SIRT1 regulates mitochondrial fission to alleviate high altitude hypoxia inducedcardiac dysfunction in rats via the PGC-1α-DRP1/FIS1/MFF pathway.

High-altitude exposure has been linked to cardiac dysfunction. Silent information regulator factor 2-related enzyme 1 (sirtuin 1, SIRT1), a nicotinamide adenine dinucleotide-dependent deacetylase, plays a crucial role in regulating numerous cardiovascular diseases. However, the relationship between SIRT1 and cardiac dysfunction induced by hypobaric hypoxia (HH) remains unexplored. This study aims to assess the impact of SIRT1 on HH-induced cardiac dysfunction and delve into the underlying mechanisms, both in vivo and in vitro. In this study, we have demonstrated that exposure to HH results in cardiomyocyte injury, along with the downregulation of SIRT1 and mitochondrial dysfunction. Upregulating SIRT1 significantly inhibits mitochondrial fission, improves mitochondrial function, reduces cardiomyocyte injury, and consequently enhances cardiac function in HH-exposed rats. Additionally, HH exposure triggers aberrant expression of mitochondrial fission-regulated proteins, with a decrease in PPARγ coactivator 1 alpha (PGC-1α) and mitochondrial fission factor (MFF) and an increase in mitochondrial fission 1 (FIS1) and dynamin-related protein 1 (DRP1), all of which are mitigated by SIRT1 upregulation. Furthermore, inhibiting PGC-1α diminishes the positive effects of SIRT1 regulation on the expression of DRP1, MFF, and FIS1, as well as mitochondrial fission. These findings demonstrate that SIRT1 alleviates HHinduced cardiac dysfunction by preventing mitochondrial fission through the PGC-1α-DRP1/FIS1/MFF pathway.

Intermittent high altitude hypoxia induced liver and kidney injury leading to hyperuricemia.

About 140 million people worldwide live at an altitude above 2500 m. Studies have showed an increase of the incidence of hyperuricemia among plateau populations, but little is known about the possible mechanisms. This study aims to assess the effects of high altitude on hyperuricemia and explore the corresponding mechanisms at the histological, inflammatory and molecular levels. This study finds that intermittent hypobaric hypoxia (IHH) exposure results in an increase of serum uric acid level and a decrease of uric acid clearance rate. Compared with the control group, the IHH group shows significant increases in hemoglobin concentration (HGB) and red blood cell counts (RBC), indicating that high altitude hyperuricemia is associated with polycythemia. This study also shows that IHH exposure induces oxidative stress, which causes the injury of liver and renal structures and functions. Additionally, altered expressions of organic anion transporter 1 (OAT1) and organic cation transporter 1 (OCT1) of kidney have been detected in the IHH exposed rats. The adenosine deaminase (ADA) expression levels and the xanthione oxidase (XOD) and ADA activity of liver of the IHH exposure group have significantly increased compared with those of the control group. Furthermore, the spleen coefficients, IL-2, IL-1ß and IL-8, have seen significant increases among the IHH exposure group. TLR/MyD88/NF-κB pathway is activated in the process of IHH induced inflammatory response in joints. Importantly, these results jointly show that IHH exposure causes hyperuricemia. IHH induced oxidative stress along with liver and kidney injury, unusual expression of the uric acid synthesis/excretion regulator and inflammatory response, thus suggesting a potential mechanism underlying IHH-induced hyperuricemia.

Resveratrol Ameliorates High Altitude Hypoxia-Induced Osteoporosis by Suppressing the ROS/HIF Signaling Pathway.

Hypoxia at high-altitude leads to osteoporosis. Resveratrol (RES), as an antioxidant, has been reported to promote osteoblastogenesis and suppress osteoclastogenesis. However, the therapeutic effect of RES against osteoporosis induced by high-altitude hypoxia remains unclear. Thus, this study was intended to investigate the potential effects of RES on high-altitude hypoxia-induced osteoporosis both in vivo and in vitro. Male Wistar rats were given RES (400 mg/kg) once daily for nine weeks under hypoxia, while the control was allowed to grow under normoxia. Bone mineral density (BMD), the levels of bone metabolism-related markers, and the changes on a histological level were measured. Bone marrow-derived mesenchymal stem cells (BMSCs) and RAW264.7 were incubated with RES under hypoxia, with a control growing under normoxia, followed by the evaluation of proliferation and differentiation. The results showed that RES inhibited high-altitude hypoxia-induced reduction in BMD, enhanced alkaline phosphatase (ALP), osteocalcin (OCN), calcitonin (CT) and runt-related transcription factor 2 (RUNX2) levels, whereas it reduced cross-linked carboxy-terminal telopeptide of type I collagen (CTX-I) levels and tartrate-resistant acid phosphatase (TRAP) activity in vivo. In addition, RES attenuated histological deteriorations in the femurs. In vitro, RES promoted osteoblastogenesis and mineralization in hypoxia-exposed BMSCs, along with promotion in RUNX2, ALP, OCN and osteopontin (OPN) levels, and inhibited the proliferation and osteoclastogenesis of RAW264.7. The promotion effects of RES on osteoblastogenesis were accompanied by the down-regulation of reactive oxygen species (ROS) and hypoxia inducible factor-1α (HIF-1α) induced by hypoxia. These results demonstrate that RES can alleviate high-altitude hypoxia-induced osteoporosis via promoting osteoblastogenesis by suppressing the ROS/HIF-1α signaling pathway. Thus, we suggest that RES might be a potential treatment with minimal side effects to protect against high-altitude hypoxia-induced osteoporosis.

Is the response rate of oral appliance therapy for subjects with and without position-dependent obstructive sleep apnoea different?

Objective To compare the effect of oral appliance (OA) treatment on non-position-dependent obstructive sleep apnoea (non-POSA) and position-dependent obstructive sleep apnoea (POSA).Methods The investigational sample was 205 patients with obstructive sleep apnoea at baseline and they were classified as non-POSA and POSA. Polygraphic registration was employed to compare the proportion of treatment responders between non-POSA and POSA groups at eight-week and one-year follow-ups. The treatment responder was defined as apnoea-hypopnoea index (AHI) <10 and/or ≥50% reduction in AHI.Results At the eight-week follow-up, the proportions of responders were 56% and 69% for the non-POSA and POSA groups (not significant), respectively. The responder proportions were increased to 68% and 77% for the two groups, respectively, at the one-year follow-up (not significant between the two groups). At the two follow-ups, there was no significant difference in absolute change in overall AHI between the two groups. However, the decrease in supine AHI was significantly greater in the POSA group, while the decrease in non-supine AHI was significantly greater in the non-POSA group.Conclusions The response rate for OA treatment did not differ between POSA and non-POSA groups. However, greater decreases in supine AHI and non-supine AHI were found among POSA and non-POSA patients, respectively.

Sox9CreER-mediated deletion of ß-catenin in palatal mesenchyme results in delayed palatal elevation accompanied with repressed canonical Wnt signaling and reduced actin polymerization.

Cleft palate is a good model to pushing us toward a deeper understanding of the molecular mechanisms of spatiotemporal patterns in tissues and organisms because of the multiple-step processes such as elevation and fusion. Previous studies have shown that the epithelial ß-catenin is crucial for palatal fusion, however, the function of the mesenchymal ß-catenin remains elusive. We investigate the role of mesenchymal ß-catenin in palatal development by generating a ß-catenin conditional knockout mouse (CKO) (Sox9CreER; Ctnnb1F/F ). We found that the CKO mice exhibited delayed palatal elevation, leading to cleft palate in both in vivo and ex vivo. Abnormal cell proliferation and repressed mesenchymal canonical Wnt signaling were found in the CKO palate. Interestingly, Filamentous actin (F-actin) polymerization was significantly reduced in the palatal mesenchyme of mutant embryos. Furthermore, overexpression of adenovirus-mediated transfection with Acta1 in the mutant could help to elevate the palatal shelves but could not prevent cleft palate in ex vivo. Our results suggest that conditionally knock out ß-catenin in the palatal mesenchyme by Sox9CreER leading to delayed palatal elevation, which results in repressed mesenchymal canonical Wnt signaling, decreased cell proliferation, and reduced actin polymerization, finally causes cleft palate.

Radiologic Evaluation of the Influence of Cleft Treatment on Nasal Dorsum Growth.

OBJECTIVE: The study addresses whether the growth of the nasal dorsum is disturbed by cleft treatments, for cleft lip only (CL) and cleft lip with cleft palate (CLP). DESIGN: A total of 576 patients with cleft (278 CL, 298 CLP) and 333 individuals without orofacial clefts were retrospectively enrolled. Cleft lip only group was treated with a modified Millard technique combined with Tajima incision for rhinoplasty at 3 to 6 months. The CLP group underwent the same lip repair technique and then underwent a Sommerlad palatoplasty at 9 to 12 months. Lateral cephalometric radiographs of all individuals were taken to evaluate the nasal length and nasal dorsum height. Dunn test was used to analyze the difference (P < .001). RESULTS: Compared with control, in CL, nasal bone angle and nasal dorsum angle increase by age similarly (5-18 years, P > .05); the total dorsum is significantly shorter (5-18 years, P < .001), while the upper nasal dorsum length is similar (except in 5-6 years), and the lower nasal dorsum is shorter (5-18 years, P < .001). In CLP, nasal bone angle develops insufficiently as children grow (8-18 years, P < .001); the nasal dorsum angle is notably smaller (5-18 years, P < .001); nasal bone length is not significantly different except 11 to 13 years (P < .05); nasal dorsal length is similar at skeletal maturity (17-18 years, P > .05), although it is shorter during 8 to 16 years (P < .05); the upper nasal dorsum is overdeveloped (14-18 years, P < .05), whereas the lower nasal dorsum is underdeveloped (5-18 years, P < .001). CONCLUSION: Treatments in both CL and CLP could be the important factors in disturbing the growth of cartilaginous portion of the nasal dorsum (including nasal tip) and the nasal dorsum height.

Quantitative Proteomics Reveals the Effects of Resveratrol on High-Altitude Polycythemia Treatment.

High-altitude polycythemia (HAPC) is a common plateau chronic disease in which red blood cells are compensatory hyperproliferative due to high altitude hypoxic environment. HAPC severely affects the physical and mental health of populations on the plateau. However, the pathogenesis and treatment of HAPC has been rarely investigated. Here, the hypoxia-induced HAPC model of rat is established, in which hemoglobin concentration significantly increases and platelets clearly decrease. The effect of resveratrol upon hypoxia enables HAPC remission and makes hemoglobin and platelet tend to a normal level. Furthermore, quantitative proteomics is applied to investigate the plasma proteome variation and the underlying molecular regulation during HAPC occurrence and treatment with resveratrol. Hypoxia promotes erythrocyte developing and differentiating and disrupts cytoskeleton organization. Notably, the resveratrol administration reverses the proteome change pattern due to hypoxia and contributes to plateau adaption. Quantitative verification of differentially expressed proteins confirms the roles of resveratrol in HAPC. Resveratrol is expected to be useful for HAPC treatment.

Downregulation of miR-541 induced by heat stress contributes to malignant transformation of human bronchial epithelial cells via HSP27.

Environmental factors are one of the important factors affecting the occurrence of lung cancer. However, few studies have been done on the relationship between hot environment and lung cancer. In the present study, we demonstrated that heat stress leads to anchorage-independent proliferation, mitochondrial apoptosis, and autophagy of Beas-2B cells, which are normal lung bronchial epithelial cells. Heat shock protein 27 (HSP27) promoted heat stress-induced anchorage-independent proliferation and autophagy, but suppressed mitochondrial apoptosis, indicating that HSP27 might act as an oncogene in the malignant transformation of lung epithelial cells. We also showed that HSP27 promoted autophagy of these cells under heat stress via autophagy related 7 (ATG7) and ETS Transcription Factor ELK1 (ELK1), a transcription factor of ATG7, under heat stress. In addition, we showed that HSP27 translation could be repressed by microRNA miR-541, and the biological effects of miR-541 were the opposite to HSP27, suggesting that HSP27 is a downstream target of miR-541. In this study, we characterized a new mechanism whereby HSP27 promotes cell transformation during the onset of lung cancer. Our studies provide new insights into the molecular mechanisms underlying the lung carcinogenic effect of heat exposure. Specifically, heat stress promotes translation of HSP27 by inhibiting miR-541 accumulation, ultimately resulting in activation of autophagy, inhibition of mitochondrial apoptotic pathway and malignant transformation of Human Bronchial Epithelial Cells. This study identifies miR-541 as a potential prognostic biomarker or therapeutic target to improve theory of environmental carcinogenesis.

PASS: A Proteomics Alternative Splicing Screening Pipeline.

Alternative splicing (AS) has been well-investigated at the trancriptome level by the application of RNA-seq technology. There is an ongoing debate on the biological importance of AS to proteome complexity. A toolkit for accurately identifying AS from proteome data is urgently needed. Here, a software called PASS is developed to comprehensively detect AS events for the proteomics mass spectrometry (MS) data. Moreover, PASS is well compatible with MS identification by the proteogenomics approach, which provides novel AS candidates for proteome identification. The workflow of PASS mainly contains five core steps: transcripts reconstruction from RNA-Seq data, novel protein sequence generation, MS data searching, proSAM file formatting, and AS detection. Access to the program from either step is supported. PASS is successfully applied to proteome data of mouse hepatocytes and 407 AS events are first identified with proteomics MS evidences. PASS is expected to be widely used to identify AS events on proteome data and provide a deeper understanding of the proteome isoforms. The PASS software is freely available at https://github.com/wupengomics/PASS.

Epigenetic upregulation of miR-126 induced by heat stress contributes to apoptosis of rat cardiomyocytes by promoting Tomm40 transcription.

TOMM40 is the channel-forming subunit of a translocase of the mitochondrial outer membrane (TOM) that is essential for protein transport into mitochondria. TOMM40 plays an important role in maintaining normal mitochondrial function. The correlation between occupational thermal exposure and mitochondria dysfunction has been demonstrated; however, nothing is known about the alteration and role of TOMM40 in response to environmental heat stress. In the present study, we showed that environmental thermal exposure upregulated microRNA miR-126, consequently reducing AU-rich element RNA-binding protein 1 (AUF1)-mediated SP1 mRNA degradation and increasing TOMM40 transcription, which in turn decreased the mitochondria membrane potential and apoptosis of cardiomyocytes. Mechanistically, miR-126 upregulation was attributed to heat stress-induced promoter demethylation via elevated TET2 (Tet methylcytosine dioxygenase 2) expression, while SP1 mRNA degradation was caused by decreased translation of AUF1 induced by miR-126. Moreover, TOMM40 transcription was upregulated via increasing its transcription factor SP1 resulting from AUF1 inhibition in the heat stress responses. The results of the present study increased our understanding of the role of miR-126 and TOMM40 in heat stressed cardiomyocytes.

SPION-Decorated Exosome Delivered BAY55-9837 Targeting the Pancreas through Magnetism to Improve the Blood GLC Response.

BAY55-9837, a potential therapeutic peptide in the treatment of type 2 diabetes mellitus (T2DM), is capable of inducing glucose (GLC)-dependent insulin secretion. However, the therapeutic benefit of BAY55-9837 is limited by its short half-life, lack of targeting ability, and poor blood GLC response. How to improve the blood GLC response of BAY55-9837 is an existing problem that needs to be solved. In this study, a method for preparing BAY55-9837-loaded exosomes coupled with superparamagnetic iron oxide nanoparticle (SPIONs) with pancreas islet targeting activity and an enhanced blood GLC response with the help of an external magnetic force (MF) is demonstrated. The plasma half-life of BAY55-9837 loaded in exosome-SPION is 27-fold longer than that of BAY55-9837. The active targeting property of SIPONs enables BAY-exosomes to gain a favorable targeting property, which improves the BAY55-9837 blood GLC response capacity with the help of an external MF. In vivo studies show that BAY-loaded exosome-based vehicle delivery enhances pancreas islet targeting under an external MF and markedly increases insulin secretion, thereby leading to the alleviation of hyperglycemia. The chronic administration of BAY-exosome-SPION/MF significantly improves glycosylated hemoglobin and lipid profiles. BAY-exosome-SPION/MF maybe a promising candidate for a peptide drug carrier for T2DM with a better blood GLC response.

The Drosophila melanogaster Genetic Reference Panel.

A major challenge of biology is understanding the relationship between molecular genetic variation and variation in quantitative traits, including fitness. This relationship determines our ability to predict phenotypes from genotypes and to understand how evolutionary forces shape variation within and between species. Previous efforts to dissect the genotype-phenotype map were based on incomplete genotypic information. Here, we describe the Drosophila melanogaster Genetic Reference Panel (DGRP), a community resource for analysis of population genomics and quantitative traits. The DGRP consists of fully sequenced inbred lines derived from a natural population. Population genomic analyses reveal reduced polymorphism in centromeric autosomal regions and the X chromosome, evidence for positive and negative selection, and rapid evolution of the X chromosome. Many variants in novel genes, most at low frequency, are associated with quantitative traits and explain a large fraction of the phenotypic variance. The DGRP facilitates genotype-phenotype mapping using the power of Drosophila genetics.

Glutathione homeostasis is significantly altered by quercetin via the Keap1/Nrf2 and MAPK signaling pathways in rats.

Previously, we showed that 0.5% quercetin simultaneously decreased serum homocysteine and glutathione (GSH) levels in rats. The aim of the present study was to investigate the effects of 0.5% quercetin on GSH metabolism, related enzymes and signal pathways in rats. Rats were fed the control diet and 0.5% quercetin-supplemented diet for 6 weeks. The results showed that quercetin reduced serum and hepatic content of GSH and the ratio of GSH and oxidized glutathione (GSSG), enhanced hepatic activity and mRNA expression of glutathione S-transferase (GST), inhibited hepatic activity and mRNA expression of glutamate cysteine ligase (GCL), and decreased hepatic glutathione reductase (GR) mRNA expression. Levels of phosphorylated p38 and extracellular signal-regulated kinase (ERK) 1/2 mitogen-activated protein kinases (MAPKs) increased, while that of nuclear factor E2-like 2 (Nrf2) protein decreased after quercetin treatment. However, no significant hepatotoxicity was noted. We concluded that quercetin treatment altered hepatic GSH metabolism by modulating GSH metabolic enzyme activities and mRNA expression in rats, and p38, ERK1/2 MAPKs, and Nrf2 were involved in modulating GSH metabolism-related enzymes.

Natural variation in genome architecture among 205 Drosophila melanogaster Genetic Reference Panel lines.

The Drosophila melanogaster Genetic Reference Panel (DGRP) is a community resource of 205 sequenced inbred lines, derived to improve our understanding of the effects of naturally occurring genetic variation on molecular and organismal phenotypes. We used an integrated genotyping strategy to identify 4,853,802 single nucleotide polymorphisms (SNPs) and 1,296,080 non-SNP variants. Our molecular population genomic analyses show higher deletion than insertion mutation rates and stronger purifying selection on deletions. Weaker selection on insertions than deletions is consistent with our observed distribution of genome size determined by flow cytometry, which is skewed toward larger genomes. Insertion/deletion and single nucleotide polymorphisms are positively correlated with each other and with local recombination, suggesting that their nonrandom distributions are due to hitchhiking and background selection. Our cytogenetic analysis identified 16 polymorphic inversions in the DGRP. Common inverted and standard karyotypes are genetically divergent and account for most of the variation in relatedness among the DGRP lines. Intriguingly, variation in genome size and many quantitative traits are significantly associated with inversions. Approximately 50% of the DGRP lines are infected with Wolbachia, and four lines have germline insertions of Wolbachia sequences, but effects of Wolbachia infection on quantitative traits are rarely significant. The DGRP complements ongoing efforts to functionally annotate the Drosophila genome. Indeed, 15% of all D. melanogaster genes segregate for potentially damaged proteins in the DGRP, and genome-wide analyses of quantitative traits identify novel candidate genes. The DGRP lines, sequence data, genotypes, quality scores, phenotypes, and analysis and visualization tools are publicly available.

Comparative validation of the D. melanogaster modENCODE transcriptome annotation.

Accurate gene model annotation of reference genomes is critical for making them useful. The modENCODE project has improved the D. melanogaster genome annotation by using deep and diverse high-throughput data. Since transcriptional activity that has been evolutionarily conserved is likely to have an advantageous function, we have performed large-scale interspecific comparisons to increase confidence in predicted annotations. To support comparative genomics, we filled in divergence gaps in the Drosophila phylogeny by generating draft genomes for eight new species. For comparative transcriptome analysis, we generated mRNA expression profiles on 81 samples from multiple tissues and developmental stages of 15 Drosophila species, and we performed cap analysis of gene expression in D. melanogaster and D. pseudoobscura. We also describe conservation of four distinct core promoter structures composed of combinations of elements at three positions. Overall, each type of genomic feature shows a characteristic divergence rate relative to neutral models, highlighting the value of multispecies alignment in annotating a target genome that should prove useful in the annotation of other high priority genomes, especially human and other mammalian genomes that are rich in noncoding sequences. We report that the vast majority of elements in the annotation are evolutionarily conserved, indicating that the annotation will be an important springboard for functional genetic testing by the Drosophila community.

Southern rice black-streaked dwarf virus alters insect vectors' host orientation preferences to enhance spread and increase rice ragged stunt virus co-infection.

In recent years, Southern rice black-streaked dwarf virus (SRBSDV), a tentative species in the genus Fijivirus (family Reoviridae), has spread rapidly and caused serious rice losses in eastern and southeastern Asia. With this virus spread, Rice ragged stunt virus (RRSV, genus Oryzavirus, family Reoviridae) became more common in southern China, usually in co-infection with the former. SRBSDV and RRSV are transmitted by two different species of planthoppers, white-backed planthopper (WBPH, Sogatella furcifera) and brown planthopper (BPH, Nilaparvata lugens), respectively, in a persistent, circulative, propagative manner. In this study, using a Y-shape olfactometer-based device, we tested the host preference of three types of macropterous WBPH adults for healthy or SRBSDV-infected rice plants. The results showed that virus-free WBPHs significantly preferred infected rice plants to healthy plants, whereas both the viruliferous and nonviruliferous WBPHs preferred healthy plants to infected plants. In additional tests, we found that the BPHs significantly preferred healthy plants when they were virus free, whereas RRSV-carrying BPHs preferred SRBSDV-infected rice plants. From these findings, we propose that plant viruses may alter host selection preference of vectors to enhance their spread and that of insects vectoring another virus to result in co-infection with more than one virus.

Brazilian green propolis improves immune function in aged mice.

Aging weakened innate and adaptive immunity both quantitatively and qualitatively. Some components in propolis could stimulate immune function in young animals or cultured immune cells in vitro. Few studies had been carried out in the aged. The present study was to evaluate the effects of Brazilian green propolis supplementation on the immunological parameters in aged mice. Eighty Kunming mice, aged 15-18 months, were randomly assigned to the control and three experimental groups supplemented with different doses (83.3, 157.4 and 352.9 mg/kg.bw respectively) of Brazilian green propolis. The experiment lasted for 4 weeks. Contents of total polyphenol, flavonoid, cinnamic acid and artepillin-C in Brazilian green propolis were analyzed. Splenic NK cytotoxic, T lymphocyte proliferation and antibody generation cells, as well as the phagocytosis of peritoneal macrophages, ear swelling, and serum contents of IgG, IgM, hemolysin and cytokines were measured. After 4 weeks of treatment, the phagocytosis of peritoneal macrophages was enhanced in 157.4 mg/kg and 352.9 mg/kg groups. Ear swelling increased in all propolis treatmented groups. Antibodies specific to sheep erythrocytes were higher in the groups receiving 157.4 and 352.9 mg/kg.bw than that of control group. IgG level dramatically increased in the groups receiving 83.3 and 157.4 mg/kg.bw in comparison to the control group. These results indicate that administration of Brazilian green propolis have a positive effect on innate and adaptive immunity in aged mice.

Orthodontic traction of a deeply impacted mandibular first molar via a ramus miniscrew through lateral-window technique: A case report.

BACKGROUND: The incidence of mandibular first molar impaction is less than 0.01%, and there are no standard treatment guidelines. Orthodontic traction can be chosen as a treatment plan in some circumstances. One miniscrew in the mandibular ramus can offer sufficient anchorage for the traction of impacted mandibular molars. CASE DESCRIPTION: A 10-year-old boy had a completely labially positioned maxillary left canine and a deeply impacted mandibular left first molar associated with a dentigerous cyst. Using 2 palatal miniscrews for distalization, space was created to align the maxillary left canine. In addition, a miniscrew in the mandibular ramus facilitated the traction of the mandibular left first molar. After 3 years and 6 months of orthodontic treatment, the integrity of the dental arch was restored, and occlusal function was reestablished. PRACTICAL IMPLICATIONS: A ramus miniscrew is an effective and viable treatment option for the orthodontic traction of deeply impacted mandibular molars.

Isoquercitrin attenuates the osteoclast-mediated bone loss in rheumatoid arthritis via the Nrf2/ROS/NF-κB pathway.

An excess of osteoclastogenesis significantly contributes to the development of rheumatoid arthritis (RA). Activation of the nuclear factor erythroid-2 related factor 2 (Nrf2) and nuclear factor kappa B (NF-κB) ligand (RANKL)-induced reactive oxygen species (ROS)-to-NF-κB signaling cascade are important mechanisms regulating osteoclastogenesis; however, whether Nrf2 is involved in RANKL-induced NF-κB activation is controversial. Isoquercitrin, a natural flavonoid compound, has been shown to have Nrf2-dependent antioxidant effects inprevious studies. We sought to verify whether isoquercitrin could modulate RANKL-induced NF-κB activation by activating Nrf2, thereby affecting osteoclastogenesis. Tartrate-resistant acid phosphatase staining, F-actin ring staining and resorption pit assay suggested that isoquercitrin significantly inhibited osteoclastogenesis and osteolytic function. Mitosox staining showed that RANKL-induced ROS generation was significantly inhibited by isoquercitrin from day 3 of the osteoclast differentiation cycle. Quantitative real-time PCR, Western blot, and immunofluorescence indicated that isoquercitrin activated the Nrf2 signaling pathway and inhibited NF-κB expression. And when we used the Nrf2-specific inhibitor ML385, the inhibition of NF-κB by isoquercitrin disappeared. Moreover, we found that Nrf2 is not uninvolved in RANKL-induced NF-κB activation and may be related to the timing of ROS regulation. When we limited isoquercitrin administration to 2 days, Nrf2 remained activated and the inhibition of NF-κB disappeared. In vivo experiments suggested that isoquercitrin attenuated RA modeling-induced bone loss. Overall, isoquercitrin-activated Nrf2 blocked the RANKL-induced ROS-to-NF-κB signaling cascade response, thereby inhibiting osteoclastogenesis and bone loss. These findings provide new ideas for the treatment of RA.