<i>In vitro</i> isolation and culture of SD rat retinal ganglion cells and establishment of a high-glucose model / 国际眼科杂志(Guoji Yanke Zazhi)

@#AIM: To isolate the retinal ganglion cells(RGCs)of neonatal Sprague-Dawley(SD)rats <i>in vitro</i>, and to establish the RGCs primary culture method and high glucose model of neonatal SD rats.<p>METHODS: The retinal tissues of SD rats from 1-3d as the materials were taken, from which the RGCs were isolated and purified for primary culture. Toluidine blue and immunofluorescence staining methods were adopted to identify the cultured cells. After 48-72h of continuous culture, RGCs were randomly divided into 6 groups and cultured in different glucose concentrations of 5.5mmol/L(normal control group), 20mmol/L, 25mmol/L, 30mmol/L, 35mmol/L and 40mmol/L for 24, 48 and 72h, respectively. Finally, the CCK8 method and TUNEL method were adopted to determine the cell survival rate and apoptosis rate. <p>RESULTS: The primary RGCs purified and cultured <i>in vitro</i> represented typical cell morphology and grew well, the cells were confluent and aggregated in small lamellar manner, while the axons crisscrossed into a network, in addition, cell halo could be seen around the cell body. Nissl bodies with clear structure were found in the cytoplasm of toluidine blue stained cells, the percentage of neurons was more than 95%. RGCs specific antibodies Thy-1 and Brn-3a were employed to identify the purified cells <i>in vitro</i>, and the positive rate reached more than 90%. The CCK8 results showed that the survival rate of cells decreased(OD value decreased)with the increase of culture time and glucose concentration. When the cells were treated with different glucose concentrations for 24h, the OD values of each group were lower than those of the normal control group, but there was no significant difference between the OD values of each group and the normal control group(all <i>P</i>>0.05). With the extension of culture time, the OD values of 35mmol/L and 40mmol/L glucose concentration intervention RGCs 48h, 30mmol/L, 35mmol/L, 40mmol/L intervention RGCs 72h were significantly lower than those of the normal control group, the difference was statistically significant compared with the normal control group(all <i>P</i><0.05). TUNEL results revealed that the apoptosis rate of RGCs increased with the increase of glucose concentration and time, among them, the apoptosis rate of RGCs cultured in glucose concentration of 30mmol/L, 35mmol/L and 40mmol/L for 48h and 72h was significantly statistical different from that of normal control group(all <i>P</i><0.05). <p>CONCLUSION: The RGCs primary culture method established in this study is capable of separating typical RGCs with high purity. With the increase of glucose concentration in the medium, the survival rate of RGCs have been decreased while the apoptosis rate increased. Notably, the 35mmol/L glucose intervention for 48h can be employed as the optimal intervention concentration and time to effectively induce RGCs to establish the high glucose model.

Effects of Huangpu Tongqiao capsule on apoptosis of Alzheimer's disease cell model / 中国中药杂志

The loss of hippocampal neurons is one of the main pathological features of Alzheimer's disease (AD), which is related to the apoptosis of hippocampal neurons. Huangpu Tongqiao capsule is used for the treatment of AD, but the underlying mechanism is still unclear. This study is to investigate the mechanism of neuroprotective effect of Huangpu Tongqiao capsule in the treatment of AD, through observing the effect of Huangpu Tongqiao capsule containing serum on cell injury of primary cultured hippocampal neurons induced by Aβ₂₅₋₃₅ via inhibiting the cell apoptosis. Primary cultured hippocampal neurons were cultured and identified by MAP-2 immunofluorescence staining, and cell growth state was observed by inverted microscope. The Huangpu Tongqiao capsule containing serum was prepared using the method of serum pharmacology. MTT assays were used to measure the optimum concentration range of Huangpu Tongqiao capsule containing serum, and optimum Aβ concentration for establishing the AD model. After primary cultured hippocampal neurons AD cell model was induced by Aβ₂₅₋₃₅, cell survival rate was detected by MTT, cell apoptosis rate was assayed by flow cytometry, and protein expressions of Bax, Cyt C and caspase-3 were determined by Western blot analysis. The results showed that the primary cultured hippocampal neurons were cultured successfully, and cells grew mature at seventh days; Compared with normal group, the survival rate of hippocampal neurons in AD cell model group was decreased, the apoptosis rate of hippocampal neurons was increased, and the protein expressions of Bax, Cyt C and caspase-3 were increased (<0.05, <0.01); Compared with AD cell model group, the survival rate of hippocampal neurons in Huangpu Tongqiao capsule containing serum group was increased, the apoptosis rate of hippocampal neurons was decreased, and the protein expressions of Bax, Cyt C and caspase-3 were decreased (<0.05, <0.01). These findings suggest that Huangpu Tongqiao capsule containing serum has a neuroprotective effect on cell injury of the primary cultured hippocampal neurons induced by Aβ₂₅₋₃₅, and its effect on the treatment of AD is associated with the inhibition the apoptosis of hippocampal neurons.

Establishment of in vitro methods for evaluation of induction and inhibition of human CYP450 enzymes by drugs / 药学学报

The aim of this study is to establish the in vitro methods for the study of induction and inhibition on CYP450 by drugs, and to validate the analytical method and incubation system. A method for the simultaneous determination of eight metabolites of seven subtypes of CYP450 enzymes probe substrates in human liver microsomes (HLM) was established and validated. The incubation system was optimized to confirm the incubation time and protein concentration of HLM, the enzyme activity of seven subtypes of CYP450 enzymes in HLM was determined, and the inhibition effects on each CYP450s were checked by positive controls. The method for the simultaneous determination of three metabolites of subtypes of CYP450 enzymes was established and validated in human primary cultured hepatocytes (HPCH) using the incubation medium. The enzyme activity of three subtypes of CYP450 enzymes in HPCH was determined, and the total RNA was extracted from HPCH after incubation. The expression of CYP450 enzymes were measured by Taqman fluorescence probe method. The induction effects on each CYP450s were examined using the positive controls. The established methods for the determination of metabolites of probe substrates were fully validated, and the results were conformed to the requirements of bioanalytical method validation. The induction and inhibition effects on each CYP450s were checked by positive controls. The established in vitro methods for the study of drug induction and inhibition on CYP450 were simple and reliable, which could be used in the investigation of enzyme induction or inhibition properties of new drug candidates and to evaluation the metabolic interactions of concomitant medication in clinical.

Effects of propofol treatment on neuroapoptosis in primary cultured cortical neurons / 临床麻醉学杂志

Objective To investigate the effect of propofol exposure on neuroapoptosis in pri-mary cultured cortical neurons and its mechanisms.Methods Cortical neurons were primarily cultured for seven days,then divided into two groups:control group (treated with equal volume of 20% in-tralipid),propofol-treated group (treated with 500 μmol/L propofol).The neurons were treated for 12 h.The neuron viability was determined by MTT.Neuroapoptosis was identified by Hoechest 33 258 dying.Mitochondrial membrane potential was measured by the fluorescent dye rhodamine 123 (Rh123).Western blot was performed to detect the level of cyt-c and cleaved-caspase-3.Results Neu-rons survival rate (54.4%±6.4%)in the propofol group was significantly lower than that of control group (99.8% ± 4.1%) (P < 0.05 ), the rate of neuronal apoptosis (46.5% ± 5.3%) was significantly higher than that of control group (7.2%±0.9%)(P <0.05),mitochondrial membrane potential (59.6%±4.3%)was significantly lower than that of the control group (99.9% ± 5.7%) (P <0.05 ),cyt-C protein level (0.38 ± 0.03 )was significantly higher than that of control group (0.1 5±0.02)(P < 0.05 ),level of cleaved-caspase-3 protein level (0.46 ± 0.04)was significantly higher than that of control group (0.13±0.02)(P <0.05).Conclusion Propofol induces neuroapo-tosis in primary cultured cortical neurons,which is associated with the decreased level of MPP and the increase levels of cyt-c and cleaved-caspase-3.

Effect and mechanism of dexmedetomidine on propofol-induced apoptosis of cortical neurons in rats / 中华行为医学与脑科学杂志

Objective To investigate the mechanisms of the protective effects of dexmedetomidine against the propofol-induced neuroapoptosis in primary cultured cortical neurons.Methods The neurons were cultured 7days and then divided into four groups: vehicle-control group (treated with equal volume of intralipid), propofoltreated group (treated with 500 μmol/L propofol), propofol plus dexmedetomidine treated group (treated with 500 μmol/L propofol and 0.1 μmol/L dexmedetomidine), and LY294002 pretreated group (treated with 500 μmol/L propofol ,0.1 μ mol/L dexmedetomidine and 10 μmol/L LY294002).12 hours after different treatments, neuron viability was measured by MTT assay,neuroapoptosis was detected by Hoechst33258 staining, and the levels of pAkt and Bcl-2 protein were detected by Western blot.Results Compared with the vehicle-reduced group,propofol reduced neuron viability greatly((53.4±4.2)％ vs (99.9±6.3)％;P＜0.01), but increased neuroapoptosis greatly((44.6±4.3)％ vs (5.8±0.4)％;P＜0.01).The levels of pAkt((0.41±0.03) vs (0.86±0.07))and Bcl-2 ((0.15±0.02) vs (0.72±0.03)) were decreased greatly (both P＜0.01).Compared with propofol treatment group, the neuron viability of propofol plus dexmedetomidine group were increased greatly((86.4±5.3) ％ , P＜0.01) ,the neu roapoptosis was decreased greatly ((23.1 ± 3.5) ％, P＜ 0.01), and the levels of pA kt (0.8 ± 0.03) and Bc1-2 (0.52 ±0.05) were increased greatly (both P＜0.01).Compared with propofol plus dexmedetomidine treated group,LY294002 inhibited the protective effects of dexmedetomidine, decreased neuron viability greatly ((64.3±5.1) ％ ,P＜0.01), increased the number of apoptotic neurons((38.8±4.9) ％, P＜0.01), and reduced the levels of pAkt (0.52±0.04) and Bcl-2(0.31±0.02) significantly (P＜0.01).Conclusion Dexmedetomidine exerts the neuroprotective effects against propofol-induced neuroapoptosis by activating the PI3K-Akt-Bcl-2 signalling pathway.

Effects of 17β-estradiol on propofol-induced rat cortical neuroapoptosis / 中国比较医学杂志

Objective To investigate the protective effects and the mechanisms of 17β-estradiol on the propofol-induced neuroapoptosis in primary cultured rat cortical neurons.Methods The neurons were cultured for 7 days and then divided into three groups: vehicle-control group ( treated with equal volume of 20% intralipid ) , propofol-treated group ( treated with 500μmol/L propofol) , and propofol plus 17β-estradiol treated group ( treated with 500μmol/L propofol and 0.1 μmol/L 17β-estradiol).12 hours after the treatment, neuroapoptosis was detected by Hoechst 33258 staining and TUNEL assay, and the levels of Bcl-2, Bax and cleaved caspase-3 proteins were detected by Western blot.Results Compared with the vehicle-control group, the neuroapoptosis increased greatly ( P<0.01 ) , Bcl-2 level reduced ( P <0.01), Bax and cleaved caspase-3 levels increased greatly (P<0.01), and Bcl-2/Bax ratio reduced significantly (P<0.01).Compared with the propofol-treatment group, the neuroapoptosis decreased greatly ( P <0.01), Bcl-2 level increased ( P<0.01 ) , Bax and cleaved caspase-3 levels reduced greatly ( P <0.01 ) , and Bcl-2/Bax ratio increased greatly ( P <0.01 ) . Conclusions 17β-estradiol can protect cortical neurons against propofol-induced cortical neuroapoptosis by regulating the expression of Bcl-2 and Bax.

Dexmedetomidine protects cortical neurons from ketamine-induced apoptosis by activating the PI3 K-Akt signaling pathway / 医学研究生学报

Objective Dexmedetomidine is known to have a neuroprotective effect.The aim of this study was to investigate the effects of dexmedetomidine on ketamine-induced apoptosis of primarily cultured cortical neurons and its action mechanisms. Methods Rat cortical neurons were primarily cultured for 7 days and treated with ketamine (100μmol/L) and different concentrations of dexmedetomi-dine (0.001, 0.01, 0.1, and 1 μmol/L) for 24 hours, followed by measurement of the viability of the neurons by MTT assay.The neurons were divided into four groups:vehicle control, ketamine ( trea-ted with 100 μmol/L ketamine), dexmedetomidine+ketamine (DD+K, treated with 0.1 μmol/L DD and 100 μmol/L ketamine), and LY294002 ( treated with 0.1 μmol/L DD, 100 μmol/L ketamine, and 10 μmol/L LY294002) .After 24 hours of treatment, the apoptosis rate of the neurons was determined by Hoechst33258 staining, and the expressions of pAkt and cleaved-caspase-3 in the neu-rons detected by Western blot. Results The apoptosis rate of neurons was dramatically increased in the LY294002 and ketamine groups in comparison with the vehicle control and DD+K groups ([36.8 ±4.4] and [43.4 ±4.5]%vs [7.5 ±1.1] and [16.4 ± 3.6]%, P<0.01), the pAkt level remarkably decreased (0.26 ±0.02 and 0.15 ±0.01 vs 0.61 ±0.05 and 0.50 ±0.04, P<0.01), and the expression of cleaved caspase-3 significantly upregulated in the former two as compared with the latter two groups (0.40 ±0.02 and 0.65 ±0.03 vs 0.10 ±0.02 and 0.12 ±0.01, P<0.01). Conclusion Dexmedetomidine exerts a neuroprotec-tive effect against ketamine-induced apoptosis of neurons by activating the PI3K-Akt signaling pathway.

Japanese Kampo Medicine, Ninjinyoeito, Inhibits the Induction of iNOS Gene Expression in Proinflammatory Cytokine-Stimulated Hepatocytes.

Background: Japanese Kampo medicine, ninjinyoeito (NYT), is used for the treatment of complaints in patients with general fatigue, anorexia and anemia, and is recently applied in patients with chronic hepatitis C. However, there is little scientific evidence to demonstrate the liver-protective effects of NYT. We examined proinflammatory cytokine stimulated hepatocytes as a simple in vitro liver injury model to determine liver-protective effects of NYT and to clarify the mechanisms involved in. Methods: Primary cultured rat hepatocytes were treated with interleukin (IL)-1β in the presence or absence of NYT. Inflammatory biomarkers including inducible nitric oxide synthase (iNOS) and tumor necrosis factor (TNF)-α were analyzed. Results: IL-1β stimulated the induction of iNOS and enhanced the production of NO. Simultaneous addition of NYT decreased the levels of iNOS protein and its mRNA expression, resulting in the reduction of NO production. NYT also reduced the expression of TNF-α mRNA and enhanced the expression of IL-10 mRNA. NYT inhibited two essential signaling pathways for iNOS induction, the activation of nuclear factor (NF)-κB and the upregulation of type I IL-1 receptor. Experiments with transfection and iNOS gene antisense transcript revealed that NYT reduced the levels of iNOS mRNA at both the promoter activation and mRNA stabilization steps. The delayed administration of NYT after IL-1β addition also inhibited iNOS induction. Conclusions: Results demonstrate that NYT can influence the induction of inflammatory mediators, such as iNOS and TNF-α, in part through the inhibition of NF-κB activation in hepatocytes. NYT may have therapeutic potential for various acute liver injuries.

Expression and Activity of the Na-K ATPase in Ischemic Injury of Primary Cultured Astrocytes

Astrocytes are reported to have critical functions in ischemic brain injury including protective effects against ischemia-induced neuronal dysfunction. Na-K ATPase maintains ionic gradients in astrocytes and is suggested as an indicator of ischemic injury in glial cells. Here, we examined the role of the Na-K ATPase in the pathologic process of ischemic injury of primary cultured astrocytes. Chemical ischemia was induced by sodium azide and glucose deprivation. Lactate dehydrogenase assays showed that the cytotoxic effect of chemical ischemia on astrocytes began to appear at 2 h of ischemia. The expression of Na-K ATPase alpha1 subunit protein was increased at 2 h of chemical ischemia and was decreased at 6 h of ischemia, whereas the expression of alpha1 subunit mRNA was not changed by chemical ischemia. Na-K ATPase activity was time-dependently decreased at 1, 3, and 6 h of chemical ischemia, whereas the enzyme activity was temporarily recovered to the control value at 2 h of chemical ischemia. Cytotoxicity at 2 h of chemical ischemia was significantly blocked by reoxygenation for 24 h following ischemia. Reoxygenation following chemical ischemia for 1 h significantly increased the activity of the Na-K ATPase, while reoxygenation following ischemia for 2 h slightly decreased the enzyme activity. These results suggest that the critical time for ischemia-induced cytotoxicity of astrocytes might be 2 h after the initiation of ischemic insult and that the increase in the expression and activity of the Na-K ATPase might play a protective role during ischemic injury of astrocytes.

Effects of Hypoxia on the Ubiquitin-proteasome System in Primary Cortical Neuronal Cell Cultures / 체질인류학회지

The ubiquitin-proteasome system is crucial in maintaining cellular growth and metabolism. Dysfunction of this system may contribute to neurodegenerative diseases, such as Parkinson's disease. But its effects on primary neurons are largely unknown. In the present study, we investigated the effects of proteasome inhibitor and hypoxia on primary neuronal cultures to determine whether proteasomal malfunction induces neuronal death. Neuronal apoptosis increased in primary cultured cortical neurons with treatment of proteasomal inhibitor in normoxic condition and in the presence or absence of proteasomal inhibitor in hypoxic condition. Also expression of PARP and activated caspase 3 increased. NF-kappaB, a key transcription factor in this system expression increased in hypoxic condition and proteasomal inhibition. Interestingly, hypoxic condition induced an expression and accumulation of alpha-synuclein in neuron, one of components of Lewy body in Parkinson's disease. Our findings determine that hypoxic condition may affect the ubiquitin-proteasome system. Furthermore, it suggests that hypoxic condition and proteasomal inhibitors are involved, at least in parts, in neurodegeneration of mouse model for Parkinson's disease.

A study on expression changes of a tumor suppressor WWOX in human lung adenocarcinoma cell line A549 / 中国癌症杂志

Purpose:To detect the abnormalities of WWOX(WW domain containing oxidoreductase) gene in human lung adenocarcinoma cell line A549. Methods:Deletion of WWOX exons 6-8 transcript was analyzed by reverse transcriptase-PCR technology; loss of heterozygosity (LOH) of WWOX gene was analyzed by PCR-based assays for dinucleotide repeat polymorphisms technology. Aberrant expression of WWOX protein was analyzed by western blot. Results:A549 cells samples showed loss of WWOX exons 6-8 transcript.This deletion was not detected in normal primary cultured human bronchial epithelial cells samples.Three microsatellites(D16S3029、D16S3096、D16S504)did not have LOH in the normal primary cultured human bronchial epithelial cells samples, but D16S2029 and D16S3096 were all found to have LOH in A549 Cells samples. We further observed that expression of WWOX protein was significantly lower in A549 cell samples compared to the normal primary cultured human bronchial epithelial cells samples. Conclusions:WWOX gene may be important during tumorigenesis in lung adenocarcinoma cancer.Deletion of exons 6-8,LOH and aberrant expression of protein are all modes of WWOX gene inactivity in lung adenocarcinoma cancer.

Protective effects of puerarin on rat primary cultured neurons damaged by glutamic acid / 中国药理学通报

Aim To study the protective effects of puerarin on the neurons damaged by glutamic acid.Method The rat primary cultured neurons were damaged by glutamic acid,the membrane lipids fluidity was studied with fluorescence polarization(P) and mean microviscosity(?) with DPH as a probe;the protective effects of puerarin on this model were evaluated by lactate dehydrogenase(LDH) efflux assay and colormetric MTT assay.Results The fluorescence polarizating and mean microviscosity of puerarin were lower than glutamic acid model group in a dose-depended manner.Treatment of puerarin resulted in the decrease of LDH release from primary cultured neurons damaged by glutamic acid and increase in the optical density(A) at 540 nm tested by colorimetric MTT assay.No proliferating effects on normal primary cultured neurons were observed.Conclusion The results suggested that puerarin had a protective effect on rat primary cultured neurons damaged by glutamic acid,it might be relative to the improvement of membrane lipids fluidity