Pharmacological inhibition of demethylzeylasteral on JAK-STAT signaling ameliorates vitiligo.

BACKGROUND: The activation of CD8+ T cells and their trafficking to the skin through JAK-STAT signaling play a central role in the development of vitiligo. Thus, targeting this key disease pathway with innovative drugs is an effective strategy for treating vitiligo. Natural products isolated from medicinal herbs are a useful source of novel therapeutics. Demethylzeylasteral (T-96), extracted from Tripterygium wilfordii Hook F, possesses immunosuppressive and anti-inflammatory properties. METHODS: The efficacy of T-96 was tested in our mouse model of vitiligo, and the numbers of CD8+ T cells infiltration and melanocytes remaining in the epidermis were quantified using whole-mount tail staining. Immune regulation of T-96 in CD8+ T cells was evaluated using flow cytometry. Pull-down assay, mass spectrum analysis, molecular docking, knockdown and overexpression approaches were utilized to identify the target proteins of T-96 in CD8+ T cells and keratinocytes. RESULTS: Here, we found that T-96 reduced CD8+ T cell infiltration in the epidermis using whole-mount tail staining and alleviated the extent of depigmentation to a comparable degree of tofacitinib (Tofa) in our vitiligo mouse model. In vitro, T-96 decreased the proliferation, CD69 membrane expression, and IFN-γ, granzyme B, (GzmB), and perforin (PRF) levels in CD8+ T cells isolated from patients with vitiligo. Pull-down assays combined with mass spectrum analysis and molecular docking showed that T-96 interacted with JAK3 in CD8+ T cell lysates. Furthermore, T-96 reduced JAK3 and STAT5 phosphorylation following IL-2 treatment. T-96 could not further reduce IFN-γ, GzmB and PRF expression following JAK3 knockdown or inhibit increased immune effectors expression upon JAK3 overexpression. Additionally, T-96 interacted with JAK2 in IFN-γ-stimulated keratinocytes, inhibiting the activation of JAK2, decreasing the total and phosphorylated protein levels of STAT1, and reducing the production and secretion of CXCL9 and CXCL10. T-96 did not significantly inhibit STAT1 and CXCL9/10 expression following JAK2 knockdown, nor did it suppress upregulated STAT1-CXCL9/10 signaling upon JAK2 overexpression. Finally, T-96 reduced the membrane expression of CXCR3, and the culture supernatants pretreated with T-96 under IFN-γ stressed keratinocytes markedly blocked the migration of CXCR3+CD8+ T cells, similarly to Tofa in vitro. CONCLUSION: Our findings demonstrated that T-96 might have positive therapeutic responses to vitiligo by pharmacologically inhibiting the effector functions and skin trafficking of CD8+ T cells through JAK-STAT signaling.

Negative associations between folate and bacterial vaginosis in the NHANES 2001 to 2004.

BACKGROUND: Bacterial vaginosis (BV) is one of the most common infections among women of reproductive age and accounts for 15-50% of infections globally. The role played by folate in the pathogenesis and progression of BV is poorly understood. The aim of this study was to investigate the association between serum folate, red blood cell (RBC) folate, and BV in American women. METHODS: 1,954 participants from the 2001-2004 National Health and Nutrition Examination Survey (NHANES) program were included in this study. Multiple logistic regression was used to analyze the association between serum folate, RBC folate, and BV, and covariates including race, age, education level, and body mass index were used to construct adjusted models. Stratified analysis was used to explore the stability of the above associations in different populations. RESULTS: In the present cross-sectional study, we found that serum folate and RBC folate were inversely associated with the risk of BV. In the fully adjusted model, the risk of BV was reduced by 35% (OR=0.65, 95% CI: 0.51~0.83, p=0.0007) in the highest serum folate group and 32% (OR=0.68, 95% CI: 0.53~0.87, p=0.0023) in the highest RBC folate group compared to the lowest group. CONCLUSIONS: The results of this study indicated that serum folate and RBC folate were inversely associated with the risk of BV folate supplementation may play an important role in the prevention and management of BV.

Metabolomics Signature and Potential Application of Serum Polyunsaturated Fatty Acids Metabolism in Patients With Vitiligo.

Vitiligo is a depigmented skin disorder caused by a variety of factors, including autoimmune, metabolic disturbance or their combined effect, etc. Non-targeted metabolomic analyses have denoted that dysregulated fatty acids metabolic pathways are involved in the pathogenesis of vitiligo. However, the exact category of fatty acids that participate in vitiligo development and how they functionally affect CD8+ T cells remain undefined. We aimed to determine the difference in specific fatty acids among vitiligo patients and healthy individuals and to investigate their association with clinical features in patients with vitiligo. Serum levels of fatty acids in 48 vitiligo patients and 28 healthy individuals were quantified by performing ultra-performance liquid chromatography-tandem mass spectrometry. Univariate and multivariate analyses were carried out to evaluate the significance of differences. Moreover, flow cytometry was used to explore the effect of indicated fatty acids on the function of CD8+ T cells derived from patients with vitiligo. We demonstrated that serological level of alpha-linolenic acid (ALA) was markedly upregulated, while that of arachidonic acid (ARA), arachidic acid (AA) and behenic acid were significantly downregulated in patients with vitiligo. Moreover, ALA levels were positively associated with vitiligo area scoring index (VASI) and ARA was a probable biomarker for vitiligo. We also revealed that supplementation with ARA or nordihydroguaiaretic acid (NDGA) could suppress the function of CD8+ T cells. Our results showed that vitiligo serum has disorder-specific phenotype profiles of fatty acids described by dysregulated metabolism of polyunsaturated fatty acids. Supplementation with ARA or NDGA might promote vitiligo treatment. These findings provide novel insights into vitiligo pathogenesis that might add to therapeutic options.

Sesamol supplementation alleviates nonalcoholic steatohepatitis and atherosclerosis in high-fat, high carbohydrate and high-cholesterol diet-fed rats.

Sesamol, a major ingredient in sesame seeds (Sesamum indicum L.) and its oil, is considered a powerful functional food ingredient. However, few studies have investigated its effects on high-fat, high carbohydrate and high-cholesterol (HF-HCC) diet-induced nonalcoholic steatohepatitis (NASH) complicated with atherosclerosis. The present study elucidates the protective effects of sesamol against NASH and atherosclerosis in HF-HCC diet-fed rats. Sprague-Dawley rats were supplemented with or without sesamol in drinking water (0.05 mg mL-1, 0.1 mg mL-1 and 0.2 mg mL-1) from the beginning to end. At the end of the experiment, sesamol supplementation suppressed HF-HCC diet-induced body weight gain and increased absolute liver and adipose tissue weights in rats. Serum biochemical analyses showed that sesamol supplementation improved HF-HCC diet-induced metabolism disorders and damaged vascular endothelial function. Histological examinations displayed that dietary sesamol not only alleviated hepatic balloon degeneration, steatosis, inflammation and fibrosis, but also mitigated lipid accumulation and fibrous elements in the aorta arch in HF-HCC diet-fed rats. In addition, sesamol supplementation inhibited hepatic NOD-like receptor protein 3 (NLRP3) expression and ERS-IRE1 signaling pathway activation. Moreover, sesamol treatment decreased uric acid levels both in serum and the liver by its effect on the inhibition of xanthine oxidase (XO) activity and/or its expression, which might be closely associated with the inhibitions of NLRP3 expression and ERS-IRE1 signaling pathway activation in HF-HCC diet-fed rats. These findings demonstrated that sesamol alleviated NASH and atherosclerosis in HF-HCC diet-fed rats, and may be a potent dietary supplement for protection against these diseases.

A fucoidan from Sargassum fusiforme with novel structure and its regulatory effects on intestinal microbiota in high-fat diet-fed mice.

A fucoidan SFP, having novel structure, was extracted from Sargassum fusiforme. It had a molecular weight of 703 kDa and was composed of fucose and galactose with the ratio of 73.16:26.84 (mol%). Structural analyses showed that it mainly consisted of 1,3-, 1,4-, 1,3,4-linked-α-l-Fucp and 1,3-, 1,6-linked-ß-d-Galp, with partial sulfation at C-4, C-3 of fucose units and C-6, C-3 of galactose units. The branches consisted of sulfated fucosyl and galactofucosyl oligosaccharides. The regulatory effects of SFP on the intestinal microbiota in high-fat diet-fed mice were investigated. The high-dosage SFP exhibited good hypolipidemic effects, especially in regulating the high-densitylipoproteincholesterol, non-esterified fatty acid levels and lipase activity. It also significantly decreased the ratio of phyla Firmicutes/Bacteroidetes (P < 0.05). Besides, SFP had certain effects on the richness and diversity of intestinal microbiota. Therefore, SFP exhibited novel structure and certain beneficial effects on the disorder of intestinal microbiota in high-fat diet-fed mice.

Self-Propelled Active Photothermal Nanoswimmer for Deep-Layered Elimination of Biofilm In Vivo.

Increasing penetration of antibacterial agents into biofilm is a promising strategy for improvement of therapeutic effect and slowdown of the progression of antibiotic resistance. Herein, we design a near-infrared (NIR) light-driven nanoswimmer (HSMV). Under NIR light irradiation, HSMV performs efficient self-propulsion and penetrates into the biofilm within 5 min due to photothermal conversion of asymmetrically distributed AuNPs. The localized thermal (∼45 °C) and thermal-triggered release of vancomycin (Van) leads to an efficient combination of photothermal therapy and chemotherapy in one system. The active motion of HSMV increases the effective distance of photothermal therapy (PTT) and also improves the therapeutic index of the antibiotic, resulting in superior biofilm removal rate (>90%) in vitro. Notably, HSMV can eliminate S. aureus biofilms grown in vivo under 10 min of laser irradiation without damage to healthy tissues. This work may shed light on therapeutic strategies for in vivo treatment of biofilm-associated infections.

Homocysteine induces melanocytes apoptosis via PERK-eIF2α-CHOP pathway in vitiligo.

Vitiligo is a depigmentation disorder that develops as a result of the progressive disappearance of epidermal melanocytes. The elevated level of amino acid metabolite homocysteine (Hcy) has been identified as circulating marker of oxidative stress and known as a risk factor for vitiligo. However, the mechanism underlying Hcy-regulated melanocytic destruction is currently unknown. The present study aims to elucidate the effect of Hcy on melanocytic destruction and its involvement in the pathogenesis of vitiligo. Our results showed that Hcy level was significantly elevated in the serum of progressive vitiligo patients. Notably, Hcy induced cell apoptosis in melanocytes via activating reactive oxygen species (ROS) and endoplasmic reticulum (ER) stress protein kinase RNA-like ER kinase (PERK)-eukaryotic translation initiation factor 2α (eIF2α)-C/EBP homologous protein (CHOP) pathway. More importantly, folic acid, functioning in the transformation of Hcy, could lower the intracellular Hcy level and further reverse the apoptotic effect of Hcy on melanocytes. Additionally, Hcy disrupted melanogenesis whereas folic acid supplementation could reverse the melanogenesis defect induced by Hcy in melanocytes. Taken together, Hcy is highly increased in vitiligo patients at progressive stage, and our in vitro studies revealed that folic acid could protect melanocytes from Hcy-induced apoptosis and melanin synthesis inhibition, indicating folic acid as a potential benefit agent for patients with progressive vitiligo.

Ginkgo biloba extract protects human melanocytes from H2 O2 -induced oxidative stress by activating Nrf2.

Vitiligo is a common skin depigmenting disorder characterized by the loss of functional melanocytes. Its pathogenesis is complicated and oxidative stress plays a critical role in the development of vitiligo. Thus, antioxidant therapy is a promising therapeutic strategy to prevent or even reverse the progression of depigmentation. Ginkgo biloba extract EGb761 has been confirmed to have protective effects on neurons against oxidative stress. Notably, several clinical trials have shown that patients with stable vitiligo achieved repigmentation after taking EGb761. However, the exact mechanism underlying the protective effects of EGb761 on melanocytes against oxidative stress has not been fully elucidated. In the present study, we found that EGb761 effectively protected melanocytes against oxidative stress-induced apoptosis and alleviated the excessive accumulation of reactive oxygen species (ROS) and lipid peroxidation by enhancing the activity of antioxidative enzymes. Furthermore, the antioxidative effect of EGb761 was achieved by activating Nrf2 and its downstream antioxidative genes. In addition, interfering Nrf2 with siRNA abolished the protective effects of EGb761 on melanocytes against oxidative damage. In conclusion, our study proves that EGb761 could protect melanocytes from H2 O2 -induced oxidative stress by activating Nrf2. Therefore, EGb761 is supposed to be a potential therapeutic agent for vitiligo.

Baicalein protects human vitiligo melanocytes from oxidative stress through activation of NF-E2-related factor2 (Nrf2) signaling pathway.

Vitiligo is a complex disorder characterized by patchy loss of skin pigmentation due to abnormal melanocyte function. Overwhelming evidences have suggested that oxidative stress plays a major role in the loss of melanocytes thereby mediating the onset and progression of vitiligo. The nuclear factor erythroid 2-like factor 2 (Nrf2) is a master regulator of cellular redox homeostasis and the activation of Nrf2 signaling pathway is impaired in the vitiligo melanocytes. Baicalein, as flavonoid extracted from the Scutellaria baicalensis, has been proved to possess the ability to activate Nrf2 signaling pathway in other cell types and mouse model. Our previous data found that baicalein exerts a cytoprotective role in H2O2-induced apoptosis in human melanocytes cell line (PIG1). Based on these founding, we hypothesized that baicalein activates Nrf2 signaling pathway, alleviates H2O2-induced mitochondrial dysfunction and cellular damage, thereby protecting human vitiligo melanocytes from oxidative stress. In the present study, we found that baicalein effectively inhibited H2O2-induced cytotoxicity and apoptosis in human vitiligo melanocytes (PIG3V). Further results demonstrated that baicalein promoted Nrf2 nucleus translocation as well as up-regulated the expression of Nrf2 and its target gene, heme oxygenase-1 (HO-1). Moreover, the protective effects of baicalein against H2O2-induced cellular damage and apoptosis as well as mitochondrial dysfunction were abolished by Nrf2 knockdown. Additionally, we observed that Nrf2 knockdown suppressed proliferation and increased the sensitivity of PIG3V cells to H2O2 treatment. Finally, we explored the mechanism of baicalein associated with Nrf2 activation and found that the phosphorylation of Nrf2 as well as ERK1/2and PI3K/AKT signaling were not involved in the baicalein-induced activation of Nrf2. Taken together, these data clearly suggest that baicalein enhances cellular antioxidant defense capacity of human vitiligo melanocytes through the activation of the Nrf2 signaling pathway, providing beneficial evidence for the application of baicalein in the vitiligo treatment.

Combined treatment with sorafenib and silibinin synergistically targets both HCC cells and cancer stem cells by enhanced inhibition of the phosphorylation of STAT3/ERK/AKT.

Silibinin, a nontoxic bioactive component in milk thistle, is used as a liver-protective drug in the clinic mainly because of its antioxidant and anti-inflammation activities. In this study, we studied the cytotoxic effects of silibinin combined with sorafenib on hepatocellular carcinoma (HCC). The results indicated that silibinin combined with sorafenib potently inhibited the proliferation of various HCC cells and induced significant apoptosis. In an HCC subcutaneous transplantation tumor model, the combination of silibinin and sorafenib significantly suppressed tumor growth compared with monotherapy. As determined by fluorescence staining and Western blots, the combination of the two drugs inhibited the phosphorylation of RAC-alpha serine/threonine-protein kinase (AKT) and signal transducer and activator of transcription 3 (STAT3) together with the expression of antiapoptotic proteins including myeloid leukemia cell differentiation protein Mcl-1 (Mcl-1) and apoptosis regulator Bcl-2 (Bcl-2), resulting in the death of cancer cells. We also found that the combination inhibited the formation and self-renewal of HCC stem cells by down-regulating the expression of stemness-related proteins, such as Homeobox protein NANOG (Nanog) and Krueppel-like factor 4 (Klf4). These results suggested that silibinin improved the efficacy of sorafenib in HCC therapy, indicating a clinical promising therapeutic strategy for HCC patients.

[Blink restoration by the functional electrical stimulation in unilateral facial nerve palsy rabbits].

OBJECTIVE: Tocompare the effects of different waveforms and parameters of electrical stimulation to elicit a blink, and construct a functional electrical stimulation (FES) system to restore synchronous blink in unilateral facial nerve palsy (FNP). METHODS: Firstly, twenty-four rabbits were surgically induced unilateral FNP and were divided into three groups, who received square, sine and triangle pulse wareforms, respectirely. Both the healthy and the paralysis eyelids of the rabbits received pulse train stimulation to produce a blink in both eyes. For each rabbit, twenty-seven combinations of frequencies (25 Hz, 50 Hz and 100 Hz) and nine pulse widths (1-9 ms) were stimulated. The threshold amplitude and electric charge to elicit a blink was compared between different waveforms and different parameters. Secondly, a FES system was constructed to treat six surgically induced unilateral FNP rabbit chosen in the twenty-four rabbits, it consisted by an electromyogram (EMG) amplifier module which record the EMG of the healthy muscle, and a stimulator which received the EMG input and output a pulse train stimulation when triggered by the EMG. RESULTS: When the carrier frequency of the pulse train was 25 Hz, it was not able to induce a smooth blink. However, when the carrier frequencies were 50 Hz and 100 Hz, a smooth blink could be induced. The voltage required by 100 Hz was lower than 50 Hz, but it cost more electric charge. The amplitude that square waveforms required was far lower than sine and triangle, but the electric charge between the three waveforms was similar. Synchronous blink could be restored in the six unilateral FNP rabbits with the FES system. CONCLUSIONS: To elicit a blink, square pulse train delivered in 50 Hz is a preferable option. The motion of the healthy eyelids as a source of information for stimulation of the paralyzed sides can restore the synchronous blink in unilateral FNP rabbits.

[Genetic transformation of Pinellia ternata with Agrobacterium tumefaciens-mediated sHSP genes].

OBJECTIVE: To establish an efficient genetic transformation system of Pinellia ternata. METHOD: With petioles from test-tube seedlings of P. ternata as explants, Agrobacterium tumefaciens mediation method was adopted to explore the effect of phenolic substances, A. tumefaciens's concentration, infection time, pre-incubation time and co-cultivation time on genetic transformation efficiency of P. ternata. RESULT AND CONCLUSION: The genetic transformation efficiency could be effectively enhanced by infecting in A. tumefaciens culture containing AS 40 mg x L(-1) for 15 min for three days. The petioles were put into the differentiation medium containing 150 mg x L(-1) Kan and 350 mg x L(-1) Carb to screening and cultivation. After around 30 days, microtubers could be observed at both sides of the petioles. Gus staining and PCR verification on the regenerated plants showed that the exogenous gene sHSP had been integrated into genome of P. ternata.