Ursolic Acid Alleviates Mitotic Catastrophe in Podocyte by Inhibiting Autophagic P62 Accumulation in Diabetic Nephropathy.

The glomerular podocyte, a terminally differentiated cell, is crucial for the integrity of the glomerular filtration barrier. The re-entry of podocytes into the mitotic phase results in injuries or death, known as mitotic catastrophe (MC), which significantly contributes to the progression of diabetic nephropathy (DN). Furthermore, P62-mediated autophagic flux has been shown to regulate DN-induced podocyte injury. Although previous studies, including ours, have demonstrated that ursolic acid (UA) mitigates podocyte injury by enhancing autophagy under high glucose conditions, the protective functions and potential regulatory mechanisms of UA against DN have not been fully elucidated. For aiming to investigate the regulatory mechanism of podocyte injuries in DN progression, and the protective function of UA treatment against DN progression, we utilized db/db mice and high glucose (HG)-induced podocyte models in vivo and in vitro, with or without UA administration. Our findings indicate that UA treatment reduced DN progression by improving biochemical indices. P62 accumulation led to Murine Double Minute gene 2 (MDM2)-regulated MC in podocytes during DN, which was ameliorated by UA through enhanced P62-mediated autophagy. Additionally, the overexpression of NF-κB p65 or TNF-α abolished the protective effects of UA both in vivo and in vitro. Overall, our results provide strong evidence that UA could be a potential therapeutic agent for DN, regulated by inhibiting podocyte MC through the NF-κB/MDM2/Notch1 pathway by targeting autophagic-P62 accumulation.

PRMT6-mediated ADMA promotes p62 phase separation to form a negative feedback loop in ferroptosis.

Purpose: Due to intrinsic defensive response, ferroptosis-activating targeted therapy fails to achieve satisfactory clinical benefits. Though p62-Keap1-Nrf2 axis is activated to form a negative feedback loop during ferroptosis induction, how p62 is activated remains largely unknown. Methods: MTS assay was applied to measure cell growth. Lipid ROS was detected with C11-BODIPY reagent by flow cytometer. Quantitative real-time PCR (qPCR) and western blotting were performed to determine mRNA and protein level. Immunofluorescence (IF) was performed to examine the distribution of proteins. Fluorescence recovery after photobleaching (FRAP) was adopted to evaluate p62 phase separation. Immunoprecipitation (IP), co-IP and Proximal ligation assay (PLA) were performed to detected protein posttranslational modifications and protein-protein interactions. Tumor xenograft model was employed to inspect in vivo growth of pancreatic cancer cells. Results: Upon ferroptosis induction, Nuclear Factor E2 Related Factor 2 (Nrf2) protein and its downstream genes such as HMOX1 and NQO1 were upregulated. Knockdown of p62 significantly reversed Nrf2 upregulation and Keap1 decrease after ferroptosis induction. Knockdown of either p62 or Nrf2 remarkably sensitized ferroptosis induction. Due to augmented p62 phase separation, formation of p62 bodies were increased to recruit Keap1 after ferroptosis induction. Protein arginine methyltransferase 6 (PRMT6) mediated asymmetric dimethylarginine (ADMA) of p62 to increase its oligomerization, promoting p62 phase separation and p62 body formation. Knockdown of p62 or PRMT6 notably sensitized pancreatic cancer cells to ferroptosis both in vitro and in vivo through suppressing Nrf2 signaling. Conclusion: During ferroptosis induction, PRMT6 mediated p62 ADMA to promote its phase separation, sequestering Keap1 to activate Nrf2 signaling and inhibit ferroptosis. Therefore, targeting PRMT6-mediated p62 ADMA could be a new option to sensitize ferroptosis for cancer treatment.

CPEB1 Controls NRF2 Proteostasis and Ferroptosis Susceptibility in Pancreatic Cancer.

Pancreatic cancer is the deadliest malignancy with a poor response to chemotherapy but is potentially indicated for ferroptosis therapy. Here we identified that cytoplasmic polyadenylation element binding protein 1 (CPEB1) regulates NRF2 proteostasis and susceptibility to ferroptosis in pancreatic ductal adenocarcinoma (PDAC). We found that CPEB1 deficiency in cancer cells promotes the translation of p62/SQSTM1 by facilitating mRNA polyadenylation. Consequently, upregulated p62 enhances NRF2 stability by sequestering KEAP1, an E3 ligase for proteasomal degradation of NRF2, leading to the transcriptional activation of anti-ferroptosis genes. In support of the critical role of this signaling cascade in cancer therapy, CPEB1-deficient pancreatic cancer cells display higher resistance to ferroptosis-inducing agents than their CPEB1-normal counterparts in vitro and in vivo. Furthermore, based on the pathological evaluation of tissue specimens from 90 PDAC patients, we established that CPEB1 is an independent prognosticator whose expression level is closely associated with clinical therapeutic outcomes in PDAC. These findings identify the role of CPEB1 as a key ferroptosis regulator and a potential prognosticator in pancreatic cancer.

Suppressed basal mitophagy drives cellular aging phenotypes that can be reversed by a p62-targeting small molecule.

Selective degradation of damaged mitochondria by autophagy (mitophagy) is proposed to play an important role in cellular homeostasis. However, the molecular mechanisms and the requirement of mitochondrial quality control by mitophagy for cellular physiology are poorly understood. Here, we demonstrated that primary human cells maintain highly active basal mitophagy initiated by mitochondrial superoxide signaling. Mitophagy was found to be mediated by PINK1/Parkin-dependent pathway involving p62 as a selective autophagy receptor (SAR). Importantly, this pathway was suppressed upon the induction of cellular senescence and in naturally aged cells, leading to a robust shutdown of mitophagy. Inhibition of mitophagy in proliferating cells was sufficient to trigger the senescence program, while reactivation of mitophagy was necessary for the anti-senescence effects of NAD precursors or rapamycin. Furthermore, reactivation of mitophagy by a p62-targeting small molecule rescued markers of cellular aging, which establishes mitochondrial quality control as a promising target for anti-aging interventions.

Immunohistochemistry analysis of autophagy-related proteins Beclin-1, p62/SQSTM1, and LC3B in breast carcinoma progression to bone metastasis.

Autophagy is a catabolic pathway involved both in tissue homeostasis and in cellular response to stress. The precise role of autophagy in cancer is still undefined and seems to depend on the tumor stage, appearing tumor-suppressive in physiological conditions and helpful to tumor progression in the established tumor. Here we analyzed by immunohistochemistry Beclin-1, p62, and LC3B, autophagic markers, in human specimens of normal breast, bone metastasis together with pair-matched invasive breast carcinoma of no special type (IBC-NST) as well as non-metastatic breast carcinoma, to disclose the possibility that they could be early prognostic indicators of the evolution of the disease toward the worst outcome. Different regions of metastatic carcinomas, i.e., areas adjacent to the tumor without signs of neoplastic growth, dysplastic lesions, and areas with invasive growth were considered. The pattern of autophagic parameters showed differences among the stages of breast carcinoma progression with a trend that indicated the activation of autophagic process in normal breast (Beclin-1 more elevated than p62), a pattern that was maintained in non-metastatic carcinoma. As the neoplasia proceeds with malignancy, the modification of the pattern of expression of autophagic markers (low ratio between Beclin-1 and p62) in areas of invasive growth of carcinomas suggested inhibition of the process. Of note, the parameters showed a different pattern in bone metastasis with respect to bone metastatic (bm)-IBC-NST, suggesting the reactivation of the autophagic process in the new growth site, helpful to the colonization. The course of autophagy markers during tumor progression could have a prognostic value towards bone metastasis and reveal different roles of the process in different phases of neoplastic growth. The understanding of the role of autophagy in bone metastasis could disclose new therapeutic targets to improve the conditions of patients.

CRHR1 antagonist alleviated depression-like behavior by downregulating p62 in a rat model of post-stroke depression.

Post-stroke depression (PSD) is a complication of cerebrovascular disease, which can increase mortality after stroke. CRH is one of the main signaling peptides released after activation of the hypothalamic-pituitary-adrenal (HPA) axis in response to stress. It affects synaptic plasticity by regulating inflammation, oxidative stress and autophagy in the central nervous system. And the loss of spines exacerbates depression-like behavior. Therefore, synaptic deficits induced by CRH may be related to post-stroke depression. However, the underlying mechanism remains unclear. The Keap1-Nrf2 complex is one of the core components of the antioxidant response. As an autophagy associated protein, p62 participates in the Keap1-NrF2 pathway through its Keap1 interaction domain. Oxidative stress is involved in the feedback regulation between Keap1-Nrf2 pathway and p62.However, whether the relationship between CRH and the Keap1-Nrf2-p62 pathway is involved in PSD remains unknown. This study found that serum levels of CRH in 22 patients with PSD were higher than those in healthy subjects. We used MCAO combined with CUMS single-cage SD rats to establish an animal model of PSD. Animal experiments showed that CRHR1 antagonist prevented synaptic loss in the hippocampus of PSD rats and alleviated depression-like behavior. CRH induced p62 accumulation in the prefrontal cortex of PSD rats through CRHR1. CRHR1 antagonist inhibited Keap1-Nrf2-p62 pathway by attenuating oxidative stress. In addition, we found that abnormal accumulation of p62 induces PSD. It alleviates depression-like behavior by inhibiting the expression of p62 and promoting the clearance of p62 in PSD rats. These findings can help explore the pathogenesis of PSD and design targeted treatments for PSD.

Selective autophagy receptor p62 promotes antibacterial and antiviral immunity in common carp (Cyprinus carpio).

Sequestosome 1 (SQSTM1/p62) is a selective autophagy adapter protein that participates in antiviral and bacterial immune responses and plays an important regulatory role in clearing the proteins to be degraded and maintaining intracellular protein homeostasis. In this study, two p62 genes were cloned from common carp (Cyprinus carpio), namely Ccp62-1 and Ccp62-2, and conducted bioinformatics analysis on them. The results showed that Ccp62s had the same structural domain (Phox and Bem1 domain, ZZ-type zinc finger domain, and ubiquitin-associated domain) as p62 from other species. Ccp62s were widely expressed in various tissues of fish, and highly expressed in immune organs such as gills, spleen, head kidney, etc. Subcellular localization study showed that they were mainly distributed in punctate aggregates in the cytoplasm. After stimulation with Aeromonas hydrophila and spring viraemia of carp virus (SVCV), the expression level of Ccp62s was generally up-regulated. Overexpression of Ccp62s in EPC cells could inhibit SVCV replication. Upon A. hydrophila challenge, the bacterial load in Ccp62s-overexpressing group was significantly reduced, the expression levels of pro-inflammatory cytokines and interferon factors were increased, and the survival rate of the fish was improved. These results indicated that Ccp62s were involved in the immune response of common carp to bacterial and viral infections.

Activation of nicotinic acetylcholine receptor α7 subunit limits Zika viral infection via promoting autophagy and ferroptosis.

Vagus nerve regulates viral infection and inflammation via the alpha 7 nicotinic acetylcholine receptor (α7 nAChR); however, the role of α7 nAChR in ZIKA virus (ZIKV) infection, which can cause severe neurological diseases such as microcephaly and Guillain-Barré syndrome, remains unknown. Here, we first examined the role of α7 nAChR in ZIKV infection in vitro. A broad effect of α7 nAChR activation was identified in limiting ZIKV infection in multiple cell lines. Combined with transcriptomics analysis, we further demonstrated that α7 nAChR activation promoted autophagy and ferroptosis pathways to limit cellular ZIKV viral loads. Additionally, activation of α7 nAChR prevented ZIKV-induced p62 nucleus accumulation, which mediated an enhanced autophagy pathway. By regulating proteasome complex and an E3 ligase NEDD4, activation of α7 nAChR resulted in increased amount of cellular p62, which further enhanced the ferroptosis pathway to reduce ZIKV infection. Moreover, utilizing in vivo neonatal mouse models, we showed that α7 nAChR is essential in controlling the disease severity of ZIKV infection. Taken together, our findings identify an α7 nAChR-mediated effect that critically contributes to limiting ZIKV infection, and α7 nAChR activation offers a novel strategy for combating ZIKV infection and its complications.

Changes in cytokine and sequestosome-1 levels during twin pregnancy progression: Association with outcome.

BACKGROUND: Twin pregnancies are associated with complications and adverse outcomes. The number of twin pregnancies has increased in the last decades, due to the use of assisted reproductive techniques and delayed childbearing. Analysis of changes that occur during twin pregnancy progression and their association with outcome will lead to improved clinical interventions. OBJECTIVE: We evaluated if the plasma concentration of select cytokines and the level of sequestosome-1 (p62) in peripheral blood mononuclear cells (PBMCs) during each trimester of twin gestations was predictive of pregnancy outcome. STUDY DESIGN: This prospective, observational study was conducted at Careggi University Hospital, Florence, Italy. Plasma from 82 women with twin pregnancies was collected in each trimester for measurement of interleukin (IL)-1ß, IL-6, IL-10, IL-12 and tumor necrosis factor (TNF)-α. The intracellular PBMC concentration of p62, a protein involved in autophagy, kinase activity and cell differentiation, was also determined. RESULTS: IL-1ß (p < 0.001), IL-6 (p < 0.001), TNF-α (p < 0.001) and p62 (p < 0.05) increased from the 1st to the 2nd to the 3rd trimester. The TNF-α level was correlated with the IL-1ß concentration in the 1st and 3rd trimesters p < 0.01) and with the IL-6 concentration in each of the three trimesters (p < 0.01). The intracellular p62 level in PBMCs was negatively correlated with the concentration of IL-1ß in the 2nd trimester (p < 0.05) and negatively correlated with the IL-6 level in the 3rd trimester (p < 0.05). The TNF-α level was significantly higher in the 2nd (p < 0.05) and 3rd (p < 0.001) trimester in women with a spontaneous preterm delivery. The TNF-α concentrations in the 2nd (p < 0.05) and 3rd (p < 0.01) trimester, respectively, and 3rd trimester IL-6 (p < 0.01), were negatively associated with gestational age at delivery. The concentration of IL-6 was highest in the 2nd (p < 0.05) and 3rd (p < 0.05) trimesters in women who utilized assisted reproductive technologies. An elevated IL-1ß level in the 3rd trimester was associated with gestational diabetes mellitus (p < 0.05). CONCLUSION: Variations in cytokine levels between individual women during the three trimesters of twin gestations are predictive of spontaneous preterm delivery and the onset of gestational diabetes.

Urolithin A promotes p62-dependent lysophagy to prevent acute retinal neurodegeneration.

BACKGROUND: Age-related macular degeneration (AMD) is the leading cause of blindness in elderly people in the developed world, and the number of people affected is expected to almost double by 2040. The retina presents one of the highest metabolic demands in our bodies that is partially or fully fulfilled by mitochondria in the neuroretina and retinal pigment epithelium (RPE), respectively. Together with its post-mitotic status and constant photooxidative damage from incoming light, the retina requires a tightly-regulated housekeeping system that involves autophagy. The natural polyphenol Urolithin A (UA) has shown neuroprotective benefits in several models of aging and age-associated disorders, mostly attributed to its ability to induce mitophagy and mitochondrial biogenesis. Sodium iodate (SI) administration recapitulates the late stages of AMD, including geographic atrophy and photoreceptor cell death. METHODS: A combination of in vitro, ex vivo and in vivo models were used to test the neuroprotective potential of UA in the SI model. Functional assays (OCT, ERGs), cellular analysis (flow cytometry, qPCR) and fine confocal microscopy (immunohistochemistry, tandem selective autophagy reporters) helped address this question. RESULTS: UA alleviated neurodegeneration and preserved visual function in SI-treated mice. Simultaneously, we observed severe proteostasis defects upon SI damage induction, including autophagosome accumulation, that were resolved in animals that received UA. Treatment with UA restored autophagic flux and triggered PINK1/Parkin-dependent mitophagy, as previously reported in the literature. Autophagy blockage caused by SI was caused by severe lysosomal membrane permeabilization. While UA did not induce lysosomal biogenesis, it did restore upcycling of permeabilized lysosomes through lysophagy. Knockdown of the lysophagy adaptor SQSTM1/p62 abrogated viability rescue by UA in SI-treated cells, exacerbated lysosomal defects and inhibited lysophagy. CONCLUSIONS: Collectively, these data highlight a novel putative application of UA in the treatment of AMD whereby it bypasses lysosomal defects by promoting p62-dependent lysophagy to sustain proteostasis.

Development and application of a colloidal-gold immunochromatographic strip for detecting Getah virus antibodies.

Getah virus (GETV) is a re-emerging mosquito-borne alphavirus that is highly pathogenic, mainly to pigs and horses. There are no vaccines or treatments available for GETV in swine in China. Therefore, the development of a simple, rapid, specific, and sensitive serological assay for GETV antibodies is essential for the prevention and control of GETV. Current antibody monitoring methods are time-consuming, expensive, and dependent on specialized instrumentation, and these features are not conducive to rapid detection in clinical samples. To address these problem, we developed immunochromatographic test strips (ICTS) using eukaryotically expressed soluble recombinant p62-E1 protein of GETV as a labelled antigen, which has good detection sensitivity and no cross-reactivity with other common porcine virus-positive sera. The ICTS is highly compatible with IFA and ELISA and can be stored for 1 month at 37 °C and for at least 3 months at room temperature. Hence, p62-E1-based ICTS is a rapid, accurate, and convenient method for rapid on-site detection of GETV antibodies. KEY POINTS: • We established a rapid antibody detection method that can monitor GETV infection • We developed colloidal gold test strips with high sensitivity and specificity • The development of colloidal gold test strips will aid in the field serologic detection of GETV.

Immunohistochemical expression in idiopathic inflammatory myopathies at a single center in Vietnam.

Background: The identification of idiopathic inflammatory myopathies (IIMs) requires a comprehensive analysis involving clinical manifestations and histological findings. This study aims to provide insights into the histopathological and immunohistochemical aspects of IIMs. Methods: This retrospective case series involved 56 patients diagnosed with IIMs at the Department of Pathology, University of Medicine and Pharmacy at Ho Chi Minh City, from 2019 to 2023. The histology and immunohistochemical expression of HLA-ABC, HLA-DR, C5b-9, Mx1/2/3, and p62 were detected. Results: We examined six categories of inflammatory myopathy, including immunemediated necrotizing myopathy (58.9%), dermatomyositis (DM; 23.2%), overlap myositis (8.9%), antisynthetase syndrome (5.4%), inclusion body myositis (IBM; 1.8%), and polymyositis (1.8%). The average age of the patients was 49.7 ± 16.1 years, with a female-to-male ratio of 3:1. Inflammatory cell infiltration in the endomysium was present in 62.5% of cases, perifascicular atrophy was found in 17.8%, and fiber necrosis was observed in 42 cases (75.0%). Rimmed vacuoles were present in 100% of cases in the IBM group. Immunohistochemistry showed the following positivity rates: HLA-ABC (89.2%), HLA-DR (19.6%), C5b-9 (57.1%), and Mx1/2/3 (10.7%). Mx1/2/3 expression was high in DM cases. p62 vacuole deposits were noted in the IBM case. The combination of membrane attack complex and major histocompatibility complex I helped detect IIMs in 96% of cases. Conclusions: The diagnosis of IIMs and their subtypes should be based on clinical features and histopathological characteristics. Immunohistochemistry plays a crucial role in the diagnosis and differentiation of these subgroups.

Curcumin inhibits PAT-induced renal ferroptosis via the p62/Keap1/Nrf2 signalling pathway.

Patulin (PAT), the most common mycotoxin, is widespread in foods and beverages which poses a serious food safety issue to human health. Our previous research confirmed that exposure to PAT can lead to acute kidney injury (AKI). Curcumin is the most abundant active ingredient in turmeric rhizome with various biological activities. The aim of this study is to investigate whether curcumin can prevent the renal injury caused by PAT, and to explore potential mechanisms. In vivo, supplementation with curcumin attenuated PAT-induced ferroptosis. Mechanically, curcumin inhibited autophagy, led to the accumulation of p62 and its interaction with Keap1, promoted the nuclear translocation of nuclear factor E2 related factor 2 (Nrf2), and increased the expression of antioxidant stress factors in the process of ferroptosis. These results have also been confirmed in HKC cell experiments. Furthermore, knockdown of Nrf2 in HKC cells abrogated the protective effect of curcumin on ferroptosis. In conclusion, we confirmed that curcumin mitigated PAT-induced AKI by inhibiting ferroptosis via activation of the p62/Keap1/Nrf2 pathway. This study provides new potential targets and ideas for the prevention and treatment of PAT.

Benzo[a]pyrene evokes epithelial-mesenchymal transition and pulmonary fibrosis through AhR-mediated Nrf2-p62 signaling.

Benzo[a]pyrene (BaP) and its metabolic end product benzo(a)pyren-7,8-dihydrodiol-9,10-epoxide (BPDE), are known toxic environmental pollutants. This study aimed to analyze whether sub-chronic BPDE exposure initiated pulmonary fibrosis and the potential mechanisms. In this work, male C57BL6/J mice were exposed to BPDE by dynamic inhalation exposure for 8 weeks. Our results indicated that sub-chronic BPDE exposure evoked pulmonary fibrosis and epithelial-mesenchymal transition (EMT) in mice. Both in vivo and in vitro, BPDE exposure promoted nuclear translocation of Snail. Further experiments indicated that nuclear factor erythroid 2-related factor 2 (Nrf2) and p62 were upregulated in BPDE-exposed alveolar epithelial cells. Moreover, Nrf2 siRNA transfection evidently attenuated BPDE-induced p62 upregulation. Besides, p62 shRNA inhibited BPDE-incurred Snail nuclear translocation and EMT. Mechanically, BPDE facilitated physical interaction between p62 and Snail in the nucleus, then repressed Snail protein degradation by p62-dependent autophagy-lysosome pathway, and finally upregulated transcriptional activity of Snail. Additionally, aryl hydrocarbon receptor (AhR) was activated in BPDE-treated alveolar epithelial cells. Dual-luciferase assay indicated activating AhR could bind to Nrf2 gene promoter. Moreover, pretreatment with CH223191 or α-naphthoflavone (α-NF), AhR antagonists, inhibited BPDE-activated Nrf2-p62 signaling, and alleviated BPDE-induced EMT and pulmonary fibrosis in mice. Taken together, AhR-mediated Nrf2-p62 signaling contributes to BaP-induced EMT and pulmonary fibrosis.

Apelin-13 improves pulmonary epithelial barrier function in a mouse model of LPS-induced acute lung injury by inhibiting Chk1-mediated DNA damage.

Apelin-13, a type of active peptide, can alleviate lipopolysaccharide (LPS)-induced acute lung injury (ALI). However, the specific mechanism is unclear. Cell cycle checkpoint kinase 1 (Chk1) plays an important role in DNA damage. Here, we investigated the regulatory effect of Apelin on Chk1 in ALI. Chk1-knockout and -overexpression mice were used to explore the role of Chk1 in LPS-induced ALI mice treated with or without Apelin-13. In addition, A549 cells were also treated with LPS to establish a cell model. Chk1 knockdown inhibited the destruction of alveolar structure, the damage of lung epithelial barrier function, and DNA damage in the ALI mouse model. Conversely, Chk1 overexpression had the opposite effect. Furthermore, Apelin-13 reduced Chk1 expression and DNA damage to improve the impaired lung epithelial barrier function in the ALI model. However, the high expression of Chk1 attenuated the protective effect of Apelin-13 on ALI. Notably, Apelin-13 promoted Chk1 degradation through autophagy to regulate DNA damage in LPS-treated A549 cells. In summary, Apelin-13 regulates the expression of Chk1 by promoting autophagy, thereby inhibiting epithelial DNA damage and repairing epithelial barrier function.

Boswellic acid and apigenin alleviate methotrexate-provoked renal and hippocampal alterations in rats: Targeting autophagy, NOD-2/NF-κB/NLRP3, and connexin-43.

The neuronal and renal deteriorations observed in patients exposed to methotrexate (MTX) therapy highlight the need for medical interventions to counteract these complications. Boswellic acid (BA) and apigenin (APG) are natural phytochemicals with prominent neuronal and renal protective impacts in various ailments. However, their impacts on MTX-provoked renal and hippocampal toxicity have not been reported. Thus, the present work is tailored to clarify the ability of BA and APG to counteract MTX-provoked hippocampal and renal toxicity. BA (250 mg/kg) or APG (20 mg/kg) were administered orally in rats once a day for 10 days, while MTX (20 mg/kg, i.p.) was administered once on the sixth day of the study. At the histopathological level, BA and APG attenuated MTX-provoked renal and hippocampal aberrations. They also inhibited astrocyte activation, as proven by the inhibition of glial fibrillary acidic protein (GFAP). These impacts were partially mediated via the activation of autophagy flux, as proven by the increased expression of beclin1, LC3-II, and the curbing of p62 protein, alongside the regulation of the p-AMPK/mTOR nexus. In addition, BA and APG displayed anti-inflammatory features as verified by the damping of NOD-2 and p-NF-κB p65 to reduce TNF-α, IL-6, and NLRP3/IL-1ß cue. These promising effects were accompanied with a notable reduction in one of the gap junction proteins, connexin-43 (Conx-43). These positive impacts endorse BA and APG as adjuvant modulators to control MTX-driven hippocampal and nephrotoxicity.

TBK1 is ubiquitinated by TRIM5α to assemble mitophagy machinery.

Ubiquitination of mitochondrial proteins provides a basis for the downstream recruitment of mitophagy machinery, yet whether ubiquitination of the machinery itself contributes to mitophagy is unknown. Here, we show that K63-linked polyubiquitination of the key mitophagy regulator TBK1 is essential for its mitophagy functions. This modification is catalyzed by the ubiquitin ligase TRIM5α and is required for TBK1 to interact with and activate a set of ubiquitin-binding autophagy adaptors including NDP52, p62/SQSTM1, and NBR1. Autophagy adaptors, along with TRIM27, enable TRIM5α to engage with TBK1 following mitochondrial damage. TRIM5α's ubiquitin ligase activity is required for the accumulation of active TBK1 on damaged mitochondria in Parkin-dependent and Parkin-independent mitophagy pathways. Our data support a model in which TRIM5α provides a mitochondria-localized, ubiquitin-based, self-amplifying assembly platform for TBK1 and mitophagy adaptors that is ultimately necessary for the recruitment of the core autophagy machinery.

Preparation of polyclonal antibodies to chicken P62 protein and its application in nephropathogenic infectious bronchitis virus-infected chickens.

p62, also known as SQSTM1, has been shown to be closely related to the coronavirus. However, it remains unclear on the relationship between p62 and NIBV infection. Moreover, there are no available antibodies against the chicken p62 protein. Thus, this study aimed to prepare p62 polyclonal antibody and investigate the correlation between the p62 protein and NIBV infection. Here, PET-32a-p62 prokaryotic fusion expression vector was constructed for prokaryotic protein expression, and then p62 polyclonal antibody was prepared by immunizing rabbits. Lastly, these antibodies were then utilized in Western blotting (WB), immunohistochemistry (IHC), and immunofluorescence (IF) assays. The results showed that we successfully prepared chicken p62 polyclonal antibody. Meanwhile, WB and IF demonstrated that the expression of p62 showed a trend of first increase and then decrease after NIBV infection. IHC showed that the expression of p62 in the spleen, lung, kidney, bursa of Fabricius and trachea of chickens infected with NIBV in 11 dpi was significantly higher than that of normal chickens. Taken together, this study successfully prepared a polyclonal antibody for chicken p62 protein and confirmed its application and expression in chickens, as well as the expression of p62 in tissues after NIBV infection.

Pentoxifylline and Norcantharidin Modify p62 Expression in 2D and 3D Cultures of B16F1 Cells.

Three-dimensional cell cultures have improved the evaluation of drugs for cancer therapy, due to their high similarity to solid tumors. In melanoma, autophagy appears to show a dual role depending on the progression of the disease. p62 protein has been proposed for the evaluation of autophagic flux since its expression is an indicator of the state of autophagy. Pentoxifylline (PTX) and Norcantharidin (NCTD) are drugs that have been shown to possess anticancer effects. In this work, we used B16F1 mouse melanoma cells in two-dimensional (2D) monolayer cultures and three-dimensional (3D) spheroids to test the effect of PTX and NCTD over the p62 expression. We analyzed the effect on p62 expression through Western blot and immunofluorescence assays. Our results indicate that PTX decreases p62 expression in both cell culture models, while Norcantharidin increases its expression in 3D cultures at 24 h. Therefore, these drugs could have a potential therapeutic use for the regulation of autophagy in melanoma, depending on the state of evolution of the disease.

Alginate Oligosaccharides Protect Gastric Epithelial Cells against Oxidative Stress Damage through Induction of the Nrf2 Pathway.

Gastric diseases represent a significant global public health challenge, characterized by molecular dysregulation in redox homeostasis and heightened oxidative stress. Although prior preclinical studies have demonstrated the cytoprotective antioxidant effects of alginate oligosaccharides (AOSs) through the Nrf2 pathway, whether such mechanisms apply to gastric diseases remains unclear. In this study, we used the GES-1 gastric cell line exposed to hydrogen peroxide (H2O2) as a damage model to investigate the impact of AOS on cell viability and its associated mechanisms. Our results revealed that pre-incubation with AOS for either 4 h or 24 h significantly improved the viability of GES-1 cells exposed to H2O2. In addition, AOS reduced the intracellular ROS levels, activating the Nrf2 signaling pathway, with increased Nrf2 protein and mRNA expression and a significant upregulation of the target genes HO-1 and NQO1. The activation of Nrf2 was correlated with decreased Keap1 protein expression and an increased level of the autophagy protein p62/SQSTM1, suggesting the activation of Nrf2 through a noncanonical pathway. This study suggests that AOS is a potential treatment for protecting gastric epithelial cells from oxidative stress by activating the p62/SQSTM1-Keap1-Nrf2 axis and laying the foundation for future investigations about its specific therapeutic mechanisms.