous cancer cell processes has been described. METHODS: In our work we investigate the anti-tumor effects of pioglitazone in in vitro models of non small cell lung cancer (NSCLC) and also, we generated ex-vivo three-dimensional (3D) cultures from human lung adenocarcinoma (ADK) as a model to test drug efficacy observed in vitro. The inhibitory effect of pioglitazone on cell proliferation, apoptosis and cell invasion in a panel of human NSCLC cell lines was evaluated by multiple assays. RESULTS: Pioglitazone reduced proliferative and invasive abilities with an IC50 ranging between 5 and 10 µM and induced apoptosis of NSCLC cells. mRNA microarray expression profiling showed a down regulation of MAPK, Myc and Ras genes after treatment with pioglitazone; altered gene expression was confirmed by protein analysis in a dose-related reduction of survivin and phosphorylated proteins levels of MAPK pathway. Interestingly mRNA microarray analysis showed also that pioglitazone affects TGFß pathway, which is important in the epithelial-to-mesenchimal transition (EMT) process, by down-regulating TGFßR1 and SMAD3 mRNA expression. In addition, extracellular acidification rate (ECAR) and a proportional reduction of markers of altered glucose metabolism in treated cells demonstrated also cell bioenergetics modulation by pioglitazone. CONCLUSIONS: Data indicate that PPAR-γ agonists represent an attractive treatment tool and by suppression of cell growth (in vitro and ex vivo models) and of invasion via blockade of MAPK cascade and TGFß/SMADs signaling, respectively, and its role in cancer bioenergetics and metabolism indicate that PPAR-γ agonists represent an attractive treatment tool for NSCLC.

Regulation of pollen lipid body biogenesis by MAP kinases and downstream WRKY transcription factors in Arabidopsis.

Signaling pathways that control the activities in non-photosynthetic plastids, important sites of plant metabolism, are largely unknown. Previously, we demonstrated that WRKY2 and WRKY34 transcription factors play an essential role in pollen development downstream of mitogen-activated protein kinase 3 (MPK3) and MPK6 in Arabidopsis. Here, we report that GLUCOSE-6-PHOSPHATE/PHOSPHATE TRANSLOCATOR 1 (GPT1) is a key target gene of WRKY2/WRKY34. GPT1 transports glucose-6-phosphate (Glc6P) into plastids for starch and/or fatty acid biosynthesis depending on the plant species. Loss of function of WRKY2/WRKY34 results in reduced GPT1 expression, and concomitantly, reduced accumulation of lipid bodies in mature pollen, which leads to compromised pollen viability, germination, pollen tube growth, and male transmission in Arabidopsis. Pollen-specific overexpression of GPT1 rescues the pollen defects of wrky2 wrky34 double mutant. Furthermore, gain-of-function activation of MPK3/MPK6 enhances GPT1 expression; whereas GPT1 expression is reduced in mkk4 mkk5 double mutant. Together, this study revealed a cytoplasmic/nuclear signaling pathway capable of coordinating the metabolic activities in plastids. High-level expression of GPT1 at late stages of pollen development drives Glc6P from cytosol into plastids, where Glc6P is used for fatty acid biosynthesis, an important step of lipid body biogenesis. The accumulation of lipid bodies during pollen maturation is essential to pollen fitness and successful reproduction.

Major compounds of red ginseng oil attenuate Aß25-35-induced neuronal apoptosis and inflammation by modulating MAPK/NF-κB pathway.

ß-Amyloid (Aß)-induced neuronal toxicity in Alzheimer's disease (AD) is associated with complex mechanisms. Thus, a multi-target approach might be suitable for AD treatment. Following our previous study on the neuroprotective effects of red ginseng oil extract, its major compounds, including linoleic acid (LA), ß-sitosterol (BS), and stigmasterol (SS), were examined to elucidate the mechanism of anti-apoptosis and anti-inflammation in Aß25-35-stimulated PC12 cells. The results showed that the three compounds mitigated Aß25-35 toxicity by regulating oxidative stress, apoptotic responses, and pro-inflammatory mediators. LA and SS strongly regulated intrinsic apoptosis markers, such as mitochondrial membrane potential, intracellular Ca2+, Bax/Bcl-2 ratio, and caspases-9, -3, and -8. However, BS blocked only the intrinsic apoptotic pathway, particularly by suppressing Ca2+ accumulation. Furthermore, all three compounds downregulated iNOS and phospho-nuclear factor-κB, but only LA and SS inhibited the expression of cyclooxygenase-2 and phospho-IκB. In assays to evaluate MAPK expression for confirming upstream signal pathways, BS decreased the phosphorylation of p38 and ERK, but not JNK, while SS markedly decreased the phosphorylation of all three MAPKs, and LA clearly decreased the phosphorylation of ERK and JNK, but not p38. These results indicate that LA, BS, and SS act as neuroprotectives against Aß25-35-induced injury by distinct molecular mechanisms, indicating their preventive and/or therapeutic potential to treat AD.

Protective effects of Coptis chinensis inflorescence extract and linarin against carbon tetrachloride-induced damage in HepG2 cells through the MAPK/Keap1-Nrf2 pathway.

Coptis chinensis inflorescence is traditionally used as tea and has been popular in the local market. C. chinensis inflorescence extract (CE) exhibits protective effects against carbon tetrachloride (CCl4)-induced damage, but the underlying mechanism remains unclear. The main chemicals of CE were detected, purified, and identified in this study. CE and linarin could reverse changes in cell viability, decrease alanine aminotransferase and aspartate transaminase levels, and reduce reactive oxygen species (ROS) generation induced by CCl4 in HepG2 cells. CE and linarin could also phosphorylate mitogen-activated protein kinases (MAPKs) and up-regulate Kelth-like ECH-associated protein (Keap1). The pathways of MAPKs and Keap1 lead to the separation of Keap1 and nuclear factor (erythroid-derived 2)-like 2 (Nrf2). Free Nrf2 transferred to the nucleus and enhanced the expression of phase II detoxification enzymes. This study provides a scientific basis for the use of C. chinensis inflorescence, which exhibits a hepatoprotective function, as a supplement in the food industry.