Quercetin ameliorates senescence and promotes osteogenesis of BMSCs by suppressing the repetitive element­triggered RNA sensing pathway.

Cell senescence impedes the self­renewal and osteogenic capacity of bone marrow mesenchymal stem cells (BMSCs), thus limiting their application in tissue regeneration. The present study aimed to elucidate the role and mechanism of repetitive element (RE) activation in BMSC senescence and osteogenesis, as well as the intervention effect of quercetin. In an H2O2­induced BMSC senescence model, quercetin treatment alleviated senescence as shown by a decrease in senescence­associated ß­galactosidase (SA­ß­gal)­positive cell ratio, increased colony formation ability and decreased mRNA expression of p21 and senescence­associated secretory phenotype genes. DNA damage response marker γ­H2AX increased in senescent BMSCs, while expression of epigenetic markers methylation histone H3 Lys9, heterochromatin protein 1α and heterochromatin­related nuclear membrane protein lamina­associated polypeptide 2 decreased. Quercetin rescued these alterations, indicating its ability to ameliorate senescence by stabilizing heterochromatin structure where REs are primarily suppressed. Transcriptional activation of REs accompanied by accumulation of cytoplasmic double­stranded (ds)RNA, as well as triggering of the RNA sensor retinoic acid­inducible gene I (RIG­I) receptor pathway in H2O2­induced senescent BMSCs were shown. Similarly, quercetin treatment inhibited these responses. Additionally, RIG­I knockdown led to a decreased number of SA­ß­gal­positive cells, confirming its functional impact on senescence. Induction of senescence or administration of dsRNA analogue significantly hindered the osteogenic capacity of BMSCs, while quercetin treatment or RIG­I knockdown reversed the decline in osteogenic function. The findings of the current study demonstrated that quercetin inhibited the activation of REs and the RIG­I RNA sensing pathway via epigenetic regulation, thereby alleviating the senescence of BMSCs and promoting osteogenesis.

Time-Lapse Live-Cell Imaging Using Fluorescent Protein Sensors in Outflow Pathway Cells Under Fluid Flow Conditions.

The role of shear stress in regulating aqueous humor (AH) outflow and intraocular pressure (IOP) in the trabecular meshwork (TM) and Schlemm's canal (SC) of the eye is an emerging field. Shear stress has been shown to activate mechanosensitive ion channels in TM cells and induce nitric oxide production in SC cells, which can affect outflow resistance and lower IOP. Live-cell imaging using fluorescent protein sensors has provided real-time data to investigate the physiological relationship between fluid flow and shear stress in the outflow pathway cells. The successful application of time-lapse live-cell imaging in primary cultured cells has led to the identification of key cellular and molecular mechanisms involved in regulating AH outflow and IOP, including the role of autophagy and primary cilia as mechanosensors. This chapter presents a detailed protocol for conducting time-lapse live-cell imaging under fluid flow conditions in the outflow pathway cells.

Study of phenanthrenes from their unique mass spectrometric behavior through quantum chemical calculations to liquid chromatographic quantitation.

Phenanthrenes and their derivatives have biological relevance owing to their antimicrobial, antioxidant, and cytotoxic effects on cancer cells. They can be efficiently analyzed through ultrahigh-performance liquid chromatography coupled to tandem high-resolution mass spectrometry (UHPLC-MS/HRMS). Herein, we first studied the unique fragmentation behavior of phenanthrenes based on direct infusion MS/HRMS analysis. As a newly described phenomenon, "organ pipe distribution", we found a structural connection linking their unique fragmentation pattern to serial H radical losses. The bonds responsible for this behavior were identified through quantum chemical calculations using a stepwise approach. Furthermore, the chromatographic aspect of this study was enhanced by developing, validating, and applying a new unscheduled targeted UHPLC-MS/HRMS method for quantifying phenanthrenes in Juncus compressus herb. Targeted compounds were efficiently separated within 4 min upon utilizing the Accucore C30 column, and the unscheduled targeted analytical approach afforded five new isomers. Compounds 1 (effususol), 3 (dehydroeffusol), and 6 (7-hydroxy-1-methyl-2-methoxy-5-vinyl-9,10-dihydrophenanthrene) had their linearity limits determined within 10-5000 nM, and Compounds 2 (effusol), 4 (juncusol), 5 (effususin A), and 7 (compressin A) within 25-5000 nM. The coefficients of variation for precision ranged from 1.4 % to 15.2 %. The obtained matrix effects and accuracy values were also within acceptable ranges. Compounds 2 (effusol) and 3 (dehydroeffusol) were present in both methanolic and dichloromethanolic extracts of Plants 1 and 3 at the highest concentrations. Furthermore, the relationship between phenanthrene fingerprints, obtained through ANOVA statistical analysis of quantitative data, and the geographical location of herbs was also established.

Golden Gate Cloning for Efficient Biosynthesis of Lycopene in Synthetic Yeast.

Golden Gate Assembly (GGA) represents a versatile method for assembling multiple DNA fragments into a single molecule, which is widely used in rapid construction of complex expression cassettes for metabolic engineering. Here we describe the GGA method for facile construction and optimization of lycopene biosynthesis pathway by the combinatorial assembly of different transcriptional units (TUs). Furthermore, we report the method for characterizing and improving lycopene production in the synthetic yeast chassis.

Disruption of the signal recognition particle pathway leading to impaired growth, sugar metabolism and acid resistance of Lactococcus lactis.

Lactococcus lactis is a well-known workhorse for dairy products, whose important industrial traits are tightly associated with numerous cytoplasmic membrane proteins. However, roles of the signal recognition particle (SRP) pathway responsible for membrane protein targeting have not been studied in L. lactis. In this work, the putative genes ffh and ftsY encoding SRP pathway components were identified in the genome of L. lactis NZ9000. Experimental evidence showed that sequence mutation in either the ffh or ftsY was not lethal, but prolonged the lag phase of the resultant mutants Δffh and ΔftsY by 2 h and lowered their biomass to 85.7 % of the wild type under static conditions, as well as deprived the mutants of improved growth capacity under aerobic respiration conditions. Besides, the speeds of glucose consumption and lactate production were significantly decreased in the mutants. Then, the impact of the SPR components on acid resistance was detected, showing that the ffh and ftsY were transcriptionally upregulated by 3.02 ± 1.21 and 8.66 ± 1.01-fold in the wild type during acid challenge at pH 3.0, and cell survival of the Δffh and ΔftsY decreased by10- and 100-fold compared with the wild type. To explore the possible mechanism about the SRP pathway involved in the above physiological traits, proteomics analysis was performed and revealed that disruption of the Ffh or FtsY led to decrease in ribosomal proteins, but increase in DnaK, GroEL and heat shock protein GrpE, indicating that the SRP pathway was closely linked to protein synthesis and folding in L. lactis. Decrease in the fructose-bisphosphate aldolase, respiratory complexes NADH dehydrogenase, as well as glutamate decarboxylase was also detected in the Δffh and ΔftsY, which is consistent with the phenomena of impaired sugar metabolism and acid resistance. Our results demonstrated the dispensable SRP pathway could contribute to the maintenance of metabolism homeostasis and acid resistance of L. lactis.

Electronic coupling of iron-cobalt in Prussian blue towards improved peroxydisulfate activation.

Prussian blue analogs (PBAs) have attracted extensive attention in the field of aqueous organic degradation due to the tremendous potential for peroxydisulfate (PDS) activation. However, the relationship between the d-band center of the catalyst and the activation behavior of PDS remained largely unexplored. Herein, a series of Fe-Co PBAs-based catalysts with different Fe/Co ratios (Fe-Co PBAs-1 = 1: 0.52; Fe-Co PBAs-2 = 1: 1.21, and Fe-Co PBAs-3 = 1: 1.48) have been prepared by a facile hydrothermal procedure and subsequent acid treatment (Fe-Co PBAs-xH). The as-prepared Fe-Co PBAs-xH exhibited superior PDS activation performance and excellent recyclability in the degradation of methylene blue (MB). Density functional theory calculations revealed that the electron-occupied state of the Fe-Co PBAs was shifted to the Fermi level, indicating a strong interaction and easier electron transfer. Moreover, the d-band center of Fe-Co PBAs was upshifted relative to that of Fe PBAs, suggesting easier adsorption of MB and PDS, which was beneficial to enhancing catalytic activation and subsequent dissociation. Radicals such as •OH, 1O2, O2•-, and SO4•- were determined by the radical quenching experiment and electron paramagnetic resonance (EPR) testing in the Fe-Co PBAs-3H/PDS system, and the order of MB degradation by the free active radical is •OH > 1O2 > O2•- > SO4•-. The degradation pathway and potential ecotoxicity of MB and its intermediates were also studied. This work can provide new insights to construct the efficient catalysts for the activation of PDS and the degradation of organic pollutants.

Melittin promotes the proliferation of Schwann cells in hyperglycemic environment by up­regulating the Crabp2/Wnt/ß­catenin signaling pathway.

The present study aimed to explore the effect of melittin (MLT) on the growth of Schwann cells (SCs) in high glucose conditions and to understand the mechanisms involved. The goal was to provide a theoretical basis for using MLT in the treatment of diabetic peripheral neuropathy (DPN). The CCK­8 assay was used to measure cell activity at different concentrations of glucose and MLT. Flow cytometry was employed to analyze the effect of MLT on cell cycle phases and apoptosis in SCs under high glucose conditions. To identify differentially expressed proteins, 4D label­free quantitative proteomics with liquid chromatography­mass spectrometry was used, followed by biological analysis to explore potential mechanisms. PCR, western blotting and immunofluorescence were conducted to confirm these mechanisms. Melittin (0.2 µg/ml) increased the proliferation of SCs in a high glucose environment. Flow cytometry showed that after MLT treatment, the proportion of cells in the G2/M+S phase increased and the combined ratio of early and late apoptosis decreased under high glucose conditions. Proteomics identified 1,784 proteins with significant changes in expression; 725 were upregulated, and 1,059 were downregulated. Kyoto Encyclopedia of Genes and Genomes analysis indicated that the differentially expressed proteins were mainly involved in metabolic pathways and neurodegenerative disease pathways. PCR, western blotting and immunofluorescence confirmed the increase in Crabp2, Wnt3a, C­Jun, CDK4, CyclinD1 and proliferating cell nuclear antigen. In high glucose conditions, MLT protects SCs from glucose toxicity by upregulating the Crabp2/Wnt/ß­catenin signaling pathway, potentially providing a new treatment for DPN.

Apoptosis and its role in postmortem meat tenderness: A comprehensive review.

Tenderness is considered a crucial attribute of postmortem meat quality, directly influencing consumers' preferences and industrial economic benefits. The degradation of myofibrillar proteins by endogenous enzymes within muscle fibers is believed to be the most effective pathway for meat tenderization. After animals are slaughtered and exsanguinated, there is a significant accumulation of reactive oxygen species (ROS), and a dramatic depletion of adenosine triphosphate (ATP) in muscle, leading to inevitable cell death. Caspases are activated in postmortem muscle cells, which disrupt the cell structure and improve meat tenderness through protein hydrolysis. In this review, we systematically summarized the three primary types of cell death studied in postmortem muscle: apoptosis, autophagy and necrosis. Furthermore, we emphasized the molecular mechanisms of apoptosis and its corresponding apoptotic pathways (mitochondrial apoptosis, death receptors, and endoplasmic reticulum stress) that affect meat tenderness during muscle conversion to meat. Additionally, factors affecting apoptosis were comprehensively discussed, such as ROS, heat shock proteins, calcium (Ca2+)/calpains, and Bcl-2 family proteins. Finally, this comprehensive review of existing research reveals that apoptosis is mainly mediated by the mitochondrial pathway. This ultimately leads to myofibrillar proteins degradation through caspase activation, improving meat tenderness. This review summarizes the research progress on postmortem muscle apoptosis and its molecular mechanisms in meat tenderization. We hope this will enhance understanding of postmortem meat tenderness and provide a theoretical basis for meat tenderization techniques development in the future.

Isolation, structural elucidation and biosynthetic pathway of bioactive prenyl quinone compounds from Panus lecomtei based on untargeted metabolomics combined with molecular networking.

Panus lecomtei is a relatively unfamiliar and undeveloped mushroom. This study generated ethyl acetate extracts of P. lecomtei intracellular (I), extracellular (E) and total fermentation broth (T). Both E and T extracts demonstrated antioxidant and antibacterial activities at 100 to 200 µg/mL. The composition differences of metabolites of these extracts were further studied based on comparative metabolomics by LS/MS and molecular network analysis. The results revealed that there were over 2000 significantly distinct metabolites among the three extracts, with abundant prenyl quinone compounds. Furthermore, the molecular network clarified the conversion relationship of P. lecomtei metabolites. Seven known prenyl quinone derivatives (1-7) were isolated from the E extract. Among them, compound 3 displayed excellent antioxidant activity and modest antibacterial activity. Compound 5 was discovered in fungi for the first time. Finally, a potential biosynthetic route for prenyl quinone in P. lecomtei was suggested.

Astragaloside IV plays a neuroprotective role by promoting PPARγ in cerebral ischemia-reperfusion rats.

BACKGROUND: Cerebral ischemia-reperfusion injury (CIRI) usually occurs during the treatment phase of ischemic disease, which is closely related to high morbidity and mortality. Promoting neurogenesis and synaptic plasticity are effective neural recovery strategies for CIRI. Astragaloside IV (AS-IV) has been shown to play a neuroprotective role in some neurological diseases. In the current study, we evaluated the effect and possible mechanism of AS-IV in CIRI rats. METHODS: The middle cerebral artery occlusion reperfusion (MCAO/R) model was established in rats to simulate the occurrence of human CIRI. First, we determined the cerebral injury on the 1st, 3rd, 5th and 7th day after cerebral ischemia-reperfusion (I/R) surgery by neurological deficit detection, TTC staining, TUNEL staining and Western blot analysis. Furthermore, rats were pre administered with AS-IV and then subjected to cerebral I/R surgery. Brains were collected on the 3rd day to evaluate the neuroprotective effect of AS-IV. RESULTS: Our results showed that on the 3rd day after I/R, the neurological impairment score and infarct volume were highest, the levels of apoptosis and expression of Caspase3 and Bax reached the peak. AS-IV treatment apparently attenuated neurological dysfunction, reduced infarct volume and pathological damage, promoted the neurogenesis, and alleviated the pathological damage caused by cerebral I/R involved in thickening and blurring of synaptic membranes, reduction of microtubules and synaptic vesicles, and loss of synaptic cleft. Our study also showed that AS-IV promoted the transcription and expression of the peroxisome proliferators-activated receptors γ (PPARγ) and brain-derived neurotrophic factor (BDNF), increased the expression of phosphorylation of tyrosine kinase receptor B (TrkB) and downstream PI3K/Akt/mTOR pathway proteins. Notably, when GW9662, an inhibitor of PPARγ was administered with AS-IV, the neuroprotective effect of AS-IV was reduced. CONCLUSIONS: These findings suggested that AS-IV has neuroprotective function in CIRI rats, and its molecular mechanism may depend on the phosphatidylinositide 3-kinase (PI3K)/protein kinase B (PKB)/Akt signalling pathway activated by PPARγ. AS-IV could be an effective therapeutic drug candidate for CIRI treatment.

Static magnetic field contributes to osteogenic differentiation of hPDLSCs through the H19/Wnt/ß-catenin axis.

BACKGROUND: Static magnetic field (SMF) as an effective physical stimulus is capable of osteogenic differentiation for multiple mesenchymal stem cells, including human periodontal ligament stem cells (hPDLSCs). However, the exact molecular mechanism is still unknown. Therefore, this study intends to excavate molecular mechanisms related to SMF in hPDLSCs using functional experiments. METHODS: hPDLSCs were treated with different intensities of SMF, H19 lentivirus, and Wnt/ß-catenin pathway inhibitor (XAV939). Changes in osteogenic markers (Runx2, Col Ⅰ, and BMP2), Wnt/ß-catenin markers (ß-catenin and GSK-3ß), and calcified nodules were examined using RT-qPCR, western blotting, and alizarin red staining in hPDLSCs. RESULTS: SMF upregulated the expression of H19, and SMF and overexpressing H19 facilitated the expression of osteogenic markers (Runx2, Col Ⅰ, and BMP2), activation of the Wnt/ß-catenin pathway, and mineralized sediment in hPDLSCs. Knockdown of H19 alleviated SMF function, and treatment with XAV939 limited SMF- and H19-mediated osteogenic differentiation of hPDLSCs. Notably, the expression of hsa-miR-532-3p, hsa-miR-370-3p, hsa-miR-18a-5p, and hsa-miR-483-3p in hPDLSCs was regulated by SMF, and may form an endogenous competitive RNA mechanism with H19 and ß-catenin. CONCLUSION: SMF contributes to the osteogenic differentiation of hPDLSCs by mediating the H19/Wnt/ß-catenin pathway, and hsa-miR-532-3p, hsa-miR-370-3p, hsa-miR-18a-5p, and hsa-miR-483-3p may be the key factors in it.

Downregulation of glucose-energy metabolism via AMPK signaling pathway in granulosa cells of diminished ovarian reserve patients.

Glucose metabolism plays a crucial role in the function of granulosa cells (GCs) and the development of follicles. In cases of diminished ovarian reserve (DOR), alterations in these processes can impact female fertility. This study aims to investigate changes in glucose-energy metabolism in GCs of young DOR patients aged 20 to 35 years and their correlation with the onset and progression of DOR. 72 DOR cases and 75 women with normal ovarian reserve (NOR) as controls were included based on the POSEIDON and Bologna criteria. Samples of GCs and follicular fluid (FF) were collected for a comprehensive analysis involving transcriptomics, metabolomics, RT-qPCR, JC-1 staining, and flow cytometry. The study identified differentially expressed genes and metabolites in GCs of DOR and NOR groups, revealing 7 common pathways related to glucose-energy metabolism, along with 11 downregulated genes and 14 metabolites. Key substances in the glucose-energy metabolism pathway, such as succinate, lactate, NADP, ATP, and ADP, showed decreased levels, with the DOR group exhibiting a reduced ADP/ATP ratio. Downregulation of genes involved in glycolysis (HK, PGK, LDH1), the TCA cycle (CS), and gluconeogenesis (PCK) was observed, along with reduced glucose content and expression of glucose transporter genes (GLUT1 and GLUT3) in DOR GCs. Additionally, decreased AMPK pathway activity and impaired mitochondrial function in DOR suggest a connection between mitochondrial dysfunction and disrupted energy metabolism. Above all, the decline in glucose-energy metabolism in DOR is closely associated with its onset and progression. Reduced glucose uptake and impaired mitochondrial function in DOR GCs lead to internal energy imbalances, hindering the AMPK signaling pathway, limiting energy production and supply, and ultimately impacting follicle development and maturation.

Lipidomics analysis reveals the effects of Schizochytrium sp. supplementation on the lipid composition of Tan sheep meat.

Schizochytrium sp. (SZ) can potentially be employed in nutritional strategies for producing high-quality sheep meat. However, the effects of SZ on the lipid composition of sheep meat are insufficiently understood. In this study, the effects of SZ supplementation on the lipid profile of Tan sheep meat were evaluated using non-targeted lipidomic techniques. Lipidomics analysis revealed 383 differential lipids (DLs) between the SZ and control groups, and there were six metabolic pathways associated with lipids, including glycerophospholipid metabolism, glycerolipid metabolism, α-linolenic acid metabolism, linoleic acid metabolism, glycine, serine and threonine metabolism, and arachidonic acid metabolism (P < 0.05). Glycerophospholipid metabolism was the core pathway of DLs; we found that phosphatidylcholine, phosphatidylserine, and lysophosphatidylcholine were the crucial lipid metabolites of this pathway. Dietary supplementation with SZ increased n-3 polyunsaturated fatty acid (PUFA), C22:6n-3, and C20:5n-3 (P < 0.05), while it decreased C18:0, saturated fatty acid (SFA), and SFA/PUFA (P < 0.05). These results indicate that SZ supplementation induces positive alterations in the lipid profile of Tan sheep meat, which is beneficial to meat quality and sheds valuable insights into the future development of functional lipids in sheep meat.

Exploring Core fermentation microorganisms, flavor compounds, and metabolic pathways in fermented Rice and wheat foods.

The unique flavors of fermented foods significantly influence consumer purchasing choices, prompting widespread scientific interest in the flavor development process. Fermented rice and wheat foods are known for their unique flavors and they occupy an important place in the global diet. Many of these are produced on an industrial scale using starter cultures, whereas others rely on spontaneous fermentation, homemade production, or traditional activities. Microorganisms are key in shaping the sensory properties of fermented products through different metabolic pathways, thus earning the title "the essence of fermentation." Therefore, this study systematically summarizes the key microbial communities and their interactions that contribute positively to iconic fermented rice and wheat foods, such as steamed bread, bread, Mifen, and rice wine. This study revealed the mechanism by which these core microbial communities affect flavor and revealed the strategies of core microorganisms and related enzymes to enhance flavor during fermentation.

T-2 toxin induces cardiac fibrosis by causing metabolic disorders and up-regulating Sirt3/FoxO3α/MnSOD signaling pathway-mediated oxidative stress.

T-2 toxin, an omnipresent environmental contaminant, poses a serious risk to the health of humans and animals due to its pronounced cardiotoxicity. This study aimed to elucidate the molecular mechanism of cardiac tissue damage by T-2 toxin. Twenty-four male Sprague-Dawley rats were orally administered T-2 toxin through gavage for 12 weeks at the dose of 0, 10, and 100 nanograms per gram body weight per day (ng/(g·day)), respectively. Morphological, pathological, and ultrastructural alterations in cardiac tissue were meticulously examined. Non-targeted metabolomics analysis was employed to analyze alterations in cardiac metabolites. The expression of the Sirt3/FoxO3α/MnSOD signaling pathway and the level of oxidative stress markers were detected. The results showed that exposure to T-2 toxin elicited myocardial tissue disorders, interstitial hemorrhage, capillary dilation, and fibrotic damage. Mitochondria were markedly impaired, including swelling, fusion, matrix degradation, and membrane damage. Metabonomics analysis unveiled that T-2 toxin could cause alterations in cardiac metabolic profiles as well as in the Sirt3/FoxO3α/MnSOD signaling pathway. T-2 toxin could inhibit the expressions of the signaling pathway and elevate the level of oxidative stress. In conclusion, the T-2 toxin probably induces cardiac fibrotic impairment by affecting amino acid and choline metabolism as well as up-regulating oxidative stress mediated by the Sirt3/FoxO3α/MnSOD signaling pathway. This study is expected to provide targets for preventing and treating T-2 toxin-induced cardiac fibrotic injury.

4-Nitrophenol at environmentally relevant concentrations mediates reproductive toxicity in Caenorhabditis elegans via metabolic disorders-induced estrogen signaling pathway.

4-Nitrophenol (4-NP), as a toxic and refractory pollutant, has generated significant concern due to its adverse effects. However, the potential toxic effects and mechanism remained unclear. In this study, the reproduction, development, locomotion and reactive oxygen species (ROS) production of Caenorhabditis elegans were investigated to evaluate the 4-NP toxicity. We used metabolomics to assess the potential damage mechanisms. The role of metabolites in mediating the relationship between 4-NP and phenotypes was examined by correlation and mediation analysis. 4-NP (8 ng/L and 8 µg/L) caused significant reduction of brood size, ovulation rate, total germ cells numbers, head thrashes and body bends, and an increase in ROS. However, the oosperm numbers in uterus, body length and body width were decreased in 8 µg/L. Moreover, 36 differential metabolites were enriched in the significant metabolic pathways, including lysine biosynthesis, ß-alanine metabolism, tryptophan metabolism, pentose phosphate pathway, pentose and glucuronate interconversions, amino sugar and nucleotide sugar metabolism, starch and sucrose metabolism, galactose metabolism, propanoate metabolism, glycerolipid metabolism, and estrogen signaling pathway. The mechanism of 4-NP toxicity was that oxidative stress caused by the perturbation of amino acid, which had effects on energy metabolism through disturbing carbohydrate and lipid metabolism, and finally affected the estrogen signaling pathway to exert toxic effects. Moreover, correlation and mediation analysis showed glycerol-3P, glucosamine-6P, glucosamine-1P, UDP-galactose, L-aspartic acid, and uracil were potential markers for the reproduction and glucose-1,6P2 for developmental toxicity. The results provided insight into the pathways involved in the toxic effects caused by 4-NP and developed potential biomarkers to evaluate 4-NP toxicity.

Shuangdan Jiedu Decoction improved LPS-induced acute lung injury by regulating both cGAS-STING pathway and inflammasome.

ETHNOPHARMACOLOGICAL RELEVANCE: Shuangdan Jiedu Decoction (SJD) is a formula composed of six Chinese herbs with heat-removing and detoxifying, antibacterial, and anti-inflammatory effects, which is clinically used in the therapy of various inflammatory diseases of the lungs including COVID-19, but the therapeutic material basis of its action as well as its molecular mechanism are still unclear. AIM OF THE STUDY: The study attempted to determine the therapeutic effect of SJD on LPS-induced acute lung injury (ALI), as well as to investigate its mechanism of action and assess its therapeutic potential for the cure of inflammation-related diseases in the clinical setting. MATERIALS AND METHODS: We established an ALI model by tracheal drip LPS, and after the administration of SJD, we collected the bronchoalveolar lavage fluid (BALF) and lung tissues of mice and examined the expression of inflammatory factors in them. In addition, we evaluated the effects of SJD on the cyclic guanosine monophosphate-adenosine monophosphate synthase -stimulator of interferon genes (cGAS-STING) and inflammasome by immunoblotting and real-time quantitative polymerase chain reaction (RT-qPCR). RESULTS: We demonstrated that SJD was effective in alleviating LPS-induced ALI by suppressing the levels of pro-inflammatory cytokines in the BALF, improving the level of lung histopathology and the number of neutrophils, as well as decreasing the inflammatory factor-associated gene expression. Importantly, we found that SJD could inhibit multiple stimulus-driven activation of cGAS-STING and inflammasome. Further studies showed that the Chinese herbal medicines in SJD had no influence on the cGAS-STING pathway and inflammasome alone at the formulated dose. By increasing the concentration of these herbs, we observed inhibitory effects on the cGAS-STING pathway and inflammasome, and the effect exerted was maximal when the six herbs were combined, indicating that the synergistic effects among these herbs plays a crucial role in the anti-inflammatory effects of SJD. CONCLUSIONS: Our research demonstrated that SJD has a favorable protective effect against ALI, and its mechanism of effect may be associated with the synergistic effect exerted between six Chinese medicines to inhibit the cGAS-STING and inflammasome abnormal activation. These results are favorable for the wide application of SJD in the clinic as well as for the development of drugs for ALI from herbal formulas.

Naoxintong capsule accelerates mitophagy in cerebral ischemia-reperfusion injury via TP53/PINK1/PRKN pathway based on network pharmacology analysis and experimental validation.

ETHNOPHARMACOLOGICAL RELEVANCE: The incidence and mortality of cerebrovascular diseases are increasing year by year. Cerebral ischemia-reperfusion injury (CIRI) is common in patients with ischemic stroke. Naoxintong (NXT) is composed of a variety of Chinese medicines and has the ability to treat CIRI. AIM OF THE STUDY: The aim of this study is to investigate whether NXT regulates mitophagy in CIRI based on network pharmacology analysis and experimental validation. MATERIALS AND METHODS: Oxygen and glucose deprivation/re-oxygenation (OGD/R, 2/22 h) model of PC12 cells and transient middle cerebral artery occlusion (tMCAO, 2/22 h) model of rats were established. Pharmacodynamic indicators include neurological deficit score, 2,3,5-triphenyte-trazoliumchloride (TTC) staining, hematoxylin-eosin (HE) staining and cell viability. Network pharmacology was used to predict pharmacological mechanisms. Pharmacological mechanism indexes include transmission electron microscopy (TEM), drug affinity responsive target stability (DARTS), cellular thermal shift assay (CETSA), immunohistochemistry (IHC), western blot (WB) and immunofluorescence (IF). Kevetrin (an agonists of p53) and pifithrin-α (an inhibitor of p53) used to detect the key role of p53 in mitophagy of NXT. RESULTS: NXT (1% serum containing NXT and 110 mg/kg) improved the damage of OGD/R PC12 cells and tMCAO rats, and this protective effect was related to the anti-oxidation and ability to promote mitophagy of NXT. NXT and pifithrin-α increased the expression of promoting-mitophagy targets (PINK1, PRKN and LC3B) and inhibited the expression of inhibiting-mitophagy targets (p52) via restraining p53, and finally accelerated mitophagy caused by CIRI. CONCLUSION: This study demonstrates that NXT promotes mitophagy in CIRI through restraining p53 and promoting PINK1/PRKN in vivo and in vitro.

Protein Kinase R (PKR) as a Novel dsRNA Sensor in Antiviral Innate Immunity.

Protein kinase R (PKR), a key double-stranded RNA (dsRNA)-activated sensor, is pivotal for cellular responses to diverse stimuli. This protocol delineates a comprehensive methodological framework employing single luciferase assays, yeast assays, immunoblot assays, and quantitative PCR (qPCR) to discern and validate PKR activities and their downstream impacts on NF-κB-activating signaling pathways. These methodologies furnish a systematic approach to unraveling the role of PKR as a dsRNA sensor and effector in antiviral innate immunity, enabling in-depth analyses of dsRNA sensor activities.

Bioactive nanocomposite hydrogel enhances postoperative immunotherapy and bone reconstruction for osteosarcoma treatment.

Osteosarcoma, a malignant bone tumor often characterized by high hedgehog signaling activity, residual tumor cells, and substantial bone defects, poses significant challenges to both treatment response and postsurgical recovery. Here, we developed a nanocomposite hydrogel for the sustained co-delivery of bioactive magnesium ions, anti-PD-L1 antibody (αPD-L1), and hedgehog pathway antagonist vismodegib, to eradicate residual tumor cells while promoting bone regeneration post-surgery. In a mouse model of tibia osteosarcoma, this hydrogel-mediated combination therapy led to remarkable tumor growth inhibition and hence increased animal survival by enhancing the activity of tumor-suppressed CD8+ T cells. Meanwhile, the implanted hydrogel improved the microenvironment of osteogenesis through long-term sustained release of Mg2+, facilitating bone defect repair by upregulating the expression of osteogenic genes. After 21 days, the expression levels of ALP, COL1, RUNX2, and BGLAP in the Vis-αPD-L1-Gel group were approximately 4.1, 5.1, 5.5, and 3.4 times higher than those of the control, respectively. We believe that this hydrogel-based combination therapy offers a potentially valuable strategy for treating osteosarcoma and addressing the tumor-related complex bone diseases.