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Secondary vascular tissue (SVT) development and regeneration are regulated by phytohormones. In this study, we used an in vitro SVT regeneration system to demonstrate that gibberellin (GA) treatment significantly promotes auxin‐induced cambium reestablishment. Altering GA content by overexpressing or knocking down ent‐kaurene synthase (KS) affected secondary growth and SVT regeneration in poplar. The poplar DELLA gene GIBBERELLIC ACID INSENSITIVE (PtoGAI) is expressed in a specific pattern during secondary growth and cambium regeneration after girdling. Overexpression of PtoGAI disrupted poplar growth and inhibited cambium regeneration, and the inhibition of cambium regeneration could be partially restored by GA application. Further analysis of the PtaDR5:GUS transgenic plants, the localization of PIN‐FORMED 1 (PIN1) and the expression of auxin‐related genes found that an additional GA treatment could enhance the auxin response as well as the expression of PIN1, which mediates auxin transport during SVT regeneration. Taken together, these findings suggest that GA promotes cambium regeneration by stimulating auxin signal transduction. [ABSTRACT FROM AUTHOR]

Ischemic brain injury impacts cardiac dysfunction depending on the part of the brain affected, with a manifestation of irregular blood pressure, arrhythmia, and heart failure. Generally called brain–heart syndrome in traditional Chinese medicine, few mechanistic understanding and treatment options are available at present. We hypothesize that considering the established efficacy for both ischemic stroke and myocardial infarction (MI), Danhong injection (DHI), a multicomponent Chinese patent medicine, may have a dual pharmacological potential for treating the brain–heart syndrome caused by cerebral ischemic stroke through its multi-targeted mechanisms. We investigated the role of DHI in the setting of brain–heart syndrome and determined the mechanism by which it regulates this process. We induced Ischemia/Reperfusion in Wistar rats and administered intravenous dose of DHI twice daily for 14 days. We assessed the neurological state, infarct volume, CT scan, arterial blood pressure, heart rhythm, and the hemodynamics. We harvested the brain and heart tissues for immunohistochemistry and western blot analyses. Our data show that DHI exerts potent anti-stroke effects (infarct volume reduction: ∗∗p < 0.01 and ∗∗∗p < 0.001 vs. vehicle. Neurological deficit correction: ∗p < 0.05 and ∗∗∗p < 0.001 vs. vehicle), and effectively reversed the abnormal arterial pressure (∗p < 0.05 vs. vehicle) and heart rhythm (∗∗p < 0.01 vs. vehicle). The phenotype of this brain–heart syndrome is strikingly similar to those of MI model. Quantitative assessment of hemodynamic in cardiac functionality revealed a positive uniformity in the PV-loop after administration with DHI and valsartan in the latter. Immunohistochemistry and western blot results showed the inhibitory effect of DHI on the β-adrenergic pathway as well as protein kinase C epsilon (PKCε) (∗∗p < 0.01 vs. model). Our data showed the underlying mechanisms of the brain–heart interaction and offer the first evidence that DHI targets the adrenergic pathway to modulate cardiac function in the setting of brain–heart syndrome. This study has made a novel discovery for proper application of the multi-target DHI and could serve as a therapeutic option in the setting of brain–heart syndrome. [ABSTRACT FROM AUTHOR]

The traditional Chinese medicine Danshensu ( DSS) has a protective effect on cardiac ischaemia/reperfusion (I/R) injury. However, the molecular mechanisms underlying the DSS action remain undefined. We investigated the potential role of DSS in autophagy and apoptosis using cardiac I/R injury models of cardiomyocytes and isolated rat hearts. Cultured neonatal rat cardiomyocytes were subjected to 6 hrs of hypoxia followed by 18 hrs of reoxygenation to induce cell damage. The isolated rat hearts were used to perform global ischaemia for 30 min., followed by 60 min. reperfusion. Ischaemia/reperfusion injury decreased the haemodynamic parameters on cardiac function, damaged cardiomyocytes or even caused cell death. Pre-treatment of DSS significantly improved cell survival and protected against I/R-induced deterioration of cardiac function. The improved cell survival upon DSS treatment was associated with activation of mammalian target of rapamycin ( mTOR) (as manifested by increased phosphorylation of S6K and S6), which was accompanied with attenuated autophagy flux and decreased expression of autophagy- and apoptosis-related proteins (including p62, LC3-II, Beclin-1, Bax, and Caspase-3) at both protein and mRNA levels. These results suggest that alleviation of cardiac I/R injury by pre-treatment with DSS may be attributable to inhibiting excessive autophagy and apoptosis through mTOR activation. [ABSTRACT FROM AUTHOR]

Epidermal growth factor ( EGF) and their receptor ( EGFR) play an important role in the development of cancer proliferation, and metastasis, although the mechanism remains unclear. The present study aimed at investigating the role of EGF- EGFR signalling pathway in the development of human hepatocellular carcinoma ( HCC) inflammatory environment. Gene profiles of inflammatory cytokines from HCC were measured. Cell bio-behaviours of HCC with low or high metastasis were detected by the live cell monitoring system. Cell proliferation was measured by CCK8. The protein level of CXCL5 and CXCL8 was measured by ELISA. The phosphorylation of PI3K, ERK, MAPK was measured by western blot. EGF significantly induced cell proliferation in HepG2 cells, but not in HCCLM3 cells. EGF prompted the cell movement in both HepG2 and HCCLM3 and regulated the production of CXCL5 and CXCL8 from HCC, which were inhibited by EGFR inhibitor, Erk inhibitor (U0126), or PI3K inhibitors ( BEZ-235 and SHBM1009). HCC proliferation, metastasis and production of inflammatory cytokines were regulated via EGF- EGFR signal pathways. CXCL5 could interact with CXCL8, possibly by CXCR2 or the cross-talk between CXCR2 and EGFR. EGF- EGFR signaling pathway can be the potential target of therapies for HCC. [ABSTRACT FROM AUTHOR]

Summary: Tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2) is a key mediator in TNF signaling. Previous studies suggested that TRAF2 functions as an adaptor in the NF-κB and AP-1 pathways. However, the precise molecular mechanisms by which TRAF2 relays signals are unknown. We previously reported that TRAF2 is phosphorylated following TNF stimulation and now identify the PKC kinases responsible for phosphorylation. Phosphorylated TRAF2 facilitates recruitment of IKKα and IKKβ to the TNF receptor. Phosphorylation also determines K63-linked polyubiquitination of TRAF2 at lysine 31. TRAF2 K63-linked ubiquitination contributes to associations with TAB2/3 and activation of the downstream IKK and JNK kinases. The combined data reveal that phosphorylation of TRAF2 plays a critical role in TNF signaling by directing the IKK complex to the membrane, promoting TRAF2 K63-linked ubiquitination, and positioning the IKKα and IKKβ chains with the TAK1/TAB kinase. [Copyright &y& Elsevier]

Epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase that instigates several signaling cascades, including the NF-κB signaling pathway, to induce cell differentiation and proliferation. Overexpression and mutations of EGFR are found in up to 30% of solid tumors and correlate with a poor prognosis. Although it is known that EGFR-mediated NF-κB activation is involved in tumor development, the signaling axis is not well elucidated. Here, we found that plakophilin 2 (PKP2) and the linear ubiquitin chain assembly complex (LUBAC) were required for EGFR-mediated NF-κB activation. Upon EGF stimulation, EGFR recruited PKP2 to the plasma membrane, and PKP2 bridged HOIP, the catalytic E3 ubiquitin ligase in the LUBAC, to the EGFR complex. The recruitment activated the LUBAC complex and the linear ubiquitination of NEMO, leading to IκB phosphorylation and subsequent NF-κB activation. Furthermore, EGF-induced linear ubiquitination was critical for tumor cell proliferation and tumor development. Knockout of HOIP impaired EGF-induced NF-κB activity and reduced cell proliferation. HOIP knockout also abrogated the growth of A431 epidermal xenograft tumors in nude mice by more than 70%. More importantly, the HOIP inhibitor, HOIPIN-8, inhibited EGFR-mediated NF-κB activation and cell proliferation of A431, MCF-7, and MDA-MB-231 cancer cells. Overall, our study reveals a novel linear ubiquitination signaling axis of EGFR and that perturbation of HOIP E3 ubiquitin ligase activity is potential targeted cancer therapy. [ABSTRACT FROM AUTHOR]

As sessile organisms, the precise development phase transitions are very important for the success of plant adaptability, survival and reproduction. The transition from juvenile to the adult phase—referred to as the vegetative phase change—is significantly influenced by numbers of endogenous and environmental signals. Here, we showed that brassinosteroid (BR), a major growth-promoting steroid hormone, positively regulates the vegetative phase change in Arabidopsis thaliana. The BR-deficient mutant det2-1 and BR-insensitive mutant bri1-301 displayed the increased ratio of leaf width to length and reduced blade base angle. The plant specific transcription factors SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) are key masters for the vegetative phase transition in plants. The expression levels of SPL9, SPL10 and SPL15 were significantly induced by BR treatment, but reduced in bri1-116 mutant compared to wild-type plants. The gain-of-function pSPL9:rSPL9 transgenic plants displayed the BR hypersensitivity on hypocotyl elongation and partially suppressed the delayed vegetative phase change of det2-1 and bri1-301. Furthermore, we showed that BRASSINAZOLE-RESISTANT 1 (BZR1), the master transcription factor of BR signaling pathway, interacted with SPL9 to cooperatively regulate the expression of downstream genes. Our findings reveal an important role for BRs in promoting vegetative phase transition through regulating the activity of SPL9 at transcriptional and post-transcriptional levels. [ABSTRACT FROM AUTHOR]

Background: To investigate the protective effects of helix B surface peptide (HBSP) on acute liver injury induced by carbon tetrachloride (CCl4).Methods: HBSP (8 nmol/kg) was intraperitoneally injected into C57 BL/6 mice 2 h after CCl4 administration. Serum and liver tissue samples were collected 24 h after injury. Liver function and histological injuries were evaluated. Inflammatory cell infiltration and cytokines were examined and hepatocytes apoptosis was measured. The human liver cell line LO2 and murine primary hepatocytes were stimulated by CCl4 with and without HBSP treatment and glutathione peroxidase activity, cell survival, and apoptosis were evaluated. In addition, we examined the PI3K/Akt/mTORC1 pathway to elucidate the mechanism underlying HBSP-mediated protection in acute liver injury.Results: HBSP significantly decreased serum alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase, and pro-inflammatory cytokines in liver tissues after CCl4 injection compared with those in the control group. Immunohistochemical staining indicated that the number of CD3-, CD8-, and CD68-positive cells and the expression of cleaved caspase-3 were significantly decreased by HBSP treatment. Additionally, HBSP reduced apoptosis in vivo. In an in vitro study, the glutathione peroxidase activity and survival rate increased, while the total apoptotic rate was reduced in the HBSP-treated group compared with that in the control group after CCl4 treatment. HBSP activated the PI3K/Akt/mTORC1 pathway, which was confirmed by the PI3K inhibitor LY294002 both in vivo and in vitro. Furthermore, HBSP increased the survival of mice with acute liver injury, and this effect was abolished by LY294002.Conclusions: HBSP is a potential therapeutic agent against acute liver injury induced by CCl4. [ABSTRACT FROM AUTHOR]

Injuring mouse corneas with alkali causes myofibroblast expression leading to tissue opacification. However, in transient receptor potential vanilloid 1 channel (TRPV1-/-) knockout mice healing results in transparency restoration. Since TGFβ is the primary inducer of the myofibroblast phenotype, we examined the mechanism by which TRPV1 affects TGFβ-induced myofibroblast development. Experiments were performed in pig corneas and human corneal fibroblasts (HCFs). Immunohistochemical staining of α-smooth muscle actin (α-SMA) stress fibers was used to visualize myofibroblasts. Protein and phosphoprotein were determined by Western blotting. siRNA transfection silenced TRPV1 gene expression. Flow cytometry with a reactive oxygen species (ROS) reporting dye analyzed intracellular ROS. [Ca2+]I was measured by loading HCF with fura2. In organ cultured corneas, the TRPV1 antagonist capsazepine drastically reduced by 75% wound-induced myofibroblast development. In HCF cell culture, TGF-β1 elicited rapid increases in Ca2+ influx, phosphorylation of SMAD2 and MAPKs (ERK1/2, JNK1/2 and p38), ROS generation and, after 72 hrs myofibroblast development. SMAD2 and p38 activation continued for more than 16 h, whereas p-ERK1/2 and p-JNK1/2 waned within 90 min. The long-lived SMAD2 activation was dependent on activated p38 and vice versa, and it was essential to generate a > 13-fold increase in α-SMA protein and a fully developed myofibroblast phenotype. These later changes were markedly reduced by inhibition of TRPV1 or reduction of the ROS generation rate. Taken together our results indicate that in corneal derived fibroblasts, TGFβ- induced myofibroblast development is highly dependent on a positive feedback loop where p-SMAD2-induced ROS activates TRPV1, TRPV1 causes activation of p38, the latter in turn further enhances the activation of SMAD2 to establish a recurrent loop that greatly extends the residency of the activated state of SMAD2 that drives myofibroblast development. [ABSTRACT FROM AUTHOR]

Hepatic fibrosis is a major consequence of liver disease. Radix Paeoniae Rubra (RPR), the dry root of Paeonia lactiflora Pall., has a long history of clinical application in traditional Chinese medicine (TCM) for the treatment of liver diseases. The researches of RPR active ingredients are mainly focused on paeoniflorin. However, the functional roles of other ingredients have not been clarified sufficiently in the treatment of hepatic fibrosis with RPR. This study was to figure out the anti-hepatic fibrosis potential and mechanisms of CS-4, one of the paeoniflorin-free subfraction of RPR. With the guide of bioassay, CS-4, a subfraction of RPR showed in vitro inhibition of hepatic stellate cell activation, was obtained using multiple chromatographic techniques. Its ingredients were determined by UPLC-Q-TOF-MS/MS. Then, the target profiles of ingredients were obtained from the HERB database, and the disease targets were collected from the DisGeNET database. Through the network pharmacology method, a protein-protein interaction network of CS-4 against hepatic fibrosis was established to analyze and excavate the potential therapeutic targets. Combined with the KEGG analysis, a series of signaling pathways were obtained, thereby validated by western blot analysis. The paeoniflorin-free subfraction of RPR, CS-4, was obtained and showed the most potential anti-fibrotic effect in vitro. A total of 20 main ingredients were identified from CS-4 and considered as its active ingredients. From HERB and DisGeNET databases, 1460 potential targets of CS-4 and 1180 disease targets were obtained, respectively. The overlapped 79 targets were considered to exert the potential anti-fibrosis effect of CS-4, such as JAK2, MYC, SMAD3, and IFNG. The gene enrichment analysis revealed that classical TGF-β/Smad signaling pathway and nonclassical TGF-β/PI3K-AKT signaling pathway may be two of the main mechanisms of CS-4 against hepatic fibrosis, which supported by western blot analysis. In this study, a paeoniflorin-free subfraction with potential anti-hepatic fibrosis activity in vitro , CS-4, was obtained from RPR. Its multiple ingredients, multiple targets, and multiple mechanisms against hepatic fibrosis were explained by network pharmacology and verified by western blot analysis to further support the clinical applications of RPR. [Display omitted] • A paeoniflorin-free subfraction of Paeonia lactiflora Pall. was first reported. • Its main ingredients were identified by UPLC-TOF-MS/MS. • Its complex mechanisms of anti-hepatic fibrosis were clarified by network pharmacology. • Parts of signaling pathways were verified by experiments. [ABSTRACT FROM AUTHOR]

The present study aimed to explore the role of oxytocin (OT) in myocardial injury induced by ischemia/reperfusion (I/R) and hyperglycemia and its underlying mechanisms. In this study, the isolated rat hearts underwent I/R in Langendorff perfusion model and H9c2 cells were subjected to hypoxia/reoxygenation (H/R) to establish an in vitro model. I/R injury was induced by exposing the rat hearts to 40 min of global ischemia followed by 60 min of reperfusion. H9c2 cells were cultured under the normoglycemic or hyperglycemic condition with or without pretreatment of OT, and then exposed to 4 h of hypoxia and 2 h of reoxygenation. Measurement indicators included myocardial infarct size assessed by triphenyltetrazolium chloride (TTC) staining and hemodynamic parameters in the ex vivo model as well as cell viability detected by cell counting kit 8 (CCK-8), apoptotic rate evaluated by flow cytometry, and the protein expressions by Western blot. The findings demonstrated that OT attenuated myocardial I/R injury. First, OT preconditioning significantly reduced hemodynamic disorders and myocardial infarct sizes. In addition, OT increased cell viability, decreased cell apoptosis and the expressions of IL-18, IL-1β, cleaved-caspase-1, NLRP3, and GSDMD following H/R. NLRP3 activator nigericin eliminated the beneficial effects of OT in H9c2 cells. Furthermore, OT also activated AMPK and decreased the expressions of pyroptosis-related proteins. Administration of AMPK inhibitor compound C blunted OT-induced AMPK phosphorylation and elevated the expressions of pyroptosis-related proteins in H9c2 cells subjected to H/R with hyperglycemia. OT alleviates myocardial I/R injury with hyperglycemia by inhibiting pyroptosis via AMPK/NLRP3 signaling pathway. [Display omitted] • Oxytocin preconditioning alleviates myocardial hyperglycemia- and ischemia/reperfusion-induced injury. • Pyroptosis is involved in myocardial hyperglycemia- and ischemia/reperfusion-induced injury. • Oxytocin preconditioning-induced cardioprotection is associated with inhibition of pyroptosis. • Oxytocin inhibits pyroptosis through activation of AMPK signaling pathway. [ABSTRACT FROM AUTHOR]

Mucin-type O-glycosylation is one of the most abundant types of O-glycosylation and plays important roles in various human carcinomas, including breast cancer. A large family of polypeptide N -acetyl-α-galactosaminyltransferases (GALNTs) initiate and define sites of mucin-type O-glycosylation. However, the specific mechanisms underlying GALNT8 expression and its roles in tumorigenesis remain poorly characterized. GALNT8 expression was assessed in 140 breast cancer patients. Immunofluorescence, immunoprecipitation, lectin blot and quantitative real-time PCR were used to investigate the expression of GALNT8 and its role in regulating estrogen receptor α (ERα) via bone morphogenetic protein (BMP) signaling. The expression of GALNT8 was associated with breast cancer patient survival. GALNT8 downregulation was associated with a reduction in ERα levels, while GALNT8 overexpression elevated the transcription and protein levels of ERα and suppressed colony formation, suggesting an important role of GALNT8 in cancer cell proliferation. Conversely, GALNT8 knockdown led to the inhibition of BMP/SMAD/RUNX2 axis, which decreased ERα transcription. Further analysis suggested that BMP receptor 1A (BMPR1A) was O-GalNAcylated. Sites mutation of BMPR1A indicated that Thr137 and Ser37/Ser39/Ser44/Thr49 of BMPR1A were the main O-glycosylation sites. Although we cannot exclude the indirect effect of GALNT8, our results demonstrated that the expression of GALNT8 and O-glycosylation of BMPR1A play key roles in regulating the activity of BMP/SMAD/RUNX2 signaling and ERα expression. These findings suggest that GALNT8 expression and abnormal O-GalNAcylation of BMPR1A increase ERα expression and suppress breast cancer cell proliferation by modulating the BMP signaling pathway. Our results identify the involvement of GALNT8 in regulating ERα expression. • GALNT8 elevated the transcription and protein levels of ERα and suppressed breast cancer cells proliferation. • O-GalNAcylation of BMP receptor 1A was modulated by GALNT8. • GALNT8 enhances expression of ERα by activating BMP/SMAD/RUNX2 signaling pathway. [ABSTRACT FROM AUTHOR]