Proteasomal and Autophagic Degradation Systems.

Autophagy and the ubiquitin-proteasome system are the two major quality control pathways responsible for cellular homeostasis. As such, they provide protection against age-associated changes and a plethora of human diseases. Ubiquitination is utilized as a degradation signal by both systems, albeit in different ways, to mark cargoes for proteasomal and lysosomal degradation. Both systems intersect and communicate at multiple points to coordinate their actions in proteostasis and organelle homeostasis. This review summarizes molecular details of how proteasome and autophagy pathways are functionally interconnected in cells and indicates common principles and nodes of communication that can be therapeutically exploited.

Activation of the p62-Keap1-Nrf2 pathway improves pulmonary arterial hypertension in MCT-induced rats by inhibiting autophagy.

Autophagy is implicated in the pathogenesis of pulmonary arterial hypertension (PAH). We aimed to investigate whether the p62-Keap1-Nrf2 pathway affects the development of PAH by mediating autophagy. A PAH rat model was established using monocrotaline (MCT). Pulmonary artery smooth muscle cells (PASMCs) were extracted, and the changes in proliferation, migration, autophagy, and oxidative stress were analyzed following overexpression or knockdown of p62. The impact of p62 on the symptoms of PAH rats was assessed by the injection of an adenovirus overexpressing p62. We found that the knockdown of p62 increased the proliferation and migration of PASMCs, elevating the oxidative stress of PASMCs and upregulating gene expression of NADPH oxidases. Co-IP assay results demonstrated that p62 interacted with Keap1. p62 knockdown enhanced Keap1 protein stability and Nrf2 ubiquitination. LC3II/I and ATG5 were expressed more often when p62 was knocked down. Treating with an inhibitor of autophagy reversed the impact of p62 knockdown on PASMCs. Nrf2 inhibitor treatment reduced the expression of Nrf2 and p62, while increasing the expression of Keap1, LC3II/I, and ATG5 in PASMCs. However, overexpressing p62 diminished mRVP, SPAP, and Fulton index in PAH rats and attenuated pulmonary vascular wall thickening. Overexpression of p62 also decreased the expression of Keap1, LC3II/I, and ATG5 and increased the nuclear expression of Nrf2 in PAH rats. Importantly, overexpression of p62 reduced oxidative stress and the NADPH oxidase expression in PAH rats. Overall, activation of the p62-Keap1-Nrf2 positive feedback signaling axis reduces the proliferation and migration of PASMCs and alleviates PAH by inhibiting autophagy and oxidative stress.

Harnessing the Noncanonical Keap1-Nrf2 Pathway for Human Cytomegalovirus Control.

Host-derived cellular pathways can provide an unfavorable environment for virus replication. These pathways have been a subject of interest for herpesviruses, including the betaherpesvirus human cytomegalovirus (HCMV). Here, we demonstrate that a compound, ARP101, induces the noncanonical sequestosome 1 (SQSTM1)/p62-Keap1-Nrf2 pathway for HCMV suppression. ARP101 increased the levels of both LC3 II and SQSTM1/p62 and induced phosphorylation of p62 at the C-terminal domain, resulting in its increased affinity for Keap1. ARP101 treatment resulted in Nrf2 stabilization and translocation into the nucleus, binding to specific promoter sites and transcription of antioxidant enzymes under the antioxidant response element (ARE), and HCMV suppression. Knockdown of Nrf2 recovered HCMV replication following ARP101 treatment, indicating the role of the Keap1-Nrf2 axis in HCMV inhibition by ARP101. SQSTM1/p62 phosphorylation was not modulated by the mTOR kinase or casein kinase 1 or 2, indicating ARP101 engages other kinases. Together, the data uncover a novel antiviral strategy for SQSTM1/p62 through the noncanonical Keap1-Nrf2 axis. This pathway could be further exploited, including the identification of the responsible kinases, to define the biological events during HCMV replication. IMPORTANCE Antiviral treatment for human cytomegalovirus (HCMV) is limited and suffers from the selection of drug-resistant viruses. Several cellular pathways have been shown to modulate HCMV replication. The autophagy receptor sequestosome 1 (SQSTM1)/p62 has been reported to interact with several HCMV proteins, particularly with components of HCMV capsid, suggesting it plays a role in viral replication. Here, we report on a new and unexpected role for SQSTM1/p62, in HCMV suppression. Using a small-molecule probe, ARP101, we show SQSTM1/p62 phosphorylation at its C terminus domain initiates the noncanonical Keap1-Nrf2 axis, leading to transcription of genes under the antioxidant response element, resulting in HCMV inhibition in vitro. Our study highlights the dynamic nature of SQSTM1/p62 during HCMV infection and how its phosphorylation activates a new pathway that can be exploited for antiviral intervention.

Momordica charantia L.-derived exosome-like nanovesicles stabilize p62 expression to ameliorate doxorubicin cardiotoxicity.

BACKGROUND: Doxorubicin (DOX) is a first-line chemotherapeutic drug for various malignancies that causes cardiotoxicity. Plant-derived exosome-like nanovesicles (P-ELNs) are growing as novel therapeutic agents. Here, we investigated the protective effects in DOX cardiotoxicity of ELNs from Momordica charantia L. (MC-ELNs), a medicinal plant with antioxidant activity. RESULTS: We isolated MC-ELNs using ultracentrifugation and characterized them with canonical mammalian extracellular vesicles features. In vivo studies proved that MC-ELNs ameliorated DOX cardiotoxicity with enhanced cardiac function and myocardial structure. In vitro assays revealed that MC-ELNs promoted cell survival, diminished reactive oxygen species, and protected mitochondrial integrity in DOX-treated H9c2 cells. We found that DOX treatment decreased the protein level of p62 through ubiquitin-dependent degradation pathway in H9c2 and NRVM cells. However, MC-ELNs suppressed DOX-induced p62 ubiquitination degradation, and the recovered p62 bound with Keap1 promoting Nrf2 nuclear translocation and the expressions of downstream gene HO-1. Furthermore, both the knockdown of Nrf2 and the inhibition of p62-Keap1 interaction abrogated the cardioprotective effect of MC-ELNs. CONCLUSIONS: Our findings demonstrated the therapeutic beneficials of MC-ELNs via increasing p62 protein stability, shedding light on preventive approaches for DOX cardiotoxicity.

Neuroprotective properties of the Lilium brownii extracts in the experimental model of Parkinson's disease.

Lilium brownii (L. brownii) is a plant that can be used for both medicine and food. Its bulbs are commonly used to treat neurological disorders like depression, insomnia, and Parkinson's disease (PD). However, the mechanism by which it treats PD is not yet fully understood. This study aims to investigate the possible mechanism of L. brownii extract in treating PD and to compare the efficacy of ethanol and aqueous extracts of L. brownii. In this study, mice with PD induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride (MPTP) were given L. brownii extracts for 30 days, and the effects of both extracts were then evaluated. Our study demonstrated that both extracts of L. brownii effectively improved motor dysfunction in PD mice induced by MPTP. Additionally, they increased the number of neurons in the substantia nigra region of the mice. Moreover, both extracts reduced levels of malondialdehyde (MDA) and ferrous ion (Fe2+), while increasing levels of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in serum. They also influenced the expression of proteins associated with the p62-Keap1-Nrf2 pathway. Interestingly, while both extracts had similar behavioral effects, the ethanol extract appeared to have a more significant impact on individual proteins in the p62-Keap1-Nrf2 pathway compared to the aqueous extract, possibly due to its higher phenolic acid glyceride content. In conclusion, L. brownii shows promise as an effective and safe treatment for PD.

Activation of the p62-Keap1-Nrf2 pathway protects against oxidative stress and excessive autophagy in ovarian granulosa cells to attenuate DEHP-induced ovarian impairment in mice.

Di-(2-ethylhexyl) phthalate (DEHP) is widely used in various plastics but has been demonstrated to cause female reproductive toxicity. However, the exact mechanism underlying the ovarian damage induced by DEHP remains unclear. In this study, DEHP was administered orally to 5-week-old female mice for 30 days at doses of 0, 250, 500, and 1000 mg/kg/day. The findings demonstrated that DEHP exposure disrupted ovarian function and follicular development as well as induced oxidative stress and autophagy in ovarian granulosa cells (GCs). Further, 200 µM mono-(2-ethylhexyl) phthalate (MEHP), the primary metabolite of DEHP in vivo, induced autophagy in both human ovarian granulosa cells line (KGN) and mouse primary GCs within 24 h in vitro. However, it did not affect the p62-dependent autophagy flux. Furthermore, MEHP-induced autophagy was inhibited by the autophagy inhibitor 3-MA and exacerbated by the autophagy activator rapamycin, indicating that MEHP induces excessive autophagy in GCs. Subsequently, we found that MEHP-induced autophagic cell death was primarily attributed to oxidative damage from elevated intracellular ROS levels. Meanwhile, MEHP exposure induced nuclear translocation of erythroid-derived factor 2-related factor (Nrf2), a key regulator of antioxidant activity resulting in activating antioxidant effects. Interestingly, we also found that MEHP-induced increase in p62 competitively binds Keap1, thereby facilitating nuclear translocation of Nrf2 and establishing a positive feedback loop in antioxidant regulation. Therefore, this study demonstrated that inhibition of Nrf2 could aggravate oxidative damage and enhance excessive autophagy caused by MEHP, while activation of Nrf2 could reverse the trend. These findings have also been reinforced in studies of cultured ovaries in vitro. Our study suggests that the p62-Keap1-Nrf2 pathway may serve as a potential protective mechanism against DEHP-induced oxidative stress and excessive autophagy in mouse GCs.

NEDD4L inhibits migration, invasion, cisplatin resistance and promotes apoptosis of bladder cancer cells by inactivating the p62/Keap1/Nrf2 pathway.

PURPOSE: This study identified the function of neural precursor cell expressed developmentally down-regulated 4-like (NEDD4L) on bladder cancer (BLCA). METHODS: NEDD4L expression in BLCA patients was scrutinized. The function of NEDD4L on the viability, apoptosis, migration and invasion of BLCA cells was evaluated by cell counting kit-8, flow cytometry and Transwell assays. The effect of NEDD4L on the cisplatin (DDP) resistance of the DDP-resistant BLCA cells was explored. The influence of NEDD4L on the p62/Keap1/Nrf2 pathway activity in BLCA cells was tested by Western blot. Rescue experiments were implemented to verify whether NEDD4L regulated BLCA cell malignant behavior by mediating the Keap1/Nrf2 pathway activity via p62. The effect of NEDD4L on the growth and the p62/Keap1/Nrf2 pathway activity in vivo was researched in xenograft tumor nude mice models. RESULTS: The down-regulated NEDD4L in BLCA patients was associated with unfavorable survival. NEDD4L suppressed the viability (inhibition rate 57.1%/49.0%), migration (inhibition rate 49.7%/77.1%), invasion (inhibition rate 50.6%/75.7%), promoted the apoptosis of T24/5637 cells (promotion rate 243.8%/201.9%), reduced IC 50 of DDP-resistant T24/5637 cells from 132.2/101.8 to 57.81/59.71 µM, respectively, and inactivated the p62/Keap1/Nrf2 pathway in T24/5637 cells. p62 up-regulation partially abrogated the inhibition of NEDD4L on the Keap1/Nrf2 pathway activity, the malignant behavior of BLCA cells, and the DDP resistance of DDP-resistant BLCA cells. NEDD4L overexpression inhibited the tumor growth and the p62/Keap1/Nrf2 pathway activity in vivo in BLCA. CONCLUSION: NEDD4L inhibits the progression of BLCA by inactivating the p62/Keap1/Nrf2 pathway. It may be an effective target for BLCA treatment.

ATP2B3 Inhibition Alleviates Erastin-Induced Ferroptosis in HT-22 Cells through the P62-KEAP1-NRF2-HO-1 Pathway.

Ferroptosis participates in the occurrence and development of neurological disorders. Modulating ferroptosis may have therapeutic potential in nervous system diseases. Therefore, TMTbased proteomic analysis in HT-22 cells was performed to identify erastin-induced differentially expressed proteins. The calcium-transporting ATP2B3 (ATP2B3) was screened as a target protein. ATP2B3 knockdown markedly alleviated the erastin-induced decrease in cell viability and elevated ROS (p < 0.01) and reversed the up-regulation of oxidative stress-related proteins polyubiquitin-binding protein p62 (P62), nuclear factor erythroid 2-related factor2 (NRF2), heme oxygenase-1 (HO-1), and NAD(P)H quinone oxidoreductase-1 (NQO1) protein expression (p < 0.05 or p < 0.01) and the down-regulation of Kelch-like ECH-associated protein 1(KEAP1) protein expression (p < 0.01). Moreover, NRF2 knockdown, P62 inhibition, or KEAP1 overexpression rescued the erastin-induced decrease in cell viability (p < 0.05) and increase in ROS production (p < 0.01) in HT-22 cells, while simultaneous overexpression of NRF2 and P62 and knockdown of KEAP1 partially offset the relief effect of ATP2B3 inhibition. In addition, knockdown of ATP2B3, NRF2, and P62 and overexpression of KEAP1 significantly down-regulated erastin-induced high expression of the HO-1 protein, while HO-1 overexpression reversed the alleviating effects of ATP2B3 inhibition on the erastin-induced decrease in cell viability (p < 0.01) and increase in ROS production (p < 0.01) in HT-22 cells. Taken together, ATP2B3 inhibition mediates the alleviation of erastin-induced ferroptosis in HT-22 cells through the P62-KEAP1-NRF2-HO-1 pathway.

Suppression of JNK/ERK dependent autophagy enhances Jaspine B derivative-induced gastric cancer cell death via attenuation of p62/Keap1/Nrf2 pathways.

Gastric cancer is one of the most common cancers with few effective treatments, a new treatment agent is desperately needed. C-2, a Jaspine B derivative, has shown anti-cancer efficacy in gastric cancer cells. The anti-cancer mechanism, however, remains unknown. As a result, we investigate the anti-cancer effect and the underlying mechanism of C-2 in gastric cancer cells. The results showed that C-2 selectively reduced the proliferation of gastric cancer cells when compared to normal epithelial gastric cells. Western blotting and flow cytometry further demonstrated that Caspase9 is involved in causing cell death. Meanwhile, C-2 triggered autophagy in gastric cancer cells, inhibition of which with LY294002 can enhance the anti-proliferative activity of C-2. Next, we found that C-2 triggered autophagy through activating JNK/ERK, and that inhibitors of these proteins exacerbated C-2 induced cell death. Mechanically, enhanced phosphorylation of JNK/ERK elevated Beclin-1 by disturbing Beclin-1/Bcl-xL or Beclin-1/Bcl-2 complexes, resulting in autophagy and up-regulation of p62. Finally, p62 binds Keap1 competitively to release Nrf2, boosting Nrf2 translocation from the cytoplasm to the nucleus and triggering expression of Nrf2 target genes, so enhancing survival. C-2 inhibited the growth of gastric cancer cells, while JNK/ERK dependent autophagy antagonized C-2 induced cell growth inhibition through p62/Keap1/Nrf2 pathway.

PM2.5 exposure triggers cell death through lysosomal membrane permeabilization and leads to ferroptosis insensitivity via the autophagy dysfunction/p62-KEAP1-NRF2 activation in neuronal cells.

PM2.5 exposure can be associated with the onset of neurodegenerative diseases, with oxidative stress-induced cellular homeostasis disruption and cell death as one of the main mechanisms. However, the exact cellular and molecular processes are still rarely investigated. Autophagy and KEAP1-NRF2 (Kelch-like ECH-Associating protein 1-nuclear factor erythroid 2 related factor 2) signaling pathway are two main cellular defense systems for maintaining cellular homeostasis and resisting oxidative stress. In this study, we primarily investigated the role of autophagy and KEAP1-NRF2 in regulating cell death resulting from PM2.5 exposure in mouse neuroblastoma N2a cells. Our results showed that PM2.5 exposure disrupted autophagic flux by impairing lysosomal function, including lysosomal alkalinization, increased lysosome membrane permeabilization (LMP), and Cathepsin B release. Furthermore, dysregulated autophagy enhances NRF2 activity in a p62-dependent manner, which then initiates the expression of a series of antioxidant genes and increases cellular insensitivity to ferroptosis. Meanwhile, autophagy dysfunction impairs the intracellular degradation of ferroptosis related proteins such as GPX4 and ferritin. As these proteins accumulate, cells also become less sensitive to ferroptosis. LMP-associated cell death may be the main mechanism of PM2.5-induced N2a cytotoxicity. Our results may provide insights into the mechanisms of PM2.5-induced neurotoxicity and predict effective prevention and treatment strategies.

Urolithin A alleviates acute kidney injury induced by renal ischemia reperfusion through the p62-Keap1-Nrf2 signaling pathway.

Acute kidney injury (AKI) induced by renal ischemia reperfusion (RIR) is typically observed in renal surgeries and is a leading cause of renal failure. However, there is still an unmet medical need currently in terms of clinical treatments. Herein, we report the effect of Urolithin A (UA) in a mouse RIR model, wherein we demonstrated its underlying mechanism both in vitro and in vivo. The expression levels of p62 and Keap1 significantly decreased, while that of nuclear Nrf2 increased in vitro in a hypoxia cell model after UA treatment. Furthermore, the apoptosis of tubular cells was attenuated and the reactive oxygen species (ROS) levels were reduced in the kidneys in a mouse RIR model after UA administration. In this study, we demonstrated that UA can alleviate oxidative stress and promote autophagy by activating the p62-Keap1-Nrf2 signaling pathway, which could protect the kidneys from ischemia reperfusion injury.

Penfluridol regulates p62 / Keap1 / Nrf2 signaling pathway to induce ferroptosis in osteosarcoma cells.

The cure rate for patients with osteosarcoma (OS) has stagnated over the past few decades. Penfluridol, a first-generation antipsychotic, has demonstrated to prevent lung and esophageal malignancies from proliferation and metastasis. However, the effect of penfluridol on OS and its underlying molecular mechanism remains unclear. This study revealed that penfluridol effectively inhibited cell proliferation and migration, and induced G2/M phase arrest in OS cells. In addition, penfluridol treatment was found to increased reactive oxygen species (ROS) levels in OS cells. Combined with the RNA-Seq results, the anti-OS effect of penfluridol was hypothesized to be attributed to the induction of ferroptosis. Western blot results showed that penfluridol promoted intracellular Fe2+ concentration, membrane lipid peroxidation, and decreased intracellular GSH level to induce ferroptosis. Further studies showed that p62/Keap1/Nrf2 signaling pathway was implicated in penfluridol-induced ferroptosis in OS cells. Overexpression of p62 effectively reversed penfluridol-induced ferroptosis. In vivo, penfluridol effectively inhibited proliferation and prolonged survival in xenograft tumor model. Therefore, penfluridol is a promising drug targeting OS in the future.

Ovatodiolide induces autophagy-mediated cell death through the p62-Keap1-Nrf2 signaling pathway in chronic myeloid leukemia cells.

Ovatodiolide is a macrocyclic diterpenoid compound with various biological activities that displays considerable anticancer potential in different tumor models. However, the underlying mechanism for this antineoplastic activity remains unclear. The aim of the present study was to investigate the anticancer effect and possible molecular mechanism of ovatodiolide in human chronic myeloid leukemia (CML). Ovatodiolide suppressed cell colony formation and induced apoptosis in the K562 and KU812 cells. We also observed that ovatodiolide enhanced the production of reactive oxygen species (ROS), activated Nrf2 signaling, and inhibited mTOR phosphorylation. Autophagic flux was shown to be enhanced after treatment with ovatodiolide in K562 cells. Furthermore, autophagy inhibition alleviated ovatodiolide-induced cell apoptosis, whereas autophagy promotion aggravated apoptosis in CML cells. These results demonstrated that ovatodiolide activates autophagy-mediated cell death in CML cells. Additionally, ovatodiolide transcriptionally activated the expression of p62, and the p62 levels were negatively regulated by autophagy. Moreover, p62-Keap1-Nrf2 signaling was confirmed to be involved in ovatodiolide-induced cell death. Accordingly, LC3B knockdown augmented the ovatodiolide-induced p62 expression, increased the p62-Keap1 interaction, and enhanced the translocation of Nrf2 into the nucleus. In contrast, p62 inhibition abolished the effects that were induced through ovatodiolide treatment. Nrf2 inhibition with ML385 diminished the protective effect of autophagy inhibition in CML cells. Collectively, our results indicate that ovatodiolide induces oxidative stress and provokes autophagy, which effectively decreases the expression of p62 and weakens the protective effect of Nrf2 signaling activation, thus contributing to apoptosis in CML cells.

Electroacupuncture improves cognitive function in APP/PS1 mice by inhibiting oxidative stress related hippocampal neuronal ferroptosis.

BACKGROUND: Electroacupuncture, recognized as a crucial non-pharmacological therapeutic approach, has demonstrated notable efficacy in enhancing cognitive function among Alzheimer's disease (AD) patients. This study aimed to investigate the neuroprotective properties of electroacupuncture in APP/PS1 mice with AD. METHODS: A total of thirty APP/PS1 mice were randomly assigned to three groups: the Alzheimer's disease group (AD), the electroacupuncture treatment group (EA), and the ferroptosis inhibitor deferasirox treatment group (DFX). Additionally, ten C57BL/6 mice were included as a control group (Control). In the EA group, mice underwent flat needling at Baihui and Yintang, as well as point needling at Renzhong, once daily for 15 min each time. In the DFX group, mice received intraperitoneal injections of deferasirox at a dosage of 100 mg/kg/day. Following the 28-day treatment period, behavioral evaluation, morphological observation of neurons, and detection of neuronal ferroptosis were conducted. RESULTS: The electroacupuncture treatment demonstrated a significant improvement in spatial learning, memory ability, and neuronal damage in mice with AD. Analysis of neuronal ferroptosis markers indicated that electroacupuncture interventions reduced the elevated levels of malondialdehyde, iron, and ptgs2 expression, while also increasing superoxide dismutase activity, Ferroportin 1 and glutathione peroxidase 4 expression. Moreover, the regulatory impact of electroacupuncture on ferroptosis may be attributed to its ability to enhance the expression and nuclear translocation of Nrf2. CONCLUSIONS: This study suggested that electroacupuncture could inhibit the neuronal ferroptosis by activating the antioxidant function in neurons through p62/Keap1/Nrf2 signal pathway, thereby improve the cognitive function of AD mice by the neuronal protection effect.

Necroptosis inhibits autophagy by regulating the formation of RIP3/p62/Keap1 complex in shikonin-induced ROS dependent cell death of human bladder cancer.

BACKGROUND: Shikonin, a natural naphthoquinone compound, has a wide range of pharmacological effects, but its anti-tumor effect and underlying mechanisms in bladder cancer remain unclear. PURPOSE: We aimed to investigate the role of shikonin in bladder cancer in vitro and in vivo in order to broaden the scope of shikonin's clinical application. STUDY DESIGN AND METHODS: We performed MTT and colony formation to detect the inhibiting effect of shikonin on bladder cancer cells. ROS staining and flow cytometry assays were performed to detect the accumulation of ROS. Western blotting, siRNA and immunoprecipitation were used to evaluate the effect of necroptosis in bladder cancer cells. Transmission electron microscopy and immunofluorescence were used to examine the effect of autophagy. Nucleoplasmic separation and other pharmacological experimental methods described were used to explore the Nrf2 signal pathway and the crosstalk with necroptosis and autophagy. We established a subcutaneously implanted tumor model and performed immunohistochemistry assays to study the effects and the underlying mechanisms of shikonin on bladder cancer cells in vivo. RESULTS: The results showed that shikonin has a selective inhibitory effect on bladder cancer cells and has no toxicity on normal bladder epithelial cells. Mechanically, shikonin induced necroptosis and impaired autophagic flux via ROS generation. The accumulation of autophagic biomarker p62 elevated p62/Keap1 complex and activated the Nrf2 signaling pathway to fight against ROS. Furthermore, crosstalk between necroptosis and autophagy was present, we found that RIP3 may be involved in autophagosomes and be degraded by autolysosomes. We found for the first time that shikonin-induced activation of RIP3 may disturb the autophagic flux, and inhibiting RIP3 and necroptosis could accelerate the conversion of autophagosome to autolysosome and further activate autophagy. Therefore, on the basis of RIP3/p62/Keap1 complex regulatory system, we further combined shikonin with late autophagy inhibitor(chloroquine) to treat bladder cancer and achieved a better inhibitory effect. CONCLUSION: In conclusion, shikonin could induce necroptosis and impaired autophagic flux through RIP3/p62/Keap1 complex regulatory system, necroptosis could inhibit the process of autophagy via RIP3. Combining shikonin with late autophagy inhibitor could further activate necroptosis via disturbing RIP3 degradation in bladder cancer in vitro and in vivo.

A polysaccharide from Pleurotus citrinopileatus mycelia enhances the immune response in cyclophosphamide-induced immunosuppressed mice via p62/Keap1/Nrf2 signal transduction pathway.

The manuscript aimed to study the immunoregulatory activity and the mechanism of the polysaccharide (CMP) from Pleurotus citrinopileatus mycelia. The mice were divided into normal group, model group, different dosage of CMP (50, 100 and 200 mg/kg, respectively) groups and levamisole hydrochloride treated group. The results showed that, compared with the model group, CMP could significantly improve the auricle swelling rate, half hemolysis value and phagocytic index in mice. The indices of immune organs were raised, and tissue damage of spleen was relieved. Splenic Th1 cells were decreased, while Th2 cells were increased, furthermore the proliferation of splenic lymphocytes and the cytotoxicity of NK cells were increased. The levels of interleukin-12 (IL-12), interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) in spleen were decreased, while interleukin-4 (IL-4) and interleukin-10 (IL-10) were increased. In serum and spleen, the levels of catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) activities were increased, while the level of malondialdehyde (MDA) was decreased. And the levels of Immunoglobulin were also increased. Western blot showed that CMP had immunoregulatory activity by activating Nrf2, Keap1, p62, HO-1, and NQO1 in the p62/Keap1/Nrf2 signaling pathway. The study proved that CMP could be used as a biological Immune regulating agent.

Crosstalk between autophagy and the Keap1-Nrf2-ARE pathway regulates realgar-induced neurotoxicity.

ETHNOPHARMACOLOGICAL RELEVANCE: Realgar, the main component of which is As2S2 or As4S4 (≥90%), is a traditional Chinese natural medicine that has been used to treat carbuncles, furuncles, snake and insect bites, abdominal pain caused by parasitic worms, and epilepsy in China for many years. Because realgar contains arsenic, chronic or excessive use of single-flavor realgar and realgar-containing Chinese patent medicine can lead to drug-induced arsenic poisoning, but the exact mechanism underlying its toxicity to the central nervous system is unclear. AIM OF THE STUDY: The aim of this study was to clarify the mechanism of realgar-induced neurotoxicity and to investigate the effects of realgar on autophagy and the Keap1-Nrf2-ARE pathway. MATERIALS AND METHODS: We used rats treated with the autophagy inhibitor 3-methyladenine (3-MA) or adeno-associated virus (AAV2/9-r-shRNA-Sqstm1, sh-p62) to investigate realgar-induced neurotoxicity and explore the specific relationship between autophagy and the Keap1-Nrf2-ARE pathway (the Nrf2 pathway) in the cerebral cortex. Molecular docking analysis was used to assess the interactions among the Nrf2, p62 and Keap1 proteins. RESULTS: Our results showed that arsenic from realgar accumulated in the brain and blood to cause neuronal and synaptic damage, decrease exploratory behavior and spontaneous movement, and impair memory ability in rats. The mechanism may have involved realgar-mediated autophagy impairment and continuous activation of the Nrf2 pathway via the LC3-p62-Keap1-Nrf2 axis. However, because this activation of the Nrf2 pathway was not sufficient to counteract oxidative damage, apoptosis was aggravated in the cerebral cortex. CONCLUSIONS: This study revealed that autophagy, the Nrf2 pathway, and apoptosis are involved in realgar-induced central nervous system toxicity and identified p62 as the hub of the LC3-p62-Keap1-Nrf2 axis in the regulation of autophagy, the Nrf2 pathway, and apoptosis.

Dysfunction of ZNF554 promotes ROS-induced apoptosis and autophagy in Fetal Growth Restriction via the p62-Keap1-Nrf2 pathway.

Fetal growth restriction (FGR) is one of the most common complications of an abnormal pregnancy. Placental dysplasia has been established as a significant contributing factor to FGR. Zinc finger protein 554 (ZNF554) is a member of the Krüppel-associated box domain zinc finger protein subfamily, primarily expressed in the placenta and essential for maintaining normal pregnancy outcomes. However, its precise role in FGR remains uncertain. In this study, we confirmed that ZNF554 was low expressed in the placenta of the FGR pregnancy. To further elucidate the impact of ZNF554 on trophoblasts, we conducted experiments using siRNA and overexpression plasmids on HTR8/SVneo and JEG3 cells. Our findings revealed that silencing ZNF554 increased apoptosis and inhibited migration and invasion, while overexpression reduced apoptosis and promoted migration and invasion. Notably, ZNF554 knockdown decreased cellular antioxidant capacity and elevated the production of reactive oxygen species (ROS). Conversely, ZNF554 activated the nuclear factor E2-related factor 2 (NRF2) signaling pathway, exerting its antioxidant effects. Additionally, ZNF554 knockdown promoted cellular autophagy by suppressing P62 and enhancing LC3-II/LC3-I expression. Importantly, the antioxidant N-acetylcysteine (NAC) partially mitigated the impact of ZNF554 knockdown on mitochondrial ROS in trophoblast cells and subsequent effects on cellular autophagy and apoptosis. In conclusion, our results suggest that ZNF554 plays a pivotal role in modulating trophoblast cell invasion and may serve as a prognostic marker and potential therapeutic target for FGR.

Suppression of endoplasmic reticulum stress-dependent autophagy enhances cynaropicrin-induced apoptosis via attenuation of the P62/Keap1/Nrf2 pathways in neuroblastoma.

Autophagy has dual roles in cancer, resulting in cellular adaptation to promote either cell survival or cell death. Modulating autophagy can enhance the cytotoxicity of many chemotherapeutic and targeted drugs and is increasingly considered to be a promising cancer treatment approach. Cynaropicrin (CYN) is a natural compound that was isolated from an edible plant (artichoke). Previous studies have shown that CYN exhibits antitumor effects in several cancer cell lines. However, it anticancer effects against neuroblastoma (NB) and the underlying mechanisms have not yet been investigated. More specifically, the regulation of autophagy in NB cells by CYN has never been reported before. In this study, we demonstrated that CYN induced apoptosis and protective autophagy. Further mechanistic studies suggested that ER stress-induced autophagy inhibited apoptosis by activating the p62/Keap1/Nrf2 pathways. Finally, in vivo data showed that CYN inhibited tumour growth in xenografted nude mice. Overall, our findings suggested that CYN may be a promising candidate for the treatment of NB, and the combination of pharmacological inhibitors of autophagy may hold novel therapeutic potential for the treatment of NB. Our paper will contribute to the rational utility and pharmacological studies of CYN in future anticancer research.

Xiaojianzhong decoction attenuates gastric mucosal injury by activating the p62/Keap1/Nrf2 signaling pathway to inhibit ferroptosis.

Gastric mucosal injury is the initial stage of the occurrence and development of gastric diseases. Oxidative stress and ferroptosis caused by the imbalance of redox and iron dynamics in gastric mucosal epithelial cells are present throughout the occurrence and development of gastric mucosal injury. Therefore, the inhibition of oxidative stress and ferroptosis is a potential target for the treatment of the gastric mucosal injury. Xiaojianzhong decoction (XJZ), which consists of six Chinese herbal medicines and extracts, is used for the treatment of diseases related to gastrointestinal mucosal injury; however, its specific mechanism of action has yet to be clarified. In this study, we clarified the protective effect of XJZ on gastric mucosa and revealed its underlying mechanism. We established a gastric mucosal injury model using aspirin and administered XJZ. Furthermore, we systematically evaluated the mucosal injury and examined the expression of genes related to oxidative stress, ferroptosis, and inflammation. The study found that XJZ significantly counteracted aspirin-induced gastric mucosal injury and inhibited oxidative stress and ferroptosis in mice. Upon examining SQSTM1/p62(p62)/Kelch-like ECH-associated protein 1 (Keap1)/Nuclear Factor erythroid 2-Related Factor 2 (Nrf2), a well-known signaling pathway involved in the regulation of oxidative stress and ferroptosis, we found that its activation was significantly inhibited by aspirin treatment and that this signaling pathway was activated after XJZ intervention. Our study suggests that XJZ may inhibit aspirin induced oxidative stress and ferroptosis via the p62/Keap1/Nrf2 signaling pathway, thereby attenuating gastric mucosal injury.