Discovery and mechanistic study of Imperatorin that inhibits HBsAg expression and cccDNA transcription.

Chronic hepatitis B virus (HBV) infection remains a significant global health challenge due to its link to severe conditions like HBV-related cirrhosis and hepatocellular carcinoma (HCC). Although current treatments effectively reduce viral levels, they have limited impact on certain HBV elements, namely hepatitis B surface antigen (HBsAg) and covalently closed circular DNA (cccDNA). This highlights the urgent need for innovative pharmaceutical and biological interventions that can disrupt HBsAg production originating from cccDNA. In this study, we identified a natural furanocoumarin compound, Imperatorin, which markedly inhibited the expression of HBsAg from cccDNA, by screening a library of natural compounds derived from Chinese herbal medicines using ELISA assay and qRT-PCR. The pharmacodynamics study of Imperatorin was explored on HBV infected HepG2-NTCP/PHHs and HBV-infected humanized mouse model. Proteome analysis was performed on HBV infected HepG2-NTCP cells following Imperatorin treatment. Molecular docking and bio-layer interferometry (BLI) were used for finding the target of Imperatorin. Our findings demonstrated Imperatorin remarkably reduced the level of HBsAg, HBV RNAs, HBV DNA and transcriptional activity of cccDNA both in vitro and in vivo. Additionally, Imperatorin effectively restrained the actions of HBV promoters responsible for cccDNA transcription. Mechanistic study revealed that Imperatorin directly binds to ERK and subsequently interfering with the activation of CAMP response element-binding protein (CREB), a crucial transcriptional factor for HBV and has been demonstrated to bind to the PreS2/S and X promoter regions of HBV. Importantly, the absence of ERK could nullify the antiviral impact triggered by Imperatorin. Collectively, the natural compound Imperatorin may be an effective candidate agent for inhibiting HBsAg production and cccDNA transcription by impeding the activities of HBV promoters through ERK-CREB axis.

Natural small-molecules reverse Xeroderma Pigmentosum Complementation Group C (XPC) deficient-mediated drug-resistance in renal cell carcinoma.

BACKGROUND: Renal cancer is insensitive to radiotherapy or most chemotherapies. While the loss of the XPC gene was correlated with drug resistance in colon cancer, the expression of XPC and its role in the drug resistance of renal cancer have not yet been elucidated. With the fact that natural small-molecules have been adopted in combinational therapy with classical chemotherapeutic agents to increase the drug sensitivity and reduce adverse effects, the use of herbal compounds to tackle drug-resistance in renal cancer is advocated. PURPOSE: To correlate the role of XPC gene deficiency to drug-resistance in renal cancer, and to identify natural small-molecules that can reverse drug-resistance in renal cancer via up-regulation of XPC. METHODS: IHC was adopted to analyze the XPC expression in human tumor and adjacent tissues. Clinical data extracted from The Cancer Genome Atlas (TCGA) database were further analysed to determine the relationship between XPC gene expression and tumor staging of renal cancer. Two types of XPC-KD renal cancer cell models were established to investigate the drug-resistant phenotype and screen XPC gene enhancers from 134 natural small-molecules derived from herbal plants. Furthermore, the identified XPC enhancers were verified in single or in combination with FDA-approved chemotherapy drugs for reversing drug-resistance in renal cancer using MTT cytotoxicity assay. Drug resistance gene profiling, ROS detection assay, immunocytochemistry and cell live-dead imaging assay were adopted to characterize the XPC-related drug resistant mechanism. RESULTS: XPC gene expression was significantly reduced in renal cancer tissue compared with its adjacent tissue. Clinical analysis of TCGA database also identified the downregulated level of XPC gene in renal tumor tissue of stage IV patients with cancer metastasis, which was also correlated with their lower survival rate. 6 natural small-molecules derived from herbal plants including tectorigenin, pinostilbene, d-pinitol, polygalasaponin F, atractylenolide III and astragaloside II significantly enhanced XPC expression in two renal cancer cell types. Combinational treatment of the identified natural compound with the treatment of FDA-approved drug, further confirmed the up-regulation of XPC gene expression can sensitize the two types of XPC-KD drug-resistant renal cancer cells towards the FDA-approved drugs. Mechanistic study confirmed that GSTP1/ROS axis was activated in drug resistant XPC-KD renal cancer cells. CONCLUSION: XPC gene deficiency was identified in patient renal tumor samples, and knockdown of the XPC gene was correlated with a drug-resistant phenotype in renal cancer cells via activation of the GSTP1/ROS axis. The 6 identified natural small molecules were confirmed to have drug sensitizing effects via upregulation of the XPC gene. Therefore, the identified active natural small molecules may work as an adjuvant therapy for circumventing the drug-resistant phenotype in renal cancer via enhancement of XPC expression.

Mutant p53R211* ameliorates inflammatory arthritis in AIA rats via inhibition of TBK1-IRF3 innate immune response.

BACKGROUND: Rheumatoid arthritis (RA) is an autoimmune inflammation disease characterized by imbalance of immune homeostasis. p53 mutants are commonly described as the guardian of cancer cells by conferring them drug-resistance and immune evasion. Importantly, p53 mutations have also been identified in RA patients, and this prompts the investigation of its role in RA pathogenesis. METHODS: The cytotoxicity of disease-modifying anti-rheumatic drugs (DMARDs) against p53 wild-type (WT)/mutant-transfected RA fibroblast-like synoviocytes (RAFLSs) was evaluated by MTT assay. Adeno-associated virus (AAV) was employed to establish p53 WT/R211* adjuvant-induced arthritis (AIA) rat model. The arthritic condition of rats was assessed by various parameters such as micro-CT analysis. Knee joint samples were isolated for total RNA sequencing analysis. The expressions of cytokines and immune-related genes were examined by qPCR, ELISA assay and immunofluorescence. The mechanistic pathway was determined by immunoprecipitation and Western blotting in vitro and in vivo. RESULTS: Among p53 mutants, p53R213* exhibited remarkable DMARD-resistance in RAFLSs. However, AAV-induced p53R211* overexpression ameliorated inflammatory arthritis in AIA rats without Methotrexate (MTX)-resistance, and our results discovered the immunomodulatory effect of p53R211* via suppression of T-cell activation and T helper 17 cell (Th17) infiltration in rat joint, and finally downregulated expressions of pro-inflammatory cytokines. Total RNA sequencing analysis identified the correlation of p53R211* with immune-related pathways. Further mechanistic studies revealed that p53R213*/R211* instead of wild-type p53 interacted with TANK-binding kinase 1 (TBK1) and suppressed the innate immune TBK1-Interferon regulatory factor 3 (IRF3)-Stimulator of interferon genes (STING) cascade. CONCLUSIONS: This study unravels the role of p53R213* mutant in RA pathogenesis, and identifies TBK1 as a potential anti-inflammatory target.

N-Acetylcysteine overcomes epalrestat-mediated increase of toxic 4-hydroxy-2-nonenal and potentiates the anti-arthritic effect of epalrestat in AIA model.

Epalrestat, an aldose reductase inhibitor (ARI), has been clinically adopted in treating diabetic neuropathy in China and Japan. Apart from the involvement in diabetic complications, AR has been implicated in inflammation. Here, we seek to investigate the feasibility of clinically approved ARI, epalrestat, for the treatment of rheumatoid arthritis (RA). The mRNA level of AR was markedly upregulated in the peripheral blood mononuclear cells (PBMCs) of RA patients when compared to those of healthy donors. Besides, the disease activity of RA patients is positively correlated with AR expression. Epalrestat significantly suppressed lipopolysaccharide (LPS) induced TNF-α, IL-1ß, and IL-6 in the human RA fibroblast-like synoviocytes (RAFLSs). Unexpectedly, epalrestat treatment alone markedly exaggerated the disease severity in adjuvant induced arthritic (AIA) rats with elevated Th17 cell proportion and increased inflammatory markers, probably resulting from the increased levels of 4-hydroxy-2-nonenal (4-HNE) and malondialdehyde (MDA). Interestingly, the combined treatment of epalrestat with N-Acetylcysteine (NAC), an anti-oxidant, to AIA rats dramatically suppressed the production of 4-HNE, MDA and inflammatory cytokines, and significantly improved the arthritic condition. Taken together, the anti-arthritic effect of epalrestat was diminished or even overridden by the excessive accumulation of toxic 4-HNE or other reactive aldehydes in AIA rats due to AR inhibition. Co-treatment with NAC significantly reversed epalrestat-induced upregulation of 4-HNE level and potentiated the anti-arthritic effect of epalrestat, suggesting that the combined therapy of epalrestat with NAC may sever as a potential approach in treating RA. Importantly, it could be regarded as a safe intervention for RA patients who need epalrestat for the treatment of diabetic complications.

Age-related self-DNA accumulation may accelerate arthritis in rats and in human rheumatoid arthritis.

The incidence of rheumatoid arthritis (RA) is increasing with age. DNA fragments is known to accumulate in certain autoimmune diseases, but the mechanistic relationship among ageing, DNA fragments and RA pathogenesis remain unexplored. Here we show that the accumulation of DNA fragments, increasing with age and regulated by the exonuclease TREX1, promotes abnormal activation of the immune system in an adjuvant-induced arthritis (AIA) rat model. Local overexpression of TREX1 suppresses synovial inflammation in rats, while conditional genomic deletion of TREX1 in AIA rats result in higher levels of circulating free (cf) DNA and hence abnormal immune activation, leading to more severe symptoms. The dysregulation of the heterodimeric transcription factor AP-1, formed by c-Jun and c-Fos, appear to regulate both TREX1 expression and SASP induction. Thus, our results confirm that DNA fragments are inflammatory mediators, and TREX1, downstream of AP-1, may serve as regulator of cellular immunity in health and in RA.

Targeting autophagy to discover the Piper wallichii petroleum ether fraction exhibiting antiaging and anti-Alzheimer's disease effects in Caenorhabditis elegans.

BACKGROUND: With population aging, the incidence of aging-related Alzheimer's disease (AD) is increasing, accompanied by decreased autophagy activity. At present, Caenorhabditis elegans (C. elegans) is widely employed to evaluate autophagy and in research on aging and aging-related diseases in vivo. To discover autophagy activators from natural medicines and investigate their therapeutic potential in antiaging and anti-AD effects, multiple C. elegans models related to autophagy, aging, and AD were used. METHOD: In this study, we employed the DA2123 and BC12921 strains to discover potential autophagy inducers using a self-established natural medicine library. The antiaging effect was evaluated by determining the lifespan, motor ability, pumping rate, lipofuscin accumulation of worms, and resistance ability of worms under various stresses. In addition, the anti-AD effect was examined by detecting the paralysis rate, food-sensing behavior, and amyloid-ß and Tau pathology in C. elegans. Moreover, RNAi technology was used to knock down the genes related to autophagy induction. RESULTS: We discovered that Piper wallichii extract (PE) and the petroleum ether fraction (PPF) activated autophagy in C. elegans, as evidenced by increased GFP-tagged LGG-1 foci and decreased GFP-p62 expression. In addition, PPF extended the lifespan and enhanced the healthspan of worms by increasing body bends and pumping rates, decreasing lipofuscin accumulation, and increasing resistance to oxidative, heat, and pathogenic stress. Moreover, PPF exhibited an anti-AD effect by decreasing the paralysis rate, improving the pumping rate and slowing rate, and alleviating Aß and Tau pathology in AD worms. However, the feeding of RNAi bacteria targeting unc-51, bec-1, lgg-1, and vps-34 abolished the antiaging and anti-AD effects of PPF. CONCLUSION: Piper wallichii may be a promising drug for antiaging and anti-AD. More future studies are also needed to identify autophagy inducers in Piper wallichii and clarify their molecular mechanisms.

Pomiferin targets SERCA, mTOR, and P-gp to induce autophagic cell death in apoptosis-resistant cancer cells, and reverses the MDR phenotype in cisplatin-resistant tumors in vivo.

Drug resistance in cancer has been classified as innate resistance or acquired resistance, which were characterized by apoptotic defects and ABC transporters overexpression respectively. Therefore, to preclude or reverse these resistance mechanisms could be a promising strategy to improve chemotherapeutic outcomes. In this study, a natural product from Osage Orange, pomiferin, was identified as a novel autophagy activator that circumvents innate resistance by triggering autophagic cell death via SERCA inhibition and activation of the CaMKKß-AMPK-mTOR signaling cascade. In addition, pomiferin also directly inhibited the P-gp (MDR1/ABCB1) efflux and reversed acquired resistance by potentiating the accumulation and efficacy of the chemotherapeutic agent, cisplatin. In vivo study demonstrated that pomiferin triggered calcium-mediated tumor suppression and exhibited an anti-metastatic effect in the LLC-1 lung cancer-bearing mouse model. Moreover, as an adjuvant, pomiferin potentiated the anti-tumor effect of the chemotherapeutic agent, cisplatin, in RM-1 drug-resistant prostate cancer-bearing mouse model by specially attenuating ABCB1-mediated drug efflux, but not ABCC5, thereby promoting the accumulation of cisplatin in tumors. Collectively, pomiferin may serve as a novel effective agent for circumventing drug resistance in clinical applications.

Tetrandrine Inhibits Cancer Stem Cell Characteristics and Epithelial to Mesenchymal Transition in Triple-Negative Breast Cancer via SOD1/ROS Signaling Pathway.

Targeting the stemness of triple-negative breast cancer (TNBC) is a potential therapeutic approach for treating TNBC. Tetrandrine, a natural plant alkaloid, has several anticancer effects. Here, we aimed to evaluate the efficacy of tetrandrine in cancer stemness and epithelial to mesenchymal transition (EMT) in TNBC, and to explore the underlying mechanisms. The effects of tetrandrine on cell growth, cell viability, cell stemness capacity, cell migration, and cell invasion, as well as the molecules involved in these processes, were investigated in a cell culture system. An in vivo xenograft tumor and lung metastasis study was performed using nude mice to verify the effects and mechanisms of tetrandrine. Tetrandrine exhibited antiproliferative and cell cycle arrest activities in TNBC cell lines, significantly reduced aldehyde dehydrogenase and CD44[Formula: see text]CD24[Formula: see text] characteristic subpopulation, and successfully prevented mammosphere formation. It suppressed migration and invasion, enhanced anoikis, and regulated the expression of proteins involved in the EMT, including E-cadherin, Vimentin, and Occludin, in both TNBC cells and MDA-MB-231 spheroid cells. Further studies revealed that tetrandrine downregulated the expression of superoxide dismutase 1 (SOD1) and catalase and induced reactive oxygen species (ROS) production, which subsequently contributed to the inhibition of cell EMT and stemness. The in vivo studies also showed that tetrandrine inhibited tumor growth and metastasis of both adherent normal cells, and flow cytometry sorted specific CD44[Formula: see text]CD24[Formula: see text] breast cancer stem cells, which could be rescued by SOD1 overexpression. The results of this study suggest that tetrandrine could effectively inhibit breast cancer stem cell characteristics and the EMT process via the SOD1/ROS signaling pathway. Therefore, tetrandrine can be considered a promising anti-TNBC agent.

Artesunate, a new antimalarial clinical drug, exhibits potent anti-AML activity by targeting the ROS/Bim and TFRC/Fe2+ pathways.

BACKGROUND AND PURPOSE: Artesunate, approved by the Food and Drug Administration in 2020 as a new treatment for severe malaria, also shows anti-tumour activity against acute myeloid leukaemia (AML). However, the underlying molecular mechanism(s) of artesunate-induced apoptosis and differentiation of AML is not clearly elucidated. EXPERIMENTAL APPROACH: The biological effects of artesunate on AML were explored in vitro, using cells from AML patients and leukaemia cell lines, and in vivo, using female C57BL/6 or nude nu/nu BALB/c mice. Underlying mechanisms in vitro were examined with the Trypan blue dye exclusion assay, western blotting and flow cytometry. Effects of artesunate in C57BL/6 mice intravenously injected with murine AML cells (C1498-GFP) were assessed by numbers of AML cells and by survival. KEY RESULTS: In vitro, artesunate promoted apoptosis and differentiation in both leukaemia cell lines and patient-derived primary leukaemia cells. Mechanistically, artesunate promoted cell apoptosis by triggering reactive oxygen species (ROS) production and increasing expression of the pro-apoptotic protein Bim. Interestingly, transferrin receptor 1 (TFRC)-mediated regulation of intracellular iron homeostasis also played an essential role in AML cell differentiation induced by artesunate. In vivo, artesunate slowed AML progression and prolonged survival in a mouse leukaemia model. Notably, artesunate displayed no apparent toxicity towards healthy haematopoietic stem cells, bone marrow mononuclear cells or experimental animals. CONCLUSION AND IMPLICATIONS: Artesunate is a safe agent with significant anti-leukaemia effects in mice and may serve as a promising chemotherapeutic strategy for patients with AML, based on two different mechanisms, targeting the ROS/Bim and the TFRC/Fe2+ pathways.

Poria cocos polysaccharides exert prebiotic function to attenuate the adverse effects and improve the therapeutic outcome of 5-FU in ApcMin/+ mice.

BACKGROUND: As a first-line chemotherapeutic agent, 5-fluorouracil (5-FU) exhibits many side effects, weakening its efficacy in cancer treatment. In this study, we hypothesize that Poria cocos polysaccharides (PCP), a traditional Chinese herbal medicine with various bioactivities and prebiotic effects, might improve the therapeutic effect of 5-FU by restoring the homeostasis of the gut microenvironment and the commensal gut microflora. METHODS: ApcMin/+ mice were employed to evaluate the anti-cancer effect of 5-FU in conjunction with PCP treatment. Body weight and food consumption were monitored weekly. Polyp count was used to assess the anti-cancer effect of PCP and 5-FU. Expressions of mucosal cytokines and gut epithelial junction molecules were measured using qRT-PCR. 16S rRNA gene sequencing of fecal DNAs was used to evaluate the compositional changes of gut microbiota (GM). Transplantation of Lactobacillus johnsonii and Bifidobacterium animalis were performed to verify the prebiotic effects of PCP in improving the efficacy of 5-FU. RESULTS: The results showed that PCP treatment alleviated the weight loss caused by 5-FU treatment and reduced the polyp burden in ApcMin/+ mice. Additionally, PCP treatment eased the cytotoxic effects of 5-FU by reducing the expressions of pro-inflammatory cytokines, increasing the anti-inflammatory cytokines; and significantly improving the gut barriers by enhancing the tight junction proteins and associated adhesion molecules. Furthermore, 16S rRNA gene sequencing data showed that PCP alone or with 5-FU could stimulate the growth of probiotic bacteria (Bacteroides acidifaciens, Bacteroides intestinihominis, Butyricicoccus pullicaecorum, and the genera Lactobacillus, Bifidobacterium, Eubacterium). At the same time, it inhibited the growth of potential pathogens (e.g., Alistipes finegoldii, Alistipes massiliensis, Alistipes putredinis., Citrobacter spp., Desulfovibrio spp., and Desulfovibrio desulfuricans). Moreover, the results showed that transplantation of L.johnsonii and B.animalis effectively reduced the polyp burden in ApcMin/+ mice being treated with 5-FU. CONCLUSION: Our study showed that PCP could effectively improve the anti-cancer effect of 5-FU by attenuating its side effects, modulating intestinal inflammation, improving the gut epithelial barrier, and modulating the gut microbiota of ApcMin/+ mice.

Antimalarial and Anticancer Activity Evaluation of Bridged Ozonides, Aminoperoxides, and Tetraoxanes.

Bridged aminoperoxides, for the first time, were investigated for the in vitro antimalarial activity against the chloroquine-resistant Plasmodium falciparum strain K1 and for their cytotoxic activities against immortalized human normal liver (LO2) and lung (BEAS-2B) cell lines as well as human liver (HepG2) and lung (A549) cancer cell lines. Aminoperoxides exhibit good cytotoxicity against lung A549 cancer cell line. Synthetic ozonides were shown to have high activity against the chloroquine-resistant P. falciparum. A cyclic voltammetry study of peroxides was performed, and most of the compounds did not show a direct correlation in oxidative capacity-activity. Peroxides were analyzed for ROS production to understand their mechanism of action. However, none of the compounds has an impact on ROS generation, suggesting that ozonides induce apoptosis in HepG2 cells through ROS-independent dysfunction pathway.

Licochalcone B, a Natural Autophagic Agent for Alleviating Oxidative Stress-Induced Cell Death in Neuronal Cells and Caenorhabditis elegans Models.

Autophagy has been implicated in the regulation of neuroinflammation and neurodegenerative disorders. Licochalcone B (LCB), a chalcone from Glycyrrhiza inflata, has been reported to have anti-cancer, anti-oxidation and anti-ß-amyloid fibrillation effects; however, its effect in autophagy remain un-investigated. In the current study, the potential neuro-protective role of LCB in terms of its anti-oxidative, anti-apoptotic, and autophagic properties upon oxidative stress-induced damage in neuronal cells was investigated. With the production of reactive oxygen species (ROS) as a hallmark of neuroinflammation and neurodegeneration, hydrogen peroxide (H2O2) was adopted to stimulate ROS-induced cell apoptosis in PC-12 cells. Our findings revealed that LCB reduced cell cytotoxicity and apoptosis of PC-12 cells upon H2O2-stimulation. Furthermore, LCB increased the level of the apoptosis-associated proteins caspase-3 and cleaved caspase-3 in H2O2-induced cells. LCB effectively attenuated the level of oxidative stress markers such as MDA, SOD, and ROS in H2O2-induced cells. Most importantly, LCB was confirmed to possess its anti-apoptotic effects in H2O2-induced cells through the induction of ATG7-dependent autophagy and the SIRT1/AMPK signaling pathway. As a novel autophagic inducer, LCB increased the level of autophagy-related proteins LC3-II and decreased p62 in both neuronal cells and Caenorhabditis elegans (C. elegans) models. These results suggested that LCB has potential neuroprotective effects on oxidative damage models via multiple protective pharmacological mechanisms.

High-throughput screening for amyloid-ß binding natural small-molecules based on the combinational use of biolayer interferometry and UHPLC-DAD-Q/TOF-MS/MS.

Discovery of drugs rapidly and effectively is an important aspect for Alzheimer's disease (AD). In this study, a novel high-throughput screening (HTS) method aims at screening the small-molecules with amyloid-ß (Aß) binding affinity from natural medicines, based on the combinational use of biolayer interferometry (BLI) and ultra-high-performance liquid chromatography coupled with diode-array detector and quadrupole/time-of-flight tandem mass spectrometry (UHPLC-DAD-Q/TOF-MS/MS) has been firstly developed. Briefly, the components in natural medicines disassociated from biotinylated Aß were collected to analyze their potential Aß binding affinity by UHPLC-DAD-Q/TOF-MS/MS. Here, baicalein was confirmed to exhibit the highest binding affinity with Aß in Scutellaria baicalensis. Moreover, polyporenic acid C (PPAC), dehydrotumulosic acid (DTA), and tumulosic acid (TA) in Kai-Xin-San (KXS) were also identified as potent Aß inhibitors. Further bioactivity validations indicated that these compounds could inhibit Aß fibrillation, improve the viability in Aß-induced PC-12 cells, and decrease the Aß content and improve the behavioral ability in Caenorhabditis elegans. The molecular docking results confirmed that PPAC, DTA, and TA possessed good binding properties with Aß. Collectively, the present study has provided a novel and effective HTS method for the identification of natural inhibitors on Aß fibrillation, which may accelerate the process on anti-AD drugs discovery and development.

Ursolic acid enhances the antitumor effects of sorafenib associated with Mcl-1-related apoptosis and SLC7A11-dependent ferroptosis in human cancer.

As a broad-spectrum oral small molecule inhibitor targeting multikinase, sorafenib is currently approved for the clinical treatment of several types of cancer as a single agent. A considerable number of clinical trial results have indicated that combination therapies involving sorafenib have been shown to improve treatment efficacy and may lead to novel therapeutic applications. Ursolic acid (UA), a natural pentacyclic triterpene compound extracted from a great variety of traditional medicinal plants and most fruits and vegetables, exhibits a wide range of therapeutic potential, including against cancer, diabetes, brain disease, liver disease, cardiovascular diseases, and sarcopenia. In the present study, we investigated the antitumor effects of sorafenib in combination with ursolic acid and found that the two agents displayed significant synergistic antitumor activity in in vitro and in vivo tumor xenograft models. Sorafenib/UA induced selective apoptotic death and ferroptosis in various cancer cells by evoking a dramatic accumulation of intracellular lipid reactive oxygen species (ROS). Mechanistically, the combination treatment promoted Mcl-1 degradation, which regulates apoptosis. However, decreasing the protein level of SLC7A11 plays a critical role in sorafenib/UA-induced cell ferroptosis. Therefore, these results suggest that the synergistic antitumor effects of sorafenib combined with ursolic acid may involve the induction of Mcl-1-related apoptosis and SLC7A11-dependent ferroptosis. Our findings may offer a novel effective therapeutic strategy for tumor treatment.

Far infrared irradiation suppresses experimental arthritis in rats by down-regulation of genes involved inflammatory response and autoimmunity.

Introduction: Far-infrared radiation (FIR) is widely used in the treatment of various diseases such as insomnia and cardiovascular risk. Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease in which the therapeutic potential of FIR in RA is unclear. Objectives: To determine the therapeutic potential and mechanistic actions of FIR in treatment of RA. Methods: Adjuvant-induced arthritis (AIA) rat models were established to assess the therapeutic potency of FIR in RA treatment. The scoring parameters such as arthritis score, swelling of the hind paw, spleen and thymus indices, micro-CT analysis indices were adopted to estimate the beneficial effects of FIR during RA treatment in AIA model. PCR gene expression arrays were used to analyze inflammatory and autoimmune genes expression profiles in rat synovium. The inflammatory and immunity genes profiling was further analyzed through transcription factor prediction using PROMO. A signaling network map of possible molecular circuits connecting the identified differential genes to the RA's pathogenesis was constructed based on extensive literature reviews, and the major signaling pathways were validated by Western blotting. Results: Thirty minutes of FIR treatment significantly improved the symptoms of AIA in rats. Gene expression profiling indicated that 27 out of 370 genes were down-regulated by FIR. AP-1, CEBPα, CEBPß, c-Fos, GR, HNF-3ß, USF-1, and USF-2 were predicted as key transcription factors that regulated the identified differential genes. In addition, MAPK, PI3K-Akt, and NF-κB signaling are the major molecular pathways down-regulated by FIR treatment. Conclusion: FIR may provide beneficial effects on the AIA rat model of arthritis by suppression of the MAPK, PI3K-Akt and NF-κB signaling pathways. Therefore, we believe that FIR may provide an alternative non-pharmacological and non-surgical therapeutic approach for the treatment of RA.

Targeting Nrf2-Mediated Oxidative Stress Response in Traumatic Brain Injury: Therapeutic Perspectives of Phytochemicals.

Traumatic brain injury (TBI), known as mechanical damage to the brain, impairs the normal function of the brain seriously. Its clinical symptoms manifest as behavioral impairment, cognitive decline, communication difficulties, etc. The pathophysiological mechanisms of TBI are complex and involve inflammatory response, oxidative stress, mitochondrial dysfunction, blood-brain barrier (BBB) disruption, and so on. Among them, oxidative stress, one of the important mechanisms, occurs at the beginning and accompanies the whole process of TBI. Most importantly, excessive oxidative stress causes BBB disruption and brings injury to lipids, proteins, and DNA, leading to the generation of lipid peroxidation, damage of nuclear and mitochondrial DNA, neuronal apoptosis, and neuroinflammatory response. Transcription factor NF-E2 related factor 2 (Nrf2), a basic leucine zipper protein, plays an important role in the regulation of antioxidant proteins, such as oxygenase-1(HO-1), NAD(P)H Quinone Dehydrogenase 1 (NQO1), and glutathione peroxidase (GPx), to protect against oxidative stress, neuroinflammation, and neuronal apoptosis. Recently, emerging evidence indicated the knockout (KO) of Nrf2 aggravates the pathology of TBI, while the treatment of Nrf2 activators inhibits neuronal apoptosis and neuroinflammatory responses via reducing oxidative damage. Phytochemicals from fruits, vegetables, grains, and other medical herbs have been demonstrated to activate the Nrf2 signaling pathway and exert neuroprotective effects in TBI. In this review, we emphasized the contributive role of oxidative stress in the pathology of TBI and the protective mechanism of the Nrf2-mediated oxidative stress response for the treatment of TBI. In addition, we summarized the research advances of phytochemicals, including polyphenols, terpenoids, natural pigments, and otherwise, in the activation of Nrf2 signaling and their potential therapies for TBI. Although there is still limited clinical application evidence for these natural Nrf2 activators, we believe that the combinational use of phytochemicals such as Nrf2 activators with gene and stem cell therapy will be a promising therapeutic strategy for TBI in the future.

Biological Evaluation in Resistant Cancer Cells and Study of Mechanism of Action of Arylvinyl-1,2,4-Trioxanes.

1,2,4-trioxane is a pharmacophore, which possesses a wide spectrum of biological activities, including anticancer effects. In this study, the cytotoxic effect and anticancer mechanism of action of a set of 10 selected peroxides were investigated on five phenotypically different cancer cell lines (A549, A2780, HCT8, MCF7, and SGC7901) and their corresponding drug-resistant cancer cell lines. Among all peroxides, only 7 and 8 showed a better P-glycoprotein (P-gp) inhibitory effect at a concentration of 100 nM. These in vitro results were further validated by in silico docking and molecular dynamic (MD) studies, where compounds 7 and 8 exhibited docking scores of -7.089 and -8.196 kcal/mol, respectively, and remained generally stable in 100 ns during MD simulation. Further experiments revealed that peroxides 7 and 8 showed no significant effect on ROS accumulations and caspase-3 activity in A549 cells. Peroxides 7 and 8 were also found to decrease cell membrane potential. In addition, peroxides 7 and 8 were demonstrated to oxidize a flavin cofactor, possibly elucidating its mechanism of action. In conclusion, apoptosis induced by 1,2,4-trioxane was shown to undergo via a ROS- and caspase-3-independent pathway with hyperpolarization of cell membrane potential.

Ferulic Acid Exerts Neuroprotective Effects via Autophagy Induction in C. elegans and Cellular Models of Parkinson's Disease.

Parkinson's disease (PD) is a complex neurological disorder characterized by motor and nonmotor features. Although some drugs have been developed for the therapy of PD in a clinical setting, they only alleviate the clinical symptoms and have yet to show a cure. In this study, by employing the C. elegans model of PD, we found that ferulic acid (FA) significantly inhibited α-synuclein accumulation and improved dyskinesia in NL5901 worms. Meanwhile, FA remarkably decreased the degeneration of dopaminergic (DA) neurons, improved the food-sensing behavior, and reduced the level of reactive oxygen species (ROS) in 6-OHDA-induced BZ555 worms. The mechanistic study discovered that FA could activate autophagy in C. elegans, while the knockdown of 3 key autophagy-related genes significantly revoked the neuroprotective effects of FA in α-synuclein- and 6-OHDA-induced C. elegans models of PD, demonstrating that FA exerts an anti-PD effect via autophagy induction in C. elegans. Furthermore, we found that FA could reduce 6-OHDA- or H2O2-induced cell death and apoptosis in PC-12 cells. Moreover, FA was able to induce autophagy in stable GFP-RFP-LC3 U87 cells and PC-12 cells, while bafilomycin A1 (Baf, an autophagy inhibitor) partly eliminated the protective effects of FA against 6-OHDA- and H2O2-induced cell death and ROS production in PC-12 cells, further confirming that FA exerts an anti-PD effect via autophagy induction in vitro. Collectively, our study provides novel insights for FA as a potent autophagy enhancer to effectively prevent neurodegenerative diseases such as PD in the future.

Dietary Plant Polyphenols as the Potential Drugs in Neurodegenerative Diseases: Current Evidence, Advances, and Opportunities.

Neurodegenerative diseases, including Alzheimer's disease (AD), Parkinson's disease (PD), and Huntington's disease (HD), are characterized by the progressive degeneration of neurons. Although the etiology and pathogenesis of neurodegenerative diseases have been studied intensively, the mechanism is still in its infancy. In general, most neurodegenerative diseases share common molecular mechanisms, and multiple risks interact and promote the pathologic process of neurogenerative diseases. At present, most of the approved drugs only alleviate the clinical symptoms but fail to cure neurodegenerative diseases. Numerous studies indicate that dietary plant polyphenols are safe and exhibit potent neuroprotective effects in various neurodegenerative diseases. However, low bioavailability is the biggest obstacle for polyphenol that largely limits its adoption from evidence into clinical practice. In this review, we summarized the widely recognized mechanisms associated with neurodegenerative diseases, such as misfolded proteins, mitochondrial dysfunction, oxidative damage, and neuroinflammatory responses. In addition, we summarized the research advances about the neuroprotective effect of the most widely reported dietary plant polyphenols. Moreover, we discussed the current clinical study and application of polyphenols and the factors that result in low bioavailability, such as poor stability and low permeability across the blood-brain barrier (BBB). In the future, the improvement of absorption and stability, modification of structure and formulation, and the combination therapy will provide more opportunities from the laboratory into the clinic for polyphenols. Lastly, we hope that the present review will encourage further researches on natural dietary polyphenols in the treatment of neurodegenerative diseases.

Natural Citrus flavanone 5-demethylnobiletin stimulates melanogenesis through the activation of cAMP/CREB pathway in B16F10 cells.

BACKGROUND: 5-demethylnobiletin is a natural polymethoxyflavone which is isolated from the extract of citrus fruits peels. It exhibits a broad spectrum of biological activities such as anti-cancer, anti-inflammatory, cardiovascular protective and neuroprotective effects, however, its effect in melanogenesis remains uninvestigated. PURPOSE: Melanin synthesis is a very important biological process in curing disease such as vitiligo with depigmentation on the skin. In the current work, we aim to confirm the bioactivity and mechanism of 5-demethylnobiletin in stimulating melanogenesis. STUDY DESIGN: To confirm the mechanistic role of 5-demethylnobiletin in enhancing melanogenesis, its effect on the activity of tyrosinase, together with the level of microphthalmia-associated transcription factor (MITF), Trp-1, Trp-2, melanocyte-specific marker protein PMEL17, Rab27a, Melanophilin and Myosin VA were studied in B16F10 melanoma cells. METHODS: Multiple biological assays on melanogenesis-associated proteins such as melanin content detection, tyrosinase activity colorimetric assay, qPCR, western blot analysis, dual-luciferase reporter assay, cAMP activity assay and Fontana-Masson ammoniacal silver staining were used to confirm the role of 5-demethylnobiletin in stimulating melanin synthesis and the transportation of melanosomes. RESULTS: As confirmed by multiple biological assays, 5-demethylnobiletin is found to stimulate dendrite structure formation in cells, melanin synthesis and the transportation of melanosomes, via inducing the phosphorylation of cAMP response element-binding protein (CREB) and increasing the intracellular levels of cAMP in vitro through the PKA-dependent pathway. CONCLUSION: The findings suggested that 5-demethylnobiletin may be considered as a potential natural product candidate for patients with pigment disorder.