6-Shogaol Inhibits the Cell Migration of Colon Cancer by Suppressing the EMT Process Through the IKKß/NF-κB/Snail Pathway.

6-Shogaol from ginger has anti-inflammatory, anti-oxidation and anti-cancer effects. Aim of the Study: To study the effects and possible mechanisms of 6-Shogaol on inhibiting the migration of colon cancer cells Caco2 and HCT116 and prove the effects on proliferation and apoptosis. Materials and methods: The cells were treated with 6-Shogaol at the concentrations of 20, 40, 60, 80, and 100 µM, the cytotoxicity was tested by Colony formation assays and 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and the Western blot was used to evaluate IKKß/NF-κB/Snail pathway and EMT-related proteins. In addition, in order to eliminate the interference of proliferation inhibition on the experiment, Caco2 cells were treated with 6-Shogaol at the concentrations of 0, 40, and 80 µM, HCT116 cells were treated with 6-Shogaol at the concentrations of 0, 20, and 40 µM, apoptosis was measured by Annex V/PI staining, and migration was measured by Wound healing assays and Transwell test. Results: 6-Shogaol significantly inhibited the growth of cells. The maximum inhibitory concentration of half of them was 86.63 µM in Caco2 cells and 45.25 µM in HCT116 cells. At 80 µM and 40 µM concentrations, 6-Shogaol significantly promoted apoptosis of colon cancer Caco2 cells and HCT116 cells, and also significantly inhibited cell migration (P < .05). In addition, Western blot analysis showed that at 80 µM dose of 6-Shogaol significantly reduced MMP-2, N-cadherin, IKKß, P-NF-κB and Snail expression in Caco2 cells (P < .05). 40 µM dose of 6-Shogaol significantly reduced VEGF, IKKß, and P-NF-κB expression, and MMP-2, N-cadherin and Snail was significantly decreased at 60 µM of 6-Shogaol in HCT116 cells(P < .05). However, there was no significant change in E-cadherin in Caco2 cells, and the expression of E-cadherin protein in HCT116 cells decreased. Conclusion: This study proposes and confirms that 6-Shogaol can significantly inhibit the migration of colon cancer cells Caco2 and HCT116, and its mechanism may be produced by inhibiting EMT through IKKß/NF-κB/Snail signaling pathway. It was also confirmed that 6-Shogaol inhibited the proliferation and promoted apoptosis of Caco2 and HCT116 cells.

Activity in MCF-7 Estrogen-sensitive Breast Cancer Cells of Capsicodendrin from Cinnamosma fragrans.

BACKGROUND/AIM: Effect of capsicodendrin on the NF-κB pathway was studied in MCF-7 cancer cells. MATERIALS AND METHODS: The transcription factor assay was used to screen for NF-κB activity. The effect on IKKß, ICAM-1, and caspase-7 were studied using western blot. Caspase-1 was studied using Promega Caspase-Glo® assay. Reactive oxygen species (ROS) were detected using the fluorescent probe DCFH-DA. The potentiometric dye JC-1 was used to assess mitochondrial membrane potential (ΔΨm) and the cell cycle was examined using a fluorescence-activated cell sorter. RESULTS: NF-κB p65 inhibitory effect was IC50=8.6 µM and cytotoxic activity was IC50=7.5 µM. The upstream IKK and the downstream ICAM-1 were down-regulated. Sub G1-phase population increased to 81% after 12 h of treatment with capsicodendrin (10 µM) and there was no loss of ΔΨM. CONCLUSION: Increased levels of intracellular ROS promoted activity of caspase-1 and induced cell death in MCF-7 cells. Capsicodendrin may be a future anticancer agent that prevents the progression of metastatic breast cancer.

Clinical efficacies, underlying mechanisms and molecular targets of Chinese medicines for diabetic nephropathy treatment and management.

Diabetic nephropathy (DN) has been recognized as a severe complication of diabetes mellitus and a dominant pathogeny of end-stage kidney disease, which causes serious health problems and great financial burden to human society worldwide. Conventional strategies, such as renin-angiotensin-aldosterone system blockade, blood glucose level control, and bodyweight reduction, may not achieve satisfactory outcomes in many clinical practices for DN management. Notably, due to the multi-target function, Chinese medicine possesses promising clinical benefits as primary or alternative therapies for DN treatment. Increasing studies have emphasized identifying bioactive compounds and molecular mechanisms of reno-protective effects of Chinese medicines. Signaling pathways involved in glucose/lipid metabolism regulation, antioxidation, anti-inflammation, anti-fibrosis, and podocyte protection have been identified as crucial mechanisms of action. Herein, we summarize the clinical efficacies of Chinese medicines and their bioactive components in treating and managing DN after reviewing the results demonstrated in clinical trials, systematic reviews, and meta-analyses, with a thorough discussion on the relative underlying mechanisms and molecular targets reported in animal and cellular experiments. We aim to provide comprehensive insights into the protective effects of Chinese medicines against DN.

8-Geranylumbelliferone isolated from Paramignya trimera triggers RIPK1/RIPK3-dependent programmed cell death upon TNFR1 ligation.

In tumor necrosis factor (TNF) signaling, IκB kinase (IKK) complex-mediated activation of NF-κB is a well-known protective mechanism against cell death via transcriptional induction of pro-survival genes occurring as a late checkpoint. However, recent belief holds that IKK functions as an early cell death checkpoint to suppress the death-inducing signaling complex by regulating receptor interacting protein kinase1 (RIPK1) phosphorylation. In this study, we propose that two major gernaylated 7-hydroxy coumarins, 6-geranyl-7-hydroxycoumarin (ostruthin) and 8-geranyl-7-hydroxycoumarin (8-geranylumbelliferone, 8-GU) isolated from Paramignya timera, facilitate RIPK1-dependent dual modes of apoptosis and necroptosis by targeting IKKß upon TNF receptor1 (TNFR1) ligation. Analysis of events upstream of NF-κB revealed that 8-GU and ostruthin drastically inhibited TNF-induced IKK phosphorylation, while having no effect on TAK1 phosphorylation and TNFR1 complex-I formation. Interestingly, 8-GU did not affect the cell death induced by Fas ligand or TNF-related apoptosis-inducing ligand or that induced by DNA-damaging agents, indicating that 8-GU sensitizes TNF-induced cell death exclusively. Moreover, 8-GU accelerated TNF-driven necroptosis by up-regulating necrosome formation in FADD deficient cancer cells harboring RIPK3. Thus, the present study provides new insights into the molecular mechanism underlying geranylated 7-hydroxy coumarin-mediated control of the RIPK1-dependent early cell death checkpoint and suggests that 8-GU is a potential anti-cancer therapeutic via an alternative apoptosis-independent strategy to overcome TNF resistance.

Triptolidenol, isolated from Tripterygium wilfordii, disrupted NF-κB/COX-2 pathway by targeting ATP-binding sites of IKKß in clear cell renal cell carcinoma.

Triptolidenol (TPD) is an epoxy diterpene lactone from Tripterygium wilfordii, which has been used for chronic nephritis in China，and possessed various pharmacological properties, such as anti-inflammatory and anti-cancer activities. However, the precise molecular antitumor mechanism of TPD remains to be elucidated. In this study, we investigated the effects of TPD on human clear cell renal cell carcinoma (ccRCC) and investigated its precise anti-tumor mechanisms. It was showed that TPD significantly suppressed ccRCC cell proliferation, cell migration, and induced cell cycle arrest at S phase. Furthermore, TPD also induced apoptosis by activating the cytochrome c (cyt c)/caspase cascade signaling pathway. Moreover, using confocal immunofluorescence, a dual-luciferase reporter assay and molecular docking study, the results showed that TPD obviously reduced the expression of COX-2 by inhibiting the kinase activity of IKKß via targeting its ATP-binding domain, and then attenuating the transactivation of NF-κB. Collectively, our study demonstrated that TPD suppressed renal cell carcinoma growth through disrupting NF-κB/COX-2 pathway by targeting ATP-binding sites of IKKß, and provided pharmacological evidence that TPD exhibits potential use in the treatment of COX-2-mediated diseases such as ccRCC.

Budlein A methylacrylate demonstrates potent activity against triple-negative breast cancer by targeting IκBα kinase and exportin-1.

Triple-negative breast cancer (TNBC) remains the most challenging breast cancer subtype to treat because there are no targeted therapies. Currently, chemotherapy is the only clinical option for TNBC despite development of resistance. New therapeutic agents with unique mechanisms of action are urgently needed; therefore, this study investigated the potential anti-TNBC effects of budlein A methylacrylate (BAM), a natural sesquiterpene lactone isolated from plants of the Helianthus genus. We discovered that BAM selectively suppressed and induced apoptosis TNBC cell growth versus other breast cancer or normal mammary epithelial cells. Mechanistically, BAM co-inhibited inhibitor of nuclear factor κBα (IκBα) kinase subunit ß (IKKß) and exportin-1 (XPO-1; chromosome region maintenance 1, CRM1), which are two dysregulated onco-related proteins in TNBC cells, by covalently modifying key functional cysteine residues (Cys179 of IKKß, Cys528 of XPO-1). Dual inhibition led to the stabilization and nuclear retention of IκBα, impairment of NF-κB transcriptional activity, and consequent induction of TNBC cell apoptosis. In conclusion, this study provides evidence that co-inhibition of IKKß and XPO-1 by BAM was effective against TNBC, demonstrating it as a representative new generation inhibitor with potential for TNBC treatment.

Chanling Gao Attenuates Bone Cancer Pain in Rats by the IKKß/NF-κB Signaling Pathway.

Cancer pain is one of the most common and serious symptoms of cancer patients. At present, the agents used for the prevention or treatment of cancer pain do not act with optimal safety and efficacy. The nuclear factor kappa B (NF-κB) signaling pathway and its downstream inflammatory factors interleukin-6 (IL-6), interleukin-1ß (IL-1ß), and tumor necrosis factor-α (TNF-α) play an important regulatory role in the developmental process of cancer pain. IKKß is a key molecule of the IκB (IKK) kinase that propagates cellular responses to inflammation. Previous studies have shown that phosphorylation and degradation of the IκBα protein promotes the activation of NF-κB and the expression of TNF-α, IL-1ß, and IL-6, participating in the formation and development of cancer pain. Chanling Gao (CLG) is a compound preparation of traditional Chinese medicine. It contains specific functions, namely nourishing Yin, activating blood circulation and relieving pain and dysfunction syndrome. It is used in the treatment of a variety of pain disorders including cancer-induced bone pain (CIBP), which has a certain relief effect. However, its mechanism of action still remains unclear. In the present study, a rat model of tibia CIBP was successfully established using the Walker 256 breast cancer cell line. The IKKß/NF-κB signaling pathway and its related factors TNF-α, IL-1ß, and IL-6 were used as the entry points to explore the effect of CLG on CIBP and their possible mechanisms of action. The results indicated that CLG improved the body mass of the CIBP rat model and increased the pain threshold in rats. CLG significantly inhibited the degradation of IκBα and the levels of p-IκBα, p-IKKß, and p-p65 NF-κB proteins in the spinal cord of CIBP rats, inhibiting the contents of TNF-α, IL-1ß, and IL-6. Therefore, we conclude that the analgesic effect of CLG in this rat model of CIBP may be related to the inhibition of the IKKß/NF-κB signaling pathway and the reduction of synthesis and release of TNF-α, IL-1ß, and IL-6.

Small molecule inhibitors of IκB kinase ß: A chip-based screening and molecular docking simulation.

A chip-based screening system for IκB kinase ß (IKKß) has been developed by physically immobilizing the substrate IκBα on a glass matrix using a calixarene linker. Phosphorylation of IκBα by IKKß and ATP was quantitated using a fluorescently labeled antibody. Using this efficient assay system a chemical library of 2000 bioactive compounds was screened against IKKß and four were identified as good inhibitors, namely, aurintricarboxylic acid, diosmin, ellagic acid, and hematein. None of them have been reported to be an inhibitor of IKKß although they were implicated in various NFκB-mediated biological processes. Our enzyme-based assay showed that IC50 of the four inhibitors is comparable with that of IKK-16, a previously known strong inhibitor. Molecular docking simulation shows that the hydrophobic moiety of an inhibitor interacts with the four hydrophobic residues (Leu21, Val29, Val152, and Ile165) of the active site. The MM-PBSA calculation suggests that these hydrophobic interactions appear to be the predominant contributor to the binding free energy. As IKKß is ubiquitously expressed in various cell types and executes many biological functions, the enzyme and cell specificity of the four inhibitors need to be rigorously tested before accepted as a drug candidate.

Modulation of LPS-induced inflammation in RAW264.7 murine cells by novel isoflavonoids from Millettia pulchra.

Millettia pulchra is a renowned anti-inflammatory herbal medicine in southeast provinces of China. However, the underlying anti-inflammation mechanism remained incompletely understood. Herein, four new isoflavones, pulvones A-D and eleven reported constituents were isolated from the stems of Millettia pulchra with their structures being elucidated by HRMS and NMR analysis. The anti-inflammatory activities of pulvones A and C were further evaluated due to the better inhibitory activity on nitric oxide production in LPS-stimulated RAW264.7 cells and no obvious cytotoxicity to RAW264.7 cells. Western blot showed that pulvones A significantly decreased the levels of iNOS and COX-2 proteins and pulvones C only decreased the level of iNOS protein. ELISA analysis demonstrated that pulvones A inhibited the production of both interleukin-6 (IL-6) and IL-1ß while pulvones C showed better suppression effect on IL-1ß production in LPS-stimulated RAW264.7 cells. Then, their potential inhibitory effects on NF-κB pathway were tested in LPS-stimulated RAW264.7 cells. Immunofluorescence and western blot assay showed that pulvones A and C reduced the nuclear translocation of NF-κB(p65) and interrupted IκB phosphorylation. The ADP-Glo™ kinase assay showed pulvones A and C could directedly inhibit the IKKß kinase activity with the inhibitory rate of 40%, which were also verified by docking study. Collectively, these results suggested that pulvones A and C's anti-inflammatory effects were relevant to the interruption of NF-κB activation by inhibiting IKKß kinase.

Alkaloids from Black Pepper (Piper nigrum L.) Exhibit Anti-Inflammatory Activity in Murine Macrophages by Inhibiting Activation of NF-κB Pathway.

Black pepper (Piper nigrum L.) has been commonly utilized in food preparation and traditional medicine in several countries. Seven new amide alkaloids, pipernigramides A-G (3, 10, 38, and 41-44), a new piperic ester, pipernigrester A (48), along with 47 known compounds were isolated from the EtOH extract of P. nigrum. The inhibitory effects on nitric oxide (NO) of all compounds were then evaluated. Among the tested compounds, three of them (42-44) significantly inhibited inducible nitric oxide synthase (iNOS)-mediated NO (IC50 = 4.74 ± 0.18, 4.08 ± 0.19, and 3.71 ± 0.32 µM, respectively), and IL-1ß, IL-6, TNF-α, and PGE2 release in RAW 264.7 cells stimulated by lipopolysaccharide. Moreover, 42-44 suppressed IκB degradation and further inhibited the cytosol-nucleus translocation of the p65 subunit by targeting IKK-ß. In the carrageenan-induced paw edema test, 42-44 demonstrated anti-inflammatory effects as well. These results indicate that all three compounds from P.nigrum have the potential anti-inflammatory effects.

Suppression of migration, invasion, and metastasis of cisplatin-resistant head and neck squamous cell carcinoma through IKKß inhibition.

We explored the role of the transcription factor, NF-κB, and its upstream kinase IKKß in regulation of migration, invasion, and metastasis of cisplatin-resistant head and neck squamous cell carcinoma (HNSCC). We showed that cisplatin-resistant HNSCC cells have a stronger ability to migrate and invade, as well as display higher IKKß/NF-κB activity compared to their parental partners. Importantly, we found that knockdown of IKKß, but not NF-κB, dramatically impaired cell migration and invasion in these cells. Consistent with this, the IKKß inhibitor, CmpdA, also inhibited cell migration and invasion. Previous studies have already shown that N-Cadherin, an epithelial-mesenchymal transition (EMT) marker, and IL-6, a pro-inflammatory cytokine, play important roles in regulation of HNSCC migration, invasion, and metastasis. We found that cisplatin-resistant HNSCC expressed higher levels of N-Cadherin and IL-6, which were significantly inhibited by CmpdA. More importantly, we showed that CmpdA treatment dramatically abated cisplatin-resistant HNSCC cell metastasis to lungs in a mouse model. Our data demonstrated the crucial role of IKKß in control of migration, invasion, and metastasis, and implicated that targeting IKKß may be a potential therapy for cisplatin-resistant metastatic HNSCC.

ACT001 modulates the NF-κB/MnSOD/ROS axis by targeting IKKß to inhibit glioblastoma cell growth.

Glioblastomas are high-grade brain tumors with poor prognoses, and new therapeutic approaches for these tumors are critically needed. This study revealed the underlying mechanisms of a new orphan drug, ACT001, that is currently in clinical trials for the treatment of advanced glioblastoma in Australia and China. ACT001 significantly suppressed glioma cell proliferation and induced apoptosis and cell cycle arrest in vitro, as determined by Cell Counting Kit-8 assays and flow cytometry. In addition, U-118 MG cells with high expression of p-IKKß were sensitive to ACT001. Changes in the oxidative stress pathway in U-118 MG cells were detected with the isobaric tags for relative and absolute quantitation (iTRAQ) method. We further verified that ACT001 elevated the levels of reactive oxygen species (ROS) by regulating NF-κB-targeted MnSOD. ACT001 markedly inhibited NF-κB activation by directly binding IKKß and inhibiting its phosphorylation. Overexpression of IKKß markedly attenuated the changes in MnSOD and NOX1, indicating that ACT001 increased the levels of ROS by reducing the protein expression of p-IKKß. Furthermore, ACT001 reduced cyclin B1/CDC2 expression and triggered G2/M phase arrest by increasing ROS production. ACT001 also upregulated the expression of Bax and Bim and induced apoptosis in a ROS-dependent manner. ACT001 effectively suppressed the growth of U-118 MG tumors in BALB/c nude mice and GL-261-luciferase tumors in C57BL/6 J mice. Finally, ACT001 downregulated the expression of p-p65, MnSOD, cyclin B1, CDC2, and Ki67 in U-118 MG tumor tissues. Patients with activated NF-κB signaling should thus be given priority for enrollment in future phase II clinical trials. KEY MESSAGES: ACT001 directly bind to IKKß and inhibited its phosphorylation. The inhibition of p-IKKß induced the generation of ROS. ACT001 promoted the generation of ROS by regulating MnSOD expression to induce G2/M phase arrest.

Triptolide dysregulates glucose uptake via inhibition of IKKß-NF-κB pathway by p53 activation in cardiomyocytes.

Triptolide (TP), a principal bioactive component extracted from traditional Chinese medicine Tripterygium wilfordii Hook. F. (TWHF), has attracted wide attention of its therapeutic effects on inflammation and autoimmune diseases. However, the therapeutic application of TP is hindered by severe cardiomyocyte toxicity and narrow therapeutic window. We previously identified that the p53 was an indispensable contributor in TP-induced myocardial injury. p53 has an inhibitory effect on IKKß-NF-κB pathway that regulates glucose transporters (GLUT) expression. Based on these evidences, we speculate that p53 mediates TP-disturbed glucose uptake by blocking IKKß-NF-κB signaling. This study focused on the effect of TP on cardiac glucose uptake and the role of p53 in glucose metabolism in cardiomyocytes, and p53 -/- mice. TP treatment depressed glucose consumption and ATP production resulting in myocardial damage. Incubation with ATP (5 mM) remarkably decreased the cellular damage. Immunoblotting and immunofluorescence identified that TP suppressed glucose uptake by restricting IKKß-NF-κB signaling activation, GLUT1 and GLUT4 expression. p53 inhibition alleviated the cell damage and the compromise of glucose uptake. Mechanistically, p53 antagonist PFTα abolished TP-induced the inhibition of IKKß, IκBα phosphorylation, p65 nuclear translocation, and GLUT1, GLUT4 expression. Consistently, in acute heart injury models, p53 deficiency upregulated IKKß-NF-κB activation and GLUT1, GLUT4 protein levels which was also indicated as amelioration of heart histological injury after 1.2 mg kg-1 TP administration. The present findings indicate that TP-induced p53 overactivation suppresses glucose uptake by inhibiting IKKß-NF-κB pathway and downregulating NF-κB-dependent GLUT1 and GLUT4 expression.

Arenobufagin, isolated from toad venom, inhibited epithelial-to-mesenchymal transition and suppressed migration and invasion of lung cancer cells via targeting IKKß/NFκB signal cascade.

ETHNOPHARMACOLOGICAL RELEVANCE: Lung cancer is the leading cause of cancer incidence and mortality worldwide. Arenobufagin (Arg), a representative natural bufadienolide compound, is one of the major bioactive components isolated from toad venom ("Chan Su"named in Chinese to treat multifarious clinical neoplasms in China). However, the underlying molecular mechanisms that Arg inhibited the metastasis of lung cancer cells remain poorly understood. MATERIALS AND METHODS: The mobility capacities of lung cancer cells treated with Arg were evaluated using wound healing assay. The anti-migratory and anti-invasive effects of Arg on lung cancer cells were investigated by transwell invasion assay and matrigel invasion assay. iTRAQ-labeled LC-MS proteomics was used to analyze the potential proteins related to metastasis in lung cancer cells treated with Arg and differentially-expressed proteins related to EMT and NFκB signaling cascade were further confirmed by Western blotting assay. The changed subcellular localization of p65 in lung cancer A549 and H1299 cells treated with Arg was detected by immunofluorescence staining. Molecular docking and molecular dynamic (MD) simulation assay were performed to verify the binding between Arg and IKKα/IKKß. siRNA knockdown was used to check whether Arg inhibited EMT of lung cancer cells via targeting NFκB signaling cascade, which was further verified by in vivo study of lung cancer cell xenograft mice model and pulmonary metastasis mice model accompanying with immunohistochemical and hematoxylin-eosin (HE) staining. RESULTS: Arg suppressed the wound closure of lung cancer cells using wound healing assay. Moreover, Arg significantly inhibited the migration and invasion of lung cancer cells by transwell invasion assay and matrigel invasion assay. 24 unique differentially-expressed proteins related to metastasis in lung cancer cells treated with Arg were identified using iTRAQ-labeled LC-MS proteomics and 14 differentially-expressed proteins related to EMT were further confirmed by Western blotting assay. Arg significantly decreased the phosphorylation of IKKß, IκBα and p65 in the cytoplasm of lung cancer cells by Western blotting assay, and remarkably reduced the release of p65 from the cytoplasm to the nucleus. Arg could be bound in the ATP binding pocket of IKKα and IKKß by molecular docking assay, and MD simulation assay further demonstrated that Arg binding to the ATP-binding pocket of IKKß was very stable in 300 ns MD simulation, compared with the binding of Arg and IKKα. IKKß/NFκB signaling cascade was also involved in the inhibitory effect of Arg on EMT of lung cancer cells by siRNA knockdown assay. The study of lung cancer cell xenograft mice model and pulmonary metastasis mice model in vivo indicated that Arg inhibited EMT and suppressed migration and invasion of lung cancer cells via downregulating IKKß/NFκB signaling cascade. CONCLUSION: In the present study, we explored the molecular mechanism of Arg prohibiting the metastasis of lung cancer cells in vitro and in vivo, which displayed Arg could target IKKß to inactive NFκB signaling cascade and further change the expression of proteins related to EMT. These results highlight the potential of toad venom as a potential chemotherapeutic agent and warrant its development as the clinical therapy for lung cancer.

Phytochemicals as potential IKK-ß inhibitor for the treatment of cardiovascular diseases in plant preservation: terpenoids, alkaloids, and quinones.

Modulation of inhibitor kappa B kinase-beta (IKK-ß) kinase activity could be useful for preventing inflammation that serves an efficient role in protection against cardiovascular diseases (CVDs). IKK-ß induces inflammation by activating transcription factor NF-kappa B (NF-κB) through phosphorylation of IκB. Therefore, IKK-ß is considered an interesting target for protecting and treating CVDs. The cardioprotective potential of terpenoids, alkaloids and quinines may be related to modulating inflammatory responses. In this study, the interactions between different classes of inhibitors and IKK-ß were investigated, through the application of SystemsDock. Molecular docking results showed that Diosgenin and Ginsenoside Re were the top docking score compounds. Diosgenin and Ginsenoside Re are the most promising IKK-ß inhibitors in terpenoids, alkaloids, and quinones. Diosgenin and Ginsenoside Re could be helpful to find the lead compounds on designing and developing novel cardioprotective agents.

Olanzapine-induced endoplasmic reticulum stress and inflammation in the hypothalamus were inhibited by an ER stress inhibitor 4-phenylbutyrate.

Antipsychotics are the most important treatment for schizophrenia. However, antipsychotics, particularly olanzapine and clozapine, are associated with severe weight gain/obesity side-effects. Although numerous studies have been carried out to identify the exact mechanisms of antipsychotic-induced weight gain, it is still important to consider other pathways. Endoplasmic reticulum (ER) stress signaling and its associated inflammation pathway is one of the most important pathways involved in regulation of energy balance. In the present study, we examined the role of hypothalamic protein kinase R like endoplasmic reticulum kinase- eukaryotic initiation factor 2α (PERK-eIF2α) signaling and the inflammatory IkappaB kinase ß- nuclear factor kappa B (IKKß-NFκB) signaling pathway in olanzapine-induced weight gain in female rats. In this study, we found that olanzapine significantly activated PERK-eIF2α and IKKß-NFκB signaling in SH-SY5Y cells in a dose-dependent manner. Olanzapine treatment for 8 days in rats was associated with activated PERK-eIF2α signaling and IKKß-NFκB signaling in the hypothalamus, accompanied by increased food intake and weight gain. Co-treatment with an ER stress inhibitor, 4-phenylbutyrate (4-PBA), decreased olanzapine-induced food intake and weight gain in a dose- and time-dependent manner. Moreover, 4-PBA dose-dependently inhibited olanzapine-induced activated PERK-eIF2α and IKKß-NFκB signaling in the hypothalamus. These results suggested that hypothalamic ER stress may play an important role in antipsychotic-induced weight gain.

Astrocytic Process Plasticity and IKKß/NF-κB in Central Control of Blood Glucose, Blood Pressure, and Body Weight.

Central regulation of metabolic physiology is mediated critically through neuronal functions; however, whether astrocytes are also essential remains unclear. Here we show that the high-order processes of astrocytes in the mediobasal hypothalamus displayed shortening in fasting and elongation in fed status. Chronic overnutrition and astrocytic IKKß/NF-κB upregulation similarly impaired astrocytic plasticity, leading to sustained shortening of high-order processes. In physiology, astrocytic IKKß/NF-κB upregulation resulted in early-onset effects, including glucose intolerance and blood pressure rise, and late-onset effects, including body weight and fat gain. Appropriate inhibition in astrocytic IKKß/NF-κB protected against chronic overnutrition impairing astrocytic plasticity and these physiological functions. Mechanistically, astrocytic regulation of hypothalamic extracellular GABA level and therefore BDNF expression were found partly accountable. Hence, astrocytic process plasticity and IKKß/NF-κB play significant roles in central control of blood glucose, blood pressure, and body weight as well as the central induction of these physiological disorders leading to disease.

Astrocyte IKKß/NF-κB signaling is required for diet-induced obesity and hypothalamic inflammation.

OBJECTIVE: Obesity and high fat diet (HFD) consumption in rodents is associated with hypothalamic inflammation and reactive gliosis. While neuronal inflammation promotes HFD-induced metabolic dysfunction, the role of astrocyte activation in susceptibility to hypothalamic inflammation and diet-induced obesity (DIO) remains uncertain. METHODS: Metabolic phenotyping, immunohistochemical analyses, and biochemical analyses were performed on HFD-fed mice with a tamoxifen-inducible astrocyte-specific knockout of IKKß (GfapCreERIkbkbfl/fl, IKKß-AKO), an essential cofactor of NF-κB-mediated inflammation. RESULTS: IKKß-AKO mice with tamoxifen-induced IKKß deletion prior to HFD exposure showed equivalent HFD-induced weight gain and glucose intolerance as Ikbkbfl/fl littermate controls. In GfapCreERTdTomato marker mice treated using the same protocol, minimal Cre-mediated recombination was observed in the mediobasal hypothalamus (MBH). By contrast, mice pretreated with 6 weeks of HFD exposure prior to tamoxifen administration showed substantially increased recombination throughout the MBH. Remarkably, this treatment approach protected IKKß-AKO mice from further weight gain through an immediate reduction of food intake and increase of energy expenditure. Astrocyte IKKß deletion after HFD exposure-but not before-also reduced glucose intolerance and insulin resistance, likely as a consequence of lower adiposity. Finally, both hypothalamic inflammation and astrocytosis were reduced in HFD-fed IKKß-AKO mice. CONCLUSIONS: These data support a requirement for astrocytic inflammatory signaling in HFD-induced hyperphagia and DIO susceptibility that may provide a novel target for obesity therapeutics.

A Polyphenol-rich Pomegranate Fruit Extract Suppresses NF-κB and IL-6 Expression by Blocking the Activation of IKKß and NIK in Primary Human Chondrocytes.

Pomegranate fruit extract (PE) rich in polyphenols has been shown to exert chondroprotective effects, but the mechanism is not established. Here, we used an in vitro model of inflammation in osteoarthritis (OA) to investigate the potential of PE to suppress interleukin 1 beta (IL-1ß)-stimulated expression of inflammatory cytokine IL-6, generation of reactive oxygen species (ROS) levels, and investigated the mechanism of NF-κB inhibition by analyzing the activation of the kinases upstream of IκBα in primary human chondrocytes. Total and phosphorylated forms of kinases and expression of IL-6 were determined at protein and mRNA levels by western immunoblotting and Taqman assay, respectively. Dihydrorhodamine 123 staining estimated ROS generation. Pomegranate fruit extract inhibited the mRNA and protein expression of IL-6, generation of ROS, and inhibited the IL-1ß-mediated phosphorylation of inhibitor of nuclear factor kappa-B kinase subunit beta (IKKß), expression of IKKß mRNA, degradation of IκBα, and activation and nuclear translocation of NF-κB/p65 in human chondrocytes. Importantly, phosphorylation of NF-κB-inducing kinase was blocked by PE in IL-1ß-treated human OA chondrocytes. Taken together, these data suggest that PE exerts the chondroprotective effect(s) by suppressing the production of IL-6 and ROS levels. Inhibition of NF-κB activation by PE was blocked via modulation of activation of upstream kinases in human OA chondrocytes. Copyright © 2017 John Wiley & Sons, Ltd.

Dihydromyricetin suppresses inflammatory responses in vitro and in vivo through inhibition of IKKß activity in macrophages.

Dihydromyricetin (DMY) a flavonoid derived from medicinal plant Ampelopsis grossedentata, possesses anti-oxidative and anti-inflammatory effects in vitro, however, the in vivo anti-inflammatory action of DMY remains unknown. In the current study, carrageenan-induced paw edema in rat, an acute inflammation model, and RAW264.7 macrophages activated by LPS were employed to evaluate the anti-inflammatory potency of DMY in vivo and in vitro. Results showed that DMY significantly attenuated rat paw edema induced by carrageenan. Also, DMY markedly inhibited NO secretion, iNOS, and COX-2 protein expression, as well as p65 phosphorylation via suppression of IKKß activity and IKKα/ß phosphorylation in RAW264.7 cells. And using high resolution Atomic Force Microscope (AFM), we also proved that DMY prevented morphological change and membrane alterations of RAW 264.7 macrophages caused by LPS stimulation. As activation of macrophages is one of major factors in carrageenan-induced paw edema of rats, the anti-inflammatory action of DMY is suggested to be closely associated with suppression of macrophage activation. These findings indicate that DMY is valuable of being further investigated as a candidate new agent for treating inflammatory conditions, and suggest that AFM could be a powerful nanotool for anti-inflammatory investigations. SCANNING 38:901-912, 2016. © 2016 Wiley Periodicals, Inc.