Dual-Channel/Localization Single-Molecule Fluorescence Probe for Monitoring ATP and HOCl in Early Diagnosis and Therapy of Rheumatoid Arthritis.

Rheumatoid arthritis (RA), a common chronic inflammatory illness, is still incurable, reducing the sufferers' quality of life significantly. Adenosine 5'-triphosphate (ATP) and hypochlorous acid (HOCl) are key indicators in RA, but their precise mechanisms in RA pathophysiology are unknown. As a result, in order to detect ATP and HOCl simultaneously, we created two new dual-channel/localization single-molecule fluorescence probes, RhTNMB and RhFNMB. Furthermore, RhFNMB outperformed RhTNMB in terms of detection performance. ATP and HOCl produce independent fluorescence responses in the light red channel (λex = 520 nm, λem = 586 nm) and deep red channel (λex = 620 nm, λem = 688 nm), respectively, without spectral crosstalk. It should be noted that the probe RhFNMB successfully imaged ATP in mitochondria and HOCl in cells. Surprisingly, the probe RhFNMB demonstrated remarkable detection ability in the diagnosis and treatment of Pseudomonas aeruginosa-induced abdominal inflammation in mice. We continued to apply the probe RhFNMB to track ATP and HOCl in RA and discovered that ATP and HOCl concentrations were considerably greater in RA joints than in normal joints. We also confirmed the therapeutic effect of methotrexate on RA. This study is the first to achieve dual-channel imaging of ATP and HOCl, which is of great value for the early diagnosis and therapy of RA.

Mitochondrial-Targeted AIE-Active Fluorescent Probe Based on Tetraphenylethylene Fluorophore with Dual Positive Charge Recognition Sites for Monitoring ATP in Cells.

Adenosine triphosphate (ATP), as an important intracellular energy currency produced in mitochondria, is closely related to various diseases in living organisms. Currently, the biological application of AIE fluorophore as a fluorescent probe for ATP detection in mitochondria is rarely reported. Herein, D-π-A and D-A structure-based tetraphenylethylene (TPE) fluorophores were employed to synthesize six different ATP probes (P1-P6), and the phenylboronic acid groups and dual positive charge sites of probes could interact with the vicinal diol of ribose and negatively charged triphosphate structure of ATP, respectively. However, P1 and P4 with a boronic acid group and a positive charge site had poor selectivity for ATP detection. In contrast, P2, P3, P5, and P6 with dual positive charge sites exhibited better selectivity than P1 and P4. In particular, P2 had more advantages of high sensitivity, selectivity, and good time stability for ATP detection than P3, P5, and P6, which was ascribed to its D-π-A structure, linker 1 (1,4-bis(bromomethyl)benzene), and dual positive charge recognition sites. Then, P2 was employed to detect ATP, and it exhibited a low detection limit of 3.62 µM. Moreover, P2 showed utility in the monitoring of mitochondrial ATP level fluctuations.

The interaction between Hsp90-mediated unfolded protein response and autophagy contributes to As3+/ Se4+ combination-induced apoptosis of acute promyelocytic leukemia cells.

The interaction between the unfolded protein response (UPR) and autophagy plays either pro-survival or pro-apoptotic roles in the treatment of acute promyelocytic leukemia (APL). Our previous study has shown that the combination therapy of arsenite (As3+) and selenite (Se4+) induces apoptosis in APL NB4 cells, although the mechanisms are not clear. Here, we demonstrate that the interaction between heat shock protein 90 (Hsp90)-mediated UPR and autophagy is the core module for As3+/Se4+ combination-induced apoptosis. Hsp90 overexpression and knockdown assays indicate that Hsp90 inhibition by PERK modulates two branches of the UPR, leading to the activation of ATF4 and CHOP, causing the degradation of IRE1α and the dephosphorylation of eIF2α, thereby contributing to switching the cytoprotective UPR into an apoptotic pathway. Assays using pretreatment with inducers and inhibitors of endoplasmic reticulum stress (ERS) and autophagy reveal that autophagy is stimulated by ERS but suppressed by As3+/Se4+ combination via the mTOR signaling pathway. However, inhibition of autophagy decreases GRP78 expression and eIF2α phosphorylation, thereby further promoting ERS-induced apoptosis. Moreover, As3+/Se4+ combination blocks hepatic infiltration in an APL-NCG mouse model of extramedullary infiltration. Taken together, these findings provide novel agents and therapeutic approaches for APL.

Inhibition of splicing factors SF3A3 and SRSF5 contributes to As3+/Se4+ combination-mediated proliferation suppression and apoptosis induction in acute promyelocytic leukemia cells.

The low-dose combination of Arsenite (As3+) and selenite (Se4+) has the advantages of lower biological toxicity and better curative effects for acute promyelocytic leukemia (APL) therapy. However, the underlying mechanisms remain unclear. Here, based on the fact that the combination of 2 µM A3+ plus 4 µM Se4+ possessed a stronger anti-leukemic effect on APL cell line NB4 as compared with each individual, we employed iTRAQ-based quantitative proteomics to identify a total of 58 proteins that were differentially expressed after treatment with As3+/Se4+ combination rather than As3+ or Se4+ alone, the majority of which were involved in spliceosome pathway. Among them, eight proteins stood out by virtue of their splicing function and significant changes. They were validated as being decreased in mRNA and protein levels under As3+/Se4+ combination treatment. Further functional studies showed that only knockdown of two splicing factors, SF3A3 and SRSF5, suppressed the growth of NB4 cells. The reduction of SF3A3 was found to cause G1/S cell cycle arrest, which resulted in proliferation inhibition. Moreover, SRSF5 downregulation induced cell apoptosis through the activation of caspase-3. Taken together, these findings indicate that SF3A3 and SRSF5 function as pro-leukemic factors and can be potential novel therapeutic targets for APL.

Red and Near-Infrared Fluorescent Probe for Distinguishing Cysteine and Homocysteine through Single-Wavelength Excitation with Distinctly Dual Emissions.

Small-molecule biothiols, including cysteine (Cys), homocysteine (Hcy), and glutathione (GSH), participate in various pathological and physiological processes. It is still a challenge to simultaneously distinguish Cys and Hcy because of their similar structures and reactivities, as well as the interference from the high intramolecular concentration of GSH. Herein, a novel fluorescent probe, CySI, based on cyanine and thioester was developed to differentiate Cys and Hcy through a single-wavelength excitation and two distinctly separated emission channels. The probe exhibited a turn-on fluorescence response to Cys at both 625 nm (the red channel) and 740 nm (the near-infrared channel) but only showed fluorescence turn-on to Hcy at 740 nm (the near-infrared channel) and no fluorescent response to GSH. With the aid of built-in self-calibration of single excitation and dual emissions, simultaneous discriminative determinations of Cys and Hcy were realized through red and near-infrared channels. CySI exhibited excellent selectivity toward Cys and Hcy with a fast response. This probe was further exploited to visualize exogenous Cys and Hcy in cells through dual emission channels under one excitation. Moreover, it could efficiently target mitochondria and was applied to monitor the endogenous Cys fluctuations independently in mitochondria through the red emission channel.

A dual-response mitochondria-targeted NIR fluorescent probe with large Stokes shift for monitoring viscosity and HOCl in living cells and zebrafish.

Mitochondria are important subcellular organelles involved in many cellular activities. Therefore, it is very important to monitor the concentration of various substances in mitochondria. In this work, we constructed a dual-response mitochondria-targeted fluorescent probe HBTN for the detection of viscosity and HOCl in vitro and in vivo. HBTN not only has a long emission wavelength with an emission peak of 680 nm, but also has a large Stokes shift of 278 nm. The fluorescence intensity of probe HBTN at 680 nm has a good linear relationship with solution viscosity, which can be used for quantitative detection of viscosity. In addition, the probe HBTN enables ratiometric detection of HOCl with a low detection limit (DL = 24.5 nM) and rapid response (<20 min). More importantly, the probe HBTN has been successfully applied to the detection of viscosity and exogenous/endogenous HOCl in the mitochondria of MCF-7 cells. Furthermore, the probe HBTN has been implemented in imaging viscosity and HOCl in zebrafish. HBTN is expected to be a practical tool for monitoring viscosity and HOCl in vivo.

Water-Soluble Conjugated Polymer as a Fluorescent Probe for Monitoring Adenosine Triphosphate Level Fluctuation in Cell Membranes during Cell Apoptosis and in Vivo.

Adenosine triphosphate (ATP) is used as the energy source in cells and plays crucial roles in various cellular events. The cellular membrane is the protective barrier for the cytoplasm of living cells and involved in many essential biological processes. Many fluorescent probes for ATP have been successfully developed, but few of these probes were appropriate for visualizing ATP level fluctuation in cell membranes during the apoptotic cell death process. Herein, we report the synthesis of a new water-soluble cationic polythiophene derivative that can be utilized as a fluorescent sensor for detecting ATP in cell membranes. Poly((3-((4-methylthiophen-3-yl)oxy)propyl)triphenylphosphonium chloride) (PMTPP) exhibits high sensitivity and good selectivity to ATP, and the detection limit is 27 nM. The polymer shows low toxicity to live cells and excellent photostability in cell membranes. PMTPP was practically utilized for real-time monitoring of ATP levels in the cell membrane through fluorescence microscopy. We have demonstrated that the ATP levels in cell membranes increased during the apoptotic cell death process. The probe was also capable of imaging ATP levels in living mice.

A direct assay of carboxyl-containing small molecules by SALDI-MS on a AgNP/rGO-based nanoporous hybrid film.

Silver nanoparticles (AgNPs) and reduced graphene oxide (rGO) hybrid nanoporous structures fabricated by the layer-by-layer (LBL) electrostatic self-assembly have been applied as a simple platform for the rapid analysis of carboxyl-containing small molecules by surface-assisted laser desorption/ionization (D/I) mass spectrometry (SALDI-MS). By the simple one-step deposition of analytes onto the (AgNP/rGO)9 multilayer film, the MS measurements of various carboxyl-containing small molecules (including amino acids, fatty acids and organic dicarboxylic acids) can be done. In contrast to other energy transfer materials relative to AgNPs, the signal interferences of a Ag cluster (Agn(+) or Agn(-)) and a C cluster (Cn(+) or Cn(-)) have been effectively reduced or eliminated. The effects of various factors, such as the pore structure and composition of the substrates, on the efficiency of D/I have been investigated by comparing with the (AgNP)9 LBL nanoporous structure, (AgNP/rGO)9/(SiO2NP)6 LBL multilayer film and AgNP/prGO nanocomposites.

A novel nanoparticle fluorescent probe based on a water-soluble conjugated polymer for real-time monitoring of ATP fluctuation and configuration of the Golgi apparatus during the inhibition of glycolysis.

BACKGROUND: Adenosine 5'-triphosphate (ATP) plays an important role in cell metabolism and has been regarded as an indicator of cell survival and damage. Golgi apparatus participates in the signal transduction processes of substance transport, ion homeostasis and stress when extracellular substances enter cells. Till now, there is no fluorescent probe for monitoring Golgi ATP level fluctuation and visualizing the configuration change of the Golgi apparatus during the inhibition of glycolysis. RESULTS: Herein, we report the synthesis of a novel water-soluble cationic polythiophene derivative (PEMTEA) that can be employed as a fluorescent sensor for measuring ATP in the Golgi apparatus. PEMTEA self-assembles into PT-NP nanoparticles in aqueous solution with a diameter of approximately 2 nm. PT-NP displays high sensitivity and superb selectivity towards ATP with a detection limit of 90 nM and a linear detection range from 0 to 3.0 µM. The nanoparticles show low toxicity to HepG2 cells and good photostability in the Golgi apparatus. With the stimulation of Ca2+, PT-NP was practically applied to real-time monitor of endogenous ATP levels in the Golgi apparatus through fluorescence microscopy. Finally, we studied the relationship between the concentration of ATP and configuration of the Golgi apparatus during the inhibition of glycolysis using PT-NP. SIGNIFICANCE: We have demonstrated that PT-NP can not only indicate the fluctuation and distribution of ATP in the Golgi apparatus, but also give the information of the configuration change of the Golgi apparatus at the single-cell level during the inhibition of glycolysis.

Water extract of earthworms mitigates mouse liver fibrosis by potentiating hepatic LKB1/Nrf2 axis to inhibit HSC activation and hepatocyte death.

ETHNOPHARMACOLOGICAL RELEVANCE: When left untreated, liver fibrosis (LF) causes various chronic liver diseases. Earthworms (Pheretima aspergillum) are widely used in traditional medicine because of their capacity to relieve hepatic diseases. AIM OF THE STUDY: This study aimed to explore the anti-LF effects of water extract of earthworms (WEE) and the underlying molecular mechanisms. MATERIALS AND METHODS: A CCl4-induced mouse model of LF was used to study the impact of WEE on LF in vivo. The anti-LF activity of WEE in mice was compared with that of silybin, which can be clinically applied in LF intervention and was used as a positive control. Activation of LX-2 hepatic stellate cells (HSCs) and apoptosis and ferroptosis of AML-12 hepatocytes induced by TGFß1 were used as in vitro models. RESULTS: WEE drastically improved LF in mice. WEE reduced markers of activated HSCs in mice and inhibited TGFß1-induced activation of LX-2 HSCs in vitro. Additionally, WEE suppressed CCl4-induced apoptosis and ferroptosis in mouse hepatocytes. Mechanistically, WEE induced Nrf2 to enter the nuclei of the mouse liver cells, and the hepatic levels of Nrf2-downstream antioxidative factors increased. LKB1/AMPK/GSK3ß is an upstream regulatory cascade of Nrf2. In the LF mouse model, WEE increased hepatic phosphorylated LKB1, AMPK, and GSK3ß levels. Similar results were obtained for the LX-2 cells. In AML-12 hepatocytes and LX-2 HSCs, WEE elevated intracellular Nrf2 levels, promoted its nuclear translocation, and inhibited TGFß1-induced ROS accumulation. Knocking down LKB1 abolished the impact of WEE on the AMPK/GSK3ß/Nrf2 cascade and eliminated its protective effects against TGFß1. CONCLUSIONS: Our findings reveal that WEE improves mouse LF triggered by CCl4 and supports its application as a promising hepatoprotective agent against LF. The potentiation of the hepatic antioxidative AMPK/GSK3ß/Nrf2 cascade by activating LKB1 and the subsequent suppression of HSC activation and hepatocyte apoptosis and ferroptosis are implicated in WEE-mediated alleviation of LF.

Rapid equilibrium kinetic analysis of arsenite methylation catalyzed by recombinant human arsenic (+3 oxidation state) methyltransferase (hAS3MT).

In the human body, arsenic is metabolized by methylation. Understanding this process is important and provides insight into the relationship between arsenic and its related diseases. We used the rapid equilibrium kinetic model to study the reaction sequence of arsenite methylation. The results suggest that the mechanism for arsenite methylation is a completely ordered mechanism that is also of general interest in reaction systems with different reductants, such as tris(2-carboxyethyl)phosphine, cysteine, and glutathione. In the reaction, cysteine residues of recombinant human arsenic (+3 oxidation state) methyltransferase (hAS3MT) coordinate with arsenicals and involve the methyl transfer step. S-Adenosyl-l-methionine (AdoMet) is the first-order reactant, which modulates the conformation of hAS3MT to a best matched state by hydrophobic interaction. As the second-order reactant, reductant reduces the disulfide bond, most likely between Cys-250 and another cysteine residue of hAS3MT, and exposes the active site cysteine residues for binding trivalent inorganic arsenic (iAs(3+)) to give monomethylarsonic dicysteine (MADC(3+)). In addition, the reaction can be extended to further methylate MADC(3+) to dimethylarsinic cysteine (DAMC(3+)). In the methylation reaction, the ß-pleated sheet content of hAS3MT is increased, and the hydrophobicity of the microenvironment around the active sites is decreased. Similarly, we confirm that both the high ß-pleated sheet content of hAS3MT and the high dissociation ability of the enzyme-AdoMet-reductant improve the yield of dimethylated arsenicals.

Indirect transformation of coordination-polymer particles into magnetic carbon-coated MN3O4 (MN3O4@C) nanowires for supercapacitor electrodes with good cycling performance.

Carbon-coated Mn3O4 nanowires (Mn3O4@C NWs) have been synthesized by the reduction of well-shaped carbon-coated bixbyite networks and characterized by TEM, X-ray diffraction, X-ray photoelectron spectroscopy, and electrochemical experiments. To assess the properties of 1D carbon-coated nanowires for their use in supercapacitors, cyclic voltammetry and galvanostatic charging-discharging measurements were performed. Mn3O4 @C NWs could be charged and discharged faster and had higher capacitance than bare Mn3O4 nanostructures and other commercial materials. The capacitance of the Mn3O4@C NWs was 92% retained after 3000 cycles at a charging rate of 5 Ag(-1). This improvement can be attributed to the carbon shells, which promote fast Faradaic charging and discharging of the interior Mn3O4 core and also act as barriers to protect the inner core. These Mn3O4@C NWs could be a promising candidate material for high-capacity, low-cost, and environmentally friendly electrodes for supercapacitors. In addition, the magnetic properties of the as-synthesized samples are also reported to investigate the influence of the carbon coating.

Distribution of microcystin-LR to testis of male Sprague-Dawley rats.

Microcystins (MCs) are a group of cyclic heptapeptide toxins produced by naturally freshwater cyanobacteria. Among more than 90 identified analogues of microcystins, microcystin-LR (MC-LR) is the most abundant and toxic. Our previous investigations indicated that MC-LR displays male reproductive toxicity, but the target of MC-LR in testes remains unclear. To this end, the present study is designed to elucidate whether microcystin-LR could be distributed to testes and explore the target cells in testes. In the in vivo study, male Sprague-Dawley rats were injected intraperitoneally with MC-LR at a dose of 300 µg/kg per day for 6 days. MC-LR was detected in testes, mainly within seminiferous tubules, which was further validated by Western blot. The concentrations of MC-LR were determined by LC-MS analysis, with a result of 0.0252 ± 0.0037 and 0.0056 ± 0.0012 µg/g dry weight in liver and testis respectively. In the in vitro study, Primary cultured spermatogonia, Sertoli cells and Leydig cells were exposed to MC-LR respectively, and MC-LR was observed to enter spermatogonia and Sertoli cells, but not Leydig cells. These results suggested that the reproductive toxicity of MC-LR were induced by its distribution in testis. Spermatogonia and Sertoli cells are important target cells.

A network pharmacology-based approach to explore the active ingredients and molecular mechanism of Shen-Kui-Tong-Mai granules on a rat model with chronic heart failure.

OBJECTIVES: This study aimed to comprehensively investigate the potential active components and therapeutic mechanisms of Shen-Kui-Tong-Mai granule (SKTMG) in the treatment of heart failure. METHODS: Network pharmacology combined with ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS), molecular docking, and in vivo validation was performed to identify the active components and the potential targets for SKTMG to improve chronic heart failure (CHF). KEY FINDINGS: The network pharmacology identified 192 active compounds and 307 potential consensus targets for SKTMG. On the other hand, network analysis discovered 10 core target genes related to the MAPK signal pathway. These genes include AKT1, STAT3, MAPK1, P53, SRC, JUN, TNF, APP, MAPK8 and IL6. The molecular docking results revealed that the SKTMG components were luteolin, quercetin, astragaloside IV and kaempferol, which could bind AKT1, MAPK1, P53, JUN, TNF and MAPK8. Additionally, SKTMG inhibited phosphorylation of AKT, P38, P53 and c-JUN, and reduced TNF-α expression in CHF rats. CONCLUSIONS: The present results demonstrated that network pharmacology combined with UHPLC-MS/MS, molecular docking and in vivo validation can facilitate the identification of active components and the potential targets for SKTMG to improve CHF.

The suppressive effects of Bursopentine (BP5) on oxidative stress and NF-ĸB activation in lipopolysaccharide-activated murine peritoneal macrophages.

BACKGROUND/AIM: Bursopentine (BP5) is a novel thiol-containing pentapeptide isolated from chicken bursa of Fabricius, and is reported to exert immunomodulatory effects on B and T lymphocytes. It has been found that some thiol compounds, such as glutathione (GSH) and N-acetylcysteine (NAC) protect living cells from oxidative stress. This led us to investigate whether BP5 had any ability to protect macrophages from oxidative stress as well as any mechanism that might underlie this process. METHODS: Murine peritoneal macrophages activated by lipopolysaccharide (LPS) (2 µg/ml) were treated with single bouts (0, 25, 50, and 100 µM) of BP5. RESULTS: BP5 potently suppressed the markers for oxidative stress, including nitric oxide (NO), reactive oxygen species (ROS), lipid peroxidation, and protein oxidation. It also decreased the expression and activity of inducible nitric oxide synthase (iNOS) and promoted a protective antioxidant state by elevating GSH content and by activating the expression and activity of certain key antioxidant and redox enzymes, including glutathione peroxidase (GPx), glutathione reductase (GR), superoxide dismutase (SOD) and catalase (CAT). This suppressive effect on oxidative stress was accompanied by down-regulated expression and activity of nuclear factor kappa B (NF-κB). CONCLUSION: These findings demonstrate that BP5 can protect LPS-activated murine peritoneal macrophages from oxidative stress. BP5 may have applications as an anti-oxidative stress reagent.

Evidence for inhibition of HIF-1α prolyl hydroxylase 3 activity by four biologically active tetraazamacrocycles.

Hypoxia inducible factor 1 (HIF-1) is central to the hypoxic response in mammals. HIF-1α prolyl hydroxylase 3 (PHD3) degrades HIF through the hydroxylation of HIF-1α. Inhibition of PHD3 activity is crucial for up-regulating HIF-1α levels, thereby acting as HIF-dependent diseases therapy. Macrocyclic polyamines which display high stability on iron-chelating may well inhibit the enzyme activity. Thus inhibition and interaction on catalytic PHD3 by four biologically active tetraazamacrocycles (1-4), which have two types of parent rings to chelate iron(ii) dissimilarly, were studied. The apparent IC(50) values of 2.56, 1.91, 5.29 and 2.44 µM, respectively, showed good inhibition potency of the four compounds. K(I) values were 7.86, 3.69, 1.59 and 2.92 µM for 1-4, respectively. Different inhibition actions of the two groups of compounds were identified. Circular dichroism (CD) and fluorescence spectrometries proved that one type of compound has significant effects on protein conformation while another type does not. Computational methodology was constructed to employ the equilibrium geometry of enzyme active site with the presence of substrate competitive inhibitor. Iron(ii) coordination in the active site by inhibitors of this kind induces conformational change of the enzyme and blocks substrate binding.

Fracture repair by IOX2: Regulation of the hypoxia inducible factor-1α signaling pathway and BMSCs.

The treatment of fracture delayed union and nonunion has become a challenging problem. Hypoxia inducible factor-1α (HIF-1α) is reported to be a key factor in fracture healing, and is degraded by hydroxylation of prolyl hydroxylase (PHDs) under normal oxygen. Small molecules could inhibit the activity of PHDs, stabilize HIF-1α protein, regulate the expression of downstream target genes of HIF-1α, and make the body adapt to hypoxia. The migration and osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) is the most promising candidate for the treatment of fracture nonunion. Here we reported that IOX2, an HIF-1α PHD inhibitor, markedly improved the proliferation and migration of BMSCs by upregulating intracellular Ca2+ and concomitant decreasing reactive oxygen species (ROS) in vitro, and facilitated the repair of bone fracture by increasing the number of BMSCs and cartilage formation in vivo. No significant influence of IOX2 on the proliferation and migration of BMSCs after silencing of the HIF-1α. Together, our findings indicated that IOX2 promoted the proliferation and migration of BMSCs via the HIF-1α pathway and further accelerated fracture healing. These results provide a deeper understanding of the mechanism by which HIF promotes fracture healing.

BP5 alleviates endotoxemia-induced acute lung injury by activating Nrf2 via dual regulation of the Keap1-Nrf2 interaction and the Akt (Ser473)/GSK3ß (Ser9)/Fyn pathway.

Oxidative stress and inflammation play a crucial role in the pathogenesis of acute lung injury (ALI). Previously, pentapeptide bursopentin (BP5, Cys-Lys-Arg-Val-Tyr) was reported to possess significant antioxidant activity and inhibit lipopolysaccharides (LPS)-induced NF-κB activation in vitro, whereas little is known about its effects in vivo. In this study, we explored the effects of BP5 on endotoxemia-induced ALI in mice and the underlying molecular mechanisms. Our studies revealed that BP5 markedly improved survival and effectively alleviated lung injury by reducing overoxidation and excessive inflammatory response in endotoxemia mice. In LPS-stimulated mouse primary macrophages and RAW 264.7 cells, BP5 also exhibited antioxidant and anti-inflammatory properties by enhancing Nrf2 activation. Importantly, these beneficial effects were abolished by Nrf2 knockdown. To further elucidate the underlying mechanisms, we performed localized surface plasmon resonance (LSPR) assays, molecular docking, together with cell-based studies, and found that BP5 inhibited the Keap1-Nrf2 interaction to promote Nrf2 nuclear translocation and activation. Moreover, BP5-induced Nrf2 activation was shown to be accompanied by an increase in the phosphorylation of Akt (at Ser473) and GSK3ß (at Ser9), and a decrease in Fyn nuclear accumulation both in vitro and in vivo. Pharmacologically inhibiting phosphorylation of Akt and GSK3ß obviously enhanced Fyn nuclear accumulation in RAW 264.7 cells, which partially attenuated the promoting effect of BP5 on Nrf2 nuclear accumulation and activation. Furthermore, In Nrf2-/- mice, the protective effects of BP5 on the endotoxemia-induced ALI in WT mice were largely vanished. Our findings indicated that BP5 effectively protected endotoxemia-induced ALI against oxidative stress and inflammatory response, which are largely dependent on activation of the Nrf2 pathway. Underlying mechanisms include dual regulation of the Keap-Nrf2 interaction and the Akt (Ser473)/GSK3ß (Ser9)/Fyn pathway.

Berberine regulates short-chain fatty acid metabolism and alleviates the colitis-associated colorectal tumorigenesis through remodeling intestinal flora.

BACKGROUND: Colitis-associated cancer (CAC) is known to be a complex combination of tumor cells, non-tumor cells and a large intestinal flora. The increasing role of intestinal flora in CAC may represent a new approach to improving CAC treatment. Berberine can reduce colorectal adenoma recurrence and inhibit colorectal carcinogenesis. PURPOSE: Berberine has demonstrated efficacy for the control and suppression of CAC. Given the low oral absorption into the blood and large intestinal excretion of berberine, intestinal flora may be one of the important targets of berberine inhibiting the occurrence of colorectal cancer (CRC). The purpose of this study was to investigate the effects of berberine on intestinal flora in CAC mice and its ability to remodel intestinal flora to improve short-chain fatty acid metabolism. STUDY DESIGN AND METHODS: The CAC model in mice was induced by Azoxymethane/Dextran sodium sulfate (AOM/DSS). Berberine was administered daily at doses of 50 and 100 mg/kg, and aspirin was used as the positive control. The effect of berberine on colitis-associated colorectal tumorigenesis was assessed by general imaging, tumor counting, and Ki67 staining. Intestinal flora changes were detected by 16S rDNA sequencing technology. Targeted short-chain fatty acid detection was performed by GC-MS/MS, and Lipopolysaccharide (LPS) levels in feces were quantified with an ELISA kit. The signaling pathway of TLR4/NF-κB P65/IL-6/p-STAT3 was evaluated by Western blotting and immunofluorescence. The expression levels of intestinal barrier functional biomarkers Occludin and ZO-1 were detected by immunohistochemistry. Fecal flora transplantation (FMT) was used to evaluate the effect of intestinal flora in inhibiting inflammatory cancer transformation by berberine. RESULTS: Berberine reduced the number and load of tumors in CAC mice. Berberine remodeled the composition of pathogenic and beneficial bacteria in mice with colitis-associated colorectal tumorigenesis. Berberine treatment resulted in increases in fecal butyric acid, acetic acid and propionic acid levels, but did not alter isobutyric acid, isovaleric acid, valeric acid and caproic acid. In addition, berberine reduced LPS content in feces in mice with colitis-associated colorectal tumorigenesis. Occludin and ZO-1 were upregulated, and the TLR4/p-NF-κB p65/IL-6/p-STAT3 inflammatory-cancer transformation pathway was inhibited with berberine. The FMT results further verified that the berberine-treated intestinal flora was sufficient to alleviate the occurrence of colonic tumors associated with colitis in mice. CONCLUSION: Our study showed that berberine alleviated the colitis-associated colorectal tumorigenesis from three equilibrium levels: (1) Pathogenic and beneficial bacteria; (2) Short-chain fatty acids and LPS produced by intestinal flora; and (3) Inflammatory cancer transformation signaling and intestinal barrier function. This study provided a new approach and experimental basis for the application of berberine in the treatment of CAC in clinical practice.

Tracking HOCl by an incredibly simple fluorescent probe with AIE plus ESIPT in vitro and in vivo.

Hypochlorous acid is an important active substance involved in a variety of physiological processes in living organisms, while abnormal concentrations of HOCl are strongly associated with a variety of diseases such as cancer, inflammation, atherosclerosis, and Alzheimer's disease. As a result, it's crucial to establish a reliable method for tracking HOCl in vivo in order to investigate its physiological consequences. In this work, we developed a fluorescent probe DFSN with both AIE and ESIPT for imaging HOCl in vivo. DFSN not only has a basic structure and is easy to synthesize, but also has superior performance. The probe responds to HOCl in less than 10 s and has good selectivity and sensitivity to HOCl (DL = 6.3 nM), with a 110-fold increase in fluorescence intensity following response. In addition, DFSN can realize the rapid detection of hypochlorous acid with naked eyes. Moreover, DFSN can be used for the detection of exogenous and endogenous HOCl in RAW264.7 cells, and additionally enables the tracking of HOCl in cancer cells (Hela cells and HepG2 cells). More notably, it has been utilized to image hypochlorous acid in zebrafish with great success. The probe DFSN will be useful in determining the physiological significance of HOCl.