| It is well known that stroke is one of the most important reasons leading to neural dysfunction, the incidence, morbidity and mortality and recurrence of stroke rate rising trend year by year. Although thrombolysis therapy is an effective treatment of obstructive stroke, the treatment time window is narrow and rt-PA thrombolysis has various side effects. There are only 3-5% of patients who achieve treatment and could benefit from it. Therefore, the stroke is still a threat to human life and the life quality of major diseases.After cerebral ischemia reperfusion, mitochondria are involved in the internal environment of a complex set of changes, including the regulation of calcium homeostasis, the generation of reactive oxygen free radical, activation of various signa ling pathways in cells, etc. Studies confirm that mitochondria are continuously carrying on the biogenesis, autophagy, fusion and fission dynamic change, which ensure the old mitochondrial and new mitochondrial alternate and maintain mitochondrial health p lay an important role in the cell. Mitochondrial biogenesis not only increase the quality and quantity of mitochondria in cells, and meet cells for energy needs, but also enhance the recovery of neurons after cerebral ischemia injury, and improve neurons function. Numerous studies have demonstrated that under cerebral ischemia condition, mitochondrial biogenesis is an endogenous neuroprotective reaction, and induction of mitochondrial biogenesis is a new neural protection strategy. At the same time, studies have shown that inhibition of glycogen synthase kinase-3?(GSK-3?) can promote mitochondrial biogenesis, reduce ischemia caused by brain damage, and activat ing cannabinoid receptor 1(CB1R) can promote GSK-3? ninth serine phosphorylation, and make its inactivation. However, the relationship between the endocannabinoids system activation and mitochondrial biogenesis is obscure after cerebral ischemia reperfusion, and the relative research is less reported.This experiment is intended to explore whether ACEA(arachidonyl-2-chloroethylamide), a high selective agonist of CB1 R, could alleviate cerebral ischemia injury through GSK-3? mediated mitochondrial biogenesis and functional improvement, thus play a role of cerebral protection. Experiment I:CB1R selective agonist ACEA relieves cerebral ischemia injury [Objective] To study the protective effect of AC EA against ischemic injury in rats and the best effective dose. [Methods] 70 adult male SD rats were randomly divided into 5 groups, 1 h after cerebral ischemia-reperfusion intraperitoneal injection of physiological saline group(vehicle) or different concentrations of AC EA(,0.5 0, 1, 2 mg/kg), by TTC staining(n = 8) and neurobehavioral scores(n = 6), another 20 adult male SD rats, and randomly divided into four groups: S ham, MC AO, AC EA and vehicle groups, using TUNEL staining(n = 5) method to evaluate brain function. [Results] Compared with the vehicle group, 1 mg/kg and 2 mg/kg ACEA can significantly reduce the infarct volume of rats(P < 0.05), and improved the rats neurobehavioral scores(P < 0.05). Therefore, in the subsequent experiments we chose 1 mg/kg dose, the lowest effectie dose for treatment. TUN EL staining indicated significantly reduce the amount of neuron apoptosis after giving ACEA(P < 0.05), only to give the vehicle would not have this effect. [Conclusion] AC EA has the protective effects against cerebral ischemic injury, and its best effective dose is 1 mg/kg. Experime nt II : ACEA alleviates cerebral ische mia injury via promoting mitochondrial biogenesis and improving mitochondrial function [Objective] To observe ACEA's effect on related mitochondrial biogenesis markers, mitochondrial quantity and volume, and mitochondrial function after cerebral ischemia injury. [Methods]1. To investigate the influence of ACEA on mitochondrial biogenesis related moleculars, 56 male SD rats were randomly divided into Sham group and MCAO group, then MCAO were randomly divided into 6 groups, namely the MC AO 2 h, AC EA 2 h, MCAO 4 h, ACEA 4 h, MCAO 24 h, AC EA 24 h, and after cerebral ischemia reperfusion according to 2 h, 4 h and 24 h take brain tissue of ischemic penumbra, and Western Blot detects the Nrf- 1 Tfam and COX IV mitochondrial bio genesis related molecules(n = 4). At the same time, 4 h after reperfusion the brain tissue assessed by immunofluorescent staining, detection of the Nrf- 1, Tfam and COX IV mitochondrial bio genesis related marks(n = 4).2. To investigate the influence of AC EA on mitochondrial quantity and volume, 9 male SD rats were randomly divided into Sham group and MCAO group, then MCAO group randomly divided into 2 groups, namely give ACEA and don't give ACEA(MC AO group), 4 h after reperfusion the cortical neuron mitochondrial number and volume were assessed by transmission electron microscopy(TEM).3. To investigate the effects of ACEA on mitochondrial functions, we cultivated the primary neurons, and build oxygen glucose deprivation(OGD)model simulated cerebral ischemia injury, CCK-8 tests the cell vitality and LDH releasing test screening the best effective dose of ACEA to treat injury neurons, and then based on the best effective dose detection each function of mitochondria. Cells can be divided into four groups: Control group, OGD group, AC EA and vehicle group, ACEA group was at the on set of reoxygenaton incubated with ACEA. By detecting adenosine triphosphate(ATP), mitochondrial permeability transition pore(m PTP), mitochondrial membrane potentials(MMP), the content of malondialdehyde(MDA) and reactive oxygen species(ROS), to evaluate the function of mitochondria l, at last, by flow cytometry to detect neuron survival situation. [Results]1. Western Blot results indicated that the given ACEA after 2 h and 4 h increased the expression of the Nrf-1(P<0.05); After giving AC EA 2 h, 4 h and 24 h, raised Tfam and COX IV expression(P<0.05). Immunofluorescence staining indicateed give AC EA after 4 h, the Nrf-1, Tfam and the expression of COX IV raised significantly(P<0.05).2. The TEM results indicated that ACEA can significantly increase the number of mitochondria(P<0.05), increased mitochondrial volume(P<0.05), suggesting AC EA promotes the formation of new mitochondria.3. Compared with the vehicle group, AC EA promoted the formation of ATP(P<0.05), and improve the mitochondrial membrane potential(P<0.05), decreased the mitochondrial permeability transition pore open(P<0.05), decreased the ROS(P<0.05) and MDA(P<0.05), so as to improve the function of mitochondria. Flow cytometry indicated AC EA significantly reduced the number of neuron apoptosis caused by OGD(P<0.05). [Conclusion] ACEA through promote the mitochondrial biogenesis, improves the function of mitochondria and thus plays a role of neuroprotection. Experime nt ?: The role of GSK-3? in ACEA promoting mitochondrial biogenesis and improving mitochondrial function [Objective] To investigate the influence of different level GSK-3? phosphralation on ACEA promoting mitochondrial biogenesis and improve mitochondrial function. [Methods]1. To detect the GSK-3? and its phosphorylation level after cerebral ischemia, 20 male SD rats were randomly divided into Sham group and MCAO group, MC AO were randomly divided into 4 groups, namely the MCAO 2 h, ACEA 2 h, MCAO 4 h and ACEA 4 h, 2 or 4 h after reperfusion, the ischemic penumbra tissue were taken(n = 4), Western Blot detection of GSK-3? after cerebral ischemia and its changes in the level of phosphorylation.2. To investigate the effects of inhibition of GSK-3? phosphorylation on mitochondrial biogenesis, 28 male SD rats were randomly divided into Sham group and MCAO group, then MCAO group were randomly divided into 6 groups, namely MC AO, ACEA, AC EA +wrt, AC EA +vehicle(W), ACEA +LY and AC EA +vehicle(L), wrt and LY are two of PI3 K inhibitors, they can block the GSK-3? translate to its phosphorylation. 4 h after reperfusion the ischemic penumbral was taken, and Western Blot detection of biomarkers of mitochondrial biogenensis, the expression of Nrf-1, Tfam and COX IV, to observe whether inhibition of GSK-3? phosphorylation can affect mitochondrial biogenesis.3. To investigate the effects of promotion of GSK-3? phosphorylation on mitochondrial biogenesis, 20 male SD rats were randomly divided into Sham group and MCAO group, then MCAO was and randomly divided into 4 groups, namely the MCAO, ACEA, AC EA + vehicle(T) and TDZD-8, TDZD-8 for GSK-3? inhibitor, which can simulate GSK-3? in its phosphorylation status. 4 h after reperfusion the ischemic penumbral was taken, and Western Blot detection of biomarkers of mitochondrial biogenesis, the expression of Nrf-1, Tfam and COX IV, to observe whether promotion GSK-3? phosphorylation affects mitochondrial biogenesis.4. To investigate the effects of GSK-3? phosphorylation on mitochondrial function, we cultivated the primary neurons, and built OGD model simulated cerebral ischemia injury, administrated PI3 K inhibitorswrt and LY, or GSK-3? inhibitor TDZD-8, to detect the content of ATP, m PTP, MMP, MDA and ROS, and evaluate the function of the mitochondrial. [Results]1. The rats after 2 h and 4 h reperfusion, the expression level of GSK-3? have no obvious change, but the phosphorylation level of GSK-3? significantly increased(P<0.05).2. Western Blot results indicated that compared with the vehicle, inhibition of GSK–3? translate to its phosphorylation, decreased the expression of mitochondrial bio genesis related molecules Nrf-1, Tfam and COX IV(P<0.05).3. Western Blot results indicated that compared with the vehicle, promote GSK-3? to its phosphorylation, increased the expression of mitochondrial bio genesis related molecules Nrf-1, Tfam and COX IV(P<0.05).4. To further confirm on the primary neurons that promote GSK-3? translate to its phosphorylation, which promoted the formation of ATP(P<0.05), and improved the mitochondrial membrane potential(P<0.05), decreased the mitochondrial permeability transition pore(P<0.05), reduced the ROS(P<0.05) and MDA(P<0.05), so as to improve the function of mitochondria. O n the contrary, inhibit GSK-3? phosphorylation lower the function of mitochondria. [Conclusion] ACEA plays a cerebral protective role by regulating the phosphorylation of GSK-3? to promote mitochondria biogenesis and improve the mitochondria function. Activation of CB1 R by AC EA is initiative factor of promoting mitochondrial biogenesis and regulation of GSK-3? phosphorylation by ACEA is an essential step of controlling mitochondrial biogenesis, which provides a new drug target and a new approach for the treatment of stroke. |
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