Duration of the preemptive analgesic effects of low- and high-frequency transcutaneous electrical nerve stimulation in rats with acute inflammatory pain.

Transcutaneous electrical nerve stimulation (TENS) activates various pathways to induce antinociceptive effects, based on the frequencies used. This study evaluates the preemptive analgesic effects and their duration of low- (LT: 4 Hz) and high-frequency TENS (HT: 100 Hz) using a rat model of acute inflammatory pain. Acute inflammation was induced by injecting 1% formalin into the hind paws of rats. LT or HT was applied for 30 min before formalin injection. Pain-related behaviors, such as licking, flinching, and lifting, were recorded for 60 min postinjection. Immunohistochemistry was used to assess the number of phosphorylated extracellular signal-regulated kinase (pERK)- and c-fos-positive cells in the spinal cord. Naloxone, a µ-opioid receptors (MORs) antagonist, and naltrindole, a δ-opioid receptors (DORs) antagonist, were administered before TENS application. Pain behavior duration and pERK- and c-fos-positive cell expression were then measured. LT and HT pretreatment significantly reduced both pain behaviors and the number of pERK- and c-fos-positive cells postformalin injection. Naloxone and naltrindole partially reversed the effects of LT and HT, respectively. Notably, HT's analgesic effect lasted up to 120 min whereas that of LT persisted for 90 min. LT and HT effectively exerted their preemptive analgesic effects on acute inflammatory pain by inhibiting pERK and c-fos expression in the spinal cord. HT presented a longer-lasting effect compared to LT. MOR and DOR activation may contribute to LT and HT's analgesic mechanisms, respectively.

Influences of Lavender Essential Oil Inhalation on Stress Responses during Short-Duration Sleep Cycles: A Pilot Study.

Lavender essential oil (LEO) was reported to improve sleep quality. We investigated the influence of aromatherapy by testing the effects of LEO on stress responses during a short-duration sleep in a single-blind, randomized, crossover trial. The subjects were twelve healthy adults who were nonsmokers without any known disease and who were not prescribed medications, and nine of these completed the study. After the subjects had fallen asleep, they were sprayed with LEO using an aroma diffuser. Before and after 90 min of sleep, α-amylase, chromogranin A (CgA), and cortisol levels in saliva were measured as objective stress indicators, and the Japanese version of the UWIST Mood Adjective Checklist was used as a subjective indicator. A comparison of changes before and after sleep, with and without LEO, revealed that the cortisol level did not significantly change; however, α-amylase (p < 0.05) and CgA (p < 0.01) levels significantly decreased after LEO inhalation. A mood test indicated no change in mood before and after sleep, with or without LEO. Since α-amylase and CgA reflect the sympathetic nervous system response, these results indicate that LEO aromatherapy during a short-duration sleep cycle suppresses the stress response, especially that of the sympathetic nervous system.

Synovial fibroblast-derived exosomal microRNA-106b suppresses chondrocyte proliferation and migration in rheumatoid arthritis via down-regulation of PDK4.

Fibroblast-derived exosomes have been reported to transfer microRNAs to recipient cells, where they regulate target gene expression, which is of interest for understanding the basic biology of inflammation, tissue homeostasis, and development of therapeutic approaches. Initial microarray-based analysis carried out in this study identified the rheumatoid arthritis (RA)-related differentially expressed gene pyruvate dehydrogenase kinase 4 (PDK4). Subsequently, the upstream regulatory microRNA-106b (miR-106b) of PDK4 was predicted with bioinformatic analyses. A collagen-induced arthritis (CIA)-induced mouse model was established, and exosomes were isolated from synovial fibroblasts (SFs) and transferred into chondrocytes to identify the role of exosomes in rheumatoid arthritis (RA). We found that PDK4 was poorly expressed in RA cartilage tissues and chondrocytes, while miR-106b was highly expressed in RA SFs and SF-derived exosomes. Notably, PDK4 was confirmed as a target gene of miR-106b. Over-expression of PDK4 promoted the proliferation and migration abilities of chondrocytes and inhibited their apoptosis as well as affected the receptor activator of nuclear factor kappa B ligand (RANKL)/RANK/osteoprotegerin (OPG) system. Meanwhile, miR-106b was delivered from SFs to chondrocytes through exosomes, which suppressed chondrocyte proliferation and migration and accelerated apoptosis as well as affected the RANKL/RANK/OPG system via down-regulation of PDK4. Furthermore, in vivo results validated that miR-106b inhibition could relieve CIA-induced RA. Taken together, SF-derived exosomal miR-106b stimulates RA initiation by targeting PDK4, indicating a physiologically validated potential approach for the prevention and treatment of RA. KEY MESSAGES: PDK4 is decreased in chondrocytes of RA, while miR-106b is increased in SFBs. PDK4 promotes proliferation and migration of chondrocytes. miR-106b could target 3'UTR of PDK4 gene. SFB-exosomal miR-106b inhibits proliferation and migration of chondrocytes. Inhibition of miR-106b attenuates RA progression in a CIA mouse model.

Kampo (Traditional Japanese Herbal) Formulae for Treatment of Stomatitis and Oral Mucositis.

Stomatitis is occasionally multiple, recurrent, and refractory. Currently, mucositis induced by chemotherapy and radiation therapy in patients with cancer has become a significant clinical problem. Effective treatments have not been established and the treatment of numerous cases remains a challenge for physicians. Traditional Japanese herbal medicines termed Kampo formulae (i.e., Hangeshashinto, Orengedokuto, Inchinkoto, Orento, Byakkokaninjinto, Juzentaihoto, Hochuekkito, and Shosaikoto) are used for treating various types of stomatitis and mucositis. Its use has been based on the Kampo medical theories-empirical rules established over thousands of years. However, recently, clinical and basic research studies investigating these formulae have been conducted to obtain scientific evidence. Clinical studies investigating efficacies of Shosaikoto and Orento for the treatment of cryptogenic stomatitis and acute aphthous stomatitis and those investigating the effects of Hangeshashinto, Orengedokuto, and Juzentaihoto on chemotherapy- or radiotherapy-induced mucositis have been conducted. The Kampo formulae comprise several crude drugs, whose mechanisms of action are gradually being clarified. Most of these drugs that are used for the treatment of stomatitis possess anti-inflammatory, analgesic, and antioxidative properties. In this review, we introduce the clinical applications and summarize the available evidence on the Kampo formulae for the treatment of stomatitis and oral mucositis.

Yokukansan (Kampo medicinal formula) prevents the development of morphine tolerance by inhibiting the secretion of orexin A.

BACKGROUND: Yokukansan (YKS), a traditional herbal (Kampo) medicine consisting of seven herbs, is effective in the treatment of pain disorders, such as headache, postherpetic neuralgia, fibromyalgia, and trigeminal neuralgia, and we have previously shown it to be effective against morphine analgesic tolerance in rats. It has been reported that orexin receptor antagonists prevent the development of morphine tolerance and that YKS inhibits the secretion of orexin A in the hypothalamus. This study examined whether the inhibition of the secretion of orexin A by YKS is one mechanism underlying its effect against morphine analgesic tolerance. METHODS: Male Wistar rats were administered a subcutaneous injection of morphine hydrochloride (10 mg/kg/day) for 5 days. One group was preadministered YKS, starting 3 days before the morphine. The withdrawal latency following thermal stimulation was measured daily using a hot plate test. On day 5, the levels of orexin A in the plasma and the midbrain were measured, and the appearance of activated astrocytes in the midbrain was examined by immunofluorescence staining. RESULTS: The preadministration of YKS prevented the development of morphine tolerance. The repeated administration of morphine significantly increased the plasma and midbrain levels of orexin A and the activation of astrocytes. These increases were significantly inhibited by the preadministration of YKS. CONCLUSION: These results suggest that the preadministration of YKS attenuated the development of antinociceptive morphine tolerance and that the inhibition of orexin A secretion may be one mechanism underlying this phenomenon.

Celastrus orbiculatus extracts induce cell cycle arrest and apoptosis in human esophageal squamous carcinoma ECA-109 cells in vitro via the PI3K/AKT/mTOR signaling pathway.

Recently, Celastrus orbiculatus ethyl acetate extracts (COE) have been investigated for their anticancer effects on digestive tract tumors. However, the therapeutic effects of COE on esophageal squamous carcinoma cells (ESCC) have not been investigated. In the present study, the effects of COE on the cell cycle and apoptosis of ESCCs were assessed in vitro, and it was revealed that COE treatment triggered G0/G1 cell cycle arrest, and induced DNA damage and apoptosis in a dose-dependent manner in ESCC. Activation of the phosphatidylinositol 3-kinase/protein kinase-B/mechanistic target of rapamycin (mTOR) pathway was also suppressed by COE. Additionally, the combined treatment with COE and rapamycin (an mTOR inhibitor) acted synergistically in ECA-109 cells compared with the treatment with COE or rapamycin alone. These findings extend the understanding of the action of COE and suggest that COE has potential as a treatment option for ESCC as a single treatment or in combination.

Time-dependent role of prefrontal cortex and hippocampus on cognitive improvement by aripiprazole in olfactory bulbectomized mice.

Dopamine (DA) modulates cognitive functions in the prefrontal cortex (PFC) and hippocampus. Olfactory bulbectomy (OBX) in mice induces cognitive dysfunctions. Recently, we reported that aripiprazole (ARI) normalizes the behavioral hyper-responsivity to DA agonists in OBX mice. However, it remains unclear whether ARI affects OBX-induced cognitive dysfunctions. To address this question we evaluated ARI-treated and untreated OBX mice in a passive avoidance test. Then, we investigated the effects of ARI on cell proliferation in the hippocampal dentate gyrus by immunohistochemistry, and on c-fos levels in the PFC and hippocampus, as well as nerve growth factor (NGF) levels in the hippocampus by western blotting. On the 14th day after surgery OBX mice showed an alteration in passive avoidance and decreases in both cell proliferation and levels of p-ERK, p-CREB and NGF in the hippocampus. The cognitive dysfunctions in OBX mice improved 30min to 24h after the administration of ARI (0.01mg/kg). C-fos levels in the PFC but not in the hippocampus was increased 30min after the administration (early response). This early response was inhibited by the selective D1 receptor antagonist SCH23390. Cell proliferation and NGF levels in the hippocampus increased 24h after ARI administration (late response), and these effects were also inhibited by SCH23390. The MEK1/2 inhibitor U0126 prevented ARI from improving the behavioral impairment as well as enhancing NGF levels in OBX mice. These findings revealed the potential of ARI to improve cognitive dysfunctions via D1 receptors with the PFC and hippocampus being affected sequentially.

Anticancer efficacy of the ethyl acetate extract from the traditional Chinese medicine herb Celastrus orbiculatus against human gastric cancer.

ETHNOPHARMACOLOGICAL RELEVANCE: The traditional Chinese medicine (TCM) herb Celastrus orbiculatus is an important folk medicinal plant in China that has been used as an anti-inflammatory, antitumor, and analgesic in various diseases. The ethyl acetate extract of C. orbiculatus (C. orbiculatus extract, COE) was reported to show significant antitumor effects. However, no study in China or abroad has reported the effect and mechanism of COE in triggering apoptosis of gastric cancer (GC) cells. AIM OF STUDY: To further uncover the molecular mechanism underlying COE's apoptotic and anti-proliferative effects and lay a foundation for the development of novel, effective antitumor TCM agents. MATERIALS AND METHODS: The effect of COE on AGS and BGC-823 GC cell viability was examined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Apoptosis of AGS and BGC-823 cells induced by COE was analyzed using flow cytometry and a mitochondrial membrane potential assay kit (JC-1). The proliferating GC cells were identified and examined using a 5-bromo-2'-deoxyuridine (BrdU) staining kit and flow cytometric analysis. A western blot assay was used to detect the effect of COE on apoptosis-related proteins, B-cell lymphoma-2 (Bcl-2), Bcl-extra-large (Bcl-xL), Bcl-2-like protein 12 (Bcl-L12), Bcl-2-associated X protein (Bax), and caspase as well as proliferation-related proteins, phosphoinositide 3-kinase (PI3K)/Akt/mechanistic target of rapamycin (mTOR)/p70s6k. Transmission electron microscopy (TEM) and an animal imaging technique were used to evaluate the microstructure of apoptotic GC cells and the effect of COE on tumor cell growth in vivo, respectively. RESULTS: The results indicate that COE significantly inhibited proliferation and induced apoptosis of GC AGS and BGC-823 cell lines both in vivo and in vitro. COE significantly decreased the cell mitochondrial membrane potential. Moreover, COE downregulated the levels of Bcl-2, Bcl-xL, and PI3K/Akt/mTOR/p70s6k while those of Bax and caspase were upregulated. More interestingly, COE altered the microstructure of the mitochondria. CONCLUSION: All these data collectively indicate that COE not only has significant antiproliferative effects but also has both in vivo and in vitro apoptotic effects. In addition, COE altered the structure and function of the mitochondria, which is another potential pathway for the antitumor activity of COE. These findings may provide a basis for the development of new anticancer TCM candidates.

Efficacy of Juzentaihoto for Tumor Immunotherapy in B16 Melanoma Metastasis Model.

Introduction. Medical care for Japanese cancer patients includes Western and Kampo medicines, and treatments with juzentaihoto (JTT) reportedly prevent cancer metastasis and recurrence. In this study, we examined the effects of JTT on natural killer (NK) cell activity and metastasis in combined treatments with anti-PD-1 antibody in a mouse model of melanoma metastasis. Methods. C57BL/6 male mice were intravenously injected with B16 melanoma cells (B16 cell) and were given chow containing 3% JTT. In subsequent in vivo experiments, we assessed serum cytokine levels and tumor colony formation in the lungs. Additionally, we assessed NK cell activity in ex vivo experiments. Results. JTT significantly suppressed B16 cell metastasis, whereas injection of anti-asialo-GM1 antibody into mice abrogated the inhibitory actions of JTT. JTT significantly increased interleukin- (IL-) 12 and interferon- (IFN-) γ levels in serum and induced NK cell activity. It increased the inhibitory actions of the anti-PD-1 antibody on B16 cell metastasis. Discussion. These data suggest that JTT inhibits B16 cell metastasis by inducing NK cell activity. Additionally, combination therapy with JTT and anti-PD-1 antibody increased treatment response rates for B16 melanoma.

Influence of Mechanical Force on Bone Matrix Proteins in Ovariectomised Mice and Osteoblast-like MC3T3-E1 Cells.

AIM: To investigate the effect of mechanical stress on periostin and semaphorin-3A expression in a murine model of postmenopausal osteoporosis and in osteoblast-like MC3T3-E1 cells. MATERIALS AND METHODS: Female mice were divided into three groups and treated with a sham operation, ovariectomy (OVX) or OVX plus treadmill training (OVX+Run). After 10 weeks, tibias were used for histological analysis. MC3T3-E1 cells were burdened by mechanical stress using a centrifuge or were treated with periostin, and the production of biologically-active semaphorin-3A was examined in vitro. RESULTS: In OVX+Run group tibias, the number of tartrate-resistant acid phosphatase-positive osteoclasts was lower than in the OVX group, and the expression of periostin and semaphorin-3A was higher. In MC3T3-E1 cells, centrifugal stress significantly increased periostin and semaphorin-3A mRNA expression. Treatment with periostin increased the semaphorin-3A level. CONCLUSION: We speculate that mechanical load may increase periostin production in osteoblasts, and periostin may inhibit osteoclast differentiation by its effects on semaphorin-3A. Our results support the concept of a positive correlation between exercise and inhibition of osteoclasts in post-menopausal osteoporosis.

Notch1 promotes vasculogenic mimicry in hepatocellular carcinoma by inducing EMT signaling.

Hypervascularity is one of the main characteristics of hepatocellular carcinoma (HCC). However, the mechanisms of angiogenesis in HCC remain controversial. In this study, we investigate the role of Notch1 in angiogenesis of HCC. We found that Notch1 expression was correlated with formation of vasculogenic mimicry (VM) and expression of biomarkers of epithelial-to-mesenchymal transition (EMT) in the tumor specimens. Two HCC cell lines, HepG2 and MHCC97-H, with low and high Notch1 expression, respectively, were used to study the mechanism of VM formation both in vitro and in vivo. It was found that MHCC97-H cells, but not HepG2 cells form VM when they grow on matrigel in vitro. HepG2 cells gained the power of forming VM when they were overexpressed with Notch1, while knockdown Notch1 expression in MHCC97-H cells led to the loss of VM forming ability of the cells. Similar results were found in in vivo study. High expression of Notch1 in HepG2 promoted xenograft growth in nude mice, with abundant VM formation in the tumor samples. Moreover, we observed Notch1 was associated with the EMT and malignant behavior of hepatocellular carcinoma by analyzing clinical specimens, models for in vitro and in vivo experiments. HepG2 presented EMT phenomenon when induced by TGF-ß1, accompanied by Notch1 activation while MHCC97-H with knockdown of Notch1 lost the responsiveness to TGF-ß1 induction. Our results suggest that Notch1 promotes HCC progression through activating EMT pathway and forming VM. Our results will guide targeting Notch1 in new drug development.

Celastrus orbiculatus extract triggers apoptosis and autophagy via PI3K/Akt/mTOR inhibition in human colorectal cancer cells.

Celastrus orbiculatus is used as a folk medicine in China for the treatment of numerous diseases. The ethyl acetate extract of Celastrus orbiculatus (COE) also displays a wide range of anti-cancer activities in the laboratory. However, the effectiveness of COE-induced autophagy and its mechanism of action in colorectal cancer cells have not been investigated thus far. The present study analyzed the effect of COE on HT-29 cell viability, apoptosis and autophagy using MTT assay, flow cytometry, transmission electron microscopy and western blotting. Additionally, the autophagy inhibitor 3-methyladenine and the autophagy inducer rapamycin were used to further explore the effects of COE-induced autophagy in HT-29 cells. The present study also examined whether the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/Akt/mechanistic target of rapamycin (mTOR)/p70 ribosomal protein S6 kinase (p70S6K) signaling pathway was involved in the regulation of COE-induced autophagy. The results revealed that COE inhibited HT-29 cell proliferation and decreased cell survival in a time- and dose-dependent manner, and that COE possessed the ability to induce both apoptosis and autophagy in HT-29 cells. Furthermore, autophagy and apoptosis induced by COE synergized to inhibit colorectal cancer growth. In addition, COE treatment decreased the phosphorylation of Akt and its downstream effectors mTOR and p70S6K. Taken together, these results demonstrate that both autophagy and apoptosis were activated during COE treatment of HT-29 cells, and that COE-induced autophagy decreases the viability of HT-29 cells via a mechanism that may depend on the PI3K/Akt/mTOR/p70S6K signaling pathway. Furthermore, compounds that induce autophagy administered in combination with COE may be an attractive strategy for enhancing the anti-tumor potency of COE in colorectal cancer.

Quercetin-induced apoptosis of HT-29 colon cancer cells via inhibition of the Akt-CSN6-Myc signaling axis.

Constitutive photomorphogenesis 9 signalosome (CSN) consists of a total of eight subunits (CSN1-CSN8) in mammalian cells. CSN6 may promote carcinogenesis by positively regulating v­myc avian myelocytomatosis viral oncogene homolog (Myc) and MDM2 proto­oncogene stability, and is regarded as a potential target for cancer therapy. Quercetin has a substantial anticancer effect on various human cancer cells. The present study investigated the effects of quercetin on HT-29 human colorectal cancer cell viability, apoptosis and cell cycle arrest using an MTT assay, flow cytometry, transmission electron microscopy and western blotting. It was determined that quercetin inhibited HT­29 cell viability in a dose­dependent manner. Cell shrinkage, chromatin condensation and nuclear collapse were observed in the 50, 100 and 200 µM quercetin groups. The exposure of HT­29 cells to quercetin led to significant cell cycle arrest in the S­phase. Western blot analysis revealed that quercetin reduced the protein expression levels of phosphorylated-Akt and increased CSN6 protein degradation; therefore, affecting the expression levels of Myc, p53, B­cell lymphoma 2 (Bcl­2) and Bcl­2 associated X protein. The overexpression of CSN6 reduced the effect of quercetin treatment on HT­29 cells, suggesting that quercetin­induced apoptosis may involve the Akt­CSN6­Myc signaling axis in HT­29 cells.

Vasculogenic mimicry in hepatocellular carcinoma contributes to portal vein invasion.

Portal vein invasion (PVI) is common in hepatocellular carcinoma (HCC) and largely contributes to tumor recurrence after radical tumor resection or liver transplantation. Vasculogenic mimicry (VM) was an independent vascular system lined with tumor cells and associated with poor prognosis of HCC. The present study was conducted to evaluate the relationship between VM and portal vein invasion. A total of 44 HCC cases receiving anatomic liver resection were included in the study and were divided into groups with and without PVI. The prevalence of VM in each group was examined by CD34-PAS dual staining. The regulatory molecules of VM formation such as Notch1, Vimentin and matrix metalloproteinases (MMPs) were investigated by immunohistochemical staining. Analysis was performed to explore the association of PVI, VM and the VM regulatory molecules. PVI was found in 40.91% (18/44) cases and VM was found in 38.64% (17/44) cases in total samples. The incidence of VM was 72.22% (13/18) in PVI group while it was 15.38% (4/26) in non-PVI group (P<0.001), VM formation was positively correlated with PVI (r=0.574, P<0.001). The VM forming regulatory molecules such as Notch1, Vimentin, MMP-2 and MMP-9 were found to be correlated with PVI in HCC patients. Taken together, our results suggested that VM formation, alone with its regulatory molecules, is the promoting factor of PVI in hepatocellular carcinoma.

Influence of Palmatine on Bone Metabolism in Ovariectomized Mice and Cytokine Secretion of Osteoblasts.

BACKGROUND/AIM: Receptor activator of nuclear factor-κB ligand (RANKL) and osteoprotegerin (OPG), regulate the cognate receptor RANK on osteoclast precursor cells. Herein we examined the inhibitory effects of palmatine on bone metabolism using ovariectomized (OVX) mice. MATERIALS AND METHODS: The first experimentaI set was designed to histologically and biochemically examine mice randomly divided into four groups: sham-operated, OVX, and OVX-palmatine intake groups (1 mg/kg and 10 mg/kg). The second experimental set examined the influence of palmatine on osteoblast-like cells in vitro. RESULTS: Palmatine caused significant suppression of osteoclast numbers in tissues. In palmatine-treated mice, RANKL and OPG expression decreased. In the culture supernatant of MC3T3-E1 cells, RANKL and OPG levels were significantly reduced by palmatine addition. CONCLUSION: Palmatine may attenuate osteoclast differentiation through inhibition of RANKL and OPG expression by osteoblasts. Therefore, palmatine might be a candidate anti-resorptive agent for osteoporosis therapy.

Antimetastatic effects of Celastrus orbiculatus on human gastric adenocarcinoma by inhibiting epithelial-mesenchymal transition and NF-κB/snail signaling pathway.

AIM OF THE STUDY: Celastrus orbiculatus has been used as a folk medicine in China for the treatment of many diseases. In the laboratory, the ethyl acetate extract of Celastrus orbiculatus (COE) displays a wide range of anticancer functions. However, the inhibition of the metastasis mechanism of COE in gastric cancer cells has not been investigated so far. The present study was undertaken to determine if the antimetastatic effects of COE were involved in inhibition of the epithelial-mesenchymal transition (EMT) of human gastric adenocarcinoma SGC-7901 cells. METHODS: The adhesion, invasion, and migration of SGC-7901 cells were determined by COE treatment in vitro, using Matrigel-coated plate, transwell membrane chamber, and wound healing models, respectively. In vivo, the growth-inhibiting and antimetastatic effects of COE on the nude mice model of gastric cancer were tested and the mechanisms were explored. The expression of EMT markers and nuclear factor κB (NF-κB)/Snail signaling pathway were evaluated by using western blotting and immunohistochemistry. RESULTS: Treatment with COE dose-dependently inhibited the proliferation, adhesion, invasion, and migration of SGC-7901 cells in vitro, which was realized by enhancing the expression of E-cadherin and reducing N-cadherin and vimentin expression. Moreover, COE suppressed the activation of NF-κB/Snail signaling pathway induced by tumor necrosis factor-α. In addition, COE effectively suppressed tumor growth and metastasis in the nude mice model due to reduced expression of N-cadherin, vimentin, NF-κB p65, and Snail and increased expression of E-cadherin in the tumor tissues. CONCLUSION: Our findings provided new evidence that COE is an effective inhibitor of metastatic potential of SGC-7901 cells through suppression of EMT and NF-κB/Snail signal pathway. Based on these findings, COE may be considered a novel anticancer agent for the treatment of metastasis in gastric cancer.

Research on the efficacy of Celastrus Orbiculatus in suppressing TGF-ß1-induced epithelial-mesenchymal transition by inhibiting HSP27 and TNF-α-induced NF-κ B/Snail signaling pathway in human gastric adenocarcinoma.

BACKGROUND: Celastrus orbiculatus has been used as a folk medicine in China for the treatment of many diseases. In the laboratory, the ethyl acetate extract of Celastrus orbiculatus (COE) displays a wide range of anticancer functions. However, the inhibition of the metastasis mechanism of COE in gastric cancer cells has not been investigated so far. METHODS: The present study was undertaken to determine if the anti-metastasis effect of COE was involved in inhibiting of epithelial-mesenchymal transition (EMT) of human gastric adenocarcinoma SGC-7901 cells. In vitro, a well-established experimental EMT model involving transforming growth factor ß1 (TGF-ß1) was applied. Viability, invasion and migration, protein and mRNA expression of tumor cells were analyzed by MTT assay, transwell assay, western blot and real-time PCR, respectively. The molecular targets of COE in SGC-7901 cells were investigated by two-dimensional gel electrophoresis (2-DE) and MALDI-TOF-TOF mass spectrometer. Overexpression of heat shock protein 27 (HSP27) was performed by transfected with the recombinant retroviral expression plasmid. In vivo, the anti-metastasis mechanisms of COE in the peritoneal gastric cancer xenograft model was explored and the effect was tested. RESULTS: The non-cytostatic concentrations of COE effectively inhibited TGF-ß1 induced EMT process in SGC-7901 cells, which is characterized by prevented morphological changes, increased E-cadherin expression and decreased Vimentin, N-cadherin expression. Moreover, COE inhibited invasion and migration induced by TGF-ß1. Using a comparative proteomics approach, four proteins were identified as differently expressed, with HSP27 protein being one of the most significantly down-regulated proteins induced by COE. Moreover, the activation of nuclear factor κB (NF-κB)/Snail signaling pathway induced by tumor necrosis factor-α (TNF-α) was also attenuated under the pretreatment of COE. Interestingly, overexpression of HSP27 significantly decreases the inhibitory effect of COE on EMT and the NF-κB/Snail pathway. Furthermore, COE significantly reduced the number of peritoneal metastatic nodules in the peritoneal gastric cancer xenograft model. CONCLUSIONS: Taken together, these results suggest that COE inhibits the EMT by suppressing the expression of HSP27, correlating with inhibition of NF-κB/Snail signal pathways in SGC-7901 cells. Based on these results, COE may be considered a novel anti-cancer agent for the treatment of metastasis in gastric cancer.

Anti-invasive effects of Celastrus Orbiculatus extract on interleukin-1 beta and tumour necrosis factor-alpha combination-stimulated fibroblast-like synoviocytes.

BACKGROUND: Invasion of fibroblast-like synoviocytes (FLSs) is critical in the pathogenesis of rheumatoid arthritis (RA). The metalloproteinases (MMPs) and activator of nuclear factor-kappa B (NF-κB) pathway play a critical role in RA-FLS invasion induced by interleukin-1 beta (IL-1ß) and tumour necrosis factor-alpha (TNF-α). The present study aimed to explore the anti-invasive activity and mechanism of Celastrus orbiculatus extract (COE) on IL-1ß and TNF-α combination-stimulated human RA-FLSs. METHODS: We investigated the effect of COE on IL-1ß and TNF-α combination-induced FLS invasion as well as MMP expression and explored upstream signal transduction. RESULTS: COE suppressed IL-1ß and TNF-α combination-stimulated FLSs invasion by inhibiting MMP-9 expression and activity. Furthermore, our results revealed that COE inhibited the transcriptional activity of MMP-9 by suppression of the binding activity of NF-κB in the MMP-9 promoter, and inhibited IκBα phosphorylation and nuclear translocation of NF-κB. CONCLUSIONS: COE inhibits IL-1ß and TNF-α combination-induced FLSs invasion by suppressing NF-κB-mediated MMP-9 expression.

PI3 kinase/Akt/HIF-1α pathway is associated with hypoxia-induced epithelial-mesenchymal transition in fibroblast-like synoviocytes of rheumatoid arthritis.

Migration and invasion of fibroblast-like synoviocytes (FLSs) are critical in the pathogenesis of rheumatoid arthritis (RA). Hypoxic conditions are present in RA joints, and hypoxia has been extensively studied in angiogenesis and inflammation. However, its effect on the migration and invasion of RA-FLSs remains unknown. In this study, we observed that RA-FLSs exposed to hypoxic conditions experienced epithelial-mesenchymal transition (EMT), with increased cell migration and invasion. We demonstrated that hypoxia-induced EMT was accompanied by increased hypoxia-inducible factor (HIF)-1α expression and activation of Akt. After knockdown or inhibition of HIF-1α in hypoxia by small interfering RNA or genistein (Gen) treatment, the EMT transformation and invasion ability of FLSs were regained. HIF-1α could be blocked by phosphatidylinositol 3-kinase (PI3K) inhibitor, LY294002, indicating that HIF-1α activation was regulated by the PI3K/Akt pathway. Administration of LY294002 (20 mg/kg, intra-peritoneally) twice weekly and Gen (25 mg/kg, by gavage) daily for 3 weeks from day 20 after primary immunization in a collagen-induced arthritis rat model, markedly alleviated the clinical signs, radiology progression, synovial hyperplasia, and inflammatory cells infiltration of joints. Thus, results of this study suggest that activation of the PI3K/Akt/HIF-1α pathway plays a pivotal role in mediating hypoxia-induced EMT transformation and invasion of RA-FLSs under hypoxia.

Moxibustion at mingmen reduces inflammation and decreases IL-6 in a collagen-induced arthritis mouse model.

The purpose of this study was to compare the effectiveness of moxibustion (MOX) treatment at the GV4 and CV12 acupoints, and to determine the correlations between MOX treatment and interleukin (IL)-6 and corticosterone levels in a collagen-induced arthritis (CIA) mouse model. CIA mice were immunized twice intradermally over a 3-week interval with bovine type II collagen. After the second immunization (day 21), MOX was applied to the mouse equivalent of the GV4 and CV12 acupoints with a 1mg moxa cone five times/day. Clinical symptoms of CIA were observed three times/week until day 35. The concentrations of IL-6 and corticosterone in the blood samples were measured by immunoassay kits. At day 35, the incidence of CIA was significantly decreased in mice treated with MOX at the GV4 acupoint (78%, n=23, p<0.05), compared to untreated CIA mice (100%) and mice treated with MOX at the CV12 acupoint (100%). IL-6 and corticosterone levels were significantly increased by immunization. IL-6 levels significantly decreased in mice treated with MOX at the GV4 acupoint. These results suggest that MOX treatment suppressed CIA at the GV4 acupoint, not at the CV12 acupoint, possibly through inhibition of IL-6 production.