Protein c-Jun Research Articles

Hyperthermia-mediated cell death via deregulation of extracellular signal-regulated kinase and c-Jun NH2-terminal kinase signaling

Hyperthermia is a promising anticancer treatment that induces heat stress, thereby stimulating various signal transduction pathways to maintain cellular homeostasis. Mitogen-activated protein kinases (MAPKs) associate various extracellular stimuli with cytoplasmic and nuclear mediators through a three-tiered cascade of kinases, including MAPKs, MAP2Ks, and MAP3Ks. In mammals, three major groups of MAPKs have been characterized: extracellular signal-regulated protein kinases (ERK1/2), p38 MAPKs (α, β, γ, and δ), and c-Jun NH2-terminal kinases (JNK1/2/3). Each group of MAPKs is activated by heat and exhibits distinct biological functions. Recent studies have indicated that in hyperthermia, MAPK signaling pathways regulate cell survival and death in unique ways. This review offers a concise overview of the MAPK signaling pathway, specifically ERK and JNK, focusing on their relevance in cancer, interplay with heat shock proteins or phosphatases, and current understanding of the MAPK signaling pathway in hyperthermia.

Astrocytes initiate autophagic flux and maintain cell viability after internalizing non-active native extracellular α-synuclein

Astrocytes are tasked with regulating the synaptic environment. Early stages of various neurodegenerative diseases are characterized by synapse loss, and astrocytic atrophy and dysfunction has been proposed as a possible cause. α-Synuclein (αS) is a highly expressed neuronal protein located in the synapse that can be released in the extracellular space. Evidence points to astrocytes as being responsible for uptake and degradation of extracellular αS. Therefore, misfolded active fibrillized αS resulting in protein inclusions and aggregates could be due to astrocytic dysfunction. Despite these pathological hallmarks and lines of evidence, the autophagic function of astrocytes in response to monomeric non-active αS to model healthy conditions has not been investigated. Human primary cortical astrocytes were treated with 100 nM of extracellular monomeric non-active αS alone, and in combination with N-terminal binding monomeric γ-synuclein (γS) as a control. Western blot analysis and super resolution imaging of HiLyte-488 labeled αS confirmed successful internalization of αS at 12, 24 and 48 h after treatment, while αS dimers were only observed at 48 h. Western blot analysis also confirmed αS's ability to induce autophagic flux by 48 h. Annexin V/PI flow cytometry results revealed increased early apoptosis at 24 h, but which resolved itself by 48 h, indicating no cell death in cortical astrocytes at all time points, suggesting astrocytes can manage the protein degradation demand of monomeric αS in healthy physiological conditions. Likewise, astrocytes reduced secretion of apolipoprotein (ApoE), a protein involved in pro-inflammatory pathways, synapse regulation, and autophagy by 12 h. Similarly, total c-JUN protein levels, a transcription factor involved in pro-inflammatory pathways increased by 12 h in the nuclear fraction. Therefore, astrocytes are able to respond and degrade αS in healthy physiological conditions, and astrocyte dysfunction could precede detrimental αS accumulation.

Molecular mechanisms of Buqing granule for the treatment of diabetic retinopathy: Network pharmacology analysis and experimental validation.

Diabetic retinopathy (DR) is a common microvascular complication of diabetes mellitus. Its blindness rate is high; therefore, finding a reasonable and safe treatment plan to prevent and control DR is crucial. Currently, there are abundant and diverse research results on the treatment of DR by Chinese medicine Traditional Chinese medicine compounds are potentially advantageous for DR prevention and treatment because of its safe and effective therapeutic effects. To investigate the effects of Buqing granule (BQKL) on DR and its mechanism from a systemic perspective and at the molecular level by combining network pharmacology and in vivo experiments. This study collected information on the drug targets of BQKL and the therapeutic targets of DR for intersecting target gene analysis and protein-protein interactions (PPI), identified various biological pathways related to DR treatment by BQKL through Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses, and preliminarily validated the screened core targets by molecular docking. Furthermore, we constructed a diabetic rat model with a high-fat and high-sugar diet and intraperitoneal streptozotocin injection, and administered the appropriate drugs for 12 weeks after the model was successfully induced. Body mass and fasting blood glucose and lipid levels were measured, and pathological changes in retinal tissue were detected by hematoxylin and eosin staining. ELISA was used to detect the oxidative stress index expression in serum and retinal tissue, and immunohistochemistry, real-time quantitative reverse transcription PCR, and western blotting were used to verify the changes in the expression of core targets. Six potential therapeutic targets of BQKL for DR treatment, including Caspase-3, c-Jun, TP53, AKT1, MAPK1, and MAPK3, were screened using PPI. Enrichment analysis indicated that the MAPK signaling pathway might be the core target pathway of BQKL in DR treatment. Molecular docking prediction indicated that BQKL stably bound to these core targets. In vivo experiments have shown that compared with those in the Control group, rats in the Model group had statistically significant (P < 0.05) severe retinal histopathological damage; elevated blood glucose, lipid, and malondialdehyde (MDA) levels; increased Caspase-3, c-Jun, and TP53 protein expression; and reduced superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) levels, ganglion cell number, AKT1, MAPK1, and MAPK3 protein expression. Compared with the Model group, BQKL group had reduced histopathological retinal damage and the expression of blood glucose and lipids, MDA level, Caspase-3, c-Jun and TP53 proteins were reduced, while the expression of SOD, GSH-Px level, the number of ganglion cells, AKT1, MAPK1, and MAPK3 proteins were elevated. These differences were statistically significant (P < 0.05). BQKL can delay DR onset and progression by attenuating oxidative stress and inflammatory responses and regulating Caspase-3, c-Jun, TP53, AKT1, MAPK1, and MAPK3 proteins in the MAPK signaling pathway mediates these alterations.

Saikosaponin A Recovers Impaired Filaggrin Levels in Inflamed Skin by Downregulating the Expression of FRA1 and c-Jun.

Filaggrin (FLG) is an essential structural protein expressed in differentiated keratinocytes. Insufficient FLG expression contributes to the pathogenesis of chronic inflammatory skin diseases. Saikosaponin A (SSA), a bioactive oleanane-type triterpenoid, exerts anti-inflammatory activity. However, the effects of topically applied SSA on FLG expression in inflamed skin remain unclear. This study aimed to evaluate the biological activity of SSA in restoring reduced FLG expression. The effect of SSA on FLG expression in HaCaT cells was assessed through various biological methods, including reverse transcription PCR, quantitative real-time PCR, immunoblotting, and immunofluorescence staining. TNFα and IFNγ decreased FLG mRNA, cytoplasmic FLG protein levels, and FLG gene promoter-reporter activity compared to the control groups. However, the presence of SSA restored these effects. A series of FLG promoter-reporter constructs were generated to investigate the underlying mechanism of the effect of SSA on FLG expression. Mutation of the AP1-binding site (mtAP1) in the -343/+25 FLG promoter-reporter abrogated the decrease in reporter activities caused by TNFα + IFNγ, suggesting the importance of the AP1-binding site in reducing FLG expression. The SSA treatment restored FLG expression by inhibiting the expression and nuclear localization of FRA1 and c-Jun, components of AP1, triggered by TNFα + IFNγ stimulation. The ERK1/2 mitogen-activated protein kinase signaling pathway upregulates FRA1 and c-Jun expression, thereby reducing FLG levels. The SSA treatment inhibited ERK1/2 activation caused by TNFα + IFNγ stimulation and reduced the levels of FRA1 and c-Jun proteins in the nucleus, leading to a decrease in the binding of FRA1, c-Jun, p-STAT1, and HDAC1 to the AP1-binding site in the FLG promoter. The effect of SSA was evaluated in an animal study using a BALB/c mouse model, which induces human atopic-dermatitis-like skin lesions via the topical application of dinitrochlorobenzene. Topically applied SSA significantly reduced skin thickening, immune cell infiltration, and the expression of FRA1, c-Jun, and p-ERK1/2 compared to the vehicle-treated group. These results suggest that SSA can effectively recover impaired FLG levels in inflamed skin by preventing the formation of the repressor complex consisting of FRA1, c-Jun, HDAC1, and STAT1.

Recent Molecular Targets and Their Ligands for the Treatment of Alzheimer Disease.

Alzheimer's disease is a multifaceted neurodegenerative disease. Cholinergic dysfunction, amyloid β toxicity, tauopathies, oxidative stress, neuroinflammation are among the main pathologies of the disease. Ligands targeting more than one pathology, multi-target directed ligands, attract attention in the recent years to tackle Alzheimer's disease. In this review, we aimed to cover different biochemical pathways, that are revealed in recent years for the pathology of the disease, as druggable targets such as cannabinoid receptors, matrix metalloproteinases, histone deacetylase and various kinases including, glycogen synthase kinase-3, mitogen-activated protein kinase and c-Jun N-terminal kinase, and their ligands for the treatment of Alzheimer's disease in the hope of providing more realistic insights into the field.

Using broadly targeted plant metabolomics technology combined with network pharmacology to explore the mechanism of action of the Yishen Gushu formula in the treatment of postmenopausal osteoporosis in vivo

Ethnopharmacological relevanceYishen Gushu Formula (YSGSF) is composed of Epimedium, prepared Rehmannia, Drynaria, Eucommia, Dodder, ginseng, Astragalus, Ligusticum wallichii, Aucklandia and Panax notoginseng. It can improve bone mineral density by regulating bone metabolism. However, the mechanism of YSGSF in the treatment of Postmenopausal osteoporosis (PMOP) remains unclear. Aim of the studyThe compounds, targets, and molecular mechanisms of YSGSF in the treatment of PMOP were investigated using broad-spectrum target metabolomics from plants, combined with network pharmacology and animal studies, leading to a discussion on a novel approach to understanding YSGSF's action in PMOP treatment. Materials and methodsUsing ultra-performance liquid chromatography coupled with triple quadrupole-linear ion trap tandem mass spectrometry (UPLC-QTRAP-MS/MS) within a comprehensive targeted metabolomics framework, the active constituents of YSGSF were identified. This, alongside network pharmacology and molecular docking, facilitated the identification of critical signaling pathways and targets pertinent to YSGSF's therapeutic effect on PMOP. Subsequently, an animal model for PMOP was developed. Following intervention grouping, rats' weight changes were recorded; serum bone metabolic factors were assessed via ELISA; bone microstructure was examined using HE staining and Micro-CT; and key signaling pathway proteins and genes were analyzed through immunohistochemistry to validate YSGSF's potential mechanism in PMOP treatment. ResultsA total of 84 main active components of YSGSF were identified. The key signaling pathways affected by YSGSF in the treatment of PMOP were the TNF and IL-7 signaling pathways, closely related to TNF-α, IL-1β, c-jun and other protein targets. The results of animal experiments showed that YSGSF could downregulate the expression of TNF-a, IL-1β and c-Jun proinflammatory factors by regulating the TNF and IL-7 signaling pathways and regulate the inflammatory response, osteocyte differentiation and apoptosis to control the development of PMOP. ConclusionYSGSF activates the TNF-α and IL-7 signaling pathways in PMOP rats, reducing TNF-α and IL-1β levels, the c-Jun inflammatory response, and osteocyte differentiation and apoptosis, thus playing a significant role in treating PMOP.

γ-Mangosteen, an autophagy enhancer, prevents skin-aging via activating KEAP1/NRF2 signaling and downregulating MAPKs/AP-1/NF-κB-mediated MMPs

BackgroundMangosteens, a naturally occurring xanthones, found abundantly in mangosteen fruits. The anti-skin aging potential of γ-mangosteen (GM) remains unexplored; therefore, we investigated the UVB-induced anti-skin aging of GM via activation of autophagy. HypothesisWe hypothesized that GM exerts antioxidant and anti-aging capabilities both in vitro and in vivo through activation of autophagy as well as control of KEAP1/NRF2 signaling and MAPKs/AP-1/NF-κB-mediated MMPs pathways. MethodsThe anti-skin aging effects of GM were studied using HDF cells and a mice model. Various assays, such as DPPH, ABTS, CUPRAC, FRAP, and ROS generation, assessed antioxidant activities. Kits measured antioxidant enzymes, SA-β-gal staining, collagen, MDA content, si-RNA experiments, and promoter assays. Western blotting evaluated protein levels of c-Jun, c-Fos, p-IκBα/β, p-NF-κB, MAPK, MMPs, collagenase, elastin, KEAP1, NRF2, HO-1, and autophagy-related proteins. ResultsGM exhibited strong antioxidant, collagenase and elastase enzyme inhibition activity surpassing α- and β-mangosteen. GM competitively inhibited elastase with a Ki value of 29.04 µM. GM orchestrated the KEAP1-NRF2 pathway, enhancing HO-1 expression, and suppressed UVB-induced ROS in HDF cells. NRF2 knockdown compromised GM's antioxidant efficacy, leading to uncontrolled ROS post-UVB. GM bolstered endogenous antioxidants, curbing lipid peroxidation in UVB-exposed HDF cells and BALB/c mice. GM effectively halted UVB-induced cell senescence, and reduced MMP-1/-9, while elevated TIMP-1 levels, augmented COL1A1, ELN, and HAS-2 expression in vitro and in vivo. Additionally, it suppressed UVB-induced MAPKs, AP-1, NF-κB phosphorylation. Pharmacological inhibitors synergistically enhanced GM's anti-skin aging potential. Moreover, GM inhibited UVB-induced mTOR activation, upregulated LC3-II, Atg5, Beclin 1, and reduced p62 in both UVB induced HDF cells and BALB/c mice, while blocking of autophagy successfully halt the GM effects against the UVB-induced increase of cell senescence, degradation of collagen through upregulation of MMP-1, underscoring GM's substantial anti-skin aging impact via autophagy induction in vitro and in vivo. ConclusionTogether, GM has potent antioxidant and anti-skin aging ingredients that can be used to formulate skin care products for both the nutraceutical and cosmeceutical industries.

Mitochondria-targeted catalase induced cell malignant transformation by the downregulation of p53 protein stability via USP28/miR-200b/PP2A-Cα axis

Antioxidants exert a paradoxical influence on cancer prevention. The latest explanation for this paradox is the different target sites of antioxidants. However, it remains unclear how mitochondria-targeted antioxidants trigger specific p53-dependent pathways in malignant transformation models. Our study revealed that overexpression of mitochondria-targeted catalase (mCAT) instigated such malignant transformation via mouse double minute 2 homolog (MDM2) -mediated p53 degradation. In mouse epithelial JB6 Cl41 cells, the stable expression of mCAT resulted in MDM2-mediated p53 degradation, unlike in catalase-overexpressed Cl41 cells. Further, we demonstrated that mCAT overexpression upregulated ubiquitin-specific protease 28 (USP28) expression, which in turn stabilized c-Jun protein levels. This alteration initiated the activation of the miR-200b promoter transcription activity and a subsequent increase in miR-200b expression. Furthermore, elevated miR-200b levels then promoted its binding to the 3′-untranslated region of protein phosphatase 2A catalytic subunit (PP2A-C) α-isoform mRNA, consequently resulting in PP2A-C protein downregulation. This cascade of events ultimately contributed to increased MDM2 phosphorylation and p53 protein degradation. Thus, the mCAT overexpression triggers MDM2/p53-dependent malignant transformation through USP28/miR-200b/PP2A-Cα pathway, which may provide a new information for understanding mitochondria-targeted antioxidants facilitate the progression to the tumorigenic state.

Signal Transduction of Transient Receptor Potential TRPM8 Channels: Role of PIP5K, Gq-Proteins, and c-Jun.

Transient receptor potential melastatin-8 (TRPM8) is a cation channel that is activated by cold and "cooling agents" such as menthol and icilin, which induce a cold sensation. The stimulation of TRPM8 activates an intracellular signaling cascade that ultimately leads to a change in the gene expression pattern of the cells. Here, we investigate the TRPM8-induced signaling pathway that links TRPM8 channel activation to gene transcription. Using a pharmacological approach, we show that the inhibition of phosphatidylinositol 4-phosphate 5 kinase α (PIP5K), an enzyme essential for the biosynthesis of phosphatidylinositol 4,5-bisphosphate, attenuates TRPM8-induced gene transcription. Analyzing the link between TRPM8 and Gq proteins, we show that the pharmacological inhibition of the βγ subunits impairs TRPM8 signaling. In addition, genetic studies show that TRPM8 requires an activated Gα subunit for signaling. In the nucleus, the TRPM8-induced signaling cascade triggers the activation of the transcription factor AP-1, a complex consisting of a dimer of basic region leucine zipper (bZIP) transcription factors. Here, we identify the bZIP protein c-Jun as an essential component of AP-1 within the TRPM8-induced signaling cascade. In summary, with PIP5K, Gq subunits, and c-Jun, we identified key molecules in TRPM8-induced signaling from the plasma membrane to the nucleus.

Mechanistic analyses reveal that Pueraria montana var. lobata (Willd.) is effective in inhibiting ovarian cancer progression.

Ovarian cancer (OC) is a common malignancies of the female genitalia. P. montana var. lobata (Willd.), a herb with anti-tumor effects, is widely used in the clinical treatment of ovarian cancer (OC), but the ingredients and molecular mechanism of action remains to be explored. In this study, we extracted the main active ingredients of P. montana var. lobata (Willd.) from the TCMSP database, and predicted its potential targets of action against OC from the DisGeNET and GeneCards databases. Protein-protein interaction (PPI) was constructed using the STRING database, while pathway enrichment analyses were performed using the DAVID database. Next, we generated an Ingredient-Target-Pathway network using Cytoscape 3.7.2, then processed the key targets of action and main active ingredients for molecular docking.The results showed that seven active ingredients of P montana var. lobata (Willd.) were associated with treating for OC, namely beta-sitosterol, coumestrol, daidzein, formononetin, genistein, puerarin and scoparone, two important targets Casp3 and Jun, and signaling pathways of P. montana var. lobata (Willd.) against the progression of OC. TUNEL staining, enzyme-linked immunosorbent assay (ELISA), and Western blot assays, the pharmacodynamic effect of puerarin in the treatment of OC and the major targets were verified. Animal experiment demonstrated that application of puerarin at different times of modeling not only upregulated expression of Casp3, Smac, and c-jun proteins, but also promoted apoptosis in tumor cells, hence inhibiting progression of OC. This study demonstrates that P. montana var. lobata (Willd.) can thereby induce apoptosis in tumor cells and inhibit malignant progression through activating expression of Casp3, smac, and c-jun proteins to regulate related apoptosis pathways, as validated by network pharmacology predictions and animal experiments, and can be verifed by large-scale clinical trials in the future. This study also provides theoretical support and new research perspectives for this disease.

Endothelin-1 stimulated human preadipocyte growth through the AMPK, ERK, cJUN, PKC, and STAT3 pathways

High level of plasma endothelin (ET)-1 was noticed in obesity, and it was found to regulate adipogenesis and the endocrine activity of fat cells. However, relatively little is known about the ET-1 signaling pathway in preadipocyte growth. Our previous reports have shown that ET-1 and ET-3 stimulated murine 3T3-L1 preadipocyte growth through different kinase signaling cascades with ET type A receptor (ETAR) but not ETBR dependency. In this study, the hypothesis arose that ET-1 may regulate human visceral white preadipocyte growth through the AMPK, ERK, cJUN, PKC, and STAT3 pathways with ET receptor-specific dependency. As indicated by an increased number of cells and greater conversion of 3-4,5-dimethylthiazol-2-yl-2,5 diphenyl tetrazolium bromide (MTT) to formazan crystals by living cells, the stimulation of human preadipocyte growth by ET-1 depended on concentration. Neither ET-2 nor ET-3 stimulated human preadipocyte growth. When signaling pathways were examined, ET-1 signaling stimulated phosphorylation of AMPK, ERK, cJUN, PKC, and STAT3, proteins but not AKT, JNK, or p38 MAPK. Treatment with an ETAR antagonist, such as BQ610, or an ETBR antagonist, such as BQ788, blocked the ET-1-induced increase in cell growth and phosphorylated levels of AMPK, PKC, and STAT3 proteins. However, treatment with BQ788 but not BQ610 blocked the ET-1-increased levels of ERK and c-JUN protein phosphorylation. In addition, pretreatment with specific inhibitors of AMPK (compound C), ERK1/2 (U0126), JNK (SP600125), JAK2/STAT3 (AG490), or PKC (Ro318220) prevented the ET-1-induced increase in cell growth and reduced the ET-1-stimulated phosphorylation of AMPK, ERK1/2, c-JUN, STAT3, and PKC. However, neither the p38 MAPK antagonist nor the ceramide synthase inhibitor altered the effect of ET-1. The results indicate that the target molecules of ETAR and ETBR signaling pathways transducing ET-1 growth signals in human visceral preadipocytes can be similar (e.g., AMPK, JAK2/STAT3, and PKC) or different (e.g., ERK1/2 and JNK/c-JUN). This work was supported in part by the National Science and Technology Council, Taiwan, Grant NSTC 112-2320-B-008-001 to YHK and the Taoyuan Armed Forces General Hospital, Taiwan, Grant AFTGH to PJH, CCK, LJS, and YHK. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Parabens promotes invasive properties of multiple human cells: A potential cancer-associated adverse outcome pathway

Parabens are esters of p-hydroxybenzoic acid, which have been used as preservatives and considered safe for nearly a century, until the last two decades when concerns began to be raised about their association with cancers. Knowledge of the mode of action of parabens on the metastatic properties of different cancer cells is still very limited. In the present study, we investigated the effects of methylparaben (MP) and propylparaben (PP) on cell invasion and/or migration in multiple human cancerous and noncancerous cells, including hepatocellular carcinoma cells (HepG2), cervical carcinoma cells (HeLa), breast carcinoma cells (MCF-7), and human placental trophoblasts (HTR-8/SVneo). MP and PP at concentrations in a range of 5–500 μg/L significantly promoted the invasion of four cell lines, with a minimum effective concentration of 5 μg/L. MP and PP up-regulated the expression levels and enzymatic activities of matrix metalloproteinase 2 and 9 (MMP2 and MMP9), as well as altered the expression of the tissue inhibitors of metalloproteinase 1 and 2 (TIMP1 and TIMP2) in four cell lines, suggesting MMPs/TIMPs as potential key events (KEs) for paraben-induced cell invasion. Activation of the p38 mitogen-activated protein kinase (p38 MAPK) and c-Jun N-terminal protein kinases 1/2 (JNK1/2) signaling pathways was required for MP- and PP-promoted invasion of four cell lines, suggesting MAPK signaling pathways as candidates for KEs in cancer or noncancerous cells response to paraben exposure. This study showed for the first time that the two widely used parabens, MP and PP, promoted invasive capacity of multiple human cells through a common mode of action. This study provides evidence for the establishment of a potential cancer-associated AOP for parabens based on pathway-specific mechanism(s), which contributes towards assessing the health risks of these environmental chemicals.

Mode of delivery alters sensitivity to thermal and chemical stimuli in adult rats: An experimental study.

The mode of delivery might prompt a long-lasting alteration in physiological and behavioral responsiveness in offspring. This study was intended to evaluate if the mode of delivery could alter sensitivity to thermal and chemical stimuli in female rats. 56 adult female Wistar rats (200-220 gr) that were born by vaginal or cesarean section (C-section) were used (n = 28/each). Inflammatory pain was induced by subcutaneous injection of formalin into the hind paw. The thermal nociceptive threshold was determined by tail-flick and hot plate tests. Besides, the Western blot test was used to evaluate the spinal cord levels of c-Fos and c-Jun proteins. Formalin-induced inflammation was significantly decreased in C-section group as compared to vaginally born rats (p 0.001). The baseline nociceptive threshed and morphine-induced analgesia were significantly increased in C-section groups in comparison to vaginally born rats. In addition, the levels of c-Fos and c-Jun proteins were significantly decreased in the spinal cord of C-section rats as compared to vaginally born animals (p 0.01). Morphine treatment could decrease the expression of c-Fos and c-Jun in the C-section group (p 0.05). Overall, C-section rats showed lower spinal nociceptive processing and neuronal activity later in life, compared to the vaginal born rats.

Local application of zoledronate inhibits early bone resorption and promotes bone formation.

Nonunion resulting from early bone resorption is common after bone transplantation surgery. In these patients, instability or osteoporosis causes hyperactive catabolism relative to anabolism, leading to graft resorption instead of fusion. Systemic zoledronate administration inhibits osteoclastogenesis and is widely used to prevent osteoporosis; however, evidence on local zoledronate application is controversial due to osteoblast cytotoxicity, uncontrolled dosing regimens, and local release methods. We investigated the effects of zolendronate on osteoclastogenesis and osteogenesis and explored the corresponding signaling pathways. In vitro cytotoxicity and differentiation of MC3T3E1 cells, rat bone marrow stromal cells (BMSCs) and preosteoclasts (RAW264.7 cells) were evaluated with different zolendronate concentrations. In vivo bone regeneration ability was tested by transplanting different concentrations of zolendronate with β-tricalcium phosphate (TCP) bone substitute into rat femoral critical-sized bone defects. In vitro, zolendronate concentrations below 2.5 × 10-7M did not compromise viability in the three cell lines and did not promote osteogenic differentiation in MC3T3E1 cells and BMSCs. In RAW264.7 cells, zoledronate inhibited extracellular regulated protein kinases and c-Jun n-terminal kinase signaling, downregulating c-Fos and NFATc1 expression, with reduced expression of fusion-related dendritic cell‑specific transmembrane protein and osteoclast-specific Ctsk and tartrate-resistant acid phosphatase (. In vivo, histological staining revealed increased osteoid formation and neovascularization and reduced fibrotic tissue with 500μM and 2000μM zolendronate. More osteoclasts were found in the normal saline group after 6weeks, and sequential osteoclast formation occurred after zoledronate treatment, indicating inhibition of bone resorption during early callus formation without inhibition of late-stage bone remodeling. In vivo, soaking β-TCP artificial bone with 500μM or 2000μM zoledronate is a promising approach for bone regeneration, with potential applications in bone transplantation.

Effects and mechanisms of negative air ions on blood pressure, oxidative stress and inflammation levels in SHR rats

To investigate the effects and possible mechanisms of negative air ions(NAIs) on blood pressure, oxidative stress, and inflammatory status in spontaneous hypertension rats(SHR). A total of 60 SHR(half male and half female) were randomly divided into one-month and three-month groups, 30 rats per groups, based on the duration of the intervention. Each group was further randomized into three groups based on the daily intervention time: SHR control group, 2 h NAIs-SHR group, and 6 h NAIs-SHR group, 10 rats per groups. In addition, 20 Wistar Kyoto(WKY)(half male and half female), were randomized into one-month WKY group and three-month WKY group, 10 rats per groups, based on the intervention time. The 2 h NAIs-SHR group and 6 h NAIs-SHR group were exposed to an environment with NAIs concentrations of 4.5×10~4-5×10~4 cm~3 per day for 2 h and 6 h. The WKY group and SHR group were exposed to normal air on a daily basis. Blood pressure of rats in each group was measured every three days, while weight was measured once a week. After sacrificing the rats in the first month and the third month of rearing, wet weight of the organs was weighed. The enzyme linked immunosorbent assay(ELISA) was used to detect 8-hydroxylated deoxyguanosine(8-OHdG), interleukin-6(IL-6), interleukin-8(IL-8), tumor necrosis factor-α(TNF-α), nitric oxide(NO) and endothelin-1(ET-1) levels. Reactive oxygen species(ROS) detection kit was used to detect ROS level. Malondialdehyde(MDA) and superoxide dismutase(SOD), glutathione(GSH) and glutathione disulfide(GSSG) were measured by colorimetric analysis. HE staining was conducted to observe the histopathological morphological changes of the thoracic aorta in each group, and Western blot was conducted to detect the thoracic aortap38 mitogen-activated protein kinase(p38 MAPK), extracellular signal-regulated kinases(ERK), c-Jun n-terminal kinase(JNK), c-fos proteins, c-jun proteins and their phosphorylated proteins level. The weight of WKY male mice in the same week age group was higher than that of SHR control group, and there was no significant difference in the weight between the other groups. The coefficient of heart in SHR control group(4.66±0.48) was higher than that in WKY group(3.73±0.15)(P&lt;0.05), while there were no significant differences in the coefficients of brain, kidney, liver and spleen among the groups. Blood pressure in WKY group at the same age was lower than that in SHR group, and blood pressure in SHR control group at 2-5 and 8-11 weeks was higher than that in 2 h NAIs-SHR and 6 h NAIs-SHR groups(P&lt;0.05). HE staining showed that the internal, middle and external membranes of thoracic aorta in 2 h NAIs-SHR group and 6 h NAIs-SHR group were improved to varying degrees compared with those in SHR control group, including disordered internal membrane structure, thickened middle membrane and broken external membrane. In terms of oxidative stress levels, compared with the SHR control group, the ROS(0.66%±0.17%, 0.49%±0.32%) and 8-OHdG((48.29±8.00) ng/mL, (33.13±14.67)ng/mL) levels were lower in the 6 h NAIs-SHR group(P&lt;0.05), while the GSH/GSSG ratio was higher in the one-month 6 h NAIs-SHR group(10.08±4.93). Compared with the 2 h NAIs-SHR group, the ROS level(0.99%±0.19%) was lower in the 6 h NAIs-SHR group(P&lt;0.05). In terms of inflammatory factor levels, compared with the SHR control group, the IL-8 levels((160.44±56.54) ng/L, (145.77±38.39) ng/L) were lower in the 6 h NAIs-SHR group(P&lt;0.05), while the ET-1 level((249.55±16.98) ng/L) was higher in the one-month WKY group. There was no significant difference in NO levels among the groups. The relative expression of p-p38 protein in the thoracic aorta of rats in the one-month SHR control group was lower than that in the WKY group(P&lt;0.05). The relative expression of p-p38 and p-c-fos proteins in the thoracic aorta of rats at three-months was higher in the SHR control group than in the 2 h NAIs-SHR and 6 h NAIs-SHR groups(P&lt;0.05). The intervention of NAIs at a concentration of 4.5×10~4-5×10~4/cm~3 may regulate the partial oxidation and inflammatory state of SHR rats through the ROS/MAPK/AP1 signaling pathway, thereby reducing their blood pressure level.

Microtubule interacting and trafficking domain containing 1 deficiency leads to poor survival via tissue factor-mediated coagulation in bladder cancer

BackgroundPatients with cancer are at an increased risk of developing a hypercoagulative phenotype and venous thromboembolism. However, no clinical trial has yet confirmed that anticoagulant therapy improves cancer prognosis, and the mechanism underlying hypercoagulation in patients with bladder cancer is not well understood. ObjectivesWe hypothesized that the prognostic genes affect tumor progression via tumor-mediated coagulation. MethodsWe detected the most significant prognostic genes of bladder cancer with The Cancer Genome Atlas dataset and validated them in 2 Gene Expression Omnibus datasets and 1 ArrayExpress dataset. Immunohistochemical tests were performed on a cohort of 80 individuals to further examine the prognostic genes. For the most reliable prognostic gene, its influence on coagulation was evaluated with gene knockdown followed by next-generation sequencing and cellular and animal experiments. ResultsDepletion of microtubule interacting and trafficking domain containing 1 (MITD1), a major prognostic gene of bladder cancer, significantly increased the tissue factor (TF) expression. MITD1 deficiency led to cytokinesis arrest, which, in turn, promoted the TF expression via unfolded protein response and c-Jun. The knockdown of IRE1, an essential kinase of unfolded protein response or the inactivation of c-Jun using c-Jun N-terminal kinase inhibitors weakened MITD1 deficiency– or dithiothreitol-induced TF upregulation. Cells lacking MITD1 promoted coagulation and metastasis in the experimental metastasis assay. ConclusionOur findings suggest the novel role of tumor prognostic genes upon the development of hypercoagulative phenotype and venous thromboembolism, thereby underlining the importance of anticoagulant therapy and shedding light on the therapeutic value of targeting MITD1 in bladder cancer.

Roles of Glutathione and AP-1 in the Enhancement of Vitamin D-Induced Differentiation by Activators of the Nrf2 Signaling Pathway in Acute Myeloid Leukemia Cells.

Active vitamin D derivatives (VDDs)-1α,25-dihydroxyvitamin D3/D2 and their synthetic analogs-are well-known inducers of cell maturation with the potential for differentiation therapy of acute myeloid leukemia (AML). However, their dose-limiting calcemic activity is a significant obstacle to using VDDs as an anticancer treatment. We have shown that different activators of the NF-E2-related factor-2/Antioxidant Response Element (Nrf2/ARE) signaling pathway, such as the phenolic antioxidant carnosic acid (CA) or the multiple sclerosis drug monomethyl fumarate (MMF), synergistically enhance the antileukemic effects of various VDDs applied at low concentrations in vitro and in vivo. This study aimed to investigate whether glutathione, the major cellular antioxidant and the product of the Nrf2/ARE pathway, can mediate the Nrf2-dependent differentiation-enhancing activity of CA and MMF in HL60 human AML cells. We report that glutathione depletion using L-buthionine sulfoximine attenuated the enhancing effects of both Nrf2 activators concomitant with downregulating vitamin D receptor (VDR) target genes and the activator protein-1 (AP-1) family protein c-Jun levels and phosphorylation. On the other hand, adding reduced glutathione ethyl ester to dominant negative Nrf2-expressing cells restored both the suppressed differentiation responses and the downregulated expression of VDR protein, VDR target genes, as well as c-Jun and P-c-Jun levels. Finally, using the transcription factor decoy strategy, we demonstrated that AP-1 is necessary for the enhancement by CA and MMF of 1α,25-dihydroxyvitamin D3-induced VDR and RXRα protein expression, transactivation of the vitamin D response element, and cell differentiation. Collectively, our findings suggest that glutathione mediates, at least in part, the potentiating effect of Nrf2 activators on VDDs-induced differentiation of AML cells, likely through the positive regulation of AP-1.

USP5 facilitates bladder cancer progression by stabilizing the c-Jun protein

BackgroundBladder cancer is the second most common genitourinary malignancy worldwide. The death rate of bladder cancer has increased every year. However, the molecular mechanism of bladder cancer is not sufficiently studied. Deubiquitinating enzymes (DUBs) play an important role in carcinogenesis. Several studies have demonstrated that USP5 associated with malignancy and pathological progression in hepatocellular carcinoma, colorectal and non-small cell lung cancer. However, the role of USP5 in bladder cancer need to be explored.MethodsThe USP5 expression was analysed using the web server GEPIA. To explore USP5 function in bladder cancer, we constructed USP5-knockout cell lines in T24 cells. A FLAG-USP5 (WT USP5) plasmid and a plasmid FLAG-USP5 C335A (catalytic-inactive mutant) used to overexpress USP5 in EJ cells. CCK8, colony formation, transwell and scratch assays were used to assess cell viability, proliferation and migration. RNA sequencing (RNA-seq) and dual-luciferase reporter assays were performed to screen the pathway. Coimmunoprecipitation and immunofluorescence were used to explore the interaction between USP5 and c-Jun. Cycloheximide (CHX) chase assays were performed to establish the effect of USP5 on c-Jun stability. Xenograft mouse model was used to study the role of USP5 in bladder cancer.ResultsUSP5 expression is increased in bladder cancer patients. Genetic ablation of USP5 markedly inhibited bladder cancer cell proliferation, viability, and migration both in vitro and in vivo. RNA-seq and luciferase pathway screening showed that USP5 activated JNK signalling, and we identified the interaction between USP5 and c-Jun. USP5 was found to activate c-Jun by inhibiting its ubiquitination.ConclusionsOur results show that high USP5 expression promotes bladder cancer progression by stabilizing c-Jun and that USP5 is a potential therapeutic target in bladder cancer.

Kechuanning Gel Plaster Exerts Anti-inflammatory and Immunomodulatory Effects on Ovalbumin-induced Asthma Model Rats via ERK Pathway.

We aimed to evaluate the therapeutic effects of Kechuanning gel plaster on ovalbumin (OVA)-induced rat model of asthma. Rats were injected with OVA to induce asthma, and Kechuanning gel plaster was administered after the OVA challenge. The immune cell counts in the bronchial alveolar lavage fluid (BALF) were calculated after Kechuanning gel plaster administration. The levels of immune factors in BALF and serum OVA-specific IgE levels were analyzed. Western blot analysis and immunohistochemistry were carried out to analyze the following proteins: C-FOS, C-JUN, RAS p21 protein activator 1 (RASA1), matrix metalloproteinase 9 (MMP9), RAF1, p-MEK1, tissue inhibitor of metalloproteinase-1 (TIMP1), and p-extracellular signal-regulated kinase 1 (ERK1). Administration of Kechuanning gel plaster led to decreased immune cell counts, inflammatory cytokines (interleukin (IL)-1β, IL13, and IL17), and OVA-specific IgE expression. Compared to the normal group, the C-FOS, C-JUN, RASA1, MMP9, RAF1, MEK1, TIMP1, and p- ERK1 expressions in the model group were significantly increased, whereas Kechuanning gel plaster administration decreased C-JUN, MMP9, TIMP1, RAF1, MEK1, p-ERK1, C-FOS, and RASA1 protein levels. Kechuanning gel plaster exerted its therapeutic effects on OVA-induced asthma model rats through the ERK signaling pathway. Kechuanning gel plaster could be considered as a potential alternative therapeutic agent for the management of asthma.

Pentadecanoic acid (C15:0, PA) induces mild maternal glucose intolerance and promotes the growth of the offspring partly through up-regulating liver PPARα and MAPK signaling pathways.

Gestational diabetes mellitus (GDM) is one of the most common metabolic disturbances during pregnancy, which poses a serious threat to both maternal and offspring health. Pentadecanoic acid (C15:0, PA) is one of the most common odd-chain saturated fatty acids (OCS-FAs). However, its safety and nutritional value are yet to be verified. Herein, we provide a systematic assessment of the effects of PA on maternal and progeny health and insulin sensitivity for the first time. Our results showed that consumption of 1% PA during pregnancy could increase the contents of PA and heptadecanoic acid (C17:0) in maternal plasma, fetal tissue and offspring plasma, but it had no effect on embryonic development. During pregnancy, PA treatment caused mild insulin resistance, while it had little effect on the maternal body composition. During lactation, PA treatment caused mild insulin resistance and oxidative stress. Maternal body fat deposition was also reduced, but the growth rate of the offspring was faster. It is worth noting that PA treatment decreased plasma and liver TG content and increased the antioxidant capacity of the offspring. The effect of PA on the transcription and expression genes in the liver of pregnant mice was investigated using RNA-seq. PPARα and MAPK signaling pathways, both closely related to lipolysis, inflammation, oxidative stress, and insulin resistance were significantly increased. The expression of c-JUN, ERK, JNK and P65 proteins was also significantly up-regulated. In conclusion, our results suggest that 1% PA can induce a mild decrease in the maternal glucose tolerance and lipolysis mainly by activated MAPK and PPARα signaling. Moreover, low concentrations of PA may be an effective nutrient to alleviate the oxidative stress and reduce blood lipid levels of offspring.