To explore the role of cell apoptosis in denervated skeletal muscle atrophy in rats and the effect of losartan on it. Methods Forty-two Sprague Dawley rats were randomly divided into 3 groups: group I (n =14, normal control group), group II (n =14, denervated group) and group III (n =14, losartan group). The rats were not treated in group I, and were made denervated gastrocnemius models in groups II and III. In group III, the rats were treated with losartan 10 mg /kg• d by gavage and with normal sal ine in groups I and II. After 4 weeks, gastrocnemius mass to body mass ratio (GAS/BM) served as the degree of muscle atrophy. Apoptotic cells in gastrocnemius were stained in situ by using TUNEL. Gastrocnemius Bcl-2 and Bax protein were quantified by immunohistochemistry and Western blot. Bax /Bcl-2 served as the degree of apoptosis. Results The ratio of apoptosis was higher in group II than that in group I (11.32% ± 4.51% vs 0.56% ± 0.21%, P lt; 0.05). The ratio of apoptosis was lower in group III than that in group II (7.21% ± 2.05% vs 11.32% ± 4.51%, P lt; 0.05). The atrophy of skeletal muscle（GAS/BM） in group II was more serious than that in group I (11.68 ± 1.98 vs 12.86 ±0.74, P lt; 0.05), there was no significant difference between group III and group II (12.11 ± 0.65 vs 11.68 ± 1.98, P gt; 0.05). The expression of Bcl-2 in group II (18.3% ± 4.9%) was significantly lower than that in group I (27.5% ± 2.8%) and group III (25.5% ± 3.5%); there was no significant difference between group III and group I (P gt; 0.05). The expression of Bax in group II (24.1% ± 3.1%) was significantly higher than that in group I (22.1% ± 3.6%) and group III (21.7% ± 2.3%); there was no significant difference between group III and group I (P gt; 0.05). Western blot results showed that: the expressions of Bcl-2 were 122.5 ± 14.6 in group II, 135.3 ± 6.2 in group I and 139.2 ± 16.2 in group III; showing significant diffeerences between group II and group I, between group III and group II (P lt;0.05). The expressions of Bax were 107.1 ± 15.8 in group II, 89.3 ± 8.4 in group I, and 94.2 ± 9.5 in group III; showing significant diffeerences between group II and group I, between group III and group II (P lt; 0.05). There was no significant difference in the expression of Bcl-2 and Bax between group Ⅲ and group I (P gt; 0.05). Conclusion Cell apoptosis plays an important role in denervated skeletal muscle atrophy in rats and may be one of the factors causing skeletal muscle atrophy. Losarton can decrease skeletal muscle cell apoptosis through regulating the ratio of Bax / Bcl-2.
Objective To evaluate the phenomena of apoptosis and its relevant mechanism during ischemia-reperfusion period. Methods The published papers to explore the apoptotic phenomena and its mechanism in organs or tissues which experienced ischemia-reperfusion injury were reviewed. Results Apoptosis was common in ischemia-reperfusioned organ or tissue. The severity of apoptosis was influenced by many factors such as ischemia, hypoxia, oxygen free radials, intracellular free calcium ion overloading, various cytokines, et al; and also was regulated by bcl-2 family, caspase family and NF-κB，et al. Conclusion Apoptosis is a common phenomenum in ischemiareperfusioned organ or tissue which is affected and regulated by various factors.
Objective To investigate inhibited effects of melatonin (MLT) on proliferative activity of retinoblastoma cell line HXORB44 and its related mechanism. Methods HXO-RB44 cells were treated by MLT of different concentration (10-10, 10-9, 10-8, 10-7 mmol/L. Cell counting and tetrazolium dyereduction assay (MTT) were used to determine the effect of MLT on the survival and proliferation of HXO-RB44 cells. Apoptotic nuclei were further analyzed by HoechstPI fluorescence staining. Flow cytometry was used to measure the fluorescent intensity of ROS, cell cycle distribution and apoptosis. Results 10 -6 mmol/L (or exceed) of MLT could inhibit the proliferation of HXO-RB44cells in vitro while 10-7 mmol/L (or below) of MLT couldn't. With the increase of MLT concentration from 10-10 mmol/L to 10-7 mmol/L, HXO-RB44 cells gradually increased the expression of ROS. Hoechst staining showed that 4, 8, 12 and 24 hours after the incubation with MLT, the nuclear pyknosis and nuclear fragmentation increased in HXORB44 cells. The extent of apoptosis was proportional to the concentrations of MLT. Flow cytometry revealed that with the increasing of MLT concentration, G0/G1 and G2/M phase cells increased, S phase cells decreased. The apoptotic rate was also increased. Conclusion 10 -6 M of MLT could inhibit the proliferation of HXO-RB44 cells. This effect may relate to the increased ROS expression, cell cycle arrest at G0/G1 phase and apoptosis of HXO-RB44 cells.
Objective To investigate the apoptosis of photoreceptor cells in experimental model of retinal detachment in mice. Methods Thirty-six adult C57Bl/6J mice were divided into 2 groups: retinal detachment model was set up in the left eyes of 18 mice by subretinal injection with 1.4% sodium hyaluronate in the experimental group, while the left eyes of other 18 mice underwent scleral puncture only as the control. The retinal sections were stained with histochemical and immunofluorescent staining and examined by confocal microscopy 1,3,7 and 28 days after injection. eye enucleated, and retinal sections studied by histochemistry, immunofluorescence labeling, and confocal microscopy. Rods, cones, and apoptotic cells were labeled by antibodies of anti-rod and anti-cone cells, and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL), respectively. Photoreceptor cell apoptosis and cell loss were assessed quantitatively by counting both surviving and apoptotic rod and cone cells. Results TUNEL-positive cells were only found in the outer nuclear layer (ONL) of the detached portion of the retina, which were detected at the 1st day after the detachment. The apoptosis of the cells reached the peak at the 3rd day and decreased sharply after 7 days. Photoreceptor cell loss of both rod and cone cells followed a similar time course after retinal detachment. Conclusion Apoptosis is a major pathological degeneration of photoreceptor cell death after retinal detachment. (Chin J Ocul Fundus Dis, 2006, 22: 124-127)
Objective To investigate the effect of N-acetylcysteine (NAC) on the apoptosis during myocardial ischemia reperfusion injury in rats’ heart transplantation, and to explore the possible role of NAC in myocardial apoptosis. Methods Sixty healthy male Lewis rats (weighing, 200-220 g) were randomly divided into 3 groups, 20 rats each group (10 donors and 10 recipients). In control group, 1 mL normal saline was infused via inferior vena cava at 30 minutes before donor harvesting; in donor preconditioning group, NAC (300 mg/kg) was infused via inferior vena cava at 30 minutes before donor harvesting, but no treatment in recipients; and in recipient preconditioning group, NAC (300 mg/kg) was infused via inferior vena cava at 30 minutes before recipient transplantation, but no treatment in donors. Heart transplantation was established in each group. Blood was drawn at 6 and 24 hours after reperfusion for analysis of aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH) as markers of graft injury; myocardial tissue was harvested to determine the superoxide dismutase (SOD) and lipid hydroperoxide (LPO) activity at 24 hours after reperfusion and to observe the histology and ultrastructural changes. Graft active Caspase-3 protein expression was measured by immunohistochemistry staining, and apoptosis index (AI) was calculated by TUNEL. Results The heart transplantation operation was successfully completed in all groups, and the rats survived to the end of the experiment. The serum levels of AST, ALT, and LDH in donor and recipient preconditioning groups were significantly lower than those in control group at 6 hours after reperfusion (P lt; 0.05); the levels of AST and ALT in donor preconditioning group and the levels of AST and LDH in recipient preconditioning group were significantly lower than those in control group at 24 hours (P lt; 0.05); and no significant difference was found between donor and recipient perconditioning groups (P gt; 0.05). The levels of AST, ALT, and LDH at 24 hours were significantly lower than those at 6 hours in each group (P lt; 0.05) except the level of ALT in recipient preconditioning group (P gt; 0.05). SOD activity and SOD/LPO in donor and recipient preconditioning groups were significantly higher than those in control group (P lt; 0.05), but no significant difference between donor and recipient preconditioning groups (P gt; 0.05); there was no significant difference in LPO activity among 3 groups (P gt; 0.05). Histological staining and transmission electron microscope showed that myocardial injury in recipient preconditioning group was obviously lighter than that in donor preconditioning group and control group. Active Caspase-3 in recipient pretreatment group was significantly higher than that in donor preconditioning group and control group (P lt; 0.05). AI of donor and recipient preconditioning groups was significantly lower than that of control group (P lt; 0.05), but no significant difference was found between donor and recipient preconditioning groups (P gt; 0.05). Conclusion NAC can relieve ischemia reperfusion injury in rats’ heart transplantation by improving myocardial SOD content, and reducing active Caspase-3 activity and AI, which has a protective effect on myocardial cell of donor heart.
ObjectiveTo investigate the endoplasmic reticulum stress associated apoptosis gene Caspase-12 expression in paraquat-induced pulmonary fibrosis. Methods30 adult healthy Sprague-Dawley(SD) rats were randomly divided into a nomal control group,two pulmonary fibrosis model groups (intragastrically administered paraquat for 14 days and 28 days,respectively).The model of pulmonary fibrosis was established through intragastrically administering paraquat at the dose of 30 mg/kg.RT-PCR was used to determine the mRNA expression of Caspase-12.Immunohistochemistry was used to determine the protein expression of Caspase-12.HE staining and Masson staining were used to determine the degree of alveolitis and pulmonary fibrosis. ResultsHE staining and Masson staining of lung tissues proved that pulmonary fibrosis model was successfully constructed.The degree of alveolitis and pulmonary fibrosis in the model group was significantly more serious than that in the control group(P<0.01).RT-PCR and Immunohistochemistry results showed that the expression of Caspase-12 were remarkably increased in the pulmonary fibrosis model group(14 d group)(P<0.01),even more elevated in 28 d group compared with the 14 d group. ConclusionThe results demonstrate that the expression of Caspase-12 in paraquat poisoned rats is up-regulated,suggesting endoplasmic reticulum stress plays an important role in paraquat induced-pulmonary fibrosis.
Epilepsy is a disorder of the brain in which sudden abnormal discharges of neurons cause transient dysfunction and is a common disorder of the nervous system. Although most patients experience remission of symptoms with medication, about 20 ~ 30% of patients still have poor outcomes with medication and progress to refractory epilepsy. The etiology of epilepsy is complex and the exact pathogenesis is not yet clear. Current research has explored the pathophysiological mechanisms underlying epileptogenesis, thus providing a basis for identifying potential therapeutic targets for epilepsy and advancing the precision treatment of epilepsy. p38 Mitogen-activated protein kinase (MAPK) signalling pathway is a conserved class of kinases involved in many physiological/pathological processes by regulating intracellular gene expression levels, cell division, differentiation and apoptosis in response to various extracellular stimuli in order to mediate intracellular signalling cascades. The p38 MAPK signalling pathway is one of the subfamilies of MAPK that mediates inflammatory responses, apoptosis, tissue edema and other biological processes involved in the development of central nervous system diseases. The p38 MAPK signalling pathway is now reviewed for its involvement in the development of epilepsy through unused pathways, in order to identify new potential targets for epilepsy treatment and provide clinical precision.
PURPOSES:To investigate the time of neuronie apoptosis in the retinas of Imman fetuses,and its relations with neuronie proliferation and differentiation, METHODS:The retinas of 27 human fetuses from 8th to 38th week of R,~til- ization age and 3 adults were studied by TdT-mediated dUTP nick end labelling(TUNEL) method. RESULTS:Tbe nuctei of labeled apoptotic cells were charaeterised by nuclear marginization,ehromatln condensation and cleseent shape,and some apoptotie bodies were visible in the specimens. The apoptosis of neuroepithelium of fetal rclina took place during 8th to 18th week, Apoptosis of ganglion cells were observed from 1256 to 18th week. The apoptos[s of pholorec, plors were formd from 14th to 2Ist week ,while thai of bipolar neurones and M~ller cells were found from ldth to 28th week. No apoptosb of ocstones were observed in the retinas after 28th week of fertilization age and within the retinas of adults. CONCLUSION:The proliferating cells of neuroepithelium and Ihe neurones which just differetiated from fetal retina might partly undergo apoptosis. The time of apoptosls of differentiated neurones was consistent with the time of the synapses formation between neurones and their targel cells. （Chin J Ocul Fundus Dis,1997,13:67 -69 ）
Objective To investigate the relationship between the expression of apoptosis-related gene Fas and recovery of neurological function after surgical decompression at different time points in acute spinal cord injury (SCI) rat model by cerclage. Methods A total of 100 13-week-old male Sprague Dawley rats (weighing, 255-376 g) were randomly divided into 4 groups (n=25). The rats only received laminectomy in group A as control; the rats were made the acute SCI models by cerclage in groups B, C, and D. The spinal cord decompression was performed in group B at 8 hours and in group C at 72 hours, no spinal cord decompression in group D. At 1, 3, 7, 14, and 21 days, Basso-Beattie-Bresnahan (BBB) score and inclined plane test were used to evaluate the recovery of neurological function; the neuronal apoptosis level of spinal cord was examined by TUNEL staining; HE staining and immunohistochemical staining were applied to analyze the expressions of Fas. Results The BBB score and inclined plane test score in group A were significantly better than those in groups B, C, and D at different time points (P lt; 0.05); group B was significantly better than groups C and D, and group C than group D at 3, 7, 14, and 21 days (P lt; 0.05). In group A, no bleeding, edema, or necrosis was found. The edema, hemorrhage, and neuron death were observed in spinal cord tissue of groups B, C, and D at 1 day after operation, especially in group D. The degree of cell degeneration in group B was lighter than that in groups C and D at 3 and 7 days after operation; few glial cells and fibroblast proliferation were found at damaged zone in group B at 14 and 21 days, but necrosis and cystic cavity in groups C and D. Fas and TUNEL expression was little in group A at different time points. Fas and TUNEL were expressed in groups B, C, and D; the expressions of Fas and TUNEL reached the maximum at 3 days, and then gradually decreased at 7 and 21 days. The number of positive cells was highest in group D, and the number of positive cells in group B was significantly less than that in groups C and D (P lt; 0.05). Conclusion Early decompression of SCI is beneficial to recovering the neurological function. The Fas signal pathway may play an important role in the apoptosis of neuron and glial cells after SCI.
Objective To study hyperthermia induced apoptosis and the effect of aspirin on hyperthermia induced apoptosis in retinoblastoma cells. Methods Retinoblastoma cells (Y79) were divided into two groups:hyperthermia groups,hyperthermia+aspirin (0.18～18mu;g/ml) groups.Heat shock condition:44℃,heat shock time:10,20,30, and 40 minutes respectively.The following events were studied after heat shock by using FAC Scan: ①cell apoptosis; ②heat shock protein 70 (HSP70) expression；③bcl-2 expression. Results Apoptosis was induced by the treatment of hyperthermia (44℃) in Y79 cells in a heat dose dependent fashion.Longer time heating (44℃，40 minutes) induced necrosis rather than apoptosis.Aspirin could rescue Y79 cells from hyperthermia induced apoptosis in a dose dependent manner．HSP70 was induced in Y79 cells after heat shock,it was further enhanced by the treatment of aspirin(＞1.8mu;g/ml).Heat shock itself showed no effect on bcl-2 expression in Y79 cells,aspirin,on the other hand,could enhance bcl-2 expression in a modest level in heat treated Y79 cells. Conclusions Hyperthermia may induce apoptosis in Y79 cells which can be protected by use of aspirin．The enhancement of HSP70 and bcl-2 expression in Y79 cells by the treatment of aspirin in heating condition may be responsible for the protective function. (Chin J Ocul Fundus Dis, 1999, 15: 143-145)