Intracellular ROS Concentration Research Articles

Ameliorative effects of osthole on acrylamide-induced neurotoxicity in PC12 cells: Role of oxidative stress, apoptosis and ERK pathways.

The possible protective effects of osthole on acrylamide-induced neurotoxicity in PC12 cells. Cells were pretreated with different concentrations of osthole (1- 25μM) for 24h and then the IC50 value of acrylamide (5mM) was added. After 24h, cell viability and intracellular ROS content were detected by MTT assay and DCF-DA methods, respectively. Also, DNA fragmentation in apoptotic cells was determined by propidium iodide assay, and apoptosis (Caspase-3, Bax, Bcl-2, ERK, and P-ERK) was measured by the western blot method. Exposing PC12 cells to acrylamide diminished cell viability, and enhanced the intracellular ROS generation and the percentage of apoptotic cells. Furthermore, acrylamide elevated the P-ERK/ERK and Bax/Bcl-2 ratio, and the level of cleaved caspase-3 protein in PC12 cells. Pretreating cells with osthole enhanced cell viability and reduced ROS generation. Also, osthole (10μM) significantly reduced P-ERK/ERK and Bax/Bcl-2 ratio, the level of cleaved caspase-3 protein, and the percentage of apoptotic cells in comparison to the acrylamide group. Osthole can exhibit a protective effect on the neurotoxicity of acrylamide through the inhibition of oxidative stress and apoptosis in PC12 cells.

Intracellular Iron Deficiency and Abnormal Metabolism, Not Ferroptosis, Contributes to Homocysteine-Induced Vascular Endothelial Cell Death.

Background/Objectives: Homocysteine (Hcy) and iron are factors co-related with the progression of cardiovascular diseases. The vascular endothelium is an important barrier for physiological homeostasis, and its impairment initiates cardiovascular injury. However, the mechanism underlying Hcy-caused vascular endothelial cell injury and the participation of iron are not fully elucidated. This study aims to investigate the Hcy-induced vascular endothelial injury and iron metabolism dysfunction as well as the underlying molecular mechanism. Methods: Human umbilical vein endothelial cells (HUVECs) were employed as the experimental model to examine the Hcy-induced endothelial injury and its underlying mechanism via various biochemical assays. Results: Hcy suppressed the cell viability and proliferation and caused cell death in a concentration-dependent manner. Hcy induced cell cycle arrest, apoptosis, and autophagy as well as impairment of intracellular energy metabolism. Hcy disrupted the intracellular antioxidant system and mitochondrial function by increasing intracellular ROS, MDA and mitochondrial content, and decreasing the SOD activity and mitochondrial membrane potential. Hcy significantly reduced the GSH-Px activity along with the accumulation of intracellular GSH in a concentration-dependent manner. Ferroptosis inhibitors, Ferrostatin-1 (Fer-1), and Deferoxamine (DFO) significantly decreased the Hcy-caused cytotoxicity accompanied by a reduction in dysregulated mitochondria content, but only DFO ameliorated the elevation of intracellular ROS, and neither Fer-1 nor DFO affected the Hcy-caused reduction in intracellular ATP. In addition, Hcy decreased the intracellular concentration of iron, and supplementing Hcy with various concentrations of Fe3+ increased the cell viability and decreased the LDH release in a concentration-dependent manner. Hcy dramatically decreased the mRNA expression level of transferrin receptor while increasing the mRNA expression levels of transferrin, ferritin light chain, ferritin heavy chain, ferroportin, and SLC7A11. Moreover, Hcy suppressed the protein expression of phospho-Akt, phospho-mTOR, Beclin-1, LC3A/B, Nrf2, HO-1, phospho-MEK1/2, phospho-ERK1/2, and Caspase-3 in concentration- and time-dependent manners. Conclusions: Hcy-induced vascular endothelial injury is likely to be associated with apoptosis and autophagy, but not ferroptosis. The key underlying mechanisms are involved in the disruption of the intracellular antioxidant system and iron metabolism via regulation of PI3K/Akt/mTOR, MAPKs, Nrf2/HO-1, and iron metabolism.

Catalpol inhibits HHcy-induced EndMT in endothelial cells by modulating ROS/NF-κB signaling

BackgroundHyperhomocysteinemia (HHcy) is an independent risk factor for atherosclerosis (AS). Endothelial mesenchymal transition (EndMT) refers to the process in which endothelial cells lose endothelial cell morphology and characteristic gene expression, and acquire phenotypic characteristics and gene expression related to mesenchymal cells. Numerous studies have confirmed that EndMT is involved in the formation of atherosclerosis. Catalpol is one of the active components of Rehmannia, which has antioxidant, anti-inflammatory, anti-tumor, neuroprotective and other biological activities. Studies have shown that catalpol can reduce atherosclerotic plaque induced by high sugar or fat. However, the effect of catalpol on HHCY-induced EndMT is unclear.Methods and resultsIn vitro HHcy-treated primary human umbilical vein endothelial cells (HUVECs) were used to construct a cell model, and the antioxidants N-acetylcysteine (NAC) and catalase alcohol were administered. In vivo C57BL/6N mice were given a diet fed with 4.4% high methionine chow to construct a HHcy mice model and were treated with catalpol. The results showed that hhcy could induce morphological transformation of endothelial cells into mesenchymal cells, increase intracellular ROS content, up-regulate α-SMA, N-cadherin, p-p65 protein expression, down-regulate VE-cadherin, CD31 protein expression, induce pathological changes of aortic root endothelium, and increase aortic endothelial ROS content. Catalpol reversed these hhcy induced outcomes.ConclusionsCatalpol inhibits HHcy-induced EndMT, and the underlying mechanism may be related to the ROS/NF-κB signaling pathway. Catalpol may be a potential drug for the treatment of HHcy-related AS.

New insights into the pro-oxidant mechanism of dehydroleucodine on Trypanosoma cruzi

Chagas disease, caused by Trypanosoma cruzi (T. cruzi), is one of the most important neglected diseases in Latin America. The limited use of the current nitro-derivative-based chemotherapy highlights the need for alternative drugs and the identification of their molecular targets. In this study, we investigated the trypanocidal effect of the sesquiterpene lactone dehydroleucodine (DhL) and its derivatives, focusing on the antioxidative defense of the parasites. DhL and two derivatives, at lesser extent, displayed antiproliferative effect on the parasites. This effect was blocked by the reducing agent glutathione (GSH). Treated parasites exhibited increased intracellular ROS concentration and trypanothione synthetase activity, accompanied by mitochondrial swelling. Although molecular dynamics studies predicted that GSH would not interact with DhL, 1H-NMR analysis confirmed that GSH could protect parasites by interacting with the lactone. When parasites overexpressing mitochondrial tryparedoxin peroxidase were incubated with DhL, its effect was attenuated. Overexpression of cytosolic tryparedoxin peroxidase also provided some protection against DhL. These findings suggest that DhL induces oxidative imbalance in T. cruzi, offering new insights into potential drug targets against this parasite.

Sertraline Alleviates Chronic Prostatitis by Regulating the TRPV1 Channel.

Although sertraline has been widely used for chronic prostatitis (CP), the mechanisms are unclear. Herein, we explored the mechanisms of sertraline in treating CP. Network pharmacology methods were used to explore the potential targets and molecular mechanisms. LPS was used to stimulate RWPE-1 cells to construct an in vitro model of CP. An experimental autoimmune prostatitis (EAP) mice model was built. CCK-8 assay, EdU assay, BrdU detection, and Tunel assay were performed to evaluate the proliferation and apoptosis process of cells or tissues, respectively. DCFH-DA and Fluo-4 fluorescence probes were used to detect intracellular ROS and calcium concentrations. Von Frey filaments and open-field tests were utilized to evaluate pain response and depressive-like behavior of mice. Histopathology was evaluated through hematoxylin and eosin staining. RT-qPCR, Western blot, immunofluorescence, and immunohistochemistry were utilized to evaluate the transcription, expression, and location of related proteins. Molecular dynamics (MD) simulation and surface plasmon resonance (SPR) assay were performed to measure the binding capacity of sertraline and related proteins. Through a network pharmacology analysis, 27 potential targets of sertraline for CP were obtained, and 5 key targets (CHRM1, ADRA1B, HTR2B, HTR2A, and TRPV1) were finally identified. Functional experiments suggested that TRPV1 was involved in the proliferation, apoptosis inhibition, and ROS production of LPS-induced RWPE-1 cells. In vitro experiments showed that sertraline significantly inhibited cell proliferation, ROS generation, and transcription of inflammation cytokines of LPS-induced RWPE-1 cells. Additionally, sertraline markedly promoted the apoptosis level of LPS-stimulated RWPE-1 cells and elevated the expression level of BAX while reducing the expression levels of Bcl2 and Caspase-3. MD simulation and SPR assay confirmed the direct binding of sertraline to TRPV1. Moreover, sertraline significantly down-regulated the expression level of TRPV1 and inhibited calcium influx of LPS-induced RWPE-1 cells. TRPV1 agonist (Capsaicin) significantly restored the effects on proliferation, apoptosis, ROS production, and calcium influx of sertraline on LPS-induced RWPE-1 cells. Mice experiments demonstrated that sertraline treatment could reduce pain response, improve depression-like symptoms, and relieve local prostate inflammation of EAP mice, as well as down-regulated the expression level of TRPV1, inhibit the proliferation, and promote apoptosis of prostate tissues in EAP mice. The results revealed the anti-inflammatory effect of sertraline for RWPE-1 cells and EAP mice, and the potential mechanism was regulating the TRPV1 channel. It indicated that sertraline might serve as a complementary anti-inflammatory agent for CP.

Modification of mesenchymal stromal cells with silibinin-loaded PLGA nanoparticles improves their therapeutic efficacy for cutaneous wound repair

The use of mesenchymal stromal cells (MSCs) for treating chronic inflammatory disorders, wounds, and ischemia-reperfusion injuries has shown improved healing efficacy. However, the poor survival rate of transplanted cells due to oxidative stress in injured or inflamed tissue remains a significant concern for MSC-based therapies. In this study, we developed a new approach to protect MSCs from oxidative stress, thereby improving their survival in a wound microenvironment and enhancing their therapeutic effect. We produced PLGA nanoparticles loaded with the cytoprotective phytochemical silibinin (SBN), and used them to modify MSCs. Upon internalization, these nanoformulations released SBN, activating the Nrf2/ARE signaling pathway, resulting in threefold reduction in intracellular ROS content and improved cell survival under oxidative stress conditions. Modification of MSCs with SBN-loaded PLGA nanoparticles increased their survival upon transplantation to full-thickness cutaneous wounds and improved wound healing. This study suggests that MSC modification with cytoprotective nanoparticles could be a promising approach for improving wound healing.

Synergistic Effect of Layered Double Hydroxides Nanodosage Form to Induce Apoptosis and Ferroptosis in Breast Cancer.

Breast cancer is the most common cancer in women and one of the leading causes of cancer death worldwide. Ferroptosis, a promising mechanism of killing cancer cells, has become a research hotspot in cancer therapy. Simvastatin (SIM), as a potential new anti-breast cancer drug, has been shown to cause ferroptosis of cancer cells and inhibit breast cancer metastasis and recurrence. The purpose of this study is to develop a novel strategy boosting ferroptotic cascade for synergistic cancer therapy. In this paper, iron base form of layered double hydroxide supported simvastatin (LDHs-SIM) was synthesized by hydrothermal co-precipitation method. The characterization of LDHs-SIM were assessed by various analytical techniques, including ultraviolet-visible (UV-vis) spectroscopy, X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy, and transmission electron microscopy (TEM). Biological activity, ferroptosis mechanism and biocompatibility were analyzed through in vivo and in vitro analysis, so as to evaluate its therapeutic effect on breast cancer. The constructed LDHs-SIM nanosystem can not only release SIM through mevalonate (MVA) pathway, inhibit the expression of glutathione peroxidase 4 (GPX4), inhibit the expression of SLC7A11 and reduce the synthesis efficiency of GSH, but also promote the accumulation of Fe2+ in cells through the release of Fe3+, and increase the intracellular ROS content. In addition, LDHs-SIM nanosystem can induce apoptosis of breast cancer cells to a certain extent, and achieve the synergistic effect of apoptosis and ferroptosis. In the present study, we demonstrated that nanoparticles of layered double hydroxides (LDHs) loaded with simvastatin were more effective than a free drug at inhibiting breast cancer cell growth, In addition, superior anticancer therapeutic effects were achieved with little systemic toxicity, indicating that LDHs-SIM could serve as a safe and high-performance platform for ferroptosis-apoptosis combined anticancer therapy.

Feasibility and mechanism of removing Microcystis aeruginosa and degrading microcystin-LR by dielectric barrier discharge plasma

Harmful cyanobacterial bloom is one of the serious environmental problems worldwide. Microcystis aeruginosa is a representative harmful alga in cyanobacteria bloom. It is of great significance to develop new technologies for the removal of Microcystis aeruginosa and microcystins. The feasibility and mechanism of removing microcystis aeruginosa and degrading microcystins by dielectric barrier discharge (DBD) plasma were studied. The suitable DBD parameters obtained in this study are DBD (41.5 W, 40 min) and DBD (41.5 W, 50 min), resulting in algae removal efficiency of 77.4% and 80.4%, respectively; scanning electron microscope and LIVE-DEATH analysis demonstrate that DBD treatment can disrupt cell structure and lead to cell death; analysis of elemental composition and chemical state indicated that there are traces of oxidation of organic nitrogen and organic carbon in microcystis aeruginosa; further intracellular ROS concentration and antioxidant enzyme activity analysis confirm that DBD damage microcystis aeruginosa through oxidation. Meanwhile, DBD can effectively degrade the microcystin-LR released after cell lysis, the extracellular microcystin-LR concentration in the DBD (41.5 W) group decreased by 88.7% at 60 min compared to the highest concentration at 20 min; further toxicity analysis of degradation intermediates indicated that DBD can reduce the toxicity of microcystin-LR. The contribution of active substances to the inactivation of microcystis aeruginosa is eaq− >•OH > H2O2 > O3 > 1O2 >•O2− >ONOO−, while on the degradation of microcystin-LR is eaq− >•OH > H2O2 > O3 >•O2− >1O2 > ONOO−. The application of DBD plasma technology in microcystis aeruginosa algae removal and detoxification has certain prospects for promotion and application.

Enhancement of Lycopene Synthesis via Low-Frequency Alternating Magnetic Field in Brassica trispora

In recent years, magnetic fields have emerged as a non-thermophysical treatment with a significant impact on microbial fermentation processes. Brassica trispora is a microorganism known for its industrial-scale production of lycopene and high yield of single cells. This study aimed to investigate the impact of low-frequency magnetic fields on lycopene synthesis by Brassica trispora and elucidate the underlying mechanism for enhancing lycopene yield. The results indicate that both the intensity and duration of the magnetic field treatment influenced the cells. Exposing the cells to a 0.5 mT magnetic field for 48 h on the second day of fermentation resulted in a lycopene yield of 25.36 mg/g, representing a remarkable increase of 244.6% compared to the control group. Transcriptome analysis revealed that the alternating magnetic field significantly upregulated genes related to ROS and the cell membrane structure, leading to a substantial increase in lycopene production. Scanning electron microscopy revealed that the magnetic field treatment resulted in a rough, loose, and wrinkled surface morphology of the mycelium, along with a few micropores, thereby altering the cell membrane permeability to some extent. Moreover, there was a significant increase in intracellular ROS content, cell membrane permeability, key enzyme activity involved in lycopene metabolism, and ROS-related enzyme activity. In conclusion, the alternating frequency magnetic field can activate a self-protective mechanism that enhances lycopene synthesis by modulating intracellular ROS content and the cell membrane structure. These findings not only deepen our understanding of the impact of magnetic fields on microbial growth and metabolism but also provide valuable insights for developing innovative approaches to enhance carotenoid fermentation.

Synergistic celecoxib and dimethyl-celecoxib combinations block cervix cancer growth through multiple mechanisms.

The synergistic inhibitory effect of celecoxib (CXB) and dimethyl-celecoxib (DMC) plus paclitaxel (PA) or cisplatin (CP) on human cervix HeLa and SiHa cells was assessed at multiple cellular levels in order to elucidate the biochemical mechanisms triggered by the synergistic drug combinations. The effect of CXB (5 μM)/CP (2 μM) or CXB (5 μM)/PA (15 μM) and DMC (15 μM)/CP (5 μM) or DMC (15 μM)/PA (20 μM) for 24 h was assayed on cancer cell proliferation, energy metabolism, mitophagy, ROS production, glycoprotein-P activity, DNA stability and apoptosis/necrosis. Drug combinations synergistically decreased HeLa and SiHa cell proliferation (>75%) and arrested cellular cycle by decreasing S and G2/M phases as well as the Ki67 content (HeLa) by 7.5-30 times. Cell viability was preserved (>90%) and no apparent effects on non-cancer cell growth were observed. Mitochondrial and glycolytic protein contents (44-95%) and ΔΨm (45-50%) in HeLa cells and oxidative phosphorylation and glycolysis fluxes (70-90%) in HeLa and SiHa cells were severely decreased, which in turn promoted a drastic fall in the ATP supply (85-88%). High levels of mitophagy proteins in HeLa cells and active mitochondrial digestion in HeLa and SiHa cells was observed. Mitochondrial fission and microtubule proteins were also affected. Intracellular ROS content (2-2.3-fold) and ROS production was stimulated (2.3-4 times), whereas content and activity of glycoprotein-P (45-85%) were diminished. DNA fragmentation was not observed and apoptosis/necrosis was not detected suggesting that cell death could be mainly associated to mitophagy induction. CXB or DMC combination with canonical chemotherapy may be a promising chemotherapy strategy against cervical cancer growth, because it can selectively block multiple cell processes including inhibition of energy pathways and in consequence ATP-dependent processes such as cell proliferation, glycoprotein-P activity, ROS production and mitophagy, with no apparent effects on non-cancer cells.

Neuroprotective Effect of Antiapoptotic URG7 Protein on Human Neuroblastoma Cell Line SH-SY5Y.

Up-regulated Gene clone 7 (URG7) is a protein localized in the endoplasmic reticulum (ER) and overexpressed in liver cells upon hepatitis B virus (HBV) infection. Its activity has been related to the attenuation of ER stress resulting from HBV infection, promoting protein folding and ubiquitination and reducing cell apoptosis overall. While the antiapoptotic activity of URG7 in HBV-infected cells may have negative implications, this effect could be exploited positively in the field of proteinopathies, such as neurodegenerative diseases. In this work, we aimed to verify the possible contribution of URG7 as a reliever of cellular proteostasis alterations in a neuronal in vitro system. Following tunicamycin-induced ER stress, URG7 was shown to modulate different markers of the unfolded protein response (UPR) in favor of cell survival, mitigating ER stress and activating autophagy. Furthermore, URG7 promoted ubiquitination, and determined a reduction in protein aggregation, calcium release from the ER and intracellular ROS content, confirming its pro-survival activity. Therefore, in light of the results reported in this work, we hypothesize that URG7 offers activity as an ER stress reliever in a neuronal in vitro model, and we paved the way for a new approach in the treatment of neurodegenerative diseases.

Modeling heterogeneity in a cell culture using a coupled population balance-oxidative stress model

Population balance models (PBMs) were formulated to describe the evolution in time of the cell population in terms of growth and oxidative stress. Flow cytometry data was used to gain insight into the distribution of important quantities (e.g., cell size, intracellular concentrations of metabolites) over the entire cell population. A coupled population balance-oxidative stress model was developed to predict distributions in cell size and intracellular glutamate, ROS, NADPH and NADP+ concentrations in shake flask cultures of B. pertussis. The major advantage of using a PBM is that it accounts for the distributions and can predict the heterogeneity of the cell population with respect to experimental conditions that are averaged out in bulk models. When comparing the coupled population balance – oxidative stress model to the bulk oxidative stress model, it is apparent that the PBM provides much better predictions of the intracellular ROS concentration. We hypothesize that due to the nonlinear relations between cell growth and oxidative stress, intracellular and cell surface quantities can be better modeled with population balance models.

All-in-One Engineering Multifunctional Nanoplatforms for Sensitizing Tumor Low-Temperature Photothermal Therapy In Vivo.

Low-temperature photothermal therapy (PTT) is a noninvasive method that harnesses the photothermal effect at low temperatures to selectively eliminate tumor cells, while safeguarding normal tissues, minimizing thermal damage, and enhancing treatment safety. First we evaluated the transcriptome of tumor cells at the gene level following low-temperature treatment and observed significant enrichment of genes involved in cell cycle and heat response-related signaling pathways. To address this challenge, we have developed an engineering multifunctional nanoplatform that offered an all-in-one strategy for efficient sensitization of low-temperature PTT. Specifically, we utilized MoS2 nanoparticles as the photothermal core to generate low temperature (40-48 °C). The nanoplatform was coated with DPA to load CPT-11 and Fe2+ and was further modified with PEG and iRGD to enhance tumor specificity (MoS2/Fe@CPT-11-PEG-iRGD). Laser- and acid-triggered release of CPT-11 can significantly increase intracellular H2O2 content, cooperate with Fe2+ ions to increase intracellular lipid ROS content, and activate ferroptosis. Furthermore, CPT-11 induced cell cycle arrest in the temperature-sensitive S-phase, and increased lipid ROS levels contributed to the degradation of HSPs protein expression. This synergistic approach could effectively induce tumor cell death by the sensitized low-temperature PTT and the combination of ferroptosis and chemotherapy. Our nanoplatform can also maximize tumor cell eradication and prolong the survival time of tumor-bearing mice in vivo. The multifunctional approach will provide more possibilities for clinical applications of low-temperature PTT and potential avenues for the development of multiple tumor treatments.

Characterization of the effect of pomegranate crude extract, and its post-harvesting preservation procedures, on redox tone, cellular growth and metabolic profile of MDA-MB-231 cell line

BackgroundPomegranate is known for its beneficial properties due to its high content in antioxidants and might constitute a natural option for preventing and treatment of different pathologies including cancer. Since mitochondria are involved in tumorigenesis through ROS production and modulation of oxidative metabolism, we investigated the biological effects of pomegranate on cellular redox state, proliferation and metabolism in the breast cancer cell line MDA-MB-231 (MDA).MethodsMDA were treated for 24 h with graded concentration of filtered Pomegranate juice (PJ) and tested for metabolic Flux Analysis with XFe96 Extracellular Flux Analyzer, for proliferation using the xCELLigence System Real-Time Cell Analyzer and for intracellular ROS content by Confocal Microscopy Imaging.ResultsCells-treatment with freshly prepared pomegranate juice (PJ) resulted in a significant reduction of the intracellular ROS content already at the lower concentration of PJ tested. Additionally, it enhanced mitochondria respiration, and decreased glycolysis at high concentrations, inhibiting at the same time cell proliferation. As pomegranate is a seasonal fruit, assessment of optimum storage conditions preserving its bio-active properties was investigated. Our results indicated that storage conditions under controlled atmosphere for 30 days was able to enhance mitochondrial respiration at the same extent than freshly extracted PJ. Conversely, freezing procedure, though retaining the antioxidant and cell-growth inhibitory property, elicited an opposite effect on the metabolic profile as compared with fresh extract.ConclusionOverall, the results of our study, on the one hand, confirms the preventive/therapeutic potential of PJ, as well as of its post-harvested processing, for cancer management. On the other hand, it highlights the intrinsic difficulties in attaining mechanistic insights when a multiplicity of effects is elicited by a crude mixture of bio-active compounds.

Effect of Bovine Milk Peptides on Cell Inflammation, Proliferation and Differentiation: Milk Potential Benefits Are Preserved in an Unconventional Cow Feeding Strategy.

Animal feeding through the reuse of agro-industrial by-products in one of the ultimate goals of sustainable agriculture. Olive oil pomace (OOP) produced as a waste product during olive oil milling has been used as an ingredient in the diet for Holstein lactating cows. Recent findings have shown no decrease in animal performance, feed intake or detrimental effect on rumen microbiota. In contrast, an improvement in C18 polyunsaturated fatty acids has been observed. In this work, the milk protein content from cows fed a commercial diet (CON) or an experimental one supplemented with OOP was determined and compared, and the peptides derived from the simulated gastrointestinal digestion of raw milk were analyzed. After fractionation via RP-HPLC, peptides were characterized for their biological activity on different cell lines. The ability to reduce both the intracellular ROS content and the expression of inflammatory markers, such as Cyclooxygenase, isoenzyme 2 (COX-2) and inducible Nitric Oxide Synthase (iNOS), as well as the remarkable properties to induce cell differentiation and to slow down the proliferation of human intestinal cancer cells, enable us to define them as bioactive peptides. In spite of there being no observed significant difference between the healthy activity of CON and OOP peptides, the results allow us to broaden the knowledge about the biological activity of these bioactive peptides and to confirm that agro-industrial by-products may be successfully incorporated into the feeding strategy of dairy cows.

Tanshinone ⅡA activates PI3K/AKT signaling pathway to inhibit the apoptosis of mice cochlear pericytes induced by high glucose

Objective: To investigate whether tanshinone ⅡA can protect the apoptosis of mice cochlear pericytes induced by high glucose and its specific protective mechanism, so as to provide experimental evidence for the prevention and treatment of diabetic hearing loss. Methods: C57BL/6J male mice were used to prepare type 2 diabetes model, which were divided into normal (NG) group, diabetic (DM) group, diabetic+tanshinone ⅡA (HG+tanshinone ⅡA) group and tanshinone ⅡA group. Each group had 10 animals. Primary cochlear pericytes were divided into NG group, HG group (high glucose 35 mmol/L), HG+tanshinone ⅡA (1, 3, 5 μmol/L) group, HG+Tanshinone ⅡA+LY294002 (PI3K/AKT pathway inhibitor) group, LY294002 group, tanshinone ⅡA group and DMSO group. Auditory brainstem response (ABR) was used to measure hearing threshold. Evans blue was used to detect the permeability of blood labyrinth barrier in each group. TBA methods were used to detect oxidative stress levels in various organs of mice. Morphological changes of stria vascularis were observed by hematoxylin-eosin staining (HE). Evans blue was used to detect the vascular labyrinth barrier permeability in cochlea. The expression of apoptosis protein in stria vascularis pericytes was observed by immunofluorescence. Pericytes apoptosis rate was observed by flow cytometry. DCFH-DA was combined with flow cytometry to detect intracellular ROS content, and Western blot was used to detect the expression of apoptotic proteins (Cleaved-caspase3, Bax), anti-apoptotic proteins (BCL-2) and pathway proteins (PI3K, p-PI3K, AKT, p-AKT). SPSS software was used for statistical analysis. Independent sample t test was performed, and P<0.05 was considered statistically significant. Results: Animal experiments: Tanshinone ⅡA decreased the hearing threshold of DM group [(35.0±3.5) dB SPL vs. (55.3±8.1) dB SPL] (t=4.899, P<0.01), decreased the oxidative stress level in cochlea (t=4.384, P<0.05), improved the structure disorder, atrophy of cochlea vascular lines, vacuole increased phenomenon. Tanshinone ⅡA alleviated the increased permeability of the blood labyrinth barrier [Evans blue leakage (6.84±0.27) AU vs. (8.59±0.85) AU] in the cochlea of DM mice (t=2.770, P<0.05), reversed the apoptotic protein: Caspase3 (t=4.956, P<0.01) and Bax (t=4.388, P<0.05) in cochlear vascularis. Cell experiments: Tanshinone ⅡA decreased intracellular ROS content in a concentration-dependent way (t=3.569, P<0.05; t=4.772, P<0.01; t=7.494, P<0.01); Tanshinone ⅡA decreased apoptosis rate and apoptotic protein, and increased the expression of anti-apoptotic protein, p-PI3K/PI3K and p-AKT/AKT in concentration-dependent manner (all P values<0.05); LY294002 reversed the protective effect of tanshinone ⅡA on pericytes apoptosis (all P values<0.05). Conclusion: Tanshinone ⅡA can inhibit the apoptosis of cochlear pericytes induced by high glucose by reducing oxidative stress level and activating PI3K/AKT signaling pathway under high glucose environment, thus playing a protective role in diabetic hearing loss.

Evaluation of the Anticancer Activity and Mechanism Studies of Glycyrrhetic Acid Derivatives toward HeLa Cells.

In this paper, a series of glycyrrhetic acid derivatives 3a-3f were synthesized via the esterification reaction. The cytotoxicity of these compounds against five tumor cells (SGC-7901, BEL-7402, A549, HeLa and B16) and normal LO2 cells was investigated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. The results showed that compound 3a exhibited high antiproliferative activity against HeLa cells (IC50 = 11.4 ± 0.2 μM). The anticancer activity was studied through apoptosis, cloning, and scratching; the levels of the intracellular ROS, GSH, and Ca2+; and the change in the mitochondrial membrane potential, cell cycle arrest and RNA sequencing. Furthermore, the effects of compound 3a on gene expression levels and metabolic pathways in HeLa cells were investigated via transcriptomics. The experimental results showed that this compound can block the cell cycle in the S phase and inhibit cell migration by downregulating Focal adhesion kinase (FAK) expression. Moreover, the compound can reduce the intracellular glutathione (GSH) content, increase the Ca2+ level and the intracellular ROS content, and induce a decrease in the mitochondrial membrane potential, further leading to cell death. In addition, it was also found that the mechanism of compounds inducing apoptosis was related to the regulation of the expression of mitochondria-related proteins B-cell lymphoma-2 (Bcl-2), Bcl-2-Associated X (Bax), and the activation of the caspase proteins. Taken together, this work provides a help for the development of glycyrrhetinic acid compounds as potential anticancer molecules.

Houttuynia cordata Thunb. crude extract inactivates Cronobacter sakazakii: Antibacterial components, antibacterial mechanism, and application as a natural disinfectant

The purpose of this study was to reveal the antibacterial components of Houttuynia cordata Thunb. crude extract (HCCE), analyze the antibacterial activity and mechanism of HCCE against Cronobacter sakazakii, and evaluate the bactericidal effect of HCCE as a natural disinfectant. Using an ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-QE-MS) non-target metabolomics technology, 15 kinds oforganic acids (18.65%), 14 kinds of phenols (16.17%), 10 kinds of flavonoids (12.32%), 5 kinds of terpenoids (5.87%), 3 kinds of alkaloids (1.62%), and 3 kinds of aldehydes (1.28%) were detected from HCCE. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of HCCE against C. sakazakii were 7.5 mg/mL and 15 mg/mL, respectively, and when the concentration was 2 MIC, C. sakazakii stagnated in sluggish phase. C. sakazakii exposed to HCCE showed hyperpolarization of cell membrane, increased intracellular ROS content, decreased protein and DNA content, impaired cell membrane integrity, improved cell membrane permeability, cell fluid leakage, and partial cell fragmentation. After treatments with 1 MIC and 2 MIC of HCCE for 5 min, C. sakazakii strains on 8 contact surfaces including stainless steel, tinplate, glass, polystyrene, bamboo, plank, ceramic, and tin foil paper were significantly inhibited (P < 0.05), and the reduction amounts ranged from 3.00 to 3.68 log CFU/100 cm2 and 3.97 to 4.53 log CFU/100 cm2, respectively. These findings demonstrate the potential of HCCE as a natural disinfectant and provide a new idea to reduce the C. sakazakii contamination caused by surface contact.

Individual and joint effects of glyphosate and cypermethrin formulations on two human cell lines

The final effect of pesticides and their mixtures on living organisms is determined by the particular toxicodynamics of the system. Oxidative stress is one of the most studied molecular mechanisms of toxicity due to increasing evidence supporting its association with the toxic effects of different agrochemicals. In the present study we evaluated the presence of redox balance alterations in the cell lines HEp-2 and A549 exposed to formulations of glyphosate (March®) and cypermethrin (Superfina®) used separately or in combination (in a proportion equivalent to that used in soybean fields). We determined the activity of catalase, superoxide dismutase, glutathione S-transferase, intracellular GSH content, content of oxidized proteins (as measure of damage) and intracellular ROS content in both cell lines at two different mixture concentrations. Additionally, we evaluated the presence of statistical interaction to determine if the effect of the mixture on the parameters evaluated was additive, synergistic, or antagonistic. For this purpose, we used the Combination Subthresholding, Cooperative Effect and Statistical Linear Interaction approaches. We found that the interaction between pesticides depended on their concentration and the cellular models studied.

Synthesis, biological evaluation of novel iridium(III) complexes targeting mitochondria toward melanoma B16 cells

A new ligand 2-(1E,3E,5E,7E)-2,6-dimethyl-8-(2,6,6-trimethylcyclohex-1-yl)octa-1,2,5,7-tetraen-1-yl)-1H-imidazo[4,5-f][1,10]phenanthroline (DTOIP) was synthesized and combined with [Ir(ppy)2Cl]2·2H2O (ppy = deprotonated Hppy: 2-phenylpyridine), [Ir(piq)2Cl]2·2H2O (piq = deprotonated Hpiq: 1-phenylisoquinoline) and [Ir(bzq)2Cl]2·2H2O (bzq = deprotonated Hbzq: benzo[h]quinolone) to form [Ir(ppy)2(DTOIP)](PF6) (Ir1), [Ir(piq)2(DTOIP)](PF6) (Ir2), and [Ir(bzq)2(DTOIP)](PF6) (Ir3), respectively. The complexes were characterized by elemental analysis, high-resolution mass spectrometry (HRMS), 1H NMR and 13C NMR. The antiproliferative activity of the complexes toward B16, BEL-7402, Eca-109 and normal LO2 cells was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. Complexes Ir1, Ir2 and Ir3 showed high antiproliferative activity against B16 cells with a low IC50 values of 0.4 ± 0.1, 2.0 ± 0.1 and 1.4 ± 0.09 μM, respectively. Three-dimensional (3D) in vitro cell models also demonstrated that the iridium(III) complexes have a remarkable cytotoxicity to B16 cells. The experiments of cellular uptake, mitochondrial localization, and intracellular distribution of the drugs proved that the three iridium(III) complexes can enter the mitochondria, leading to the loss of mitochondrial membrane potential (MMP), decreased glutathione (GSH) levels, causing an increase of intracellular ROS content, and DNA damage, finally inducing apoptosis. RNA-sequence and bioinformatics analyses were used to analyze the differentially expressed genes and enriched biology processes. Antitumor in vivo demonstrated that complex Ir1 (5 mg/kg) exhibits a high efficacy to inhibit the tumor growth with an inhibitory rate of 71.67%. These results show that the complexes may be potent anticancer candidate drugs.