| Radix curcumae is a plant of Curcuma genera, there are four botanic origin of Radix curcumae: Curcuma longs L. (Huangsi Radix curcumae), C.wenRadix curcumae Y H. Chen et. C. Ling, C.kwangsiensis S.G Lee et C. R Liang (Gui Radix curcumae), G phaeocaulis Val. (Lvsi Radix curcumae).Radix curcumae,one of traditional Chinese medicine, has the function of activating blood and Qi circulation, alleviating pain, alleviating depression, clearing away heart-fire, promoting the function of the gallbladder to alleviate jaundice and cooling blood. In clinic it has wide pharmacological functions such as anti-tumor, anti-inflammation, anti-virus and liver protection.Objective:To observe the effect and explore its possible mechanism of Radix curcumae on cerebral function in hypoxic mice.Methods:1. The measurement of mice survival time under acute hypoxic experimental setting:To reproduce acute hypoxic mice models, 60 mice were divided into five groups, namely model group, positive group, high dosage Radix curcumae group, middle dosage Radix curcumae group and low dosage Radix curcumae group, 12 in each group, half male and half female. In Radix curcumae groups, the mice were treated with Radix curcumae at the doses of 10g·kg-1, 20g·kg-1, 40g·kg-1 respectively through theintragastric tubes everyday. 6 days later, Radix curcumae groups, Radix curcumae three dose groups respectively. Positive drug group and model group with the same volume of saline gavage. Positive drug group were treated with 0.1% chlorpromazine 0.1ml/10g by intraperitoneal injection. The mice were placed in 250ml confined wide-mouth bottles with sodium lime, observe and record the survival time of mice which identified with the last breath.2. Observing and recording the number of mice open-mouth breathing and breathing time:From 72 mice were randomly divided into six groups: normal control group, model group, positive drug group, and Radix curcumae high, medium and low dose groups, and 12 in each group, half male and half female. The drug administration methods wre the same as above and the positive drug group were treated with 0.1% chlorpromazine 0.1ml/10g by intraperitoneal injection. After treatment, the mice were placed in 250ml confined wide-mouth bottles with sodium lime. At last, observed the times of mice open-mouth and difficult–breathing time after rapid decapitation.3. Determination of cerebral index:After decapitation, quickly isolated and weigh brain on cool plate, then calculate brain index. Brain index = brain weight (mg)×100/body weight (10g).4. Brain SOD, MDA and NO Determination:Weigh the brain after decapitation, and made 10% brain homogenate from the brain tissue. Pack and frozen at -80°C refrigerator.Determination methods are shown in the kit manual.5. Pathological observation of brain tissue:The brain tissue were fixed in 10% formaldehyde solution, then sliced and HE stained, and observed the morphological changes under optical microscopy.6. Immunohistochemical staining:The immunohistochemical stainings were carried with AQP4, IL-18, caspase-3 and bcl-2 antibodis.7. Statistical Analysis:All data were shown as mean±standard deviation (x±s), using SPSS 10.0 software for statistical analysis, P <0.05 for a significant difference.Results:1. The impact on acute hypoxic mice survival time of Radix curcumae:Compared with the model group, Radix curcumae high and medium-dose groups mice are significantly prolonged the survival time (P <0.05, P <0.01) and showed a dose-dependent manner.2. The number of mice open-mouth breathing and breathing time: Radix curcumae can increase the number of mice open-mouth breathing and breathing time after rapid decapitation(P<0.05,P<0.01)。3. Brain SOD, MDA and NO Determination:Compared with the model group, the brain tissue SOD activity was significantly increased (P <0.01) and showed a dose-dependent manner; MDA and NO levels decreased (P <0.05, P <0.01) Radix curcumaegroups.4. The effects of Radix curcumae on cerebral index:Compared with the control group, model mice cerebral index was significantly increased (P <0.05); Compared with model group, Radix curcumae high dose group of mice cerebral index decreased significantly (P <0.05), Radix curcumae medium and low-dose groups mice cerebral indice have a downward trend.5. Pathological observation of brain tissue:The mice cerebral tissues of model group are obvious perivascular inflammatory edema and meningeal vascular dilation and bleeding, however in Radix curcumae groups are without other lesions except for occasionally meningeal artery dilatation.6. Immunohistochemistry results:Compared with the model group, the AQP4,IL-18 and caspase-3 expression down-regulated and the bcl-2 expression up-regulated in the mice cerebral tissues of Radix curcumae groups.When the mice were placed in sealed containers, with their constantly breathing, the oxygen will be less and less, carbon dioxide will not be significantly increased due to the absorption of sodium lime. The mice are hypoxic; the brain tissue blood supply stpping will increased the mouse brain hypoxia. Because the brain is a high oxygen consumption organ, it is extremely sensitive to hypoxia. The hypoxia will damage to brain cells with ATP production decreasing, oxidation disturbance, ion transport dysfunction, increased intracellular Ca2 + and the activation of calcium-dependent protease, promoting formation of xanthine oxidase,and a lot of oxygen free radicals produing. Through the lipid peroxidation reaction, the membrane and sun-membrane will be destroyed, the function will be losed. The modern medical research has proved Radix curcumae have anti-inflammatory, anti-tumor, anti-oxidation, anti-virus extensive pharmacological effects. Some scientists have confirmed that the major components of Radix curcumae can inhibit the lipid peroxidation in adult mice and older rats. This conclusion is accord with our experimental results. Radix curcumae can increase the mice hypoxia tolerance ability which is associated with enhancing the SOD activity and decreasing the contents of SOD, MDA and NO. Further more, it is also concerned with inhibition of inflammation and cell apoptosis.Conclusions:1. Radix curcumae can lessen the acute cerebral hypoxic condition.2. Radix curcumae will enhance free radical scavenging enzymes SOD activity and inhibit the lipid peroxidation reaction, reduce NO-mediated neurotoxicity and suppress the role of free radicals.3. Radix curcumae can decrease the mice cerebral hypoxia-induced brain edema.4. The role of decreasing the mice cerebral hypoxia-induced brain edema of Radix curcumae is associated with the expression of AQP4,IL-18 proteins.5. The function of hypoxia tolerance of Radix curcumae on mice cerebral tissue is also concerned with the inhibition of cell apoptosis in some degree. |
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