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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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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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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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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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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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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.
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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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Objective To explore the neuroprotective effect of ginsenoside Rg1 against PC12 cell apoptosis induced by 1-methyl-4-phenylpyridinum (MPP++). Methods MPP++-induced apoptosis in PC12 cells, with the characteristics of dopaminergic neuron, were taken as the model of Parkinson disease in vitro. The cells were divided into control group, MPP++ group and 3 ginsenoside Rg1 pretreatment groups (concentrations 10, 20, and 50 μmol/L). MTT assay was used for detecting the cell viability, FCM for apoptosis ratio, TUNEL enzyme labelling for DNA fragment of the cell nuclear, and Western blotting analysis for cytochrome C protein. Results Ginsenoside Rg1 (10, 20, and 50 μmol/L) showed protective effect against MPP++-induced PC12 cells injury. Compared with MPP++-treated cells([52±4.7]%), pretreatment with 10, 20, and 50 μmol/ L ginsenoside Rg1 increased the cell viability to (64 ± 3. 4) %, (72 ± 5.2) % and (83±6.2)%, respectively (P<0.05 or P< 0.01). FCM analysis indicated that apoptosis rates decreased by ginsenoside Rg1 pretreatment, with the apoptosis rates in the control, MPP++ and 3 ginsenoside Rg1 groups (10, 20, 50 μmol/L) being 1.8%, 44.5%, 32.9%, 21.1% and 14.2%, respectively. We also found that ginsenoside Rg1 pretreatment greatly decreased DNA fragment of PC12 cells. Western blotting analysis indicated that the cytochrome C was depressed by the ginsenoside Rg1 pretreatment. Conclusion Ginsenoside Rg1 can protect PC12 cells against MPP++-induced apoptosis in a concentration-dependent manner, which may be closely related to down- regulation of cytochrome C over-expression in the mitochondria.
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Objective To explore the neuroprotective effect of ginsenoside Rg1 against PC12 cell apoptosis inducedby 1-methyl-4- phenylpyridinum (MPP+). Methods MPP+-induced apoptosis in PC12 cells, with the characteristics of dopaminergic neuron, were taken as the model of Parkinson disease in vitro. The cells were divided into control group, MPP+ group and 3 ginsenoside Rg1 pretreatment groups (concentrations 10, 20, and 50 μmol/L). MTT assay was used for detecting the cell viability, FCM for apoptosis ratio, TUNEL enzyme labelling for DNA fragment of the cell nuclear, and Western blotting analysis for cytochrome C protein. Results Ginsenoside Rg1 (10, 20, and 50 μmol/L) showed protective effect against MPP+-induced PC12 cells injury. Compared with MPP+-treated cells([52±4. 7]%), pretreatment with 10, 20, and 50 |imol/ L ginsenoside Rg1 increased the cell viability to (64 ± 3. 4) %, (72 ± 5. 2) % and (83±6.2)%, respectively (P<0. 05 or P< 0. 01). FCM analysis indicated that apoptosis rates decreased by ginsenoside Rg1 pretreatment, with the apoptosis rates in the control, MPP+ and 3 ginsenoside Rg1 groups (10, 20, 50 μmol/L) being 1. 8%, 44. 5%, 32. 9%, 21. 1% and 14. 2%, respectively. We also found that ginsenoside Rg1 pretreatment greatly decreased DNA fragment of PC12 cells. Western blotting analysis indicated that the cytochrome C was depressed by the ginsenoside Rg1 pretreatment. Conclusion Ginsenoside Rg1 can protect PC12 cells against MPP+-induced apoptosis in a concentration-dependent manner, which may be closely related to down-regulation of cytochrome C over-expression in the mitochondria.
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Objective To investigate the effects of inhalation of different concentrations of isoflurane on the expression of cytochrome c ( Cyt c) in hippocampus in aged rats.Methods Sixty-three aged male SD rats (20 months) weighing 500-600 g were randomly divided into 3 groups(n=21each):control group inhaling 30%O2 for 2h (group C) and 2 isoflurane groups anesthetized with 0.75 % and 1.5 % isoflurane in 30 % O2 for 2 h respectively (group Ⅰ1 and Ⅰ2 ).Arterial blood samples were obtained from 5 rats at 30 min, 1 and 2 h of anesthesia for blood gas analysis. Eight animals were killed at 24 h after anesthesia in each group.Their hippocampi were immediately removed for determination of Gyt c expression by immuno-histochemistry and Western blot analysis.Cognitive function was assessed by Morris water maze test the day before experiment and once a day for 6 consecutive days starting from the 1st postoperative day.Results The Cyt c expression in hippocampus was significantly increased in Ⅰ1 and Ⅰ2 groups in a concentration-dependent manner as compared with group C.The escape latency was significantly prolonged and the frequency of crossing the original platform and the duration of staying at the original platform quadrant were decreased in group Ⅰ1 and Ⅰ2 compared with group C.Conclusion Inhalation of isoflurane anesthesia can decrease cognitive function through up-regulating the Gyt c expression in hippocampus in aged rats.
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Objective To investigate Smac/DIABLO and cytochrome c(cyt-c)mRNA levels in liver tissue of rats with acute hepatic failure treated by microencapsulated hepatocyte.Methods Acute hepatic failure were induced by intraperitoneal injection of D-galactosamine in rats.and the rats were treated with microencapsulated hepatocytes,free hepatocytes and physical saline(contr01),respectively.Smac/DIABLO and cyt-c mRNA in liver tissue was detected by RT-PCR and the mRNA expression levels among three groups were compared.Results Smac/DIABLO and cyt-c mRNA levels in liver tissues of rats with acute hepatic failure were higher than those of normal rata(F=4.345,14.821,47.565,42.178 and 62.961,P<0.05).The peak values of Smac/DIABLO and cyt-C mRNA expressions in free hepatocytes and control groups were at 48 h.while that in microencapsulated hepatoeytes group was at 24 h.Conclusion Smac/DIABLO and cyt-c mRNA expression is an indicator of apoptosis of hepatocytes.
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Os autores descrevem pela primeira vez no país um caso de criança do sexo feminino, com 10 anos de idade, atendida no ambulatório de Oftalmologia do Hospital Universitário Clementino Fraga Filho - UFRJ, portadora da síndrome de Leigh, que faz parte de um grupo de enfermidades metabólicas conhecidas como encefalomiopatias mitocondriais. É doença hereditária transmitida por diferentes modos de herança: mitocondrial, autossômica recessiva e recessiva ligada ao X. O início das manifestações clínicas é variado, ocorrendo em geral, dentro dos primeiros dois anos de vida, com evolução insidiosa, progressiva e com períodos de exacerbações. O diagnóstico é difícil pelo pleomorfismo de sua apresentação, sendo baseado nos achados clínicos e estudos complementares relacionados à deficiência na produção mitocondrial de ATP e da citocromo C oxidase. Como não há tratamento específico, este é baseado em medidas paliativas, portanto a identificação desta síndrome é importante como diagnóstico correto permitindo condutas adequadas à melhor qualidade de vida de seus portadores.
The authors describe for the first time in the Country a case of a 10-year-old female child, assisted at the Ophthalmology Clinic of the Hospital Universitário Clementino Fraga Filho UFRJ, with Leigh's syndrome that is part of a metabolic disease group known as mitochondrial encephalomyopathies. It is an hereditary disease transmitted by a different mode of inheritance: mitochondrial, X-linked recessive and autosomal recessive. The beginning of clinical manifestations is varied and occurs usually in the first two years of life, with progressive and insidious evolution and exacerbation periods. Diagnosis is difficult because pleomorphic presentation, based on clinical findings and complementary study related to mitochondrial production of ATP and cytochrome c oxidase deficiencies. Considering that there is no specific treatment, this is based on a palliative procedure. So, the identification of this syndrome is very important to keep it under control, since its evolution is progressive.
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Child , Female , Humans , Leigh Disease/diagnosisABSTRACT
Norcantharidin (NCTD) is the demethylated form of cantharidin, which is the active substance of mylabris. To examine the pathway of NCTD-induced A375-S2 cell death, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-dipheyltetrazolium bromide (MTT) assay, photomicroscopical observation, DNA agarose gel electrophoresis, caspase activity assay and Western blot analysis were carried out. A375-S2 cells treated with NCTD exhibited several typical characteristics of apoptosis. The inhibitory effect of NCTD on human melanoma, A375-S2 cells, was partially reversed by the inhibitors of pan-caspase, caspase-3 and caspase-9. The activities of caspase-3 and -9 were significantly increased after treatment with NCTD at different time. The expression of inhibitor of caspase-activated DNase was decreased in a time-dependent manner, simultaneously, the ratio of Bcl-2/Bax or Bcl-xL/Bax was decreased and the expression ratio of proteins could be reversed by caspase-3 inhibitor. The expression of cytochrome c in cytosol was increased after NCTD treatment and caspase- 3 inhibitor had no significant effect on the up-regulation of cytochrom c. These results suggest that NCTD induced A375-S2 cell apoptosis and the activation of caspase and mitochondrial pathway were involved in the process of NCTD-induced A375-S2 cell apoptosis.
Subject(s)
Animals , Humans , Apoptosis/physiology , Bridged Bicyclo Compounds, Heterocyclic/chemistry , Caspases/antagonists & inhibitors , Cell Line, Tumor/drug effects , Cell Shape , DNA Fragmentation , Enzyme Activation , Mitochondria/metabolism , Molecular Structure , Proto-Oncogene Proteins c-bcl-2/metabolism , Signal Transduction/physiologyABSTRACT
Objective To probe the mechanism of hepatocarcinoma cell apoptosis promoted by human telomerase reverse transcriptase (hTERT) RNA interference (RNAi) by mitochondrial pathway in vitro. Methods Westernblot was employed to detect the intracellular expressions of caspase-9, hTERT, Bcl-2 and Bax, and mitochondrial and cytoplastic cytochrome C (cyt C) in HepG2 cells transfected by pSliencer 3.1-H1 neo-shTERT (small hairpin RNA hTERT). Result In the HepG2 cells transfected by pSliencer 3.1-H1 neo-shTERT, expressions of hTERT, Bcl-2 and mitochondrial cyt C were significantly down-regulated, while Bax and cytoplastic cyt C were obviously up-regulated, and active caspase-9 was found in addition to procaspase-9 compared with that in negative control cells and untransfected cells. Conculsion hTERT RNAi may suppress hTERT expression to result in reduction of Bcl-2 and increase of Bax, and then induce hepatocarcinoma HepG2 cell apoptosis by mitochondrial pathway subsequent to cyt C release from mitochondria to cytoplast.