The Modulation of Phospho-Extracellular Signal-Regulated Kinase and Phospho-Protein Kinase B Signaling Pathways plus Activity of Macrophage-Stimulating Protein Contribute to the Protective Effect of Stachydrine on Acetaminophen-Induced Liver Injury.

Stachydrine, a prominent bioactive alkaloid derived from Leonurus heterophyllus, is a significant herb in traditional medicine. It has been noted for its anti-inflammatory and antioxidant characteristics. Consequently, we conducted a study of its hepatoprotective effect and the fundamental mechanisms involved in acetaminophen (APAP)-induced liver injury, utilizing a mouse model. Mice were intraperitoneally administered a hepatotoxic dose of APAP (300 mg/kg). Thirty minutes after APAP administration, mice were treated with different concentrations of stachydrine (0, 2.5, 5, and 10 mg/kg). Animals were sacrificed 16 h after APAP injection for serum and liver tissue assays. APAP overdose significantly elevated the serum alanine transferase levels, hepatic pro-inflammatory cytokines, malondialdehyde activity, phospho-extracellular signal-regulated kinase (ERK), phospho-protein kinase B (AKT), and macrophage-stimulating protein expression. Stachydrine treatment significantly decreased these parameters in mice with APAP-induced liver damage. Our results suggest that stachydrine may be a promising beneficial target in the prevention of APAP-induced liver damage through attenuation of the inflammatory response, inhibition of the ERK and AKT pathways, and expression of macrophage-stimulating proteins.

Trichosanthis Semen Exerts Neuroprotective Effects in Alzheimer's Disease Models by Inhibiting Amyloid-ß Accumulation and Regulating the Akt and ERK Signaling Pathways.

Background: Alzheimer's disease (AD), the most common form of dementia, is characterized by memory loss and the abnormal accumulation of senile plaques composed of amyloid-ß (Aß) protein. Trichosanthis Semen (TS) is a traditional herbal medicine used to treat phlegm-related conditions. While TS is recognized for various bioactivities, including anti-neuroinflammatory effects, its ability to attenuate AD remains unknown. Objective: To evaluate the effects of TS extract (TSE) on neuronal damage, Aß accumulation, and neuroinflammation in AD models. Methods: Thioflavin T and western blot assays were used to assess effects on Aß aggregation in vitro. TS was treated to PC12 cells with Aß to assess the neuroprotective effects. Memory functions and histological brain features were investigated in TSE-treated 5×FAD transgenic mice and mice with intracerebroventricularly injected Aß. Results: TSE disrupted Aß aggregation and increased the viability of cells and phosphorylation of both protein kinase B (Akt) and extracellular signal-regulated kinase (ERK) in vitro. TSE treatment also suppressed the accumulation of Aß plaques in the brain of 5×FAD mice, protected neuronal cells in both the subiculum and medial septum, and upregulated Akt/ERK phosphorylation in the hippocampus. Moreover, TSE ameliorated the memory decline and glial overactivation observed in 5×FAD mice. As assessing whether TS affect Aß-induced neurotoxicity in the Aß-injected mice, the effects of TS on memory improvement and neuroinflammatory inhibition were confirmed. Conclusions: TSE disrupted Aß aggregation, protected neurons against Aß-induced toxicity, and suppressed neuroinflammation, suggesting that it can suppress the development of AD.

Capsicum annuum var. abbreviatum Induces a Macrophage M2-Like Phenotype Mediated by Nrf2/HO-1 in LPS-Stimulated Macrophages.

Capsicum annuum var. abbreviatum (CAAE), which is in the genus Capsicum L. (Solanaceae), was found to be richer in polyphenols and flavonoids than other prevalent peppers of Capsicum annuum var. angulosum and Capsicum annuum. L. Yet, it is still unclear how CAAE reduces inflammation. In this study, we used the lipopolysaccharide-stimulated RAW264.7 macrophage cell line and bone marrow-derived macrophages to assess its anti-inflammatory activities. Initially, we discovered that CAAE decreased the levels of nitric oxide and inducible nitric oxide synthase. In addition, CAAE decreased the intracellular reactive oxygen species levels and increased the nuclear factor-erythroid 2-related factor 2 and heme oxygenase-1 compared with the phenotype of M2 macrophages. CAAE inhibited the activation of mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinases, c-Jun N-terminal kinases, and p38 MAPKs. CAAE also inhibited the translocation of nuclear factor kappa B into nuclear, hence preventing the production of proinflammatory cytokines. Therefore, we suggest that CAAE might have potential as a candidate therapeutic agent for inflammatory diseases.

Peppermint as a promising treatment agent in inflammatory conditions: A comprehensive systematic review of literature.

Inflammation, a type of the body's defense against injury or infection, causes many chronic disorders including diabetes, cardiovascular disease, and cancer. Therefore, discovering natural compounds with numerous biological activities for the management of inflammation is highly recommended. Out of natural compounds, peppermint and its main component, menthol, has been suggested to possess antiinflammatory potential. Four databases including Web of Sciences, PubMed, Scopus, and Embase were searched to identify articles about peppermint and its antiinflammatory effects up to March 2023. Out of 3805 records screened, 14 articles met the study criteria. The evidence reviewed here proposed peppermint as an antiinflammatory agent. Peppermint may suppress inflammation by activating the AMP-activated protein kinase/unc-51 like kinase 1/nuclear factor-E2 associated factor 2 autophagy pathway, downregulating extracellular signal-regulated kinase-nuclear factor kappa B and mitogen activated protein kinases pathways, attenuating oxidative stress, suppressing the production of pro-inflammatory mediators and nitric oxide, and inducing the production of antiinflammatory prostaglandins. Due to the promising antiinflammatory effects of peppermint and the lack of human studies in this regard, future randomized clinical trials examining the effects of peppermint on inflammation and its related maladies are warranted.

Leaves and pseudostems extract of Curcuma longa attenuates immunoglobulin E/bovine serum albumin-stimulated bone marrow-derived cultured mast cell activation and passive cutaneous anaphylaxis in BALB/c mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Curcuma longa, known as turmeric, is an herbaceous perennial plant belonging to the genus Curcuma. It is dispersed throughout tropical and subtropical regions worldwide. Since ancient times, turmeric has been used as an ethnomedicinal plant in the Ayurvedic system, particularly in Asian countries. Rhizomes of turmeric possess several pharmacological properties that give high value as a medicinal remedy for treating a range of conditions, including inflammation, pain, allergies, and digestive issues. Moreover, turmeric leaves and pseudostems also contain a variety of health-enhancing secondary metabolites, such as curcumin, flavonoids, and other phenolic compounds, which exhibit anti-inflammatory, antitumor, antibacterial, and antioxidant properties. AIM OF THE STUDY: Allergic diseases are a group of immune-mediated disorders mainly caused by an immunoglobulin E (IgE)-dependent immunological response to an innocuous allergen. Therefore, this study aimed to investigate the effect of leaves and pseudostems extract of turmeric (TLSWE-8510) on IgE/bovine serum albumin (BSA)-stimulated allergic responses in mouse bone marrow-derived cultured mast cells (BMCMCs) and passive cutaneous anaphylaxis (PCA) in BALB/c mice. MATERIALS AND METHODS: The effect of TLSWE-8510 on mast cell degranulation has been evaluated by investigating the release of ß-hexosaminidase and histamine in IgE/BSA-stimulated BMCMCs. Additionally, anti-allergic properties of TLSWE-8510 on IgE/BSA-stimulated BMCMCs were investigated using suppression of nuclear factor-kappa B (NF-κB), and spleen tyrosine kinase (Syk)-linker for T-cell activation (LAT)-extracellular-signal-regulated kinase (ERK)-GRB2 associated binding protein 2 (Gab2) signaling pathway and downregulation of allergy-related cytokines and chemokines expression. Furthermore, in vivo, studies were conducted using IgE-mediated PCA in BALB/c mice. RESULTS: TLSWE-8510 treatment significantly inhibited the degranulation of IgE/BSA-stimulated BMCMCs by inhibiting the release of ß-hexosaminidase and histamine dose-dependently. Additionally, TLSWE-8510 reduced the expression of high-affinity IgE receptors (Fc epsilon receptor I-FcεRI) on the surface of BMCMCs and the binding of IgE to FcεRI. Besides, the expression of cytokines and chemokines is triggered by IgE/BSA stimulation via activating the allergy-related signaling pathways. TLSWE-8510 dose-dependently downregulated the mRNA expression and the production of allergy-related cytokines (interleukin (IL)-1ß, IL-3, IL-4, IL-5, IL-6, IL-13, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ), and chemokines (thymus and activation-regulated chemokine (TARC), and regulated upon activation, normal T cell expressed and secreted (RANTES)) by regulating the phosphorylation of downstream signaling molecules, NF-κB, and Syk, LAT, ERK and Gab2 in IgE/BSA-stimulated BMCMCs. Moreover, PCA reaction in IgE/BSA-stimulated BALB/c mice ears was effectively decreased by TLSWE-8510 treatment in a dose-dependent manner. CONCLUSIONS: These results collectively demonstrated that TLSWE-8510 suppressed mast cell degranulation by inhibiting the release of chemical mediators related to allergies. TLSWE-8510 downregulated the allergy-related cytokines and chemokines expression and phosphorylation of downstream signaling molecules in IgE/BSA-stimulated BMCMCs. Furthermore, in vivo studies with IgE-mediated PCA reaction in the BALB/c mice ears were attenuated by TLSWE-8510 treatment. These findings revealed that TLSWE-8510 has the potential as a therapeutic agent for the treatment of allergic diseases.

Identification of intracellular activation mechanism of rhamnogalacturonan-I type polysaccharide purified from Panax ginseng leaves in macrophages and roles of component sugar chains on activity.

This study aimed to investigate the mechanisms underlying intracellular signaling pathways in macrophages in relation to the structural features of rhamnogalacturonan (RG) I-type polysaccharide (PGEP-I) purified from Panax ginseng leaves. For this investigation, we used several specific inhibitors and antibodies against mitogen-activated protein kinase (MAPK), nuclear factor-kappa B (NF-κB), and pattern recognition receptors (PRRs). Furthermore, we investigated the roles of component sugar chains on immunostimulating activity through a sequential enzymatic and chemical degradation steps. We found that PGEP-I effectively induced the phosphorylation of several MAPK- and NF-κB-related proteins, such as p38, cJun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and p65. Particularly, immunocytochemistry analysis confirmed the PGEP-I-induced translocation of p65 into the nucleus. Furthermore, the breakdown of PGEP-I side chains and main chain during sequential enzymatic and chemical degradation reduced the PGEP-I-induced macrophage cytokine secretion activity. IL-6, TNF-α, and NO secreted by macrophages are associated with several signaling pathway proteins such as ERK, JNK, and NF-κB and several PRRs such as dectin-1, CD11b, CD14, TLR2, TLR4, and SR. Thus, these findings suggest that PGEP-I exerts potent macrophage-activating effects, which can be attributed to its typical RG-I structure comprising arabinan, type II arabinogalactan, and rhamnose-galacturonic acid repeating units in the main chain.

LPC20K modified from krill oil ameliorates the scopolamine-induced cognitive impairment.

Alzheimer's disease (AD) is characterized by cognitive impairment. It is common in the elderly. Etiologically, dysfunction of cholinergic neurotransmitter system is prominent in AD. However, disease modifying drug for AD is still unavailable. We hypothesized that krill oil and modified krill oil containing 20 % lysophosphatidylcholine-docosahexaenoic acid (LPC-DHA, LPC20K) could play a crucial role in AD by improving cognitive functions measured by several behavioral tests. We found that LPC20K could ameliorate short-term, long-term, spatial, and object recognition memory under cholinergic hypofunction states. To find the underlying mechanism involved in the effect of LPC20K on cognitive function, we investigated changes of signaling molecules using Western blotting. Expression levels of protein kinase C zeta (PKCζ) and postsynaptic density protein 95 (PSD-95), and phosphorylation levels of extracellular signal-regulated kinase (ERK), Ca2+/calmodulin-dependent protein kinase Ⅱ (CaMKⅡ), and cAMP response element-binding protein (CREB) were significantly increased in LPC20K-administered group compared to those in the memory impairment group. Moreover, the expression levels of BDNF were temporally increased especially 6 or 9 h after administration of LPC20K compared with the control group. These results suggest that LPC20K could ameliorate memory impairment caused by hypocholinergic state by enhancing the expression levels of PKCζ and PSD-95, and phosphorylation levels of ERK, CaMKⅡ and CREB and increasing BDNF expression levels. Therefore, LPC20K could be used as a dietary supplement against cognitive impairment observed in diseases such as AD with a hypocholinergic state.

Forsythiaside A Activates AMP-Activated Protein Kinase and Regulates Oxidative Stress via Nrf2 Signaling.

Forsythiaside A (FA) is an active constituent isolated from Forsythia suspensa, a beneficial herb used in traditional medicine known for its antioxidant and anti-inflammatory properties. Although various studies have suggested that FA has the protective effects, its impacts on arachidonic acid (AA) plus iron in vitro models and carbon tetrachloride (CCl4)-induced mouse liver damage in vivo have not been explored. In this study, HepG2 cells were subjected to AA + iron treatment to induce apoptosis and mitochondrial impairment and determine the molecular mechanisms. FA exhibited protective effects by inhibiting cell damage and reactive oxygen species (ROS) production induced by AA + iron, as assessed via immunoblot and flow cytometry analyses. Further molecular investigations revealed that FA resulted in the activation of extracellular-signal-related protein kinase (ERK), which subsequently triggered the activation of AMP-activated protein kinase (AMPK), a critical regulator of cellular oxidative stress. Additionally, FA modulated the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway, which is a significant antioxidant transcription factor regulated by the AMPK pathway. For in vivo studies, mice were orally administered FA and then subjected to induction of CCl4-based hepatotoxicity. The protective effect of FA was confirmed via blood biochemistry and immunohistochemical analyses. In conclusion, our findings demonstrated the protective effects of FA against oxidative stress both in vitro and in vivo, thus indicating that FA is a potential candidate for liver protection. Our study sheds light on the mechanistic pathways involved in the antioxidant effects of FA, highlighting the hepatoprotective potential of naturally occurring compounds in traditional herbs, such as FA.

Spent hops extract (Humulus Lupulus L.) attenuates inflammation and angiogenesis of the retina via the nuclear factor-kappaB and protein kinase B/extracellular signal-regulated kinase pathways.

Spent hops extract (SHE) is a plant extract containing compounds with proven anti-inflammatory and anti-angiogenic activities. However, extract may exert synergic effects compared to its individual polyphenol components. Inflammatory diseases of the retina may lead to visual impairment, a reduction of the comfort of life, and even blindness due to the formation of new pathological blood vessels. More effective therapeutic options are being sought. The goal of the present study was to investigate the anti-inflammatory and anti-angiogenic potentials of SHE on human retinal pigment epithelial cells (ARPE-19) stimulated by lipopolysaccharide (LPS) or tumor necrosis factor alpha (TNF-α). The SHE (250 µg/mL) was found to downregulate the gene expression of interleukin 6 (IL-6) to 33% in LPS-triggered cells; it also reduced both matrix metalloproteinase 2 (MMP-2) and 9 (MMP-9) mRNA expression to 13% and 43% respectively, and their activity to 82% (MMP-2) and 57% (MMP-9), compared to TNF-α-stimulated cells. Also, SHE modulated the TNF-α-induced expression of vascular endothelial growth factor (VEGF) and endothelial growth factor receptor 2 (VEGFR2). It is possible that SHE inhibited retinal inflammation and angiogenesis by suppressing the nuclear factor kappa B (NF-κB), protein kinase B (Akt) and extracellular signal-regulated kinase (ERK) pathways. Our results demonstrate that SHE has anti-inflammatory and anti-angiogenic potential against retinal diseases. This is the first such study to report on the efficacy of SHE on retinal inflammatory diseases.

Platycodin D induces apoptotic cell death through PI3K/AKT and MAPK/ERK pathways and synergizes with venetoclax in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a highly heterogeneous and rapidly progressive hematopoietic neoplasm characterized by frequent relapses and variable prognoses. The development of new treatment options, therefore, is of crucial importance. Platycodin D (PD) is a triterpenoid saponin, extracted from the roots of the traditional Chinese herbal medicine Platycodon grandiflorum (Jacq.) A. DC., which has been reported to exhibit therapeutic potential against a broad range of cancers. Although the effects of PD on AML remain unclear, in the present study, we observed a concentration-dependent reduction in the viability of multiple human AML cell lines in response to treatment with PD. In addition to triggering mitochondria-dependent apoptosis via the upregulation of BAK and BIM, treatment with PD also induced cell cycle arrest at the G0/G1 phase. Western blot analyses revealed marked suppression of the phosphorylation of protein kinase B (AKT), glycogen synthase kinase-3ß, ribosomal protein S6, and extracellular signal-regulated kinase (ERK) by PD, in turn implying the participation of the phosphoinositide 3-kinase (PI3K)/AKT and mitogen-activated protein kinase (MAPK)/ERK pathways. Pre-incubation with LY294002, MK2206, AR-A014418, or U0126 was consistently found to significantly aggravate PD-induced inhibition of viability. Additionally, PD combined with the B-cell lymphoma 2 (BCL2) inhibitor venetoclax elicited synergistically enhanced cytotoxic effects. The anti-leukemic activity of PD was further validated using primary samples from de novo AML patients. Given the results of the present study, PD may be a potent therapeutic candidate for the treatment of AML.

Effects of Artemisia annua L. on postmenopausal syndrome in ovariectomized mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Artemisia annua L. (Asteraceae) has been used as an antipyretic and anti-parasitic drug in traditional medicine for more than 2000 years. It has also been prescribed to treat symptoms caused by deficiency of Yin, which might be observed in menopausal state from the point of view of traditional medicine. AIM OF THE STUDY: We hypothesized that A. annua might be useful for treating menopausal disorders with less adverse effects than hormone replacement therapy. Thus, the aim of the present study was to investigate effects of A. annua on postmenopausal symptoms of ovariectomized (OVX) mice. MATERIALS AND METHODS: OVX mice were employed as a model for postmenopausal disorders. Mice were treated with a water extract of A. annua (EAA; 30, 100 or 300 mg/kg, p.o.) or 17ß-estradiol (E2; 0.5 mg/kg, s.c.) for 8 weeks. Open field test (OFT), novel object recognition task (NOR), Y-maze test, elevated plus maze test (EPM), splash test and tail suspension test (TST) were conducted to determine whether EAA could ameliorate postmenopausal symptoms. Phosphorylated levels of extracellular signal-regulated kinase (ERK), protein kinase B (Akt), and glycogen synthase kinase-3ß (GSK-3ß), ß-catenin and expression level of synaptophysin in the cortex and hippocampus were evaluated by Western blot analysis. RESULTS: EAA treatment significantly increased the discrimination index in NOR, decreased the time in closed arm than in open arm in EPM, increased grooming time in splash test, and decreased immobility time in TST, as did E2 treatment. In addition, decreased phosphorylation levels of ERK, Akt, GSK-3ß, and ß-catenin and expression levels of synaptophysin in the cortex and hippocampus after OVX were reversed by administration of EAA and E2. CONCLUSION: These results suggest that A. annua can ameliorate postmenopausal symptoms such as cognitive dysfunction, anxiety, anhedonia, and depression by activating ERK, Akt, and GSK-3ß/ß-catenin signaling pathway and hippocampal synaptic plasticity, and that A. annua would be a novel treatment for postmenopausal symptoms.

Shaoyao Gancao decoction alleviates the central hyperalgesia of recurrent NTG-induced migraine in rats by regulating the NGF/TRPV1/COX-2 signal pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Shaoyao Gancao Decoction (SGD) is well known as an effective prescription for analgesia composed of two herbs, and is noted as traditional Chinese medicine morphine. It is widely used in various conditions causing pain, including migraine. However, there is currently no research exploring the mechanism of action in the treatment of migraines. AIM OF THE STUDY: The current research was devised to determine the underlying regulatory mechanism of SGD, by verifying its role in the NGF/TRPV1/COX-2 signal pathway. MATERIALS AND METHODS: The active components in SGD were identified by UHPLC-MS. A migraine model was prepared by subcutaneous (s.c.) injection of nitroglycerin (NTG) into the neck to detect migraine-like behavior, orbital hyperalgesia threshold changes, and the therapeutic effect of SGD. The mechanism of SGD in remedying migraine was studied through transcriptome sequencing (RNA-seq), which was further validated utilizing Elisa, Reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blotting (WB) experiments. RESULTS: In the SGD chemical composition analysis, 45 components were identified including gallic acid, paeoniflorin and albiforin. In the behavioral experiments, SGD treatment significantly decreased the score of migraine-like head scratching in the NTG-induced migraine model (Mod) rats, while the hyperalgesia threshold increased outstandingly on days 10, 12, and 14 (P < 0.01, P < 0.001 or P < 0.0001). In migraine biomarkers experiment, compared with the Mod group, the 5-hydroxytryptamine (5-HT) contents were outstandingly enhanced by SGD treatment, while nitric oxide (NO) contents were markedly declined (P < 0.01). In the RNA-seq test, the down-regulated genes of SGD inhibiting hyperalgesia migraine included the neurotrophic factor (NGF) and transient receptor potential vanillic acid subfamily protein 1 receptor (TRPV1). The down-regulation pathway is the inflammatory mediator regulation of TRP channels. In gene set enrichment analysis (GSEA), SGD decreased the over-expression of protooncogene tyrosine-protein kinase Src (SRC) and TRPV1 in this pathway, and the two genes clustered at its lower end, with similar functions. PPI network results show that NGF interacts with TRPV1. Further verification shows that when compared with Mod group, the plasma cyclooxygenase-2 (COX-2), prostaglandin E2 (PGE2) protein expression levels and the dura mater calcitonin gene-related peptide (CGRP), extracellular signal-regulated kinase (ERK), p-ERK, SRC and NGF protein expression levels in the SGD group were remarkably decreased (P < 0.01, P < 0.001 or P < 0.0001), and the expression level of TRPV1 protein showed a downward trend (P = 0.06). The expression levels of COX-2, NO, CGRP, TRPV1, SRC and NGF mRNA in the dura mater was overtly down-regulated (P < 0.05, P < 0.01 or P < 0.001). CONCLUSIONS: SGD has a significant inhibitory effect on the NGF/TRPV1/COX-2 signaling pathway that mediates central hyperalgesia migraine, thus suggesting the molecular mechanism of SGD in improving the symptoms of migraine may be related to the central hyperalgesia neurotransmitter that regulates the pathogenesis of migraine.

Kisspeptin induces Kiss-1 and GnRH gene expression in mHypoA-55 hypothalamic cell models: Involvement of the ERK and PKA signaling pathways.

mHypoA-55 cells are kisspeptin-expressing neuronal cells originating from the arcuate nucleus of the mouse hypothalamus. These cells are called KNDy neurons because they co-express kisspeptin, neurokinin B, and dynorphin A. In addition, they express gonadotropin-releasing hormone (GnRH). Here, we found that kisspeptin 10 (KP10) increased Kiss-1 (encoding kisspeptin) and GnRH gene expression in kisspeptin receptor (Kiss-1R)-overexpressing mHypoA-55 cells. KP10 greatly increased serum response element (SRE) promoter activity, which is a target of extracellular signal-regulated kinase (ERK) (20.0 ± 2.54-fold). KP10 also increased cAMP-response element (CRE) promoter activity in these cells (2.32 ± 0.36-fold). KP10-increased SRE promoter activity was significantly prevented in the presence of PD098095, a MEK kinase (MEKK) inhibitor, and KP10-induced CRE promoter activity was also inhibited by PD098059. Similarly, H89, a protein kinase A (PKA) inhibitor, significantly inhibited the KP10 induction of SRE and CRE promoters. KP10-induced Kiss-1 and GnRH gene expressions were inhibited in the presence of PD098059. Likewise, H89 significantly inhibited the KP10-induced increase in Kiss-1 and GnRH. Transfection of mHypoA-55 cells with constitutively active MEKK (pFC-MEKK) increased SRE and CRE promoter activities by 9.75 ± 1.77- and 1.36 ± 0.12-fold, respectively. Induction of constitutively active PKA (pFC-PKA) also increased SRE and CRE promoter activities by 2.41 ± 0.42- and 40.71 ± 7.77-fold, respectively. Furthermore, pFC-MEKK and -PKA transfection of mHypoA-55 cells increased both Kiss-1 and GnRH gene expression. Our current observations suggest that KP10 increases both the ERK and PKA pathways and that both pathways mutually interact in mHypoA-55 hypothalamic cells. Activation of both ERK and PKA signaling might be necessary to induce Kiss-1 and GnRH gene expressions.

AAV-glycine receptor α3 alleviates CFA-induced inflammatory pain by downregulating ERK phosphorylation and proinflammatory cytokine expression in SD rats.

BACKGROUND: Glycine receptors (GlyRs) play key roles in the processing of inflammatory pain. The use of adeno-associated virus (AAV) vectors for gene therapy in human clinical trials has shown promise, as AAV generally causes a very mild immune response and long-term gene transfer, and there have been no reports of disease. Therefore, we used AAV for GlyRα1/3 gene transfer in F11 neuron cells and into Sprague-Dawley (SD) rats to investigate the effects and roles of AAV-GlyRα1/3 on cell cytotoxicity and inflammatory response. METHODS: In vitro experiments were performed using plasmid adeno-associated virus (pAAV)-GlyRα1/3-transfected F11 neurons to investigate the effects of pAAV-GlyRα1/3 on cell cytotoxicity and the prostaglandin E2 (PGE2)-mediated inflammatory response. In vivo experiment, the association between GlyRα3 and inflammatory pain was analyzed in normal rats after AAV-GlyRα3 intrathecal injection and after complete Freund's adjuvant (CFA) intraplantar administration. Intrathecal AAV-GlyRα3 delivery into SD rats was evaluated in terms of its potential for alleviating CFA-induced inflammatory pain. RESULTS: The activation of mitogen-activated protein kinase (MAPK) inflammatory signaling and neuronal injury marker activating transcription factor 3 (ATF-3) were evaluated by western blotting and immunofluorescence; the level of cytokine expression was measured by ELISA. The results showed that pAAV/pAAV-GlyRα1/3 transfection into F11 cells did not significantly reduce cell viability or induce extracellular signal-regulated kinase (ERK) phosphorylation or ATF-3 activation. PGE2-induced ERK phosphorylation in F11 cells was repressed by the expression of pAAV-GlyRα3 and administration of an EP2 inhibitor, GlyRαs antagonist (strychnine), and a protein kinase C inhibitor. Additionally, intrathecal AAV-GlyRα3 administration to SD rats significantly decreased CFA-induced inflammatory pain and suppressed CFA-induced ERK phosphorylation, did not induce obvious histopathological injury but increased ATF-3 activation in dorsal root ganglion (DRGs). CONCLUSIONS: Antagonists of the prostaglandin EP2 receptor, PKC, and glycine receptor can inhibit PGE2-induced ERK phosphorylation. Intrathecal AAV-GlyRα3 administration to SD rats significantly decreased CFA-induced inflammatory pain and suppressed CFA-induced ERK phosphorylation, did not significantly induce gross histopathological injury but elicited ATF-3 activation. We suggest that PGE2-induced ERK phosphorylation can be modulated by GlyRα3, and AAV-GlyRα3 significantly downregulated CFA-induced cytokine activation.

Valtrate, an iridoid compound in Valeriana, elicits anti-glioblastoma activity through inhibition of the PDGFRA/MEK/ERK signaling pathway.

BACKGROUND: Valtrate, a natural compound isolated from the root of Valeriana, exhibits antitumor activity in many cancers through different mechanisms. However, its efficacy for the treatment of glioblastoma (GBM), a tumor type with a poor prognosis, has not yet been rigorously investigated. METHODS: GBM cell lines were treated with valtrate and CCK-8, colony formation and EdU assays, flow cytometry, and transwell, 3D tumor spheroid invasion and GBM-brain organoid co-culture invasion assays were performed to assess properties of proliferation, viability, apoptosis and invasion/migration. RNA sequencing analysis on valtrate-treated cells was performed to identify putative target genes underlying the antitumor activity of the drug in GBM cells. Western blot analysis, immunofluorescence and immunohistochemistry were performed to evaluate protein levels in valtrate-treated cell lines and in samples obtained from orthotopic xenografts. A specific activator of extracellular signal-regulated kinase (ERK) was used to identify the pathways mediating the effect. RESULTS: Valtrate significantly inhibited the proliferation of GBM cells in vitro by inducing mitochondrial apoptosis and suppressed invasion and migration of GBM cells by inhibiting levels of proteins associated with epithelial mesenchymal transition (EMT). RNA sequencing analysis of valtrate-treated GBM cells revealed platelet-derived growth factor receptor A (PDGFRA) as a potential target downregulated by the drug. Analysis of PDGFRA protein and downstream mediators demonstrated that valtrate inhibited PDGFRA/MEK/ERK signaling. Finally, treatment of tumor-bearing nude mice with valtrate led to decreased tumor volume (fivefold difference at day 28) and enhanced survival (day 27 vs day 36, control vs valtrate-treated) relative to controls. CONCLUSIONS: Taken together, our study demonstrated that the natural product valtrate elicits antitumor activity in GBM cells through targeting PDGFRA and thus provides a candidate therapeutic compound for the treatment of GBM.

Nobiletin inhibits breast cancer cell migration and invasion by suppressing the IL-6-induced ERK-STAT and JNK-c-JUN pathways.

BACKGROUND: Breast cancer is one of the most common cancers in women, affecting more than 2 million women worldwide annually. However, effective treatments for breast cancer are limited. Nobiletin is a flavonoid present in the dried mature pericarp of mandarin orange (Citrus reticulata Blanco), which is used to prepare Citri Renetulatae Pericarpium and can inhibit tumour growth and progression according to modern pharmacological studies. However, whether nobiletin exhibits an antimetastatic role in breast cancer and its potential mechanism need to be further investigated. PURPOSE: This study aims to evaluate the inhibitory effect of nobiletin on breast cancer and to elucidate potential mechanisms against invasion and migration. METHODS: Cell viability was determined by cell counting kit-8 and colony formation assays. Wound healing and Boyden chamber assays detected cancer cell migration and invasion capabilities. Immunoblotting and qPCR were applied to determine the protein and mRNA expression levels of extracellular signal-regulated kinases (ERK) and the c-Jun N-terminal kinase (JNK) signalling pathways. Molecular docking was used to assess the degree of nobiletin binding to phosphatidylinositol 3-kinase (PI3K). Xenografts and liver metastases were constructed in BALB/c nude mice to evaluate the anticancer effect of nobiletin in vivo. H&E staining and immunohistochemistry were used to detect proliferation and the expression of related proteins. RESULTS: Nobiletin induced cell death in a concentration- and time-dependent manner and possessed anti-invasion and anti-migration effects on MCF-7 and T47D cells by suppressing the interleukin-6-induced ERK and JNK signalling pathways. In addition, nobiletin docked with the binding site of PI3K, and the binding score was -8.0 kcal/mol. Furthermore, the inhibition of breast cancer growth and metastasis by nobiletin was demonstrated by constructing xenografts and liver metastases in vivo. CONCLUSION: Nobiletin inhibited liver metastasis of breast cancer by downregulating the ERK-STAT and JNK-c-JUN pathways, and its safety and efficacy were verified, indicating the potential of nobiletin as an anticancer agent.

Warm acupuncture therapy alleviates neuronal apoptosis after spinal cord injury via inhibition of the ERK signaling pathway.

PURPOSE: Warm acupuncture (WA) therapy has been applied to treat spinal cord injury (SCI), but the underlying mechanism is unclear. The current study attempted to explore the WA therapy on neuronal apoptosis of SCI and the relationship with the extracellular signal-regulated kinase (ERK) signaling pathway. METHODS: The rat SCI models were established by the impact method. SCI rat models were subjected to WA treatment at Dazhui (GV14) and Jiaji points (T10), Yaoyangguan (GV3), Zusanli (ST36), and Ciliao (BL32). The rat SCI models were established by the impact method. WA and U0126 treatments were performed on the SCI rats. Motor function and neuronal apoptosis were detected. The relative mRNA of tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), and interleukin-6 (IL-6), the phosphorylation level of ERK 1/2 and levels of B-cell lymphoma-2 (Bcl-2), BCL2-Associated X (Bax), and caspase-3 in spinal cord tissue were tested. RESULTS: After WA treatment, the Basso, Beattie & Bresnahan locomotor rating scale (BBB scale) of SCI rats in the WA treatment was significantly raised from 7 to 14 days after SCI. WA and U0126 treatment significantly diminished apoptotic cells and preserved the neurons in the injured spinal cord. WA and U0126 treatment alleviated the production of inflammatory cytokines in the spinal cord. The distinct increase of p-ERK 1/2 induced by SCI was reversed in WA and U0126 treatment groups. WA and U0126 treatment augmented the level of Bcl-2 and reversed the elevated cleaved caspase-3 protein level after SCI. CONCLUSION: Our study demonstrated that WA might be associated with the downregulation of the ERK signaling pathway. In summary, our findings indicated that WA promotes the recovery of SCI via the protection of nerve cells and the prevention of apoptosis. Meanwhile, the anti-apoptotic effect of WA might be associated with the downregulation of the ERK signaling pathway, which could be one of the mechanisms of WA in the treatment of SCI.

Ligularia fischeri ethanol extract: An inhibitor of alpha-melanocyte-stimulating hormone-stimulated melanogenesis in B16F10 melanoma cells.

BACKGROUND: Ligularia fischeri is a perennial herb isolated from plants of the Asteraceae family. Ligularia fischeri is distributed throughout Korea, Japan, eastern Siberia, and China. AIMS: The aim of this study is to examine the intracellular inhibitory effect of Ligularia fischeri ethanol extract on melanin synthesis and expression of tyrosinase and tyrosinase-related protein 1 and 2. In addition, we analyzed the mitogen-activated protein kinase signaling pathway and microphthalmia-associated transcription factor in alpha-melanocyte-stimulating hormone-stimulated B16F10 melanoma cells. METHODS: To assess the inhibition of melanogenesis in alpha-melanocyte-stimulating hormone-stimulated B16F10 melanoma cells, the expression of melanogenesis-related genes was investigated by quantitative real-time polymerase chain reaction, while western blotting was performed to determine protein expression levels. RESULTS: We confirmed that the ethanol extract of Ligularia fischeri inhibited melanin synthesis in vitro by decreasing tyrosinase and tyrosinase-related protein 1 and 2 expression. Furthermore, we revealed that tyrosinase expression was regulated by the suppression of microphthalmia-associated transcription factor expression and activation of extracellular signal-regulated kinase phosphorylation. The ethanol extract of Ligularia fischeri inhibited melanogenesis by activating extracellular signal-regulated kinase phosphorylation and suppressing microphthalmia-associated transcription factor and tyrosinase expression. CONCLUSIONS: Ligularia fischeri ethanol extract may be used as an effective skin whitening agent in functional cosmetics.

Reduction in Insulin Mediated ERK Phosphorylation by Palmitate in Liver Cells Is Independent of Fatty Acid Induced ER Stress.

Saturated free fatty acids (FFAs) such as palmitate in the circulation are known to cause endoplasmic reticulum (ER) stress and insulin resistance in peripheral tissues. In addition to protein kinase B (AKT) signaling, extracellular signal-regulated kinase (ERK) has been implicated in the development of insulin resistance. However, there are conflicting data regarding role of ERK signaling in ER stress-induced insulin resistance. In this study, we investigated the effects of ER stress on insulin resistance and ERK phosphorylation in Huh-7 cells and evaluated how oleate prevents palmitate-mediated ER stress. Treatment with insulin resulted in an increase of 38-45% in the uptake of glucose in control cells compared to non-insulin-treated control cells, along with an increase in the phosphorylation of AKT and ERK. We found that treatment with palmitate increased the expression of ER stress genes, including the splicing of X box binding protein 1 (XBP1) mRNA. At the same time, we observed a decrease in insulin-mediated uptake of glucose and ERK phosphorylation in Huh-7 cells, without any change in AKT phosphorylation. Supplementation of oleate along with palmitate mitigated the palmitate-induced ER stress but did not affect insulin-mediated glucose uptake or ERK phosphorylation. The findings of this study suggest that palmitate reduces insulin-mediated ERK phosphorylation in liver cells and this effect is independent of fatty-acid-induced ER stress.

The analgesic effects of ß-elemene in rats with neuropathic pain by inhibition of spinal astrocytic ERK activation.

Neuropathic pain takes a heavy toll on individual well-being, while current therapy is far from desirable. Herein, we assessed the analgesic effect of ß-elemene, a chief component in the traditional Chinese medicine Curcuma wenyujin, and explored the underlying mechanisms at the level of spinal dorsal horn (SDH) under neuropathic pain. A spared nerve injury (SNI)-induced neuropathic pain model was established in rats. Intraperitoneal injection (i.p.) of ß-elemene was administered for 21 consecutive days. Mechanical allodynia was explored by von Frey filaments. The activation of the mitogen-activated protein kinase (MAPK) family (including ERK, p38, and JNK) in spinal neurons, astrocytes, and microglia was evaluated using immunostaining 29 days after SNI surgery. The expression of GFAP, Iba-1, p-ERK, p-JNK, and p-p38 within the SDH was measured using immunoblotting. The levels of proinflammatory cytokines (including TNF-α, IL-1ß, and IL-6) were measured with ELISA. The levels of oxidative stress indicators (including MDA, SOD, and GSH-PX) were detected using biochemical tests. Consecutive i.p. administration of ß-elemene relieved SNI-induced mechanical allodynia (with an EC50 of 16.40 mg/kg). SNI significantly increased the expression of p-ERK in spinal astrocytes but not microglia on day 29. ß-elemene reversed spinal astrocytic ERK activation and subsequent upregulation of proinflammatory cytokines in SNI rats, with no effect on the expression of p38 and JNK in spinal glia. ß-elemene also exerted antioxidative effects by increasing the levels of SOD and GSH-PX and decreasing the level of MDA. Our results suggest that SNI induces robust astrocytic ERK activation within the SDH in the late phase of neuropathic pain. ß-elemene exerts remarkable analgesic effects on neuropathic pain, possibly by inhibiting spinal astrocytic ERK activation and subsequent neuroinflammatory processes. Our findings suggest that ß-elemene might be a promising analgesic for the treatment of chronic pain.