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.

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.

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.

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.

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.

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.

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.

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.

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.

Chemoproteomics reveals berberine directly binds to PKM2 to inhibit the progression of colorectal cancer.

Colorectal cancer is one of the most serious tumors and berberine can inhibit the recurrence and transformation of colorectal adenoma into colorectal cancer. However, the direct binding target proteins of berberine in inhibiting colorectal cancer remain unclear. In this study, the chemical proteomics method was used and demonstrated that berberine is directly bound to pyruvate kinase isozyme type M2 (PKM2) in colorectal cancer cells. The triangular N-O-O triangular structure of berberine contributed to hydrophobic interaction with I119 amino acid residues and π-π interaction with F244 amino acid residues of PKM2 protein. Moreover, berberine was shown to inhibit the reprogramming of glucose metabolism and the phosphorylation of STAT3, down regulate the expression of Bcl-2 and Cyclin D1 genes, ultimately inhibiting the progression of colorectal cancer. This study uncovered the direct binding target protein and mechanism of berberine to improve metabolic reprogramming in colorectal cancer, which is helpful to guide the optimization of berberine.

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.

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.

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.

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.

HIF/Ca2+/NO/ROS is critical in roxadustat treating bone fracture by stimulating the proliferation and migration of BMSCs.

AIMS: Fracture site is regionally hypoxic resulting from vasculature disruption. HIF-1αplays an essential role in fracture repair. This study aims to investigate the influence of FG4592 on the femur fracture of SD rats and the proliferation, migration of BMSCs. MATERIALS AND METHODS: After the femoral fracture model was established, computed tomography imaging and histological analyses were used to quantify bone healing and the expression of CD90, HIF-1α, VEGF were observed by means of immunohistochemistry method on Day 10 and Day 20. In addition, CCK-8 assay, transwell, flow cytometric analysis, laser confocal microscopy assay, western blot and rT-PCR were performed to text the proliferation and migration of BMSCs using FG4592. KEY FINDINGS: In vivo, FG4592 facilitated the repair of bone fracture by increasing the number of BMSCs and cartilage formation. In vitro, FG4592 markedly improved the proliferation, migration of BMSCs via upregulation of intracellular Ca2+, NO and concomitant decrease of ROS. Gene silencing of HIF-1α resulted in the opposite phenomenon in BMSCs with the treatment of FG4592. SIGNIFICANCE: The transplantation of BMSCs is the most promising candidate for the treatment of fracture non-union. We illustrated that FG4592 promoted the proliferation, migration of BMSCs via the HIF/Ca2+/NO/ROS pathway and further accelerated fracture healing. These results provide a deeper understanding for the mechanism of HIF in promoting fracture healing.

Targeted copper supplementation oriented theranostic for fluorescence and 19F NMR detection of tumors.

Alteration of the levels of copper is a promising approach for cancer therapy. Herein, we develop a dual-mode copper vehicle, M985. The biotin-tailed M985 can exert tumor-directed copper supplementation and undergo self-immolative cleavage in living cancerous cells, resulting in the liberation of F542 along with the generation of excess reactive oxygen species. Thus, fluorescence and 19F NMR detection is realized to specifically discriminate cancer cells. F542 acts as a fluorescence reporter and a potent cytotoxic agent, facilitating the visualization of molecular release and distribution, as well as confirming the ER autophagy-induced apoptosis. Therefore, we present a promising dual-mode theranostic M985 for the efficient detection and therapy of cancer.

Mn-TAT PTD-Ngb ameliorates inflammation through the elimination of damaged mitochondria and the activation of Nrf2-antioxidant signaling pathway.

Inflammation, mitochondrial dysfunction and oxidative stress are closely associated with neurological diseases. In this study, Mn-TAT PTD-Ngb, a novel artificial recombinant protein, exerted inhibitory effects on the inflammatory response and inflammasome activation. During the lipopolysaccharide (LPS)-induced inflammatory response, Mn-TAT PTD-Ngb suppressed the nuclear translocation of nuclear factor kappa B (NF-κB) and the release of proinflammatory cytokines and attenuated the phosphorylation of mitogen-activated protein kinase (MAPK). Furthermore, the recombinant protein blocked reactive oxygen species (ROS) production, abated mitochondrial dysfunction and significantly suppressed the assembly of the inflammasome, which led to the overproduction of proinflammatory cytokines IL-1ß and IL-18. Mn-TAT PTD-Ngb increased the level of nuclear factor-erythroid 2 -related factor 2 (Nrf2), which protected against oxidative stress and improved pyroptosis. Mn-TAT PTD-Ngb might be a promising drug for curing neurological diseases.

A novel bimacrocyclic polyamine-based fluorescent probe for sensitive detection of Hg2+ and glutathione in human serum.

Detection of glutathione in human serum is of great importance for clinical diagnosis of various diseases, such as AIDS, diabetes mellitus, Alzheimer disease and cancer. In this work, a new water-soluble bismacrocyclic polyamine-derived compound, namely L, which contains two molecules of 4-nitro-1,2,3-benzoxa-diazole as the fluorophores, was designed and prepared. The experiments of selectivity of L toward metal ions showed it could rapidly and sensitively detect Hg2+ with a detection limit of 27 nM. Furthermore, the cell imaging and co-staining experiments in HeLa cells demonstrated that the L-Hg2+ probe had selectivity for the Golgi apparatus to a certain degree. Moreover, it had excellent selectivity for biothiols, especially for glutathione. Finally, the probe was successfully applied to sensitively detect glutathione (GSH) in human serum and fetal bovine serum.

ITRAQ-based quantitative proteomic analysis of MG63 in response to HIF-1α inducers.

Non-healing fractures constitute a serious clinical problem. HIF-1α is a crucial regulator in response to hypoxia and is proven to be pivotal in bone growth; however, the mechanism still needs further research. In this study, iTRAQ was used to study the effects of two HIF-1α inducers on the expression of proteins in MG63 cells. A total of 841 proteins were significantly changed after treatment with HIF-1α inducers. Among these, 12 proteins were functionally involved in the HIF-1 and VEGF signaling pathways. We then studied the protein and gene expression of the twelve proteins by western blot and RT-PCR, respectively. The results confirmed that VEGF, TFRC, ERK1/2, iNOS, GLUT1, ALDOA, ENO1 and IP3R1 were markedly upregulated, while NF-κB, RCN1, PLCγ1 and CaMKII were significantly downregulated upon treatment with HIF-1α inducers. Meanwhile, the intracellular levels of Ca2+, NO and ROS were closely related and significantly changed. Up-regulation of HIF can maintain high levels of Ca2+ and NO while reducing ROS and protect cells from apoptosis induced by low serum. This study presents a new way to study the regulation of HIF on bone growth by investigating the Ca2+, NO and ROS levels. SIGNIFCANCE: We found that the regulation of Ca2+ and NO proteins are tightly associated with HIF pathway using iTRAQ method. Furthermore, the concentration of Ca2+, NO and ROS are closely related in low serum cultured cells. Up-regulation of HIF pathway can maintain high levels of Ca2+ and NO while reducing ROS damage.