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Objective To investigate the inhibitory effect of ursolic acid in Hippophae rhamnoides L. on hepatocyte apoptosis in rats with alcoholic liver disease based on the mitochondria-cytochrome c pathway. Methods A total of 50 specific pathogen-free male Wistar rats were divided into normal control group, alcohol model group, and low-, middle-, and high-dose ursolic acid groups using a random number table, with 10 rats in each group. The rats in the normal control group were given normal saline by gavage once a day for 8 weeks; the rats in the alcohol model group were given alcohol at increasing concentrations by gavage for 8 consecutive weeks; the rats in the low-, middle-, and high-dose ursolic acid groups were given ursolic acid at a dose of 50, 100, and 150 mg/kg, respectively, followed by an equal volume of alcohol as the model group 1 hour later. Serum liver function parameters were measured for each group; HE staining was used to observe liver histopathology; an electron microscope was used to observe hepatocyte ultrastructure; the TUNEL method was used to measure hepatocyte apoptosis; Western Blotting was used to measure the protein expression levels of cytochrome c and activated caspase-3 in hepatocyte mitochondria and cytoplasm. A one-way analysis of variance was used for comparison of continuous data between multiple groups, and the least significant difference t -test was used for further comparison between two groups. Results Compared with the alcohol model group, the middle- and high-dose ursolic acid groups had significant reductions in the serum level of alanine aminotransferase, aspartate aminotransferase, and cholinesterase (all P < 0.05). The rats in the alcohol model group had disordered arrangement of hepatic cords with marked hepatocyte edema and fatty degeneration, while those in the middle- and high- dose ursolic acid groups had basically normal arrangement of hepatic cords and a significant improvement in hepatocyte fatty degeneration, as well as a significant increase in the number of hepatocyte mitochondria and a significant improvement in morphology. Compared with the alcohol model group, the middle- and high-dose ursolic acid groups had significantly lower hepatocyte apoptosis rate and protein expression levels of cytochrome c and caspase-3 in cytoplasm (all P < 0.05). Conclusion Ursolic acid in Hippophae rhamnoides L. can improve the liver function and histomorphology of rats with alcoholic liver disease, possibly by inhibiting the release of cytochrome c in hepatocyte mitochondria, the activation of caspase-3, and the apoptosis of hepatocytes via the mitochondria-cytochrome c pathway.
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Objective:To investigate the expression and clinical significance of Beclin-1 and cytochromes C (CytC) in patients with hand, foot and mouth disease (HFMD).Methods:Sixty children with HFMD were classified into two groups of severe group and common group with 30 cases in each group. Another thirty children who underwent circumcision and had no underlying disease were selected as control group. Serum Beclin-1, CytC and S100B levels were detected before and after treatment. The levels of Beclin-1 and CytC in cerebrospinal fluid (CSF) of children with severe HFMD were detected before and after treatment. Receiver operating characteristic (ROC) curve was used to evaluate the prediction efficiency of Beclin-1 and CytC for the severity of HFMD.Results:Serum Beclin-1 and CytC levels in the severe group were higher than those in the other two groups ( P<0.01), and the common group showed significantly increased serum Beclin-1 and CytC levels as compared with the control group ( P<0.01). After treatment, the serum Beclin-1 and CytC levels decreased in both severe and common groups ( P<0.05). Compared with the common group, the severe group had remarkable increases in the levels of Beclin-1 and CytC in CSF ( P<0.01), which decreased significantly after treatment ( P<0.01). Serum Beclin-1 and CytC levels were positively correlated with the level of S100B protein. In the prediction of severe HFMD, serum CytC had the highest Youden value of 0.533 at the cut-off value of 38.785 ng/ml with a sensitivity of 56.67% and a specificity of 96.67%; serum Beclin-1 had the highest Youden value of 0.467 at the cut-off value of 6.560 ng/ml with a sensitivity of 46.67% and a specificity of 100.00%. Combined measurements of these two parameters had the highest predictive value for severe HFMD with a sensitivity of 76.67% and a specificity of 96.67%. Conclusions:Serum Beclin-1 and CytC levels were conducive to predict the severity and treatment outcomes of HFMD. Combined measurements of these two parameters had a higher predictive value for severe HFMD.
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Abstract: Objective: The feasibility of the use of WHO impregnated paper and biochemical assays to determine lethal concentrations (LC50 and LC99) and insecticide metabolic enzyme levels of Triatoma dimidiata. Materials and methods: LC50 and LC99 were calculated with WHO papers impregnated at different concentrations of malathion, propoxur and deltamethrin; the percentage of insensitive acetylcholinesterase (iAChE); and the levels of esterases, glutathione S-transferases, and monooxygenases in laboratory nymphs of the first stage (5 to 7 days), were undertaken using the WHO biochemical assays. Results: Respectively the LC50 and LC99 µg/cm2 obtained for malathion were 43.83 and 114.38, propoxur 4.71 and 19.29, and deltamethrin 5.80 and 40.46. A 30% of the population had an iAChE, and only a few individuals had high P450 and β-eterase levels. Conclusion: Impregnated papers and biochemical tests developed by WHO for other insects, proved to be feasible methods in monitoring insecticide resistance and metabolic enzymes involved in T. dimidiata.
Resumen: Objetivo: La factibilidad de usar los papeles impregnados y ensayos bioquímicos según la OMS para determinar concentraciones letales (CL50 y CL99) y niveles enzimáticos en la resistencia a insecticidas en Triatoma dimidiata. Material y métodos: Se calcularon la CL50 y CL99 con papeles impregnados según la OMS a diferentes concentraciones de malatión, propoxur y deltametrina; el porcentaje de acetilcolinesterasa insensible (iAChE); y los niveles de esterasas, glutatión S-transferasas, y monooxigenasas en ninfas de laboratorio del estadio I (5-7 días) se determinaron usando los ensayos bioquímicos según la OMS. Resultados: Se obtuvieron las CL50 y CL99 µg / cm2 respectivamente para malatión 43.83 y 114.38, propoxur 4.71 y 19.29, y deltametrina 5.80 y 40.46. Un 30% de las chinches tuvo iAChE, y sólo pocos individuos tuvieron niveles superiores de P450 y β-eterasas. Conclusión: Los papeles impregnados y ensayos bioquímicos que describe la OMS para otros insectos demostraron ser métodos factibles para monitorear la resistencia a insecticidas y las enzimas metabólicas involucradas en T. dimidiata.
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Animals , Propoxur/toxicity , Pyrethrins/toxicity , Triatoma/drug effects , Insecticide Resistance , Insecticides/toxicity , Malathion/toxicity , Nitriles/toxicity , Acetylcholinesterase/analysis , Triatoma/enzymology , World Health Organization , Feasibility Studies , Cytochrome P-450 Enzyme System/analysis , Esterases/analysis , Glutathione Transferase/analysis , Mixed Function Oxygenases/analysis , Lethal Dose 50 , Nymph/drug effects , Nymph/enzymologyABSTRACT
Objective To investigate the protective effect of ginkgolide A and ginkgolide B (GKAB) mixture on neurons of rats with permanent middle cerebral artery occlusion (pMCAO) and related molecular mechanisms. Methods Sixty male Sprague-Dawley rats were randomly divided into sham group, pMCAO permanent focal cerebral ischemia group and GKAB-treated low-, medium- and high-dose groups. In addition to the sham group (only isolated without interruption of the arteries), the rats in the remaining four groups were induced pMCAO by blocking the right middle cerebral artery. Rats in the GKAB-treated low-, medium- and high-dose groups were injected with GKAB 12.5, 25, and 50 mg/kg through sublingual vein at 10 min after pMCAO, while the sham and pMCAO groups were injected with saline of the same volume as the medium-dose group. After 12 h of treatment, the neuronal apoptosis was determined by TUNEL method, the level of phosphorylated c-Jun N-terminal kinase (p-JNK) was determined by immunohistochemistry, the expressions of p-JNK, Bcl-2, Bax, cytochrome C (Cyt C), caspase-9, caspase-3, cleaved caspase-9, and cleaved caspase-3 in brain tissues were detected by Western blotting. Results Compared with the sham group, the apoptosis rate and p-JNK expression of neurons in the pMCAO group were significantly increased (P<0.01), and the expressions of apoptosis-related proteins Bax, cleaved caspase-9 and cleaved caspase-3 in brain tissues were significantly increased (P<0.01), while the expressions of Bcl-2, caspase-9 and caspase-3 in brain tissues were significantly decreased (P<0.01). Compared with the pMCAO group, the apoptosis rate and p-JNK expression of neurons in GKAB-treated low-, medium- and high-dose groups were significantly decreased (P<0.01), the expressions of Bax, cleaved caspase-9 and cleaved caspase-3 protein were significantly decreased (P<0.01), and the expressions of Bcl-2, caspase-9 and caspase-3 were significantly increased (P<0.01) in a dose-dependent manner. Compared with the sham group, the expression of Cyt C in cytoplasm in the pMCAO group was significantly increased, and the expression of mitochondrial Cyt C was significantly decreased (P<0.01). Compared with the pMCAO group, the expressions of Cyt C in cytoplasm in the GKAB-treated low-, medium- and high-dose groups were significantly decreased in a dose-dependent manner, and the expressions of mitochondrial Cyt C were significantly increased (P<0.05, P<0.01). Conclusion GKAB can inhibit neuronal apoptosis after pMCAO in rats, and its mechanism may be related to the inhibition of JNK phosphorylation and JNK signaling pathway and the block of mitochondrial apoptosis pathway.
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Objective To investigate the effect of CYP3A5 on the proliferation of pancreatic cancer cells and its underlying mechanisms.Methods The protein expression of CYP3A5 in five pancreatic cancer cell lines BxPC-3,FG,MDA28,8902 and PANC1 was detected by Western blotting.The PANC1 cells with the lowest protein expression of CYP3A5 and the BxPC-3 cells with highest expression of CYP3A5 were transfected with CYP3A5 overexpression plasmid and CYP3A5 targeted-siRNA (siRNA-CYP3A5),respectively.CCK-8 and cloning formation assay were used to investigate the role of CYP3A5 overexpression and knockdown in the proliferation of pancreatic cancer cells.The changes of the protein and mRNA expression of cell cycle regulating gene cyclin E,cyclin D1 and apoptosis related gene Bcl-2 were detected by Western blotting and PCR,respectively.Results CYP3A5 protein expression in PANC1 cells increased significantly after the transfection of CYP3A5 overexpression plasmid (1.66 ± 0.14 to 1,P =0.0021),which greatly decreased in BxPC-3 cells transfected with siRNA CYP3A5 (0.18 ± 0.02 to 1,P ＜0.0001).A450 values of the CYP3A5 overexpression group and the empty plasmid group in PANC1 cells cultured for 48 and 72 h were 1.36 ±0.05 vs 1.15 ± 0.03,2.1 ± 0.09 vs 1.42 ± 0.03,respectively,which were significantly higher in CYP3A5 overexpression group than empty plasmid group,and the differences were statistically significant (P value ＜ 0.005 or 0.001).The A450 values of BxPC-3 cells in CYP3A5-siRNA transfected group and siRNA-NC transfected group were 0.62 ±0.01 vs 0.77 ± 0.03、0.83 ± 0.01 vs 1.18 ± 0.02,respectively,which in The CYP3A5-siRNA transfection group was significantly lower than that of siRNA-NC transfection group,and the difference was statistically significant (P ＜ 0.05 or ＜ 0.001).The clone formation rate of PANC1 cells in the overexpression group was (19.33 ± 0.58)％,which was significantly higher than that in the empty plasmid group (9.67±0.63) ％,and the clone formation rate in CYP3A5-siRNA group was (8.5± 0.8)％,which was significantly lower than that of group siRNA-NC (16± 0.6)％,and the differences were statistically significant (P ＜0.01).The protein expression of cyclin D1 in CYP3A5 overexpression PANC1 cells was 2.00 ± 0.11,which was obviously higher than 1.00 in empty plasmid group (P ＜0.01).The protein expression of cyclin D1 in siRNA CYP3A5 BxPC cells was 0.45 ±0.04,which was obviously lower than 1.00 in siRNA NC group,and the difference was statistically significant (P＜0.01).However,CYP3A5 overexpression or inhibition did not influence the relative expression of cyclin D1 mRNA and cyclin E,Bcl-2 pretein expression.Conclusions CYP3A5 can promote the proliferation of pancreatic cancer cells by up-regulating cyclin D1 protein expression.
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Objective To investigate the effects of inhibition of adenosine monophosphate -activated protein kinase (AMPK) on expressions of cytochrome c (CytC) and caspase -3 and apoptosis in the cerebral cortex after cerebral ischemia-reperfusion injury in mice. Methods Thirty-six male C57BL/6 mice w ere randomly divided into three groups, a sham operation group, a ischemia -reperfusion group, and a AMPK inhibitor group, 12 in each group. A model of middle cerebral artery occlusion w as induced by suture method. The AMPK inhibitor compound C ( 20 mg/kg) w as injected intraperitonealy in the AMPK inhibitor group, the equal volume normal saline w as injected intraperitonealy in the sham operation group and the ischemia-reperfusion group w hen a thread w as inserted. Immunohistochemical staining w as used to detect the expression levels of CytC and caspase-3 and TUNEL method w as used to detect apoptosis at 24 h after ischemia-reperfusion. Results Compared w ith the ischemia-reperfusion group, the numbers of CytC (28.86 ±9.65/HP vs.58.86 ±9.65/HP; t = 7.615, P = 0.030 ) and caspase-3 (7.16 ±5.85/HP vs. 14.36 ±7.85/HP; t =2.548, P =0.035), and TUNEL (67.14 ±8.55/HP vs.95.00 ±13.51/HP; t = 6.891, P = 0.030) positive cels in the cerebral cortex w ere reduced significantly in the AMPK inhibitor group. Conclusion Inhibition of AMPK activity after cerebral ischemia-reperfusion may decrease apoptosis by dow nregulating the expressions of CytC and caspase -3, and play a neuroprotective effect.
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BACKGROUND:Previous studies have shown that apoptosis, a central feature of articular chondrocytes, plays a dominant role in cartilage damage, which is one of the pathological factors of articular cartilage degeneration. OBJECTIVE:To observe the effects of meditcated serum containingDuhuojisheng Decoction on the expression of cytochrome C, proCaspase-9 and proCaspase-3 in rat degenerative chondrocytes in vitro and to investigate the possible molecular biological mechanism ofDuhuojisheng Decoction in the treatment of knee osteoarthritis. METHODS:A cultivation system of degenerative chondrocytes in vitro was established. After treatment with meditcated serum containingDuhuojisheng Decoction or blank serum for 24 and 48 hours, the protein expression of cytochrome C, proCaspase-9 and proCaspase-3 was measured by western blot assay. RESULTS AND CONCLUSION: In the cytoplasm, the release of cytochrome C was reduced gradualy in both groups in a time-dependent manner, and the release amount of cytochrome C was significantly lower in the medicated serum group than the blank serum group (P < 0.05). In mitochondria, cytochrome C leakage was gradualy decreased in both groups, and it was decreased significantly in the medicated serum group compared with the blank serum group (P < 0.05). The protein expression of proCaspase-9 and proCaspase-3 was gradualy increased in both groups, especialy in the medicated serum group; the medicated serum containingDuhuojisheng Decoction could promote the protein expression of proCaspase-9 and proCaspase-3 in a time-dependent manner, and there was a significant difference at 24 and 48 hours (P< 0.01). These findings indicate that the medicated serum containingDuhuojisheng Decoction can inhibit the apoptosis of osteoarthritis chondrocytes through inhibiting the release of cytochrome C and the activation of Caspase-9 and Caspase-3.
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AIM:To investigate the interaction of polymorphisms of resistin gene promoter -420C/G, cyto-chromes P4501A1-MspI and cigarette smoking in nonalcoholic fatty liver disease (NAFLD).METHODS: The genetic polymorphisms in resistin gene promoter -420C/G and CYP1A1-MspI were analyzed by the technique of polymerase chain reaction ( PCR) in peripheral blood leukocytes of 900 NAFLD cases and 900 healthy persons.RESULTS:The frequencies of -420C/G (GG) and CYP1A1-MspI (m2/m2) were 49.75%and 50.08%in NAFLD cases and 24.00%and 24.25%in healthy controls, respectively.Statistical tests showed a significant difference in the frequencies between the 2 groups ( P<0.01).The risk of NAFLD with -420C/G (GG) was significantly higher than that of controls.Individuals who carried with CYP1A1-MspI (m2/m2) had a high risk of NAFLD.Combined analysis of the polymorphisms showed that the per-centages of -420C/G (GG)/CYP1A1-MspI (m2/m2) in NAFLD and control groups were 39.83% and 12.83%, re-spectively (P<0.01).The people who carried with -420C/G (GG)/CYP1A1-MspI(m2/m2) had a high risk in NAFLD group.The cigarette smoking rate in NAFLD group was signi-ficantly higher than that in control group ( P<0.01) , and the statistic analysis suggested an interaction between cigarette smoking and -420C/G (GG) and CYP1A1-MspI (m2/m2), which increased the risk of NAFLD.CONCLUSION: -420C/G (GG), CYP1A1-MspI (m2/m2) and cigarette smoking are the risk factors in NAFLD.The interactions between genetic polymorphisms in -420C/G, CYP1A1-MspI ( m2/m2) and cigarette smoking increase the risk of NAFLD.
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Las manifestaciones clínicas de la leishmaniosis son variables y están relacionadas con la especie infectante, su relación con el medioambiente y con la respuesta inmune del hospedero. Se presenta un caso de leishmaniosis andina cutánea tardía con una manifestación extensa. El caso se confirmó a través de estudios microbiológicos e inmunológicos, la identificación se realizó mediante secuenciamiento del gen del citocromo b, determinándose la especie como Leishmania (Leishmania) amazonensis. La paciente recibió tratamiento con estibogluconato sódico y al término de la terapia, mostró mejoría clínica de las lesiones. Se recomienda considerar a la leishmaniosis en el diagnóstico diferencial cuando se atienda ulceras crónicas dermatológicas atípicas...
Clinical manifestations of leishmaniasis are diverse and related to the infecting species, its relationship with the environment and the host immune response. A case of late Andean cutaneous leishmaniasis with extensive manifestation is presented. The case was confirmed through microbiological and immunological studies; identification was performed by cytochrome b gene sequencing and the species was determined as Leishmania (Leishmania) amazonensis. The patient was treated with sodium stibogluconate and at the end of therapy the patient showed clinical improvement of the lesions. It is recommended to consider leishmaniasis in differential diagnosis when treating atypical dermatological chronic ulcers...
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Humans , Adult , Female , Cytochromes b , Leishmania , Leishmaniasis, Cutaneous , PeruABSTRACT
Aim: The aim of this study was to evaluate the synergistic effect of low radiation dose with the chemotherapeutic drug in order to find possible way to lessen the harmful effects during chemo-radiotherapy. Study Design: Randomized controlled experiment. Place and Duration of Study: Experimental Animal Unit, Drug Radiation Research Department, National Center for Radiation Research and Technology, Cairo Egypt. Methodology: Estimation of antioxidant activity of low radiation dose on oxidative stress induced by cisplatin administration at a dose of 10 mg/kg b. wt. in male albino rat. Results: Results of experiment revealed that cisplatin administration caused a significant increase in serum alanine transaminase (GPT) activity (38.58±2.060) and FSH level (8.162±1.424) accompanied with a decrease in serum albumin (3.492±0.253), and Butyry Cholein Esterase (BChE) (65.35 12.61). In Liver and testis, GSH content (68.00±2.391 & 24.93±4.778) as well as cytochromes P450 levels (0.3875±0.0727 & 0.2167±0.0459) showed a significant decrease as compared to the normal control level respectively. In addition the level of Fe, Cu and Zn showed no significant changes in liver and appeared to be significantly decrease as in case of corresponding trace elements in testis organs. On the other hand, exposing to low dose of radiation (0.5 Gy) post-cisplatin treatment effectively prevented these alterations and maintained the antioxidant status. Conclusion: Data from present results revealed that low radiation dose have the existence as an antioxidant and antitumor agents which may be useful to use as a synergistic agents with the chemotherapeutic drug.
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Objective To explore the impact and significance of hypoxia preconditioning on the expression of cytochrome C and caspase 3 protein in rats after hepatic resection.Methods A hepatectomy model was used to study the ischemia reperfusion injury in hepatic resection.Sprague-Dawley rats were randomly divided into the following three groups:normal control (NC) group,hepatic resection(HR) group,and hypoxia preconditioning (HP) group,there were twenty four rats in each group.HP Group was given an 10％ oxygen-mixed gas for 90 minutes before the operation.At 1,6,12 and 24 hours after the operation,the rats were killed and the following tests were conducted:(1) Liver tissue was sampled to observe the expression of cytochrome C and caspase 3 protein; (2) blood was drawn to conduct a chemical examination; (3) Liver tissue and morphology was observed by transmission electron microscopy.Results The serum levels of ALT and AST in HP group were significantly lower than that of HR group (P＜0.05) at 1,6,12 and 24 hours after the hepatic resection.In each time,liver function of the HP group was significantly better than the HR group; The expression of cytochrome C and caspase 3 protein was decreased significantly in HP group at each measurement point.Hepatic cells in HR group showed typical apoptosis signs under transmission electron microscopy (TEM),but no apoptosis was found in HP group.Conclusion HP has marked inhibition to apoptosis by down-regulating the expression of Cyt C and Caspase-3protein and protection to chondrosomes after a hepatic resection.
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BACKGROUND:Studies have shown that dibutyl phthalate has certain toxicity to the reproductive system of male animals. OBJECTIVE:To investigate the effects of dibutyl phthalate on secretion of rat ovarian granulose cells. METHODS:Ovarian granulosa cells from 25-day-old female Sprague-Dawley rats were cultured in vitro and exposed to different concentrations of dibutyl phthalate (0, 5, 20, 80μmol/L) for 24 hours. Estradiol and progesterone in ovarian granulosa cells were measured by the method of radio-immunity. Expression of the cytochrome P450aromatase and P450 side chain cleavage enzyme mRNA in granulosa cells were examined by real-time PCR. RESULTS AND CONCLUSION:The levels of estradiol and progesterone in granulosa cells decreased after exposure to dibutyl phthalate. With the increase of the dibutyl phthalate concentration, the expression of P450arom and P450scc mRNA decreased gradual y. Dibutyl phthalate could induce reproductive dysfunction and toxicity to female rats.
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Objective To investigate the protective effect of Ebselen on mitochondrial damage and its influence to Cytochrome C expression and the neuronal apoptosis after spinal cord injury in rats. Methods Sixty adult SD rats were ran-domly divided into 5 groups (12 each group). Spinal cord injury model was made using Allen's method. Sham operation group received only laminectomy;SCI group received laminectomy and spinal trauma;Saline group received saline injection intraperitoneally (0.1%DMSO) after injury;methylprednisolone group received 30 mg/kg methylprednisolone injection intra-peritoneally, ebselen group received 10 mg/kg ebselen injection intraperitoneally. The malonaldehyde (MDA) and glutathi-one (GSH)level at the injured sites of the spinal cord were detected 24 hours after trauma, and the expression level of Cyto-chrome C was also observed. Finally, neuronal apoptosis was identified by TUNEL staining. Results MDA level in the Eb-selen group was significantly lower than that in the SCI group, and GSH level was significantly elevated in the Ebselen group compared with SCI group (P<0.01). Expression of Cytochrome C in Ebselen group was lower than that in SCI group shown by Western blot, and the neuronal apoptosis in Ebselen group reduced significantly too compared with SCI group (P<0.01). Conclusion Ebselen can alleviate peroxidation,prohibit expression of Cytochrome C and inhibit neuronal apoptosis,thus it shows a protective effect to experimental acute SCI.
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Objective To detect the effects of the selective mitochondrial fission inhibitor-Mdivi-1 on the malondi-alolehyde (MDA), glutathione (GSH) as well as cytochrome C (Cyt-C) in neuronal mitochondria and neuronal apoptosis. Methods Thirty-six adult female SD rats (250-300 g) were randomly divided into 3 groups (n=12):sham operation (Sham) group, single spinal cord injury (SCI) group and Mdivi-1 pretreatment (1.20 mg/kg, Mdivi-1) group. In sham group, the rats’ spinal cord was exposed, but no hit. The rat model of spinal cord injury was established by Allen’s method in SCI group and Mdivi-1 group. In Mdivi-1 group, rats were given Mdivi-1 through the tail vein 15 min before spinal cord injury, and SCI group received the same amount of dimethyl sulfoxide (DMSO). Rats in Sham group were sacrificed 8 h after exposing spinal cord. Rats in SCI group and Mdivi-1 group were sacrificed at 8 h after the spinal cord injury, then were removed the spinal cord T9-11. The contents of MDA and GSH in mitochondria of spinal cord tissues were detected with spectrophotometer. The expressions of Cyt-C protein in the mitochondria and cytoplasm were detected by Western blot assay. The neuronal apoptosis was assessed by TUNEL staining. Results Compared with Sham group, levels of Cyt-C and GSH in mitochondria were decreased significantly (P<0.01), while levels of MDA in mitochondria, Cyt-C in cytoplasm and the neuronal apopto-sis were increased significantly in SCI group (P<0.01). Compared with SCI group, Cyt-C and GSH levels in mitochondria were increased significantly in Mdivi-1 group (P<0.01), however, MDA in mitochondria,Cyt-C in cytoplasm and the neuro-nal apoptosis were significantly reduced (P<0.01). Conclusion Mdivi-1 can relieve neurons from mitochondrial oxidative damage, inhibit the release of cytochrome C and neuronal apoptosis after acute spinal cord injury, which plays a role in pro-moting the recovery of spinal cord function.
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Objective To investigate the possible mechanism of mitochondrial in chronic myeloid leukemia cells K562/G01 cells apoptosis induced by triptolide.Methods K562/G01 cells were treated with different concentrations of triptolide.MTT assay was used to assess cytotoxic effect.FCM was used to determine apoptosis rate,mitochondrial membrane potential and the activity of Caspase-9 of each experimental group.Real-time quantitative PCR assay was used to quantify mRNA levels of Caspase-9 and cytochrome C and Western blot assay was used to determine protein levels of cytochrome C.Results Triptolide inhibited the growth and proliferation of K562/G01 cells in a time-and dose-dependent manner (both P ＜ 0.001).Meantime,triptolide could make the mitochondria membrane potential fade away and enhance the activity of Caspase-9 (F =566.431,2 555.485,P ＜ 0.001).In addition,triptolide could dose-dependently up-regulated the transcription of Caspase-9 and cytochrome C (F =61 007.702,452 121.760,P ＜ 0.001),and the protein expression of cytochrome C,whose gray value in each experimental group was 21.54±0.59,39.63±0.58,53.29± 1.47 and 75.68±1.87 (F =5 677.928,P ＜ 0.001) respectively.Conclusion Triptolide could potently inhibit the growth and proliferation of K562/G01 cells,and the mitochondria apoptosis pathway might be one of the important apoptosis mechanisms in chronic myeloid leukemia cells induced by triptolide.
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Objective To investigate the effect of hydrogen peroxide (H2O2) pretreatment on free mitochondrial cyto-chrome c (Cyt-c) release in mitochondrial levels, and reveal the mechanism of the ischemic preconditioning (IPC) on the ischemia reperfusion (IR) injury thereof. Methods The rat liver mitochondria was isolated and made free mitochondria. Free mitochondria were divided into 5 groups:control group (C) and different concentrations of Ca2+groups (12.5, 25, 50 and 100 μmol/L). The levels of Cyt-c and second mitochondria-defived activator of caspase (Smac) were detected after 10 min stimulation of free mitochondria. The free mitochondrial IPC reperfusion model was made and divided into seven groups:C group, IR group and different concentrations of H2O2 groups (2 μL H2O2 in 200 μL system respectively, final concentration of 1, 2, 5, 20 and 100 μmol/L respectively). 100 μmol/L Ca2+was used again on the simulation of IR group. The level of Cyt-c release was detected. The changes in the activity of cardiolipin were detected in IR group and H2O2 (1 and 2 μmol/L of final concentration) groups.Results Compared with C group, there were significantly higher levels of Cyt-c and Smac emission in 25, 50, and 100 μmol/L Ca2+groups (P<0.05). Compared with IR group, there was significantly decreased level of Cyt-c emission in H2O2 (1 and 2 μmol/L) groups (P < 0.05). The activity of cardiolipin was changed when reducing release of Cyt-c. Conclusion Cyt-c was bonded with cardiolipin more closely when the low concentration of H2O2 pretreatment in mitochondria. There was a lower level of Cyt-c emission in mitochondria after stimulation with high concentration of Ca 2+(100 μmol/L Ca2+). The blocking apoptotic pathway plays a key fact in the effect of IPC on IR injury.
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Objective To investigate the efficacy and safety of warfarin anticoagulation in Chinese elderly patients based on vitamin K epoxide reductase complex 1 (VKORC1) and cytochrome P450 2C9 (CYP2C9) genetic polymorphisms.Methods Clinical data of 41 elderly patients with initial anticoagulation therapy in our emergency department and respiratory department were collected.Patients were divided into observation group (n=20,patients treated with warfarin based on genetic polymorphisms) and control group (n =21,patients treated based on clinical experience).The international normalized ratio (INR),the time of INR stabilized within target range (2.0-3.0) and the incidence of bleeding episodes in 6-month follow up were compared between groups.Results INR within target range at day 3,4,5 and 7 were 0.0％,42.1％,52.6％,68.4％ in observation group and 0.0％,10.0％,25.0％,35.0％ in control group,respectively.There were significant differences in INR within target range at day 4,7 between the two groups (both P＜0.05),while no significant difference was found in INR within target range at day 5 (P＞0.05).The time of INR stabilized within target range was shorter in observation group than in control group [(9.5±2.4) d vs.(12.3± 4.8) d,P＜0.05].Bleeding complication occurred in 3 patients in observation group and 5 patients in control group,and there was no significant difference between the two groups.Conclusions Warfarin therapy based on VKORC1 and CYP2C9 gene polymorphisms may shorten the time of first INR reaching the target value and INR within target range in elderly patients.However,the risk of bleeding complications should be alerted.
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Objective To observe the changes in expressions of apoptosis-promoting gene Bax, apoptosis-inhibiting gene Bcl-2, and cytochrome C in the renal tissue of diabetic rats. Methods Twenty-four male Sprague-Dawley rats were randomly divided into 2 groups (n=12): normal control group and diabetic group. Diabetic models were induced by single intraperitoneal injection of 2% streptozotocin (dissolved in pH 4. 4,0. 1 mol/L citric acid sodium buffer, 65 mg/kg). Normal control group was only injected with same volume of folie buffer. Animals were sacrificed at the 4th and 12th week, and body mass, 24-hour urine protein, blood glucose, blood urine nitrogen and serum creatinine were determined. The changes of the renal morphology were observed by H-E staining. Immunohistochemical method was used to investigate the expressions of Bax, Bcl-2 and cytochrome C protein. The apoptosis of renal cortex cells was determined by TUNEL method. Results Compared with normal control group, the 24-hour urine protein, blood glucose, blood urine nitrogen and serum creatinine were significantly increased in the diabetic group (P<0. 05, P<0. 01). The size of glomerulus was increased in diabetic rats during the 4th week; hyperplasia of renal glomerulus mesangial matrix, glomerular sclerosis, and vacuolar degeneration in renal tubular epithelial cells were observed during the 12th week. With disease progression in the diabetic group, the expressions of Bax and cytochrome C were increased and the expression of Bcl-2 was decreased. Apoptosis tests showed increased apoptotic cells in the 4th week, mostly in both the distant tubular epithelial cells; in the 12th week, apoptotic cells were seen in both the distant tubular and proximal tubules. Conclusion Renal expression of Bax and cytochrome C gradually increases with the progression of diabetes, inducing apoptosis of more cells and leading to renal dysfunction, which may partly contribute to the diabetic nephropathy in diabetic rats.
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ObjectiveTo investigate the effects of different time administration of propofol on cytochrome c (Cyt c) in the cytoplasm in rat hippocampal neurons with hypoxia-reoxygenation (H/R) injury.MethodsPrimary cultured hippocampal neurons were randomly divided into 5 groups ( n =5 each):control group (group C),model of H/R injury group (group M),and different time administration of propofol groups (group Ⅰ,Ⅱ,Ⅲ ).In groups M,Ⅰ,Ⅱ and Ⅲ,the neurons were exposed to 95％ N2 + 5％ CO2 for6 h followed by 12 h reoxygenation.In groups Ⅰ, Ⅱ,Ⅲ,propofol was added to the culture medium before hypoxia,immediately after reoxygenation and at 2 h of reoxygenation (T0-2) respectively,with the final concentration of 20 μmol/L.The cell apoptosis was observed at T1,2 and at 24 h of reoxygenation ( T3 ) and the concentration of Cyt c in the cytoplasm was detected at T1-3.ResultsCompared with group C,the concentration of Cyt c in the cytoplasm was significantly increased at T1-3 in group M and at T1,2 in groups Ⅰ,Ⅱ,Ⅲ (P ＜ 0.05).Compared with group M,the concentration of Cyt c in the cytoplasm was significantly decreased at T1-3 in group I and at T1,2 in groups Ⅱ and Ⅲ ( P ＜0.05).The concentration of Cyt c in the cytoplasm was significantly higher at T1,2 in groups Ⅱ and Ⅲ than in group Ⅰ,and at T2 in group Ⅲ than in group Ⅱ ( P ＜ 0.05).The neuronal apoptosis was significantly decreased in groups Ⅰ,Ⅱ and Ⅲ as compared with group M.ConclusionDifferent time administration of propofol can reduce the mitochondrial Cyt c release to the cytoplasm,inhibit apoptosis in hippocampal neurons,and reduce H/R injury in rats,with better effect when given before hypoxia.
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Objective To investigate the effects of Inula Britannica on myocardial caspase-3 and cytochrome c ( cyt c) following overtraining-induced acute myocardial injury in rats.Methods Forty-eight male Wistar rats weighing 200-220 g were randomly divided into 3 groups:group control (group C,n =8) ; group exhausting swim (group E,n =24) and group Inula Britannica (group IB,n =16).The animal model of overtraining-indnced acute myocardial injury was developed by exhausting swim.The animals were forced to swim until they were exhausted.The animals sank to the bottom and no righting reflex or escape response was elicited when they were taken out of water in groups E and IB.In group IB oral Inula Britannica 25 ml/kg was given 24 h and immediately before overtraining.Blood samples were taken from inferior vena cava immediately and at 6,24 h after overtraining in group E and at 6,24 h after overtraining in group IB for determination of serum cardiac troponin I (cTnI) concentration (by ELISA).The animals were sacrificed after blood sampling and myocardial specimens were obtained for microscopic examination and determination of caspase-3 and cyt c expression (by immuno-histochemistry).Results Overtraining significantly increased serum cTnI concentration and up-regulated myocardial caspase-3 and cyt c expression in group E as compared with group C.Oral Inula Britannica significantly attenuated overtraining-induced increase in serum cTnI concentration and myocardial caspase-3 and cyt c expression in group IB as compared with group E.Conclusion Inula Britannica can reduce overtraining-induced acute myocardial injury by down-regulating caspase-3 and cyt c expression.