Computational analysis to define efficacy & molecular mechanisms of 7, 4'- Dihydroxyflavone on eosinophilic esophagitis: Ex-vivo validation in human esophagus biopsies.

Introduction: Eosinophilic Esophagitis (EoE) is a chronic condition characterized by eosinophilic inflammation of the esophagus which leads to esophageal dysfunction with common symptoms including vomiting, feeding difficulty, dysphagia, abdominal pain. Current main treatment options of EoE include dietary elimination and swallowed steroids. Diet elimination approach could lead to identifying the trigger food(s), but it often requires repeated upper endoscopy with general anesthesia and potentially could negatively affect nutrition intake and growth of the child and individuals' quality of life. Although the swallowed steroid treatment of effective, the EoE will universally recur after discontinuation of the treatment. Digestive Tea formula (DTF) has been used by the Traditional Chinese Medicine (TCM) practice to improve GI symptoms in EoE patients, including abdominal pain, GE reflux, and abnormal bowel movement. Previously, a flavonoid small molecule compound 7, 4 dihydroxy flavone (DHF) from Glycyrrhiza uralensis in DTF inhibited eotaxin, Th2 cytokine and IgE production in vitro and in vivo. Method: This study comprehensively evaluates the potential therapeutic and immunological mechanisms underlying DHF improvement of symptoms related to EoE using computational modeling, including target mining, gene ontology enrichment, pathway analyses, protein-protein interaction analyses, in silico molecular docking and dynamic simulation followed by ex-vivo target validation by qRT-PCR using cultured human esophagus biopsy specimen with or without DHF from patients with EoE. Results: Computational analyses defined 29 common targets of DHF on EoE, among which TNF-α, IL-6, IL1ß, MAPK1, MAPK3 and AKT1 were most important. Docking analysis and dynamic simulation revealed that DHF directly binds TNF-α with a free binding energy of -7.7 kcal/mol with greater stability and flexibility. Subsequently, in the human esophagus biopsy culture system, significant reduction in levels of TNF-α, IL-6, IL-8 and IL1-ß was found in the supernatant of biopsy sample cultured with DHF. Furthermore, the gene expression profile showed significant reduction in levels of TNF-α, IL1-ß, IL-6, CCND and MAPK1 in the esophagus biopsy sample cultured with DHF. Discussion: Taken together, the current study provides us an insight into the molecular mechanisms underlying multi-targeted benefits of DHF in the treatment of EoE and paves the way for facilitating more effective EoE therapies.

Anti-IgE effect of small-molecule-compound arctigenin on food allergy in association with a distinct transcriptome profile.

BACKGROUND: Excessive production of IgE plays a major role in the pathology of food allergy. In an attempt to identify anti-IgE natural products, Arctium Lappa was one of the most effective herbs among approximately 300 screened medicinal herbs. However, little is known about its anti-IgE compounds. OBJECTIVE: To identify compounds from Arctium Lappa for targeted therapy on IgE production and explore their underlying mechanisms. METHODS: Liquid-liquid extraction and column chromatographic methods were used to purify the compounds. IgE inhibitory effects were determined on IgE-producing human myeloma U266 cells, peanut-allergic murine model and PBMCs from food-allergic patients. Genes involved in IgE inhibition in PBMCs were studied by RNA sequencing. RESULTS: The main compounds isolated were identified as arctiin and arctigenin. Both compounds significantly inhibited IgE production in U266 cells, with arctigenin the most potent (IC50=5.09µg/mL). Arctigenin (at a dose of 13 mg/kg) markedly reduced peanut-specific IgE levels, blocked hypothermia and histamine release in a peanut-allergic mouse model. Arctigenin also significantly reduced IgE production and Th2 cytokines (IL-5, IL-13) by PBMCs. We found 479 differentially expressed genes in PBMCs with arctigenin treatment (p < .001 and fold-change ≥1.5), involving 24 gene ontology terms (p < .001, FDR <0.05); cell division was the most significant. Eleven genes including UBE2C and BCL6 were validated by qPCR. CONCLUSION: Arctigenin markedly inhibited IgE production in U266 cells, peanut-allergic murine model and PBMCs from allergic patients by down-regulating cell division, cell cycle-related genes and up-regulating anti-inflammatory factors.

Butanol Purified Food Allergy Herbal Formula-2 Has an Immunomodulating Effect ex-vivo in Pediatric Crohn's Disease Subjects.

Background: TNF-α has a major role in the pathogenesis of Crohn's disease (CD). In contrast, GM-CSF may be beneficial for its anti-inflammatory role in a subset of patients with CD with antibodies against GM-CSF as seen in prior trials of GM-CSF which resulted in clinical improvement in CD. We developed butanol purified Food Allergy Herbal Formula-2 (B-FAHF-2) by refining FAHF-2. FAHF-2 suppressed TNF-α production by human peripheral blood mononuclear cells (PBMCs) and colonic mucosa, and abrogated colitis in a murine model. We sought to examine the effect of B-FAHF-2 and the herbs that comprise it on TNF-α and GM-CSF production as a potential herbal therapy for the treatment of CD. Methods: B-FAHF-2 was examined using high pressure liquid chromatography (HPLC) and compared to the original formulation, FAHF-2. PBMCs from pediatric patients with CD were cultured with lipopolysaccharide and B-FAHF-2, individual herbs or medium alone. Colonic biopsy specimens were cultured with or without B-FAHF-2. TNF-α and GM-CSF were measured by enzyme-linked immunosorbent assay (ELISA). B-FAHF-2 efficacy was tested in vivo in the CD45Rbhi transfer model. Results: B-FAHF-2 had a similar HPLC fingerprint as FAHF-2 but decreased TNF-α production by PBMCs and colonic mucosa from pediatric CD subjects at 20% of the FAHF-2 dose. B-FAHF-2 increased GM-CSF production by PBMCs and colonic mucosa from pediatric CD subjects including those with antibodies to GM-CSF. Of B-FAHF-2's herbal constituents, only Huang Bai suppressed TNF-α and increased GM-CSF production. In the murine model, B-FAHF-2 treatment alleviated colitis. Conclusions: B-FAHF-2 decreased TNF-α production by PBMCs and colonic mucosa from pediatric subjects at a lower dose than FAHF-2. B-FAHF-2 also increased GM-CSF production by PBMCs independent of antibodies. B-FAHF-2 may have a benefit in CD patients.

Sophora flavescens Alkaloids and Corticosteroid Synergistically Augment IL-10/IL-5 Ratio with Foxp3-Gene-Epigenetic Modification in Asthma PBMCs.

BACKGROUND: It has been demonstrated that ASHMI (antiasthma-simplified herbal medicine intervention) can improve airway function and reduce inflammation in human asthmatic patients with high safety and tolerability. In addition, ASHMI significantly suppresses Th2 cytokine production and increases Th1 cytokine production in treating asthma. OBJECTIVE: Allergic asthma is associated with dysregulation of cytokines. We focused on IL-5 and IL-10 as signature Th2 and Treg cytokines to characterize ASHMI immunomodulatory components. METHODS: The effects of ASHMI and individual herbal constituents on IL-5 and IL-10 production by PBMCs from asthmatic subjects were determined ex vivo. Sophora flavescens (SF)-F2, containing alkaloid compounds, effects on PBMC IL-10 and IL-5 production in the presence or absence of dexamethasone (Dex), and on DNA methylation levels at the foxp3 gene promoter were determined. RESULTS: The ratio of anti-CD3/CD28 stimulated IL-10/IL-5 production by PBMCs from asthmatic subjects was significantly reduced compared to healthy subjects. In PBMCs from asthmatic subjects, ASHMI significantly reduced IL-5 production and increased IL-10 secretion in a dose-dependent manner (p < 0.05-0.01). SF-F2 was most effective in increasing IL-10, whereas SF-F4 (flavonoid compounds) was most effective in suppressing IL-5 production. Dex-treated PBMCs from asthma subjects showed a trend of increasing ratio of IL-10/IL-5 while demonstrating reduced levels in both IL-5 and IL-10 (p < 0.05). Co-culture with Dex and SF-F2 significantly prevented Dex suppression of IL-10, while retained Dex-suppression of IL-5 production, and increased IL-10/IL-5 ratio by Dex. Co-culture with SF-F2 and Dex significantly reduced DNA methylation levels at the foxp3 gene promoter at CpG-126. CONCLUSION: The SF alkaloid-rich fraction may be responsible for ASHMI induction of IL-10 production by PBMCs and plays a synergistic effect with Dex for augmenting IL-10/IL-5 ratio.

Tumor Necrosis Factor Inhibitor-Induced Psoriasis in a Pediatric Crohn's Disease Patient Successfully Treated with Ustekinumab.

BACKGROUND: Tumor necrosis factor (TNF) inhibitors are widely used in pediatric patients with inflammatory bowel disease, as well as psoriasis. However, there is growing evidence that these medications can also paradoxically induce a psoriasiform skin reaction in a subset of patients. GOALS: We seek to share our experience in treating severe TNF inhibitor-induced psoriasis in a pediatric patient with Crohn’s disease. STUDY: We report a case of a 10-year-old female with Crohn’s disease, who developed psoriasis after twelve months of infliximab therapy. Her skin disease was recalcitrant to topical therapies, methotrexate, and phototherapy. RESULTS: The patient was transitioned to ustekinumab with significant improvement in her symptoms and maintenance of remission of her bowel disease. CONCLUSION: This is the first reported case of a school-age pediatric patient with TNF inhibitor-induced psoriasis treated with ustekinumab. Controlled trials are warranted to fully assess the safety and efficacy of ustekinumab for treating TNF inhibitor-induced psoriasis in the pediatric population.J Drugs Dermatol. 2020;19(3): doi:10.36849/JDD.2020.2106.

Ganoderic acid C1 isolated from the anti-asthma formula, ASHMI™ suppresses TNF-α production by mouse macrophages and peripheral blood mononuclear cells from asthma patients.

Asthma is a heterogeneous airway inflammatory disease, which is associated with Th2 cytokine-driven inflammation and non-Th2, TNF-α mediated inflammation. Unlike Th2 mediated inflammation, TNF-α mediated asthma inflammation is generally insensitive to inhaled corticosteroids (ICS). ASHMITM, aqueous extract of three medicinal herbs-Ganoderma lucidum (G. lucidum), Sophora flavescens Ait (S. flavescens) and Glycyrrhiza uralensis Fischer (G. uralensis), showed a high safety profile and was clinically beneficial in asthma patients. It also suppresses both Th2 and TNF-α associated inflammation in murine asthma models. We previously determined that G. uralensis flavonoids are the key active compounds responsible for ASHMITM suppression of Th2 mediated inflammation. Until now, there are limited studies on anti-TNF-α compounds presented in ASHMITM. The objective of this study was to isolate and identify TNF-α inhibitory compounds in ASHMITM. Here we report that G. lucidum, but not the other two herbal extracts, S. flavescens or G. uralensis inhibited TNF-α production by murine macrophages; and that the methylene chloride (MC)-triterpenoid-enriched fraction, but not the polysaccharide-enriched fraction, contained the inhibitory compounds. Of the 15 triterpenoids isolated from the MC fraction, only ganoderic acid C1 (GAC1) significantly reduced TNF-α production by murine macrophages (RAW 264.7 cells) and peripheral blood mononuclear cells (PBMCs) from asthma patients. Inhibition was associated with down-regulation of NF-κB expression, and partial suppression of MAPK and AP-1 signaling pathways. Ganoderic acid C1 may have potential for treating TNF-α mediated inflammation in asthma and other inflammatory diseases.

Anti-inflammatory Effects of Ganoderma lucidum Triterpenoid in Human Crohn's Disease Associated with Downregulation of NF-κB Signaling.

BACKGROUND: Crohn's disease (CD) is a chronic inflammatory disease of the gastrointestinal tract. Current medications have potentially serious side effects. Hence, there is increasing interest in alternative therapies. We previously demonstrated the anti-inflammatory effects of Food Allergy Herbal Formula-2 in vitro on peripheral blood mononuclear cells (PBMCs) and mucosa from CD subjects. Here, we investigated the anti-inflammatory effects of a bioactive compound isolated from Ganoderma lucidum (G. lucidum), a key herbal constituent of Food Allergy Herbal Formula-2, in CD in vitro. METHODS: Triterpene ganoderic acid C1 (GAC1) was isolated from G. lucidum. Stimulated RAW 264.7 macrophages were treated with GAC1. Human PBMCs and colonic biopsies were obtained from children with CD and cultured with or without GAC1. TNF-α and other proinflammatory cytokine levels were measured in the culture supernatant. NF-κB signaling was investigated in PBMCs and colonic mucosa treated with GAC1 by In-Cell Western and Western blot analysis. RESULTS: GAC1 decreased TNF-α production by macrophages and PBMCs from CD subjects. GAC1 significantly decreased TNF-α, IFN-γ, and IL-17A production by inflamed colonic biopsies from CD subjects. These effects were due to downregulation of the NF-κB signaling pathway. CONCLUSIONS: GAC1 inhibited production of TNF-α and other proinflammatory cytokines by PBMCs and inflamed CD colonic mucosa due to blockage of NF-κB activation. GAC1 is a key beneficial constituent in G. lucidum and the Food Allergy Herbal Formula-2 in suppressing the inflammatory cytokines found in CD and warrants clinical investigation for the treatment of CD.

Complementary and alternative medicine use at a single pediatric inflammatory bowel disease center.

The aim of this study was to determine the use of complementary and alternative medicine (CAM) in an inflammatory bowel disease population at a single pediatric center. The secondary aims were to determine predictors of CAM use and assess parental attitude to CAM use. A survey was developed that was distributed electronically and given out in the clinic. Two hundred thirty-five surveys were analyzed. Thirty-six percent of respondents reported that their children had used CAM, while 19.6% were current users. Sixty-three percent of respondents were "extremely" or "very supportive" of CAM and 57.6% would have been "extremely" or "very supportive" at the time of their children's diagnosis. The most commonly used CAM modalities were fish oil (48.8%), probiotics (22.5%), acupuncture/pressure (17.5%), aloe (16.3%), yoga/meditation (16.3%), chiropractic (12%), and herbal medicine (13.8%). Multivariate analysis revealed 2 independent factors predictive of subjects using CAM: use of biologics (odds ratio of 2.8; p = .008) and subjects' parent using CAM (odds ratio of 10.9; p ≤ .001). More than one third of children in this study and their parents have used CAM. Families are supportive of CAM both at the time of diagnosis and as an ongoing component of their child's treatment even if they were not past or current users of CAM. Predictors of CAM use were treatment with a biologic and having a parent who used CAM.

Effect of Antiasthma Simplified Herbal Medicine Intervention on neutrophil predominant airway inflammation in a ragweed sensitized murine asthma model.

BACKGROUND: Neutrophil-predominant asthma is less responsive to steroids and associated with poorer disease control. The effects of Antiasthma Simplified Herbal Medicine Intervention (ASHMI), a traditional Chinese medicine formula reported to be efficacious in asthmatic patients and murine asthma models, on neutrophil predominant asthma are unknown. OBJECTIVE: To determine the effects of standard ASHMI and refined formula ASHMI (ASHMI(II)) in a neutrophil-predominant murine model of ragweed (RW) asthma and explore underlying mechanisms. METHODS: BALB/c mice were systemically sensitized, intranasally challenged with RW extract, and orally treated with ASHMI, ASHMI(II), or vehicle (water). In a separate experiment, some RW sensitized mice were treated with dexamethasone before challenge. After RW challenge, airway hyperreactivity (AHR), total and differential bronchoalveolar lavage fluid leukocyte counts, lung histologic features, and bronchoalveolar lavage fluid cytokine and chemokine levels were assessed. RW stimulation of the murine macrophage cell line RAW264.7 was used to determine effects of ASHMI active compound ganoderic acid C1 (GAC1) on tumor necrosis factor α (TNF-α) production and regulation of phosphorylated IκB and histone deacetylase 2 (HDAC2) levels. RESULTS: ASHMI and ASHMI(II) markedly reduced AHR, mucous production, neutrophilic inflammation, and TNF-α, interleukin 8, and interleukin 17 levels and decreased eosinophilic inflammation and TH2 responses in vivo (P < .01-.001 for all). GAC1 inhibited TNF-α production in RW-stimulated RAW264.7 cells in association with suppression of phosphorylated IκB and increased HDAC2 expression. Dexamethasone failed to reduce AHR and neutrophilic inflammation. CONCLUSION: ASHMI treatment was efficacious in a murine model of neutrophil-predominant asthma via modulation of innate chemokines, TH2 responses, nuclear factor-κB, and HDAC2. ASHMI, and/or its constituent GAC1, may be a valuable option for treating neutrophil-predominant asthma.

Anti-inflammatory effects of the Chinese herbal formula FAHF-2 in experimental and human IBD.

BACKGROUND: Crohn's disease (CD) is a chronic inflammatory disease with increasing incidence in children. Current medications have potentially serious side effects, hence increasing interest in alternative therapies. We previously developed an herbal formula, FAHF-2, based on a classical traditional Chinese herbal formula Wu Mei Wan that has long been used in China to treat colitis. We investigated FAHF-2's potential anti-inflammatory effects. METHODS: FAHF-2 efficacy was tested in vivo in the CD45RbRAG1 transfer colitis model. Weight loss, colonic histology, and cytokine production from mesenteric lymph nodes were assessed. Human peripheral blood mononuclear cells (PBMCs) and colonic biopsies were obtained from children newly diagnosed with CD and controls and cultured with or without FAHF-2. Cytokine levels were measured by multiplex immunoassay. The effect of FAHF-2 on TNF-α-producing cells was determined by flow cytometry. NF-κB signaling was investigated in human lamina propria mononuclear cells upon FAHF-2 treatment by In-Cell Western. RESULTS: FAHF-2-treated mice had decreased weight loss, improved histology, and reduced TNF-α, IL-17, IL-6, and IFN-γ production. In vitro treated PBMCs produced less TNF-α, IFN-γ, and IL-12. FAHF-2 reduced the TNF-α-producing monocytes and T cells. Inflamed CD biopsies produced less TNF-α, IL-17, IL-6, and IL-1ß. These effects are because of decreased NF-κB activation. CONCLUSIONS: FAHF-2 inhibited both adaptive and innate immune proinflammatory cytokine responses in PBMCs and inflamed CD mucosa due in part to blockage of NF-κB activation. FAHF-2 was effective in halting progression of colitis in a murine model. This study shows that FAHF-2 has potential as a novel treatment of CD.