Protective Effects of Repetitive Transcranial Magnetic Stimulation Against Streptozotocin-Induced Alzheimer's Disease.

Repetitive transcranial magnetic stimulation (rTMS) is a non-invasive brain stimulation under investigation for treatment of a wide range of neurological disorders. In particular, the therapeutic application of rTMS for neurodegenerative diseases such as Alzheimer's disease (AD) is attracting attention. However, the mechanisms underlying the therapeutic efficacy of rTMS have not yet been elucidated, and few studies have systematically analyzed the stimulation parameters. In this study, we found that treatment with rTMS contributed to restoration of memory deficits by activating genes involved in synaptic plasticity and long-term memory. We evaluated changes in several intracellular signaling pathways in response to rTMS stimulation; rTMS treatment activated STAT, MAPK, Akt/p70S6K, and CREB signaling. We also systematically investigated the influence of rTMS parameters. We found an effective range of applications for rTMS and determined the optimal combination to achieve the highest efficiency. Moreover, application of rTMS inhibited the increase in cell death induced by hydrogen peroxide. These results suggest that rTMS treatment exerts a neuroprotective effect on cellular damage induced by oxidative stress, which plays an important role in the pathogenesis of neurological disorders. rTMS treatment attenuated streptozotocin (STZ)-mediated cell death and AD-like pathology in neuronal cells. In an animal model of sporadic AD caused by intracerebroventricular STZ injection, rTMS application improved cognitive decline and showed neuroprotective effects on hippocampal histology. Overall, this study will help in the design of stimulation protocols for rTMS application and presents a novel mechanism that may explain the therapeutic effects of rTMS in neurodegenerative diseases, including AD.

Anti-inflammatory effects of Allium cepa L. peel extracts via inhibition of JAK-STAT pathway in LPS-stimulated RAW264.7 cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Allium cepa L. (A. cepa) is one of the oldest cultivated plants in the world. A. cepa has been used in traditional folk medicine to treat inflammatory disease in several regions, such as Palestine and Serbia. A. cepa peel has a higher content of flavonoids, such as quercetin, than the edible parts. These flavonoids alleviate inflammatory diseases. However, the anti-inflammatory effects of A. cepa peel extract-obtained using various extraction methods-and their underlying mechanisms require further investigation. AIM OF THE STUDY: Although research to find safe anti-inflammatory substances in various natural products has been actively conducted for many years, it is important to continue identifying potential anti-inflammatory effects in natural materials. The purpose of this study was to investigate the ethnopharmacological properties of the A. cepa peel extract, whose efficacy when obtained through different extraction methods and underlying action mechanisms is not well known. The present study specifically aimed to observe the anti-inflammatory effects of the A. cepa peel extracts obtained using various extraction methods and the related detailed mechanisms of A. cepa peel extracts in lipopolysaccharide (LPS)-induced RAW264.7 cells. MATERIALS AND METHODS: The total flavonoid content of the A. cepa peel extracts was determined the diethylene glycol colorimetric method and measured using a calibration curve prepared using quercetin as a standard solution. The antioxidant activity was evaluated using the ABTS assay, and cytotoxicity was measured using the MTT assay. NO production was measured using Griess reagent. Protein levels were measured by western blotting, and mRNA expression was measured by RT-qPCR. Secreted cytokines were analyzed using ELISA or cytokine arrays. In the GSE160086 dataset, we calculated Z-scores for individual genes of interest and displayed using a heat map. RESULTS: Of the three A. cepa peel extracts obtained using different extraction methods, the A. cepa peel 50% EtOH extract (AP50E) was the most effective at inhibiting LPS-induced nitric oxide (NO) and inducible nitric oxide synthase (iNOS). Furthermore, AP50E significantly reduced the levels of pro-inflammation cytokines interleukin (IL)-1α, IL-1ß, IL-6, and IL-27. Additionally, AP50E directly inhibited the Janus kinase-signaling transducer and activator of transcription (JAK-STAT) pathway. CONCLUSIONS: These results showed that AP50E exhibited an anti-inflammatory effect in LPS-induced RAW264.7 mouse macrophages by directly inhibiting JAK-STAT signaling. Based on these findings, we propose AP50E as a potential candidate for the development of preventive or therapeutic agents against inflammatory diseases.

Pharmacological anti-tumor effects of natural Chamaecyparis obtusa (siebold & zucc.) endl. Leaf extracts on breast cancer.

ETHNOPHARMACOLOGICAL RELEVANCE: Chamaecyparis obtusa (C. obtusa, cypress species) is a plant that grows mainly in the temperate Northern Hemisphere and has long been used as a traditional anti-inflammatory treatment in East Asia. C. obtusa contains phytoncides, flavonoids, and terpenes, which have excellent anti-cancer effects and have been reported to prevent the progression of various cancers. However, the detailed mechanisms underlying the anti-cancer effects of C. obtusa extracts are unknown. AIM OF THE STUDY: We sought to confirm the anti-cancer effects of C. obtusa leaf extracts and to reveal the mechanism of action, with the possibility of its application in the treatment or prevention of cancer. MATERIAL &METHODS: The cytotoxicity of C. obtusa leaf extracts was confirmed using an MTT assay. Intracellular changes in protein levels were measured by immunoblotting, and mRNA levels were measured with qRT-PCR. Wound healing assay and transwell migration assay were used to evaluate the metastatic potential of breast cancer cells. The extract-induced apoptosis was observed using IncuCyte Annexin V Red staining analysis. A syngeneic breast cancer mouse model was established by injecting 4T1-Luc mouse breast cancer cells into the fat pad of female BALB/c mice, and the extract was administered orally. Luciferin solution was injected intraperitoneally to assess primary tumor development and metastasis by bioluminescence. RESULTS: C. obtusa leaf extracts were extracted with boiling water, 70% EtOH, and 99% EtOH. Among the extracts, the 99% EtOH extract of C. obtusa leaf (CO99EL) most clearly inhibited the tyrosine phosphorylation of Signal Transducer and Activator of Transcription 3 (pY-STAT3) in MDA-MB-231 breast cancer cells at a concentration of 25 and 50 µg/mL. In addition, CO99EL strongly inhibited not only endogenous pY-STAT3 levels but also IL-6-induced STAT3 activation in various types of cancer cells, including breast cancer. CO99EL inhibited metastatic potential by downregulating the expression of N-cadherin, fibronectin, TWIST, MMP2, and MMP9 in MDA-MB-231 breast cancer cells. CO99EL also induced apoptotic cell death by increasing cleaved caspase-3 and decreasing anti-apoptotic proteins Bcl-2 and Bcl-xL. In an in vivo syngeneic breast cancer mouse model, 100 mg/kg CO99EL suppressed tumor growth and induced apoptosis of cancer cells. Moreover, CO99EL significantly inhibited lung metastasis from primary breast cancer. CONCLUSIONS: Our study demonstrated that 100 mg/kg CO99EL has potent anti-tumor effects against breast cancer, thus suggesting that 100 mg/kg CO99EL has potential applications in the treatment and prevention of breast cancer.

Gochujang Ameliorates Hepatic Inflammation by Improving Dysbiosis of Gut Microbiota in High-Fat Diet-Induced Obese Mice.

Abnormal fat accumulation with gut microbiota dysbiosis results in hepatic inflammation by upregulating the release of lipopolysaccharide (LPS) and inflammatory cytokine. Gochujang, a traditional fermented condiment, has beneficial effects, such as anti-colonic inflammatory effects. However, Gochujang has been controversial because of its high salt content (the Korean Paradox). Thus, the present study aimed to investigate the preventative effects of Gochujang on hepatic inflammation and related gut microbiota through discussing the Korean Paradox. The mice were divided into groups including a normal diet (ND), high-fat diet (HD), HD with salt (SALT), HD with a high percentage of beneficial microbiota Gochujang (HBM), and HD with diverse beneficial microbiota Gochujang (DBM). Gochujang markedly reduced lipid accumulation, hepatic injury, and inflammation response. Furthermore, Gochujang attenuated protein expression involved in the JNK/IκB/NF-κB pathway. Additionally, Gochujang regulated the gut microbiota-derived LPS production and Firmicutes/Bacteroidetes ratio. Gochujang regulated the levels of gut microbiota such as Bacteroides, Muribaculum, Lactobacillus, and Enterorhabdus, which were correlated with hepatic inflammation. Salt did not have foregoing effects, meaning that the salt content in Gochujang did not affect its anti-inflammatory effect. In conclusion, Gochujang showed anti-hepatic inflammation effects via reduced lipid accumulation, hepatic injury, and inflammatory response together with reorganization of gut microbiota dysbiosis regardless of salt content and the difference of micro bacteria composition.

Loss of phospholipase Cγ1 suppresses hepatocellular carcinogenesis through blockade of STAT3-mediated cancer development.

Phospholipase C gamma 1 (PLCγ1) plays an oncogenic role in several cancers, alongside its usual physiological roles. Despite studies aimed at identifying the effect of PLCγ1 on tumors, the pathogenic role of PLCγ1 in the tumorigenesis and development of hepatocellular carcinoma (HCC) remains unknown. To investigate the function of PLCγ1 in HCC, we generated hepatocyte-specific PLCγ1 conditional knockout (PLCγ1f/f ; Alb-Cre) mice and induced HCC with diethylnitrosamine (DEN). Here, we identified that hepatocyte-specific PLCγ1 deletion effectively prevented DEN-induced HCC in mice. PLCγ1f/f ; Alb-Cre mice showed reduced tumor burden and tumor progression, as well as a decreased incidence of HCC and less marked proliferative and inflammatory responses. We also showed that oncogenic phenotypes such as repressed apoptosis, and promoted proliferation, cell cycle progression and migration, were induced by PLCγ1. In terms of molecular mechanism, PLCγ1 regulated the activation of signal transducer and activator of transcription 3 (STAT3) signaling. Moreover, PLCγ1 expression is elevated in human HCC and correlates with a poor prognosis in patients with HCC. Our results suggest that PLCγ1 promotes the pathogenic progression of HCC, and PLCγ1/STAT3 axis was identified as a potential therapeutic target pathway for HCC.

The acidic tumor microenvironment enhances PD-L1 expression via activation of STAT3 in MDA-MB-231 breast cancer cells.

Tumor acidosis, a common phenomenon in solid cancers such as breast cancer, is caused by the abnormal metabolism of cancer cells. The low pH affects cells surrounding the cancer, and tumor acidosis has been shown to inhibit the activity of immune cells. Despite many previous studies, the immune surveillance mechanisms are not fully understood. We found that the expression of PD-L1 was significantly increased under conditions of extracellular acidosis in MDA-MB-231 cells. We also confirmed that the increased expression of PD-L1 mediated by extracellular acidosis was decreased when the pH was raised to the normal range. Gene set enrichment analysis (GSEA) of public breast cancer patient databases showed that PD-L1 expression was also highly correlated with IL-6/JAK/STAT3 signaling. Surprisingly, the expression of both phospho-tyrosine STAT3 and PD-L1 was significantly increased under conditions of extracellular acidosis, and inhibition of STAT3 did not increase the expression of PD-L1 even under acidic conditions in MDA-MB-231 cells. Based on these results, we suggest that the expression of PD-L1 is increased by tumor acidosis via activation of STAT3 in MDA-MB-231 cells.

Chamaecyparis obtusa (Siebold & Zucc.) Endl. leaf extracts prevent inflammatory responses via inhibition of the JAK/STAT axis in RAW264.7 cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Chamaecyparis obtusa (Siebold & Zucc.) Endl. (C. obtusa) has been used as folk medicine in East Asia and has been reported to alleviate inflammatory diseases. However, the detailed mechanisms for the anti-inflammatory effects of C. obtusa remain unclear. AIM OF THE STUDY: Although the anti-inflammatory mechanisms of natural products have been studied for decades, it is still important to identify the potential anti-inflammatory effects of natural sources. In this study, we investigated the anti-inflammatory effects and underlying mechanism of C. obtusa leaf extracts. MATERIAL &METHODS: The cell viability was determined by MTT and crystal violet staining. NO production in the supernatant was measured using Griess reagent. The cell lysates were analyzed by immunoblotting and RT-qPCR. Secreted cytokines were analyzed using ELISA kit and cytokine array kit. mRNA expression from the GSE9632 database set. Z-scores were calculated for each gene and visualized by heat map. RESULTS: Among the extracts of C. obtusa obtained with different extraction methods, the 99% ethanol leaf extract (CO99EL) strongly inhibited lipopolysaccharide (LPS)-induced inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression and Janus kinase/signaling transducer and activator of transcription (JAK/STAT) phosphorylation in RAW264.7 cells. In addition, CO99EL strongly inhibited LPS-induced interleukin (IL)-1ß, IL-6, IL-27, and C-C motif chemokine ligand (CCL)-1 production and directly inhibited LPS-induced JAK/STAT phosphorylation in RAW264.7 cells. CONCLUSIONS: These findings demonstrate that CO99EL significantly prevents LPS-induced macrophage activation by inhibiting the JAK/STAT axis. Therefore, we suggest the use of C. obtusa extracts as therapeutic approach for inflammatory diseases.

Extracellular Acidosis Promotes Metastatic Potency via Decrease of the BMAL1 Circadian Clock Gene in Breast Cancer.

Circadian oscillation is an essential process that influences many physiological and biological mechanisms and a decrease of circadian genes is associated with many diseases such as cancer. Despite many efforts to identify the detailed mechanism for decreasing circadian genes and recovering reduced circadian genes in cancer, it is still largely unknown. We found that BMAL1 was reduced in tumor hypoxia-induced acidosis, and recovered by selectively targeting acidic pH in breast cancer cell lines. Surprisingly, BMAL1 was reduced by decrease of protein stability as well as inhibition of transcription under acidosis. In addition, melatonin significantly prevented acidosis-mediated decrease of BMAL1 by inhibiting lactate dehydrogenase-A during hypoxia. Remarkably, acidosis-mediated metastasis was significantly alleviated by BMAL1 overexpression in breast cancer cells. We therefore suggest that tumor hypoxia-induced acidosis promotes metastatic potency by decreasing BMAL1, and that tumor acidosis could be a target for preventing breast cancer metastasis by sustaining BMAL1.

T Cell-Specific Knockout of STAT3 Ameliorates Dextran Sulfate Sodium-Induced Colitis by Reducing the Inflammatory Response.

Signal transducer and activator of transcription 3 (STAT3) has a crucial role in various autoimmune disorders including, inflammatory bowel disease (IBD). Our previous study demonstrated that STAT3 activation by IL-6 in colonic epithelial cells exacerbates experimental ulcerative colitis. Activated T lymphocytes are also found in ulcerative colitis patients with intestinal inflammation, but the role of STAT3 in T cells remains elusive. To determine the STAT3 function of T cells in intestinal inflammation, we generated T cell-specific STAT3 knockout (KO) mice and used dextran sulfate sodium (DSS) to induce colitis. In this study, we demonstrated that T cell-specific STAT3 deletion alleviated DSS-induced colitis in mice, resulting in reduced histological scores and myeloperoxidase (MPO) activity. Importantly, the population of T cells in the spleen and lymph nodes was significantly decreased in the control and DSS-induced groups of STAT3 KO mice. In addition, STAT3 deficiency in T cells markedly reduced the production of interferon (IFN)-γ, IL-6, and IL-17A, whereas IL-10 secretion was increased. Collectively, the results suggest that STAT3 in T cells may be a therapeutic target in ulcerative colitis by balancing the immune response through T cell homeostasis.