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@#Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related deaths, characterized by highly hypoxic tumor microenvironment. Hypoxia-inducible factor-1α (HIF-1α) is a major regulator involved in cellular response to changes of oxygen levels, supporting the adaptation of tumor cells to hypoxia. Bruceine D (BD) is an isolated natural quassinoid with multiple anti-cancer effects. Here, we identified BD could significantly inhibit the HIF-1α expression and its subsequently mediated HCC cell metabolism. Using biophysical proteomics approaches, we identified inhibitor of β-catenin and T-cell factor (ICAT) as the functional target of BD. By targeting ICAT, BD disrupted the interaction of β-catenin and ICAT, and promoted β-catenin degradation, which in turn induced the decrease of HIF-1α expression. Furthermore, BD could inhibit HCC cells proliferation and tumor growth in vivo, and knockdown of ICAT substantially increased resistance to BD treatment in vitro. Our data highlight the potential of BD as a modulator of β-catenin/HIF-1α axis mediated HCC metabolism.
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OBJECTIVE@#To observe the impacts of electroacupuncture (EA) on neurological function, the pathological morphology in brain tissue, apoptosis level and the protein expressions of apoptosis-related cytochrome C (Cyt-C) and cysteine aspartic acid protease-9 (Caspase-9) in the rats with traumatic brain injury (TBI) and explore the potential mechanism of EA in treatment of TBI.@*METHODS@#A total of 70 clean-grade SD mice were randomized into a blank group (8 rats), a sham-operation group (8 rats), a model group (27 rats) and an EA group (27 rats). In terms of interventions of 3, 7 and 14 days, 3 subgroups were divided in the model group and the EA group successively, 9 rats in each subgroup. The modified Feeney free-fall percussion method was adopted to establish TBI models of rats. In the sham-operation group, only the skull was exposed and drilled and no free-fall percussion was exerted. One day after modeling, EA was given in the rats of EA group at "Shuigou" (GV 26), "Baihui" (GV 20) and "Neiguan" (PC 6) and "Zusanli" (ST 36) on the affected side, with intermittent wave, 2 Hz in frequency, once daily, 10 min each time, for 3, 7 and 14 days successively. Separately, on the day 3, 7 and 14 of intervention, the modified neurological severity scale (mNSS) was used to evaluate the degree of neurological function injury in the rats, HE staining and Nissl staining were to observe the pathological and morphological changes in brain tissue, TUNEL method was to observe the level of apoptosis in brain tissue and immunohistochemistry (IHC) method and Western blot were to determine the protein expressions of Cyt-C and Caspase-9 in brain tissue.@*RESULTS@#Compared with the sham-operation group, on the day 3, 7 and 14 of intervention, mNSS scores were increased obviously in the rats of the model group respectively (<0.01). Compared with the model group, on the day 3, 7 and 14 of intervention, mNSS scores were reduced in the rats of the EA group respectively (<0.05). On day 3 of intervention, in brain injury region of the rats in the model group and the EA group, gross tissue necrosis, nuclear fragmentation, consolidation and obvious vacuolar changes, reduced Nissl bodies and scattered arrangement were found. On day 7 and 14 of intervention, in the model group and the EA group, the new connective tissue filling and normal cells were visible and Nissl bodies increased. The overall repair and Nissl body quantity in the EA group were better than the model group. Compared with the sham-operation group, on day 3, 7 and 14 of intervention, the numbers of apoptotic cells were increased obviously in the model group (<0.01) and they were reduced in the EA group as compared with the model group (<0.05). Compared with the sham-operation group, on day 3, 7 and 14 of intervention, the protein expressions of Cyt-C and Caspase-9 in damaged brain tissue were all increased obviously in the model group (<0.01) and they were all reduced in the EA group as compared with the model group successively (<0.05).@*CONCLUSION@#Electroacupuncture remarkably improves the condition in the neurological function injury and reduces apoptosis degree in TBI model rats, which is likely related to the down-regulation of the protein expressions of Cyt-C and Caspase-9 in damaged brain tissue and further to bring the impacts on mitochondria mediated apoptosis process.
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Animals , Rats , Apoptosis , Brain Injuries, Traumatic , Therapeutics , Caspase 9 , Metabolism , Cytochromes c , Metabolism , Electroacupuncture , Random Allocation , Rats, Sprague-DawleyABSTRACT
OBJECTIVE@#To observe the effect of electroacupuncture (EA) on neuronal apoptosis in rats with traumatic brain injury (TBI), and to explore the action mechanism of EA on improving the brain nerve function of TBI.@*METHODS@#A total of 88 6-week-old SD rats were randomly divided into a sham operation group, a model group, an EA group and a LY294002+EA group, 22 rats in each group. The TBI model on the left side was established by the improved Feeney's free fall method. After modeling for 24 h, the rats in the EA group and LY294002+EA group were treated with acupuncture at "Baihui" (GV 20) for 10 min and pricking acupuncture at "Shuigou" (GV 26) for 20 s; EA was applied at "Neiguan" (PC 6) and "Zusanli" (ST 36) on the right side (discontinuous wave, 2 Hz of frequency, 1 mA of intensity) for 10 min, once a day for 3 days. After 3 days of intervention, the TUNEL method was used to detect the level of neuron apoptosis in left cerebral cortex; the Western blot method was used to detect the expression of Akt, p-Akt, Bcl-2, Bax, Cyt-C and Caspase-9 in the left cerebral cortex.@*RESULTS@#After 3-day treatment, compared with the sham group, the number of neuronal apoptosis in the left cortex was increased in the model group (<0.01), and the expression of Bax, Cyt-C and Caspase-9 protein was increased (<0.01), and the expression of p-Akt/Akt, Bcl-2 was decreased (<0.01). Compared with the model group, the number of neuronal apoptosis in the left cortex was decreased in the EA group (<0.01), and the expression of Bax, Cyt-C and Caspase-9 was decreased (<0.01), and the expression of p-Akt/Akt and Bcl-2 was increased (<0.01). Compared with the LY294002+EA group, the number of neuronal apoptosis in the left cortex was decreased in the EA group (<0.01), and the expression of Bax, Caspase-9 and Cyt-C was decreased (<0.01, <0.05), and the expression of p-Akt/Akt and Bcl-2 was increased (<0.01).@*CONCLUSION@#EA could significantly reduce the neuronal apoptosis in rats with TBI, and its mechanism may be related to the activation of PI3K/Akt signaling pathway.
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Objective:To explore the effect of Anmeidan （AMD） on the learning and memory levels of sleep deprived rats through mitochondrial mediated hippocampal neuronal apoptosis pathway. Method:Forty-eight SD rats were randomly divided into blank group， model group， low， medium， high-dose AMD groups （4.86， 9.72， 19.44 g·kg-1·d-1） and estazolam group （0.1 mg·kg-1·d-1）. Insomnia model was prepared by self-made sleep deprivation box for 14 days. Morris water maze was used to detect learning and memory levels， enzyme-linked immunosorbent assay （ELISA） was used to detect the expressions of cytochrome C （Cyt-C）， cysteine aspartic acid protease-3 （Caspase-3） in hippocampus. Transmission electron microscopy （TEM） was used to observe the morphological structure of mitochondria in hippocampus. Protein and mRNA expressions of Cyt-C， Caspase-3， Bcl-2， Bax were detected by immunofluorescence （IF） and Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） respectively. Result:In the model group， the incubation period of the platform and the total distance of swimming and the time of first arriving platform were prolonged， the number of platform crossing and the time of target quadrant movement were reduced， protein and mRNA expressions of Bcl-2 dropped， protein and mRNA expressions of Bax increased （P<0.01）， and mitochondrial structure was abnormal with crista fracture， swelling and deformation. And protein and mRNA expressions of Cyt-C， Caspase-3 increased significantly （P<0.01）. Low， medium and high-dose AMD groups could improve levels of space exploration and navigation of SD rats （P<0.01）， increase protein and mRNA expressions of Bcl-2， decrease protein and mRNA expressions of Bax， improve the damage of mitochondria， and decrease the protein and mRNA expressions of Cyt-C， Caspase-3 （P<0.01）. Conclusion:AMD can improve the learning and memory levels of SD rats， the effect is related to the mitochondrial mediated hippocampal neuronal apoptosis pathway and decrease of Cyt-C and Caspase-3 expressions.
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Objective To discuss the effect of α-asarone on the expression level of Cyt-c,Smac,Caspase3 mRNA and protein in human esophageal carcinoma Eca-109 cell mitochondria. Methods The Eca-109 cells were cultured in vitro,and divided into the negative control group and the α-asarone treatment groups(final concentration:25,50,100 μg·mL-1).After 48 h,the morphological changes of Eca-109 cells were observed by fluorescence inversion microscope.The total RNA of cells were extracted by TRIzol method,the expressions of Cyt-c、Smac and Caspase3 were measured by RT-PCR and Western blotting. Results After Eca-109 cells were treated with different concentrations of α-asarone for 48 h,and obvious changes in the morphology were observed,the expressions of Cyt-c,Smac and Caspase3 genes and protein were increased significantly compared to the negative control group( P<0.05). Conclusion α-asarone can induce the human Eca-109 cells apoptosis by regulating expressions of mitochondrial apoptosis pathway correlation genes such as Cyt-c,Smac and Caspase3.
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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.
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OBJECTIVE: To investigate the mitochondrial damage and the possible mechanism in the process of MCF-7 cell apoptosis which induced by quercetin. METHODS: The cell apoptosis model was established with MCF-7 cells induced by quercetin. The morphological characteristics of apoptotic MCF-7 cells were observed by transmission electron microscope. Fluorescent probes DCFH-DA and Fluo-8-AM were used to detect the invaded cells' reactive oxygen species (ROS) and intracellular Ca2+concentration, respectively. The calcium channel blockers of extracellular and intracellular were used to inhibit apoptosis induced by quercetin in MCF-7 cells, the cellular apoptosis rates of which were measured by flow cytometry. Western blot assay was used to detect the expressions of Cyt C. RESULTS: Quercetin can induce MCF-7 cell mitochondria apoptosis. The spherical swelling, mitochondrial crest disappear and the formation of cavity were happened to those cells. Intracellular ROS and Ca2+ increased. The apoptosis induced by quercetin can be inhibit by intracellular calcium blockers ryanodine. After incubation with ryanodine, apoptosis rates fell by 55.4% at 24 h, 55.4% at 48 h and 39.9% at 72 h, respectively. The cell apoptosis induced by quercetin was not inhibited by EDTA. The expression of Cyt C was increased in the cytoplasm but not mitochondria after cultured the MCF-7 cell with EDTA. CONCLUSION: The mitochondrial damage plays an important role in the MCF-7 apoptosis which induced by quercetin, and its possible mechanism may related to the elevated levels of ROS and the release of Ca2+ and Cyt C in the cytoplasm. Meanwhile, Cyt C in the cytosol is potential critical signaling molecular in the process of apoptosis.
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ObjectiveTo investigate the effect of jin’s three-needle scalp electroacupuncture on Cyt-c and Caspase-3 expressions in the cerebral cortex in Intrauterine distress-induced HIBD rats and reveal the mechanism of its protective action on the brain. MethodThe abdomen was incised in a female SD rat at 21 days of pregnancy. Bilateral uterine horn blood vessels were tightly clamped with a hemostat for five minutes. An infant rat was then taken out by a cesarean section. A rat model of hypoxic-ischemic brain damage was confirmed by the use of a behavior test and braintissue sections. The rats were randomized into model and acupuncture groups. The model group was not needled. Acupuncture groups 1, 2, 3 and 4 began to be needled at 3, 5, 7 and 14 days after birth, respectively. Acupuncture treatment was given for seven consecutive days. The normal control group was naturally delivered, not used to make a model and not needled. All groups of rats were decapitated to take the brain tissue at 21 daysafter birth. Cyt-c and Caspase-3 expressions in the cerebral cortex were detected.ResultCyt-c optical density value decreased somewhat in the four acupuncture groups; there was a statistically significant difference between acupuncture group 1 or 2 and the modelgroup (P<0.05). Caspase-3 expression was significantly down-regulated in acupuncture group 1; there was a statistically significant difference compared with the model group (P<0.05).ConclusionThe protective effect of acupuncture beginning at 3 days after birthon the brain is related to the down-regulation of Cyt-c and Caspase-3 expressions in intrauterine distress-induced HIBD rats.
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Objective To preliminary investigate the cytoactive of HELA cell after PUMA gene promoting HELA cell apoptosis and whether or not the protein of cyt-c,AIF in the chondrosome have participated in the process of PUMA gene inducing HELA cell.Methods we Used AO/EB dyeing to detect the Morphologic change of cell induces by PUMA gene and Western Blot to detect whether or not the position of cyt-c,AIF in the chondrosome have transferred after PUMA gene induces HELA cell apoptosis.Result ①Fluorescence microscope and inverted phase contrast microscope display HELA cell have a series of apoptosis Morphologic change after transfecting PUMA into HELA cell,and this Morphologic change of cell is more obviously after transfecting 48h than after 24h.;②Western Blot display the protein cyt-c and AIF transferred toward to kytoplasm after HELE cell took place apoptosis,the mount of transfecting protein is more obviously after transfecting 48h than after 24h.Conclusion ① PUMA gene have the effect of promoting HELA cell apoptosis;②cyt-c and AIF take part in the process of promoting HELA cell apoptosis,but during this process if the membrane potential of chondrosome was changing need to do some more study to confirm it.
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Objective:To discuss the effect ofChinese herbal medicine ofremoving toxic and blood stasis and nourishing yin on lymphocyte apoptosis in spleen ofMRL/lpr mouse and expression ofCyt-C and Bcl-2.Methods:50 2-month-old male MRL/lpr mouse and 10 2-month-old male C57BL/6 mouse were randomly divided into six groups:high, middle and low dosage groups, pre-treatment group, model group and control group.After 30days treatment, the lymphocyte apoptosis in spleen oflupus mouse and expression ofCyt-C mRNA and Bcl-2 mRNA were detected.Results:The percentages oflymphocyte apoptosis in model group were(57.18?1.72)% lower than that in normal group(78.99?3.76)%(P
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Objective To investigate the damaging effect of sulfur mustard on the mitochondria in rat lymphocytes in vitro.Methods Rat spleen lymphocytes were isolated by density gradient centrifugation and cultured with 100 ?mol/L sulfur mustard in vitro.DNA ladderring was used to detect the cell apoptosis.Cyt-c release was measured by Western blotting.The mitochondria function was detected by MTT method.Fluorescent probe labeled with Rhodamine 123 was used to study mitochondria potential.Results The mitochondria potential and cell viability decreased at 4 h after treated with 100 ?mol/L sulfur mustard.There was a liner correlation between the mitochondria potential and cell viability in lymphocytes.The content of Cyt-c increased significantly at 4 h as compared with control group in Western blotting.Conclusion Sulfur mustard could damage the rat lymphocyte mitochondria significantly,and mitochondria participates in cytotoxic effect induced by the sulfur mustard.