| Ischemia reperfusion is the body or an organ blood supply interruption or insufficient, then perfusion, tissues and organs to refill a condition caused by oxygen. Is one of the more common is the artery blood flow is embolus block, lead to serious metabolites supply imbalance, eventually leading to hypoxia. Restore blood flow and oxygen, can accentuate tissue damage, and associated with more severe inflammation, called the reperfusion injury. Cerebral ischemia reperfusion injury in the process of a large number of free radicals, reactive oxygen species and other traumatic material make the mitochondrial membrane lipid peroxidation, damage to the mitochondrial membrane structure and enzyme complex electronic chain, further damage to the mitochondrial DNA, trigger and induced neuronal apoptosis. As a result of the nerve cells irrefragable, so the brain ischemia reperfusion injury consequences are especially serious.At present for the treatment of ischemia-reperfusion injury mainly in ischemia pretreatment, metabolic strategies to increase tolerance, gas treatment, nucleotides and nucleoside signals and treated by Micro RNAs. But all these methods have some defects and shortcomings, in terms of the effects of the treatment is not very satisfactory, so looking for other effective treatments or a treatment is especially important.Catalpol is a newly from the base of traditional Chinese medicine radix rehmanniae isolated from a glycoside, is one of the main effective ingredient rehmannia, separation and purification for the first time in 1969, it can through the blood brain barrier to play to the anti-inflammatory, analgesic, antitumor, Huan Xie, fall blood sugar, protect liver and anti-aging and other pharmacological effects. This experiment adopts the traditional middle cerebral artery obstruction(middle cerebral artery occlusion MCAO) preparation of focal ischemia model of rats, to explore the Catalpol on ischemia-reperfusion injury of rats after protection.Experiment I: The influence of Catalpol against focal cerebral ischemia / reperfusion injury in ratsObjective: To investigate the influence of Catalpol against focal cerebral ischemia / reperfusion injury in rats.Methods: Male SD rats were selected(280-320g) 24,and wererandomly divided into 4 groups(n=6): ①MCAO group: The MCAO model(ischemic for 120 minutes); ②solvent control group(Vehicle):ischemic for 120 minutes, reperfuse 0.1mol/l of DMSO after intraperitoneal injection;③ Catalpol group of 1mg/kg:ischemic for 120 minutes,reperfuse 0.1mol/l of Catalpol after intraperitoneal injection; ④Catalpol group of 5mg/kg group : ischemic for 120 minutes, reperfuse 0.1mol/l of Catalpol after intraperitoneal injection.After reperfusion for 48 h, test NDS and TTC stainingon on four groups of rats.Results:After the Ischemia-reperfusion injury for 48 h,compared with the MCAO group,NDS of Catalpol 5mg/kg group are significantly lower(P<0.05).After 48 h Catalpol 1mg / kg and 5 mg / kg group the percentage of rat damage infarct volume decreased significantly compared with MCAO group.Conclusion:Catalpol can significantly reduce the cerebral ischemia / reperfusion injury in the function of rat brain systems and can protect rats’ brain nervous system very well.Experiment Ⅱ Effect of Catalpol on neuronal apoptosis and degeneration after focal cerebral I/R injury in rats.Objective: To explore the effects of Catalpol on apoptosis of neurons after injury in rats with focal cerebral ischemia / reperfusion.Methods: 24 male SD rats were selected(280-320 g)and were randomly divided into 4 groups(n=6):Sham operation group(group Sham);MCAO group; Solvent control group(Vehicle); Catalpol group. Treat to the animals in Sham group with simple operation, not for the construction of MCAO model;The MCAO model building method of Four groups in rats is the same to the experimental one.After 48 h,detect the apoptosis of neurons in rats by TUNEL immunofluorescence,and detect the function of apoptosis related protein by using Western blot.Results: The test of injury of neuronal apoptosis after focal cerebral I/R by TUNEL staining shows, there was almost no positive staining cells in Sham group, and compared with the Sham group, the other three group apoptosis obviously( P<0.05).TUNEL staining positive was significantly higher than that of the Catalpol group.Western blot analysis found that compared with sham group, I / R after 48 h Cataipol group and MCAO group bcl-2 expression decreased significantly, the expression of Bax and cleaved caspase-3 were significantly higher(P < 0.05); and compared with MCAO group, Bcl-2 levels in Cataipol group were significantly higher(P < 0.05), Bax and cleaved caspase-3 levels significantly less(P < 0.05).Conclusion: Catalpol can protect rats with focal cerebral ischemia / reperfusion injury by suppressing neuronal apoptosis after cerebral ischemia reperfusion.Experiment Ⅲ Effect of Catalpol on antioxidant enzymes activies and MDA content of brain tissue after focal cerebral I/R injury in rats.Objective: To investigate the influence of Catalpol in the level of antioxidant enzymes, GSH-PX and MDA after focal cerebral ischemia / reperfusion injury in rats. Methods: Male SD rats were selected(280-320 g) 24 and were randomly divided into 4 groups(n=6): Sham operation group(group Sham);MCAO group; solvent control group(Vehicle); Catalpol group. Treat to the animals in Sham group with simple operation, not for the construction of MCAO model; The MCAO model building method of four groups in rats is the same as the experimental one.48 h after reperfusion, detect the level of SOD、Catalase、GSH-PX and MDA in brain tissue by Elisa.Results: After 48 h reperfusion,compared with MCAO group, activities of GSH-PX in brain were significantly lower in Catalpol group(P < 0.05),,while the MDA content in brain tissue after I/R was significantly lower(P < 0.05), and activity of SOD and catalase in plasma were significantly enhanced(P < 0.05). However, after 48 h reperfusion, activity of GSH-PX,SOD and Catalase in MCAO group were significantly lower than Sham groupConclusion: Catalpol can protect the neuroprotective by scavenging free radicals.It also has dose-dependent antioxidant capacity.Its mechanism supposed to be treating the rats with focal cerebral ischemia / reperfusion injury by increasing the activity of GSH-PX and decrease the level of MDA.Experiment IV :Study on the relationship between NADPH oxidase and Catalpol cerebral protective mechanism.Objective: To explore the relationship between cerebral protective mechanism of alcohol Ziand NADPH-oxidase.Methods: 24 male SD rats were selected(280-320 g) and were randomly divided into 4 groups(n=6):Sham operation group(group Sham);MCAO group; Solvent control group(Vehicle); Catalpol group. Treat to the animals in Sham group with simple operation, not for the construction of MCAO model;The MCAO model building method of Four groups in rats is the same to the experimental one.After 48 h,detect the content of NOX2 by using Western blot.Choose another 36 male SD rats(280-320 g) and randomly divide into 6 groups(n=6): ①MCAO group: the MCAO model(ischemia for 120min); ②solvent control group(Vehicle group): MCAO was intraperitoneally injected with PBS 2 L;③Group DMSO:anterior abdominal injection of DMSO 0.1mol/l MCAO;④TBCA group:MCAO anterior abdominal injection of 60 min TBCA(20nmol in 2 L of 50%(v/v) DMSO in PBS), the oxidase agonist of NADPH. ⑤Catalpol group: Catalpol injection in anterior abdominal of 30 min 5mg/kg before MCAO. ⑥Catalpol+TBCA group: intraperitoneal injection of TBCA(20nmol in2 L) 60 min before TBCA, intraperitoneal injection of Catalpol 5mg/kg 30 min before MCAO.Treat the rats with the neurological behavior score and detect the solvent of infarction, the level of MDA and the activity of antioxidant enzyme in plasma and brain tissue.Results:After 48 hours of reperfusion,western blot analysis found that compared with sham group, Catalpol group and MCAO group of NOX2 protein expression were significantly higher(P < 0.05), Cataipol group increased compared with MCAO group less(P < 0.05).After 48 h rats neural function defect score Catalpol was significantly lower than that of MCAO group(P < 0.05), and Catalpol+ TBCA significantly higher than Catalpol group(P < 0.05), confirmed the role TBCA reversal of Catalpol induced by cerebral ischemia in rats after nerve functional recovery. Reperfusion 48 h after cerebral infarction volume in rats in group Catalpol significantly lower than MCAO group(P < 0.05), and vehicle group, DMSO group and MCAO group showed no significant difference(P > 0.05), and Catalpol+ TBCA significantly higher than Catalpol group(P < 0.05). 48 h after reperfusion, either in plasma or brain tissue, compared with the MCAO group, Catalpol can increase the activity of SOD and Catalase, while compared with the MCAO group, Catalpol content reduced tissue or plasma MDA(P < 0.05); compared with Catalpol group, in Catalpol +TBCA group, the activity of SOD and Catalase was significantly reduced, and the content of MDA had a significantly increased(P < 0.05).Conclusion:The mechanisms of Catalpol’s protection to brain suggest to be that it can increase the activity of dantioxidant enzymes SOD, reduce the level of MDA to increase the capacity of antioxidant.It can also effect cell apoptosis by changing the level of intracellular Bcl-2, Bax, Cleaved Caspase-3.NOX2 agonist TBCA could reverse the Catalpol’s protection to neuron,therefore we can speculate that the mechanism of nervous system protection of Catalpol after reperfusion associated with inhibition of NOX2’s biological effects.SummaryCatalpol could significantly reduce neurological injury induced by transient MCAO in rats. The therapeutic effect of Catalpol on cerebral ischemia-reperfusion injury in rats might be mediated, at least partially,by up-regulating of the activity of antioxident enzymes, GSH-PX, decrease level of MDA,and can inhibite the level of NOX2. In addition Catalpol can content brain tissue and prevent apoptosis by influencing the level of Bcl-2, Bax, Cleaved caspase-3 and degeration of neurons in the ischemic brain.,... |
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