During the final preparation of Figure S2I and S9A, some panels were erroneously assembled by the au-thors. In Figure S2I, the images for "AAV9-Scramble"control actually represent "AAV9-LacZ"control. In Figure S9A, the images for the "Ad-FGF13 OE+Ad-IKB OE"actually represent "Ad-FGF13 NLS-OE."These errors do not affect the results or the conclusions of the paper. The authors apologize for any incon-venience caused to the readers.

FGF13 is an intracellular FGF factor. Its role in cardiomyopathies has been rarely investigated. We revealed that endogenous FGF13 is up-regulated in cardiac hypertrophy accompanied by increased nuclear localization. The upregulation of FGF13 plays a deteriorating role both in hypertrophic cardiomyocytes and mouse hearts. Mechanistically, FGF13 directly interacts with p65 by its nuclear localization sequence and co-localizes with p65 in the nucleus in cardiac hypertrophy. FGF13 deficiency inhibits NF-κB activation in ISO-treated NRCMs and TAC-surgery mouse hearts, whereas FGF13 overexpression shows the opposite trend. Moreover, FGF13 overexpression alone is sufficient to activate NF-κB in cardiomyocytes. The interaction between FGF13 and p65 or the effects of FGF13 on NF-κB have nothing to do with IκB. Together, an IκB-independent mechanism for NF-κB regulation has been revealed in cardiomyocytes both under basal and stressful conditions, suggesting the promising application of FGF13 as a therapeutic target for pathological cardiac hypertrophy and heart failure.

Fibroblast growth factor-18 (FGF18) has diverse organ development and damage repair roles. However, its role in cardiac homeostasis following hypertrophic stimulation remains unknown. Here we investigate the regulation and function of the FGF18 in pressure overload (PO)-induced pathological cardiac hypertrophy. FGF18 heterozygous (Fgf18+/-) and inducible cardiomyocyte-specific FGF18 knockout (Fgf18-CKO) male mice exposed to transverse aortic constriction (TAC) demonstrate exacerbated pathological cardiac hypertrophy with increased oxidative stress, cardiomyocyte death, fibrosis, and dysfunction. In contrast, cardiac-specific overexpression of FGF18 alleviates hypertrophy, decreased oxidative stress, attenuates cardiomyocyte apoptosis, and ameliorates fibrosis and cardiac function. Tyrosine-protein kinase FYN (FYN), the downstream factor of FGF18, was identified by bioinformatics analysis, LC-MS/MS and experiment validation. Mechanistic studies indicate that FGF18/FGFR3 promote FYN activity and expression and negatively regulate NADPH oxidase 4 (NOX4), thereby inhibiting reactive oxygen species (ROS) generation and alleviating pathological cardiac hypertrophy. This study uncovered the previously unknown cardioprotective effect of FGF18 mediated by the maintenance of redox homeostasis through the FYN/NOX4 signaling axis in male mice, suggesting a promising therapeutic target for the treatment of cardiac hypertrophy.

Studies of diabetic glomerular injury have raised the possibility of developing useful early biomarkers and therapeutic approaches for the treatment of type 2 diabetic nephropathy (T2DN). In this study, we found that FGF13 expression is induced in glomerular endothelial cells (GECs) during T2DN progression. Endothelial-specific deletion of Fgf13 potentially alleviates T2DN damage, while Fgf13 overexpression has the opposite effect. Mechanistically, Fgf13 deficiency results in improved mitochondrial homeostasis and endothelial barrier integrity in T2DN. Moreover, FGF13-sensitive alteration of Parkin safeguards mitochondrial homeostasis in endothelium of T2DN through promotion of mitophagy and inhibition of apoptosis. Additionally, it is confirmed that the beneficial effects of Fgf13 deficiency on T2DN are abolished by endothelial-specific double deletion of Fgf13 and Prkn. The effects of Fgf13 deficiency on mitophagy and apoptosis through Parkin-dependent regulation may be distinct and separable events under diabetic conditions. These data show that the bifunctional role of Fgf13 deficiency in promoting mitophagy and inhibiting apoptosis through Parkin can shape mitochondrial homeostasis regulation in GECs and T2DN progression. As a potential therapeutic target for prevention and control of T2DN, a mechanistic understanding of the biofunction of FGF13 may also be relevant to the pathogenesis of other FGF13- and Parkin-associated diseases.

Skin wound healing is a complex process involving intricate molecular mechanisms that remain unknown. Restoration of homeostasis after wounding requires the remodeling function of fibroblasts. In this study, we show that phosphorylation of α-cateninS641 was upregulated in fibroblasts during wounding, which accelerated their proliferation and migration to restore the skin barrier. At the wound edge, phosphorylated α-cateninS641 stabilized IκBα and thereby impaired the expression of NF-κB target genes to promote proliferation and migration of fibroblasts. Mechanically, phosphorylated α-cateninS641 blocked K48-linked polyubiquitination and proteasomal degradation of IκBα. Moreover, we also showed that EGF/EGFR/CK2α functioned as key upstream signaling of α-catenin by phosphorylating α-catenin at S641. Wound repair was significantly disrupted in the skin of mice in which α-catenin phosphorylation and CK2α kinase activity were perturbed in fibroblasts. These findings provide insights into the molecular control of fibroblast proliferation and migration in response to wounding and identify potential targets for the treatment of defective wound repair.

The mechanism underlying endothelial dysfunction leading to cardiovascular disease in type 2 diabetes mellitus (T2DM) remains unclear. Here, we show that inhibition of histone deacetylase 3 (HDAC3) reduced inflammation and oxidative stress by regulating nuclear factor-E2-related factor 2 (Nrf2), which mediates the expression of anti-inflammatory- and pro-survival-related genes in the vascular endothelium, thereby improving endothelial function. Nrf2 knockout (Nrf2 KO) C57BL/6 background mice, diabetic db/db mice, and control db/m mice were used to investigate the relationship between HDAC3 and Nrf2 in the endothelium in vivo. Human umbilical vein endothelial cells (HUVECs) cultured under high glucose-palmitic acid (HG-PA) conditions were used to explore the role of Kelch-like ECH-associated protein 1 (Keap1) –Nrf2–NAPDH oxidase 4 (Nox4) redox signaling in the vascular endothelium in vitro. Activity assays, immunofluorescence, western blotting, qRT-PCR, and immunoprecipitation assays were used to examine the effect of HDAC3 inhibition on inflammation, reactive oxygen species (ROS) production, and endothelial impairment, as well as the activity of Nrf2-related molecules. HDAC3 activity, but not its expression, was increased in db/db mice. This resulted in de-endothelialization and increased oxidative stress and pro-inflammatory marker expression in cells treated with the HDAC3 inhibitor RGFP966, which activated Nrf2 signaling. HDAC3 silencing decreased ROS production, inflammation, and damage-associated tube formation in HG-PA-treated HUVECs. The underlying mechanism involved the Keap1–Nrf2–Nox4 signaling pathway. The results of this study suggest the potential of HDAC3 as a therapeutic target for the treatment of endothelial dysfunction in T2DM.

Isosinensetin is a polymethoxyflavone existing in various kinds of citrus. It has exhibited significant antiproliferative activity and herb-drug interaction. To date, a specific determination method to quantify isosinensetin concentration in biological matrix has not been developed. In the present study, a highly specific, simple and sensitive ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) approach was developed and validated for quantification of isosinensetin in rat plasma with subsequent application to a pharmacokinetic study. Isosinensetin and lysionotin (internal standard, IS) were extracted from rat plasma by a single step protein precipitation using acetonitrile as precipitation agent. The chromatographic separation was conducted using an Agilent C18 column with a gradient elution system (0.1 % formic acid aqueous solution and acetonitrile) within 3.5 min. An electrospray ionization (ESI) source operating in positive mode and multiple reaction monitoring (MRM) were used to monitor the transitions of m/z 373.1 -> 343.1 for isosinensetin and m/z 345.1 -> 315.1 for IS. The developed method was linear within the range of 1-1000 ng/mL and fully validated according to FDA guidelines. The accuracy values reported as relative errors were between 2.0 and 10.0 % for three quality control levels (2, 400 and 800 ng/mL) and lower limit of quantification (LLOQ). The precisions were <= 11.1 % for quality controls and <= 18.1 % for LLOQ, The recoveries and matrix effects of isosinensetin were in the range of 83.4-87.7 % and 105.6-108.8 %, respectively. Other parameters such as selectivity, carryover effect, dilution integrity and stability were also validated and met the acceptance criteria. The method was applied to a pharmacokinetic study in rats following oral and intravenous administration of isosinensetin. Isosinensetin was rapidly absorbed with a poor bioavailability of 2.19 % and quickly eliminated with mean half-life of 1.40 h and 1.76 h for oral and intravenous route, respectively. (C) 2020 Elsevier B.V. All rights reserved.

Vascular complications of diabetes are a serious challenge in clinical practice, and effective treatments are an unmet clinical need. Acidic fibroblast growth factor (aFGF) has potent anti-oxidative properties and therefore has become a research focus for the treatment of diabetic vascular complications. However, the specific mechanisms by which aFGF regulates these processes remain unclear. The purpose of this study was to investigate whether aFGF alleviates diabetic endothelial dysfunction by suppressing mitochondrial oxidative stress. We found that aFGF markedly decreased mitochondrial superoxide generation in both db/db mice and endothelial cells incubated with high glucose (30 mM) plus palmitic acid (PA, 0.1 mM), and restored diabetes-impaired Wnt/β-catenin signaling. Pretreatment with the Wnt/β-catenin signaling inhibitors IWR-1-endo (IWR) and ICG-001 abolished aFGF-mediated attenuation of mitochondrial superoxide generation and endothelial protection. Furthermore, the effects of aFGF on endothelial protection under diabetic conditions were suppressed by c-Myc knockdown. Mechanistically, c-Myc knockdown triggered mitochondrial superoxide generation, which was related to decreased expression and subsequent impaired mitochondrial localization of hexokinase 2 (HXK2). The role of HXK2 in aFGF-mediated attenuation of mitochondrial superoxide levels and EC protection was further confirmed by si-Hxk2 and a cell-permeable form of hexokinase II VDAC binding domain (HXK2VBD) peptide, which inhibits mitochondrial localization of HXK2. Taken together, these findings suggest that the endothelial protective effect of aFGF under diabetic conditions could be partly attributed to its role in suppressing mitochondrial superoxide generation via HXK2, which is mediated by the Wnt/β-catenin/c-Myc axis.

The molecular signaling mechanisms of Coenzyme Q10 (CoQ10) in diabetic nephropathy (DN) remain poorly understood. We verified that mitochondrial abnormalities, like defective mitophagy, the generation of mitochondrial reactive oxygen species (mtROS) and the reduction of mitochondrial membrane potential, occurred in the glomerulus of db/db mice, accompanied by reduced PINK and parkin expression and increased apoptosis. These changes were partially reversed following oral administration of CoQ10. In inner fenestrated murine glomerular endothelial cells (mGECs), high glucose (HG) also resulted in deficient mitophagy, mitochondrial dysfunction and apoptosis, which were reversed by CoQ10. Mitophagy suppression mediated by Mdivi-1 or siPINK abrogated the renoprotective effects exerted by CoQ10, suggesting a beneficial role for CoQ10-restored mitophagy in DN. Mechanistically, CoQ10 restored the expression, activity and nuclear translocation of Nrf2 in HG-cultured mGECs. In addition, the reduced PINK and parkin expression observed in HG-cultured mGECs were partially elevated by CoQ10. CoQ10-mediated renoprotective effects were abrogated by the Nrf2 inhibitor ML385. When ML385 abolished mitophagy and the renoprotective effects exerted by CoQ10, mGECs could be rescued by treatment with mitoTEMPO, which is a mtROS-targeted antioxidant. These results suggest that CoQ10, as an effective antioxidant in mitochondria, exerts beneficial effects in DN via mitophagy by restoring Nrf2/ARE signaling. In summary, CoQ10-mediated mitophagy activation positively regulates DN through a mechanism involving mtROS, which influences the activation of the Nrf2/ARE pathway.

Background/Aims: Diabetic non-healing skin ulcers represent a serious challenge in clinical practice, in which the hyperglycemia-induced disturbance of angiogenesis, and endothelial dysfunction play a crucial role. Resveratrol (RES), a silent information regulator 1 (SIRT1) agonist, can improve endothelial function and has strong pro-angiogenic properties, and has thus become a research focus for the treatment of diabetic non-healing skin ulcers; however, the underlying mechanism by which RES regulates these processes remains unclear. Therefore, the present study was intended to determine if RES exerts its observed protective role in diabetic wound healing by alleviating hyperglycemia-induced endothelial dysfunction and the disturbance of angiogenesis. Methods: We investigated the effects of RES on cell migration, cell proliferation, apoptosis, tube formation, and the underlying molecular mechanisms in 33 mM high glucose-stimulated human umbilical vein endothelial cells (HUVECs) by semi-quantitative RT-PCR, western blot analysis, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining, and immunofluorescence in vitro. We further explored the role of RES on endothelial dysfunction and wound healing disturbance in db/db mice by TUNEL staining, immunofluorescence, and photography in vivo. Results: We observed an obvious inhibition of hyperglycemia-triggered endothelial dysfunction and a disturbance of angiogenesis, followed by the promotion of diabetic wound healing via RES, along with restoration of the activity of the hyperglycemia-impaired SIRT1 signaling pathway. Pretreatment with EX-527, a SIRT1 inhibitor, abolished the RES-mediated endothelial protection and pro-angiogenesis action, and then delayed diabetic wound healing. Furthermore, examination of the overexpression of forkhead box O1 (FOXO1), a transcription factor substrate of SIRT1, in HUVECs and db/db mice revealed that RES activated SIRT1 to restore hyperglycemia-triggered endothelial dysfunction and disturbance of angiogenesis, followed by the promotion of diabetic wound healing in a c-Myc-dependent manner. Pretreatment with 10058-F4, a c-Myc inhibitor, repressed RES-mediated endothelial protection, angiogenesis, and diabetic wound healing. Conclusion: Our findings indicate that the positive role of RES in diabetic wound healing via its SIRT1-dependent endothelial protection and pro-angiogenic effects involves the inhibition of FOXO1 and the de-repression of c-Myc expression.

Aloin-A (also known as barbaloin), the main bioactive anthraquinone-C-glycoside of Aloe species, exhibits various beneficial pharmacological effects. However, the determination and pharmacokinetic study of aloin-A in rat plasma need to be improved and systematically demonstrated. In the present study, a simple, robust and sensitive ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for rapid quantification of aloin-A in rat plasma was developed. Plasma preparation was conducted by a single step protein precipitation with obtusin serving as an internal standards (IS) followed by separation of the analytes using an Agilent C18 column with a gradient mobile phase comprised of acetonitrile and formic acid aqueous solution. Negative ion electrospray was used and multiple reaction monitoring transitions were m/z 417.1 → 297.0 for aloin-A and m/z 343.1 → 328.1 for IS, respectively. The developed method was validated with linear range of 1-1000 ng/mL. All validation parameters were well within the acceptance criteria based on the guidance of FDA. The validated approach was successfully applied to analyze samples from a pharmacokinetic study in healthy rats following intravenous and oral administration. Aloin-A was found to be quickly absorbed, extensively distributed and rapidly eliminated. The absolute bioavailability of aloin-A was 5.79%.

Studies regarding macroautophagic/autophagic regulation in endothelial cells (ECs) under diabetic conditions are very limited. Clinical evidence establishes an endothelial protective effect of metformin, but the underlying mechanisms remain unclear. We aimed to investigate whether metformin exerts its protective role against hyperglycemia-induced endothelial impairment through the autophagy machinery. db/db mice were treated with intravitreal metformin injections. Human umbilical vein endothelial cells (HUVECs) were cultured either in normal glucose (NG, 5.5 mM) or high glucose (HG, 33 mM) medium in the presence or absence of metformin for 72 h. We observed an obvious inhibition of hyperglycemia-triggered autophagosome synthesis in both the diabetic retinal vasculature and cultured HUVECs by metformin, along with restoration of hyperglycemia-impaired Hedgehog (Hh) pathway activity. Specifically, deletion of ATG7 in retinal vascular ECs of db/db mice and cultured HUVECs indicated a detrimental role of autophagy in hyperglycemia-induced endothelial dysfunction. Pretreatment with GANT61, a Hh pathway inhibitor, abolished the metformin-mediated downregulation of autophagy and endothelial protective action. Furthermore, GLI-family (transcription factors of the Hh pathway) knockdown in HUVECs and retinal vasculature revealed that downregulation of hyperglycemia-activated autophagy by the metformin-mediated Hh pathway activation was GLI1 dependent. Mechanistically, GLI1 knockdown-triggered autophagy was related to upregulation of BNIP3, which subsequently disrupted the association of BECN1/Beclin 1 and BCL2. The role of BNIP3 in BECN1 dissociation from BCL2 was further confirmed by BNIP3 overexpression or BNIP3 RNAi. Taken together, the endothelial protective effect of metformin under hyperglycemia conditions could be partly attributed to its role in downregulating autophagy via Hh pathway activation. Abbreviations: 3-MA = 3-methyladenine; 8×GLI BS-FL = 8×GLI-binding site firefly luciferase; AAV = adeno-associated virus; AAV-Cdh5-sh-Atg7 = AAV vectors carrying shRNA against murine Atg7 under control of murine Cdh5 promoter; AAV-Cdh5-sh-Gli1 = AAV vectors carrying shRNA against murine Gli1 under control of murine Cdh5 promoter; AAV-Cdh5-Gli1 = AAV vectors carrying murine Gli1 cDNA under the control of murine Cdh5 core promoter; ACAC = acetyl-CoA carboxylase; Ad-BNIP3 = adenoviruses harboring human BNIP3`; Ad-GLI1 = adenoviruses harboring human GLI1; Ad-sh-ATG7 = adenoviruses harboring shRNA against human ATG7; Ad-sh-BNIP3 = adenoviruses harboring shRNA against human BNIP3; Ad-sh-GLI = adenoviruses harboring shRNA against human GLI; AGEs = advanced glycation end products; ATG = autophagy-related; atg7flox/flox mice = mice bearing an Atg7flox allele, in which exon 14 of the Atg7 gene is flanked by 2 loxP sites; BafA1 = bafilomycin A1; BECN1 = beclin 1; CDH5/VE-cadherin = cadherin 5; CASP3 = caspase 3; CASP8 = caspase 8; CASP9 = caspase 9; ECs = endothelial cells; GAPDH = glyceraldehyde-3-phosphate dehydrogenase; GCL = ganglion cell layer; GFP-LC3B = green fluorescent protein labelled LC3B; HG = high glucose; Hh = Hedgehog; HHIP = hedgehog interacting protein; HUVECs = human umbilical vein endothelial cells; IB4 = isolectin B4; INL = inner nuclear layer; i.p. = intraperitoneal; MAP1LC3/LC3 = microtubule-associated protein 1 light chain 3; MAN = mannitol; MET = metformin; NG = normal glucose; ONL = outer nuclear layer; p-ACAC = phosphorylated acetyl-CoA carboxylase; PECAM1/CD31= platelet/endothelial cell adhesion molecule 1; PRKAA1/2 = protein kinase AMP-activated catalytic subunits alpha 1/2; p-PRKAA1/2 = phosphorylated PRKAA1/2; PTCH1 = patched 1; RAPA = rapamycin; RL = Renilla luciferase; SHH = sonic hedgehog; shRNA = short hairpin RNA; sh-PRKAA1/2 = short hairpin RNA against human PRKAA1/2; scrambled shRNA = the scrambled short hairpin RNA serves as a negative control for the target-specific short hairpin RNA, which has the same nucleotide composition as the input sequence and has no match with any mRNA of the selected organism database; SMO = smoothened, frizzled class receptor; sqRT-PCR = semi-quantitative RT-PCR; TEK/Tie2 = TEK receptor tyrosine kinase; Tek-Cre (+) mice = a mouse strain expressing Cre recombinase under the control of the promoter/enhancer of Tek, in a pan-endothelial fashion; TUNEL = terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling.

Baicalin is the major component found in Scutellaria baicalensis root, a widely used herb in traditional Chinese medicine, which exhibits strong anti-inflammatory, anti-viral and anti-tumor activities. The present work was devoted to elucidate the molecular and cellular mechanisms underlying the protective effects of Baicalin against diabetes-induced oxidative damage, inflammation and endothelial dysfunction. Diabetic mice, induced by streptozotocin (STZ), were treated with intraperitoneal Baicalin injections. Human umbilical vein endothelial cells (HUVECs) were cultured either in normal glucose (NG, 5.5 mM) or high glucose (HG, 33 mM) medium in the presence or absence of Baicalin for 72 h. We observed an obvious inhibition of hyperglycemia-triggered oxidative damage and inflammation in HUVECs and diabetic aortal vasculature by Baicalin, along with restoration of hyperglycemia-impaired nuclear factor (erythroid-derived 2)-like 2 (Nrf2) pathway activity. However, the protective effects of Baicalin almost completely abolished in HUVECs transduced with shRNA against Nrf2, but not with nonsense shRNA. Mechanistic studies demonstrated that HG decreased Akt and GSK3B phosphorylation, restrained nuclear export of Fyn and nuclear localization of Nrf2, blunted Nrf2 downstream target genes and subsequently induced oxidative stress in HUVECs. However, those destructive cascades were well prevented by Baicalin in HUVECs. Furthermore, LY294002 and ML385 (inhibitor of PI3K and Nrf2) attenuated Baicalin-mediated Nrf2 activation and the ability of facilitates angiogenesis in vivo and ex vivo. Taken together, the endothelial protective effect of Baicalin under hyperglycemia condition could be partly attributed to its role in downregulating reactive oxygen species (ROS) and inflammation via the Akt/GSK3B/Fyn-mediated Nrf2 activation.

Binge drinking is associated with increased cardiac autophagy, and often triggers heart injury. Given the essential role of autophagy in various cardiac diseases, this study was designed to investigate the role of autophagy in ethanol‐induced cardiac injury and the underlying mechanism. Our study showed that ethanol exposure enhanced the levels of LC3‐II and LC3‐II positive puncta and promoted cardiomyocyte apoptosis in vivo and in vitro. In addition, we found that ethanol induced autophagy and cardiac injury largely via the sequential triggering of reactive oxygen species (ROS) accumulation, activation of c‐Jun NH2‐terminal kinase (JNK), phosphorylation of Bcl‐2, and dissociation of the Beclin 1/Bcl‐2 complex. By contrast, inhibition of ethanol‐induced autophagic flux with pharmacologic agents in the hearts of mice and cultured cells significantly alleviated ethanol‐induced cardiomyocyte apoptosis and heart injury. Elimination of ROS with the antioxidant N‐acetyl cysteine (NAC) or inhibition of JNK with the JNK inhibitor SP600125 reduced ethanol‐induced autophagy and subsequent autophagy‐mediated apoptosis. Moreover, metallothionein (MT), which can scavenge reactive oxygen and nitrogen species, also attenuated ethanol‐induced autophagy and cell apoptosis in MT‐TG mice. In conclusion, our findings suggest that acute ethanol exposure induced autophagy‐mediated heart toxicity and injury mainly through the ROS‐JNK‐Bcl‐2 signaling pathway.

Kangfuxin (KFX) is an oral liquid derived from Periplaneta americana, with complex components. KFX has been demonstrated to exhibit anticancer activity in a variety of different types of tumor, including gastric cancer; however, its underlying molecular mechanism remains unclear. The present study was designed to investigate the pro-apoptotic effects of KFX on SGC-7901 cells, in order to provide a theoretical basis for clinical application. In order to clarify the pro-apoptotic effects of KFX on SGC-7901 cells, MTT analysis was conducted. To evaluate the anticancer effect of KFX, peroxisome proliferator-activated receptor (PPAR)-γ was analyzed by reverse transcription-polymerase chain reaction. Western blot analysis was used to determine the effects of KFX on the expression of cleaved caspase-3, phosphorylated extracellular signal-regulated kinase (p-ERK), ERK, tumor protein p53 (p53), B-cell lymphoma 2 (Bcl-2), Bcl-2 associated X, interleukin (IL)-6 and IL-1β. In addition, terminal deoxynucleotidyl-transferase-mediated dUTP nick-end labeling (TUNEL) analysis was used to detect apoptosis in SGC-7901 cells. It was revealed that PPAR-γ was increased in SGC-7901 cells following treatment with KFX, shown by an increase in mRNA expression. Furthermore, western blot analysis identified that KFX treatment groups exhibited markedly inhibited levels of Bcl-2, IL-6, IL-1β and p-ERK, and induced p53 protein expression. Additionally, TUNEL and MTT assays demonstrated that treatment with KFX may induce SGC-7901 cell apoptosis and inhibit proliferation. In conclusion, to the best of our knowledge, the results of the present study demonstrated for the first time that KFX may induce SGC-7901 cell apoptosis and inhibit its proliferation, and this may be primarily attributed to its role in mitogen-activated protein kinase/extracellular-signal-regulated kinase kinase/ERK signaling pathway inhibition.

We report here a rapid and sensitive technique for negative visualization of protein in 1D and 2D SDS‐PAGE by using 2′, 7′‐dichlorofluorescein (DCF), which appeared as transparent and colorless bands in an opaque gel matrix background. For DCF stain, down to 0.1–0.2 ng protein could be easily visualized within 7 min by only two steps, and the staining is fourfold more sensitive than that of Eosin Y (EY) negative stain and glutaraldehyde (GA) silver stain, and eightfold more sensitive than that of the commonly used imidazole‐zinc (IZ) negative stain. Furthermore, DCF stain provided good reproducibility, linearity, and MS compatibility compared with those of IZ stain. In addition, the potential staining mechanism was investigated by colorimetric experiment and molecular docking, and the results demonstrated that the interaction between DCF and protein occurs mainly via van der waals force, electrostatic interaction, and hydrogen bonding.

Skin wound healing is a complex process requiring the coordinated behavior of many cell types, especially in the proliferation and migration of fibroblasts. Basic fibroblast growth factor (bFGF) is a member of the FGF family that promotes fibroblast migration, but the underlying molecular mechanism remains elusive. The present RNA sequencing study showed that the expression levels of several canonical Wnt pathway genes, including Wnt2b, Wnt3, Wnt11, T-cell factor 7 (TCF7), and Frizzled 8 (FZD8) were modified by bFGF stimulation in fibroblasts. Enzyme-linked immunosorbent assay (ELISA) analysis also showed that Wnt pathway was activated under bFGF treatment. Furthermore, treatment of fibroblasts with lithium chloride or IWR-1, an inducer and inhibitor of the Wnt signaling pathway, respectively, promoted and inhibited cell migration. Also, levels of cytosolic glycogen synthase kinase 3 beta phosphorylated at serine9 (pGSK3β Ser9) and nuclear β-catenin were increased upon exposure to bFGF. Molecular and biochemical assays indicated that phosphoinositide 3-kinase (PI3K) signaling activated the GSK3β/β-catenin/Wnt signaling pathway via activation of c-Jun N-terminal kinase (JNK), suggesting that PI3K and JNK act at the upstream of β-catenin. In contrast, knock-down of β-catenin delayed fibroblast cell migration even under bFGF stimulation. RNA sequencing analysis of β-catenin knock-down fibroblasts demonstrated that β-catenin positively regulated the transcription of bFGF and FGF21. Moreover, FGF21 treatment activated AKT and JNK, and accelerated fibroblast migration to a similar extent as bFGF does. In addition, ELISA analysis demonstrated that both of bFGF and FGF21 were auto secretion factor and be regulated by Wnt pathway stimulators. Taken together, our analyses define a feedback regulatory loop between bFGF (FGF21) and Wnt signaling acting through β-catenin in skin fibroblasts.

A Schiff base compound derived from naphthalene has been synthesized and characterized as an Al3+ -selective fluorescent probe. The chemosensor (L) exhibits high selectively for Al3+ in aqueous solution, even in the presence of biologically relevant cations such as Na+ , K+ , Ca2+ , Mg2+ , Pb2+ and several transition metal ions. There was no observed interference from anions like Br- , Cl- , HSO3- , SO32- , S2 O32- , NO2- , CO32- and AC- . The lowest detection limit for the chemosensor L was found to be 1.89 × 10-8  M with a linear response towards Al3+ over a concentration range of 5 × 10-6 to 4 × 10-5  M. Furthermore, the proposed chemosensor has been used for imaging of Al3+ in two different types of cells with satisfying results, which further demonstrates its value for practical application in biological systems.

This study was designed to evaluate the gastroprotective effect of Kangfuxin (KFX), a Chinese patent medicine constituent isolated from American cockroach, on ethanol-induced gastric ulcer in mice and to elucidate the potential mechanisms of the effect involved. According to the results, mice treated with alcohol appeared obvious gastric mucosal injury, while treatment with Cimetidine (a positive control) and KFX significantly relieved the damage, along with decreased oxidative stress and apoptosis indexes. Subsequently, we conducted a label-free quantitative proteomic (LFQ) and found that NF-κB and PI3K/AKT signaling pathway participated in gastroprotective effect of KFX. Furthermore, Western blot analysis revealed that KFX treatment inhibited the expression of TNF-α, IL-1β, greatly reduced the phosphorylation level of IκB and repressed the nuclear translocation of NF-κB p65, which demonstrated that KFX inhibited the activation of NF-κB pathway. Meanwhile, the PI3K/AKT pathway was also involved in regulating the anti-inflammation effect. These findings define for the first time that the gastroprotective effects of KFX against gastric ulcer can be attributed to its role in NF-κB inhibition.