JNK Gene Research Articles

Yak DEFB123 alleviates lung injury caused by Klebsiella pneumoniae through MAPKs signaling pathway

Beta-defensins, such as β-defensin 123 (DEFB123), are vital components of the immune system's defense against infections due to their strong antimicrobial properties and capacity for modulating the body's immunological responses. In this study, we successfully cloned and analyzed the yak DEFB123 gene sequence. Subsequently, we obtained recombinant protein DEFB123 (rDEFB123) through prokaryotic expression. Our results demonstrate that rDEFB123 effectively inhibits the growth of Escherichia coli, Klebsiella pneumoniae, and Staphylococcus aureus. Furthermore, rDEFB123 enhances the phagocytic activity of macrophages by regulating specific factors. In a mouse model infected with Klebsiella pneumoniae, the administration of rDEFB123 showed significantly lower levels of serum ALT and AST compared to the control group. Moreover, IFN-γ and IgG were significantly increased in the rDEFB123-treated groups, indicating an enhanced immune response. In the MAPKs signaling pathway of the infected mouse lungs, the expressions of JNK, TRAF2, TRAF6, MIF, and IL-1β genes were downregulated in the rDEFB123-treated groups. Moreover, the levels of p-JNK protein were significantly decreased in these groups as well. Klebsiella pneumoniae caused systemic infection with organ damage in mice. However, the administration of rDEFB123 suppressed the expressions of inflammatory factors, thereby mitigating organ injury and regulating the activity of apoptosis-related factors to enhance immunity. These findings provide valuable theoretical data for future exploration of the functionality and potential applications of DEFB123 in yak.

Role of bone marrow mesenchymal stem cell-derived exosomes in reducing neurotoxicity and depression-like behaviors induced by doxorubicin in rats.

Doxorubicin (DOX) is a broad-spectrum antitumor drug while its use is limited nowadays due to its neurobiological side effects associated with depression. Bone marrow mesenchymal stem cells (BM-MSCs) derived exosomes are a promising regenerative therapy. In this study, we investigated the therapeutic potentiality of BM-MSCs derived exosomes against the neurotoxicity induced by DOX. Twenty-four male albino rats were divided equally in to three groups as follow: group 1 (control), group 2 (rats injected intraperitoneally (i.p|) with DOX at a dose 2.5mg/Kg), and group 3 (rats injected with DOX and BM-MSCs derived exosomes i.p at a dose 1.5ml/Kg). During the experiment the behavior tests were noted, after three weeks rats were sacrificed, serum and brain samples were collected for biochemical, molecular and histopathological examinations. The results revealed that DOX causing impairment of the locomotor and increasing the anxiety like behavior of rats, marked neuropathological changes, significant elevation of MDA content and TNF-α concentration, reduction of phospholipase (PLD) and acetylcholinesterase (AChE) protein concentration in addition, there were up regulation of JNK, NF-κB and p38 genes and down regulation of Erk1. Exosomal therapy improved the substantial neurotoxicity of DOX through modulating the markers involved in the neurotoxic signalling pathway of DOX that resulting in improving the pathological lesions and the animal behaviours.

Differential Regulation of Wingless-Wnt/c-Jun N-Terminal Kinase Crosstalk via Oxidative Eustress in Primary and Metastatic Colorectal Cancer Cells.

In the tumor microenvironment (TME), ROS production affects survival, progression, and therapy resistance in colorectal cancer (CRC). H2O2-mediated oxidative stress can modulate Wnt/β-catenin signaling and metabolic reprogramming of the TME. Currently, it is unclear how mild/moderate oxidative stress (eustress) modulates Wnt/β-catenin/APC and JNK signaling relationships in primary and metastatic CRC cells. In this study, we determined the effects of the H2O2 concentration inducing eustress on isogenic SW480 and SW620 cells, also in combination with JNK inhibition. We assessed cell viability, mitochondrial respiration, glycolysis, and Wnt/β-catenin/APC/JNK gene and protein expression. Primary CRC cells were more sensitive to H2O2 eustress combined with JNK inhibition, showing a reduction in viability compared to metastatic cells. JNK inhibition under eustress reduced both glycolytic and respiratory capacity in SW620 cells, indicating a greater capacity to adapt to TME. In primary CRC cells, H2O2 alone significantly increased APC, LEF1, LRP6, cMYC and IL8 gene expression, whereas in metastatic CRC cells, this effect occurred after JNK inhibition. In metastatic but not in primary tumor cells, eustress and inhibition of JNK reduced APC, β-catenin, and pJNK protein. The results showed differential cross-regulation of Wnt/JNK in primary and metastatic tumor cells under environmental eustress conditions. Further studies would be useful to validate these findings and explore their therapeutic potential.

Characterization of c-Jun N-terminal kinase (JNK) gene reveals involvement of immune defense against Vibrio splendidus infection in Apostichopus japonicus

The c-Jun N-terminal kinase (JNK) constitutes an evolutionarily conserved family of serine/threonine protein kinases, pivotal in regulating various physiological processes in vertebrates, encompassing apoptosis and antibacterial immunity. Nevertheless, the involvement of JNK in the innate immune response remains largely unexplored in pathogen-induced echinoderms. We isolated and characterized the JNK gene from Apostichopus japonicus (AjJNK) in our investigation. The full-length cDNA sequences of AjJNK spanned 1806 bp, comprising a 1299 bp open reading frame (ORF) encoding 432 amino acids, a 274 bp 5′-untranslated region (UTR), and a 233 bp 3′-UTR. Structural analysis revealed the presence of a classical S_TKc domain (37–335 amino acids) within AjJNK and contains several putative immune-related transcription factor-binding sites, including Elk-1, NF-κB, AP-1, and STAT5. Spatial expression analysis indicated ubiquitous expression of AjJNK across all examined tissues, with the highest expression noted in coelomocytes. The mRNA, protein, and phosphorylation levels of AjJNK were obviously induced in coelomocytes upon V. splendidus challenge and lipopolysaccharide stimulation. Immunofluorescence analysis demonstrated predominant cytoplasmic localization of AjJNK in coelomocytes with subsequent nuclear translocation following the V. splendidus challenge in vivo. Moreover, siRNA-mediated knockdown of AjJNK led to a significant increase in intracellular bacterial load, as well as elevated levels of Ajcaspase 3 and coelomocyte apoptosis post V. splendidus infection. Furthermore, the phosphorylation levels of AjJNK inhibited by its specific inhibitor SP600125 and also significantly suppressed the expression of Ajcaspase 3 and coelomocyte apoptosis during pathogen infection. Collectively, these data underscored the pivotal role of AjJNK in immune defense, specifically in the regulation of coelomocyte apoptosis in V. splendidus-challenged A. japonicus.

Hexavalent chromium damages intestinal cells and coelomocytes and impairs immune function in the echiuran worm Urechis unicinctus by causing oxidative stress and apoptosis

Hexavalent chromium (Cr(VI)) is a common pollutant in the marine environment, which impairs immunity and causes reproductive and heredity disorders in organisms. To clarify the immunotoxic effects of Cr (VI) on the marine worm Urechis unicinctus, we analyzed tissue damage and immune dysfunction caused by Cr (VI) in this organism at histopathologic, zymologic, apoptotic and molecular levels. The results indicated that the bioaccumulation of Cr (VI) bioaccumulation levels in coelomocytes was significantly higher than in the intestines and muscles. Pathological observation showed that Cr (VI) caused damage to the respiratory intestine, stomach and midgut. Cr (VI) also increased the replication of goblet cells and a reduction in the replication of epithelial cells. Meanwhile, Cr (VI) induced apoptosis of intestinal cells and coelomocytes, accompanied by an increase in the expression of Caspase-3, COX-2, and MyD88 in the intestine and coelomocytes. At the same time, Cr (VI) significantly affected the activities of antioxidant enzymes such as SOD, ACP, CAT, CAT, and GST, and increased H2O2 and MDA contents in U. unicinctus. Moreover, Cr (VI) exposure also up-regulated the transcription of hsc70, mt and jnk genes but decreased that of sod in the intestines. In contrast, Cr (VI) down-regulated the expression of sod, hsc70, mt, and jnk genes in coelomocytes. Collectively, Cr (VI) bioaccumulated in U. unicinctus cells and tissues, causing several histopathological changes, oxidative stress, and apoptosis of several cells in the organism, resulting in intestinal and coelomocyte damage and immune dysfunctioning.

The Role of Interferon Regulatory Factors in Liver Diseases.

The interferon regulatory factors (IRFs) family comprises 11 members that are involved in various biological processes such as antiviral defense, cell proliferation regulation, differentiation, and apoptosis. Recent studies have highlighted the roles of IRF1-9 in a range of liver diseases, including hepatic ischemia-reperfusion injury (IRI), alcohol-induced liver injury, Con A-induced liver injury, nonalcoholic fatty liver disease (NAFLD), cirrhosis, and hepatocellular carcinoma (HCC). IRF1 is involved in the progression of hepatic IRI through signaling pathways such as PIAS1/NFATc1/HDAC1/IRF1/p38 MAPK and IRF1/JNK. The regulation of downstream IL-12, IL-15, p21, p38, HMGB1, JNK, Beclin1, β-catenin, caspase 3, caspase 8, IFN-γ, IFN-β and other genes are involved in the progression of hepatic IRI, and in the development of HCC through the regulation of PD-L1, IL-6, IL-8, CXCL1, CXCL10, and CXCR3. In addition, IRF3-PPP2R1B and IRF4-FSTL1-DIP2A/CD14 pathways are involved in the development of NAFLD. Other members of the IRF family also play moderately important functions in different liver diseases. Therefore, given the significance of IRFs in liver diseases and the lack of a comprehensive compilation of their molecular mechanisms in different liver diseases, this review is dedicated to exploring the molecular mechanisms of IRFs in various liver diseases.

Hepatoprotective effects of Elaeagnus latifolia fruit extract against acetaminophen-induced hepatotoxicity in mice: Mechanistic insights

Elaeagnus latifolia fruit extract (EE) has been reported to contain antioxidant and anti-inflammatory phytochemicals. The present study aimed to examine the anti-hepatotoxic effects of EE in acetaminophen (APAP)-injected mice. Pretreatment with EE for 7 days in APAP mice showed significant reductions in the levels of AST and ALT and necrotic area, accompanied by decreases in liver oxidative stress, nitric oxide, and infiltrating inflammatory cells, compared to the APAP-only group. EE markedly down-regulated expression of CYP2E1, JNK, Bax, NOX subunit, and NF-κB target genes, and up-regulated expression of Bcl-2 and Nrf2 target genes in the livers. Furthermore, EE effectively lowered serum and hepatic lipids, and down-regulated expression of SREBP-1c/2 genes. EE was found to contain phenolic compounds, flavonoids, and proanthocyanidins. Therefore, mechanistic insights into EE's anti-hepatotoxic effects may be associated with preventing the abnormal expression of genes involved in toxic metabolite generation, hepatotoxic signaling, apoptosis, oxidative stress, inflammation, and lipid synthesis.

Transcriptome and proteomic analysis of mpox virus F3L-expressing cells.

Monkeypox or mpox virus (mpox) is a double-stranded DNA virus that poses a significant threat to global public health security. The F3 protein, encoded by mpox, is an apoenzyme believed to possess a double-stranded RNA-binding domain (dsRBD). However, limited research has been conducted on its function. In this study, we present data on the transcriptomics and proteomics of F3L-transfected HEK293T cells, aiming to enhance our comprehension of F3L. The gene expression profiles of pCAGGS-HA-F3L transfected HEK293T cells were analyzed using RNA-seq. Proteomics was used to identify and study proteins that interact with F3L. Real-time PCR was used to detect mRNA levels of several differentially expressed genes (DEGs) in HEK293T cells (or Vero cells) after the expression of F3 protein. A total of 14,822 genes were obtained in cells by RNA-Seq and 1,672 DEGs were identified, including 1,156 up-regulated genes and 516 down-regulated genes. A total of 27 cellular proteins interacting with F3 proteins were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS), and 19 cellular proteins with large differences in abundance ratios were considered to be candidate cellular proteins. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses showed that the DEGs were significantly enriched in immune-related pathways, including type I interferon signaling pathway, response to virus, RIG-I-like receptor signaling pathway, NOD-like receptor signaling pathway, etc. Moreover, some selected DEGs were further confirmed by real-time PCR and the results were consistent with the transcriptome data. Proteomics data show that cellular proteins interacting with F3 proteins are mainly related to RNA splicing and protein translation. Our analysis of transcriptomic and proteomic data showed that (1) F3L up-regulates the transcript levels of key genes in the innate immune signaling pathway, such as RIGI, MDA5, IRF5, IRF7, IRF9, ISG15, IFNA14, and elicits a broad spectrum of antiviral immune responses in the host. F3L also increases the expression of the FOS and JNK genes while decreasing the expression of TNFR2, these factors may ultimately induce apoptosis. (2) F3 protein interacts with host proteins involved in RNA splicing and protein translation, such as SNRNP70, POLR2H, HNRNPA1, DDX17, etc. The findings of this study shed light on the function of the F3 protein.

Isolation and Identification of a Tibetan Pig Porcine Epidemic Diarrhoea Virus Strain and Its Biological Effects on IPEC-J2 Cells.

Porcine epidemic diarrhoea virus (PEDV) is a coronavirus that can cause severe watery diarrhoea in piglets, with high morbidity and mortality rates, seriously hindering the healthy development of the global swine industry. In this study, we isolated a strain of PEDV from Tibetan pigs and named it CH/GS/2022. Subsequently, we screened the apoptosis signals of PEDV-infected IPEC-J2 cells and studied the correlation between apoptosis signals and cell apoptosis. The results showed that different infections of PEDV induced different degrees of apoptosis in cells, and PEDV-induced cell apoptosis was dose-dependent. We then detected the expression of the p53, p38, JNK, Bax, and Bcl-2 genes in the apoptosis signal pathway. The results showed that 24 h after PEDV infection, the expression of the p53, p38, JNK, and Bax genes in IPEC-J2 cells increased significantly, while the expression of the Bcl-2 gene decreased significantly (p < 0.05). Subsequently, we used Western blot to detect the protein levels of these five genes, and the results showed that PEDV infection upregulated the expression of p53, p38, JNK, and Bax proteins (p < 0.05) while downregulating the expression of Bcl-2 protein (p < 0.05). Thus, it was initially inferred that PEDV infection could regulate cell apoptosis by activating the p53, p38, and JNK signalling pathways. Finally, we further investigated the apoptosis of the cells through the use of inhibitors. The results indicated that the p53 inhibitor Pifithrin-α has a significant inhibitory effect on the expression of the p53 protein after PEDV infection and can reverse the expression levels of Bax and Bcl-2 proteins. This suggested that p53 is involved in PEDV-induced cell apoptosis. Similarly, the p38 MAPK inhibitor SB203580 has an inhibitory effect on the expression of the p38 protein and can reverse the expression levels of Bax and Bcl-2 proteins. This suggested that p38 is also involved in PEDV-induced cell apoptosis. On the other hand, the JNK inhibitor SP600125 has no inhibitory effect on the expression of the JNK protein after PEDV infection, but the expression levels of Bax and Bcl-2 proteins have changed. Furthermore, it is noteworthy that SP600125 can inhibit the activity of apoptotic proteins but not their levels, resulting in reduced cell apoptosis. These preliminary results indicated that JNK may be involved in PEDV-induced IPEC-J2 cell apoptosis.

Assessment of the effectiveness of the peptide inhibitor homologous to the transforming growth factor β cytokine blocking the TGFβRI/TGFβRII receptor complex-pilot study.

A key player in the fibrotic process is the transforming growth factor β (TGF-β) which enhances extracellular matrix production by increasing the transcription of matrix proteins. The cytokine TGF-β first binds to the TGFβRII receptor (dimer), resulting in the recruitment of the TGFβRI receptor (dimer). The complex thus formed leads to the phosphorylation of the kinase domain of TGFβRI, which in turn results in activation of the Smad pathway. This is therefore a targeted pathway for research into the application of peptide inhibitors in blocking the TGF-β-Smad signaling pathway. The aim of this study was to design a peptide inhibitor (homologous to the cytokine TGF-β) which, after binding to the TGFβRI/TGFβRII receptor, would block the cytokine binding and thus prevent the formation of an activating complex. Preliminary work on the design and synthesis of inhibitors for TGFβRI/TGFβRII has allowed us to identify and describe five key regions of the TGF-β-TGFβRI/TGFβRII interface. The following five peptide inhibitors were synthesized for Region 1: 1.1 ALDAAYCFR, 1.2 LDAAYCFRN, 1.3 DAAYCFRNV, 1.4 AAYCFRNVQ, 1.5 AYCFRNVQD. The expression of the SEAP reporter gene, Smad2, Smad3, Smad4, and JNK1 gene was measured using quantitative real-time polymerase chain reaction. For Region 1 peptide inhibitors tested for TGFβRI/TGFβRII, reduced SEAP (reporter gene) expression was observed in cells of the MFB-F11 line, which suggests inhibited the formation of cytokine-receptor complexes. For IP1_2, 1_3 and 1_5 Region 1 peptides tested for TGFβRI/TGFβRII, reduced cytokine-receptor signal by adding newly designed inhibitors. The study revealed an impact of these peptide inhibitors on the reduction of mRNA expression of Smad2, Smad3, Smad4 and JNK1 genes.

Toxic effects of triclocarban on the histological morphology, physiological and immune response in the gills of the black tiger shrimp Penaeus monodon

Triclocarban (TCC) is a widely used broad-spectrum antimicrobial agent that has become a pollutant threatening the health of aquatic animals. However, the toxic effects of TCC on Penaeus monodon are still lacking. In this study, we exposed P. monodon to 1 μg/L (TCC-1) and 10 μg/L TCC (TCC-10) for 14 days, and the changes of histological morphology, physiological and immune responses in the gills were investigated. The results showed that TCC exposure caused the deformation of the gill vessels and the disordered arrangement of the gill filaments. Oxidative stress biochemical indexes such as H2O2 content, CAT and GPx activity and the relative expression levels of antioxidant-related genes (SOD, GPx and Nrf2) were increased in the TCC-1 and TCC-10 groups; the levels of CAT and HSP70 genes were increased but POD activity was decreased in the TCC-10 group. The relative expression levels of endoplasmic reticulum (ER) stress indexes such as ERP15 and ATF-6 genes were increased in the TCC-10 group, while the level of GRP78 gene was decreased in the TCC-1 and TCC-10 groups. The relative expression levels of apoptosis indexes such as p53 and JNK genes were increased, but CytC and Casp-3 genes were decreased in the TCC-1 and TCC-10 groups. Furthermore, the relative expression levels of detoxification metabolism-related genes (cytP450 and GST) and osmotic regulation-related genes (NKA-α, NKA-β, CA, AQP, CLC and CCP) were increased in the TCC-10 group. The results showed that TCC exposure could affect the physiological homeostasis in the gills of P. monodon, probably via damaging histological morphology, inducing oxidative stress, and disordering ER stress, apoptosis, detoxification and osmotic regulation.

Taif Rose Oil Ameliorates UVB-Induced Oxidative Damage and Skin Photoaging in Rats via Modulation of MAPK and MMP Signaling Pathways.

Short-wave UVB (ultraviolet B) causes rapid oxidative damage to the skin. Rose water is obtained mainly from the petals of Rosa damascena Mill. (Rosaceae) and used traditionally to hydrate dry skin and reduce signs of aging. This work aimed at evaluating the possible protective potential of the prepared eco-friendly Taif rose oil nanoemulsion (ROSE-NANO) against UVB-induced photoaging in adult male Wistar rats. Taif rose oil (ROSE) was obtained from R. damascene by classical steam distillation and formulated in emulgel (100 mg/g). In addition, the oil was formulated in ROSE-NANO-loaded emulgel (50 and 100 mg/g) to enhance the effect of ROSE. All prepared formulas were tested topically for their potential protective effect in UV-induced skin photoaging. The obtained results demonstrated that application of ROSE-NANO-loaded emulgel resulted in superior antiaging potency over ROSE emulgel based on histological studies as well as biochemical evaluations via amendment in CAT and SOD activities, decreasing the concentration of the inflammatory markers and preventing collagen fragmentation through reduction of MMP-9 content in fibroblasts. Moreover, a significant decrease in mRNA expression of NF-KB, JNK, ERK1/2, and p38 MAPK genes was observed. In conclusion, the current study provides scientific evidence for the traditional use of rose oil in skin aging. Moreover, the NANO formula showed promising efficacy as a skin photoprotector against UV-induced oxidative damage and skin aging.

Cold Stress Induces Apoptosis in Silver Pomfret via DUSP-JNK Pathway.

We cultured silver pomfret for 20 days, decreasing water temperature from 18 to 8 ℃, and sampled muscle every 5 days. Muscle fiber degeneration and apoptosis began to increase at 13 ℃ detected by HE and TUNEL staining. Further analysis of transcriptome revealed that several apoptosis-related pathways were highly enriched by differentially expressed genes (DEGs). We analyzed 10 DEGs from these pathways by RT-qPCR during the temperature-decreasing process. JNK1, PIDD, CytC, Casp 3, and GADD45 were up-regulated after 15 and 20 days, while DUSP3, JNK2, and PARP genes were down-regulated after 15 and 20 days. DUSP5 was up-regulated from 10 to 20 days, and C-JUN was up-regulated after 20 days. We analyzed apoptosis in PaM cells under different temperatures (26 ℃, 23 ℃, 20 ℃, 17 ℃, and 14 ℃). The cell viability significantly declined from 14 to 20 ℃; the TUNEL and IHC results showed that the apoptosis signal increased with the temperature dropping, especially in 17 ℃ and 14 ℃; DUSP5, JNK1, CytC, C-JUN, Casp 3, and GADD45 were up-regulated at 17 ℃ and 14 ℃, and PIDD was up-regulated at 20 ℃, 17 ℃, and 14 ℃. DUSP3 was up-regulated at 20 ℃ but down-regulated at 17 ℃ and 14 ℃, and PARP was down-regulated at 17 ℃ and 14 ℃. JNK2 was up-regulated at 20 ℃ but down-regulated at 17 ℃ and 14 ℃. Our results suggest that DUSP could help inhibit apoptosis in the initial stage of cold stress, but low temperature could down-regulate it and up-regulate JNK-C-JUN, inducing apoptosis in a later stage. These data provide a basis for the study of the response mechanism of fish to cold.

JNKs protect from cholestatic liver disease progression by modulating Apelin signalling

Cholestatic liver injury is associated with c-Jun N-terminal kinases (JNK) activation in distinct cell types. Its hepatocyte-specific function during cholestasis, however, has not yet been established. Therefore, in our present study, we investigated the role of JNK1/2 during cholestasis and dissected its hepatocyte-specific function. A cohort of patients with primary biliary cholangitis (n= 29) and primary sclerosing cholangitis (n= 37) was examined. Wild-type, hepatocyte-specific knockout mice for Jnk2 (Jnk2Δhepa) or Jnk1 and Jnk2 (Jnk1Δhepa/2Δhepa) were generated. Mice were subjected to bile duct ligation (BDL) or carbon tetrachloride (CCl4) treatment. Finally, Apelin signalling was blocked using a specific inhibitor. As an interventional approach, Jnk1/2 were silenced in wild-type mice using lipid nanoparticles for small interfering RNA delivery. JNK activation was increased in liver specimens from patients with chronic cholestasis (primary biliary cholangitis and primary sclerosing cholangitis) and in livers of Mdr2-/- and BDL-treated animals. In Jnk1Δhepa/2Δhepa animals, serum transaminases increased after BDL, and liver histology demonstrated enhanced cell death, compensatory proliferation, hepatic fibrogenesis, and inflammation. Furthermore, microarray analysis revealed that hepatocytic Jnk1/2 ablation induces JNK-target genes involved in oxidative stress and Apelin signalling after BDL. Consequently, blocking Apelin signalling attenuated BDL-induced liver injury and fibrosis in Jnk1Δhepa/2Δhepa mice. Finally, we established an interventional small interfering RNA approach of selective Jnk1/2 targeting in hepatocytes invivo, further demonstrating the essential protective role of Jnk1/2 during cholestasis. Jnk1 and Jnk2 work together to protect hepatocytes from cholestatic liver disease by controlling Apelin signalling. Dual modification of JNK signalling in hepatocytes is feasible, and enhancing its expression might be an attractive therapeutic approach for cholestatic liver disease. The cell-specific function of Jnk genes during cholestasis has not been explicitly explored. In this study, we showed that combined Jnk1/2, but not Jnk2 deficiency, in hepatocytes exacerbates liver damage and fibrosis by enhancing Apelin signalling, which contributes to cholestasis progression. Combined cell-specific Jnk targeting may be a new molecular strategy for treating cholestatic liver disease.

Bifurcaria bifurcata extract exerts antioxidant effects on human Caco-2 cells

The present research study investigated the potential protective effect of Bifurcaria bifurcata extract on cell viability and antioxidant defences of cultured human Caco-2 cells submitted to oxidative stress induced by tert-butylhydroperoxide (tert-BOOH). Aqueous extracts were firstly characterized in terms of total phenolic contents. Concentrations of reduced glutathione (GSH) and malondialdehyde (MDA), generation of reactive oxygen species (ROS), nitric oxide (NO) production, antioxidant enzymes activities [NADPH quinone dehydrogenase 1 (NQO1) and glutathione S-transferase (GST)], caspase 3/7 activity and gene expression linked to apoptosis, proinflammation and oxidative stress signaling pathways were used as markers of cellular oxidative status. B. bifurcata extract prevented the cytotoxicity, the decrease of GSH, the increase of MDA levels and the ROS generation induced by tert-BOOH. B. bifurcata extract prevented the significant decrease of NQO1 and GST activities, and the significant increase of caspase 3/7 activity induced by tert-BOOH. B. bifurcata extract also caused an over-expression of GSTM2, Nrf2 and AKT1 transcriptors, as well as reduced ERK1, JNK1, Bax, BNIP3, NFκB1, IL-6 and HO-1 gene expressions induced by tert-BOOH suggesting an increase in cellular resistance against oxidative stress. The results of the biomarkers analyzed show that treatment of Caco-2 cells with B. bifurcata extract enhance antioxidant defences, which imply an improved cell response to an oxidative challenge. B. bifurcata extract possesses strong antioxidant properties and may be a potential effective alternative to oxidant agents in the functional food industry.

Light Intensity and Photoperiod Interaction Affects the Survival, Development, Molting and Apoptosis-Related Genes of Swimming Crab Portunus trituberculatus Larvae

A 19-day experiment was conducted to investigate the optimal light intensity and photoperiod combination for larval swimming crabs Portunus trituberculatus in terms of survival, development, and apoptosis-related gene expression. Two photoperiods, i.e., 12 and 18 h photophases, and three light intensities, i.e., 126.08, 173.17, and 191.53 μmol m−2 s−1, were used in the study. The results showed that the cumulative survival rate (CSR) of larvae decreased with the increasing light intensity, and the adverse effect of high light intensity was only observed in long photophase groups. On the contrary, a long photophase and low light intensity elevated the CSR from zoea III to juvenile crabs. A long photophase also accelerated the development of zoea III larvae and upregulated the molting-related (ecr and rxr) and apoptosis-related (jnk, p53, and bcl-2) gene expressions. Taken together, the present study suggested that the light intensity and the photoperiod had a combined effect on P. trituberculatus larvae. The optimal light intensity and photoperiod for P. trituberculatus larvae were 126.08 μmol m−2 s−1 and an 18 h photophase, respectively.

A simple, rapid, and efficient method for generating multigene-knockout culture cells by the CRISPR/Cas9 system.

We evaluated the efficacy of simultaneous multiple-gene knockout in human culture cells. By simple co-transfection of HeLa cells with a mixture of pX330-based targeting plasmids together with a puromycin resistance plasmid, followed by transient selection of puromycin-resistant cells, Cas9/single-guide RNA (sgRNA)-transduced polyclonal cell populations were selected and grown. Western blot analyses revealed co-transfection of up to seven targeting plasmids for p38α, p38β, JNK1, JNK2, Mnk1, ERK1, and mLST8 genes, drastically reduced protein expression of these genes in the polyclonal population. Analyses of a randomly isolated group of 25 clones revealed knockout efficiencies for the seven targeted genes ranging between 68% and 100%, and in six clones (24%), all targeted genes were disrupted. Deep sequencing analyses of the individual target sites revealed that, in most cases, Cas9/sgRNA-induced nonhomologous end joining resulted in deletion or insertion of only a few base pairs at the break points. These results demonstrate that simple co-transfection-based simultaneous targeting offers an easy, rapid, and efficient method to generate multiplex gene-knockout cell lines.

JNK/c-Jun pathway activation is essential for HBx-induced IL-35 elevation to promote persistent HBV infection.

Immunoregulation plays pivotal roles during chronic hepatitis B virus (HBV) infection. Studies have shown that Interleukin (IL)-35 is an important molecule associated with inadequate immune response against HBV. However, the mechanisms involved in the up-regulation of IL-35 expression during persistent HBV infection remain unknown. In this study, we constructed a plasmid expressing the HBV X protein (pCMV-HBx) to evaluate the relationship between HBx and IL-35. Activation of the JNK/c-Jun pathway was analyzed and chromatin immunoprecipitation followed by sequencing and luciferase reporter assays were performed to determine whether c-Jun could regulate IL-35 transcription. HBx can significantly activate IL-35 promoter in both LO2 and HepG2 cells compared to the control plasmid (pCMV-Tag2) using the dual-luciferase assay. Whereas other viral proteins, such as S, preS1, the core protein, had no significant effect on IL-35 expression. Similarly, WB and qRT-PCR also showed that HBx can significantly promote IL-35 expression at protein and mRNA levels in the aforementioned cells. The relevant pathway mechanism showed that the expression of JNK and c-Jun genes was significantly higher in transfected cells carrying pCMV-HBx than in the pCMV-Tag2-transfected and -untransfected cells. WB analysis revealed that phosphorylated JNK and c-Jun were overexpressed after HBx action. Conversely, the addition of the JNK/c-Jun signaling pathway inhibitor could significantly suppress HBx-induced IL-35 expression in a dose-dependent manner. A novel molecular mechanism of HBV-induced IL-35 expression was revealed, which involves JNK/c-Jun signaling in up-regulating IL-35 expression via HBx, resulting in transactivation of the IL-35 subunit EBI3 and p35 promoter.

Abstract PD9-07: Role of immunosuppressive JNK pathway in the tumor microenvironment among Triple Negative Breast Cancer subtypes in IBCSG Trial 22-00

Abstract BACKGROUND: Although the triple-negative breast cancer (TNBC) tumor microenvironment (TME) has been deeply characterized, much remains unknown about pathways attributing to an immunosuppressive TME in this disease. The phosphorylation of JNK (c-Jun N-terminal kinase) pathway has been associated with promoting an immunosuppressive phenotype, enhancing TNBC aggressiveness. Here, we aimed to explore the role of the JNK pathway in TNBC using a gene signature inferring the level of JNK phosphorylation. For this purpose, we used the TNBC cohort from the phase III adjuvant IBCSG 22-00 trial, which evaluated low-dose cyclophosphamide and methotrexate (CM) chemotherapy showing no clinical benefit in the overall population. METHODS: JNK gene signature was developed in TNBC samples by integrating RNA-seq gene expression data and phospho-JNK-targeted proteomic data in The Cancer Genome Atlas. Stochastic subsampling was performed to select the candidate markers. LASSO regression was performed to determine the final transcriptional signature to reflect JNK phosphorylation. The signature was then applied in a cohort composed of 498 TNBCs selected using stratified 1:3 relapse cases and non-relapse subcohort ratio from the IBCSG 22-00 trial. RNA-seq data from FFPE tumor samples were available for 347 patients. The JNK signature was calculated as the mean of the products between gene expression and signature coefficients, defining high and low levels using the median cut-off. Immune hot tumors were defined according to TNBC molecular subtypes or tumor-infiltrating lymphocytes (TILs) levels higher than 30%. Multivariable Cox models were used for disease-free survival (DFS) analysis. Wilcoxon test was used to evaluate the association between gene signatures and the levels of JNK. RESULTS: Tumors with either immunomodulatory (IM) phenotype or high TILs showed better DFS when presenting low levels of JNK compared to high levels (HR = 0.75, 95% CI, 0.57 to 0.99; P-value = 0.024 and HR = 0.62, 95% CI, 0.43 to 0.89, P-value = 0.0013). No significant differences in DFS were observed in other TNBC subtypes or tumors with low TILs, further highlighting the relevance of JNK signaling pathway in tumors presenting immune infiltration. Moreover, immune hot tumors with high levels of JNK were enriched for angiogenesis and eosinophils signatures and linked to immunosuppression. The immune targets B7-H3, CSF1R, GITR, and GARP were significantly associated with high levels of JNK. Of note, these genes are involved in the immune escape, activation of macrophages, and regulation of Tregs population. On the other hand, low levels of JNK were associated with higher levels of activated CD8+ T cells, pointing to an anti-tumor immune response, as well as with higher levels of the immune targets PDL-1, CTLA4, CD47, DCIR, and TIGIT. Of interest, a significant DFS benefit was found in IM tumors with high levels of JNK when treated with low-dose CM, compared to those who were not (HR = 0.52, 95% CI, 0.28 to 0.99; P interaction = 0.045). CONCLUSIONS: We developed a JNK gene signature to estimate the phosphorylation level of JNK from gene expression data in TNBC, and validated the associated biological and prognostic value in the IBCSG 22-00 trial. Our results are in line with the immunosuppressive effect described by the JNK gene signature and highlight the heterogeneity of immune response in immune hot TNBCs. Of note, high levels of JNK were associated with worse DFS, as well as with a benefit from low-dose CM potentially related to the immunomodulatory effect described for metronomic regimens. Overall, our findings suggest a potential role of the JNK signature in identifying TNBCs with an immunosuppressive TME and provide the rationale to explore its role as a biomarker for immunotherapy. Further validation of these findings is required. Citation Format: Andrea Joaquin Garcia, Takashi Semba, Mattia Rediti, Daniel J. McGrail, Xuemei Xie, Xiaoping Wang, Dileep R. Rampa, David Venet, Samira Majjaj, Roswitha Kammler, Marco Colleoni, Sherene Loi, Giuseppe Viale, Meredith Regan, Françoise Rothé, Christos Sotiriou, Naoto T. Ueno. Role of immunosuppressive JNK pathway in the tumor microenvironment among Triple Negative Breast Cancer subtypes in IBCSG Trial 22-00 [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD9-07.

Baicalein prevents capecitabine-induced heart damage in female Wistar rats and enhances its anticancer potential in MCF-7 breast cancer cells

AimWe investigated the ability of baicalein (BAI) to enhance the anticancer potential of capecitabine (CAP) in the MCF-7 cell line and its protective effect on CAP-induced cardiotoxicity in female Wistar rats. Methods and key findingsIn vitro study involved evaluating the effect of BAI and/or CAP on cell viability, cell cycle progression, and BAX and Bcl2 gene expression in MCF-7 cells. Co-treatment of BAI with CAP significantly reduced the viability of MCF-7 cells, improved their cytotoxic effect, markedly elevated the percentage of the sub-G1 population, drastically reduced the G2/M population, and significantly altered the mRNA expression of BAX and Bcl2 genes compared with each treatment alone. In vivo study revealed that the oral administration of CAP (140 mg/kg BW) to adult female rats significantly elevated the levels of serum creatine kinase-myocardial band (CK-MB), lactate dehydrogenase (LDH), tumor necrosis factor (TNF)-α, and interleukin (IL)-1β and cardiac TNF-α, IL-1β malondialdehyde (MDA) concentration, whereas it reduced the serum and cardiac total antioxidant capacity (TAC), level of cardiac glutathione (GSH) and activity of glutathione peroxidase (GPx) with a vast array of circulatory, inflammatory, degenerative, and necrotic alterations in the cardiac tissue. Furthermore, CAP administration significantly upregulated the mRNA expression of NF-κB, TLR4, MyD88, ATF6, CHOP, and JNK genes. Concurrent administration of BAI (200 mg/kg BW) and CAP significantly improved the biochemical alterations and cardiac oxidant/antioxidant status and architecture. In addition, it modulated the TLR4/MyD88/NF-κB pathway and endoplasmic reticulum stress. SignificanceAltogether, BAI can augment the anticancer potential of CAP and alleviate its cardiotoxic effects during cancer treatment.