OBJECTIVE: To investigate the effects of asperuloside on cervical cancer based on endoplasmic reticulum (ER) stress and mitochondrial pathway. METHODS: Different doses (12.5-800 µg/mL) of asperuloside were used to treat cervical cancer cell lines Hela and CaSki to calculate the half maximal inhibitory concentration (IC₅₀) of asperuloside. The cell proliferation was analyzed by clone formation assay. Cell apoptosis, intracellular reactive oxygen species (ROS) and mitochondrial membrane potential were determined by flow cytometry. The protein expressions of cleaved-caspase-3, Bcl-2, Bax, Cyt-c, cleaved-caspase-4 and glucose-regulated protein 78 (GRP78) were analyzed by Western blot. And the inhibitor of ER stress, 4-phenyl butyric acid (4-PBA) was used to treat cervical cancer cells to further verify the role of ER stress in the apoptosis of cervical cancer cells induced by asperuloside. RESULTS: Asperuloside of 325, 650, and 1300 µg/mL significantly inhibited the proliferation and promoted apoptosis of Hela and CaSki cells (P<0.01). All doses of asperuloside significantly increased intracellular ROS levels, reduced mitochondrial membrane potential, significantly reduced Bcl-2 protein expression level, and increased Bax, Cyt-c, GRP78 and cleaved-caspase-4 expressions (P<0.01). In addition, 10 mmol/L 4-PBA treatment significantly promoted cell proliferation and reduced apoptosis (P<0.05), and 650 µg/mL asperuloside could reverse 4-PBA-induced increased cell proliferation, decreased apoptosis and cleaved-caspase-3, -4 and GRP78 protein expressions (P<0.05). CONCLUSION Our study revealed the role of asperuloside in cervical cancer, suggesting that asperuloside promotes apoptosis of cervical cancer cells through ER stress-mitochondrial pathway.
Female ; Humans ; Uterine Cervical Neoplasms/metabolism* ; Caspase 3/metabolism* ; bcl-2-Associated X Protein/metabolism* ; Reactive Oxygen Species/metabolism* ; Endoplasmic Reticulum Chaperone BiP ; HeLa Cells ; Proto-Oncogene Proteins c-bcl-2/metabolism* ; Apoptosis ; Endoplasmic Reticulum Stress ; Cell Line, Tumor

NAD(P)H: quinone oxidoreductase 1 (NQO1) is a flavin protease highly expressed in various cancer cells. NQO1 catalyzes a futile redox cycle in substrates, leading to substantial reactive oxygen species (ROS) production. This ROS generation results in extensive DNA damage and elevated poly (ADP-ribose) polymerase 1 (PARP1)-mediated consumption of nicotinamide adenine dinucleotide (NAD⁺), ultimately causing cell death. Nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in the NAD⁺ salvage synthesis pathway, emerges as a critical target in cancer therapy. The concurrent inhibition of NQO1 and NAMPT triggers hyperactivation of PARP1 and intensive NAD⁺ depletion. In this study, we designed, synthesized, and assessed a novel series of proqodine A derivatives targeting both NQO1 and NAMPT. Among these, compound T8 demonstrated potent antitumor properties. Specifically, T8 selectively inhibited the proliferation of MCF-7 cells and induced apoptosis through mechanisms dependent on both NQO1 and NAMPT. This discovery offers a promising new molecular entity for advancing anticancer research.
Humans ; NAD/metabolism* ; Cell Line, Tumor ; Reactive Oxygen Species/metabolism* ; Nicotinamide Phosphoribosyltransferase/metabolism* ; Cytokines/metabolism* ; Quinones ; Oxidoreductases

BACKGROUND: Pancreatic β-cells elevate insulin production and secretion through a compensatory mechanism to override insulin resistance under metabolic stress conditions. Deficits in β-cell compensatory capacity result in hyperglycemia and type 2 diabetes (T2D). However, the mechanism in the regulation of β-cell compensative capacity remains elusive. Nuclear factor-Y (NF-Y) is critical for pancreatic islets' homeostasis under physiological conditions, but its role in β-cell compensatory response to insulin resistance in obesity is unclear. METHODS: In this study, using obese ( ob/ob ) mice with an absence of NF-Y subunit A (NF-YA) in β-cells ( ob , Nf-ya βKO) as well as rat insulinoma cell line (INS1)-based models, we determined whether NF-Y-mediated apoptosis makes an essential contribution to β-cell compensation upon metabolic stress. RESULTS: Obese animals had markedly augmented NF-Y expression in pancreatic islets. Deletion of β-cell Nf-ya in obese mice worsened glucose intolerance and resulted in β-cell dysfunction, which was attributable to augmented β-cell apoptosis and reactive oxygen species (ROS). Furthermore, primary pancreatic islets from Nf-ya βKO mice were sensitive to palmitate-induced β-cell apoptosis due to mitochondrial impairment and the attenuated antioxidant response, which resulted in the aggravation of phosphorylated c-Jun N-terminal kinase (JNK) and cleaved caspase-3. These detrimental effects were completely relieved by ROS scavenger. Ultimately, forced overexpression of NF-Y in INS1 β-cell line could rescue palmitate-induced β-cell apoptosis, dysfunction, and mitochondrial impairment. CONCLUSION Pancreatic NF-Y might be an essential regulator of β-cell compensation under metabolic stress.
Rats ; Mice ; Animals ; Reactive Oxygen Species/metabolism* ; Diabetes Mellitus, Type 2/metabolism* ; Insulin Resistance ; Insulin ; Insulin-Secreting Cells/metabolism* ; Apoptosis ; Stress, Physiological ; Transcription Factors/metabolism* ; Palmitates/pharmacology* ; Obesity/metabolism*

The widespread of tigecycline resistance gene tet(X4) has a serious impact on the clinical efficacy of tigecycline. The development of effective antibiotic adjuvants to combat the looming tigecycline resistance is needed. The synergistic activity between the natural compound β-thujaplicin and tigecycline in vitro was determined by the checkerboard broth microdilution assay and time-dependent killing curve. The mechanism underlining the synergistic effect between β-thujaplicin and tigecycline against tet(X4)-positive Escherichia coli was investigated by determining cell membrane permeability, bacterial intracellular reactive oxygen species (ROS) content, iron content, and tigecycline content. β-thujaplicin exhibited potentiation effect on tigecycline against tet(X4)-positive E. coli in vitro, and presented no significant hemolysis and cytotoxicity within the range of antibacterial concentrations. Mechanistic studies demonstrated that β-thujaplicin significantly increased the permeability of bacterial cell membranes, chelated bacterial intracellular iron, disrupted the iron homeostasis and significantly increased intracellular ROS level. The synergistic effect of β-thujaplicin and tigecycline was identified to be related to interfere with bacterial iron metabolism and facilitate bacterial cell membrane permeability. Our studies provided theoretical and practical data for the application of combined β-thujaplicin with tigecycline in the treatment of tet(X4)-positive E. coli infection.
Humans ; Tigecycline/pharmacology* ; Escherichia coli/metabolism* ; Reactive Oxygen Species/therapeutic use* ; Plasmids ; Anti-Bacterial Agents/metabolism* ; Escherichia coli Infections/microbiology* ; Bacteria/genetics* ; Microbial Sensitivity Tests

To investigate the protective effect and the potential mechanism of leonurine(Leo) against erastin-induced ferroptosis in human renal tubular epithelial cells(HK-2 cells), an in vitro erastin-induced ferroptosis model was constructed to detect the cell viability as well as the expressions of ferroptosis-related indexes and signaling pathway-related proteins. HK-2 cells were cultured in vitro, and the effects of Leo on the viability of HK-2 cells at 10, 20, 40, 60, 80 and 100 μmol·L~(-1) were examined by CCK-8 assay to determine the safe dose range of Leo administration. A ferroptosis cell model was induced by erastin, a common ferroptosis inducer, and the appropriate concentrations were screened. CCK-8 assay was used to detect the effects of Leo(20, 40, 80 μmol·L~(-1)) and positive drug ferrostatin-1(Fer-1, 1, 2 μmol·L~(-1)) on the viability of ferroptosis model cells, and the changes of cell morphology were observed by phase contrast microscopy. Then, the optimal concentration of Leo was obtained by Western blot for nuclear factor erythroid 2-related factor 2(Nrf2) activation, and transmission electron microscope was further used to detect the characteristic microscopic morphological changes during ferroptosis. Flow cytometry was performed to detect reactive oxygen species(ROS), and the level of glutathione(GSH) was measured using a GSH assay kit. The expressions of glutathione peroxidase 4(GPX4), p62, and heme oxygenase 1(HO-1) in each group were quantified by Western blot. RESULTS:: showed that Leo had no side effects on the viability of normal HK-2 cells in the concentration range of 10-100 μmol·L~(-1). The viability of HK-2 cells decreased as the concentration of erastin increased, and 5 μmol·L~(-1) erastin significantly induced ferroptosis in the cells. Compared with the model group, Leo dose-dependently increased cell via-bility and improved cell morphology, and 80 μmol·L~(-1) Leo promoted the translocation of Nrf2 from the cytoplasm to the nucleus. Further studies revealed that Leo remarkably alleviated the characteristic microstructural damage of ferroptosis cells caused by erastin, inhibited the release of intracellular ROS, elevated GSH and GPX4, promoted the nuclear translocation of Nrf2, and significantly upregulated the expression of p62 and HO-1 proteins. In conclusion, Leo exerted a protective effect on erastin-induced ferroptosis in HK-2 cells, which might be associated with its anti-oxidative stress by activating p62/Nrf2/HO-1 signaling pathway.
Humans ; Ferroptosis ; Reactive Oxygen Species/metabolism* ; NF-E2-Related Factor 2/metabolism* ; Sincalide/pharmacology* ; Signal Transduction ; Epithelial Cells/metabolism* ; Glutathione

This study aimed to explore the effects and mechanisms of total flavones of Abelmoschus manihot(TFA), the extracts from traditional Chinese medicine indicated for kidney diseases, on insulin resistance(IR) and podocyte epithelial-mesenchymal transition(EMT) in diabetic kidney disease(DKD), and further to reveal the scientific connotation. Thirty-two rats were randomly divided into a normal group, a model group, a TFA group, and a rosiglitazone(ROS) group. The modified DKD model was induced in rats by methods including high-fat diet feeding, unilateral nephrectomy, and streptozotocin(STZ) intraperitoneal injection. After modeling, the rats in the four groups were given double-distilled water, TFA suspension, and ROS suspension correspondingly by gavage every day. At the end of the 8th week of drug administration, all rats were sacrificed, and the samples of urine, blood, and kidney tissues were collected. The parameters and indicators related to IR and podocyte EMT in the DKD model rats were examined and observed, including the general condition, body weight(BW) and kidney weight(KW), the biochemical parameters and IR indicators, the protein expression levels of the key signaling molecules and structural molecules of slit diaphragm in the renal insulin receptor substrate(IRS) 1/phosphatidylinositol 3-kinase(PI3K)/serine-threonine kinase(Akt) pathway, foot process form and glomerular basement membrane(GBM) thickness, the expression of the marked molecules and structural molecules of slit diaphragm in podocyte EMT, and glomerular histomorphological characteristics. The results showed that for the DKD model rats, both TFA and ROS could improve the general condition, some biochemical parameters, renal appearance, and KW. The ameliorative effects of TFA and ROS were equivalent on BW, urinary albumin(UAlb)/urinary creatinine(UCr), serum creatinine(Scr), triglyceride(TG), and KW. Secondly, they could both improve IR indicators, and ROS was superior to TFA in improving fast insulin(FIN) and homeostasis model assessment of insulin resistance(HOMA-IR). Thirdly, they could both improve the protein expression levels of the key signaling molecules in the IRS1/PI3K/Akt pathway and glomerulosclerosis in varying degrees, and their ameliorative effects were similar. Finally, both could improve podocyte injury and EMT, and TFA was superior to ROS. In conclusion, this study suggested that podocyte EMT and glomerulosclerosis could be induced by IR and the decreased activation of the IRS1/PI3K/Akt pathway in the kidney in DKD. Similar to ROS, the effects of TFA in inhibiting podocyte EMT in DKD were related to inducing the activation of the IRS1/PI3K/Akt pathway and improving IR, which could be one of the scientific connotations of TFA against DKD. This study provides preliminary pharmacological evidence for the development and application of TFA in the field of diabetic complications.
Rats ; Animals ; Diabetic Nephropathies/drug therapy* ; Proto-Oncogene Proteins c-akt/metabolism* ; Phosphatidylinositol 3-Kinases/metabolism* ; Abelmoschus/chemistry* ; Podocytes ; Rats, Sprague-Dawley ; Epithelial-Mesenchymal Transition ; Flavones/pharmacology* ; Insulin Resistance ; Reactive Oxygen Species ; Diabetes Mellitus

Renal tubular injury in patients with diabetic kidney disease(DKD) may be accompanied by glomerular and microvascular diseases. It plays a critical role in the progression of renal damage in DKD, and is now known as diabetic tubulopathy(DT). To explore the multi-targeted therapeutic effects and pharmacological mechanisms in vivo of total flavones of Abelmoschus manihot(TFA), an extract from traditional Chinese medicine for treating kidney disease, in attenuating DT, the authors randomly divided all rats into four groups: a normal control group(normal group), a DT model group(model group), a DT model+TFA-treated group(TFA group) and a DT model+rosiglitazone(ROS)-treated group(ROS group). The DT rat model was established based on the DKD rat model by means of integrated measures. After successful modeling, the rats in the four groups were continuously given double-distilled water, TFA suspension, and ROS suspension, respectively by gavage every day. After 6 weeks of treatment, all rats were sacrificed, and the samples of their urine, blood, and kidneys were collected. The effects of TFA and ROS on various indicators related to urine and blood biochemistry, renal tubular injury, renal tubular epithelial cell apoptosis and endoplasmic reticulum stress(ERS), as well as the activation of the protein kinase R-like endoplasmic reticulum kinase(PERK)-eukaryotic translation initiation factor 2α(eIF2α)-activating transcription factor 4(ATF4)-C/EBP homologous protein(CHOP) signaling pathway in the kidney of the DT model rats were investigated. The results indicated that hypertrophy of renal tubular epithelial cells, renal tubular hyperplasia and occlusion, as well as interstitial extracellular matrix and collagen deposition occurred in the DT model rats. Moreover, significant changes were found in the expression degree and the protein expression level of renal tubular injury markers. In addition, there was an abnormal increase in tubular urine proteins. After TFA or ROS treatment, urine protein, the characteristics of renal tubular injury, renal tubular epithelial cell apoptosis and ERS, as well as the activation of the PERK-eIF2α-ATF4-CHOP signaling pathway in the kidney of the DT model rats were improved to varying degrees. Therein, TFA was superior to ROS in affecting the pathological changes in renal tubule/interstitium. In short, with the DT model rats, this study demonstrated that TFA could attenuate DT by multiple targets through inhibiting renal tubular ERS-induced cell apoptosis in vivo, and its effect and mechanism were related to suppressing the activation of the PERK-eIF2α-ATF4-CHOP signaling pathway in the kidney. These findings provided preliminary pharmacological evidence for the application of TFA in the clinical treatment of DT.
Rats ; Animals ; Abelmoschus ; Reactive Oxygen Species/metabolism* ; Flavones/pharmacology* ; Endoplasmic Reticulum Stress ; Diabetic Nephropathies/drug therapy* ; Apoptosis ; Diabetes Mellitus

This study aims to explore the neuroprotective mechanism of ginsenoside Re(GS-Re) on drosophila model of Parkinson's disease(PD) induced by rotenone(Rot). To be specific, Rot was used to induce PD in drosophilas. Then the drosophilas were grouped and respectively treated(GS-Re: 0.1, 0.4, 1.6 mmol·L~(-1); L-dopa: 80 μmol·L~(-1)). Life span and crawling ability of drosophilas were determined. The brain antioxidant activity [content of catalase(CAT), malondialdehyde(MDA), reactive oxygen species(ROS), superoxide dismutase(SOD)], dopamine(DA) content, and mitochondrial function [content of adenosine triphosphate(ATP), NADH:ubiquinone oxidoreductase subunit B8(NDUFB8) Ⅰ activity, succinate dehydrogenase complex, subunit B(SDHB) Ⅱ activity] were detected by enzyme-linked immunosorbent assay(ELISA). The number of DA neurons in the brains of drosophilas was measured with the immunofluorescence method. The levels of NDUFB8 Ⅰ, SDHB Ⅱ, cytochrome C(Cyt C), nuclear factor-E2-related factor 2(Nrf2), heme oxygenase-1(HO-1), B-cell lymphoma/leukemia 2(Bcl-2)/Bcl-2-assaciated X protein(Bax), and cleaved caspase-3/caspase-3 in the brain were detected by Western blot. The results showed that model group [475 μmol·L~(-1) Rot(IC_(50))] demonstrated significantly low survival rate, obvious dyskinesia, small number of neurons and low DA content in the brain, high ROS level and MDA content, low content of SOD and CAT, significantly low ATP content, NDUFB8 Ⅰ activity, and SDHB Ⅱ activity, significantly low expression of NDUFB8 Ⅰ, SDHB Ⅱ, and Bcl-2/Bax, large amount of Cyt C released from mitochondria to cytoplasm, low nuclear transfer of Nrf2, and significantly high expression of cleaved caspase-3/caspase-3 compared with the control group. GS-Re(0.1, 0.4, and 1.6 mmol·L~(-1)) significantly improved the survival rate of PD drosophilas, alleviated the dyskinesia, increased DA content, reduced the loss of DA neurons, ROS level, and MDA content in brain, improved content of SOD and CAT and antioxidant activity in brain, maintained mitochondrial homeostasis(significantly increased ATP content and activity of NDUFB8 Ⅰ and SDHB Ⅱ, significantly up-regulated expression of NDUFB8 Ⅰ, SDHB Ⅱ, and Bcl-2/Bax), significantly reduced the expression of Cyt C, increased the nuclear transfer of Nrf2, and down-regulated the expression of cleaved caspase-3/caspase-3. In conclusion, GS-Re can significantly relieve the Rot-induced cerebral neurotoxicity in drosophilas. The mechanism may be that GS-Re activates Keap1-Nrf2-ARE signaling pathway by maintaining mitochondrial homeostasis, improves antioxidant capacity of brain neurons, then inhibits mitochondria-mediated caspase-3 signaling pathway, and the apoptosis of neuronal cells, thereby exerting the neuroprotective effect.
Animals ; Reactive Oxygen Species/metabolism* ; Antioxidants/pharmacology* ; Oxidative Stress ; NF-E2-Related Factor 2/metabolism* ; Caspase 3/metabolism* ; Parkinson Disease/genetics* ; bcl-2-Associated X Protein/metabolism* ; Neuroprotective Agents/pharmacology* ; Kelch-Like ECH-Associated Protein 1/metabolism* ; Drosophila/metabolism* ; Proto-Oncogene Proteins c-bcl-2/metabolism* ; Apoptosis ; Superoxide Dismutase/metabolism* ; Adenosine Triphosphate/pharmacology*

OBJECTIVE: To summarize the role of chondrocyte mitochondrial homeostasis imbalance in the pathogenesis of osteoarthritis (OA) and analyze its application prospects. METHODS: The recent literature at home and abroad was reviewed to summarize the mechanism of mitochondrial homeostasis imbalance, the relationship between mitochondrial homeostasis imbalance and the pathogenesis of OA, and the application prospect in the treatment of OA. RESULTS: Recent studies have shown that mitochondrial homeostasis imbalance, which is caused by abnormal mitochondrial biogenesis, the imbalance of mitochondrial redox, the imbalance of mitochondrial dynamics, and damaged mitochondrial autophagy of chondrocytes, plays an important role in the pathogenesis of OA. Abnormal mitochondrial biogenesis can accelerate the catabolic reaction of OA chondrocytes and aggravate cartilage damage. The imbalance of mitochondrial redox can lead to the accumulation of reactive oxygen species (ROS), inhibit the synthesis of extracellular matrix, induce ferroptosis and eventually leads to cartilage degradation. The imbalance of mitochondrial dynamics can lead to mitochondrial DNA mutation, decreased adenosine triphosphate production, ROS accumulation, and accelerated apoptosis of chondrocytes. When mitochondrial autophagy is damaged, dysfunctional mitochondria cannot be cleared in time, leading to ROS accumulation, which leads to chondrocyte apoptosis. It has been found that substances such as puerarin, safflower yellow, and astaxanthin can inhibit the development of OA by regulating mitochondrial homeostasis, which proves the potential to be used in the treatment of OA. CONCLUSION The mitochondrial homeostasis imbalance in chondrocytes is one of the most important pathogeneses of OA, and further exploration of the mechanisms of mitochondrial homeostasis imbalance is of great significance for the prevention and treatment of OA.
Humans ; Reactive Oxygen Species/metabolism* ; Chondrocytes/metabolism* ; Osteoarthritis/metabolism* ; Homeostasis ; Mitochondria/metabolism* ; Cartilage, Articular/metabolism*

Objective To study the effect and mechanism of blueberry on regulating the mitochondrial inner membrane protein mitofilin/Mic60 in an in vitro model of metabolic dysfunction-associated liver disease (MAFLD). Methods L02 human hepatocytes were induced by free fatty acids (FFA) to establish MAFLD cell model. A normal group, a model group, an 80 μg/mL blueberry treatment group, a Mic60 short hairpin RNA (Mic60 shRNA) transfection group, and Mic60 knockdown combined with an 80 μg/mL blueberry treatment group were established. The intracellular lipid deposition was observed by oil red O staining, and the effect of different concentrations of blueberry pulp on the survival rate of L02 cells treated with FFA was measured by MTT assay. The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglyceride (TG), total cholesterol (TC), superoxide dismutase (SOD) activity, glutathione (GSH) and malondialdehyde (MDA) contents were measured by visible spectrophotometry. The expression of reactive oxygen species (ROS) in hepatocytes was observed by fluorescence microscopy, and the mRNA and protein expression of Mic60 were detected by real-time quantitative PCR and Western blot analysis, respectively. Results After 24 hours of FFA stimulation, a large number of red lipid droplets in the cytoplasm of L02 cells was observed, and the survival rate of L02 cells treated with 80 μg/mL blueberry was higher. The results of ALT, AST, TG, TC, MDA and the fluorescence intensity of ROS in blueberry treated group were lower than those in model group, while the levels of SOD, GSH, Mic60 mRNA and protein in blueberry treated group were higher than those in model group. Conclusion Blueberry promotes the expression of Mic60, increases the levels of SOD and GSH in hepatocytes, and reduces the production of ROS, thus alleviating the injury of MAFLD hepatocytes and regulating the disorder of lipid metabolism.
Humans ; Blueberry Plants/chemistry* ; Hepatocytes/metabolism* ; Liver/metabolism* ; Liver Diseases/metabolism* ; Reactive Oxygen Species/metabolism* ; Superoxide Dismutase/metabolism* ; Superoxides/metabolism* ; Mitochondrial Membranes/metabolism* ; Mitochondrial Proteins/metabolism* ; Plant Extracts/pharmacology*