Levels Of Apoptotic Markers Research Articles

Novel Pathways of Oxidative and Nitrosative Inactivation of the Human MGMT Protein in Colon Cancer and Glioblastoma Cells: Increased Efficacy of Alkylating Agents In Vitro and In Vivo

Background: O6-Methylguanine-DNA methyltransferase (MGMT) is a unique antimutagenic DNA repair protein that plays a crucial role in conferring resistance to various alkylating agents in brain tumor therapy. In this study, we exploited the susceptibility of the active site Cys145 of MGMT for thiolation and nitrosylation, both of which inactivate the enzyme. Methods: We designed a redox perturbing glutathione mimetic, a platinated homoglutathione disulfide (hGTX) by adding small amounts of cisplatin (1000:10) and used a nitric oxide-donor spermine NONOate. N6022, a potent inhibitor of S-nitrosoglutathione reductase was used to extend the retention of nitrosylated MGMT in tumor cell culture and subcutaneous xenografts. Results: Both hGTX and spermine NONOate inhibited MGMT activity in HT29, SF188, T98G, and other brain tumor cells. There was a robust increase in the alkylation-induced DNA interstrand cross-linking, G2/M cell cycle arrest, cytotoxicity, and the levels of apoptotic markers when either of the agents was used with alkylating agents. In the nude mice bearing T98G and HT29-luc2 xenografts, combinations of hGTX and spermine NONOate with alkylating agents produced a marked reduction in MGMT protein and tumor growth delay and regressions. N6022 treatment increased the presence of nitrosylated MGMT for a longer time, thereby extending the DNA-repair deficient state both in cell culture and preclinical settings. Conclusions: Our findings highlight the options for redox-driven therapeutic strategies for MGMT and suggest that oxidative and/or nitrosative inactivation of DNA repair in combination with alkylating agents could be exploited.

Phellodendrine inhibits oxidative stress and promotes autophagy by regulating the AMPK/mTOR pathway in burn sepsis-induced intestinal injury.

Intestinal injury is an important complication of burn sepsis with limited therapeutic choices. Phellodendrine is a promising compound for gastrointestinal inflammatory diseases and is extracted from the traditional Chinese medicine phellodendron bark. The study aimed to explore the role of phellodendrine against oxidative stress and autophagy in burn sepsis-induced intestinal injury. A mouse model of burn sepsis model was established by intraperitoneally injecting 10mg/kg lipopolysaccharide (LPS) to mice burned by boiled water. Phellodendrine (30mg/kg) was injected into mice in the drug group after scalding and before LPS injection. Hematoxylin and eosin staining was performed to observe histopathological changes in murine small intestines. TdT-mediated dUTP Nick-End Labeling (TUNEL) assay was performed to evaluate intestinal cell apoptosis. Immunofluorescence staining was performed to measure the expression and distribution of autophagy markers, light chain 3II (LC3II) and p62 in intestinal tissues. Oxidative stress indicators were detected using corresponding commercial kits. Protein levels of apoptotic markers, autophagy markers, and factors involved in adenosine monophosphate-activated protein kinase (AMPK)/mechanistic target of rapamycin (mTOR) pathway in intestines were quantified by western blotting. Phellodendrine attenuated bun sepsis-induced intestinal pathological changes. Meanwhile, aggravated cell apoptosis, reduction of antioxidant enzymes, and downregulation of autophagy markers in intestinal tissues of burn sepsis group were all improved by phellodendrine. In addition, phellodendrine activated the phosphorylation (p) of AMPK and inhibited p-mTOR signaling in intestines of burn septic mice. In conclusion, phellodendrine suppresses oxidative stress and activates autophagy in burn sepsis-induced intestinal injury by activating AMPK and inhibiting mTOR signaling.

FABP4 Inhibitor Alleviates Lipopolysaccharide-Induced HUVEC Injury by Inactivating NF-κB and Activating PPARγ.

The study aims to examine the effect of FABP4 on inflammatory response and angiogenesis in the cell model of atherosclerosis and to explore its potential mechanism.Using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting, the mRNA and protein levels of FABP4 in human umbilical vein endothelial cells (HUVECs) treated with lipopolysaccharide (LPS) or oxidized low-density lipoprotein for 6, 12, 24, or 48 hours were measured. To silence FABP4 expression and NF-κB signaling in HUVECs, FABP4 inhibitor and NF-κB inhibitor were utilized. To assess cell survival rate and apoptosis, methyl thiazolyl tetrazolium assays and flow cytometry analysis were conducted. Genes related to cholesterol metabolism, including LOX-1, ABCA1, and ABCG1, were subjected to RT-qPCR and western blotting. Moreover, protein levels of apoptotic markers (Bcl-2 and Bax), PPARγ, the phosphorylated levels of NF-κB p65, and angiogenetic markers (ICAM1, VCAM1, and VEGF) were quantified via western blotting. Using an enzyme-linked immunosorbent assay, concentrations of inflammatory cytokines in HUVECs were measured.FABP4 expression was upregulated in LPS-stimulated HUVECs. The silencing of FABP4 lowered LOX-1 and p65 levels while upregulating ABCA1 and ABCG1 expression in the context of LPS. Furthermore, the inhibition of FABP4 or NF-κB signaling promoted the growth of HUVECs, upregulated Bcl-2 and PPARγ protein levels, and reduced Bax levels, angiogenetic markers, and inflammatory cytokines in the context of LPS. The combination of FABP4 inhibitor and NF-κB inhibitor treatment amplified the above-mentioned effects on cell growth, angiogenesis, and inflammation.Inhibition of FABP4 reduces LPS-induced HUVEC cell damage via the inactivation of NF-κB p65 and activation of PPARγ signaling.

Validating anti-inflammatory and cytotoxic properties of Fagonia cretica L. through metabolic, in vitro, and in silico profiling

BackgroundFagonia cretica L. (Family: Zygophyllaceae), is a wild shrub mostly found in Mediterranean districts and extensively used in folk medicine for a vast array of purposes such as antidiabetic and anticancer during the early stages. The goal of the current study was to validate the antioxidant, anti-inflammatory, and cytotoxic properties of Egyptian F. cretica using in vitro studies, metabolic profiling, and in silico approaches.MethodsThe plant was collected from the Egyptian desert and the alcoholic extract was prepared from its aerial parts, total phenolic and total flavonoid contents were evaluated spectrophotometrically. Antioxidant potential was assessed via 1,1 diphenyl-2-picrylhydrazyl (DPPH) scavenging activity. Anti-inflammatory activity was validated through in vitro COX-2, COX-1, and nitric oxide inhibition. Cytotoxicity was tested against liver (HepG2), breast (MCF-7), and intestinal (CACO2) carcinoma cell lines followed by assessment of its impact on the levels of apoptotic markers namely topoisomerase I and caspase 9 enzymes. Chemical profiling of the extract was performed using LC-HRMS technique. Saponin rich extract was prepared and tested for affecting topo I and caspase 9 enzymes. In silico studies were conducted on anti-inflammatory (COX-2 and COX-1) and cytotoxicity (topoisomerases I, IIα, and IIβ) targets using Autodock vina in PyRx platform.ResultsTotal phenolic and total flavonoid content of the extract were 2.4 ± 0.12 mg GAE/g and 0.18 ± 0.01 mg RE/g, respectively. In vitro results revealed antioxidant activity calculated as 1.4 ± 0.1 mg AEAC/g. In vitro anti-inflammatory assays unveiled inhibition of COX-2 and COX-1 enzymes with IC50 values of 13.02 ± 0.61 and 26.51 ± 0.83 µg/ml, respectively and nitric oxide with IC50 of 147.05 ± 9.61 µg/ml. Cytotoxicity on MCF-7, HepG2, and CACO2 cell line with IC50 values of 6.9 ± 0.53, 7.6 ± 0.42, and 9.2 ± 0.35 µg/ml, respectively, in addition to in vitro topoisomerase I inhibition (IC50 = 13.57 ± 0.71 µg/ml) and caspase 9 induction by 5.66 folds. Metabolic profiling using LC-HRMS technique resulted in dereplication of 21 compounds including triterpenoid saponins, flavonoids, diterpenoids, etc. Interestingly, saponin rich fraction and non-saponin fraction exhibited similar effects on topoisomerase I and caspase 9. In silico investigation unveiled high binding affinities of almost all the detected metabolites to the active sites of COX-2, COX-1, topo I, IIα, and IIβ enzymes.ConclusionCollectively, we can conclude that F. cretica is a new source of many phytochemicals, and a significant natural source as cytotoxic and anti-inflammatory agent.

Cimifugin Alleviates Chronic Constriction Injury of the Sciatic Nerve by Suppressing Inflammatory Response and Schwann Cell Apoptosis.

Inflammation and Schwann cell apoptosis play critical roles in neuropathic pain after sciatic nerve injury. This study aimed to explore the function and mechanism of cimifugin in lipopolysaccharide (LPS)-stimulated rat Schwann cells and sciatic nerves of rats treated with chronic constriction injury (CCI). Thermal, mechanical and cold hyperalgesia of rats in response to cimifugin or mecobalamin (the positive drug control) treatment were evaluated through behavioral tests. H&E staining of sciatic nerves was performed for pathological observation. ELISA was conducted to assess concentrations of inflammatory cytokines in rat serum and sciatic nerves. The intensity of S100β in sciatic nerves was determined using immunohistochemistry. Flow cytometry analysis was conducted for detection of Schwann cell apoptosis. RT-qPCR was performed to measure mRNA levels of inflammatory factors in Schwann cells. Immunofluorescence staining was performed to detect cellular p65/NF-κB activity. Western blotting was performed to quantify protein levels of apoptotic markers and factors associated with the NF-κB and MAPK pathways in rat nerves and Schwann cells. As shown by experimental data, cimifugin mitigated thermal, mechanical and cold hyperalgesia of CCI rats. Cimifugin repressed inflammatory cell infiltration, reduced proinflammatory cytokine levels while increasing anti-inflammatory factor (IL-10) level in serum or sciatic nerves of CCI rats. Cimifugin enhanced S100β expression and downregulated apoptotic markers in vivo. The anti-inflammatory and anti-apoptotic properties of cimifugin were verified in the LPS-stimulated Schwann cells. Moreover, cimifugin suppressed nuclear translocation of p65 NF-κB in vitro and repressed the phosphorylation of IκB, p65 NF-κB, p38 MAPK, ERK1/2, as well as JNK in CCI rats. In conclusion, cimifugin alleviates neuropathic pain after sciatica by suppressing inflammatory response and Schwann cell apoptosis via inactivation of NF-κB and MAPK pathways.

The protective effects of selenium and boron on cyclophosphamide-induced hepatic oxidative stress, inflammation, and apoptosis in rats

Cyclophosphamide (CP) is an alkylating anticancer drug with broad clinical application that is highly effective in the treatment of cancer and non-malignant diseases. However, the main limiting effect of CP is multi-organ toxicity due to damage to normal tissues. The aim of this study is to compare the hepatoprotective potential of selenium (Se) and boron (B) in CP-induced liver injury in experimental rats. The rats were randomly divided into six equal groups: Control (saline), 200 mg/kg CP (administered once on the fourth day of the experiment), 1.5 mg/kg Se (administered once/time daily for 6 days), 20 mg/kg B (administered once/time daily for 6 days), Se + CP and B + CP administered intraperitoneally (i.p.). Administration of CP leads to an increase in the levels of apoptotic markers (Bax, caspase-3), the apoptotic signaling pathway (Nrf2), oxidative stress indicators (TOS, OSI), lipid peroxidation markers (MPO, MDA), inflammation levels (NF-kB, TNF-α, IL-1β, IL -6), liver function markers (ALT, AST, ALP), while apoptosis markers (Bcl-2), apoptosis pathway (Keap-1), oxidative stress indicator (TAS), inflammation (IL -10) and intracellular antioxidant defense system (SOD, CAT, GPx and GSH) decreased. In addition, degeneration of hepatocytes and congestion in the central veins were observed. In contrast, in the groups administered Se and B with CP, the changes that occurred were reversed. However, it was found that Se protects the liver slightly better against CP damage than B. The protective effect of Se and B against the toxic effects of CP on the antioxidant markers SOD, CAT and GPx1 was also investigated in silico. The in silico results were consistent with the in vivo results for SOD and CAT, but not for GPx1.

Synergistic anti-tumorigenic effect of diosmetin in combination with 5-fluorouracil on human colon cancer xenografts in nude mice

5-Fluorouracil (5-FU) is frequently used to treat colorectal cancer (CRC), but its clinical application is limited by its toxicity. Natural compounds have been combined with chemotherapeutic drugs to reduce chemotherapy-related toxicity. Diosmetin, a natural flavonoid, has demonstrated anticancer effects against CRC. This study investigated diosmetin's potential in combination with 5-FU using a murine model of HCT-116 colon cancer xenografts in nu/nu nude mice. HCT-116 cells were injected into the right flanks of mice, and once tumors reached a size of 50 mm3, the mice were treated with diosmetin (100 mg/kg), 5-FU (30 mg/kg), or a combination of both at two dose levels (100 + 30 mg/kg and 50 + 15 mg/kg) for 4 weeks. Blood and tumors were collected on the final day for further analysis. Mice treated with the higher combination dose exhibited the smallest tumor volume (330.91 ± 88.49 mm3). Biochemistry and histology analysis showed no toxicity or abnormalities in the liver, kidney, and heart with the combination therapy. Immunohistochemistry results revealed a notable reduction in the proliferation marker (Ki67) and inflammation marker (TLR4) in tumors from high-dose combination-treated mice. Moreover, immunofluorescence data indicated increased levels of apoptotic markers (Bax, Caspase-3, p53, p21) and downregulation of anti-apoptotic protein (Bcl-2) in the high-dose combination group. The findings suggest that 100 mg/kg of diosmetin combined with 30 mg/kg 5-FU significantly reduced tumor volume and had a less toxic effect on the heart compared to 5-FU monotherapy.

MiRNA-133a-3p Attenuates Renal Tubular Epithelial Cell Injury via Targeting MALM1 and Suppressing the Notch Signaling Pathway in Diabetic Nephropathy.

Diabetic nephropathy (DN) is a serious microvascular complication of diabetes characterized by structural and functional changes of kidneys. Human renal tubular epithelial (HK-2) cells are important for kidney recovery post injury and usually used for establishment of DN cell models. The study explored the role of microRNA (miR)-133a-3p in DN cell model and animal model. A cell model for DN was established via high glucose (HG) stimulation to HK-2 cells. Cell viability and apoptotic rate were measured by cell counting kit 8 and flow cytometry. Polymerase chain reaction was performed to quantify levels of miR-133a-3p and targets. Luciferase reporter assay was conducted to verify the binding of miR-133a-3p and MAML1. After establishment of a mouse model of DN, levels of renal function indicators were measured by biochemical analysis. Hematoxylin-eosin and periodic acid-schiff staining of kidney samples were performed to analyze histological changes. Western blotting was conducted to quantify levels of apoptotic markers, MAML1, and factors related to Notch signaling. Results showed that HG induced HK-2 cell apoptosis and the reduction of cell viability. MiR-133a-3p was lowly expressed in HG-stimulated HK-2 cells. Overexpressed miR-133a-3p improved HK-2 cell injury by increasing cell viability and hampering apoptosis under HG condition. In addition, miR-133a-3p directly targets MAML1 3'-untranslated region. MAML1 overexpression countervailed the repressive impact of miR-133a-3p on cell apoptosis in the context of HG. Moreover, miR-133a-3p inhibited the activity of Notch pathway by downregulating MAML1. MiR-133a-3p inhibits DN progression in mice, as evidenced by reduced fasting blood glucose level, improved levels of renal function parameters, and alleviation of kidney atrophy. In conclusion, miR-133a-3p improves HG-induced HK-2 cell injury and inhibits DN progression by targeting MAML1 and inactivating Notch signaling.

Tannic acid protects neuroblastoma cells against hydrogen peroxide – triggered oxidative stress by suppressing oxidative stress and apoptosis

Recent investigations indicate that tannic acid is associated with a decrease in oxidative damage. Growing evidence supports the protective effects of tannic acid on the central nervous system (CNS). However, uncertainties persist regarding its influence on hydrogen peroxide (H2O2)-triggered oxidative impairment in nerve cells and its interaction with apoptosis. Hence, the objective of this work was to examine the neuroprotective impact of tannic acid on SH-SY5Y cell impairment following H2O2-induced oxidative stress, particularly concerning apoptotic pathways. The control group received no treatment, while the H2O2 group underwent treatment with 0.5 mM H2O2 for a duration of 24 h. The tannic acid group received treatment with different concentrations of tannic acid for a duration of 24 h. Meanwhile, the tannic acid + H2O2 group underwent pre-treatment with tannic acid for one hour and was subsequently subjected to 0.5 mM H2O2 for one day. Within the tannic acid + H2O2 group, the cell viability in SH-SY5Y cells was notably enhanced by tannic acid at concentrations of 2.5, 5, and 10 μM. It also resulted in a considerable rise in TAS (Total Antioxidant Status) levels and a concurrent decline in TOS (Total Oxidant Status) levels, serving as indicators of reduced oxidative stress. Additionally, tannic acid treatment resulted in decreased levels of apoptotic markers (Bax, cleaved PARP, and cleaved caspase 3) and oxidative DNA damage marker (8-oxo-dG), while increasing the anti-apoptotic marker Bcl-2. The findings from flow cytometry also revealed a significant reduction in the apoptosis rate following pretreatment with tannic acid. In summary, tannic acid demonstrates protective effects on SH-SY5Y cells in the face of H2O2-triggered oxidative damage by suppressing both oxidative stress and apoptosis. Nevertheless, additional research is warranted to assess the neuroprotective potential of tannic acid.

Cilostazol attenuates mirabegron-induced hepatic and renal toxicity in rats: regulation of metabolic, oxidative, and inflammatory pathways

Background Mirabegron (MIRG) is a type of β3 adrenoceptor agonist that is considered an alternative therapy for the treatment of overactive bladder (OAB) symptoms. Cilostazol (CITZ) is a selective inhibitor of phosphodiesterase (III) that has various pharmacological effects. Objectve The current study aimed to highlight the regulatory effects of CITZ on MIRG-induced toxicity. Materials and Methods Male rats were divided into six groups. Blood samples were collected to determine different hepatic and kidney function levels along with serum protein electrophoresis and inflammatory factor levels. Histopathological studies and oxidative stress (OS) were also assessed. Kidney and hepatic damage were detected following the administration of MIRG, especially at high doses, due to elevated OS, inflammation, and apoptotic marker levels. Results Rats receiving CITZ exhibited significant improvements in both hepatic and kidney functions, with decreased inflammation and OS. Conclusion CITZ administration plays a beneficial role in alleviating hepatic and nephrotoxicity induced by MIRG by inhibiting OS and inflammation.

The protective effect of parthenolide in an in vitro model of Parkinson's disease through its regulation of nuclear factor-kappa B and oxidative stress.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by motor and non-motor symptoms, and is due to the degeneration of dopaminergic neurons. It is multifactorial, caused by genetic and environmental factors and currently has no definitive cure. We have investigated the protective effects of parthenolide (PTN), a compound with known anti-inflammatory and antioxidant properties, in an in vitro model of PD, that is induced by 6-OHDA, and that causes neurotoxicity in SH-SY5Y human neuroblastoma cells. SH-SY5Y cells were pretreated with PTN to assess its protective effects in 6-OHDA-induced cellular damage. Cell viability was measured using Alamar blue. Apoptosis was evaluated using an Annexin V-FITC/PI kit. Reactive oxygen species (ROS) levels were quantified, and expression levels of apoptotic markers (Bax, Bcl-2, p53) and NF-κB were analyzed via Western blotting and Quantitative real-time- (qRT-) PCR. We found that 6-OHDA reduced cell viability, that was inhibited significantly by pre-treatment with PTN (p < 0.05). Flow cytometry revealed that PTN reduced apoptosis induced by 6-OHDA. PTN also reduced the ROS levels raised by 6-OHDA (p < 0.05). Moreover, PTN decreased the expression of Bax, p53, NF-κB, and p-NF-κB that were increased by treatment with 6-OHDA. These findings indicate the potential beneficial effects of PTN in an in vitro model of PD via mitigating oxidative stress and inflammation, suggested PTN as a promising agent to be used for PD therapy, warranting further investigation in preclinical and clinical studies.

Protective Effects of Allium senescens Extract against 6-Hydroxydopamine in Neurons

Oxidative neurodegeneration causes various neuronal diseases, including Alzheimer’s, Parkinson’s, and Huntington’s diseases. This study aimed to demonstrate the protective effect of Allium senescens leaf extracts on 6-hydroxydopamine (6-OHDA)-stressed SH-SY5Y cells, which are known to be optimal for neurotoxic research. The levels of apoptotic markers were evaluated using quantitative polymerase chain reaction (qPCR) and flow cytometry. The localization of apoptotic cells in vivo was analyzed using whole-mount immunochemistry and terminal deoxynucleotidyl transferase dUTP nick end labeling assay. Additionally, the production of reactive oxygen species (ROS) was estimated using flow cytometry. 6-OHDA induced ROS production in neuroblasts and in vivo, but treatment with the extract protected against the 6-OHDA-induced increase in ROS levels. Under oxidative stress, the extract performs three protective functions: decreasing ROS production, preventing mitochondrial apoptosis, and protecting the central and ventral nervous systems. These results also suggest that the extract can be useful in the development of functional foods for the prevention of neural damage caused by oxidative stress.

Autophagy is involved in granulosa cell death and follicular atresia in ewe ovaries

In mammalian ovaries, most follicles do not ovulate and are eliminated by atresia, which primarily depends on granulosa cell (GC) apoptosis. Autophagy is an alternative mechanism involved in follicle depletion in mammals through independent or tandem action with apoptosis. However, follicular autophagy has not yet been investigated in sheep; therefore, the present study aimed to investigate the involvement of autophagy in atresia among a pool of growing antral follicles in ewe ovaries. The abundance of the autophagic marker LC3B-II was determined using western blotting in GCs collected from ewe antral follicles. The antral follicles were classified as healthy or atretic based on morphological criteria and steroid measurements in follicular fluid (FF). Immunofluorescence and confocal microscopy analyses were performed on GCs to evaluate the presence of autophagic proteins and their subcellular localisation. Caspase-3 and DNA fragmentation were assessed using western blotting and TUNEL assays, respectively, in the same GC population to investigate the simultaneous apoptosis. The novel results of this study demonstrated enhanced LC3B-II protein expression in GCs of atretic follicles compared to that of healthy ones (1.3-fold increase; P = 0.0001, ANOVA), indicating a correlation between autophagy enhancement in GCs and antral follicular atresia. Autophagy, either functioning independently or in tandem with apoptosis, may be involved in the atresia of growing antral follicles in ewe ovaries because atretic GCs also showed high levels of apoptotic markers. The findings of this study might have important implication on scientific understanding of ovarian follicle dynamics.

Diosgenin inhibits proliferation and migration of ovarian cancer cells and induce apoptosis via upregulation of PTEN.

Diosgenin, a natural steroidal sapogenin, has recently attracted a high amount of attention, as an effective anticancer agent in ovarian cancer. However, diosgenin mediated anticancer impacts are still not completely understood. Thus, the present study evaluated the effect of diosgenin on the proliferation, apoptosis, and metastasis of ovarian cancer cells. OVCAR-3 and SKOV-3 cells were treated with diosgenin, cellular viability was assessed by MTT assay and apoptosis was measured by ELISA and evaluated the protein expression levels of apoptotic markers through western blotting. Cell migration was examined by measuring the mRNA levels of genes involved in the cell invasion. The protein expression levels of main components of PI3K signaling were evaluated via western blotting. Diosgenin led to significant inhibition of cellular proliferation in a dose-dependent manner. It also induced apoptosis through upregulating pro-apoptotic markers and downregulating antiapoptotic mediators. In addition, OVCAR-3 cells exposure to diosgenin decreased cell migration and invasion. More importantly, diosgenin downregulated the expression levels of main proteins in PI3K signaling including PI3K, Akt, mTOR, and GSK3. Diosgenin inhibited the proliferation and migration of OVCAR-3 ovarian cancer cells and induced apoptosis, which may be mediated by targeting PI3K signaling.

Insulin Ameliorates Neurodegeneration in an Experimental Animal Model of Brain Aging by a Decrease of GFAP Expression and Apoptosis Inhibition

Background: Aging is one of the factors that will cause gradual changes in brain tissue and its destruction. One of the protective factors against aging is insulin. Objectives: Our study investigated insulin's neuroprotective effect by preventing oxidative stress-mediated neuronal damage. Methods: A total of 48 adult male NMRI (the Naval Medical Research Institute) mice were divided into 2 groups: Control and insulin. Insulin was administered intraperitoneally (IP) for 8 weeks to the control group and for 12 weeks to the insulin group. In this study, animals were treated with insulin, and insulin's role in the aging process was evaluated. Then, brain tissue was extracted for evaluation of oxidative stress by assessment of glutathione disulfide (GSH) and reactive oxygen species (ROS) levels, the expression of the glial fibrillary acidic protein (GFAP) as a biomarker for astrogliosis, stereological study, and the real-time polymerase chain reaction (PCR) technique evaluated the level of apoptosis biomarker. Results: The results of stereological parameters show that the volumes of the brain cortex and the number of neurons improved in the insulin group by aging as compared to the control group. The results of real-time PCR showed that the expression level of apoptotic markers decreased in the insulin group compared to the control group. In the insulin group, GSH levels were higher than in the control group, and the control group produced more ROS than the insulin group. As compared with insulin, the control group expressed more GFAP protein and increased the number of glial cells by aging. Conclusions: The results showed that insulin could have neuroprotective effects against aging changes and reduce apoptosis and cell death.

Celastrus paniculatus seed extract exhibits neuroprotective effects against MPP+‑induced apoptotic cell death via GSK‑3β in a Parkinson's disease model.

Parkinson's disease (PD) is a common neurodegenerative disease induced by the death of dopaminergic neurons. Seed oil of Celastrus paniculatus (CP) Willd. has protective and antioxidant properties; however, to the best of our knowledge, no studies have analyzed the neuroprotective effect of CP seeds on PD. The present study aimed to investigate the neuroprotective effects and mechanism of CP seed extract (CPSE) using an in vitro 1-methyl-4-phenylpyridinium ion (MPP+)-induced PD model. The effect of CPSE on the expression levels of apoptotic marker proteins, such as Bcl-2 and its upstream pathway protein, glycogen synthase kinase-3β (GSK-3β), was investigated in human neuroblastoma SH-SY5Y cells. The effect of CPSE on the viability of SH-SY5Y cells was evaluated using MTT assay. To investigate the potential neuroprotective effect of CPSE, SH-SY5Y cells were treated with MPP+ to induce PD-associated cytotoxicity. SH-SY5Y cells were treated with 2 mM MPP+ before or after CPSE treatment to determine the protective effect of CPSE against MPP+-induced neurotoxicity using MTT, WST-1 and lactate dehydrogenase assays. Moreover, it was investigated whether CPSE could promote survival signals by regulating the protein expression levels of apoptotic markers (Bcl-2 and GSK-3β) using western blotting. High concentrations and prolonged treatment of CPSE did not have any adverse effect on SH-SY5Y cell viability. Furthermore, MPP+-induced dopaminergic neuron damage was ameliorated by CPSE treatment. CPSE also showed anti-apoptotic activity by reversing the inhibitory effects of MPP+ on Bcl-2 expression. Moreover, CPSE abolished MPP+-induced decreases in phosphorylated-GSK-3β (Ser9) expression. Taken together, the present findings suggested that CPSE may exert a neuroprotective effect in PD.

TNFAIP3 Overexpression Inhibits Diffuse Large B-Cell Lymphoma Progression by Promoting Autophagy through TLR4/MyD88/NF-κB Signaling Pathway.

Tumor necrosis factor alpha induced protein 3 (TNFAIP3) is reportedly to have significant implications for autophagy regulation in various cancers. The current study aimed to decipher the role and mechanism of TNFAIP3 in diffuse large B-cell lymphoma (DLBCL) by modulating autophagy. Information pertaining to the differential expression and prognostic role of TNFAIP3 in DLBCL was gleaned from the Gene Expression Omnibus (GEO) database. The TNFAIP3 expression levels in human DLBCL cells were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting. Cell counting kit-8 (CCK-8) and colony formation assays were employed to determine cell proliferation. Transwell assay and flow cytometry were applied to detect cell migration and apoptosis, respectively. Immunofluorescence and transmission electron microscope were used for the assessment of cell autophagy. The levels of apoptotic markers (caspase-3, cleaved-caspase-3, Bcl-2 Associated X (Bax), and B cell lymphoma-2 (Bcl-2)), autophagy indicators (the ratio of microtubule-associated proteins 1A/1B light chain 3 II and I (LC3II/LC3I), Sequestosome (p62)), and pathway proteins (toll-like receptor 4 (TLR4), myeloid differentiation primary response 88 (MyD88), Transcription Factor NF-Kappa-B P65 Subunit (p65), and phosphorylated-p65 (p-p65)) were assessed via Western blotting. Immunohistochemistry was employed to detect Ki67 expression in tumor tissues. TNFAIP3 expression in DLBCL samples was downregulated, correlating with poor prognosis. TNFAIP3 expression was also downregulated in DLBCL cells. It was found that TNFAIP3 impeded cell proliferation and migration, and enhanced apoptosis of OCI-LY3 cells. Intervention with autophagy inhibitor 3-methyladenine (3-MA) markedly reversed apoptosis of OCI-LY3 cells induced by TNFAIP3. Besides, TNFAIP3 induced autophagy via modulating the TLR4/MyD88/nuclear factor kappa B (NF-κB) signaling pathway. In vivo experiments showed that TNFAIP3 expression in DLBCL was downregulated, and upregulation of TNFAIP3 could inhibit tumor growth. TNFAIP3 inhibits DLBCL progression by inducing TLR4/MyD88/NF-κB pathway-mediated autophagy.

Berberrubine protects against cisplatin-induced ototoxicity by promoting folate biosynthesis.

This research investigated the possible shielding properties of BB (Berberrubine) against the harmful auditory effects of cisplatin, preliminarily delving into the underlying mechanisms responsible for this protection. HEI-OC1 cell viability was determined using a Cell Counting Kit-8 (CCK-8). The impact of BB on cochlear hair cells was studied through in vitro cochlear explants culture. Apoptosis levels were measured through Annexin V-PI, Cleaved Caspase-3, and TUNEL staining. The level of ROS (reactive oxygen species) was measured through the application of DCFH-DA, MitoSOX, and JC-1 fluorescent dyes for staining. Immunofluorescence analysis of cochlear samples from mice was conducted to quantify the hair cell count, and concurrently, ABR (Auditory Brainstem Response) testing was utilized to evaluate auditory function. The mechanism of action of BB was explored using RNA-Seq and qRT-PCR analysis. BB significantly improved cell survival rates under cisplatin treatment, reduced levels of apoptotic markers (TUNEL, Cleaved Caspase-3, Annexin V-PI), decreased ROS and MitoSOX levels, and improved JC-1 signals in both HEI-OC1 cells and cochlear hair cells in cochlear explants culture. Animal studies demonstrated that treatment with BB enhanced the survival of cochlear hair cells, reduced hearing impairment caused by cisplatin in mice. RNA-seq and qRT-PCR analysis revealed that BB influenced the expression levels of multiple genes (Ccnd2, Reln, Pgf, Mylk3, Ppplr12c, Thbsl), by promoting folate biosynthesis for hearing protection. Our findings suggest that BB protects against cisplatin-induced hearing damage by enhancing folate biosynthesis, decreasing intracellular ROS levels, and inhibiting apoptosis.

Fucoidan alleviated autoimmune diabetes in NOD mice by regulating pancreatic autophagy through the AMPK/mTOR1/TFEB pathway.

The present study investigated the effect and its underlying mechanisms of fucoidan on Type 1 diabetes mellitus (T1DM) in non-obese diabetic (NOD) mice. Twenty 7-week-old NOD mice were used in this study, and randomly divided into two groups (10 mice in each group): the control group and the fucoidan treatment group (600 mg/kg. body weight). The weight gain, glucose tolerance, and fasting blood glucose level in NOD mice were detected to assess the development of diabetes. The intervention lasted for 5 weeks. The proportions of Th1/Th2 cells from spleen tissues were tested to determine the anti-inflammatory effect of fucoidan. Western blot was performed to investigate the expression levels of apoptotic markers and autophagic markers. Apoptotic cell staining was visualized through TdT-mediated dUTP nick-end labeling (TUNEL). The results suggested that fucoidan ameliorated T1DM, as evidenced by increased body weight and improved glycemic control of NOD mice. Fucoidan down-regulated the Th1/Th2 cells ratio and decreased Th1 type pro-inflammatory cytokines' level. Fucoidan enhanced the mitochondrial autophagy level of pancreatic cells and increased the expressions of Beclin-1 and LC3B II/LC3B I. The expression of p-AMPK was up-regulated and p-mTOR1 was inhibited, which promoted the nucleation of transcription factor EB (TFEB), leading to autophagy. Moreover, fucoidan induced apoptosis of pancreatic tissue cells. The levels of cleaved caspase-9, cleaved caspase-3, and Bax were up-regulated after fucoidan treatment. Fucoidan could maintain pancreatic homeostasis and restore immune disorder through enhancing autophagy via the AMPK/mTOR1/TFEB pathway in pancreatic cells.

Phenomenon of suppression of estrogen signaling in breast cancer cells under ultraviolet irradiation: role of Snail proteins

Introduction. The study of the effect of irradiation or any other DNA-damaging agents on the sensitivity of tumors to conservative therapy, drug or hormonal, is among the most imporant tasks that determine the efficiency of combined therapy of cancer patients.Aim. To investigate the effect of irradiation on the activity of key signaling proteins and the level of hormone dependence of breast cancer cells.Materials and methods. The experiments were performed on in vitro cultured estrogen-dependent MCF-7 breast cancer cells. Ultraviolet (UV) irradiation in the range of 254 nm with the intensity of 25–50 J/m2 was used as an experimental model to study the response of tumor cells to DNA damage. Cell growth rate was determined using the MTT test, cell survival after irradiation was analyzed using the colony-forming test. Estrogen receptor transcriptional activity was determined by reporter assay; cellular protein expression was determined by immunoblotting.Results. Single UV irradiation of MCF-7 cells leads to a marked increase in the level of apoptotic markers: p53, cPARP, suppression of expression of growth signaling proteins: CDK4/6 and estrogen receptor α (ERα). The above changes are accompanied with an increase in phosphorylation of Akt protein kinase and a marked increase in the expression of Snail1, one of the key proteins of epithelial-mesenchymal transition. In UV-resistant MCF-7/UVR cell subline obtained under repeated irradiation cycles, the levels of apoptotic and growth signaling proteins (p53, cPARP, CDK4/6) return to control levels, except for the phosphorylated form of Akt and Snail1, whose content remains high. Transfection of Snail1-expressing plasmid into MCF-7 cells is accompanied by activation of apoptotic signaling, suppression of ERα activity, and development of partial hormone resistance; however, the sensitivity of cells to irradiation is practically unchanged. Transfection of microRNA-181a-2, one of the microRNAs associated with cell resistance, simultaneously activates Akt and Snail1 and leads to the development of cross-resistance of cells to irradiation and hormonal drugs.Conclusion. The obtained data allow us to consider irradiation-induced Snail1 activation as one of the factors involved in deregulation of estrogen signaling and formation of cell resistance to hormonal drugs, while simultaneous activation of Akt and Snail1 is accompanied by the development of cross-resistance to irradiation and hormonal drugs.