Sustained silencing peanut allergy by xanthopurpurin is associated with suppression of peripheral and bone marrow IgE-producing B cell.

Introduction: Peanut allergy is an immunoglobulin E (IgE) mediated food allergy. Rubia cordifolia L. (R. cordifolia), a Chinese herbal medicine, protects against peanut-induced anaphylaxis by suppressing IgE production in vivo. This study aims to identify IgE-inhibitory compounds from the water extract of R. cordifolia and investigate the underlying mechanisms using in vitro and in vivo models. Methods: Compounds were isolated from R. cordifolia water extract and their bioactivity on IgE production was assessed using a human myeloma U266 cell line. The purified active compound, xanthopurpurin (XPP), was identified by LC-MS and NMR. Peanut-allergic C3H/HeJ mice were orally administered with or without XPP at 200µg or 400µg per mouse per day for 4 weeks. Serum peanut-specific IgE levels, symptom scores, body temperatures, and plasma histamine levels were measured at challenge. Cytokines in splenocyte cultures were determined by ELISA, and IgE + B cells were analyzed by flow cytometry. Acute and sub-chronic toxicity were evaluated. IL-4 promoter DNA methylation, RNA-Seq, and qPCR analysis were performed to determine the regulatory mechanisms of XPP. Results: XPP significantly and dose-dependently suppressed the IgE production in U266 cells. XPP significantly reduced peanut-specific IgE (>80%, p <0.01), and plasma histamine levels and protected the mice against peanut-allergic reactions in both early and late treatment experiments (p < 0.05, n=9). XPP showed a strong protective effect even 5 weeks after discontinuing the treatment. XPP significantly reduced the IL-4 level without affecting IgG or IgA and IFN-γ production. Flow cytometry data showed that XPP reduced peripheral and bone marrow IgE + B cells compared to the untreated group. XPP increased IL-4 promoter methylation. RNA-Seq and RT-PCR experiments revealed that XPP regulated the gene expression of CCND1, DUSP4, SDC1, ETS1, PTPRC, and IL6R, which are related to plasma cell IgE production. All safety testing results were in the normal range. Conclusions: XPP successfully protected peanut-allergic mice against peanut anaphylaxis by suppressing IgE production. XPP suppresses murine IgE-producing B cell numbers and inhibits IgE production and associated genes in human plasma cells. XPP may be a potential therapy for IgE-mediated food allergy.

Marketable 1,3-dimethylamylamine and caffeine-based thermogenic supplements: Regulatory genotoxicity assessment through in vitro and in silico approaches.

The consumption of dietary supplements to enhance physical performance has increased significantly in the last century, especially thermogenic pre-workout supplements. Nevertheless, this industry has faced criticism for inadequate safety measures surveillance in regulatory issues regarding their products. The aims of our study were to investigate two pre-workout supplements with respect to (1) mutagenicity utilizing Salmonella/microsome assay; (2) genotoxicity employing cytokinesis-block micronucleus (CBMN) assay protocols; and (3) hepatocytoxicity using WST cell proliferation, activities of lactate dehydrogenase (LDH) and alkaline phosphatase using human liver carcinoma (HepG2) and mouse fibroblast (F C3H) cells. Oxidative stress was determined through glutathione (GSH) measurement and in silico for predictions of pharmacokinetics and toxicity for the most abundant isolated substances present in these supplements. Both supplements induced mutagenicity in all examined bacterial strains, especially in the presence of exogenous metabolism. Further, tested supplements significantly elevated the formation of micronuclei (MN) as well as other cellular phenomena. Concentration- and time-dependent curves were observed for hepatotoxicity in both studied cell lines. In addition, both supplements decreased levels of intracellular and extracellular GSH. In silico predictions showed that the isolated individual compounds failed to induce the observed outcomes. Our findings provide contributions to the molecular mechanisms underlying two pre-workout supplement-induced toxicity and the need for surveillance.

Sciatic-Vagal Nerve Stimulation by Electroacupuncture Alleviates Inflammatory Arthritis in Lyme Disease-Susceptible C3H Mice.

Lyme disease is caused by Borrelia burgdorferi, and the pathogenesis of the disease is complex with both bacterial and host factors contributing to inflammatory responses. Lyme disease affects different organs including joints and results in arthritis. Immune responses stimulated by B. burgdorferi through toll-like receptors cause infiltration of leukocytes, which produce inflammatory cytokines and facilitate spirochete clearance. However, arthritic manifestations and chronic fatigue syndrome-like symptoms persist long after completion of antibiotic treatment regimens in a significant number of patients. To counter the effects of inflammation, treatment by non-steroidal anti-inflammatory drugs, hydroxychloroquine, or synovectomy to eradicate inflammatory arthritis in the involved joint could be employed; however, they often have long-term consequences. Acupuncture has been used for a long time in Asian medicine to diminish pain during various ailments, but the effects and its mechanism are just beginning to be explored. Control of inflammation by neuronal stimulation has been exploited as a systemic therapeutic intervention to arrest inflammatory processes. Our objective was to determine whether activation of the sciatic-vagal network by electroacupuncture on ST36 acupoint, which is used to control systemic inflammation in experimental models of infectious disorders such as endotoxemia, can also alleviate Lyme arthritis symptoms in mice. This aim was further strengthened by the reports that sciatic-vagal neuronal network stimulation can lead to dopamine production in the adrenal medulla and moderate the production of inflammatory factors. We first assessed whether electroacupuncture affects spirochete colonization to attenuate Lyme arthritis. Interestingly, bioluminescent B. burgdorferi burden detected by live imaging and qPCR were similar in electroacupuncture- and mock-treated mice, while electroacupuncture induced a lasting anti-inflammatory effect on mice. Despite the discontinuation of treatment at 2 weeks, the simultaneous decrease in neutrophils in the joints and inflammatory cytokine levels throughout the body at 4 weeks suggests a systemic and persistent effect of electroacupuncture that attenuates Lyme arthritis. Our results suggest that electroacupuncture-mediated anti-inflammatory responses could offer promising healthcare benefits in patients suffering from long-term Lyme disease manifestations.

The Efficacy of Radiation is Enhanced by Metformin and Hyperthermia Alone or Combined Against FSaII Fibrosarcoma in C3H Mice.

The effects of mild-temperature hyperthermia (MTH) and metformin, alone or in combination, on the efficacy of high-dose hypofractionated radiation against experimental tumors were investigated. FSaII fibrosarcoma grown subcutaneously in the hind legs of C3H mice was irradiated with a single 15 Gy dose using a 60Co irradiator. The radio frequency capacitive method was used to heat the tumors at 41.0°C for 30 min. Metformin was intraperitoneally (i.p.) administered daily to tumor-bearing mice at a dose of 150 mg/kg. The expression levels of hypoxia-inducible factor-1α (HIF-1α), vascular endothelial growth factor (VEGF), and programmed cell death-ligand 1 (PD-L1) were determined by immunohistochemical staining of the excised tumor tissues. The apoptosis of tumor cells in vivo was quantified by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining and cleaved caspase-3 staining of the excised tumor tissues. Irradiation of tumors markedly increased the expression of HIF-1α, VEGF, and PD-L1, and MTH and metformin used either alone or in combination significantly abrogated the radiation-induced upregulation of these proteins. MTH and metformin alone or combined increased the radiation-induced apoptosis in tumor cells and enhanced the radiation-induced suppression of tumor growth. The findings indicated that the increased tumor response to 15 Gy irradiation by MTH and metformin alone or in combination was due, in part, to the abrogation of the radiation-induced upregulation of HIF-1α and its downstream targets VEGF and PD-L1.

Modulation of pacemaker channel function in a model of thalamocortical hyperexcitability by demyelination and cytokines.

A consensus is yet to be reached regarding the exact prevalence of epileptic seizures or epilepsy in multiple sclerosis (MS). In addition, the underlying pathophysiological basis of the reciprocal interaction among neuroinflammation, demyelination, and epilepsy remains unclear. Therefore, a better understanding of cellular and network mechanisms linking these pathologies is needed. Cuprizone-induced general demyelination in rodents is a valuable model for studying MS pathologies. Here, we studied the relationship among epileptic activity, loss of myelin, and pro-inflammatory cytokines by inducing acute, generalized demyelination in a genetic mouse model of human absence epilepsy, C3H/HeJ mice. Both cellular and network mechanisms were studied using in vivo and in vitro electrophysiological techniques. We found that acute, generalized demyelination in C3H/HeJ mice resulted in a lower number of spike-wave discharges, increased cortical theta oscillations, and reduction of slow rhythmic intrathalamic burst activity. In addition, generalized demyelination resulted in a significant reduction in the amplitude of the hyperpolarization-activated inward current (Ih) in thalamic relay cells, which was accompanied by lower surface expression of hyperpolarization-activated, cyclic nucleotide-gated channels, and the phosphorylated form of TRIP8b (pS237-TRIP8b). We suggest that demyelination-related changes in thalamic Ih may be one of the factors defining the prevalence of seizures in MS.

Cordycepin prevents and ameliorates experimental autoimmune encephalomyelitis by inhibiting leukocyte infiltration and reducing neuroinflammation.

Multiple sclerosis (MS) is a neuroinflammatory autoimmune disease characterized by multifocal perivascular infiltration of immune cells in the central nervous system (CNS). Cordycepin (3'-deoxyadenosine), an adenosine analogue initially extracted from the fungus Cordyceps militarisa, is one of the candidates that has multiple actions. We investigated that cordycepin attenuated the activation of LPS-induced mouse bone marrow-derived dendritic cells (BMDCs) and human monocyte-derived dendritic cells (MoDCs) through the inhibition of the AKT, ERK, NFκB, and ROS pathways and impaired the migration of BMDCs through the downregulation of adhesion molecules and chemokine receptors in vitro. In experimental autoimmune encephalomyelitis (EAE) model, preventive treatment with cordycepin decreased the expression of trafficking factors in the CNS, inhibited the secretion of inflammatory cytokines (IFN-γ, IL-6, TNF-α, and IL-17), and attenuated disease symptoms. A chemokine array indicated that cordycepin treatment reversed the high levels of CCL6, PARRES2, IL-16, CXCL10, and CCL12 in the brain and spinal cord of EAE mice, consistent with the RNA-seq data. Moreover, cordycepin suppressed the release of neuroinflammatory cytokines by activated microglial cells, macrophages, Th17 cells, Tc1 cells, and Th1 cells in vitro. Furthermore, cordycepin treatment exerted therapeutic effects on attenuating the disease severity in the early disease onset stage and late disease progression stage. Our study suggests that cordycepin treatment may not only prevent the occurrence of MS by inhibiting DC activation and migration but also potentially ameliorates the progression of MS by reducing neuroinflammation, which may provide insights into the development of new approaches for the treatment of MS.

Evaluation of Berberine as an Adjunct to TB Treatment.

Tuberculosis (TB) is the global health problem with the second highest number of deaths from a communicable disease after COVID-19. Although TB is curable, poor health infrastructure, long and grueling TB treatments have led to the spread of TB pandemic with alarmingly increasing multidrug-resistant (MDR)-TB prevalence. Alternative host modulating therapies can be employed to improve TB drug efficacies or dampen the exaggerated inflammatory responses to improve lung function. Here, we investigated the adjunct therapy of natural immune-modulatory compound berberine in C57BL/6 mouse model of pulmonary TB. Berberine treatment did not affect Mtb growth in axenic cultures; however, it showed increased bacterial killing in primary murine bone marrow-derived macrophages and human monocyte-derived macrophages. Ad libitum berberine administration was beneficial to the host in combination with rifampicin and isoniazid. Berberine adjunctive treatment resulted in decreased lung pathology with no additive or synergistic effects on bacterial burdens in mice. Lung immune cell flow cytometry analysis showed that adjunctive berberine treatment decreased neutrophil, CD11b+ dendritic cell and recruited interstitial macrophage numbers. Late onset of adjunctive berberine treatment resulted in a similar phenotype with consistently reduced numbers of neutrophils both in lungs and the spleen. Together, our results suggest that berberine can be supplemented as an immunomodulatory agent depending on the disease stage and inflammatory status of the host.

Selenium Modulates the Allergic Response to Whey Protein in a Mouse Model for Cow's Milk Allergy.

Cow's milk allergy is a common food allergy in infants, and is associated with an increased risk of developing other allergic diseases. Dietary selenium (Se), one of the essential micronutrients for humans and animals, is an important bioelement which can influence both innate and adaptive immune responses. However, the effects of Se on food allergy are still largely unknown. In the current study it was investigated whether dietary Se supplementation can inhibit whey-induced food allergy in an animal research model. Three-week-old female C3H/HeOuJ mice were intragastrically sensitized with whey protein and cholera toxin and randomly assigned to receive a control, low, medium or high Se diet. Acute allergic symptoms, allergen specific immunoglobulin (Ig) E levels and mast cell degranulation were determined upon whey challenge. Body temperature was significantly higher in mice that received the medium Se diet 60 min after the oral challenge with whey compared to the positive control group, which is indicative of impaired anaphylaxis. This was accompanied by reductions in antigen-specific immunoglobulins and reduced levels of mouse mast cell protease-1 (mMCP-1). This study demonstrates that oral Se supplementation may modulate allergic responses to whey by decreasing specific antibody responses and mMCP-1 release.

Combination of Ferulic Acid, Ligustrazine and Tetrahydropalmatine attenuates Epithelial-mesenchymal Transformation via Wnt/ß-catenin Pathway in Endometriosis.

Previously the potential therapeutic action of ferulic acid, ligustrazine and tetrahydropalmatine (FLT) are discovered with unclear mechanism in rat autograft endometriosis. However, the effect of FLT on endometrial cells and allograft endometriosis is still unclear. This study is designed to elucidate the influence of FLT on epithelial-mesenchymal transformation in allograft endometriosis and endometrium cells. In vivo, fluorescent xenogeneic endometriosis model was established. In vitro, invasion and metastasis were analyzed after treating FLT. Epithelial-mesenchymal transformation and Wnt/ß-catenin pathway were inspected in vitro and in vivo. Activator or inhibitor of Wnt/ß-catenin signaling was performed to inspect mechanism of epithelial-mesenchymal transformation. In vivo, FLT not only decreased fluorescent intensity and volume of ectopic lesion, but also ameliorated pathological morphology. E2 and PROG levels in serum were reduced by FLT. In endometrial cells, FLT significantly inhibited the invasion and metastasis. Meantime, epithelial-mesenchymal transformation was reversed, accompanied by suppression of Wnt/ß-catenin pathway. In-depth study, activation of Wnt/ß-catenin pathway lead to promotion of epithelial-mesenchymal transformation, which was reversed by FLT. FLT prevented fluorescent allograft endometriosis and endometrium cells, which was related to suppress epithelial-mesenchymal transformation through inactivating Wnt/ß-catenin pathway. The findings disclose molecular mechanism of epithelial-mesenchymal transformation in endometriosis by FLT, and contribute to further application.

Plinia cauliflora (Mart.) Kausel (Jaboticaba) leaf extract: In vitro anti-Trypanosoma cruzi activity, toxicity assessment and phenolic-targeted UPLC-MSE metabolomic analysis.

ETHNOPHARMACOLOGICAL RELEVANCE: Plinia cauliflora (Mart.) Kausel, known as Brazilian grape or jaboticaba, is widely used in Brazilian traditional medicine to treat infectious and inflammatory disorders. However, several aspects of its biological potential remain unclear, such as toxicity and effects on pathogenic protozoa. AIM OF THE STUDY: Investigate the phenolic composition, the in vitro and in silico toxicity profile, and the anti-Trypanosoma cruzi activity of the phenolics-enriched hydromethanolic extract of P. cauliflora leaf. MATERIAL AND METHODS: Phytochemical analysis was performed ultra-performance liquid chromatography-mass spectrometry (UPLC-MSE). Mutagenicity, genotoxicity and eukaryotic cytotoxicity was evaluated by Ames test, cytokinesis-block micronucleus and colorimetric assays, respectively, alongside with a computational prediction of the major compound's pharmacokinetics and toxicity. Anti-T. cruzi activity was investigated on T. cruzi bloodstream trypomastigotes. RESULTS: A total of 14 phenolic compounds were identified, including 11 flavonoids and 2 phenolic acids. No positive response regarding mutagenic potential was detected in Salmonella strains TA97, TA98, TA100, TA102, TA104, both in absence or presence of metabolic activation. The extract induced significant dose-response reduction on nuclear division indexes of HepG2 cells, suggesting cytostatic effects, with no micronuclei induction on cytokinesis-block micronucleus assay. Likewise, it also presented cytotoxic effects, inducing HepG2 and F C3H dose and time dependently cell death through cell membrane damage and more evidently by mitochondrial dysfunction. A dose-response curve of in vitro trypanocidal activity was observed against T. cruzi bloodstream trypomastigotes after 2 and 24 h of exposure. In silico predictions of most abundant compounds' structural alerts, pharmacokinetics and toxicity profile indicates a moderately feasible druglikeness profile and low toxicity for them, which is compatible with in vitro results. CONCLUSIONS: The present study demonstrated that P. cauliflora leaf extract is a potential source of antiparasitic bioactive compounds, however it presents cytotoxic effects in liver cell lines.

Longer-Term Omega-3 LCPUFA More Effective Adjunct Therapy for Tuberculosis Than Ibuprofen in a C3HeB/FeJ Tuberculosis Mouse Model.

Advancement in the understanding of inflammation regulation during tuberculosis (TB) treatment has led to novel therapeutic approaches being proposed. The use of immune mediators like anti-inflammatory and pro-resolving molecules for such, merits attention. Drug repurposing is a widely used strategy that seeks to identify new targets to treat or manage diseases. The widely explored nonsteroidal anti-inflammatory drug (NSAID) ibuprofen and a more recently explored pharmaconutrition therapy using omega-3 long-chain polyunsaturated fatty acids (n-3 LCPUFAs), have the potential to modulate the immune system and are thus considered potential repurposed drugs in this context. These approaches may be beneficial as supportive therapy to the already existing treatment regimen to improve clinical outcomes. Here, we applied adjunct ibuprofen and n-3 LCPUFA therapy, respectively, with standard anti-TB treatment, in a C3HeB/FeJ murine model of TB. Bacterial loads, lung pathology, lung cytokines/chemokines and lung lipid mediators were measured as outcomes. Lung bacterial load on day 14 post-treatment (PT) was lower in the n-3 LCPUFA, compared to the ibuprofen group (p = 0.039), but was higher in the ibuprofen group than the treated control group (p = 0.0315). Treated control and ibuprofen groups had more free alveolar space initially as compared to the n-3 LCPUFA group (4 days PT, p= 0.0114 and p= 0.002, respectively); however, significantly more alveolar space was present in the n-3 LCPUFA group as compared to the ibuprofen group by end of treatment (14 days PT, p = 0.035). Interleukin 6 (IL-6) was lower in the ibuprofen group as compared to the treated control, EPA/DHA and untreated control groups at 4 days PT (p = 0.019, p = 0.019 and p = 0.002, respectively). Importantly, pro-resolving EPA derived 9-HEPE, 11-HEPE, 12-HEPE and 18-HEPE lipid mediators (LMs) were significantly higher in the EPA/DHA group as compared to the ibuprofen and treated control groups. This suggests that n-3 LCPUFAs do improve pro-resolving and anti-inflammatory properties in TB, and it may be safe and effective to co-administer as adjunct therapy with standard TB treatment, particularly longer-term. Also, our results show host benefits upon short-term co-administration of ibuprofen, but not throughout the entire TB treatment course.

Immunostimulatory Activity of Polysaccharides Extracted from Celosia cristata Flowers.

Macrophages play a major role in innate immune responses by producing a variety of immune mediators and cytokines. The stimulation of macrophages by natural products may lead to an enhanced innate immune system. This study evaluated the immunostimulatory effects of a polysaccharide-rich crude fraction of Celosia cristata L. flowers (CCP) on murine macrophages. CCP treatment induced the production of inducible nitric oxide synthase, cyclooxygenase-2, and cytokines by macrophages. Mechanistically, the activation of mitogen-activated protein kinases, NF-κB and toll-like receptor 4 were found to be associated with the stimulatory functions of CCP. CCP was found to be primarily composed of galacturonic acid and glucose in addition to small amounts of arabinose and galactose. This study demonstrated that CCP may enhance the innate immune responses and potentially improve the immune functions in the body.

Characterization, prebiotic and immune-enhancing activities of rhamnogalacturonan-I-rich polysaccharide fraction from molokhia leaves.

Plant-derived polysaccharides possess potential health benefits that improve intestinal health and the immune system. Molokhia leaves have a large amount of mucilage polysaccharide; in the present study, crude polysaccharide extract was prepared from molokhia leaves. The molecular weight of molokhia leaf polysaccharide fraction (MPF) was estimated to be 51.2 × 103 Da. Polysaccharide was methylated and the structure of MPF was mainly composed of rhamnogalacturonan-I structure with side chains, such as galactans and linear glucan (starch), as shown by GC-MS analysis. To study the biofunctional effects of MPF, its prebiotic and intestinal immune-enhancing activities were assayed in vitro. MPF exhibited good prebiotic activity, as shown by its high prebiotic scores, and increased contents of total short-chain fatty acids on five probiotic strains. In addition, MPF showed immune-enhancing activity on Peyer's patches, as revealed by the high bone marrow cell proliferating activity and production of immunoglobulin A and cytokines. These results demonstrate that MPF may be a potential beneficial prebiotic and intestinal immune-enhancer, which may have wide implications in the food industry.

Does the combination of hyperthermia with low LET (linear energy transfer) radiation induce anti-tumor effects equivalent to those seen with high LET radiation alone?

INTRODUCTION: The combination of hyperthermia with low LET (linear energy transfer) radiation may have similar anti-tumor effects as high LET radiation alone. This pre-clinical study determined the optimal heating temperature and time interval between radiation and heat to achieve this equivalent effect. METHODS: C3H mammary carcinomas (200 mm3 in size) growing in the right rear foot of CDF1 mice was used in all experiments. Tumors were locally irradiated with graded doses of either 240 kV ortho- or 6 MV mega-voltage X-rays to produce full dose-response curves. Heating (41.0-43.5 °C; 60 min) was achieved by immersing the tumor bearing foot in a water-bath applied at the same time, or up to 4-hours after, irradiating. The endpoint was the percentage of mice showing local tumor control at 90 days, with enhancements calculated from the ratios of the radiation doses causing 50% tumor control (± 95% confidence intervals). RESULTS: Previous published results in this tumor model reported that carbon ions were 1.3-1.7 times more effective than low LET radiation at inducing tumor control. Similar enhancements occurred with a temperature of only 41.0 °C with a simultaneous heat and radiation treatment. However, higher temperatures were needed with the introduction of any interval; at 42.5 °C, the enhancement was 2.5 with a simultaneous treatment, decreasing to a value within the carbon ion range with a 4-hour interval. CONCLUSIONS: Combining hyperthermia with low LET radiation can be as effective as high LET at inducing tumor control, but the temperature needed depended on the time interval between the two modalities.

Evaluation of the nephrotoxicity and safety of low-dose aristolochic acid, extending to the use of Xixin (Asurum), by determination of methylglyoxal and d-lactate.

ETHNOPHARMACOLOGICAL RELEVANCE: Most Aristolochiaceae plants are prohibited due to aristolochic acid nephropathy (AAN), except Xixin (Asarum spp.). Xixin contains trace amounts of aristolochic acid (AA) and is widely used in Traditional Chinese Medicine. Methylglyoxal and d-lactate are regarded as biomarkers for nephrotoxicity. AIM OF THE STUDY: The use of Xixin (Asarum spp.) is essential and controversial. This study aimed to evaluate tubulointerstitial injury and interstitial renal fibrosis by determining urinary methylglyoxal and d-lactate after withdrawal of low-dose AA in a chronic mouse model. MATERIALS AND METHODS: C3H/He mice in the AA group (n = 24/group) were given ad libitum access to distilled water containing 3 µg/mL AA (0.5 mg/kg/day) for 56 days and drinking water from days 57 to 84. The severity of tubulointerstitial injury and fibrosis were evaluated using the tubulointerstitial histological score (TIHS) and Masson's trichrome staining. Urinary and serum methylglyoxal were determined by high-performance liquid chromatography (HPLC); urinary d-lactate were determined by column-switching HPLC. RESULTS: After AA withdrawal, serum methylglyoxal in the AA group increased from day 56 (429.4 ± 48.3 µg/L) to 84 (600.2 ± 99.9 µg/L), and peaked on day 70 (878.3 ± 171.8 µg/L; p < 0.05); TIHS and fibrosis exhibited similar patterns. Urinary methylglyoxal was high on day 56 (3.522 ± 1.061 µg), declined by day 70 (1.583 ± 0.437 µg) and increased by day 84 (2.390 ± 0.130 µg). Moreover, urinary d-lactate was elevated on day 56 (82.10 ± 18.80 µg) and higher from day 70 (201.10 ± 90.82 µg) to 84 (193.28 ± 61.32 µg). CONCLUSIONS: Methylglyoxal is induced after AA-induced tubulointerstitial injury, so methylglyoxal excretion and metabolism may be a detoxification and repair strategy. A low cumulative AA dose is the key factor that limits tubulointerstitial injury and helps to repair. Thus, AA-containing herbs, especially Xixin, should be used at low doses for short durations (less than one month).

2,3,5,6-Tetramethylpyrazine protects retinal photoreceptors against endoplasmic reticulum stress by modulating ATF4-mediated inhibition of PRP aggregation.

Endoplasmic reticulum (ER) stress is a common threat to photoreceptors during the pathogenesis of chronic retinopathies and often results in irreversible visual impairment. 2,3,5,6-Tetramethylpyrazine (TMP), which possesses many beneficial pharmacological activities, is a potential drug that could be used to protect photoreceptors. In the present study, we found that the cellular growth rate of 661 W cells cultured under low glucose conditions was lower than that of control cells, while the G2/M phase of the cell cycle was longer. We further found that the mitochondrial membrane potential (ΔΨm) was lower and that ER stress factor expression was increased in 661 W cells cultured under low glucose conditions. TMP reversed these trends. Visual function and cell counts in the outer nuclear layer (ONL) were low and the TUNEL-positive rate in the ONL was high in a C3H mouse model of spontaneous retinal degeneration. Similarly, visual function was decreased, and the TUNEL-positive rate in the ONL was increased in fasted C57/BL6j mice compared with control mice. On the other hand, ER stress factor expression was found to be increased in the retinas of both mouse models, as shown by reverse transcription real-time PCR (RT-qPCR) and western blotting. TMP reversed the physiological and molecular biological variations observed in both mouse models, and ATF4 expression was enhanced again. Further investigation by using western blotting illustrated that the proportion of insoluble prion protein (PRP) versus soluble PRP was reduced both in vitro and in vivo. Taken together, these results suggest that TMP increased the functions of photoreceptors by alleviating ER stress in vitro and in vivo, and the intrinsic mechanism was the ATF4-mediated inhibition of PRP aggregation. TMP may potentially be used clinically as a therapeutic agent to attenuate the functional loss of photoreceptors during the pathogenesis of chronic retinopathies. KEY MESSAGES: • Already known: TMP is a beneficial drug mainly used in clinic to enhance organ functions, and the intrinsic mechanism is still worthy of exploring. • New in the study: We discovered that TMP ameliorated retinal photoreceptors function via ER stress alleviation, which was promoted by ATF4-mediated inhibition of PRP aggregation. • Application prospect: In prospective clinical practices, TMP may potentially be used in the clinic as a therapeutic agent to attenuate the photoreceptors functional reduction in chronic retinopathies.

Exercise Performance Upregulatory Effect of R-α-Lipoic Acid with γ-Cyclodextrin.

α-Lipoic acid (ALA) is a vitamin-like substance that is an indispensable supporting factor for a large number of enzymes. Due to its optical activity, ALA has optical isomers RALA and SALA. The major role of RALA is in energy metabolism. However, RALA cannot be used as a pharmaceutical or nutraceutical because it is sensitive to heat and acid conditions. Previous studies have shown that RALA complexed with γ-cyclodextrin (CD) has a higher antioxidant capacity than that of free RALA. The antioxidant enzyme system protects against intense exercise-induced oxidative damage and is related to the physical status of athletes. The aim of this study was to examine the effect of CD/RALA complex supplementation on antioxidant activity and performance during high-intensity exercise. Twenty-four male C3H/HeSlc mice were divided into four groups (n = 6): swimming+distilled water administration (C), swimming+CD/RALA supplementation (CD/RALA), swimming+RALA suplementation (RALA), and swimming+CD supplementation (CD). Blood ammonia elevation due to exercise stress was repressed by CD/RALA supplementation. The oxidative stress in the kidney increased after exercise and was reduced by CD/RALA supplementation. Our findings suggest that CD/RALA supplementation may be useful for improving the exercise performance in athletes.

Catechin-Rich Green Tea Extract and the Loss-of-TLR4 Signaling Differentially Alter the Hepatic Metabolome in Mice with Nonalcoholic Steatohepatitis.

SCOPE: Catechin-rich green tea extract (GTE) limits inflammation in nonalcoholic steatohepatitis (NASH) consistent with a Toll-like receptor 4 (TLR4)-dependent mechanism. It is hypothesized that GTE supplementation during NASH will shift the hepatic metabolome similar to that attributed to the loss-of-TLR4 signaling. METHODS AND RESULTS: Wild-type (WT) and loss-of-function TLR4-mutant (TLR4mut ) mice are fed a high-fat diet containing 0% or 2% GTE for 8 weeks prior to performing untargeted mass spectrometry-based metabolomics on liver tissue. The loss-of-TLR4 signaling and GTE shift the hepatic metabolome away from that of WT mice. However, relatively few metabolites are altered by GTE in WT mice to the same extent as the loss-of-TLR4 signaling in TLR4mut mice. GTE increases acetyl-coenzyme A precursors and spermidine to a greater extent than the loss-of-TLR4 signaling. Select metabolites associated with thiol metabolism are similarly affected by GTE and the loss-of-TLR4 signaling. Glycerophospholipid catabolites are decreased by GTE, but are unaffected in TLR4mut mice. Conversely, the loss-of-TLR4 signaling but not GTE increases several bile acid metabolites. CONCLUSION: GTE limitedly alters the hepatic metabolome consistent with a TLR4-dependent mechanism. This suggests that the anti-inflammatory activities of GTE and loss-of-TLR4 signaling that regulate hepatic metabolism to abrogate NASH are likely due to distinct mechanisms.

Polysaccharides extract from Vaccaria segetalis seeds inhibits kidney infection by regulating cathelicidin expression.

ETHNOPHARMACOLOGICAL RELEVANCE: According to the Chinese Pharmacopoeia, the seeds of Vaccaria segetalis, a traditional medicinal herb, can be used for treating urinary diseases. The polysaccharides extract from V. segetalis seeds (VSP) has been shown to prevent urinary tract infections (UTIs). AIM OF THE STUDY: Investigate the effects of VSP on treating kidney infection induced by uropathogenic Escherichia coli (UPEC) and the underlying mechanisms. MATERIALS AND METHODS: Both in vivo and in vitro infection models were established with the UPEC strain CFT073. After oral administration of VSP, the levels of bacterial load, cathelicidin (CRAMP), Toll-like receptors (TLRs) in the kidney were evaluated. The expression of cathelicidin (LL-37) in human renal cell carcinoma cell line (A498) was tested after the treatment of VSP. RESULTS: In the kidneys of infection models, high-titer bacteria was detected. In the kidney of rat model, the expression of CRAMP was down-regulated, no significant change was observed in the levels of TLRs. After oral administration of VSP, the bacterial load was significantly decreased in rat and mouse models, and the levels of CRAMP and TLRs were significantly up-regulated in rat model. In vitro, the expression of LL-37 was significantly inhibited by CFT073. VSP up-regulated the expression of LL-37 in A498 cells. CONCLUSIONS: The up-regulation of cathelicidin expression may contribute to the therapeutic effects of VSP on kidney infection.

The antiinflammatory effects of Xuefu Zhuyu decoction on C3H/HeJ mice with alopecia areata.

BACKGROUND: As a traditional and typical prescription of prominently activating blood circulation to remove blood stasis, Xuefu Zhuyu decoction (XZD) consists of 15 kinds of herbal medicine. Clinical investigations have showed that XZD could significantly promote the new hair generation of alopecia areata (AA) patients characterized by Qi stagnation and blood stasis. PURPOSE: The purpose of this study was executed to determine whether the mechanisms by which XZD stimulated newborn hair were related to its anti-inflammatory effects. METHODS: Clinical AA individuals were recruited to confirm the efficies of XZD. High performance liquid chromatography (HPLC) analysis was performed to qualitatively and quantitatively determine the contents of 15 compounds in XZD. Schrodinger molecular docking and in vivo surface plasmon resonance (SPR) techniques were used to evaluate the potential binding properties of compounds to target proteins. C3H/HeJ mice were randomly assigned to groups control, AA, and the XZD administration (6.5, 13.0 and 26.0 g/kg/d). Except for mice in control group, all the mice in the other groups were treated with a 21-day chronic unpredictable mild stress (CUMS) induced AA. Hematoxylin-eosin (H&E) staining was performed to determine the degree of pathological damage to the skin. Enzyme-linked immunosorbent assay (ELISA) was performed to detect levels of interleukin-6 (IL-6), interleukin-1 beta (IL-1ß) and tumor necrosis factor alpha (TNF-α) and in serum and skin tissues. Western blot, immunohistochemistry and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) were used to examine the expression levels of IL-6, IL-1ß, TNF-α and osteopontin proteins and genes in skin tissues. RESULTS: XZD could visibly promote hair regeneration of AA patients. The potential active ingredients in XZD prescription included at least amygdalin, hydroxysafflor yellow A, kaempferide, ferulic acid, catalpol, verbascoside, ß-ecdysone, platycodin D, paeoniflorin, naringin, neohesperidin, liquiritin, glycyrrhizic acid, saikosaponin A and saikosaponin D. The results of molecular docking and SPR analysis showed that verbascoside, liquiritin, kaempferide and amygdalin showed the best potential binding properties with IL-6, IL-1ß, TNF-α and osteopontin, respectively. Pathological evaluation showed that compared with the CUMS group, the administration of XZD significantly promoted hair regeneration, evidenced by increased number of skin hair follicles in C3H/HeJ AA mice. Compared with control group, ELISA data showed that the levels of IL-6, IL-1ß and TNF-α in serum and skin tissues of CUMS induced AA mice were significantly increased, while XZD administration dramatically restrained the contents of the three pro-inflammatory factors. Western blot, immunohistochemistry, and qRT-PCR results further demonstrated that XZD administration notably down-regulated the protein and gene expression levels of osteopontin, IL-6, IL-1ß and TNF-α in comparation with CUMS group. CONCLUSION: XZD could dramatically ameliorate CUMS-induced AA damage in the skin of C3H/HeJ mice, possibly by suppressing the levels of IL-6, IL-1ß, TNF-α and osteopontin.