Cell Damage Model Research Articles

Gastrodin Attenuates Neuronal Apoptosis and Neurological Deficits after Experimental Intracerebral Hemorrhage

Objective: Gastrodin, a glucoside of gastrodigenin, inhibits cerebral oxidant stress and apoptosis in multiple central nervous system injury, but its effect in intracerebral hemorrhage (ICH) remains unclear. This study investigated the effect of gastrodin on neuronal apoptosis and neurological deficits in rat ICH model. Methods: In vitro experiments were performed using hematoma lysate-induced cell damage model in primary cortical neurons. Rat ICH model was produced by a caudatum injection of collagenase. Gastrodin was intraperitoneal injected after 2 hours following ICH. Cell viability, brain water content, neurological score, western blot, and immunofluorescence experiments were performed. Results: Gastrodin significantly decreased hematoma lysate-induced reduction of cell viability and cell apoptosis in primary cortical neurons. Gastrodin significantly improved brain edema and neurological deficits post-ICH. Moreover, gastrodin administration significantly reduced levels of ROS, 8-OHDG, 3-Nitrotyrosine and MDA, while increased GSH-Px and SOD activity, and stimulated the upregulation of Keap1, Nrf2, and HO-1 signaling at 72 hours post-ICH. Furthermore, gastrodin significantly increased Bcl-2 expression, while reduced level of Bax, active caspase-3 and active caspase-9, also reduced the number of active caspase-3 or TUNEL positive neurons at 72 hours post-ICH. Conclusion: These results suggest that gastrodin is neuroprotective after ICH and the mechanism may be associated with the inhibition of oxidative stress and neuronal apoptosis.

Autophagy, lysosome dysfunction and mTOR inhibition in MNU-induced photoreceptor cell damage

Progressive photoreceptor death is the main cause of retinal degeneration diseases. Determining the underlying mechanism of this process is essential for therapy improvement. Autophagy has long been considered to be involved in neuronal degeneration diseases, and the regulation of autophagy is thought to have potential implications for neurodegenerative disease therapies. However, whether autophagy is protective or destructive varies among diseases and is controversial. In the present study, we established an N-methyl-N-nitrosourea (MNU)-induced photoreceptor cell damage model in vitro that faithfully replicated photoreceptor cell death in retinal degeneration diseases. Cell viability was tested by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy-methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assays. Reactive oxygen species (ROS) levels were assessed through 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescence. Autophagy was confirmed by observing autophagosomes using transmission electron microscopy (TEM). A lysosome tracker was used to identify acidic lysosomes in cells. We also measured the expression of some proteins related to autophagy, apoptosis and lysosomal degradation by western blot and immunofluorescence assays. We found that MNU could decrease photoreceptor cell viability in a time- and dose-dependent manner, and this change was accompanied by concomitant increases in ROS and the expression of the apoptosis-inducing protein cleaved caspase-3. Moreover, autophagy was activated by MNU treatment during this process. Inhibition of autophagy with 3-methyladenine accelerated cell damage. Lysosome dysfunction was confirmed by autophagosome enlargement and increased cathepsin expression, which was accompanied by mTOR dephosphorylation. In conclusion, autophagy was activated through inhibition of the PI3K/mTOR pathway in the context of MNU-induced photoreceptor cell death. Prolonged mTOR dephosphorylation and autophagy activation resulted in autophagic vacuole accumulation, as indicated by inefficient degradation in lysosomes, and further led to apoptosis.

Paper-based 3D culture device integrated with electrochemical sensor for the on-line cell viability evaluation of amyloid-beta peptide induced damage in PC12 cells

In this communication, a paper-based 3D cell culture device integrated with electrochemical biosensor was applied to on-line monitoring of dopamine release from PC12 cell damage models induced by amyloid-beta peptide (Aβ25-35) and cell intervene models protected by curcumin (Cur) and marrow mesenchymal stem cells (MSC) supernatant. The adhesion and proliferation of PC12 cells cultured on the paper scaffold was characterized by scanning electron microscopy and laser scanning confocal microscopy, which verify unique biocompatibility and 3D microarchitecture similar to human body microenvironment of paper substrate, so an artificial model simulating 3D microenvironment in vivo was constructed easily. The PC12 cells in paper-based devices consisted of four groups containing control group, Aβ25-35 group, Aβ25-35+Cur group and Aβ25-35+MSC supernatant group. Under optimal conditions, this proposed device displayed a wide linear range from 0.05 to 1 μmol/L with a detection limit of 0.009 μmol/L (S/N = 3) and exhibited high sensitivity, good selectivity and excellent reproducibility. Furtherly, electrochemcial analysis and MTT assay gave a clue that the cell viability of Aβ25-35+MSCs supernatant group was higher than that of Aβ25-35+Cur group. Therefore, the detachable paper-based 3D device paves the way to a direct detection of exocytosis DA from neuron cells for on-line cell viability evaluation of neurodegenerative disease cell damage models.

Baicalein Exerts Neuroprotective Effects in FeCl3-Induced Posttraumatic Epileptic Seizures via Suppressing Ferroptosis.

Posttraumatic epilepsy (PTE) is a prevalent type of acquired epilepsy secondary to traumatic brain injury, and is characterized by repeated seizures. Traditional antiepileptic drugs have minimal response in preventing posttraumatic epileptic seizures. It is essential for the development of new therapeutic strategy. Our previous work disclosed a potent neuroprotective role of baicalein, a flavonoid extracted from Scutellaria baicalensis Georgi, against inherited epilepsy in rats. Whether baicalein has protective potential in posttraumatic epileptic seizures and the possible molecular mechanism remain elusive. Additionally, the brain is vulnerable to lipid peroxidation-induced damage due to high consumption of oxygen and abundant polyunsaturated fatty acids in neuronal membranes. Our present investigation aimed to elucidate whether baicalein exerts neuroprotective effects on posttraumatic epileptic seizures by inhibiting ferroptosis, a newly discovered lipid peroxidation-dependent cell death modality. We found that baicalein significantly reduced seizure score, number of seizures, and average seizure duration in an iron chloride (FeCl3)-induced PTE mouse model. The neuroprotective effect of baicalein was also validated in a ferric ammonium citrate (FAC)-induced HT22 hippocampal neuron damage model. Moreover, in vitro, baicalein could remarkably decrease ferroptotic indices (lipid reactive oxygen species, 4-hydroxynonenal, and prostaglandin endoperoxide synthase 2) and inhibit the expression of 12/15-lipoxygenase (12/15-LOX) in an iron-induced HT22 cell damage model. These findings were also validated in a mouse PTE model. It was concluded that baicalein exerted neuroprotective effects against posttraumatic epileptic seizures via suppressing ferroptosis and 12/15-LOX was likely to be involved in baicalein’s neuroprotection.

Identification of a Quality Marker of Vinegar-Processed Curcuma Zedoaria on Oxidative Liver Injury.

Curcuma zedoaria (dry stenophora of Curcuma phaeocaulis Val., Curcuma kwangsiensis S. G. Lee et C. F. Liang, or Curcuma wenyujin Y. H. Chen et C.Ling) is a representative herb with clinical effects on liver diseases after being vinegar-processed. The crude Curcuma zedoaria and the processed Curcuma zedoaria (vinegar-boil) have been widely used as mixtures, but their equivalence has not been fully investigated. In this manuscript, quality markers of processed (vinegar-boil) Curcuma zedoaria were investigated by comparison of the compounds and hepatoprotective activities with the crude (three spices) ones. First, GC-MS-based untargeted metabolomics were applied to reveal the discriminatory components and discover potential markers. As a result, a total of six components were identified as potential markers. Then, the hepatoprotective activities were evaluated by dual cell damage models induced by a certain concentration of H2O2 or tertbutyl hydfroperoxide (t-BHP) (55 μM H2O2 or 40 μM t-BHP), which highlighted the potential of the processed Curcuma zedoaria on oxidative stress. Finally, epicurzerenone was identified as its quality marker on oxidative liver injury based on the above results and the cell-based biological assay. Overall, vinegar-processed Curcuma zedoaria was more suitable for the treatment of oxidative liver diseases, and epicurzerenone could be considered as its quality marker.

Antioxidant and Antiproliferative Activities of Cyanidin‐3‐O‐Glucoside (C3G) Liposome in Caco‐2 Cells Cultivated in 2D and 3D Cell Culture Models

The aim of this study was to obtain adequate and detailed information about the antioxidant and antiproliferative activities of C3G and C3G liposomes in different cell culture models. The Caco-2 cells were cultured in 2D and 3D cell culture models, the H2 O2 was used to construct the cell damage model and then the cells treated with C3G and C3G liposomes. The antioxidant activity and antiproliferative activities of C3G liposomes on Caco-2 cells were investigated. We observed the morphology of cells and measured the cell viability, the activity of glutathione (GSH), superoxide dismutase (SOD), total antioxidant capacity (T-AOC), and the content of malondialdehyde (MDA) in Caco-2 cells treated with H2 O2 , C3G, and C3G liposomes. The results showed that the Caco-2 cells cultured in the 3D culture model formed a 3D structure and tight spheroids and showed the increase of cell activity in 3D cell culture model, compared with the 2D cell culture model. The C3G and C3G liposomes can enhance the activities of GSH, SOD, and T-AOC but decrease the MDA content after H2 O2 treatment, while the changes were different in 2D and 3D cells culture models. This study revealed that the results obtained from the 2D cell model may be inaccurate compared with the results obtained from the 3D cell model. PRACTICAL APPLICATION: The results of this study showed that the results obtained from the 2D cell model may be inaccurate compared with the results obtained from the 3D cell model. Our work provides a method for evaluating antioxidant activity of C3G liposomes in different cell models and provided certain theoretical basis for the follow-up research.

Safranal protects against beta-amyloid peptide-induced cell toxicity in PC12 cells via MAPK and PI3 K pathways.

Alzheimer's disease is a type of cerebrovascular problem with progressive mental disabilities for the patient. This study aimed to investigate the protective effect of safranal on toxicity and oxidative damage induced by beta-amyloid (Aβ) and hydrogen peroxide (H2O2) in PC12 cells as an appropriate model of Alzheimer's cell damage. PC12 cells pretreated with saffron extract (2.5-40μg/ml), essential oil (2.5-40μg/ml), safranal (2.5-5-40μM) and donepezil (5, 10 and 20μM) for 120min. Then exposed to either Aβ (25μM) for 48h or H2O2 (150μM) for 24h. In the end, the cell survival and intracellular reactive oxygen species (ROS) production analyzed. The anti-apoptotic effects of safranal in PC12 cells were studied using flow cytometry after PI staining. Also, western blot analysis of Cyt c, survivin, p44/42 MAPK (ERK1/2), Phospho-p44/42 MAPK (ERK1/2), PI3 Kinase P85, Phospho-PI3 Kinase P85, phospho SAPK/JNK, SAPK/JNK and caspase 3 performed for detection of apoptosis. Safranal (2.5 and 5μM) and donepezil (10 and 20μM) significantly decreased the Aβ toxicity. The ROS significantly attenuated when cells pretreated with essential oil, saffron extract, safranal, and donepezil. Cell apoptosis significantly increased after treatment with Aβ (25-35) (25μM) compared to control. However, after pretreatment with safranal (2.5μM) apoptosis was significantly reduced. Western blot analysis of PC12 cells showed that 25μM Aβ (25-35) could increase proteins involved in apoptosis signaling and pretreatment with safranal (2.5μM) could decrease the apoptosis. According to the results, safranal showed anti-apoptotic and antioxidant effects and may exert promising potential for the prevention of Alzheimer's disease.

Investigation of the effect of natural tumor cell death on radiotherapy outcomes

The aim of the work is to investigate the impact of radiation-independent (natural or spontaneous) tumor cell death on tumor control probability (TCP) during and following fractionated external-beam radiotherapy employing both analytical and numerical methods. The analytical method solves a TCP model accounting for tumor repopulation and non-radiation tumor cell death during fractionated external-beam radiotherapy. The numerical method is based on a Monte Carlo simulation of the processes of radiation-induced cell kill, as well as cell division and natural cell death randomly taking place in the time interval between fractions. Distributions of the number of surviving cells are constructed using the Monte Carlo method for cases with and without natural cell death. The analytically and numerically calculated values of TCP were found to be in excellent agreement (as shown in the Method and materials section), thereby validating both methods. The TCP model is then fitted to two different experimental data sets with the aim of determining the model parameter values, primarily the natural death rate. Two versions of the linear-quadratic model of cell damage—with and without assumed re-sensitization of the tumor cells during treatment—are used. In two of the fits a strong correlation between the repopulation and spontaneous cell death rates is observed. It was possible to determine separately the values of the two rates only in the fit of the model with resensitization to the most diversified data set consisting of seven different fractionation regimes. The observed correlation together with a theoretical consideration leads to the conclusion that in most cases it is the net effect of the two processes of birth and death rather than the processes separately that determines treatment outcome. However, depending on the values of the rates of the two processes and the duration of the treatment, the treatment outcome may be more accurately determined by the absolute values of the two rates rather than just by their difference.

Characterization of Cell Damage and Proliferative Ability during and after Bioprinting.

When a biomaterial solution containing living cells is subject to bioprinting, the cells experience process-induced stresses, including shear and extensional stresses. These process-induced stresses breach cell membranes and can lead to cell damage, thus reducing cell viability and functioning within the printed constructs. Studies have been conducted to determine the influence of shear stress on cell damage; however, the effect of extensional stress has been typically ignored in the literature until the recently collected evidence of its importance. This paper presents a novel method to characterize and quantify the cell damage caused by both shear and extensional stresses in bioprinting. In this method, cell damage law is first established to relate cell damage to shear stress based on the experiments with a rheometer; the process-induced shear stress experienced by cells in bioprinting is represented, and the established cell damage model is applied to calculate the degree of cell damage caused by shear stress in bioprinting; then cell damage caused by extensional stress is inferred from the difference between the total cell damage and the amount of cell damage attributed to shear stress. With the obtained magnitude of extensional stress from fluidic simulation, the model that relates extensional stress to cell damage is established; the bioprinting process-induced cell damage attributed to both shear and extensional stresses is therefore presented. Schwann cells and myoblasts were used as examples to validate the models. Comparison between experimental and simulation results shows the effectiveness of the models presented in this paper. Moreover, the viability and proliferative ability of cells in the first 72 h after bioprinting is investigated, with the results illustrating that the process-induced forces affect not only cell viability but also their proliferative ability after bioprinting.

New evidences for a role of mGluR7 in astrocyte survival: Possible implications for neuroprotection

A specific activation of metabotropic glutamate receptor 7 (mGluR7) has been shown to be neuroprotective in various models of neuronal cell damage, however, its role in glia cell survival has not been studied, yet. Thus, we performed comparative experiments estimating protective effects of the mGluR7 allosteric agonist AMN082 in glia, neuronal and neuronal-glia cell cultures against various harmful stimuli. First, the transcript levels of mGluR7 and other subtypes of group II and III mGluRs in cortical neuronal, neuronal-glia and glia cell cultures have been measured by qPCR method. Next, we demonstrated that AMN082 with similar efficiency attenuated the glia cell damage evoked by staurosporine (St) and doxorubicin (Dox). The AMN082-mediated glioprotection was mGluR7-dependent and associated with decreased DNA fragmentation without involvement of caspase-3 inhibition. Moreover, the inhibitors of PI3K/Akt and MAPK/ERK1/2 pathways blocked the protective effect of AMN082. In neuronal and neuronal-glia cell cultures in the model of glutamate (Glu)- but not St-evoked cell damage, we showed a significant glia contribution to mGluR7-mediated neuroprotection. Finally, by using glia and neuronal cells derived from mGluR7+/+ and mGluR7−/− mice we demonstrated a higher cell-damaging effect of St and Dox in mGluR7-deficient glia but not in neurons (cerebellar granule cells). Our present data showed for the first time a glioprotective potential of AMN082 underlain by mechanisms involving the activation of PI3K/Akt and MAPK/ERK1/2 pathways and pro-survival role of mGluR7 in glia cells. These findings together with the confirmed neuroprotective properties of AMN082 justify further research on mGluR7-targeted therapies for various CNS disorders.

Effects of BNN27, a novel C17-spiroepoxy steroid derivative, on experimental retinal detachment-induced photoreceptor cell death

Retinal detachment (RD) leads to photoreceptor cell death secondary to the physical separation of the retina from the underlying retinal pigment epithelium. Intensifying photoreceptor survival in the detached retina could be remarkably favorable for many retinopathies in which RD can be seen. BNN27, a blood-brain barrier (BBB)-permeable, C17-spiroepoxy derivative of dehydroepiandrosterone (DHEA) has shown promising neuroprotective activity through interaction with nerve growth factor receptors, TrkA and p75NTR. Here, we administered BNN27 systemically in a murine model of RD. TUNEL+ photoreceptors were significantly decreased 24 hours post injury after a single administration of 200 mg/kg BNN27. Furthermore, BNN27 increased inflammatory cell infiltration, as well as, two markers of gliosis 24 hours post RD. However, single or multiple doses of BNN27 were not able to protect the overall survival of photoreceptors 7 days post injury. Additionally, BNN27 did not induce the activation/phosphorylation of TrkAY490 in the detached retina although the mRNA levels of the receptor were increased in the photoreceptors post injury. Together, these findings, do not demonstrate neuroprotective activity of BNN27 in experimentally-induced RD. Further studies are needed in order to elucidate the paradox/contradiction of these results and the mechanism of action of BNN27 in this model of photoreceptor cell damage.

Lycium barbarum Polysaccharide Supplementation Improves Alcoholic Liver Injury in Female Mice by Inhibiting Stearoyl-CoA Desaturase 1.

Lycium barbarum polysaccharide (LBP) is a water fraction of wolfberry, which has been demonstrated to possess a hepatoprotective effect in several liver disease models. However, the anti-alcoholic liver disease (anti-ALD) mechanism of LBP has not been investigated thoroughly. Its protective effects on both male and femal mice are investigated in the current study. A chronic ethanol-fed ALD in vivo model is applied to study the effect of LBP in both male and female mice. It is observed that ethanol causes more severe liver injury in female than male mice, and the ameliorative effects of LBP are also more significant in female mice, which are impaired after complete bilateral oophorectomy. The hepatic SCD1 expression is found to be positively correlated with the severity of the liver damage and the main mediator of LBP inducer of protection. The AMPK-CPT pathway is also activated by LBP to rebalance the dysregulated lipid metabolism during ALD development. By using concurrent sodium palmitate and an ethanol-induced in vitro cell damage model in AML-12 cell line, it is characterized that LBP directly interacts with ERα instead of ERβ to activate the SCD1-AMPK-CPT pathway. LBP is an effective and safe hepatoprotective agent against ALD primarily through the SCD1-AMPK-CPT pathway after ERα agonist.

Molecular mechanism of Nifedipine inducing liver injury in children

Objective To establish the Nifedipine-induced liver cell damage model, and to investigate the cellular or molecular mechanism for children′s liver cell damage. Methods The HepG2 cells were utilized to establish liver cell damage models.The optimal concentration and the optimal pretreatment time of Nifedipine-induced liver cell injury were confirmed.Western blot and real-time PCR (RT-PCR) were used to check the alteration in proteins and mRNAs level of the liver function-associated classic markers, which contained alkaline phosphatase (ALP), aspartate amino transferase (AST), glutamyl transpeptidase (γ-GT) and alanine aminotransferase (ALT). Additionally, flow cytometry (FCM), colony formation assay (CFA) and cell wound healing assay (CWHA) were utilized to check the effect of Nifedipine on the cell cycle progression and proliferation of HepG2. Results (1)The optimal concentration of Nifedipine was 20 mg/L and the optimal treatment period was 21 days for liver damage.(2)Western blot: intracellular ALT protein content after Nifedipine group was less than that of control group, while the protein levels of AST, γ-GT and ALP in the culture medium after Nifedipine addition (3.55±0.05, 4.91±0.055, 3.51±0.05, 3.08±0.08) were higher than those of control group(0.96±0.02, 1.03±0.02, 1.00±0.05, 0.90±0.13), and the differences were all significant (t =-85.695, -117.582, -47.371, -33.260, all P<0.05). (3)The findings of RT-PCR showed that the mRNA levels of intracellular ALT, γ-GT and ALP in Nifedipine group (0.26±0.02, 0.05±0.04, 0.05±0.02) were lower than those of control group (1.13±0.21, 0.94±0.10, 1.03±0.06), and the differences were all significant (t=7.233, 127.436, 25.687, all P<0.05). However, the mRNAs levels in purified culture me-dium in Nifedipine group (5.95±0.05, 3.13±0.10, 3.32±0.08) were higher than those in control group (1.01±0.08, 1.00±0.05, 1.00±0.05), and the differences were all significant (t=-92.339, -31.250, -43.007, all P<0.05). (4)The portion of G0/G1 phase in Nifedipine group [(84.09±0.43)%] was more than that of control group[(30.93±0.32)%], which had statistical significance (t=173.084, P=0.000). (5)In contrast with control group, the colony formation of cells in Nifedipine group declined from (97.10±1.17)% to (38.56±1.51)% (t=92.088, P=0.000) and the migration rate of cells wound healing was (56.37±2.06)%, (25.00±1.71)% separately( t=20.285, P=0.000). Conclusion Nifedipine may promote children′s liver injury through regulating cell cycle related proteins. Key words: Nifedipine; HepG2; Liver function; Cell proliferation

Flavonoids of Kudzu Root Fermented by Eurtotium cristatum Protected Rat Pheochromocytoma Line 12 (PC12) Cells against H₂O₂-Induced Apoptosis.

Novel bioactive components have greatly attracted attention as they demonstrate health benefits. Reversed-phase high performance liquid chromatography (RP-HPLC) showed that isoflavonoid compounds of kudzu root (Pueraria lobata) fermented by Eurtotium cristatum and extracted using de-ionized water were higher active compared with non-fermented. A model of H2O2-inducd cell damage was built using rat pheochromocytoma line 12 (PC12) cell to observe the protective effect of non-fermented kudzu root (Pueraria lobata) (NFK) and fermented kudzu root (Pueraria lobata) (FK). Cell viability and apoptosis were analyzed through inverted microscopy and flow cytometry. The level of lactate dehydrogenase, catalase activity, superoxide dismutase, glutathione, and reactive oxygen species (ROS) were evaluated. Results showed that NFK and FK could significantly protect PC12 cell against damage caused by H2O2-induced oxidative stress. The intracellular antioxidant system was increased, protected the cell membrane inhibit H2O2-induced apoptosis by scavenging of ROS. Moreover, NFK and FK regulated the cell cycle to prevent cell apoptosis. Isoflavonoid from the kudzu root especially fermented kudzu root with E. cristatum are potentially therapeutic drugs against diseases induced by oxidative damage.

Orthogonal Array composite design to study and optimize antioxidant combinations in the prevention of UVB-induced HSF damage

Excessive exposure to ultraviolet (UV) B radiation may lead to skin damage, photosensitivity, or even tumorigenesis via induction of oxidative stress. Naturally derived antioxidants could play significant roles in cancer therapy due to their multi-targeted actions and lack of substantial toxicity. Drug combinations target at diverse pathway of cells and make cells export meticulous biological outcomes through the multifaceted signaling network. The UVB protective effects of combinations of naturally derived antioxidants- curcumin, resveratrol, proanthocyanidins, baicalein, and beta-nicotinamide adenine dinucleotide (NADH) were investigated. An oxidative cell damage model was established to study the ultraviolet irradiation system. An orthogonal array composite design (OACD) was employed in the optimization of antioxidants combinations. Combination of resveratrol (0.1μM) and baicalein in medium concentration (0.2μM), with NADH in high concentration (0.8μM) was found to be the most efficacious combination among all the 30 runs performed using OACD. The findings suggested that UVB exposure-inflicted cell apoptosis can be significantly reduced by naturally-derived antioxidant combinations. These results provide an insight into the discovery of synergistic antioxidant combinations in skin cancer, using orthogonal array composite design (OACD). The results also have practical implications in the understanding of drug mechanisms in skin cancer, which can assist clinical practice by recommending better drug combinations.

Mechanism of endogenous digitalis-like factor‑induced vascular endothelial cell damage in patients with severe preeclampsia.

Although endogenous digitalis‑like factor (EDLF) is associated with the development of various physical disorders, the role in preeclampsia remains unclear. This study investigated the effects of EDLF on vascular endothelial cell damage in patients with preeclampsia and the potential mechanisms. From July2014 to July2015, 120 singleton pregnancy cases underwent a prenatal examination, inpatient delivery and had normal blood pressure were included in the study, either as patients with severe preeclampsia or the control patients. Serum EDLF levels were compared in these two groups, and an invitro hypoxic trophocyte‑induced vascular endothelial cell damage model was established to explore the changes in hypoxic trophocyte EDLF level and the subsequent effects on human umbilical vein endothelial cells(HUVECs). Nuclear factor‑κB(NF‑κB) p65 gene expression was silenced in hypoxic trophocytes, and EDLF levels and HUVEC damage were subsequently assessed. Serum EDLF levels were significantly higher in the severe preeclampsia cases than in the controls at the same gestational week (P<0.001). EDLF levels in hypoxic trophocytes increased with the increasing co‑culture duration. Damage to the biofunctions of HUVECs co‑cultured with hypoxic trophocytes also increased with co‑culture duration. However, silencing of NF‑κBp65 in the hypoxic trophocytes reduced the EDLF levels. AnnexinA2 was highly expressed in HUVECs, and no biofunctions were significantly damaged (P<0.05) compared with the group without receiving NF‑κBp65 silencing. Serum EDLF levels were significantly higher in patients with severe preeclampsia compared with the controls. The results of the current study indicate that NF‑κBp65 has a role in regulating EDLF production in hypoxic trophocytes.

Neuroprotective effect of really interesting novel gene finger protein 146/Iduna and its pro-survival activity in disease

Background The really interesting new gene (RING) finger protein 146/Iduna is a kind of endogenous pro-survival protein and can combine with poly ADP-ribose (PAR) to effectively interfere the PARP1-dependent nerve cell parthanatos death caused by excitotoxicity and oxidative stress. Some researches also found that Iduna had the E3 ubiquitin ligase activity and participated in the DNA repair, which played the regulating effect in many diseases through the Wnt pathway. Objective To understand the biological nature of RING finger 146/Iduna protein and its application in diseases. Content Review the neuroprotective effect and possible mechanism of endogenous pro-survival activity in different diseases and cell damage models of RING finger protein 146/Iduna. Trend With the further research on the biological function of RING finger protein 146/Iduna, its role in the disease will be revealed constantly. Key words: Really interesting new gene finger protein 146; Iduna; Neuroprotective; Pro-survival activity

MicroRNA-25 inhibits high glucose-induced apoptosis in renal tubular epithelial cells via PTEN/AKT pathway

Diabetic nephropathy (DN) has become the major cause of end-stage renal disease (ESRD). It has been demonstrated that apoptosis of renal tubular epithelial cells induced by hyperglycemia contributes to the pathogenesis of DN. Recent researches have corroborated the critical roles of microRNAs (miRNAs) in the apoptosis of various types of cells including renal tubular epithelial cells. However, the eff ; ;ect of miRNAs on the hyperglycemia-induced apoptosis of renal tubular epithelial cells remains unclear. The aim of this study is to explore the eff ; ;ect of miRNAs on the hyperglycemia-induced apoptosis of renal tubular epithelial cells and its molecular mechanism. Using a miRNA microarray, miRNAs putatively associated with DN were examined in renal biopsy tissue samples from DN patients and healthy controls. Validation analysis of miR-25 level in serum samples and renal biopsy tissue samples was performed using quantitative reverse transcription PCR (qRT-PCR). Then, gain- and loss- of function experiments were performed to determine the protective roles of miR-25 in high glucose-induced damage to renal tubular epithelial cells. Furthermore, the target gene of miR-25 and the downstream signaling pathway were also investigated. Microarray analysis and qRT-PCR revealed that miR-25 was significantly downregulated in renal biopsy tissue and serum samples from DN patients. We also observed that an inverse relationship between serum miR-25 level and proteinuria in DN patients. Meanwhile, miR-25 was decreased in human kidney (HK-2) cells subjected to HG treatment in a time dependent manner. Its overexpression reduced production of reactive oxygen species (ROS), suppressed cell apoptosis in HG-induced cell damage model, which was coupled with the decreased expression of cleaved caspase-3 and activity of caspase-3. Subsequent analyses demonstrated that phosphatase and tensin homolog deleted on chromosome ten (PTEN) was a direct and functional target of miR-25, which was validated by the dual luciferase reporter assay. Most importantly, the overexpression of PTEN effectively reversed the protective effects of miR-25 mimics on renal tubular epithelial cell injury. We also found that the anti-apoptotic effects of miR-25 are dependent on the activation of PTEN/Akt pathway. In addition, we observed that PTEN was upregulated in renal biopsy tissue samples from patients with DN, and an inverse relationship was found between PTEN and miR-25 expression, suggesting that miR-25 may exert its function through regulation of PTEN in DN. Taken together, our study proved that overexpression of miR-25 could ameliorate HG-induced oxidative stress and apoptosis in renal tubular epithelial cells through activation of PTEN/AKT pathway, suggesting that overexpression of miR-25 might provide a potential therapeutic approach for DN.

The ATM kinase inhibitor KU-55933 provides neuroprotection against hydrogen peroxide-induced cell damage via a γH2AX/p-p53/caspase-3-independent mechanism: Inhibition of calpain and cathepsin D

The role of the kinase ataxia-telangiectasia mutated (ATM), a well-known protein engaged in DNA damage repair, in the regulation of neuronal responses to oxidative stress remains unexplored. Thus, the neuroprotective efficacy of KU-55933, a potent inhibitor of ATM, against cell damage evoked by oxidative stress (hydrogen peroxide, H2O2) has been studied in human neuroblastoma SH-SY5Y cells and compared with the efficacy of this agent in models of doxorubicin (Dox)- and staurosporine (St)-evoked cell death. KU-55933 inhibited the cell death induced by H2O2 or Dox but not by St in undifferentiated (UN-) and retinoic acid-differentiated (RA)-SH-SY5Y cells, with a more pronounced effect in the latter cell phenotype. Furthermore, this ATM inhibitor attenuated the Dox- but not H2O2-induced caspase-3 activity in both UN- and RA-SH-SY5Y cells. Although KU-55933 inhibited the H2O2- and Dox-induced activation of ATM, it attenuated the toxin-induced phosphorylation of the proteins H2AX and p53 only in the latter model of cell damage. Moreover, the ATM inhibitor prevented the H2O2-evoked increases in calpain and cathepsin D activity and attenuated cell damage to a similar degree as inhibitors of calpain (MDL28170) and cathepsin D (pepstatin A). Finally, we confirmed the neuroprotective potential of KU-55933 against the H2O2- and Dox-evoked cell damage in primary mouse cerebellar granule cells and in the mouse hippocampal HT-22 cell line. Altogether, our results extend the neuroprotective portfolio of KU-55933 to a model of oxidative stress, with this effect not involving inhibition of the γH2AX/p-p53/caspase-3 pathway and instead associated with the attenuation of calpain and cathepsin D activity.

Effects of phenolic constituents of daylily flowers on corticosterone- and glutamate-treated PC12 cells

BackgroundDaylily flowers, the flower and bud parts of Hemerocallis citrina or H. fulva, are well known as Wang-You-Cao in Chinese, meaning forget-one’s sadness plant. However, the major types of active constituents responsible for the neurological effects remain unclear. This study was to examine the protective effects of hydroalcoholic extract and fractions and to identify the active fractions.MethodsThe extract of daylily flowers was separated with AB-8 resin into different fractions containing non-phenolic compounds, phenolic acid derivatives and flavonoids as determined using UPLC-DAD chromatograms. The neuroprotective activity was measured by evaluating the cell viability and lactate dehydrogenase release using PC12 cell damage models induced by corticosterone and glutamate. The neurological mechanisms were explored by determining their effect on the levels of dopamine (DA), 5-hydroxy tryptamine (5-HT), γ-aminobutyric acid (GABA), noradrenaline (NE) and acetylcholine (ACh) in the cell culture medium measured using an LC-MS/MS method.ResultsPretreatment of PC12 cells with the extract and phenolic fractions of daylily flowers at concentrations ranging from 0.63 to 5 mg raw material/mL significantly reversed corticosterone- and glutamate-induced neurotoxicity in a dose-dependent manner. The fractions containing phenolic acid derivatives (0.59% w/w in the flowers) and/or flavonoids (0.60% w/w) exerted similar dose-dependent neuroprotective effect whereas the fractions with non-phenolic compounds exhibited no activity. The presence of phenolic acid derivatives in the corticosterone- and glutamate-treated PC12 cells elevated the DA level in the cell culture medium whereas flavonoids resulted in increased ACH and 5-HT levels.ConclusionPhenolic acid derivatives and flavonoids were likely the active constituents of daylily flowers and they conferred a similar extent of neuroprotection, but affected the release of neurotransmitters in a different manner.