Targeting ROS/NF-κB sigaling pathway by the seedless black Vitis vinifera polyphenols in CCl4-intoxicated kidney, lung, brain, and spleen in rats.

Carbon tetrachloride (CCl4) is an abundant environmental pollutant that can generate free radicals and induce oxidative stress in different human and animal organs like the kidney, lung, brain, and spleen, causing toxicity. The present study evaluated the alleviative mechanism of the isolated polyphenolic fraction from seedless (pulp and skin) black Vitis vinifera (VVPF) on systemic oxidative and necroinflammatory stress in CCl4-intoxicated rats. Here, we found that the administration of VVPF to CCl4-intoxicated rats for ten days was obviously ameliorated the CCl4-induced systemic elevation in ROS, NO and TBARS levels, as well as MPO activity. Also, it upregulated the cellular activities of the enzymatic (SOD, and GPx) and non-enzymatic (TAC and GSH) antioxidants. Furthermore, the gene expression of the ROS-related necroinflammatory mediators (NF-κB, iNOS, COX-2, and TNF-α) in the kidney, brain, and spleen, as well as IL-1ß, and IL-8 in the lung were greatly restored. The histopathological studies confirmed these biochemical results and showed a noticeable enhancing effect in the architecture of the studied organs after VVPF intake. Thus, this study indicated that VVPF had an alleviative effect on CCl4-induced necroinflammation and oxidative stress in rat kidney, lung, brain, and spleen via controlling the ROS/NF-κB pathway.

Phytochemical Profile and Anti-Inflammatory Activity of the Fraction from Artemisia lavandulaefolia.

Artemisia lavandulaefolia, a traditional herbal medicine, has been utilized as anti-inflammatory and analgesia agent in clinic. Bioassay-guided fractionation resulted in a fraction (ALDF) with anti-inflammatory effect obtained from A. lavandulaefolia. Its main constituents were analyzed and identified by UPLC-ESI-Q-TOF-MS technology. ALDF showed the strong inhibitory activity on the nitrogen oxide (NO) production in LPS-induced RAW 264.7 macrophages with an IC50 value of 1.64±0.41 µg/mL. Further results displayed that ALDF also significantly suppressed the secretion of key pro-inflammatory mediators, including tumor necrosis factor-α (TNF-α), prostaglandin E2 (PGE2 ) and interleukin-1ß (IL-1ß), and the increase of the inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein expression induced by LPS stimulation. Mechanism study indicated that ALDF was able to block NF-κB signaling pathway through inhibiting IκB and p65 phosphorylation, as well as NF-κB p65 nuclear translocation. Furthermore, in vivo results in mice revealed that treatments with ALDF evoked significant inhibition on ear edema induced by xylene and on the writhing responses induced by acetic acid. These results suggest that ALDF holds great potential in the prevention and treatment of inflammatory disorders.

Assessment of the anti-rheumatoid arthritis activity of Gastrodia elata (tian-ma) and Radix aconitic lateralis preparata (fu-zi) via network pharmacology and untargeted metabolomics analyses.

AIM: Gastrodia elata and Radix aconiti lateralis preparrata are respectively named as Tian-Ma and Fu-Zi (TF) in Chinese. We explored the active components against rheumatoid arthritis (RA) from an extensively used couplet of Chinese herbs, Gastrodia elata and Radix aconiti lateralis preparata (TF) via untargeted metabolomics and network pharmacological approaches. METHODS: Water extracts of TF were mixed at ratios 1:1, 3:2 and 2:3 (w/w). Ultra-performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) was then utilized as metabolomics screening. Human Metabolome (http://www.hmdb.ca/) and Lipidmaps (http://www.lipidmaps.org/) databases were used to annotate detected compounds. Further identification of vital genes and important pathways associated with the anti-RA properties of the TF preparations was done via network pharmacology, and verified by real-time quantitative polymerase chain reaction (RT-qPCR). RESULTS: Four key compounds involved in unsaturated fatty acid biosynthesis and isoflavonoid biosynthesis were identified through metabolomics analyses. Three key components of TF associated with anti-RA activity were linoleic acid, daidzein, and daidzin. Results of RT-qPCR revealed that all 3 tested TF couplets (1:1, 3:2, and 2:3) markedly suppressed the transcription of PTGS2. These results were consistent with our network pharmacological predictions. CONCLUSIONS: The anti-RA properties of Tian-Ma and Fu-Zi are associated with the inhibition of arachidonic acid metabolism pathway.

Effect of Ergothioneine on 7-Ketocholesterol-Induced Endothelial Injury.

Ergothioneine (ET) is a naturally occurring antioxidant that is synthesized by non-yeast fungi and certain bacteria. ET is not synthesized by animals, including humans, but is avidly taken up from the diet, especially from mushrooms. In the current study, we elucidated the effect of ET on the hCMEC/D3 human brain endothelial cell line. Endothelial cells are exposed to high levels of the cholesterol oxidation product, 7-ketocholesterol (7KC), in patients with cardiovascular disease and diabetes, and this process is thought to mediate pathological inflammation. 7KC induces a dose-dependent loss of cell viability and an increase in apoptosis and necrosis in the endothelial cells. A relocalization of the tight junction proteins, zonula occludens-1 (ZO-1) and claudin-5, towards the nucleus of the cells was also observed. These effects were significantly attenuated by ET. In addition, 7KC induces marked increases in the mRNA expression of pro-inflammatory cytokines, IL-1ß IL-6, IL-8, TNF-α and cyclooxygenase-2 (COX2), as well as COX2 enzymatic activity, and these were significantly reduced by ET. Moreover, the cytoprotective and anti-inflammatory effects of ET were significantly reduced by co-incubation with an inhibitor of the ET transporter, OCTN1 (VHCL). This shows that ET needs to enter the endothelial cells to have a protective effect and is unlikely to act via extracellular neutralizing of 7KC. The protective effect on inflammation in brain endothelial cells suggests that ET might be useful as a nutraceutical for the prevention or management of neurovascular diseases, such as stroke and vascular dementia. Moreover, the ability of ET to cross the blood-brain barrier could point to its usefulness in combatting 7KC that is produced in the CNS during neuroinflammation, e.g. after excitotoxicity, in chronic neurodegenerative diseases, and possibly COVID-19-related neurologic complications.

Anti-inflammatory and Cytoprotective Effect of Clinacanthus nutans Leaf But Not Stem Extracts on 7-Ketocholesterol Induced Brain Endothelial Cell Injury.

Clinacanthus nutans (Lindau) (C. nutans) has diverse uses in traditional herbal medicine for treating skin rashes, insect and snake bites, lesions caused by herpes simplex virus, diabetes mellitus and gout in Singapore, Malaysia, Indonesia, Thailand and China. We previously showed that C. nutans has the ability to modulate the induction of cytosolic phospholipase A2 (cPLA2) expression in SH-SY5Y cells through the inhibition of histone deacetylases (HDACs). In the current study, we elucidated the effect of C. nutans on the hCMEC/D3 human brain endothelial cell line. Endothelial cells are exposed to high levels of the cholesterol oxidation product, 7-ketocholesterol (7KC), in patients with cardiovascular disease and diabetes, and this process is thought to mediate pathological inflammation. 7KC induced a dose-dependent loss of hCMEC/D3 cell viability, and such damage was significantly inhibited by C. nutans leaf extracts but not stem extracts. 7KC also induced a marked increase in mRNA expression of pro-inflammatory cytokines, IL-1ß IL-6, IL-8, TNF-α and cyclooxygenase-2 (COX-2) in brain endothelial cells, and these increases were significantly inhibited by C. nutans leaf but not stem extracts. HPLC analyses showed that leaf extracts have a markedly different chemical profile compared to stem extracts, which might explain their different effects in counteracting 7KC-induced inflammation. Further study is necessary to identify the putative phytochemicals in C. nutans leaves that have anti-inflammatory properties.

Brazilian berry extract (Myrciaria jaboticaba): A promising therapy to minimize prostatic inflammation and oxidative stress.

BACKGROUND: Brazilian berry is a fruit popularly known as "Jaboticaba," rich in bioactive compounds with antioxidant and anti-inflammatory properties. Senescence and overweight are increasing worldwide and are considered risk factors to prostatic pathogenesis mainly due to oxidative and inflammatory processes induction. Thus, this study aimed to evaluate the effect of two increasing doses of the patented jaboticaba peel extract (PJE) on oxidative-stress and inflammation in the prostate of aging or high-fat-fed aging mice. METHODS: PJE and/or high-fat diet (HFD) treatments started with 11-month-old mice and lasted 60 days. The levels or the immunoexpression of different inflammatory (nuclear factor κB [NFκB], CD3+, cyclooxygenase 2 [COX-2], toll-like receptor 4 [TLR4], phosphorylated signal transducers and activators of transcription 3 [pSTAT-3], tumor necrosis factor α [TNF-α], interleukin 6 [IL-6], and IL-1ß) and oxidative-stress (catalase, superoxide dismutase 2 [SOD2], glutathione reductase [GSR], reduced glutathione, and glutathione peroxidase 3 [GPx3]) related molecules were analyzed by western-blotting, immunohistochemistry, and enzyme-linked immunosorbent assays. RESULTS: Both PJE doses reduced the levels of oxidative-stress-related molecules (GPx3, GSR, catalase), lipid peroxidation (4-hydroxynonenal), inflammatory mediators (COX-2, TNF-α, and pSTAT-3) and CD3+ T cells number, which were associated with the maintenance of the glandular morphological integrity in aging and HFD-fed-aging mice. Nevertheless, only the high PJE dose reduced the NFκB and TLR4 levels in aging mice; and SOD2, IL-6, and IL-1ß levels in HFD-aging mice. Aging itself promoted an oxidative inflammation in the prostate, interfering in the levels of the different oxidative-stress, lipid peroxidation, and inflammatory mediators evaluated, in association with high incidence of prostate epithelial and stromal damages. The HFD intake intensified aging alterations, showing an unfavorable prostatic microenvironment prone to oxidative and inflammatory damages. CONCLUSIONS: PJE exerted a dose-dependent effect controlling inflammation and oxidative-stress in aging and HFD-fed aging mice prostate. This fact contributed to prostate microenvironment balance recovery, preserving the tissue architecture of this gland. Thus, the PJE emerges as a potential therapy to prevent inflammation and oxidative stress in the prostate.

The influence of probiotic diet and chondroitin sulfate administration on Ptgs2, Tgfb1 and Col2a1 expression in rat knee cartilage during monoiodoacetate-induced osteoarthritis.

BACKGROUND: Osteoarthritis (OA) is a common worldwide disease induced by a wide range of biochemical processes, mainly inflammation and degradation of collagen. The aim of this study, was to describe the effect of a multistrain probiotic (PB) and chondroitin sulfate (CS), administered separately or in combination, on the expression of Ptgs2, Tgfb1 and Col2a1 during monoiodoacetate-induced OA in male rats. METHODS: OA was induced in male rats by injecting monoiodoacetate in right hind knee. Therapeutic groups received 3 mg/kg of CS for 28 days and/or 1.4 g/kg of multistrain PB for 14 days. Knee cartilage were taken 30 days after monoiodoacetate injection. RNA was extracted and the expression of Ptgs2, Tgfb1 and Col2a1 were analyzed using SYBR Green 1-step real-time quantitative polymerase chain reaction. RESULTS: Induction of OA caused an upregulation in Ptgs2, Tgfb1 expression, and downregulation of Col2a1. Separate administration of PB and CS reduced Ptgs2 and Tgfb1 expressions. Their combined administration significantly decreased the expression of these pro-inflammatory cytokines, comparable to controls. Expression of Col2a1 showed similar behavior, with upregulation in therapeutic group with separate administration and the cumulative effects in case of co-administration. CONCLUSIONS: The multistrain PB diet may offer a perspective to improve the standard treatment of OA and, necessitates further investigation with clinical trials.

Madhuca indica Inhibits Breast Cancer Cell Proliferation by Modulating COX-2 Expression.

BACKGROUND: Madhuca indica belongs to the family sapotaceae, commonly known as Mahua. It is primarily known for alcoholic beverage production and is reported to have anti-inflammatory, analgesic and antipyretic properties. Madhuca indica has also been reported to be effective in several diseases. OBJECTIVE: This study was undertaken to check the anticancer efficacy and chemopreventive effect of methanolic extract of Mahua flower (ME) on human breast cancer cell lines MCF-7 and MDA-MB-468. METHOD: The cytotoxic and anti-proliferative effects on MCF-7 and MDA-MB-468 cells were studied by MTT, hexosaminidase and colony formation assay. Expression of caspase 3/7 was assessed by flow cytometry and western blot analysis. Expression of COX-2 was evaluated by western blot analysis, luciferase assay and mRNA analysis. RESULTS: ME inhibited the proliferation of breast cancer cells by inducing apoptosis through up-regulating the expression of Caspase 3/7 (P < 0.0001). Our results showed a decrease in the expression of COX-2 mRNA and COX-2 protein in both MCF-7 and MDA-MB-468 cells with an increase in ME concentration. Furthermore synergistic effect of ME and chemotherapeutic drug paclitaxel was also studied in MCF-7 and MDA-MB- 468 cells which were found to be more effective (P < 0.0001) than treatment of either ME or paclitaxel alone. Results were analyzed by ANOVA and Pearson correlation analysis. CONCLUSION: All these experiments suggest that ME inhibits breast cancer cell proliferation and apoptosis by inhibiting the expression of COX-2 in MCF-7 and MDAMB- 468 cells. This work further highlighted that ME may enhance the potentiality of paclitaxel in breast cancer treatment.

Higher baseline expression of the PTGS2 gene and greater decreases in total colonic fatty acid content predict greater decreases in colonic prostaglandin-E2 concentrations after dietary supplementation with ω-3 fatty acids.

This study evaluated whether mRNA expression of major genes regulating formation of prostaglandin (PG)E2 in the colon and colonic fatty acid concentrations are associated with the reduction in colonic mucosal PGE2 after dietary supplementation with omega-3 (ω-3) fatty acids. Supplementation with ω-3 fatty acids was done for 12 weeks using personalized dosing that was expected to reduce colonic PGE2 by 50%. In stepwise linear regression models, the ω-3 fatty acid dose and baseline BMI explained 16.1% of the inter-individual variability in the fold change of colonic PGE2 post-supplementation. Increases in mRNA gene expression after supplementation were, however, modest and were not associated with changes in PGE2. When baseline expression of PTGS1, PTGS2 and HPGD genes was included in the linear regression model containing dose and BMI, only PTGS2, the gene coding for the inducible form cyclooxygenase, was a significant predictor. Higher relative expression of PTGS2 predicted greater decreases in colonic PGE2, accounting for an additional 13.6% of the inter-individual variance. In the final step of the regression model, greater decreases in total colonic fatty acid concentrations predicted greater decreases in colonic PGE2, contributing to an additional 18.7% of the variance. Overall, baseline BMI, baseline expression of PTGS2 and changes in colonic total fatty acids together accounted for 48% of the inter-individual variability in the change in colonic PGE2. This is consistent with biochemical data showing that fatty acids which are not substrates for cyclooxygenases can activate cyclooxygenase-2 allosterically. Further clinical trials are needed to elucidate the factors that regulate the fatty acid milieu of the human colon and how this interacts with key lipid metabolizing enzymes. Given the central role of PGE2 in colon carcinogenesis, these pathways may also impact on colon cancer prevention by other dietary and pharmacological approaches.

Safflower polysaccharide inhibits the development of tongue squamous cell carcinoma.

BACKGROUND: Safflower polysaccharide (SPS) is one of the most important active components of safflower (Carthamus tinctorius L.), which has been confirmed to have the immune-regulatory function and antitumor effect. This study aimed to explore the effects of safflower polysaccharide (SPS) on tongue squamous cell carcinoma (TSCC). METHODS: HN-6 cells were treated with 5 µg/mL cisplatin and various concentrations of SPS (0, 0.02, 0.04, 0.08, 0.16, 0.32, 0.64, and 1.28 mg/mL), and cell proliferation was measured. After treatment with 5 µg/mL cisplatin and 0.64 mg/mL SPS, the induction of apoptosis and the protein and mRNA expression of Bax, Bcl-2, COX-2, and cleaved caspase-3 in HN-6 cells were quantified. In addition, HN-6 cells were implanted into mice to establish an in vivo tumor xenograft model. Animals were randomly assigned to three groups: SPS treatment, cisplatin treatment, and the model group (no treatment). The body weight, tumor volume, and tumor weight were measured, and the expression of the above molecules was determined. RESULTS: SPS treatment (0.02-0.64 mg/mL) for 24-72 h inhibited HN-6 cell proliferation. In addition, 0.64 mg/mL SFP markedly induced apoptosis in HN-6 cells and arrested the cell cycle at the G0/G1 phase. Compared with the control group, the expression of Bcl-2 and COX-2 was markedly reduced by SPS treatment, whereas the expression of Bax and cleaved caspase-3 was increased. Moreover, SPS significantly inhibited the growth of the tumor xenograft, with similar changes in the expression of Bcl-2, COX-2, Bax, and cleaved caspase-3 in the tumor xenograft to the in vitro analysis. CONCLUSIONS: Our results indicated that SPS may inhibit TSCC development through regulation of Bcl-2, COX-2, Bax, and cleaved caspase-3 expression.

Resveratrol Improves Neuroimmune Dysregulation Through the Inhibition of Neuronal Toll-Like Receptors and COX-2 Signaling in BTBR T+ Itpr3tf/J Mice.

Autism is a neurodevelopmental disorder characterized by deficits in qualitative impairments in communication, repetitive and social interaction, restricted, and stereotyped patterns of behavior. Resveratrol has been extensively studied pharmacologically and biologically and has anti-inflammatory, antioxidant, and neuroprotective effects on neuronal damage in neurodegenerative disorders. The BTBR T+ Itpr3tf/J (BTBR) autistic mouse model has been explored for treatment of autism, which shows low reciprocal social interactions, impaired juvenile play, and decreased social approach. Here, we explored whether resveratrol treatment decreases neuroimmune dysregulation mediated through toll-like receptor (TLR4) and nuclear factor-κB (NF-κB) signaling pathway in BTBR mice. We investigated the effect of resveratrol treatment on TLR2, TLR3, TLR4, NF-κB, and inducible nitric oxide synthase (iNOS or NOS2) levels in CD4 spleen cells. We also assessed the effect of resveratrol treatment on TLR2, TLR3, TLR4, NF-κB, iNOS, and cyclooxygenase (COX-2) mRNA expression levels in the brain tissue. We further explored TLR2, TLR4, NF-κB, iNOS, and COX-2 protein expression levels in the brain tissue. Resveratrol treatment on BTBR mice significantly decreased CD4+TLR2+, CD4+TLR3+, CD4+TLR4+ CD4+NF-κB+, and CD4+iNOS+ levels in spleen cells. Resveratrol treatment on BTBR mice decreased TLR2, TLR3, TLR4, NF-κB, iNOS, and COX-2 mRNA expression levels in brain tissue. Moreover, resveratrol treatment resulted in decreased protein expression of TLR2, TLR3, TLR4, NF-κB, iNOS, and COX-2 in brain tissue. Taken together, these results indicate that resveratrol treatment improves neuroimmune dysregulation through the inhibition of proinflammatory mediators and TLRs/NF-κB transcription factor signaling, which might be help devise future therapies for neuroimmune disorders.

Effects of electroacupuncture on luteal regression and steroidogenesis in ovarian hyperstimulation syndrome model rat.

AIMS: Electroacupuncture (EA) is an effective and safe therapeutic method widely used for treating clinical diseases. Previously, we found that EA could decrease serum hormones and reduce ovarian size in ovarian hyperstimulation syndrome (OHSS) rat model. Nevertheless, the mechanisms that contribute to these improvements remain unclear. MATERIALS AND METHODS: HE staining was used to count the number of corpora lutea (CL) and follicles. Immunohistochemical and ELISA were applied to examine luteal functional and structural regression. Immunoprecipitation was used for analyzing the interaction between NPY (neuropeptide Y) and COX-2; western blotting and qRT-PCR were used to evaluate the expressions of steroidogenic enzymes and PKA/CREB pathway. KEY FINDINGS: EA treatment significantly reduced the ovarian weight and the number of CL, also decreased ovarian and serum levels of PGE2 and COX-2 expression; increased ovarian PGF2α levels and PGF2α/PGE2 ratio; decreased PCNA expression and distribution; and increased cyclin regulatory inhibitor p27 expression to have further effect on the luteal formation, and promote luteal functional and structural regression. Moreover, expression of COX-2 in ovaries was possessed interactivity increased expression of NPY. Furthermore, EA treatment lowered the serum hormone levels, inhibited PKA/CREB pathway and decreased the expressions of steroidogenic enzymes. Hence, interaction with COX-2, NPY may affect the levels of PGF2α and PGE2 as well as impact the proliferation of granulosa cells in ovaries, thus further reducing the luteal formation, and promoting luteal structural and functional regression, as well as the ovarian steroidogenesis following EA treatment. SIGNIFICANCE: EA treatment could be an option for preventing OHSS in ART.

Screening a small molecule library to identify inhibitors of NF-κB inducing kinase and pro-labor genes in human placenta.

The non-canonical NF-κB signaling (RelB/p52) pathway drives pro-labor genes in the human placenta, including corticotropin-releasing hormone (CRH) and cyclooxygenase-2 (COX-2), making this a potential therapeutic target to delay onset of labor. Here we sought to identify small molecule compounds from a pre-existing chemical library of orally active drugs that can inhibit this NF-κB signaling, and in turn, human placental CRH and COX-2 production. We used a cell-based assay coupled with a dual-luciferase reporter system to perform an in vitro screening of a small molecule library of 1,120 compounds for inhibition of the non-canonical NF-κB pathway. Cell toxicity studies and drug efflux transport MRP1 assays were used to further characterize the lead compounds. We have found that 14 drugs have selective inhibitory activity against lymphotoxin beta complex-induced activation of RelB/p52 in HEK293T cells, several of which also inhibited expression of CRH and COX-2 in human term trophoblast. We identified sulfapyridine and propranolol with activity against CRH and COX-2 that deserve further study. These drugs could serve as the basis for development of orally active drugs to affect length of gestation, first in an animal model, and then in clinical trials to prevent preterm birth during human pregnancy.

Heme Oxygenase-1 Induction and Anti-inflammatory Actions of Atractylodes macrocephala and Taraxacum herba Extracts Prevented Colitis and Was More Effective than Sulfasalazine in Preventing Relapse.

BACKGROUND/AIMS: In inflammatory bowel disease (IBD), repeated bouts of remission and relapse occur in patients and can impose a risk of colitis-associated cancer. We hypothesized that plant extracts of Atractylodes macrocephala (AM) or Taraxacum herba (TH) may be better than sulfasalazine for treating this disease because these extracts can promote additional regeneration. METHODS: Murine intestinal epithelial IEC-6 cells were pretreated with AM or TH before a lipopolysaccharide (LPS)-induced challenge. Acute colitis was induced with 7 days of dextran sulfate sodium (DSS) in male C57BL/6 mice, and extracts of AM and TH were administered for 2 weeks before DSS administration. RESULTS: In vitro studies demonstrated that AM or TH treatment reduced LPS-induced COX-2 and tumor necrosis factor-α mRNA levels but increased heme oxygenase-1 (HO-1). Oral preadministration of AM and TH rescued mice from DSS-induced colitis by inhibiting inflammatory mediators via inactivated extracellular signal regulated kinase and repressed nuclear factor κB and signal transducer and activator of transcription 3, but the effect was weaker for sulfasalazine than that for the extracts. Anti-inflammatory activities occurred via the inhibition of macrophage and T lymphocyte infiltrations. Unlike sulfasalazine, which did not induce HO-1, TH extracts afforded significant HO-1 induction. CONCLUSIONS: Because the AM or TH extracts were far superior in preventing DSS-induced colitis than sulfasalazine, AM or TH extracts can be considered natural agents that can prevent IBD relapse.

Rhizoma Smilacis Glabrae inhibits pathogen-induced upper genital tract inflammation in rats through suppression of NF-κB pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Rhizoma Smilacis Glabrae (RSG) is traditionally used to treat gynecological disease, which is simply recorded in Chinese Pharmacopoeia. However, whether it has effect on upper genital tract inflammation (UGTI) is unclear. AIM OF THE STUDY: To evaluate the pharmacological effect of RSG on UGTI in rats and analyze its phytochemistry characteristics. MATERIALS AND METHODS: The substances in RSG extract was qualified by LC-Q-TOF-MS method, and 11 substances were further quantified. The RSG extract, at dose of 241, 482 (clinical dose) and 964mg/kg/day, was orally administered to UGTI rats whose upper genital tracts were multi-infected with pathogens. Infiltrations of neutrophil and lymphocyte and productions of IL-1ß, IL-6, CXCL-1, MCP-1, RANTES, PGE2, COX-2, NF-κB p65 and IκB-α in upper genital tract were examined to evaluate the effects of RSG and its potential mechanism. RESULTS: A total of 77 substances were detected in RSG extract, with 50 substances putatively identified, most of which were flavonoids, phenolic acids and phenylpropanoids. The quantification analysis showed flavonoid had a relative high amount. In pharmacological study, RSG extract suppressed infiltrations of inflammatory cells, reduced over-productions of factors involved in inflammation and pelvic pain. A potential mechanism of these effects was blocking NF-κB signal pathway. CONCLUSIONS: The RSG extract exhibited anti-inflammatory effect on UGTI, with a potential mechanism of blocking the activation of NF-κB signal pathway. The effect may be involved in the presence of substances, such as flavonoids and phenolic acids.

Inhibition of cancer migration and invasion by knocking down delta-5-desaturase in COX-2 overexpressed cancer cells.

We recently reported that knockdown of delta-5-desaturase (a key enzyme that converts dihomo-γ-linolenic acid, DGLA, to the downstream ω-6 arachidonic acid) promotes formation of an anti-cancer byproduct 8-hydroxyoctanoic acid from cyclooxygenase (COX)-catalyzed DGLA peroxidation. 8-hydroxyoctanoic acid can exert its growth inhibitory effect on cancer cells (e.g. colon and pancreatic cancer) by serving as a histone deacetylase inhibitor. Since histone deacetylase inhibitors have been well-known to suppress cancer cell migration and invasion, we thus tested whether knockdown of delta-5-desaturase and DGLA treatment could also be used to inhibit cancer migration and invasion of colon cancer and pancreatic cancer cells. Wound healing assay, transwell assay and western blot were used to assess cell migration and invasion as well as the associated molecular mechanisms. Formation of threshold level of 8-hydroxyoctanoic acid was quantified from COX-catalyzed DGLA peroxidation in the cancer cells that overexpress COX-2 and their delta-5-desaturases were knocked down by shRNA transfection. Our results showed that knockdown of delta-5-desaturase along with DGLA supplement not only significantly inhibited cell migration, but also improved the efficacies of 5-flurouracil and gemcitabine, two frontline chemotherapy drugs currently used in the treatment of colon and pancreatic cancer, respectively. The molecular mechanism behind these observations is that 8-hydroxyoctanoic acid inhibits histone deacetylase, resulting in downregulation of cancer metastasis promotors, e.g., MMP-2 and MMP-9 as well as upregulation of cancer metastasis suppressor, e.g. E-cadherin. For the first time, we demonstrated that we could take the advantage of the common phenomenon of COX-2 overexpression in cancers to inhibit cancer cell migration and invasion. With the shifting paradigm of COX-2 cancer biology, our research outcome may provide us a novel cancer treatment strategy.

Gastrodin ameliorates subacute phase cerebral ischemia­reperfusion injury by inhibiting inflammation and apoptosis in rats.

Gastrodin (GAS), which is extracted from the Chinese herbal medicine Gastrodia elata Blume, has long been used to improve stroke, epilepsy, dizziness and dementia. However, the effects and underlying mechanisms of GAS on subacute phase cerebral ischemia­reperfusion (I/R) injury remain unknown. The aim of the present study was to investigate the effects and mechanisms of GAS on cerebral I/R injury in rats. The rats were pretreated with GAS by gavage for 7 days followed by I/R surgery, and were then treated with GAS for 7 days after I/R surgery. Neurological deficits were assessed on days 1, 3 and 7 post­cerebral I/R injury. 2,3,5­Triphenyltetrazolium chloride staining was using to measure the infarct volume; morphological alterations were observed by hematoxylin and eosin staining under an optical microscope; apoptosis in the hippocampus and cortex was observed by terminal deoxynucleotidyl transferase dUTP nick end labeling staining; and the level of mRNA and protein expression was tested by reverse transcription­quantitative polymerase chain reation and western blot analysis, respectively. GAS markedly attenuated I/R­induced disability and histological damage, alleviated neuronal apoptosis, and reduced the mRNA and protein expression levels of inflammatory and proapoptotic factors, including interleukin­1ß, cyclooxygenase­2, inducible nitric oxide synthase and cleaved caspase­3. These findings suggested that GAS may ameliorate subacute phase cerebral I/R injury by inhibiting inflammation and apoptosis in rats; therefore, GAS may be considered a potential candidate for the treatment of cerebral ischemia.

Anti-Inflammatory Effects of Agrimoniin-Enriched Fractions of Potentilla erecta.

Potentilla erecta (PE) is a small herbaceous plant with four yellow petals belonging to the Rosaceae family. The rhizome of PE has traditionally been used as an antidiarrheal, hemostatic and antihemorrhoidal remedy. PE contains up to 20% tannins and 5% ellagitannins, mainly agrimoniin. Agrimoniin is a hydrolyzable tannin that is a potent radical scavenger. In this study we tested the anti-inflammatory effect of four PE fractions with increasing amounts of agrimoniin obtained by Sephadex column separation. First, we analyzed in HaCaT keratinocytes the expression of cyclooxygenase-2 (COX-2) induced by ultraviolet-B (UVB) irradiation. As COX-2 catalyzes the metabolism of arachidonic acid to prostanoids such as PGE2, we also measured the PGE2 concentration in cell culture supernatants. PE inhibited UVB-induced COX-2 expression in HaCaT cells and dose-dependently reduced PGE2. The PE fraction with the highest agrimoniin amount (PE4) was the most effective in this experiment, whereas fraction PE1 containing mainly sugars had no effect. PE4 also dose dependently inhibited the phosphorylation of the epidermal growth factor receptor (EGFR) which plays a crucial role in UVB-mediated COX-2 upregulation. A placebo-controlled UV-erythema study with increasing concentrations of PE4 demonstrated a dose dependent inhibition of UVB-induced inflammation in vivo. Similarly, PE4 significantly reduced UVB-induced PGE2 production in suction blister fluid in vivo. In summary, PE fractions with a high agrimoniin content display anti-inflammatory effects in vitro and in vivo in models of UVB-induced inflammation.

2,8-Decadiene-1,10-Diol Inhibits Lipopolysaccharide-Induced Inflammatory Responses Through Inactivation of Mitogen-Activated Protein Kinase and Nuclear Factor-κB Signaling Pathway.

Amomum tsao-ko (A. tsao-ko) has been used as a traditional medicine for the treatment of infectious and digestive disorders. In the present study, we report the anti-inflammatory activity and molecular mechanism of 2,8-decadiene-1,10-diol (DDO) isolated from the extract of A. tsao-ko in lipopolysaccharide-stimulated RAW 264.7 cells. DDO treatment inhibited the production of nitric oxide and prostaglandin E2 by downregulating inducible nitric oxide synthase and cyclooxygenase-2 expression, respectively. Moreover, DDO suppressed the production of pro-inflammatory cytokines such as interleukin-6 and tumor necrosis factor-α. These inhibitory effects of DDO on the expression of inflammatory proteins were found to be mediated through the inactivation of mitogen-activated protein kinases (MAPKs) such as extracellular signal-regulated kinase, c-Jun-N-terminal kinase and p38(MAPK), and inhibition of nuclear factor-κB (NF-κB) pathways including degradation of inhibitor of κB-α and nuclear localization of NF-κB. Taken together, these findings demonstrate the pharmacological roles and molecular mechanisms of DDO in regulating inflammatory responses, and suggest further evaluation and development of DDO as a potent therapeutic agent for the treatment of inflammatory disorders.

A fraction from Dojuksan 30% ethanol extract exerts its anti-inflammatory effects through Nrf2-dependent heme oxygenase-1 expression.

Dojuksan is a traditional herbal medicine used in Korea and China to treat urinary diseases. In the present study, we aimed to examine the anti-inflammatory effects of an ethanol solvent extract of Dojuksan and a fraction (by bioassay-guided fractionation) derived from this extract, and to elucidate the specific mechanisms involved. The Dojuksan 30% ethanol extract (DEE) had a more significant and potent anti-inflammatory effect than the Dojuksan water extract (DWE). DEE markedly inhibited the production of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), nitric oxide (NO), prostaglandin E2 (PGE2), tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß), as well as nuclear factor-κB (NF-κB) binding activity. We found that the anti-inflammatory effects of DEE were mediated by the induction of nuclear factor E2-related factor 2 (Nrf2)-dependent heme oxygenase-1 (HO-1). To further explore the anti-inflammatory effects of DEE, we generated 6 different fractions of DEE. Of these, DEE-5 decreased the production of NO more significantly than the other fractions. DEE-5 also significantly decreased the expression of iNOS and COX-2, and the production of NO, PGE2, TNF-α and IL-1ß. In addition, DEE-5 also significantly increased HO-1 levels; HO-1 significanlty contributed to the inhibitory effects of DEE-5 on the production of pro-inflammatory mediators. In this study, we determined whether the choice of extraction solvent affects the biological activity of Dojuksan, a traditional herbal formula. Our findings demonstrate that DEE and a fraction derived from this extract exerts anti-inflammatory effects through Nrf2­dependent HO-1 expression, and that DEE may thus have greater potential therapeutic application than DWE.