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The molecular identity of the mitochondrial megachannel (MMC)/permeability transition pore (PTP), a key effector of cell death, remains controversial. By combining highly purified, fully active bovine F-ATP synthase with preformed liposomes we show that Ca 2+ dissipates the H + gradient generated by ATP hydrolysis. After incorporation of the same preparation into planar lipid bilayers Ca 2+ elicits currents matching those of the MMC/PTP. Currents were fully reversible, were stabilized by benzodiazepine 423, a ligand of the OSCP subunit of F-ATP synthase that activates the MMC/PTP, and were inhibited by Mg 2+ and adenine nucleotides, which also inhibit the PTP. Channel activity was insensitive to inhibitors of the adenine nucleotide translocase (ANT) and of the voltage-dependent anion channel (VDAC). Native gel-purified oligomers and dimers, but not monomers, gave rise to channel activity. These findings resolve the long-standing mystery of the MMC/PTP and demonstrate that Ca 2+ can transform the energy-conserving F-ATP synthase into an energy-dissipating device. The molecular identity of the mitochondrial megachannel (MMC)/permeability transition pore (PTP), a key effector of cell death, remains controversial. Here authors demonstrate that the membrane embedded bovine F-ATP synthase elicits Ca2 + -dependent currents matching those of the MMC/PTP.

Changes in mitochondrial membrane permeability are closely associated with mitochondria-mediated apoptosis. Antimicrobial peptides (AMPs), which have been found to enter cells to exert physiological effects, cause damage to the mitochondria. This paper reviews the molecular mechanisms of AMP-mediated apoptosis by changing the permeability of the mitochondrial membrane through three pathways: the outer mitochondrial membrane (OMM), inner mitochondrial membrane (IMM), and mitochondrial permeability transition pore (MPTP). The roles of AMPs in inducing changes in membrane permeability and apoptosis are also discussed. Combined with recent research results, the possible application prospects of AMPs are proposed to provide a theoretical reference for the development of AMPs as therapeutic agents for human diseases.

Polydatin is thought to protect mitochondria in different cell types in various diseases. Mitochondrial dysfunction is a major contributing factor in secondary brain injury resulting from traumatic brain injury. To investigate the protective effect of polydatin after traumatic brain injury, a rat brain injury model of lateral fluid percussion was established to mimic traumatic brain injury insults. Rat models were intraperitoneally injected with polydatin (30 mg/kg) or the SIRT1 activator SRT1720 (20 mg/kg, as a positive control to polydatin). At 6 hours post-traumatic brain injury insults, western blot assay was used to detect the expression of SIRT1, endoplasmic reticulum stress related proteins and p38 phosphorylation in cerebral cortex on the injured side. Flow cytometry was used to analyze neuronal mitochondrial superoxide, mitochondrial membrane potential and mitochondrial permeability transition pore opened. Ultrastructural damage in neuronal mitochondria was measured by transmission electron microscopy. Our results showed that after treatment with polydatin, release of reactive oxygen species in neuronal mitochondria was markedly reduced; swelling of mitochondria was alleviated; mitochondrial membrane potential was maintained; mitochondrial permeability transition pore opened. Also endoplasmic reticulum stress related proteins were inhibited, including the activation of p-PERK, spliced XBP-1 and cleaved ATF6. SIRT1 expression and activity were increased; p38 phosphorylation and cleaved caspase-9/3 activation were inhibited. Neurological scores of treated rats were increased and the mortality was reduced compared with the rats only subjected to traumatic brain injury. These results indicated that polydatin protectrd rats from the consequences of traumatic brain injury and exerted a protective effect on neuronal mitochondria. The mechanisms may be linked to increased SIRT1 expression and activity, which inhibits the p38 phosphorylation-mediated mitochondrial apoptotic pathway. This study was approved by the Animal Care and Use Committee of the Southern Medical University, China (approval number: L2016113) on January 1, 2016.

Sorafenib resistance limits its survival benefit for treatment of hepatocellular carcinoma (HCC). Cholesterol metabolism is dysregulated in HCC, and its role in sorafenib resistance of HCC has not been fully elucidated. Aiming to elucidate this, in vitro and in vivo sorafenib resistant models were established. Sterol regulatory element binding transcription factor 2 (SREBF2), the key regulator of cholesterol metabolism, was activated in sorafenib resistant HepG2 and Huh7 cells. Knockdown of SREBF2 resensitized sorafenib resistant cells and xenografts tumors to sorafenib. Further study showed that SREBF2 positively correlated with StAR related lipid transfer domain containing 4 (STARD4) in our sorafenib resistant models and publicly available datasets. STARD4, mediating cholesterol trafficking, not only promoted proliferation and migration of HepG2 and Huh7 cells, but also increased sorafenib resistance in liver cancer. Mechanically, SREBF2 promoted expression of STARD4 by directly binding to its promoter region, leading to increased mitochondrial cholesterol levels and inhibition of mitochondrial cytochrome c release. Importantly, knockdown of SREBF2 or STARD4 decreased mitochondrial cholesterol levels and increased mitochondrial cytochrome c release, respectively. Moreover, overexpression of STARD4 reversed the effect of SREBF2 knockdown on mitochondrial cytochrome c release and sorafenib resistance. In conclusion, SREBF2 promotes STARD4 transcription, which in turn contributes to mitochondrial cholesterol transport and sorafenib resistance in HCC. Therefore, targeting the SREBF2–STARD4 axis would be beneficial to a subset of HCC patients with sorafenib resistance. SREBF2 promotes STARD4 transcription, which in turn contributes to mitochondrial cholesterol transport and sorafenib resistance in HCC.

Paraquat (PQ) as a Parkinsonian mimetic has been demonstrated to impair dopaminergic (DAergic) neurons and is highly correlated with the etiology of Parkinson’s disease (PD) where the death of DAergic neurons has been mainly attributed to impaired mitochondrial functioning. In this study, PQ-induced cytotoxicity focusing on mitochondrial membrane permeability (MMP), which has been implicated to play a part in neurodegeneration, was investigated. Primarily, PQ-induced cytotoxicity and reactive oxygen species (ROS) were inhibited by an inhibitor of NADPH oxidase (NOX), indicating the toxic effect of PQ redox cycling. Further, dibucaine and cyclosporin A which respectively inhibit mitochondrial apoptosis-induced channels (MAC) and mitochondrial permeability transition pores (mPTP) were used and found to prevent PQ-induced mitochondrial dysfunction, such as decreased mitochondrial membrane potential and increased MMP, mitochondrial ROS, and pro-apoptotic factor release. Knockdown of bax and/or bak blocked PQ-induced mitochondrial clusterization of Bax and/or Bak and cytotoxicity, demonstrating the significance of MAC which is composed of Bax and/or Bak. This clusterization coincided with the release of mitochondrial apoptotic factors before there was an increase in inner MMP, indicating that MAC may precede mPTP formation. Besides, NOX inhibitor but not dibucaine attenuated the earlier PQ-induced cytosolic ROS formation or Bax and/or Bak clusterization indicating PQ redox cycling may account for MAC formation. In this model, we have resolved for the first that PQ cytotoxicity through redox cycling may sequentially result in increased outer (MAC) and inner (mPTP) MMP and suggested MMP could be implicated as a therapeutic target in treating neurodegenerative diseases like PD.

To observe whether downhill running can lead to DNA damage in skeletal muscle cells and changes in mitochondrial membrane permeability and to explore whether the DNA damage caused by downhill running can lead to changes in mitochondrial membrane permeability by regulating the components of the endoplasmic reticulum mitochondrial coupling structure (MAM). A total of 48 male adult Sprague–Dawley rats were randomly divided into a control group (C, n = 8) and a motor group (E, n = 40). Rats in Group E were further divided into 0 h (E0), 12 h (E12), 24 h (E24), 48 h (E48) and 72 h (E72) after prescribed exercise, with 8 rats in each group. At each time point, flounder muscle was collected under general anaesthesia. The DNA oxidative damage marker 8-hydroxydeoxyguanosine (8-OHdG) was detected by immunofluorescence. The expression levels of the DNA damage-related protein p53 in the nucleus and the EI24 protein and reep1 protein in whole cells were detected by Western blot. The colocalization coefficients of the endoplasmic reticulum protein EI24 and the mitochondrial protein Vdac2 were determined by immunofluorescence double staining, and the concentration of Ca2+ in skeletal muscle mitochondria was detected by a fluorescent probe. Finally, the opening of the mitochondrial membrane permeability transition pore (mPTP) was detected by immunofluorescence. Twelve hours after downhill running, the mitochondrial membrane permeability of the mPTP opened the most (P < 0.05), the content of 8-OHdG in skeletal muscle peaked (P < 0.05), and the levels of the regulatory protein p53, mitochondrial Ca2+, and the EI24 and reep1 proteins peaked (P < 0.01). Moreover, the colocalization coefficients of EI24 and Vdac2 and the Mandes coefficients of the two proteins increased first and then recovered 72 h after exercise (P < 0.05). (1) Downhill running can lead to DNA damage in skeletal muscle cells, overload of mitochondrial Ca2+ and large opening of membrane permeability transformation pores. (2) The DNA damage caused by downhill running may result in p53 promoting the transcriptional activation of reep1 and EI24, enhancing the interaction between EI24 and Vdac2, and then leading to an increase in Ca2+ in skeletal muscle mitochondria and the opening of membrane permeability transition pores.

In the previous work, we found PRG, a polysaccharide from Phellinus ribis, exhibited neurotrophic activity. To obtain an active structural unit with lower molecular weight, PRG was degraded to prepare the degraded PRG (DPRG) using ascorbic acid and H2O2. The aim of the paper was to obtain DPRG by optimizing the degradation conditions using response surface methodology (RSM) and to study its protective effects of PC12 cells induced by Aβ25–35. The optimum conditions were as follows; the concentration of H2O2-Vc was 17 mM and degradation temperature was 50 °C; when degradation time was 1.6 h, the experimental response value of PC12 cell viability was 83.4 ± 0.15%, which was in accordance with the predicted value (83.5%). We also studied the protective effects of DPRG against the Aβ25–35-induced neurotoxicity and explored the underlying mechanism. The results showed that treatment with DPRG could attenuate PC12 cells death. The mechanism was relative to the inhibition of cell apoptosis by increasing the MMP level and decreasing the protein expression of cytochrome C (Cytc) in PC12 cells. In conclusion, DPRG with lower molecular weight was obtained successfully. It possessed neuroprotective properties and might be a candidate for neurodegenerative disease treatment.

Background/Objectives: Necroptosis is a critical process in the pathogenesis of myocardial ischemia–reperfusion injury (MIRI). Tilianin (Til), a natural flavonoid glycoside derived from Dracocephalum moldavica L., exhibits significant therapeutic potential in cardiovascular diseases. However, its efficacy and mechanisms in mitigating necroptosis-induced MIRI remain incompletely understood. This study aimed to elucidate the molecular mechanisms by which Til regulates cardiomyocyte necroptosis to alleviate MIRI. Methods: A rat model of MIRI was established by ligating the left anterior descending coronary artery. Necroptosis in H9c2 cardiomyocytes was induced by oxygen–glucose deprivation/reoxygenation (H/R) combined with Z-VAD-FMK. Myocardial infarct size was assessed using 2,3,5-triphenyltetrazolium chloride (TTC) staining. Histopathological injury in cardiac tissue was examined by hematoxylin–eosin (HE) staining. Fluorescent probes were used to detect reactive oxygen species (ROS) and mitochondria. The molecular mechanics Poisson–Boltzmann surface area (MM-PBSA) method was used to predict the binding energy between Til and RIP3. Furthermore, RIP3 overexpression and knockdown, along with inhibition of the downstream protein CaMKII, were used to further investigate the mechanism. Results: Til treatment significantly reduced MIRI in rats, decreased myocardial infarct size, histopathological injury, and regulated myocardial enzyme levels. Til pretreatment effectively inhibited necroptosis in H9c2 cells induced by H/R and Z-VAD-FMK, as evidenced by reduced necroptosis rates, decreased inflammatory cytokine release, improved mitochondrial function, and suppressed phosphorylation of the necroptosis marker MLKL. Molecular docking and dynamics simulation demonstrated stable binding of Til to RIP3, which was verified through Western blot. The protective effects of Til on necroptosis were reversed by RIP3 overexpression. Furthermore, the CaMKII inhibitor KN93 abolished Til’s effect on mitochondria. Conclusions: Til alleviates MIRI by targeting RIP3 to inhibit the necroptosis pathway and mPTP opening. These findings provide a new therapeutic strategy for MIRI and necroptosis-related diseases.

Purpose Antibiotic resistance poses a global health threat. It reduces the effectiveness of antibiotics against bacterial infections. Antimicrobial peptides, including thionins, offer a promising alternative due to their broad-spectrum activity and lower likelihood of resistance development. Methods This study focuses on the production of thionin from barley cell suspension cultures, induced with biotic and abiotic agents, and its evaluation for antibacterial, anticancer, and anti-inflammatory activities. Barley calli were initiated and subcultured in media containing various elicitors, such as Fusarium metabolites, polyethylene glycol, and nano-chitosan. Results The combination of nano-chitosan and Fusarium metabolites yielded the highest thionin production and optimal growth. The purified thionin demonstrated strong antibacterial activity, with a minimum inhibitory concentration of approximately 1.2 µg/mL, surpassing that of ciprofloxacin. This activity is likely due to increased bacterial membrane permeability, indicated by a high electrolyte leakage rate of around 70%, and DNA disruption, evidenced by an 81% reduction in DNA content in thionin-treated bacteria. Additionally, thionin treatment inhibited nearly 100% of biofilm formation. In anticancer assays, thionin exhibited significant cytotoxicity against multiple cancer cell lines, with low IC50 values of 0.29 µg/mL against liver cancer cells and 0.17 µg/mL against colon cancer cells. The anticancer mechanism involved the induction of apoptosis markers, including increased nuclear intensity, membrane permeability, mitochondrial membrane permeability, and cytochrome c release. Importantly, thionin showed lower hemolytic activity compared to doxorubicin. Moreover, thionin displayed superior anti-inflammatory activity relative to aspirin. Conclusion These findings underscore the potential of barley-derived thionin as a versatile therapeutic agent against antibiotic-resistant infections, cancer, and inflammation.

Few researches have explored the production of pharmaceuticals from aquatic plants. Therefore, this study explored, for the first time, the phytochemical composition and bioactivities of ten aquatic plants. Aquatic plant shoots from various Nile River canals were collected, dried, and ground for aqueous extract preparation. Phytochemical composition and antioxidant capacity were assessed using DPPH assays. Extracts were tested for antiparasitic, antibacterial, anti-biofilm, and anticancer activities through standard in vitro assays, measuring IC50 values, and evaluating mechanisms of action, including cell viability and high-content screening assays. The results showed that the aquatic plants were rich in pharmaceutical compounds. The antioxidant capacity of these extracts exceeded that of vitamin C. The extracts showed promising antiparasitic activity against pathogens like Opisthorchis viverrini and Plasmodium falciparum, with IC50 values between 0.7 and 2.5 µg/mL. They also demonstrated low MICs against various pathogenic bacteria, causing DNA damage, increased plasma membrane permeability, and 90% biofilm inhibition. In terms of anticancer activity, extracts were effective against a panel of cancer cell lines, with Ludwigia stolonifera exhibiting the highest efficacy. Its IC50 ranged from 0.5 µg/mL for pancreatic, esophageal, and colon cancer cells to 1.5 µg/mL for gastric cancer cells. Overall, IC50 values for all extracts were below 6 µg/mL, showing significant apoptotic activity, increased nuclear intensity, plasma membrane permeability, mitochondrial membrane permeability, and cytochrome c release, and outperforming doxorubicin. This study highlights the potential of aquatic plants as sources for new, safe, and effective drugs with strong antiparasitic, antibacterial, and anticancer properties.