[Xixin Decoction regulates Th17/Treg balance and transcription factor expression in senescence-accelerated mouse-prone].

This study aims to investigate the effect of Xixin Decoction on the T helper 17 cell(Th17)/regulatory T cell(Treg) ba-lance of intestinal mucosa and the expression of related transcription factors in the senescence-accelerated mouse-prone 8(SAMP8) model. Fifty 14-week male mice of SAMP8 were randomized by the random number table method into model group, probiotics group, and high-, medium-, and low-dose Xixin Decoction groups, with 10 mice in each group. Ten 14-week male mice of senescence-acce-lerated mouse-resistant 1(SAMR1) served as control group. After 10 weeks of feeding, the mice were administrated with correspon-ding drugs for 10 weeks. Morris water maze test was carried out to examine the learning and memory abilities of mice. Enzyme-linked immunosorbent assay(ELISA) was employed to determine the content of secretory immunoglobulin A(SIgA) in the intestinal mucosa, and flow cytometry to detect the percentage content of Th17 and Treg in the intestinal mucosa. Western blot was performed to determine the protein levels of retinoid-related orphan receptor gamma t(RORγt) and forkhead box p3(Foxp3) in the mouse colon tissue. Compared with control group, the escape latency of mice in model group was significantly prolonged(P<0.01), and the number of times of crossing the platform and the residence time in the target quadrant were significantly reduced within 60 s(P<0.01), intestinal mucosal SIgA content was significantly decreased(P<0.01), Th17 content was increased(P<0.05), Treg content was decreased(P<0.01), the expression of RORγt protein was increased and Foxp3 protein was decreased in colon(P<0.01). Compared with the model group, high-dose Xixin Decoction group improved the learning and memory ability(P<0.05 or P<0.01). Probiotics group and high-and medium-dose Xixin Decoction group increased the content of SIgA in intestinal mucosa(P<0.05 or P<0.01), decreased percentage content of Th17 and increased the percentage content of Treg in intestinal mucosa(P<0.05 or P<0.01). Furthermore, they down-regulated the protein level of RORγt and up-regulated the protein level of Foxp3 in the intestinal mucosa(P<0.01). In conclusion, Xixin Decoction may act on intestinal mucosal immune barrier, affect gut-brain information exchange, and improve the learning and memory ability of SAMP8 by promoting SIgA secretion and regulating the Th17/Treg balance and the expression of RORγt and Foxp3.

The muscle regeneration marker FOXP3 is associated with muscle injury in Duchenne muscular dystrophy.

BACKGROUND: In Duchenne muscular dystrophy (DMD), the immune system cells (ISC) synthesize molecules to regulate inflammation, a process needed to regenerate muscle. The relationship between those molecules and the muscle injury is unknown. Monocytes belonging to ISC are regulated by omega-3 fatty acids (ω-3 LCPUFAs) in DMD, but whether those fatty acids influence other ISC like T-cells is unknown. OBJECTIVE: We analyzed the expression of the muscle regeneration markers (FOXP3 and AREG) in circulating leukocytes of DMD patients with different lower limb muscle functions and whether ω-3 LCPUFAs regulate the expression of those markers, and the populations of circulating T-cells, their intracellular cytokines, and disease progression (CD69 and CD49d) markers. METHODS: This placebo-controlled, double-blind, randomized study was conducted in DMD boys supplemented with ω-3 LCPUFAs (n = 18) or placebo (sunflower oil, n = 13) for six months. FOXP3 and AREG mRNA expression in leukocytes, immunophenotyping of T-cell populations, CD49d and CD69 markers, and intracellular cytokines in blood samples were analyzed at baseline and months 1, 2, 3, and 6 of supplementation. RESULTS: Patients with assisted ambulation expressed higher (P = 0.015) FOXP3 mRNA levels than ambulatory patients. The FOXP3 mRNA expression correlated (Rho = -0.526, P = 0.03) with the Vignos scale score at month six of supplementation with ω-3 LCPUFAs. CD49d + CD8 + T-cells population was lower (P = 0.037) in the ω -3 LCPUFAs group than placebo at month six of supplementation. CONCLUSION: FOXP3 is highly expressed in circulating leukocytes of DMD patients with the worst muscle function. Omega-3 LCPUFAs might modulate the synthesis of the adhesion marker CD49d + CD8 + T-cells, but their plausible impact on FOXP3 needs more research.

Distal renal tubular acidosis as presenting manifestation of Wilson disease in a 11-year-old girl.

A 11-year-old girl was referred to the pediatric nephrology services of our hospital for evaluation of vitamin-D-refractory rickets. She was born to second-degree consanguineous parents. On examination, she had wrist widening and bilateral genu varum. She had normal anion gap metabolic acidosis, hypokalemia, and hyperchloremia. The fractional excretion of bicarbonate was 3% and the urine anion gap was positive. She also had hypercalciuria, but no phosphaturia, glucosuria or aminoaciduria. In view of a family history of an elder sister having rigidity with cognitive and speech impairment, an ophthalmic evaluation by slit lamp examination was performed in the index case that revealed bilateral Kayser-Fleischer rings. Serum ceruloplasmin was low and 24-h urine copper was elevated in the index case. Whole exome sequencing unveiled a novel pathogenic variant in exon 2 of the ATP7B gene (chr13: c.470del; Depth: 142x) (homozygous) that resulted in a frameshift and premature truncation of the protein, 15 amino acids downstream to codon 157 (p. Cys157LeufsTer15; NM_000053.4) confirming Wilson disease. There were no mutations in the ATP6V0A4, ATP6V1B1, SLC4A1, FOXI1, WDR72 genes or other genes that are known to cause distal RTA. Therapy with D-penicillamine and zinc supplements was initiated. A low dose of 2.5 mEq/kg/day of potassium citrate supplementation normalized the serum bicarbonate levels. This case was notable for the absence of hepatic or neurological involvement at admission. Wilson disease is well known to cause proximal renal tubular acidosis and Fanconi syndrome, with relatively lesser involvement of the distal renal tubules in the literature. However, isolated distal renal tubular involvement as presenting manifestation of Wilson disease (without hepatic or neurological involvement) is rare and can lead to diagnostic confusion.

Thalamic Foxp2 regulates output connectivity and sensory-motor impairments in a model of Huntington's Disease.

BACKGROUND: Huntington's Disease (HD) is a disorder that affects body movements. Altered glutamatergic innervation of the striatum is a major hallmark of the disease. Approximately 30% of those glutamatergic inputs come from thalamic nuclei. Foxp2 is a transcription factor involved in cell differentiation and reported low in patients with HD. However, the role of the Foxp2 in the thalamus in HD remains unexplored. METHODS: We used two different mouse models of HD, the R6/1 and the HdhQ111 mice, to demonstrate a consistent thalamic Foxp2 reduction in the context of HD. We used in vivo electrophysiological recordings, microdialysis in behaving mice and rabies virus-based monosynaptic tracing to study thalamo-striatal and thalamo-cortical synaptic connectivity in R6/1 mice. Micro-structural synaptic plasticity was also evaluated in the striatum and cortex of R6/1 mice. We over-expressed Foxp2 in the thalamus of R6/1 mice or reduced Foxp2 in the thalamus of wild type mice to evaluate its role in sensory and motor skills deficiencies, as well as thalamo-striatal and thalamo-cortical connectivity in such mouse models. RESULTS: Here, we demonstrate in a HD mouse model a clear and early thalamo-striatal aberrant connectivity associated with a reduction of thalamic Foxp2 levels. Recovering thalamic Foxp2 levels in the mouse rescued motor coordination and sensory skills concomitant with an amelioration of neuropathological features and with a repair of the structural and functional connectivity through a restoration of neurotransmitter release. In addition, reduction of thalamic Foxp2 levels in wild type mice induced HD-like phenotypes. CONCLUSIONS: In conclusion, we show that a novel identified thalamic Foxp2 dysregulation alters basal ganglia circuits implicated in the pathophysiology of HD.

CangFu Daotan decoction improves polycystic ovarian syndrome by downregulating FOXK1.

Objective: Polycystic ovarian syndrome (PCOS) is a prevalent gynecologic disorder, often associated with abnormal follicular development. Cangfu Daotan decoction (CFD) is a traditional Chinese medicine formula that is effective in alleviating PCOS clinically, but the specific mechanism remains unclear. Forkhead box K1 (FOXK1) is associated with cellular function. This study aimed to explore the effects of CFD and FOXK1 on PCOS.Methods: High-fat diet and letrozole were combined to establish PCOS rat models. Next, primary GCs were extracted from those PCOS rats. Then, GC cells were transfected with si-FOXK1 or oe-FOXK1. CFD-contain serum was prepared, and experiments were conducted to investigate the regulation of FOXK1 by CFD.Results: FOXK1 was highly expressed in GCs of PCOS rats. Further investigation revealed that FOXK1 overexpression resulted in inhibition of proliferation and DNA synthesis, along with promotion of apoptosis and autophagy in GCs. Additionally, it was found that FOXK1 promoted the expressions of the AMP-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway-related proteins. Interestingly, treatment with CFD reversed all the effects of FOXK1 overexpression in GCs. Conclusion: This study demonstrated that CFD exerted a protective role in PCOS by inhibiting FOXK1, which provided a research basis for the application of CFD in PCOS, and suggested that FOXK1 is a novel therapeutic target in PCOS treatment.

Yin Yang 1 impacts upon preeclampsia by regulating Treg/TH17 cells and PI3K/AKT pathway.

Preeclampsia (PE) is a common obstetric syndrome with an unclear etiology and pathogenesis. The study here aimed to investigate the role of Yin Yang 1 (YY1) in PE, and to reveal any YY1-regulated mechanisms in PE. Peripheral blood, placenta, and endometrial tissues of PE patients, healthy volunteers, and patients who had undergone an elective Cesarean section and had a scarred uterus (control group) were collected for analyses. Rat PE models were established by lipopolysaccharide induction. Subsets of these rats were then made to over-express YY1. At 18 d after the PE was established, urine, blood, and placental tissues from all rats were collected. Levels of regulatory-T (Treg) and helper T-type 17 (TH17) cells in both human and rat blood were measured by flow cytometry. ELISA kits were used to evaluate blood levels of inflammatory factors (i.e. IL-6, IL-10, and IL-17) as well. RT-qPCR and Western blot assays were performed to quantify levels of forkhead box P3 (Foxp3), retinoic acid-related orphan receptor C (RORc), and YY1 in the human and rat placenta and endometrial tissues. Expressions of PI3K/AKT pathway-related proteins were also evaluated by Western blots. The results indicated that the PE patients, relative to levels in control group and the healthy control subjects, had decreased circulating levels of Treg cells/increased TH17 cells; tissues from these patients also had relatively-decreased FoxP3 mRNA and protein expressions and elevated RORc mRNA and protein expressions. YY1 was expressed only at low levels in the PE patient placenta and endometrial tissues. In rats, PE rats treated with over-expressed YY1 had (relative to in PE rats without over-induced YY1) increased circulating levels of Treg cells/decreased TH17 cells; tissues from these rats had elevated FoxP3 mRNA and protein expressions and reduced mRNA and protein RORc expressions, as well as indications of alleviated inflammation. In the rat placenta samples, YY1 was also determined to activate the PI3K/AKT pathway. In summary, YY1 regulates the balance among Treg/TH17 cells and so affect the PE process in part through activation of the PI3K/AKT pathway.

Magnesium increases numbers of Foxp3+ Treg cells and reduces arthritis severity and joint damage in an IL-10-dependent manner mediated by the intestinal microbiome.

BACKGROUND: Rheumatoid arthritis (RA) is a common autoimmune disease with emerging environmental and microbiome risk factors. The western diet is typically deficient in magnesium (Mg), and there is some evidence suggesting that Mg may have anti-inflammatory properties. But the actual role of Mg supplementation in arthritis or in T cell subsets has not been explored. METHODS: We investigated the role of a high Mg diet in two different mouse models of RA induced with the KRN serum, and collagen-induced arthritis. We also characterized the phenotypes of splenocytes, gene expression, and an extensive intestinal microbiome analyses including fecal material transplantation (FMT). FINDINGS: The high Mg diet group was significantly protected with reduced arthritis severity and joint damage, and reduced expression of IL-1ß, IL-6, and TNFα. The high Mg group also had increased numbers of Foxp3+ Treg cells and IL-10-producing T cells. The high Mg protective effect disappeared in IL-10 knockout mice. FMT from the high Mg diet mice recreated the phenotypes seen in the diet-treated mice, with reduced arthritis severity, increased Foxp3+ Treg, and increased IL-10-producing T cells. Intestinal microbiome analyses using 16S rDNA sequencing revealed diet-specific changes, including reduced levels of RA-associated Prevotella in the high Mg group, while increasing levels of Bacteroides and other bacteria associated with increased production of short-chain fatty acids. Metagenomic analyses implicated additional pathways including L-tryptophan biosynthesis and arginine deiminase. INTERPRETATION: We describe a new role for Mg in suppressing arthritis, in expanding Foxp3+ T reg cells and in the production of IL-10, and show that these effects are mediated by the intestinal microbiome. Our discoveries suggest a novel strategy for modifying the intestinal microbiome to treat RA and other autoimmune and inflammatory diseases. FUNDING: None.

[Effect of electroacupuncture on Treg/Th17 balance in spleen and serum inflammatory factors in obese mice].

OBJECTIVE: To observe the therapeutic effect of electroacupuncture(EA) on obese mice, and to explore the underlying mechanism of EA in treating obesity by focusing on the balance of regulatory T cells (Treg) and T helper 17 cells (Th17) and related inflammatory factors. METHODS: C57BL/6J male mice were randomly divided into normal group, model group and EA group, with 10 mice in each group. The obesity model was established by feeding the mice with high-fat diet. Mice in the EA group was treated with EA at "Zhongwan"(CV12), "Guanyuan"(CV4), "Zusanli"(ST36) and "Fenglong"(ST40) for 20 min every time, 3 times every week, for a total of 8 weeks. The food intake and body weight of mice were observed and recorded, and Lee's index was calculated; the contents of interleukin 2(IL-2), IL-4, IL-6, IL-10, IL-17A, gamma interferon (IFN-γ) and tumor necrosis factor(TNF)-α in serum were detected by multiplex liquid chip quantitative technique; the levels of Treg and Th17 cells in mice spleen tissues were detected by flow cytometry; and the expression levels of foxhead box p3(Foxp3) and retinoic acid related orphan receptor γt(ROR-γt) mRNA in spleen were detected by real-time quantitative PCR. RESULTS: Compared with the normal group, the food intake, body weight, Lee's index, the contents of IL-2, IL-6, IL-17A, IFN-γ and TNF-α in the serum, and the percentage of Th17 and expression of ROR-γt mRNA in the spleen tissues were significantly increased (P<0.01, P<0.001), while the contents of IL-4 and IL-10 in the serum, the percentage of Treg and expression of Foxp3 mRNA in the spleen tissues were significantly decreased (P<0.001, P<0.01) in the model group. Compared with the model group, the food intake, body weight, Lee's index, the contents of IL-2, IL-6, IL-17A, IFN-γ, and TNF-α in the serum, the percentage of Th17 and expression of ROR-γt mRNA in the spleen tissues were significantly decreased (P<0.01), while the contents of IL-4 and IL-10 in serum, the percentage of Treg and expression of Foxp3 mRNA in the spleen tissues were significantly increased(P<0.01, P<0.05) in the EA group. CONCLUSION: EA may improve the obese state of mice by regulating the balance of Treg/Th17 in spleen and the expression of inflammatory factors in serum.

Black rice anthocyanin extract enhances the antioxidant capacity in PC12 cells and improves the lifespan by activating IIS pathway in Caenorhabditis elegans.

Black rice is rich in anthocyanins, and the antioxidant effect of anthocyanins is recognized by consumers. The aim of this study was to identify the molecular mechanisms underlying the antioxidant activity of black rice anthocyanin extract (BRAE) in PC12 cells and C. elegans. Results showed that BRAE increased antioxidant enzyme activities and decreased the accumulation of reactive oxygen species (ROS) and malondialdehyde in PC12 cells induced by H2O2. Meanwhile, BRAE extended the lifespan, enhanced resistance to stress, increased antioxidant enzyme activities, and reduced lipofuscin, ROS, and MDA accumulation in wild-type C. elegans. The polyQ40 aggregation in AM141, paralysis in CL4176, and chemotaxis deficit in CL2355 were alleviated by BRAE administration. BRAE downregulated the mRNA expression of age-1 and daf-2, while upregulated the daf-16 mRNA level and SOD-3, CTL-1, and GST-4 protein expression. Mutational lifespan tests and molecular docking showed that insulin pathway might be involved in the mechanism of lifespan extension.

Gancao Fuzi decoction regulates the Th17/Treg cell imbalance in rheumatoid arthritis by targeting Foxp3 via miR-34a.

ETHNOPHARMACOLOGICAL RELEVANCE: During the Eastern Han Dynasty, Zhang Zhongjing first recorded the Gancao Fuzi decoction (GCFZD) formula in the "Synopsis of the Golden Chamber", which is reportedly an effective and safe treatment for rheumatoid arthritis (RA). However, the mechanism underlying the observed improvement in the T helper 17 (Th17)/regulatory T (Treg) cell imbalance in RA obtained with GCFZD has not been reported. AIM OF THE STUDY: This study aimed to demonstrate whether GCFZD ameliorated RA by modulating the Th17/Treg imbalance in RA mice. MATERIALS AND METHODS: Collagen was used to induce a model of collagen-induced arthritis (CIA) in mice. GCFZD was administered by gavage, and the arthritis index score, imaging and histopathological changes of the ankle joints, and the levels of the immunoglobulin G (IgG) class antibodies and proinflammatory factors in serum were determined. In addition, the frequencies of Th17 and Treg cells, the levels of relevant transcription factors and functional factors and the miR-34a gene in the spleen and the levels of interleukin-17A (IL-17A) and IL-10 in serum were determined. RESULTS: GCFZD significantly reduced the arthritis score, improved joint swelling and bone damage, reduced the pathological score, and decreased the serum levels of IgG class antibody (IgG and IgG2a) and proinflammatory factor [tumour necrosis factor-alpha (TNF-α), IL-1ß and IL-6]. Moreover, the Th17-cell proportion, the expression level of the Th17-specific transcription factor retinoic acid-related orphan receptor γt (RORγt) and functional factor IL-17A in the spleen, and the serum IL-17A level were decreased, whereas the Treg cell proportion, expression levels of the Treg-specific transcription factor forkhead box P3 (Foxp3) and functional factor IL-10 in the spleen, and the serum IL-10 level were increased. Furthermore, GCFZD inhibited miR-34a gene expression while promoting Foxp3 protein expression. CONCLUSIONS: The findings of this study demonstrate the therapeutic effect of GCFZD on mice with CIA, and the mechanism is related to an improvement in the Th17/Treg cell imbalance by targeting Foxp3 via miR-34a.

Multistrain Probiotics Supplement Alleviates Asthma Symptoms via Increasing Treg Cells Population: A Randomized, Double-Blind, Placebo-Controlled Trial.

INTRODUCTION: The favorable effects of probiotics have been demonstrated in allergic disorders. However, the underlying immunological mechanisms are poorly understood. In the present study, we investigated the improvement of clinical symptoms and immunological balance after receiving probiotics in patients with asthma. METHODS: The present study was a randomized, double-blind, placebo-controlled trial in which 40 patients with asthma were enrolled. They were treated with probiotics or placebo: 1 capsule/day for 8 weeks. Pulmonary function test, percentage of CD4+ CD25+ FoxP3+ Tregs, and gene expression of T-bet, GATA-3, RORγt, and Foxp3 in PBMCs were assessed at baseline and after treatment. RESULTS: Our results showed a significant increase in the expression of FoxP3 and CD4+ CD25+ FoxP3+ Tregs population, while RORγt and GATA3 expression were reduced. In addition, pulmonary function tests showed a significant improvement in forced expiratory volume and forced vital capacity after receiving probiotics. DISCUSSION/CONCLUSION: Our findings demonstrate that 8-week treatment with probiotic supplementation can control T-helper 2-predominant and Th17 pro-inflammatory responses and improve forced vital and forced expiratory volume in asthmatic patients. It seems probiotics can be used besides common treatments for patients with asthma.

Xiehuo Xiaoying decoction inhibits Tfh cell expansion and promotes Tfr cell amplification to ameliorate Graves' disease.

ETHNOPHARMACOLOGICAL RELEVANCE: Xiehuo Xiaoying decoction (XHXY) has shown great potential in the treatment of GD, but its mechanism remains obscure. Increase of follicular helper T (Tfh) cells and reduction of follicular regulatory T (Tfr) cells contribute to a high thyrotropin receptor antibodies (TRAb) level and possible Graves' disease (GD). Oxidative stress (OS) disrupts T helper cell differentiation and aggravates autoimmunity. AIM OF THE STUDY: This study aimed to investigate whether XHXY decoction can ameliorate autoimmunity in GD via inhibiting OS and regulating Tfh and Tfr cells. MATERIALS AND METHODS: The main XHXY bioactive compounds were identified using high-performance liquid chromatography quadrupole time-of-flight mass spectrometry. GD was induced in the mice through three intramuscular injections of adenovirus expressing the TSH receptor. Then, the mice received oral gavage of XHXY (17 g/kg·d) and 34 g/kg·d) for 4 weeks. OS indicators were assessed. Flow cytometry was used to confirm the proportion of Tfh and Tfr cells in the lymph nodes and spleens of the mice. Cytokine expression levels were determined using enzyme-linked immunosorbent assay. Factors including interleukin-21, B-cell lymphoma-6, and forkhead box P3 (Foxp3) were detected using quantitative polymerase chain reaction. The mRNA and protein expression levels of Kelch-like ECH-associated protein 1 (Keap1), nuclear factor erythroid-2-related factor 2 (Nrf2), and haem oxygenase 1 (HO-1) were detected using quantitative polymerase chain reaction and Western blotting, respectively. RESULTS: Twelve main ingredients of XHXY were identified. XHXY relieved GD by lowering thyroxine (p < 0.01) and TRAb levels (p < 0.01). XHXY ameliorated OS by decreasing the levels of NADPH oxidase 2 (p < 0.05), 4-hydroxynonenal (p < 0.01), and 8-oxo-2'-deoxyguanosine (p < 0.001). It inhibited Tfh cell expansion (p < 0.05), as well as the production of cytokine interleukin -21 (p < 0.01), interleukin -4 (p < 0.01) and transcription factor B-cell lymphoma 6 (p < 0.05). XHXY also induced Tfr cell amplification (p < 0.05), increased the production of interleukin -10 (p < 0.05) and transforming growth factor ß (p < 0.05) and the mRNA levels of Foxp3 (p < 0.05). Finally, the Tfh/Tfr ratio returned to normal. In addition, XHXY activated Nrf2 and HO-1 expression, but inhibited Keap1 activation. CONCLUSIONS: XHXY relieves autoimmunity in GD via inhibiting Tfh cell amplification and Tfr cell reduction, a mechanism which probably involves the Keap1/Nrf2 signaling pathway.

The Role of FOXP3 on Tumor Metastasis and Its Interaction with Traditional Chinese Medicine.

Forkhead box protein 3 (FOXP3) is an important transcription factor for regulatory T cells (Tregs) and plays an important role in their immunosuppressive function. In recent years, studies have found that FOXP3 is expressed in many kinds of tumors and plays different roles in tumors' biological behaviors, including tumor proliferation, metastasis, drug resistance, and prognosis. However, the effects of FOXP3 on tumor metastasis and its interaction with traditional Chinese medicine (TCM) remain unclear. Therefore, in this review, we focus on the effects of FOXP3 on tumor metastasis and its relationship with TCM, which can provide evidence for further research and therapy in clinical settings.

Black mulberry (Morus nigra) fruit extract alleviated AD-Like symptoms induced by toxic Aß protein in transgenic Caenorhabditis elegans via insulin DAF-16 signaling pathway.

Alzheimer's disease (AD) is one of the most severe neurodegenerative disorders. Recently, there is no effective treatment drug for AD. Morus nigra (M. nigra) is a black mulberry and widely distributed fruit in the Moraceae family with various undiscovered biological activities. The study aimed to investigate the potential anti-AD effect of M. nigra. Mulberry fruit extract (MF) was obtained from M. nigra and treated up to 1.00 mg/mL on transgenic AD Caenorhabditis elegans (C. elegans) models. MF inhibited Amyloid-ß (Aß)-induced paralysis symptoms by about 55.65 %, reduced Aß accumulation more than 50 % via immunoblotting, and suppressed over-sensitivity to exogenous serotonin in C. elegans. Furthermore, MF decreased the Aß oligomeric depositions in worm CL2006. MF activated the DAF-16 nuclear translocation and its downstream SOD-3 and GST-4. AD is a major age-related disorder. Therefore, MF treated for an aging test and proved to be expanded the lifespan of the worms up to 34.7 %. Besides, we have evaluated the MF in vivo antioxidative properties, where MF reduced reactive oxygen species (ROS) generations in C. elegans and remitted the activation of HSP-16.2 induced by the oxidative action of Juglone. Gene knockout and extended the lifespan of AD worms. However, RNA interference (RNAi) successfully silenced the daf-16 on the Aß phenotypic paralysis proved by MF effect. Our results indicate that MF alleviates AD-Like symptoms by activating the DAF-16 insulin signal pathway in C. elegans. Therefore, this MF study may provide new insights for mulberry application in safe AD treatment and clinical study.

Sodium selenite inhibits cervical cancer growth via ROS mediated AMPK/FOXO3a /GADD45a axis.

Selenium is a trace element that has been shown to inhibit the growth of various cancer cell types. However, its role in cervical cancer and its underlying mechanisms remains largely unknown. Herein, we explored the anti-cervical cancer effect of selenium and its potential mechanisms through xenograft and in vitro experiments. HeLa cell xenografts in female nude mice showed tumor growth retardation, with no obvious liver and kidney toxicity, after being intraperitoneally injected with 3 mg/kg sodium selenite (SS) for 14 days. Compared to the control group, selenium levels in the tumor tissue increased significantly after SS treatment. In vitro experiments, SS inhibited the viability of HeLa and SiHa cells, blocked the cell cycle at the S phase, and enhanced apoptosis. RNA-sequencing, Kyoto encyclopedia of genes and genomes pathway analysis showed that forkhead box protein O (FOXO) was a key regulatory signaling pathway for SS to exhibit anticancer effects. Gene Ontology analysis filtered multiple terms associated with apoptosis, anti-proliferation, and cell cycle arrest. Further research revealed that SS increased intracellular reactive oxygen species (ROS) and impaired mitochondrial function, which activated adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) via phosphorylation at Thr172, resulting in activation of FOXO3a and its downstream growth arrest and DNA damage-inducible alpha (GADD45a). In summary, SS exhibited anti-cervical cancer effects, and their mechanisms may be that SS is involved in inducing cell cycle arrest and potentiating cell apoptosis caused by ROS-dependent activation of the AMPK/FOXO3a/GADD45a axis.

Longevity-promoting properties of ginger extract in Caenorhabditis elegans via the insulin/IGF-1 signaling pathway.

Ginger is a traditional medicinal and edible plant with multiple health-promoting properties. Nevertheless, the effects and potential mechanism of ginger on antiaging remain unknown. The aim of this study was to comprehend the antiaging effects and potential mechanism of ginger in Caenorhabditis elegans (C. elegans). The current findings showed that the lifespan of C. elegans was prolonged by 23.16% with the supplementation of 60 µg mL-1 ginger extract (GE), and the extension of lifespan was mainly attributed to the major bioactive compounds in GE, 6-, 8-, 10-gingerol and 6-, 8-, 10-shogaol. Subsequently, GE promoted healthy aging by improving nematode movement and attenuating lipofuscin accumulation, and enhanced stress tolerance by up-regulating the expression of stress-related genes and activating DAF-16 and SKN-1. Moreover, lifespan assays of relative mutants revealed that GE mediated extension of lifespan via the insulin/IGF-1 signaling (IIS) pathway. In summary, GE endowed nematodes (C. elegans) with longevity and stress resistance in an IIS pathway dependent manner.

Therapeutic effects of Hirudo medicinalis extract antigens on modulation of CD4+CD25+Foxp3 T cell activity in murine eimeriosis.

Eimeriosis is a common parasitic disease in the chicken industry. The aim of this study was to assess the protective role of Hirudo extract antigens (HEA) against murine eimeriosis induced by Eimeria papillate. The oocyst output, developmental stages, goblet cells and oxidative stress, were investigated. Immunohistochemistry was used to detect anti-apoptotic Bcl2 marker and the number of both CD4+ and CD25+ cells in jejunal tissue, while ELISA was used to quantify TGF-ß, IL-10 and IL-22 in jejunal tissue homogenate. Real-time PCR was also used to detect mRNA expression of mucin 2 (MUC2), inducible nitric oxide synthase (iNOS), IL-1ß, IFN-γ, TNF-α, IL-6, and FoxP3. The most effective dose (5 µg/mice) reduced the oocyst output by 82.95 ± 1.02% (P ˂ 0.001). Similarly, the same dose reduced the jejunal developmental stages by 66.67 ± 0.49% (P ˂ 0.001). Furthermore, HEA therapy increased the number of jejunal goblet cells by 12.8 ± 1 (P ˂ 0.001) and the expression of MUC2 by 0.83 ± 0.06 (P ˂ 0.001). In contrast, TNF-α, IFN-γ, IL-6, iNOS, and IL-1ß expression as well as apoptosis were reduced. The number of CD4+ and CD25+ in the jejunal tissue was increased (14.6 ± 1.2 (P ˂ 0.001), 6.84 ± 1 (P ˂ 0.01), respectively) after HEA therapy. The molecular analysis showed an increased expression of intestinal Foxp3 (3.2 ± 0.13 (P ˂ 0.001), while IL-22 was reduced (124 ± 10 (P ˂ 0.001)) versus an increase in TGF-ß (250 ± 17 (P ˂ 0.01)) and IL-10 (236 ± 16 (P ˂ 0.001)) after HEA treatment in comparison to the non-treated infected group. With respect to the infected group, HEA reduced lipid peroxidation (LPO) (15.7 ± 1.12 (P ˂ 0.001)) and nitric oxide (NO) (13 ± 1.3 (P ˂ 0.001)) but increased reduced glutathione (GSH) (3.7 ± 0.26 (P ˂ 0.001)). In conclusion, HEA therapy protected against intestinal tissue damage by activation of CD4+CD25+Foxp3 cells which showed anti-inflammatory action. Hence, HEA can be recommended as a therapeutic treatment for eimeriosis.

Stress Buffering and Longevity Effects of Amber Extract on Caenorhabditis elegans (C. elegans).

Amber is a fossilized tree resin historically used in wound healing and stress relief. Unfortunately, there is no concrete scientific evidence supporting such efficacy. Here, the stress buffering and longevity effect of Amber extract (AE) in Caenorhabditis elegans (C. elegans) was investigated. Survival assays, health span assays, Enzyme-Linked Immunosorbent Assay (ELISA), Stress biomarker detection assays, Green Fluorescence Proteins (GFP), Real Time quantitative PCR (RT-qPCR) and C. elegans mutants were employed to investigate the stress buffering and longevity effect of AE. In the study, it was observed that AE supplementation improved health span and survival in both normal and stressed worms. Additionally, AE positively regulated stress hormones (cortisol, oxytocin, and dopamine) and decreased fat and reactive oxygen species (ROS) accumulation. Through the Insulin/IGF-1 signaling (IIS) pathway, AE enhanced the nuclear localization of DAF-16 and the expression of heat shock proteins and antioxidant genes in GFP-tagged worms and at messenger RNA levels. Finally, AE failed to increase the survival of daf-16, daf-2, skn-1 and hsf-1 loss-of-function mutants, confirming the involvement of the IIS pathway. Evidently, AE supplementation relieves stress and enhances longevity. Thus, amber may be a potent nutraceutical for stress relief.

Folic acid intervention changes liver Foxp3 methylation and ameliorates the damage caused by Th17/Treg imbalance after long-term alcohol exposure.

Folic acid, as a key source of methyl donor in DNA methylation, has been proved to play a beneficial role in inflammation modulation, which is usually impaired in alcoholic liver disease (ALD). However, the role of folic acid in alcoholic liver inflammation and injury remain elusive. In this study, we sought to uncover the potential protective mechanism by which folic acid ameliorates alcoholic liver injury. 100 male C57BL/6J mice were randomly divided into 5 groups: normal saline group, folic acid control group (5 mg per kg BW), ethanol model group (56% v/v, 10 mL per kg BW), folic acid + ethanol group, and 5-Aza + ethanol group (0.1 mL per 20 g BW). Liquor (10 mL per kg BW) was orally administered 1 h after the folic acid treatment for 10 consecutive weeks. The results showed that folic acid-inhibited ethanol-induced serum TG, TC, and LDL elevation attenuated hepatic fat accumulation and maintained ALT at a normal level. 10 weeks of ethanol administration simultaneously upregulated the hepatic proportion of Th17 and Treg cells to different extents and broke the homeostasis of liver immunization. Folic acid limited ethanol-induced inflammatory injury by increasing the frequency of hepatic Treg cells. Importantly, this effect may be caused by decreased DNMT3a, which in turn downregulates the methylated levels of CPG2 and CPG3 in the Foxp3 promoter region, changing the abundance of Foxp3 expression and improving the Th17/Treg imbalance. In summary, our findings demonstrated that folic acid supplementation may relieve ethanol-induced Th17/Treg disbalance through altering Foxp3 promoter methylation patterns, suggesting that folic acid may be a feasible preventive strategy for ALD.

Protective effects of Wuwei Xiaodu Drink against chronic osteomyelitis through Foxp3+CD25+CD4+ Treg cells via the IL-2/STAT5 signaling pathway.

To explore the effectiveness and safety of a Chinese medicinal decoction Wuwei Xiaodu Drink (WWXDD) in inhibiting chronic osteomyelitis via regulatory T cells signaling. The effective constitutes of WWXDD and osteomyelitis related genes were screened. Target proteins were cross-validated using the Venny database. GO function and KEGG pathway analysis were performed for target proteins, while pharmacological network was constructed. The bone properties were analyzed by HE staining and the concentrations of immune factors were measured by ELISA. The expression of CTLA-4 and Foxp3 mRNA and STAT5, p-STAT5, CTLA-4 and Foxp3 protein were detected using Real-time PCR and Western blot, respectively. FACS was used to analyze the percentages of cells. A total of 117 genes overlapped between 785 target genes of the active compounds of WWXDD and 912 osteomyelitis related genes. Inflammation-related genes, including IL-6, TNFα, IL-1ß and IL-2 showed high connection degree in the drug-compound-disease-target network. GO function and KEGG pathway analysis revealed that 117 intersection genes mainly enriched in virus infection related pathways, immune related pathways and chemokine signaling pathway. Furthermore, the development of chronic osteomyelitis was suppressed in model rats after treatment with WWXDD. Meanwhile, the concentrations of IL-2 and CD4+CD25+Foxp3 Treg percentages together with the levels of p-STAT5, CTLA-4 and Foxp3 were also down-regulated. Furthermore, IL-2 and WWXDD drug-containing serum exhibited opposite effects on regulating IL-2, IL-10, TGF-ß1, Foxp3, CTLA4 and STAT5. In addition, a STAT5 phosphorylation inhibitor suppressed the expression of Foxp3 and CTLA-4. WWXDD can treat chronic osteomyelitis through suppressing the main regulating factors of Tregs and interfere its immunodepression. Our results bring a new solution for chronic osteomyelitis.