P65 Protein Research Articles

Probiotic-Derived P8 Protein: Promoting Proliferation and Migration in Stem Cells and Keratinocytes.

Probiotics exert various effects on the body and provide different health benefits. Previous reports have demonstrated that the P8 protein (P8), isolated from Lactobacillus rhamnosus, has anticancer properties. However, its efficacy in stem cells and normal cells has not been reported. In this study, the effect of P8 on cell proliferation and wound healing was evaluated, investigating its underlying mechanism. Based on scratch assay results, we demonstrated that P8 treatment significantly increases wound healing by activating the cell cycle and promoting stem cell stemness. Cellular mechanisms were further investigated by culturing stem cells in a medium containing Lactobacillus-derived P8 protein, revealing its promotion of cell proliferation and migration. Also, it is found that P8 enhances the expression of stemness markers, such as OCT4 and SOX2, along with activation of the mitogen-activated protein kinase (MAPK) signaling and Hippo pathways. These results indicate that P8 can promote cell growth by increasing stem cell proliferation, migration, and stemness in a manner associated with MAPK and Hippo signaling, which could contribute to the increased wound healing after P8 treatment. Furthermore, P8 could promote wound healing in keratinocytes by activating the MAPK signaling pathways. These results suggest that P8 might be a promising candidate to enhance stem cell culture efficiency by activating cell proliferation, and enhance therapeutic effects in skin diseases.

How Does African Swine Fever Virus Evade the cGAS-STING Pathway?

African swine fever (ASF), a highly infectious and devastating disease affecting both domestic pigs and wild boars, is caused by the African swine fever virus (ASFV). ASF has resulted in rapid global spread of the disease, leading to significant economic losses within the swine industry. A significant obstacle to the creation of safe and effective ASF vaccines is the existing knowledge gap regarding the pathogenesis of ASFV and its mechanisms of immune evasion. The cyclic GMP–AMP synthase (cGAS)–stimulator of interferon genes (STING) pathway is a major pathway mediating type I interferon (IFN) antiviral immune response against infections by diverse classes of pathogens that contain DNA or generate DNA in their life cycles. To evade the host’s innate immune response, ASFV encodes many proteins that inhibit the production of type I IFN by antagonizing the cGAS-STING signaling pathway. Multiple proteins of ASFV are involved in promoting viral replication by protein–protein interaction during ASFV infection. The protein QP383R could impair the function of cGAS. The proteins EP364R, C129R and B175L could disturb the function of cyclic guanosine monophosphate-adenosine monophosphate (cGAMP). The proteins E248R, L83L, MGF505-11R, MGF505-7R, H240R, CD2v, E184L, B175L and p17 could interfere with the function of STING. The proteins MGF360-11L, MGF505-7R, I215L, DP96R, A151R and S273R could affect the function of TANK Binding Kinase 1 (TBK1) and IκB kinase ε (IKKε). The proteins MGF360-14L, M1249L, E120R, S273R, D129L, E301R, DP96R, MGF505-7R and I226R could inhibit the function of Interferon Regulatory Factor 3 (IRF3). The proteins MGF360-12L, MGF505-7R/A528R, UBCv1 and A238L could inhibit the function of nuclear factor kappa B (NF-Κb).

Enhancing the potency of anti-P32-active targeted PEGylated liposomal doxorubicin using various spacers: in silico, in vitro and in vivo studies

Even though nanoliposomal drug delivery systems are superior to conventional drug delivery methods, studies indicate that they may deliver small quantities of drugs through passive targeting. Specific peptides are used to decorate the surface of the liposomes for active targeting of tumors. Among these, CGKRK is the tumor homing peptide to target p32 protein on the surface of endothelial and tumor cells. In the present study, serine-glycine (SG) and glycine-glycine-glycine (GGG) spacer were added to CGKRK, an anti-P32 peptide and in silico molecular docking studies were conducted. PEGylated liposomal doxorubicin (PLD) was then surface modified with these peptides. All liposomal formulations had the sizes between 96-110 nm, negative zeta potentials and there were spherical in shape. The release of the doxorubicin (Dox) followed the same pattern for all formulations during 24 h. The cytotoxic effects of the formulations during 1, 3 and 6 h of the incubation, indicated the time-dependent cytotoxicity of formulations, while the PLD-CGGGKRK was the most potent. Apoptosis and antiangiogenic assays were also demonstrated the ability of PLD-CGGGKRK in inducing apoptosis and decreasing vessels densities. The in vivo anti-tumor study demonstrated the efficacy of CGGGKRK surface-modified PLDs in delaying tumor growth and increasing the median survival. Our findings indicated that the GGG spacer could improve the efficacy of anti-P32 peptide, CGKRK, in targeting breast cancer tumor models in mice and merit further investigation.

Network pharmacology and experimental verification unraveled the mechanism of Bailing Capsule against asthma.

Asthma is a serious public health challenge around the world. Recent studies into traditional Chinese medicine preparations for asthma have yielded promising findings regarding Bailing Capsule's potential in bronchial asthma prevention and treatment. This study aims to initially clarify the potential mechanism of Bailing Capsule in the treatment of asthma using network pharmacology and in vitro experimental approaches. Network pharmacology was adopted to detect the active ingredients of Bailing Capsule via Traditional Chinese Medicine Systems Pharmacology Database, and the key targets and signaling pathways in the treatment of asthma were predicted. Docking and molecular dynamics simulations were conducted to verify the most important interactions formed by these probes within different regions of the binding site. The predicted targets were validated in lipopolysaccharide-induced 16HBE cell experiment. Seven active ingredients were screened from Bailing Capsule, 294 overlapping targets matched with asthma were considered potential therapeutic targets, such as SRC, TP53, STAT3, and E1A binding protein P300. The main functional pathways involving these key targets include phosphatidylinositol 3-kinase/protein kinase B, mitogen-activated protein kinase, renin-angiotensin system and other signaling pathways, which were mainly involved in the inflammatory response, apoptosis, and xenobiotic stimulus. Moreover, molecular docking showed that Cerevisterol have higher affinity for SRC, TP53, STAT3, and E1A binding protein P300 than other main active components, which is close to the docking results of the co-crystallized ligands to proteins. Consequently, Cerevisterol was selected for molecular dynamics simulation and the results show that Cerevisterol can bind most tightly to SRC, TP53, and STAT3. Bailing Capsule can promote the growth of 16HBE cell, reduce the production of IL-4, TNF-α and IL-6, and down-regulate the levels of SRC and STAT3 mRNA. This study preliminarily reveals the potential mechanism of Bailing Capsule against asthma with the aid of network pharmacology and in vitro cell experiment, which provided reference and guidance for in-depth research and clinical application.

FOXP2 overexpression upregulates LAMA4 expression and thereby alleviates preeclampsia by regulating trophoblast behavior

Preeclampsia (PE) is a common pregnancy disorder characterized by hypertension and proteinuria. Trophoblast behavior severely affect PE progression. Transcription factor Forkhead box protein P2 (FOXP2) was involved in cell migration and invasion, but its role in PE progression remains unknown. Laminin subunit alpha 4 (LAMA4) was predicted as a downstream gene of FOXP2 and related to PE. Thus, we supposed that FOXP2 might regulate PE by regulating LAMA4. We found the decreased FOXP2 expression in patients with PE compared with healthy pregnant women. The rat model of PE was induced by L-NAME oral gavage. FOXP2 overexpression lowered systolic and diastolic blood pressure and restored pathological changes of rats with PE. Trophoblasts under the hypoxia/reoxygenation (H/R) treatment were used to mimic PE in vitro. The results revealed that FOXP2 overexpression inhibited apoptosis but promoted migration, invasion, and angiogenesis of H/R-treated trophoblasts. Dual luciferase and chromatin immunoprecipitation-polymerase chain reaction assays confirmed that FOXP2 transcriptionally upregulated the LAMA4 expression in trophoblasts. LAMA4 knockdown reversed the migration and invasion-promoting role of FOXP2 overexpression in trophoblasts with H/R treatment. Collectively, our findings suggest that the FOXP2/LAMA4 axis regulates PE by suppressing trophoblast apoptosis and promoting its migration, invasion, and angiogenesis.

IFI27 inhibits HIV-1 replication by degrading Gag protein through the ubiquitin-proteasome pathway.

Type I interferon (IFN-I) and its downstream genes play a profound role in HIV infection. In this study, we found that an IFN-inducible gene, IFI27, was upregulated in HIV-1 infection, which in turn efficiently suppressed HIV-1 replication, specially degraded the viral gag protein, including p24 and p55 subunits. Notably, the anti-HIV-1 activity of IFI27 in Old World monkeys surpassed that in New World monkeys, and IFI27 has a higher potentially inhibitory effect on HIV-1 than simian immunodeficiency virus (SIV). Our initial observations showed that NPM-IFI27, the IFI27 variant in northern pig-tailed macaque (Macaca leonina, NPM), exhibited a strong anti-HIV-1 activity. Further investigation demonstrated that NPM-IFI27 degraded p24 and p55 via the ubiquitin-proteasome pathway, with NPM-IFI27-37-115 interacting with the p24-N domain, and the NPM-IFI27-76-122 domain was closely associated with K48 ubiquitin recruitment. Additionally, Skp2 was identified as the probable E3 ubiquitin ligase responsible for the degradation of p24 and p55. Similarly, human IFI27 (Hu-IFI27) showed a mechanism similar to NPM-IFI27 in HIV-1 inhibition. These findings underscore the pivotal role of NPM-IFI27 in HIV-1 infection and provide a potential strategy for clinical anti-HIV-1 therapy.IMPORTANCEHIV-1 infection can trigger the production of IFN-I, which subsequently activates the expression of various IFN-stimulated genes (ISGs) to antagonize the virus. Therefore, discovering novel host antiviral agents for HIV-1 treatment is crucial. Our previous study revealed that IFI27 can influence HIV-1 replication. In this study, we observed that the NPM-IFI27 complex specifically inhibited HIV-1 by targeting its Gag protein. Further exploration demonstrated that IFI27 interacted with the HIV-1 p24 and p55 proteins, leading to their degradation through the ubiquitin-proteasome pathway. Notably, the NPM-IFI27-37-122 variant exhibited potent anti-HIV-1 activity, comparable to that of SAMHD1. These findings highlight the critical role and inhibitory mechanism of NPM-IFI27 in HIV-1 infection, providing a potential strategy for clinical antiviral therapy.

SR-16234, a Unique Selective Estrogen Receptor Modulator, Suppressed Proliferation and Pain-Related Factor Expression by Inhibition of the Nuclear Factor-kappa B Pathway in Endometriotic Stromal Cells.

What is the effect of SR-16234 (SR), a selective estrogen receptor (ER) modulator, on human endometriotic stromal cells (ESCs)? Endometriotic tissues were obtained from 21 patients undergoing laparoscopic surgery for ovarian endometriomas (OEs). Normal eutopic endometrium during the luteal phase was obtained from 18 patients without endometriosis. ESCs isolated from OEs and normal eutopic endometrial stromal cells (NESCs) were cultured with SR and subsequently exposed to tumor necrosis factor (TNF)-α. After 48 h of incubation, the effect of SR on cell proliferation was evaluated by the WST-8 assay. The gene expressions of inflammatory and pain-related factors, including interleukin (IL)-6, IL-8, cyclooxygenase (COX)-2, transient receptor potential vanilloid (TRPV)1, ESR1, and ESR2, were evaluated by real-time RT-PCR. The phosphorylation of Inhibitor κBα (IκBα), extracellular signal-regulated kinase (ERK)1/2, and Protein Kinase B (AKT) were evaluated by western blot analysis. ILs, prostaglandin (PG) E2, and intranuclear p65 syntheses were assessed by ELISA. SR treatment repressed TNF-α-induced proliferation by 20% in ESCs but not NESCs. SR also reduced IL-6, IL-8, COX-2, TRPV1, ESR1, and ESR2 mRNA expressions and ILs protein, and PGE2 synthesis in ESCs, whereas in NESCs, only TRPV1 mRNA expression was decreased. SR suppressed TNF-α-induced phosphorylated IκBα levels by approximately 50%, and intranuclear p65 protein was reduced by 30% compared to addition of only TNF-α in ESCs. However, SR did not affect the phosphorylation of AKT and ERK1/2. SR appears to be a potential therapeutic agent for endometriosis by suppressing inflammatory and pain-related factor expressions by inhibiting the nuclear factor-kappa B pathway.

The Forgotten Tobamovirus Genes Encoding the 54 kDa Protein and the 4–6 kDa Proteins

This article reviews the literature concerning the largely forgotten tobamovirus gene products for which no functions have been ascribed. One of these gene products is the 54 kDa protein, representing the RNA-dependent RNA polymerase segment of the 183 kDa protein translated from the I1-subgenomic mRNA, but which has been found only by in vitro translation and not in plants. The other is a collection of small proteins, expressed from alternative reading frames (likely from internal ribosome entry sites) in either or both the movement protein gene or the capsid protein gene. Previously, two small proteins were referred to as the 4–6 kDa proteins, since only single proteins of such size had been characterized from tobacco mosaic virus and tomato mosaic virus genomes. Such putative proteins will be referred to here as P6 proteins, since many new proposed P6 open reading frames could be discerned, from an analysis of 45 of 47 tobamovirus genomes, with a coding capacity of >15 amino acids up to 94 amino acids, whereas other peptides with ≤15 amino acids were not considered here. The distribution of the putative P6 proteins among these tobamoviruses is described, as well as the various classes they fall into, based on their distribution with regard to the organization of other genes in the viral genomes. Models also are presented for possible functions of the 54 kDa protein and the P6 proteins, based on data in the literature.

Lactiplantibacillus plantarum NMGL2 exopolysaccharide ameliorates DSS-induced IBD in mice mainly by regulation of intestinal tight junction and NF-κB p65 protein expression

Treatment of inflammatory bowel disease (IBD), a common chronic intestinal disease, by exopolysaccharides (EPSs) produced by lactic acid bacteria has raised increasing concerns. Here, the EPS produced by Lactiplantibacillus plantarum NMGL2 was evaluated for its ameliorating effect on dextran sodium sulfate (DSS)-induced IBD in mice. Administration of the EPS was shown to decrease the body weight loss and the values of disease activity index (DAI) and alleviate the colon damage as evidenced by an improvement in colonic length shortening, a reduction in colonic coefficient, and a reduction in colonic mucosal architecture and inflammatory infiltration. Cytokine assay of the blood and colon tissue samples showed that the EPS could decrease the levels of pro-inflammatory TNF-α and IL-1β, and increase anti-inflammatory IL-10. Oxidative stress assay of the colon tissue showed that the nitric oxide (NO) and malondialdehyde (MDA) levels decreased significantly (p < 0.05), while superoxide dismutase (SOD) and glutathione (GSH) levels increased significantly (p < 0.05) after the EPS intervention. These results were further confirmed by the significantly (p < 0.05) down-regulated levels of NF-κB p65, p-IKKβ, and p-IκBα, and significantly (p < 0.05) enhanced expression of ZO-1 and occludin, as evaluated by Western-blot analysis of these proteins expressed in colonic tissue. The EPS produced by L. plantarum NMGL2 alleviated IBD by suppressing the NF-κB signaling pathway, suggesting its potential as a functional food agent in the prevention of IBD.

A Novel Subset of Regulatory T Cells Induced by B Cells Alleviate the Severity of Immunological Diseases.

Regulatory T (Treg) cells are crucial for maintaining immune tolerance by suppressing response to self-antigens and harmless antigens to prevent autoimmune diseases and uncontrolled immune responses. Therefore, using Treg cells is considered a therapeutic strategy treating inflammatory diseases. Based on their origin, Treg cells are classified into thymus-derived, peripherally induced, and in vitro induced Treg cells. Our group discovered a novel Treg cell subset, namely, Treg-of-B (Treg/B) cells, generated by culturing CD4+CD25- T cells with B cells, including Peyer's patch B cells, splenic B cells and peritoneal B1a cells, for 3days. Treg/B cells express CD44, OX40 (CD134), cytotoxic T-lymphocyte-associated antigen-4 (CD152), glucocorticoid-induced tumor necrosis factor receptor family-related protein (CD357), interleukin-10 receptor, lymphocyte activation gene-3 (CD223), inducible co-stimulator (CD278), programmed-death 1 (CD279), tumor necrosis factor receptor II, and high levels of IL-10, but not forkhead box protein P3, similar to type 1 Treg (Tr1) cells. However, unlike Tr1 cells, Treg/B cells do not express CD103, CD226, and latency-associated peptide. Treg/B cells have been applied for the treatment of some murine models of inflammatory diseases, including allergic asthma, inflammatory bowel disease, collagen-induced arthritis, gout, psoriasis and primary biliary cholangitis. This review summarizes the current knowledge of Treg/B cells.

Electroacupuncture improves cognitive function by modulating hippocampal lactate-mediated HIF-1α signaling pathway and inhibiting inflammation response in vascular dementia rats

To observe the effects of electroacupuncture (EA) stimulation of "Sishencong"(EX-HN1) and "Fengchi"(GB20) on lactate (Lac) content, expression of proline hydroxylase 2 (PHD2), hypoxia-inducible factor-1α (HIF-1α)/nuclear transcription factor- κB (NF- κB)/NOD-like receptor thermoprotein structural domain-associated protein 3 (NLRP3) signaling pathway, and inflammatory factors in hippocampal tissue of vascular dementia (VD) rats, so as to explore its mechanisms underlying improvement of VD. Male SD rats screened by Morris water maze tests were randomly divided into blank control, sham-operation, VD model and EA groups (12 rats in each group). The VD model was replicated using the 4-vessel occlusion (VO) method. EA (2 Hz, 1 mA) was applied to EX-HN1 and bilateral GB20 for 30 min, once daily for consecutive 21 days. Morris water maze test was employed to test the rat's memory learning ability before and after modeling and after the intervention. The hippocampal tissue was sampled for observing histopathologic changes with H.E. staining；and detecting Lac content with colorimetric method, and the contents of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-18 (also in serum) by using ELISA, respectively. The immunoactivity levels of PHD2, HIF-1α, and p-NF-κB p65 in hippocampal tissue were detected by immunohistochemistry, and the expression levels of PHD2, HIF-1α, NF-κB p65, p-NF-κB p65 and NLRP3 proteins in hippocampal tissue were detected by Western blot. Compared with the blank control and sham-operation groups, the escaping latency, Lac content in hippocampus, the TNF-α, IL-1β, and IL-18 contents in both hippocampus and serum, the immunoactivity of HIF-1α and p-NF-κB p65 and expression levels of HIF-1α, NF-κB p65, p-NF-κB p65, and NLRP3 proteins were significantly increased (P<0.01), while the number of original platform crossing, and PHD2 immunoactivity and protein expression level were significantly decreased (P<0.05, P<0.01) in the model group. Following EA intervention, modeling induced increase and decrease of the indexes mentioned above were all reversed in the EA group (P<0.05, P<0.01). H.E. staining showed disordered arrangement of neurons, uneven cytoplasm stain, blurred nucleolus or disappearance of nucleolus, dilated capillaries, many apoptotic bodies and increased inflammatory cells in the hippocampus tissue of the model group, which was improved to a certain extent in the EA group, including relatively regular arrangement of neurons, reduced apoptotic bodies and inflammatory cells, etc. in the hippocampus. EA stimulation of EX-HN1 and GB20 can improve the cognitive function in VD rats, which may be related to its functions in reducing Lac content, regulating the expression of HIF-1α pathway related proteins, and inhibiting inflammatory responses in the hippocampus tissue.

Ellagic Acid Reduces Cadmium Exposure-Induced Apoptosis in HT22 Cells via Inhibiting Oxidative Stress and Mitochondrial Dysfunction and Activating Nrf2/HO-1 Pathway

Exposure to cadmium sulfate (CdSO4) can lead to neurotoxicity. Nevertheless, the precise molecular mechanisms underlying this phenomenon remain unclear, and effective treatment strategies are scarce. This study explored the protective effects of ellagic acid (EA), a natural polyphenolic compound, against CdSO4 exposure-induced neurotoxicity in HT22 cells and the underlying molecular mechanisms. Our findings demonstrated that exposure of HT22 cells to CdSO4 resulted in apoptosis, which was effectively reversed by EA in a dose-dependent manner. EA supplementation also decreased reactive oxygen species (ROS) and mitochondrial ROS production, reduced malondialdehyde (MDA) levels, and restored the activities of superoxide dismutase (SOD) and catalase (CAT). Additionally, EA supplementation at 5–20 μM significantly counteracted Cd-induced the loss of mitochondrial membrane potential and the decrease of ATP and reduced the ratio of Bax/Bcl-2 and cleaved-caspase-3 protein expression. Furthermore, EA supplementation resulted in the upregulation of Nrf2 and HO-1 protein and mRNAs while simultaneously downregulating the phosphorylation of JNK and p38 proteins. The pharmacological inhibition of c-Jun N-terminal kinase (JNK) partially attenuated the activation of the Nrf2/HO-1 pathway induced by CdSO4 and exacerbated its cytotoxic effects. In conclusion, our findings suggest that ethyl acetate (EA) supplementation offers protective effects against CdSO4-induced apoptosis in HT22 cells by inhibiting oxidative stress and activating the Nrf2 signaling pathway. Furthermore, the activation of the JNK pathway appears to play a protective role in CdSO4-induced apoptosis in HT22 cells.

Exploring the Effect of Active Components in Oil Tree Peony Seed Meal on Swine Disease Resistance and its Potential Mechanisms Based on Network Pharmacology and Molecular Docking.

This study aims to explore the feasibility of using network pharmacology and molecular docking technology to predict the effects of active components from oil tree peony seed meal (PSM) on swine diseases. Ten active components of PSM were screened Screening through literature search and network pharmacology standards, including Betulinic acid, Quercetin, Kaempferol, Luteolin, Isorhamnetin, Hydroxygenkwanin, Hederagenin, Benzoyl Paeoniflorin, Albiflorin, Paeoniflorin. Ten types of swine diseases were selected, including African Swine Fever, Aftosa, Swine Vesicular Disease, Transmissible Gastroenteritis, Swine Streptococcal Infection, Blue Aural Disease, Swine Infectious Atrophic Rhinitis, Swine Influenza, Swine Erysipelas, Swine Epidemic Encephalitis. The results showed that the average number of cross genes between the potential target genes of PSM active components and each swine disease target gene accounted for 7.64 % of the total number of swine disease target genes. The GO enrichment analyses showed that putative targets exist in endosomes, lysosomes, cell membranes, nerves, growth factor activity, receptor tyrosine kinase binding, enzyme binding, growth factor binding, transcription coactivator binding, oxidoreductase activity, prostaglandin E receptor activity and insulin receptor substrate binding. The KEGG enrichment analysis results showed that these putative genes were involved in various cancer progression pathways, signaling pathways, and hormone regulatory pathways. A total of 8 core targets were obtained through protein-protein interaction networks analysis, including Protein Kinase CAMP-Activated Catalytic Subunit Alpha (PRKACA), Non-Receptor Tyrosine Kinase (SRC), Mitogen-Activated Protein Kinase 1 (MAPK1), E1A Binding Protein P300 (EP300), Hypoxia Inducible Factor 1 Subunit Alpha (HIF1A), Phosphatidylinositol-4,5-Bisphosphate 3-Kinase Catalytic Subunit Beta (PIK3CB), C-X-C chemokine receptor type 4 (CXCR4) and Estrogen Receptor 2 (ESR2). The HIF-1 signaling pathway was found to be associated with all 10 selected swine diseases. The PD-L1 expression, and PD-1 checkpoint pathway in cancer, and thyroid hormone signaling pathway were not only enriches the core target with a quantity of 7, but also associated with 9 Swine diseases. In addition, the molecular docking results indicate that the core ingredients have strong affinity with hub genes. The research suggests that the active components of PSM may intervene in swine diseases through multiple components, targets, and pathways.

Indigo alleviates psoriasis through the AhR/NF-κB signaling pathway: an in vitro and in vivo study.

Psoriasis is a chronic inflammatory skin disease. A strong association between the AhR/ NFκB axis and the inflammatory response in psoriasis. Indigo (IDG) has demonstrated significant anti-inflammatory properties. This study aimed to assess the anti-psoriatic efficacy of IDG while investigating the underlying mechanisms involved. In the in vitro experiments, cell viability was assessed using the CCK-8. qRT-PCR was employed to measure the mRNA levels of NF-κB, TNF-α, IL-1β, AhR, and CYP1A1. Western blotting was conducted to examine alterations in cytoplasmic and nuclear AhR protein levels. Additionally, an IDG nanoemulsion (NE) cream was prepared for the in vivo experiments. A psoriasis-like skin lesion mice model was induced using IMQ (62.5 mg/day for 7 days). The severity of psoriasis was evaluated using PASI, and skin lesions were scored while epidermal thickness was assessed via HE staining. The expression of inflammatory markers, including IL-6, IL-13, IL-17A, MCP-1, and TNF-α, was detected in skin lesions using Luminex. The levels of CYP1A1, p65, and p-p65 proteins were determined by Western blotting. LPS stimulation significantly elevated TNF-α, IL-6, and NF-κB mRNA levels, which were notably reduced by IDG treatment. Additionally, IDG significantly enhanced the expression of AhR and CYP1A1 mRNA. Further investigation revealed that IDG facilitated AhR translocation from the cytoplasm to the nucleus. In the IMQ-induced psoriasis-like mouse model, IDG NE substantially ameliorated the severity of skin lesions. Moreover, IDG NE treatment reduced the upregulation of inflammatory cytokines such as IL-6, IL-17A, MCP-1, and TNF-α in IMQ-induced skin lesions. It was also observed that IDG NE treatment increased CYP1A1 protein expression while inhibiting p65 and p-p65 protein expression. IDG emerges as a promising treatment for psoriasis, demonstrating effective therapeutic outcomes. Its mechanism of action is likely linked to the modulation of the AhR/NFκB signaling pathway.

N4BP3 facilitates NOD2-MAPK/NF-κB pathway in inflammatory bowel disease through mediating K63-linked RIPK2 ubiquitination

The NOD2 signaling pathway, which plays an important role in the mechanisms of inflammatory bowel disease (IBD) development, has been closely associated with ubiquitination. It was revealed in this study that NOD2 receptor activation could obviously affect the expression of 19 ubiquitination-related genes, with N4BP3 being the most prominently expressed and upregulated. In addition, N4BP3 knockdown was found to reduce the mRNA levels of MDP-induced inflammatory factors, while N4BP3 overexpression elevated their mRNA levels as well as the levels of phospho-ERK1/2, phospho-JNK, phospho-P38 and phospho-NF-κB P65 proteins. Immunoprecipitation tests showed that N4BP3 could pull down RIPK2 and promote its K63-linked ubiquitination. In human tissue specimen assays and mouse experiments, we found that the expression of N4BP3 was significantly elevated in Crohn’s disease (CD) patients and IBD mice, and N4BP3 knockdown reduced the dextran sulfate sodium-induced pathological score and the expression of inflammatory factors in the mouse colon tissue. In conclusion, N4BP3 is able to interact with RIPK2 and promote its K63-linked ubiquitination, to further promote the NOD2-MAPK/NF-κB pathway, thereby increasing promoting the release of inflammation factors and the degree of IBD inflammation.

Pueraria lobata extract ameliorates D-GalN/LPS-induced liver injury through targeting TLR4/NF-κB signalling pathway and regulating gut microbiota

AbstractKudzu root (Pueraria lobata), known for its dual role as a medicinal herb and food ingredient, has proven anti-inflammatory and antioxidant properties. This study explored the effects of P. lobata extract (PLE) on D-galactose/lipopolysaccharide (D-GalN/LPS)-induced liver inflammation and oxidative stress in mice, along with its impact on gut microbiota. The in vivo studies indicated that PLE significantly reduced hepatic levels of pro-inflammatory cytokines (tumour necrosis factor-α, interleukin-1β, and interleukin-6) and increased the anti-inflammatory cytokine interleukin-10. It also lowered serum aspartate aminotransferase and alanine aminotransferase activities, indicating reduced liver damage, and mitigated oxidative stress by decreasing malondialdehyde levels while restoring superoxide dismutase activity. Western blot analysis revealed that PLE inhibited the phosphorylation of NF-κB pathway proteins (p65 and IκB) and suppressed TLR4 expression, highlighting its role in this inflammatory pathway. Additionally, PLE ameliorated D-GalN/LPS-induced gut microbiota dysbiosis, reducing Proteobacteria abundance and promoting beneficial genera such as Lactobacillus, Bifidobacterium, and Akkermansia. These findings suggest that PLE protects against liver inflammation and oxidative stress, while improving gut microbiota composition, offering potential therapeutic strategies for liver-related diseases.

MTORC2 inhibition by JR-AB2-011 improves IL-1β-induced inflammation, catabolic response, and apoptosis in human chondrocytes through IκB-α/NF-κB p65.

Osteoarthritis (OA) is a very common chronic joint condition marked by inflammation and cartilage loss. mTOR is a well-known mediator of inflammation, cell survival, and aging; however, its role in OA has not been determined. To explore the role of mTORC2 in OA-and associated pathological changes, we examined the contribution of mTORC2-mediated Akt, rictor and IκB-α/NF-κB p65 pathway in interleukin (IL)-1β-treated human chondrocytes. We focused on the protein expression of proinflammatory cytokines and catabolic and apoptotic factors, including TNF-α, IL-6, iNOS, MMP13, Bax, and caspase3, which may occur through this signalling pathway in IL-1β-treated chondrocytes. Chondrocytes were cultured and treated with either 2 ng/mL IL‑1β alone or in combination with increasing concentrations of JR-AB2-011 (50, 100, or 250 µM), a selective mTORC2 inhibitor. The protein levels of phosphorylated (p)‑Akt, Akt, rictor, p-NF-κB p65, NF-κB p65, IκB-α, p-IκB-α, iNOS, MMP13, Bax, and caspase3 were evaluated by Western blotting. In IL-1β-stimulated chondrocytes, mTORC2 activity was increased with increased phosphorylation of Akt and expression of rictor. IL-1β increased the expression of p-IκBα, p-NF-κB p65, NF-κB p65, IL-6, TNF-α, iNOS, Bax, and caspase3 proteins and decreased the expression of IκB-α. All of these IL-1β-induced alterations were prevented by JR-AB2-011. The main novel finding in the present study is that selective mTORC2 inhibition by JR-AB2-011 prevents the inflammatory, catabolic, and apoptotic responses induced by IL-1β via modulation of IκB-α/NF-κB activity. Therefore, we demonstrated a previously unknown function of mTORC2 inhibition that seems to be a potential therapeutic target for OA.

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.

7872 Adiponectin Enhances Hepatic Insulin Sensitivity in a Rat Model of Polycystic Ovary Syndrome: Role of PI3K/Akt/mTORC2 Pathway

Abstract Disclosure: A. Abdelhameed: None. S. Rezq: None. R.M. Vinson: None. J. Flaherty: None. B. Kim: None. D.G. Romero: None. L.L. Yanes Cardozo: None. Background: Polycystic ovary syndrome (PCOS) is the most common endocrine disorder in reproductive-age women and is associated with a high prevalence of obesity and insulin resistance (IR). Phosphoinositide-3-kinase (PI3K)/Akt/ the mammalian target of rapamycin complex 2 (mTORC2) pathway is one major insulin signaling pathway and its impairment is involved in multiple metabolic disorders by inducing IR. Phosphorylation of protein kinase B (Akt) at (Ser)-473 by mTORC2 leads to its full activation and subsequently enhances insulin sensitivity. Adiponectin is a peptide secreted by adipocytes that increases insulin sensitivity. PEG-BHD1028 is a novel potent peptide adiponectin receptor agonist that enhances insulin sensitivity in different models of diabetes. However, its potential to attenuate androgen-mediated hepatic IR by modulating PI3K/Akt/mTORC2 pathway remains unknown in PCOS. Hypothesis: We tested the hypothesis that PEG-BHD1028 attenuates hepatic IR via modulating PI3K/Akt/mTORC2 pathway in a rat model of PCOS. Methods: To induce PCOS, four-week-old female Sprague Dawley rats were implanted with dihydrotestosterone (DHT) silastic tubes (7.5mg) or placebo (PBO) for 90 days (n=4-8/group). During the final three weeks of DHT treatment, rats received PEG-BHD1028 (25μg/kg/day) subcutaneously. Body weight (BW) by gravimetry, fat and lean mass were measured by Echo-MRI. Fasting plasma insulin was measured by ELISA while fasting plasma glucose was measured with a clinical chemistry analyzer. HOMA-IR, an index of systemic IR, was calculated. Protein levels of insulin signaling markers including IRS1, p-IRS1 (Ser1101), PI3 kinase p110, Akt, p-Akt (Ser473), mTOR, p-mTOR (Ser2481) and mTORC2 regulatory protein Rictor were quantified in the liver by Western-blot. Results: DHT significantly (p&amp;lt;0.05) increased BW, fat mass and lean mass by 1.4-, 2.2-, 1.4- folds, respectively compared to PBO group. DHT significantly (p&amp;lt;0.05) increased HOMA-IR by 3.8- fold, compared to PBO group. DHT downregulated hepatic p-Akt, Akt and mTOR protein expression by 20% to 40% while upregulating IRS-1 by 1.6- fold with no impact on PI3 kinase, p-mTOR, Rictor and p-IRS1 compared to the PBO group. PEG-BHD1028 significantly decreased BW, fat mass and lean mass while attenuating IR (34.7% reduction in HOMA-IR) in PCOS rats. Interestingly, PEG-BHD1028 significantly (p&amp;lt;0.05) increased PI3K, active Akt (pAKT), mTOR and Rictor levels by 1.6-, 2.2-, 1.5-, 1.8- folds, respectively with no effect on the other insulin signaling markers compared to vehicle-treated group. Conclusion: Our findings suggest that PEG-BHD1028 is a novel and effective therapeutic agent to attenuate hepatic IR in PCOS via activating PI3K/Akt/mTORC2 pathway. Significance: Targeting adiponectin signaling could be a novel and effective therapeutic approach to mitigate metabolic dysfunction in females with excess androgens. Presentation: 6/2/2024

Berberine modulates ovarian cancer autophagy and glycolysis through the LINC01123/P65/MAPK10 signaling axis

BackgroundBerberine, a readily accessible natural compound known for its ease of synthesis and low toxicity, exhibits anti-tumor properties by modulating inflammatory responses. Recent studies have revealed that berberine can also treat malignant tumors by influencing tumor metabolic reprogramming, making it a potential candidate for metabolic therapy in ovarian cancer. MethodsThe anti-proliferative and anti-metastatic effects of berberine on ovarian cancer cells were investigated using CCK-8 assays, scratch assays, EDU proliferation assays, and assays related to glycolysis and autophagy. Differentially expressed lncRNAs in ovarian cancer were identified using data from the TCGA database. A specific lncRNA's role was delineated through RNA pulldown assays, silver staining, mass spectrometry analysis, CHIP assays, and immunoprecipitation experiments, focusing on its involvement in glycolysis and autophagy regulation in ovarian cancer. Additionally, the inhibitory mechanism of berberine on ovarian cancer cells was validated through cell thermal shift assays and cycloheximide protein degradation experiments to confirm its interaction with key targets. ResultsIn vitro experiments revealed that berberine reduces glycolysis and autophagy levels, leading to the inhibition of ovarian cancer cell proliferation and metastasis. Bioinformatics analysis of TCGA data identified LINC00123 as associated with poor prognosis in ovarian cancer. Experimental validation, including RNA pulldown assays, confirmed that the LINC00123/P65/MAPK10 signaling axis regulates glycolysis and autophagy in ovarian cancer. Furthermore, at the molecular level, berberine inhibits the interaction between LINC00123 and P65, thereby reducing P65 protein stability and impeding its transcriptional regulation of downstream MAPK10. These findings were further validated in animal models. ConclusionOur study highlights berberine's dual benefits of anti-inflammatory effects and inhibition of ovarian cancer proliferation and metastasis by modulating autophagy and glycolysis levels. Mechanistically, berberine targets the LINC00123/P65/MAPK10 signaling pathway to regulate glycolysis and autophagy in ovarian cancer. These insights not only expand the potential of berberine in ovarian cancer therapy but also provide new targets and therapeutic strategies for metabolic therapy in this cancer type.