Effect of Qingkailing injection on rat embryonic neuronal apoptosis and mitochondrial membrane potential

He Pang; Lingqun Zhu; Shuoren Wang; Fuing Niu; Wei Cui

摘要：BACKGROUND:The decrease of mitochondrial membrane potential(MMP)is an irreversible marker of neuronal apoptosis during ischemla/reperfusion(I/R)injury of brain tissue.Qingkaiing injection is proved to have protective effect on neuronal ischemic injury.Whether inhibiting the decrease of MMP can inhibit apoptosis when I/R injury of brain tissue occurs is unclear.OBJECTIVE:To observe the effect of Qingkaiing injection on rat embryonic hippocampal neuronal apoptosis,MMP and mitochondroal activity after hypoxia/hypoglycamia and reoxygenation,and make a comparison of therapeutic effect on I/R injury between Oingkaiing injection and nimodipine.DESIGN:Observation and controlled trial.SETTING:Peropheral Vascular Center,Dongzhimen Hospital, Beijing University of Chinese Medicine;the Key Laboratory of Chinese Internal Medicine of Ministry of Education and Beijing Key Laboratory.Dongzhimen Hospital,Beijing University of Chinese Medicine.MATERIALS:Eight Wistar rats at embryonic 18 days,provided by Breeding Farm of Experimental Animals,Chinese Academy of Medical Sciences(Permission No.SCXK-11-00-0006) were employed in this trial.Qingkaiing injection (Pharmaceutical Factory of Beijing University of Chinese Medicine,Batch No.213710A,10 Ml each,baicalin 50 g and total nitrogen 25 mg included)and nimodipine(ICN company,USA)were also used.METHODS:This experiment was carried out in the Key Laboratory of Chinese Internal Medicine of Ministry of Education,Dongzhimen Hospital,Beijing University of Chinese Medicine and Beijing Key Laboratory from January 2003 to December 2005.①The pregnant rats were anesthetized and fetal rats were isolated for culturong fetal rat hippocampal neurons.The neurons cultured for 10 days were used for expedment.The neurons were divided into 5 groups:model group,control group,nimodipine group.Qingkailing high-dose group and Oingkailing low-dose group.Hypoxia/hypoglycemia and reoxygenation models served as model group,and they were used to simulate reperfusion injury,Le.glucose-free Earie's solution was used to replace UMEM medium containing serum,then neurons were placed in the hypoxic container.afterwards.which was filled with 0.95 volume fraction of N2 and 0.05 volume fraction of C02.Oxygen volume in the container was less than 0.01 volume fraction.The container was placed at 37℃for 5 hours.Hypoxia/hypoglycemia treatment was not given to the control group.10 μmol/L nimodipine was added to the medium in the nimedipine group when hypoxia/hypoglycemia occurred.Final concentration of 15 and 30 mL/L Qingkailing iniection was added to the Qingkailing low-dose group and Qingkailing high-dose group respectively 12 hours before experiment,and the other procedures were the same as model group.②Morphological changes were observed under a fluorescence microscope immediately after cell culture.Neurons were collected at the 3rd.12th and 24th hours after culture in each group.Mitochondrial activity was detected by MTT assay.neuronal apoptosis rate and MMP were detected with flow cytometry,and necrotic neurons were observed under a fluorescence microscope and necrosis rate was calculated.③Analysis of variance was used for comparison of difference of measurement data.MAIN OUTCOME MEASURES: Comparison of neuronal mitochondrial activity, apoptosis rate, morphological change,necrosis rate and MMP.RESULTS:①Mitochondrial activity(absorbance,A)at hypoxia/hypoglycemia 5 hours and reoxygenation 3.12 and 24 hours:Mitochondrial activity was respectively(0.513±0.063),(0.507±0.042)and(0.499±0.075)in the Qingkailing high-dose group and(0.464±0.088),(0.415±0.047)and(0.392±0.063)in the Qingkailinglow-dose group,which was significantly higher than that in the model group,respectively[(O.328±0.1 17),(0.276±O.033),(0.203±0.051),P＜0.05-0.01].Mitochondrial activity in the nimodipine group(0.539±0.026.0.527±0.052,0.513±0.038)was higher than that in the model group(P＜0.05),and mitochondrial activity was close between Qingkailing groups and nimodipine group(P＞0.05).②Neuronal apoptosis rate at hypoxia/hypoglycamia 5 hours and reoxygenation 3,12 and 24 hours:neuronaL apoptosis rate was(11.61±2.55)%.(17.99±3.71)%,(21.22±8.90)%in the Qingkailing high-does group and(14.72±3.36)%,(27.79±5.97)%.(23.01±5.14)%in the Qingkailing low-dose group,which was significantly lower than that in the model group [(19.06±5.01)%,(45.73±11.57)%,(49.85±10.19)%,P＜0.05-0.01] Neuronal apoptosis rate in the nimodipine group [(8.05±2.64)%,(13.90±4.88)%,(16.24±4.19)%]was lower than that in the model group(P＜0.05).Neuronal apoptosis was close between Qingkailing groups and nimodipine group(P＞0.05).③Neuronal morphological detection at hypoxia/hypoglycamia 5 hours and reoxygenation:Considerable apoptotic cells and a few necrotic cells appeared.Apoptotic and necrotic cells were increased with the elongation of time of reoxygenation.but significantly reduced after adding Qingkailing injection and nimodipine.④Detection of apoptosis rate at hypoxi a/hypoglycemia 5 hours and reoxygenation 3,12 and 24 hours:neuronal apoptosis rate was(3.77±0.49)%,(2.94±0.43)%,(5.02±0.86)%in the Qingkailing high-dose group and(4.36±0.41)%,(4.72±0.59)%"6.51±0.79)%in the Qingkailing low-dose group,which was significantly lower than that in the model group [(6.83±1.53)%.(7.57±1.33)%,(11.49±1.50)%,P＜0.05-0.01].Neuronal apoptosis rate in the nimodipine group was (2.44±0.61)%,(2.97±0.46)%,(4.35±0.53)%,which was lower than that in the model group(P＜0.05).Neuronal apontosis rate was close between Qingkailing groups and nimodipine group(P＞0.05). ⑤Detection of MPP at hyrpoxia,hypoglycemia 5 hours and reoxygenation 3,12 and 24 hours:MPP was(88.17±6.16),(73.14±7.68).(66:5g±6.03)in the Qingkailing high-dose group and(79.68±6.07),(66.95±8.11),(64.83±5.01)in the Qingkailing low-dose group.which was significantly higher than that of model group [(41.91±9.64),(29.12±5.11).(20.41±6.54),P＜0.05-0.01].MPP in the nimodipine group was 95.26±9.32,87.41±8.83,81.22±8.02.which was higher than that in the model group(P＜0.05).MPP was close between Qingkailing groups and nimodipine group(P＞0.05).CONCLUSION: Qingkailing injection can inhibit hypoxia/hypoglycemia and reoxygenation-induced decrease of MPP and mitochondrial activity.It can also inhibit apoptosis,and has protective effect on hippocampal neurons.Improving effect of Qingkailing injection weakens with the elongation of the time of reoxygenation.comporably with the effect of nimodipine.