Antioxidant and Anti-Inflammatory Properties of Hydrolyzed Royal Jelly Peptide in Human Dermal Fibroblasts: Implications for Skin Health and Care Applications.

Hydrolyzed royal jelly peptide (RJP) has garnered attention for its health-promoting functions. However, the potential applications of RJP in skincare have not been fully explored. In this study, we prepared RJP through the enzymatic hydrolysis of royal jelly protein with trypsin and investigated its antioxidant and anti-inflammatory properties on primary human dermal fibroblasts (HDFs). Our results demonstrate that RJP effectively inhibits oxidative damage induced by H2O2 and lipid peroxidation triggered by AAPH and t-BuOOH in HDFs. This effect may be attributed to the ability of RJP to enhance the level of glutathione and the activities of catalase and glutathione peroxidase 4, as well as its excellent iron chelating capacity. Furthermore, RJP modulates the NLRP3 inflammasome-mediated inflammatory response in HDFs, suppressing the mRNA expressions of NLRP3 and IL-1ß in the primer stage induced by LPS and the release of mature IL-1ß induced by ATP, monosodium urate, or nigericin in the activation stage. RJP also represses the expressions of COX2 and iNOS induced by LPS. Finally, we reveal that RJP exhibits superior antioxidant and anti-inflammatory properties over unhydrolyzed royal jelly protein. These findings suggest that RJP exerts protective effects on skin cells through antioxidative and anti-inflammatory mechanisms, indicating its promise for potential therapeutic avenues for managing oxidative stress and inflammation-related skin disorders.

Regulation of hepatocyte phospholipid peroxidation signaling by a Chinese patent medicine against psychological stress-induced liver injury.

BACKGROUND: Psychological stress is associated with various diseases including liver dysfunction, yet effective intervention strategies remain lacking due to the unrevealed pathogenesis mechanism. PURPOSE: This study aims to explore the relevance between BMAL1-controlled circadian rhythms and lipoxygenase 15 (ALOX15)-mediated phospholipids peroxidation in psychological stress-induced liver injury, and to investigate whether hepatocyte phospholipid peroxidation signaling is involved in the hepatoprotective effects of a Chinese patent medicine, Pien Tze Huang (PZH). METHODS: Restraint stress models were established to investigate the underlying molecular mechanisms of psychological stress-induced liver injury and the hepatoprotective effects of PZH. Redox lipidomics based on liquid chromatography-tandem mass spectrometry was applied for lipid profiling. RESULTS: The present study discovered that acute restraint stress could induce liver injury. Notably, lipidomic analysis confirmed that phospholipid peroxidation was accumulated in the livers of stressed mice. Additionally, the essential core circadian clock gene Brain and Muscle Arnt-like Protein-1 (Bmal1) was altered in stressed mice. Circadian disruption in mice, as well as BMAL1-overexpression in human HepaRG cells, also appeared to have a significant increase in phospholipid peroxidation, suggesting that stress-induced liver injury is closely related to circadian rhythm and phospholipid peroxidation. Subsequently, arachidonate 15-lipoxygenase (ALOX15), a critical enzyme that contributed to phospholipid peroxidation, was screened as a potential regulatory target of BMAL1. Mechanistically, BMAL1 promoted ALOX15 expression via direct binding to an E-box-like motif in the promoter. Finally, this study revealed that PZH treatment significantly relieved pathological symptoms of psychological stress-induced liver injury with a potential mechanism of alleviating ALOX15-mediated phospholipid peroxidation. CONCLUSION: Our findings illustrate the critical role of BMAL1-triggered phospholipid peroxidation in psychological stress-induced liver injury and provide new insight into treating psychological stress-associated liver diseases by TCM intervention.

Membrane phospholipid peroxidation promotes loss of dopaminergic neurons in psychological stress-induced Parkinson's disease susceptibility.

Parkinson's disease (PD) is a neurodegenerative disorder associated with α-synuclein aggregation and dopaminergic neuron loss in the midbrain. There is evidence that psychological stress promotes PD progression by enhancing glucocorticoids-related oxidative damage, however, the mechanisms involved are unknown. The present study demonstrated that plasma membrane phospholipid peroxides, as determined by phospholipidomics, triggered ferroptosis in dopaminergic neurons, which in turn contributed to stress exacerbated PD-like motor disorder in mice overexpressing mutant human α-synuclein. Using hormonomics, we identified that stress stimulated corticosteroid release and promoted 15-lipoxygenase-1 (ALOX15)-mediated phospholipid peroxidation. ALOX15 was upregulated by α-synuclein overexpression and acted as a fundamental risk factor in the development of chronic stress-induced parkinsonism and neurodegeneration. Further, we demonstrated the mechanism by which corticosteroids activated the PKC pathway and induced phosphatidylethanolamine-binding protein-1 (PEBP1) to form a complex with ALOX15, thereby facilitating ALOX15 to locate on the plasma membrane phospholipids. A natural product isolated from herbs, leonurine, was screened with activities of inhibiting the ALOX15/PEBP1 interaction and thereby attenuating membrane phospholipid peroxidation. Collectively, our findings demonstrate that stress increases the susceptibility of PD by driving membrane lipid peroxidation of dopaminergic neurons and suggest the ALOX15/PEBP1 complex as a potential intervention target.

Midbrain dopamine oxidation links ubiquitination of glutathione peroxidase 4 to ferroptosis of dopaminergic neurons.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the gradual loss of midbrain dopaminergic neurons in association with aggregation of α-synuclein. Oxidative damage has been widely implicated in this disease, though the mechanisms involved remain elusive. Here, we demonstrated that preferential accumulation of peroxidized phospholipids and loss of the antioxidant enzyme glutathione peroxidase 4 (GPX4) were responsible for vulnerability of midbrain dopaminergic neurons and progressive motor dysfunctions in a mouse model of PD. We also established a mechanism wherein iron-induced dopamine oxidation modified GPX4, thereby rendering it amenable to degradation via the ubiquitin-proteasome pathway. In conclusion, this study unraveled what we believe to be a novel pathway for dopaminergic neuron degeneration during PD pathogenesis, driven by dopamine-induced loss of antioxidant GPX4 activity.

GPX4 deficiency-dependent phospholipid peroxidation drives motor deficits of ALS.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by oxidative stress that triggers motor neurons loss in the brain and spinal cord. However, the mechanisms underlying the exact role of oxidative stress in ALS-associated neural degeneration are not definitively established. Oxidative stress-generated phospholipid peroxides are known to have extensive physiological and pathological consequences to tissues. Here, we discovered that the deficiency of glutathione peroxidase 4 (GPX4), an essential antioxidant peroxidase, led to the accumulation of phospholipid peroxides and resulted in a loss of motor neurons in spinal cords of ALS mice. Mutant human SOD1G93A transgenic mice were intrathecally injected with neuron-targeted adeno-associated virus (AAV) expressing GPX4 (GPX4-AAV) or phospholipid peroxidation inhibitor, ferrostatin-1. The results showed that impaired motor performance and neural loss induced by SOD1G93A toxicity in the lumbar spine were substantially alleviated by ferrostatin-1 treatment and AAV-mediated GPX4 delivery. In addition, the denervation of neuron-muscle junction and spinal atrophy in ALS mice were rescued by neural GPX4 overexpression, suggesting that GPX4 is essential for the motor neural maintenance and function. In comparison, conditional knockdown of Gpx4 in the spinal cords of Gpx4fl/fl mice triggered an obvious increase of phospholipid peroxides and the occurrence of ALS-like motor phenotype. Altogether, our findings underscore the importance of GPX4 in maintaining phospholipid redox homeostasis in the spinal cord and presents GPX4 as an attractive therapeutic target for ALS treatment.

Phospholipid peroxidation inhibits autophagy via stimulating the delipidation of oxidized LC3-PE.

Phospholipid peroxidation of polyunsaturated fatty acids at the bis-allylic position drives ferroptosis. Here we identify a novel role for phospholipid peroxidation in the inhibition of autophagy. Using in vitro and in vivo models, we report that phospholipid peroxidation induced by glutathione peroxidase-4 inhibition and arachidonate 15-lipoxygenase overexpression leads to overload of peroxidized phospholipids and culminate in inhibition of autophagy. Functional and lipidomics analysis further demonstrated that inhibition of autophagy was associated with an increase of peroxidized phosphatidylethanolamine (PE) conjugated LC3. We further demonstrate that autophagy inhibition occurred due to preferential cleavage of peroxidized LC3-PE by ATG4B to yield delipidated LC3. Mouse models of phospholipid peroxidation and autophagy additionally supported a role for peroxidized PE in autophagy inhibition. Our results agree with the recognized role of endoplasmic reticulum as the primary source for autophagosomal membranes. In summary, our studies demonstrated that phospholipid peroxidation inhibited autophagy via stimulating the ATG4B-mediated delipidation of peroxidized LC3-PE.

Phospholipase iPLA2ß averts ferroptosis by eliminating a redox lipid death signal.

Ferroptosis, triggered by discoordination of iron, thiols and lipids, leads to the accumulation of 15-hydroperoxy (Hp)-arachidonoyl-phosphatidylethanolamine (15-HpETE-PE), generated by complexes of 15-lipoxygenase (15-LOX) and a scaffold protein, phosphatidylethanolamine (PE)-binding protein (PEBP)1. As the Ca2+-independent phospholipase A2ß (iPLA2ß, PLA2G6 or PNPLA9 gene) can preferentially hydrolyze peroxidized phospholipids, it may eliminate the ferroptotic 15-HpETE-PE death signal. Here, we demonstrate that by hydrolyzing 15-HpETE-PE, iPLA2ß averts ferroptosis, whereas its genetic or pharmacological inactivation sensitizes cells to ferroptosis. Given that PLA2G6 mutations relate to neurodegeneration, we examined fibroblasts from a patient with a Parkinson's disease (PD)-associated mutation (fPDR747W) and found selectively decreased 15-HpETE-PE-hydrolyzing activity, 15-HpETE-PE accumulation and elevated sensitivity to ferroptosis. CRISPR-Cas9-engineered Pnpla9R748W/R748W mice exhibited progressive parkinsonian motor deficits and 15-HpETE-PE accumulation. Elevated 15-HpETE-PE levels were also detected in midbrains of rotenone-infused parkinsonian rats and α-synuclein-mutant SncaA53T mice, with decreased iPLA2ß expression and a PD-relevant phenotype. Thus, iPLA2ß is a new ferroptosis regulator, and its mutations may be implicated in PD pathogenesis.

Oxygenated phosphatidylethanolamine navigates phagocytosis of ferroptotic cells by interacting with TLR2.

During cancer therapy, phagocytic clearance of dead cells plays a vital role in immune homeostasis. The nonapoptotic form of cell death, ferroptosis, exhibits extraordinary potential in tumor treatment. However, the phagocytosis mechanism that regulates the engulfment of ferroptotic cells remains unclear. Here, we establish a novel pathway for phagocytic clearance of ferroptotic cells that is different from canonical mechanisms by using diverse ferroptosis models evoked by GPX4 dysfunction/deficiency. We identified the oxidized phospholipid, 1-steaoryl-2-15-HpETE-sn-glycero-3-phosphatidylethanolamine (SAPE-OOH), as a key eat-me signal on the ferroptotic cell surface. Enriching the plasma membrane with SAPE-OOH increased the efficiency of phagocytosis of ferroptotic cells by macrophage, a process that was suppressed by lipoprotein-associated phospholipase A2. Ligand fishing, lipid blotting, and cellular thermal shift assay screened and identified TLR2 as a membrane receptor that directly recognized SAPE-OOH, which was further confirmed by TLR2 inhibitors and gene silencing studies. A mouse mammary tumor model of ferroptosis verified SAPE-OOH and TLR2 as critical players in the clearance of ferroptotic cells in vivo. Taken together, this work demonstrates that SAPE-OOH on ferroptotic cell surface acts as an eat-me signal and navigates phagocytosis by targeting TLR2 on macrophages.

Inhibitory effects of baicalein against herpes simplex virus type 1.

Herpes simplex virus type 1 (HSV-1) is a ubiquitous and widespread human pathogen, which gives rise to a range of diseases, including cold sores, corneal blindness, and encephalitis. Currently, the use of nucleoside analogs, such as acyclovir and penciclovir, in treating HSV-1 infection often presents limitation due to their side effects and low efficacy for drug-resistance strains. Therefore, new anti-herpetic drugs and strategies should be urgently developed. Here, we reported that baicalein, a naturally derived compound widely used in Asian countries, strongly inhibited HSV-1 replication in several models. Baicalein was effective against the replication of both HSV-1/F and HSV-1/Blue (an acyclovir-resistant strain) in vitro. In the ocular inoculation mice model, baicalein markedly reduced in vivo HSV-1/F replication, receded inflammatory storm and attenuated histological changes in the cornea. Consistently, baicalein was found to reduce the mortality of mice, viral loads both in nose and trigeminal ganglia in HSV-1 intranasal infection model. Moreover, an ex vivo HSV-1-EGFP infection model established in isolated murine epidermal sheets confirmed that baicalein suppressed HSV-1 replication. Further investigations unraveled that dual mechanisms, inactivating viral particles and inhibiting IκB kinase beta (IKK-ß) phosphorylation, were involved in the anti-HSV-1 effect of baicalein. Collectively, our findings identified baicalein as a promising therapy candidate against the infection of HSV-1, especially acyclovir-resistant strain.

Novel insights into stress-induced susceptibility to influenza: corticosterone impacts interferon-ß responses by Mfn2-mediated ubiquitin degradation of MAVS.

Although stress has been known to increase the susceptibility of pathogen infection, the underlying mechanism remains elusive. In this study, we reported that restraint stress dramatically enhanced the morbidity and mortality of mice infected with the influenza virus (H1N1) and obviously aggravated lung inflammation. Corticosterone (CORT), a main type of glucocorticoids in rodents, was secreted in the plasma of stressed mice. We further found that this stress hormone significantly boosted virus replication by restricting mitochondrial antiviral signaling (MAVS) protein-transduced IFN-ß production without affecting its mRNA level, while the deficiency of MAVS abrogated stress/CORT-induced viral susceptibility in mice. Mechanistically, the effect of CORT was mediated by proteasome-dependent degradation of MAVS, thereby resulting in the impediment of MAVS-transduced IFN-ß generation in vivo and in vitro. Furthermore, RNA-seq assay results indicated the involvement of Mitofusin 2 (Mfn2) in this process. Gain- and loss-of-function experiments indicated that Mfn2 interacted with MAVS and recruited E3 ligase SYVN1 to promote the polyubiquitination of MAVS. Co-immunoprecipitation experiments clarified an interaction between any two regions of Mfn2 (HR1), MAVS (C-terminal/TM) and SYVN1 (TM). Collectively, our findings define the Mfn2-SYVN1 axis as a new signaling cascade for proteasome-dependent degradation of MAVS and a 'fine tuning' of antiviral innate immunity in response to influenza infection under stress.

(+)-Clausenamide protects against drug-induced liver injury by inhibiting hepatocyte ferroptosis.

Drug-induced liver injury is the major cause of acute liver failure. However, the underlying mechanisms seem to be multifaceted and remain poorly understood, resulting in few effective therapies. Here, we report a novel mechanism that contributes to acetaminophen-induced hepatotoxicity through the induction of ferroptosis, a distinctive form of programmed cell death. We subsequently identified therapies protective against acetaminophen-induced liver damage and found that (+)-clausenamide ((+)-CLA), an active alkaloid isolated from the leaves of Clausena lansium (Lour.) Skeels, inhibited acetaminophen-induced hepatocyte ferroptosis both in vivo and in vitro. Consistently, (+)-CLA significantly alleviated acetaminophen-induced or erastin-induced hepatic pathological damages, hepatic dysfunctions and excessive production of lipid peroxidation both in cultured hepatic cell lines and mouse liver. Furthermore, treatment with (+)-CLA reduced the mRNA level of prostaglandin endoperoxide synthase 2 while it increased the protein level of glutathione peroxidase 4 in hepatocytes and mouse liver, confirming that the inhibition of ferroptosis contributes to the protective effect of (+)-CLA on drug-induced liver damage. We further revealed that (+)-CLA specifically reacted with the Cys-151 residue of Keap1, which blocked Nrf2 ubiquitylation and resulted in an increased Nrf2 stability, thereby leading to the activation of the Keap1-Nrf2 pathway to prevent drug-induced hepatocyte ferroptosis. Our studies illustrate the innovative mechanisms of acetaminophen-induced liver damage and present a novel intervention strategy to treat drug overdose by using (+)-CLA.

Autophagic degradation of PML promotes susceptibility to HSV-1 by stress-induced corticosterone.

Rationale: Herpes simplex virus type 1 (HSV-1) is a neurotropic virus that can cause a variety of clinical syndromes including mucocutaneous disease and HSV-1 encephalitis (HSE). Here, we characterize the molecular mechanisms underlying the susceptibility to HSV-1 under stressful conditions. Methods: Restraint stress and corticosterone (CORT, a primary stress hormone) were respectively used to establish HSV-1 susceptible model in vivo and in vitro. Viral titers were determined by plaque assay. Western blotting, immunofluorescence, transmission electron microscopy (TEM), qRT-PCR, H&E staining, IHC staining and flow cytometry were employed to evaluate virus-related protein expressions and detect the activation of autophagy. Loss- and gain-function assays, co-immunoprecipitation (co-IP) technique and autophagy agonist/antagonist treatments were applied in mechanistic experiments. Results: Restraint stress increased the susceptibility of mouse brain to HSV-1. Similarly, CORT treatment enhanced the susceptibility of neural cells to HSV-1. Furthermore, PML protein level in HSV-1 infected brain tissues and neural cells was remarkably decreased by stress treatment in vivo or CORT treatment in vitro, while its transcriptional level was not affected. Notably, a striking decline in protein expressions of ICP27 and gB was observed in PML-overexpressing cells, which was reversed by CORT treatment. By contrast, protein expression of gB was increased by knockdown with si-PML in virus-infected SH-SY5Y cells. We further discovered that CORT-driven PML degradation was dependent on the activation of autophagy in a ULK1-independent manner, rather than proteasome pathway. Bafilomycin A1 (BaF1) attenuated the augmentation effect of CORT on HSV-1 infection. The expressions of viral proteins were reduced in LC3-depleted cells, and the degradation of PML by CORT-induced autophagy was prevented in cells with LC3 knockdown by RNAi. Interestingly, PML was revealed to interact with the autophagic cargo receptor P62 and the autophagic effector protein LC3. Additionally, CORT failed to increase gB protein level when PML was silenced, providing direct evidence linking autophagic degradation of PML and CORT-induced virus susceptibility. Conclusion: Our results revealed that restraint stress/CORT increased HSV-1 susceptibility by delivering PML into autolysosomes for degradation. The results obtained from in vitro and in vivo models not only demonstrated the adverse effects of stress on HSV-1 infection, but also systematically investigated the underlying molecular mechanisms. These discoveries broaden our understanding of the interplay between host and viruses, and a comprehensive understanding of the role of autophagy in viral infection will provide information for future development of innovative drugs against viral infection.

Identification and characterization of N9-methyltransferase involved in converting caffeine into non-stimulatory theacrine in tea.

Caffeine is a major component of xanthine alkaloids and commonly consumed in many popular beverages. Due to its occasional side effects, reduction of caffeine in a natural way is of great importance and economic significance. Recent studies reveal that caffeine can be converted into non-stimulatory theacrine in the rare tea plant Camellia assamica var. kucha (Kucha), which involves oxidation at the C8 and methylation at the N9 positions of caffeine. However, the underlying molecular mechanism remains unclear. Here, we identify the theacrine synthase CkTcS from Kucha, which possesses novel N9-methyltransferase activity using 1,3,7-trimethyluric acid but not caffeine as a substrate, confirming that C8 oxidation takes place prior to N9-methylation. The crystal structure of the CkTcS complex reveals the key residues that are required for the N9-methylation, providing insights into how caffeine N-methyltransferases in tea plants have evolved to catalyze regioselective N-methylation through fine tuning of their active sites. These results may guide the future development of decaffeinated drinks.