Electropositive Citric Acid-Polyethyleneimine Carbon Dots Carrying the PINK1 Gene Regulate ATP-Related Metabolic Dysfunction in APP/PS1-N2a Cells.

(1) Background: Alzheimer's disease (AD) is characterized by ß-amyloid (Aß) peptide accumulation and mitochondrial dysfunction during the early stage of disease. PINK1 regulates the balance between mitochondrial homeostasis and bioenergy supply and demand via the PINK1/Parkin pathway, Na+/Ca2+ exchange, and other pathways. (2) Methods: In this study, we synthesized positively charged carbon dots (CA-PEI CDs) using citric acid (CA) and polyethyleneimine (PEI) and used them as vectors to express PINK1 genes in the APP/PS1-N2a cell line to determine mitochondrial function, electron transport chain (ETC) activity, and ATP-related metabolomics. (3) Results: Our findings showed that the CA-PEI CDs exhibit the characteristics of photoluminescence, low toxicity, and concentrated DNA. They are ideal biological carriers for gene delivery. PINK1 overexpression significantly increased the mitochondrial membrane potential in APP/PS1-N2a cells and reduced reactive-oxygen-species generation and Aß1-40 and Aß1-42 levels. An increase in the activity of NADH ubiquinone oxidoreductase (complex I, CI) and cytochrome C oxidase (complex IV, CIV) induces the oxidative phosphorylation of mitochondria, increasing ATP generation. (4) Conclusions: These findings indicate that the PINK gene can alleviate AD by increasing bioenergetic metabolism, reducing Aß1-40 and Aß1-42, and increasing ATP production.

VDR restores the expression of PINK1 and BNIP3 in TECs of streptozotocin-induced diabetic mice.

Defective mitophagy in renal tubular epithelial cells is one of the main drivers of renal fibrosis in diabetic kidney disease. Our gene sequencing data showed the expression of PINK1 and BNIP3, two key molecules of mitophagy, was decreased in renal tissues of VDR-knockout mice. Herein, streptozotocin (STZ) was used to induce renal interstitial fibrosis in mice. VDR deficiency exacerbated STZ-induced renal impairment and defective mitophagy. Paricalcitol (pari, a VDR agonist) and the tubular epithelial cell-specific overexpression of VDR restored the expression of PINK1 and BNIP3 in the renal cortex and attenuated STZ-induced kidney fibrosis and mitochondrial dysfunction. In HK-2 cells under high glucose conditions, an increased level of α-SMA, COL1, and FN and a decreased expression of PINK1 and BNIP3 with severe mitochondrial damage were observed, and these alterations could be largely reversed by pari treatment. ChIP-qPCR and luciferase reporter assays showed VDR could positively regulate the transcription of Pink1 and Bnip3 genes. These findings reveal that VDR could restore mitophagy defects and attenuate STZ-induced fibrosis in diabetic mice through regulation of PINK1 and BNIP3.

Acupotomy alleviates knee osteoarthritis in rabbit by regulating chondrocyte mitophagy Pink1-Parkin pathway.

OBJECTIVE: To investigate the effect of acupotomy, on mitophagy and the Pink1-Parkin pathway in chondrocytes from rabbits with knee osteoarthritis (KOA). METHODS: A KOA model was established via the modified Videman method. Rabbits were randomly divided into a control group (CON), KOA group and KOA + acupotomy group (Acu). Rabbits in the acupotomy group were subjected to acupotomy for 4 weeks after model establishment. The behavior of the rabbits before and after intervention was recorded. Cartilage degeneration was evaluated by optical microscopy and fluorescence microscopy. The level of mitophagy was evaluated by transmission electron microscopy, immunofluorescence and enzyme-linked immunosorbent assay (ELISA). The expression of phosphatase and tensin homolog (PTEN)-induced kinase 1 (Pink1)-Parkin mitophagy pathway components was evaluated by immunofluorescence, Western blotting and real-time polymerase chain reaction. RESULTS: In rabbits with KOA, joint pain, mobility disorders and cartilage degeneration were observed, the Mankin score was increased, collagen type Ⅱ (Col-Ⅱ) expression was significantly decreased, mitophagy was inhibited, mitochondrial function was impaired, and factors associated with the Pink1-Parkin pathway were inhibited. Acupotomy regulated the expression of Pink1-Parkin pathway-related proteins, the mitophagy-related protein microtubule-associated protein-1 light chain-3, the translocase of the outer membrane, and the inner mitochondrial membrane 23; increased the colocalization of mitochondria and autophagosomes; promoted the removal of damaged mitochondria; restored mitochondrial adenosine-triphosphate (ATP) production; and alleviated cartilage degeneration in rabbits with KOA. CONCLUSIONS: Acupotomy played a role in alleviating KOA in rabbits by activating mitophagy in chondrocytes via the regulation of proteins that are related to the Pink1-Parkin pathway.

Gaining Insights into Key Structural Hotspots within the Allosteric Binding Pockets of Protein Kinases.

Protein kinases are essential regulators of cell function and represent one of the largest and most diverse protein families. They are particularly influential in signal transduction and coordinating complex processes like the cell cycle. Out of the 518 human protein kinases identified, 478 are part of a single superfamily sharing catalytic domains that are related in sequence. The dysregulation of protein kinases due to certain mutations has been associated with various diseases, including cancer. Although most of the protein kinase inhibitors identified as type I or type II primarily target the ATP-binding pockets of kinases, the structural and sequential resemblances among these pockets pose a significant challenge for selective inhibition. Therefore, targeting allosteric pockets that are beside highly conserved ATP pockets has emerged as a promising strategy to prevail current limitations, such as poor selectivity and drug resistance. In this article, we compared the binding pockets of various protein kinases for which allosteric (type III) inhibitors have already been developed. Additionally, understanding the structure and shape of existing ligands could aid in identifying key interaction sites within the allosteric pockets of kinases. This comprehensive review aims to facilitate the design of more effective and selective allosteric inhibitors.

Mitochondrial transfer mediates endothelial cell engraftment through mitophagy.

Ischaemic diseases such as critical limb ischaemia and myocardial infarction affect millions of people worldwide1. Transplanting endothelial cells (ECs) is a promising therapy in vascular medicine, but engrafting ECs typically necessitates co-transplanting perivascular supporting cells such as mesenchymal stromal cells (MSCs), which makes clinical implementation complicated2,3. The mechanisms that enable MSCs to facilitate EC engraftment remain elusive. Here we show that, under cellular stress, MSCs transfer mitochondria to ECs through tunnelling nanotubes, and that blocking this transfer impairs EC engraftment. We devised a strategy to artificially transplant mitochondria, transiently enhancing EC bioenergetics and enabling them to form functional vessels in ischaemic tissues without the support of MSCs. Notably, exogenous mitochondria did not integrate into the endogenous EC mitochondrial pool, but triggered mitophagy after internalization. Transplanted mitochondria co-localized with autophagosomes, and ablation of the PINK1-Parkin pathway negated the enhanced engraftment ability of ECs. Our findings reveal a mechanism that underlies the effects of mitochondrial transfer between mesenchymal and endothelial cells, and offer potential for a new approach for vascular cell therapy.

Identification of cysteine-rich receptor-like kinase gene family in potato: revealed StCRLK9 in response to heat, salt and drought stresses.

The investigation into cysteine-rich receptor-like kinases (CRLKs) holds pivotal significance as these conserved, upstream signalling molecules intricately regulate fundamental biological processes such as plant growth, development and stress adaptation. This study undertakes a comprehensive characterisation of CRLKs in Solanum tuberosum (potato), a staple food crop of immense economic importance. Employing comparative genomics and evolutionary analyses, we identified 10 distinct CRLK genes in potato. Further categorisation into three major groups based on sequence similarity was performed. Each CRLK member in potato was systematically named according to its chromosomal position. Multiple sequence alignment and phylogenetic analyses unveiled conserved gene structures and motifs within the same groups. The genomic distribution of CRLKs was observed across Chromosomes 2-5, 8 and 12. Gene duplication analysis highlighted a noteworthy trend, with most gene pairs exhibiting a Ka/Ks ratio greater than one, indicating positive selection of StCRLKs in potato. Salt and drought stresses significantly impacted peroxidase and catalase activities in potato seedlings. The presence of diverse cis -regulatory elements, including hormone-responsive elements, underscored their involvement in myriad biotic and abiotic stress responses. Interestingly, interactions between the phytohormone auxin and CRLK proteins unveiled a potential auxin-mediated regulatory mechanism. A holistic approach combining transcriptomics and quantitative PCR validation identified StCRLK9 as a potential candidate involved in plant response to heat, salt and drought stresses. This study lays a robust foundation for future research on the functional roles of the CRLK gene family in potatoes, offering valuable insights into their diverse regulatory mechanisms and potential applications in stress management.

Exploring the neuroprotective role of artesunate in mouse models of anti-NMDAR encephalitis: insights from molecular mechanisms and transmission electron microscopy.

BACKGROUND: The pathway involving PTEN-induced putative kinase 1 (PINK1) and PARKIN plays a crucial role in mitophagy, a process activated by artesunate (ART). We propose that patients with anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis exhibit insufficient mitophagy, and ART enhances mitophagy via the PINK1/PARKIN pathway, thereby providing neuroprotection. METHODS: Adult female mice aged 8-10 weeks were selected to create a passive transfer model of anti-NMDAR encephalitis. We conducted behavioral tests on these mice within a set timeframe. Techniques such as immunohistochemistry, immunofluorescence, and western blotting were employed to assess markers including PINK1, PARKIN, LC3B, p62, caspase3, and cleaved caspase3. The TUNEL assay was utilized to detect neuronal apoptosis, while transmission electron microscopy (TEM) was used to examine mitochondrial autophagosomes. Primary hippocampal neurons were cultured, treated, and then analyzed through immunofluorescence for mtDNA, mtROS, TMRM. RESULTS: In comparison to the control group, mitophagy levels in the experimental group were not significantly altered, yet there was a notable increase in apoptotic neurons. Furthermore, markers indicative of mitochondrial leakage and damage were found to be elevated in the experimental group compared to the control group, but these markers showed improvement following ART treatment. ART was effective in activating the PINK1/PARKIN pathway, enhancing mitophagy, and diminishing neuronal apoptosis. Behavioral assessments revealed that ART ameliorated symptoms in mice with anti-NMDAR encephalitis in the passive transfer model (PTM). The knockdown of PINK1 led to a reduction in mitophagy levels, and subsequent ART intervention did not alleviate symptoms in the anti-NMDAR encephalitis PTM mice, indicating that ART's therapeutic efficacy is mediated through the activation of the PINK1/PARKIN pathway. CONCLUSIONS: At the onset of anti-NMDAR encephalitis, mitochondrial damage is observed; however, this damage is mitigated by the activation of mitophagy via the PINK1/PARKIN pathway. This regulatory feedback mechanism facilitates the removal of damaged mitochondria, prevents neuronal apoptosis, and consequently safeguards neural tissue. ART activates the PINK1/PARKIN pathway to enhance mitophagy, thereby exerting neuroprotective effects and may achieve therapeutic goals in treating anti-NMDAR encephalitis.

Melatonin Ameliorates Post-Stroke Cognitive Impairment in Mice by Inhibiting Excessive Mitophagy.

Post-stroke cognitive impairment (PSCI) remains the most common consequence of ischemic stroke. In this study, we aimed to investigate the role and mechanisms of melatonin (MT) in improving cognitive dysfunction in stroke mice. We used CoCl2-induced hypoxia-injured SH-SY5Y cells as a cellular model of stroke and photothrombotic-induced ischemic stroke mice as an animal model. We found that the stroke-induced upregulation of mitophagy, apoptosis, and neuronal synaptic plasticity was impaired both in vivo and in vitro. The results of the novel object recognition test and Y-maze showed significant cognitive deficits in the stroke mice, and Nissl staining showed a loss of neurons in the stroke mice. In contrast, MT inhibited excessive mitophagy both in vivo and in vitro and decreased the levels of mitophagy proteins PINK1 and Parkin, and immunofluorescence staining showed reduced co-localization of Tom20 and LC3. A significant inhibition of mitophagy levels could be directly observed under transmission electron microscopy. Furthermore, behavioral experiments and Nissl staining showed that MT ameliorated cognitive deficits and reduced neuronal loss in mice following a stroke. Our results demonstrated that MT inhibits excessive mitophagy and improves PSCI. These findings highlight the potential of MT as a preventive drug for PSCI, offering promising therapeutic implications.

Receptor-like cytoplasmic kinases of different subfamilies differentially regulate SOBIR1/BAK1-mediated immune responses in Nicotiana benthamiana.

Cell-surface receptors form the front line of plant immunity. The leucine-rich repeat (LRR)-receptor-like kinases SOBIR1 and BAK1 are required for the functionality of the tomato LRR-receptor-like protein Cf-4, which detects the secreted effector Avr4 of the pathogenic fungus Fulvia fulva. Here, we show that the kinase domains of SOBIR1 and BAK1 directly phosphorylate each other and that residues Thr522 and Tyr469 of the kinase domain of Nicotiana benthamiana SOBIR1 are required for its kinase activity and for interacting with signalling partners, respectively. By knocking out multiple genes belonging to different receptor-like cytoplasmic kinase (RLCK)-VII subfamilies in N. benthamiana:Cf-4, we show that members of RLCK-VII-6, -7, and -8 differentially regulate the Avr4/Cf-4-triggered biphasic burst of reactive oxygen species. In addition, members of RLCK-VII-7 play an essential role in resistance against the oomycete pathogen Phytophthora palmivora. Our study provides molecular evidence for the specific roles of RLCKs downstream of SOBIR1/BAK1-containing immune complexes.

Identification and structural characterization of small molecule inhibitors of PINK1.

Mutations in PINK1 and Parkin cause early-onset Parkinson's Disease (PD). PINK1 is a kinase which functions as a mitochondrial damage sensor and initiates mitochondrial quality control by accumulating on the damaged organelle. There, it phosphorylates ubiquitin, which in turn recruits and activates Parkin, an E3 ubiquitin ligase. Ubiquitylation of mitochondrial proteins leads to the autophagic degradation of the damaged organelle. Pharmacological modulation of PINK1 constitutes an appealing avenue to study its physiological function and develop therapeutics. In this study, we used a thermal shift assay with insect PINK1 to identify small molecules that inhibit ATP hydrolysis and ubiquitin phosphorylation. PRT062607, an SYK inhibitor, is the most potent inhibitor in our screen and inhibits both insect and human PINK1, with an IC50 in the 0.5-3 µM range in HeLa cells and dopaminergic neurons. The crystal structures of insect PINK1 bound to PRT062607 or CYC116 reveal how the compounds interact with the ATP-binding pocket. PRT062607 notably engages with the catalytic aspartate and causes a destabilization of insert-2 at the autophosphorylation dimer interface. While PRT062607 is not selective for PINK1, it provides a scaffold for the development of more selective and potent inhibitors of PINK1 that could be used as chemical probes.

Luteolin-7-O-ß-d-glucuronide Ameliorates Cerebral Ischemic Injury: Involvement of RIP3/MLKL Signaling Pathway.

Luteolin-7-O-ß-d-glucuronide (LGU) is a major active flavonoid glycoside compound that is extracted from Ixeris sonchifolia (Bge.) Hance, and it is a Chinese medicinal herb mainly used for the treatment of coronary heart disease, angina pectoris, cerebral infarction, etc. In the present study, the neuroprotective effect of LGU was investigated in an oxygen glucose deprivation (OGD) model and a middle cerebral artery occlusion (MCAO) rat model. In vitro, LGU was found to effectively improve the OGD-induced decrease in neuronal viability and increase in neuronal death by a 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and a lactate dehydrogenase (LDH) leakage rate assay, respectively. LGU was also found to inhibit OGD-induced intracellular Ca2+ overload, adenosine triphosphate (ATP) depletion, and mitochondrial membrane potential (MMP) decrease. By Western blotting analysis, LGU significantly inhibited the OGD-induced increase in expressions of receptor-interacting serine/threonine-protein kinase 3 (RIP3) and mixed lineage kinase domain-like protein (MLKL). Moreover, molecular docking analysis showed that LGU might bind to RIP3 more stably and firmly than the RIP3 inhibitor GSK872. Immunofluorescence combined with confocal laser analyses disclosed that LGU inhibited the aggregation of MLKL to the nucleus. Our results suggest that LGU ameliorates OGD-induced rat primary cortical neuronal injury via the regulation of the RIP3/MLKL signaling pathway in vitro. In vivo, LGU was proven, for the first time, to protect the cerebral ischemia in a rat middle cerebral artery occlusion (MCAO) model, as shown by improved neurological deficit scores, infarction volume rate, and brain water content rate. The present study provides new insights into the therapeutic potential of LGU in cerebral ischemia.

Effects of carbamazepine on BDNF expression in trigeminal ganglia and serum in rats with trigeminal neuralgia. / å¡é©¬è¥¿å¹³å¯¹ä¸‰å‰ç¥žç»ç—›å¤§é¼ ä¸‰å‰ç¥žç»èŠ‚åŠè¡€æ¸ ä¸­BDNF表达变化的影响.

OBJECTIVES: Trigeminal neuralgia (TN) is a severe chronic neuropathic pain that mainly affects the distribution area of the trigeminal nerve with limited treating efficacy. There are numerous treatments for TN, but currently the main clinical approach is to suppress pain by carbamazepine (CBZ). Brain-derived neurotrophic factor (BDNF) is closely related to chronic pain. This study aims to determine the effects of CBZ treatment on BDNF expression in both the trigeminal ganglion (TG) and serum of TN via a chronic constriction injury of the infraorbital nerve (ION-CCI) rat model. METHODS: The ION-CCI models were established in male Sprague-Dawley rats and were randomly divided into a sham group, a TN group, a TN+low-dose CBZ treatment group (TN+20 mg/kg CBZ group), a TN+medium-dose CBZ treatment group (TN+40 mg/kg CBZ group), and a TN+high-dose CBZ treatment group (TN+80 mg/kg CBZ group). The mechanical pain threshold in each group of rats was measured regularly before and after surgery. The expressions of BDNF and tyrosine kinase receptor B (TrkB) mRNA in TGs of rats in different groups were determined by real-time PCR, and the expression of BDNF protein on neurons in TGs was observed by immunofluorescence. Western Blotting was used to detect the protein expression of BDNF, TrkB, extracellular regulated protein kinases (ERK), and phospho-extracellular regulated protein kinases (p-ERK) in TGs of rats in different groups. The expression of BDNF in the serum of rats in different groups was detected by enzyme-linked immunosorbent assay (ELISA). RESULTS: The results of mechanical pain sensitivity showed that there was no significant difference in the mechanical pain threshold in the right facial sensory area of the experimental rats in each group before surgery (all P>0.05). From the 3rd day after operation, the mechanical pain threshold of rats in the TN group was significantly lower than that in the sham group (all P<0.01), and the mechanical pain threshold of rats in the TN+80 mg/kg CBZ group, the TN+40 mg/kg CBZ group, and the TN+20 CBZ mg/kg group was higher than that in the TN group (all P<0.05). The BDNF and TrkB mRNA and protein expressions in TGs of rats in the TN group were higher than those in the sham group (all P<0.05), and those in the TN+80 mg/kg CBZ group, the TN+40 mg/kg CBZ group, and the TN+20 mg/kg CBZ group were lower than the TN group (all P<0.05). The p-ERK levels in TG of rats in the TN+80 mg/kg CBZ group, the TN+40 mg/kg CBZ group, and the TN+20 mg/kg CBZ group were significantly decreased compared with the TN group (all P<0.05). The BDNF and neuron-specific nuclear protein (NeuN) were mainly co-expressed in neuron of TGs in the TN group and they were significantly higher than those in the sham group (all P<0.05). The co-labeled expressions of BDNF and NeuN in TGs of the TN+ 80 mg/kg CBZ group, the TN+40 mg/kg CBZ group, and the TN+20 mg/kg CBZ group were lower than those in the TN group (all P<0.05). The results of ELISA showed that the level of BDNF in the serum of the TN group was significantly higher than that in the sham group (P<0.05). The levels of BDNF in the TN+80 mg/kg CBZ group, the TN+40 mg/kg CBZ group, and the TN+20 mg/kg CBZ group were lower than those in the TN group (all P<0.05). Spearman correlation analysis showed that the BDNF level in serum was negatively correlated with mechanical pain threshold (r=-0.650, P<0.01). CONCLUSIONS: CBZ treatment can inhibit the expression of BDNF and TrkB in the TGs of TN rats, reduce the level of BDNF in serum of TN rats and the phosphorylation of ERK signaling pathway, so as to inhibit TN. The serum level of BDNF can be considered as an indicator for the diagnosis and prognosis of TN.

Mono-quinoxaline-induced DNA structural alteration leads to ZBP1/RIP3/MLKL-driven necroptosis in cancer cells.

Evading the cellular apoptosis mechanism by modulating multiple pathways poses a sturdy barrier to effective chemotherapy. Cancer cell adeptly resists the apoptosis signaling pathway by regulating anti and pro-apoptotic proteins to escape cell death. Nevertheless, bypassing the apoptotic pathway through necroptosis, an alternative programmed cell death process, maybe a potential therapeutic modality for apoptosis-resistant cells. However, synthetic mono-quinoxaline-based intercalator-induced cellular necroptosis as an anti-cancer perspective remains under-explored. To address this concern, we undertook the design and synthesis of quinoxaline-based small molecules (3a-3l). Our approach involved enhancing the π-surface of the mandatory benzyl moiety to augment its ability to induce DNA structural alteration via intercalation, thereby promoting cytotoxicity across various cancer cell lines (HCT116, HT-29, and HeLa). Notably, the potent compound 3a demonstrated the capacity to induce DNA damage in cancer cells, leading to the induction of ZBP1-mediated necroptosis in the RIP3-expressed cell line (HT-29), where Z-VAD effectively blocked apoptosis-mediated cell death. Interestingly, we observed that 3a induced RIP3-driven necroptosis in combination with DNA hypomethylating agents, even in the RIP3-silenced cell lines (HeLa and HCT116). Overall, our synthesized compound 3a emerged as a promising candidate against various cancers, particularly in apoptosis-compromised cells, through the induction of necroptosis.

Combination of bone marrow mesenchymal stem cells and moxibustion restores cyclophosphamide-induced premature ovarian insufficiency by improving mitochondrial function and regulating mitophagy.

BACKGROUND: Premature ovarian insufficiency (POI) is a major cause of infertility. In this study, we aimed to investigate the effects of the combination of bone marrow mesenchymal stem cells (BMSCs) and moxibustion (BMSCs-MOX) on POI and evaluate the underlying mechanisms. METHODS: A POI rat model was established by injecting different doses of cyclophosphamide (Cy). The modeling of POI and the effects of the treatments were assessed by evaluating estrous cycle, serum hormone levels, ovarian weight, ovarian index, and ovarian histopathological analysis. The effects of moxibustion on BMSCs migration were evaluated by tracking DiR-labeled BMSCs and analyzing the expression of chemokines stromal cell-derived factor 1 (Sdf1) and chemokine receptor type 4 (Cxcr4). Mitochondrial function and mitophagy were assessed by measuring the levels of reactive oxygen species (ROS), mitochondrial membrane potential (MMP), ATP, and the mitophagy markers (Drp1, Pink1, and Parkin). Furthermore, the mitophagy inhibitor Mdivi-1 and the mitophagy activator CCCP were used to confirm the role of mitophagy in Cy-induced ovarian injury and the underlying mechanism of combination therapy. RESULTS: A suitable rat model of POI was established using Cy injection. Compared to moxibustion or BMSCs transplantation alone, BMSCs-MOX showed improved outcomes, such as reduced estrous cycle disorders, improved ovarian weight and index, normalized serum hormone levels, increased ovarian reserve, and reduced follicle atresia. Moxibustion enhanced Sdf1 and Cxcr4 expression, promoting BMSCs migration. BMSCs-MOX reduced ROS levels; upregulated MMP and ATP levels in ovarian granulosa cells (GCs); and downregulated Drp1, Pink1, and Parkin expression in ovarian tissues. Mdivi-1 significantly mitigated mitochondrial dysfunction in ovarian GCs and improved ovarian function. CCCP inhibited the ability of BMSCs-MOX treatment to regulate mitophagy and ameliorate Cy-induced ovarian injury. CONCLUSIONS: Moxibustion enhanced the migration and homing of BMSCs following transplantation and improves their ability to repair ovarian damage. The combination of BMSCs and moxibustion effectively reduced the excessive activation of mitophagy, which helped prevent mitochondrial damage, ultimately improving ovarian function. These findings provide a novel approach for the treatment of pathological ovarian aging and offer new insights into enhancing the efficacy of stem cell therapy for POI patients.

The mitochondria-targeted antioxidant MitoQ ameliorates inorganic arsenic-induced DCs/Th1/Th2/Th17/Treg differentiation partially by activating PINK1-mediated mitophagy in murine liver.

Inorganic arsenic is a well-established environmental toxicant linked to acute liver injury, fibrosis, and cancer. While oxidative stress, pyroptosis, and ferroptosis are known contributors, the role of PTEN-induced kinase 1 (PINK1)-mediated mitophagy in arsenic-induced hepatic immunotoxicity remains underexplored. Our study revealed that acute arsenic exposure prompts differentiation of hepatic dendritic cells (DCs) and T helper (Th) 1, Th2, Th17, and regulatory T (Treg) cells, alongside increased transcription factors and cytokines. Inorganic arsenic triggered liver redox imbalance, leading to elevated alanine transaminase (ALT), hydrogen peroxide (H2O2), malondialdehyde (MDA), and activation of nuclear factor erythroid 2-related factor (Nrf2)/heme oxygenase-1 (HO-1) pathway. PINK1-mediated mitophagy was initiated, and its inhibition exacerbates H2O2 accumulation while promoting DCs/Th1/Th2/Treg differentiation in the liver of arsenic-exposed mice. Mitoquinone (MitoQ) pretreatment relieved arsenic-induced acute liver injury and immune imbalance by activating Nrf2/HO-1 and PINK1-mediated mitophagy. To our knowledge, this is the first report identifying PINK1-mediated mitophagy as a protective factor against inorganic arsenic-induced hepatic DCs/Th1/Th2 differentiation. This study has provided new insights on the immunotoxicity of inorganic arsenic and established a foundation for exploring preventive and therapeutic strategies targeting PINK1-mediated mitophagy in acute liver injury. Consequently, the application of mitochondrial antioxidant MitoQ may offer a promising treatment for the metalloid-induced acute liver injury.

Tobacco toxins trigger bone marrow mesenchymal stem cells aging by inhibiting mitophagy.

Smoking disrupts bone homeostasis and serves as an independent risk factor for the development and progression of osteoporosis. Tobacco toxins inhibit the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs), promote BMSCs aging and exhaustion, but the specific mechanisms are not yet fully understood. Herein, we successfully established a smoking-related osteoporosis (SROP) model in rats and mice through intraperitoneal injection of cigarette smoke extract (CSE), which significantly reduced bone density and induced aging and inhibited osteogenic differentiation of BMSCs both in vivo and in vitro. Bioinformatics analysis and in vitro experiments confirmed that CSE disrupts mitochondrial homeostasis through oxidative stress and inhibition of mitophagy. Furthermore, we discovered that CSE induced BMSCs aging by upregulating phosphorylated AKT, which in turn inhibited the expression of FOXO3a and the Pink1/Parkin pathway, leading to the suppression of mitophagy and the accumulation of damaged mitochondria. MitoQ, a mitochondrial-targeted antioxidant and mitophagy agonist, was effective in reducing CSE-induced mitochondrial oxidative stress, promoting mitophagy, significantly downregulating the expression of aging markers in BMSCs, restoring osteogenic differentiation, and alleviating bone loss and autophagy levels in CSE-exposed mice. In summary, our results suggest that BMSCs aging caused by the inhibition of mitophagy through the AKT/FOXO3a/Pink1/Parkin axis is a key mechanism in smoking-related osteoporosis.

OSW-1 triggers necroptosis in colorectal cancer cells through the RIPK1/RIPK3/MLKL signaling pathway facilitated by the RIPK1-p62/SQSTM1 complex.

BACKGROUND: Necroptosis has emerged as a novel molecular pathway that can be targeted by chemotherapy agents in the treatment of cancer. OSW-1, which is derived from the bulbs of Ornithogalum saundersiae Baker, exerts a wide range of pharmacological effects. AIM: To explore whether OSW-1 can induce necroptosis in colorectal cancer (CRC) cells, thereby expanding its range of clinical applications. METHODS: We performed a sequence of functional experiments, including Cell Counting Kit-8 assays and flow cytometry analysis, to assess the inhibitory effect of OSW-1 on CRC cells. We utilized quantitative proteomics, employing tandem mass tag labeling combined with liquid chromatography-tandem mass spectrometry, to analyze changes in protein expression. Subsequent bioinformatic analysis was conducted to elucidate the biological processes associated with the identified proteins. Transmission electron microscopy (TEM) and immunofluorescence studies were also performed to examine the effects of OSW-1 on necroptosis. Finally, western blotting, siRNA experiments, and immunoprecipitation were employed to evaluate protein interactions within CRC cells. RESULTS: The results revealed that OSW-1 exerted a strong inhibitory effect on CRC cells, and this effect was accompanied by a necroptosis-like morphology that was observable via TEM. OSW-1 was shown to trigger necroptosis via activation of the RIPK1/RIPK3/MLKL pathway. Furthermore, the accumulation of p62/SQSTM1 was shown to mediate OSW-1-induced necroptosis through its interaction with RIPK1. CONCLUSION: We propose that OSW-1 can induce necroptosis through the RIPK1/RIPK3/MLKL signaling pathway, and that this effect is mediated by the RIPK1-p62/SQSTM1 complex, in CRC cells. These results provide a theoretical foundation for the use of OSW-1 in the clinical treatment of CRC.

SIRT3 alleviates painful diabetic neuropathy by mediating the FoxO3a-PINK1-Parkin signaling pathway to activate mitophagy.

INTRODUCTION: Painful diabetic neuropathy (PDN) is a common complication of diabetes. Previous studies have implicated that mitochondrial dysfunction plays a role in the development of PDN, but its pathogenesis and mechanism have not been fully investigated. METHODS: In this study, we used high-fat diet/low-dose streptozotocin-induced rats as a model of type 2 diabetes mellitus. Behavioral testing, whole-cell patch-clamp recordings of dorsal root ganglion (DRG) neurons, and complex sensory nerve conduction velocity studies were used to assess peripheral neuropathy. Mitochondrial membrane potential (MMP), ATP, tissue reactive oxygen species, and transmission electron microscopy were used to evaluate the function and morphology of mitochondria in DRG. Real-time PCR, western blot, and immunofluorescence were performed to investigate the mechanism. RESULTS: We found that damaged mitochondria were accumulated and mitophagy was inhibited in PDN rats. The expression of sirtuin 3 (SIRT3), which is an NAD+-dependent deacetylase in mitochondria, was inhibited. Overexpression of SIRT3 in DRG neurons by intrathecally administered LV-SIRT3 lentivirus ameliorated neurological and mitochondrial dysfunctions. This was evidenced by the reversal of allodynia and nociceptor hyperexcitability, as well as the restoration of MMP and ATP levels. Overexpression of SIRT3 restored the inhibited mitophagy by activating the FoxO3a-PINK1-Parkin signaling pathway. The effects of SIRT3 overexpression, including the reversal of allodynia and nociceptor hyperexcitability, the improvement of impaired mitochondria and mitophagy, and the restoration of PINK1 and Parkin expression, were counteracted when FoxO3a siRNA was intrathecally injected. CONCLUSION: These results showed that SIRT3 overexpression ameliorates PDN via activation of FoxO3a-PINK1-Parkin-mediated mitophagy, suggesting that SIRT3 may become an encouraging therapeutic strategy for PDN.

TROP2 promotes PINK1-mediated mitophagy and apoptosis to accelerate the progression of senile chronic obstructive pulmonary disease by up-regulating DRP1 expression.

Chronic obstructive pulmonary disease (COPD) is a chronic airway inflammatory disease characterised by irreversible airflow limitation. The elderly are a vulnerable population for developing COPD. With the growth of age, physiological degenerative changes occur in the thorax, bronchus, lung and vascular wall, which can lead to age-related physiological attenuation of lung function in the elderly, so the prevalence of COPD increases with age. Its pathogenesis has not yet been truly clarified. Mitophagy plays an important role in maintaining the stability of mitochondrial function and intracellular environment by scavenging damaged mitochondria. Currently, studies have shown that trophoblast antigen 2 (TROP2) expression is up-regulated in airway basal cells of patients with COPD, suggesting that TROP2 is involved in the progression of COPD. However, whether it is involved in disease progression by regulating mitochondrial function remains unclear. In this study, compared with non-smoking non-COPD patients, the expression of TROP2 in lung tissues of smoking non-COPD patients and patients with COPD increased, and TROP2 expression in patients with COPD was higher than that in smoking non-COPD patients. To further explore the role of TROP2, we stimulated BEAS-2B with cigarette smoke to construct an in vitro model. We found that TROP2 expression increased, whereas TROP2 silencing reversed the cigarette smoke extract-induced decrease in mitochondrial membrane potential, increased reactive oxygen species content, decreased adenosine triphosphate (ATP) production, increased inflammatory factor secretion and increased apoptosis. In addition, we searched online bioinformatics and screened the gene dynamin-related protein 1 (DRP1) related to mitophagy as the research object. Co-IP assay verified the binding relationship between DRP1 and TROP2. Further study found that TROP2 promoted mitophagy and apoptosis of BEAS-2B cells by up-regulating the expression of DRP1. In addition, PTEN-induced putative kinase 1 (PINK1) is a potential binding protein of DRP1, and DRP1 accelerated mitophagy and apoptosis of BEAS-2B cells by promoting the expression of PINK1. We established a COPD SD rat model by cigarette smoke exposure and LPS instillation and treated it by intraperitoneal injection of si-TROP2. The results showed that TROP2 silencing restored lung function and reduced the secretion of inflammatory factors in bronchoalveolar lavage fluid. In conclusion, TROP2 can be used as a new reference for COPD treatment.

Data-oriented protein kinase drug discovery.

The continued growth of data from biological screening and medicinal chemistry provides opportunities for data-driven experimental design and decision making in early-phase drug discovery. Approaches adopted from data science help to integrate internal and public domain data and extract knowledge from historical in-house data. Protein kinase (PK) drug discovery is an exemplary area where large amounts of data are accumulating, providing a valuable knowledge base for discovery projects. Herein, the evolution of PK drug discovery and development of small molecular PK inhibitors (PKIs) is reviewed, highlighting milestone developments in the field and discussing exemplary studies providing a basis for increasing data orientation of PK discovery efforts.