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

摘要

BACKGROUND: The decrease of mitochondrial membrane potential (MMP) is an irreversible marker of neuronal apoptosis during ischemia/reperfusion (I/R) injury of brain tissue. Qingkailing 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 Qingkailing injection on rat embryonic hippocampal neuronal apoptosis, MMP and mitochondrial activity after hypoxia/hypoglycemia and reoxygenation, and make a comparison of therapeutic effect on I/R injury between Qingkailing injection and nimodipine. DESIGN: Observation and controlled trial. SETTING: Peripheral 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. Qingkailing 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 culturing fetal rat hippocampal neurons. The neurons cultured for 10 days were used for experiment. The neurons were divided into 5 groups: model group, control group, nimodipine group, Qingkailing high-dose group and Qingkailing low-dose group. Hypoxia/hypoglycemia and reoxygenation models served as model group, and they were used to simulate reperfusion injury, i.e. glucose-free Earle’s solution was used to replace DMEM 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 CO2. 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 nimodipine group when hypoxia/ hypoglycemia occurred. Final concentration of 15 and 30 mL/L Qingkailing injection 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. ③ Analysis 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: ① Mitochondrial 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 Qingkailing low-dose group , which was significantly higher than that in the model group , respectively [(0.328±0.117),(0.276±0.033),(0.203±0.051),P 0.05). ② Neuronal apoptosis rate at hypoxia/hypoglycemia 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). ③ Neuronal morphological detection at hypoxia/hypoglycemia 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. ④ Detection of apoptosis rate at hypoxia/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). ⑤Detection of MPP at hypoxia/hypoglycemia 5 hours and reoxygenation 3,12 and 24 hours: MPP was (88.17±6.16),(73.14±7.68),(69.59±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). 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, comparably with the effect of nimodipine.