| Cerebrovascular disease, especially ischemic cerebrovascular disease(ICVD), is the leading reason for residents' death and disability in China.Recently, there is an increase in the incidence of ICVD, which seriously threatens people's health. Nowadays, thrombolysis and neuroprotectant are mainly used to treat ICVD clinically. However, on account of the narrow time limitations of thrombolysis and the deficit of the neuroprotective agents, the search for safe and effective agents is greatly required for the treatment of ICVD.Orientin, belonging to flavonoid C-glycoside, is the main active components in Chinese traditional medicines Trollius chinensis Bunge,Polygonum orientale L. and Bamboo leaves. Orientin have various pharmacological effects(such as anti-inflammatory, antioxidant, inhibition of thrombosis, etc.) and usually has been used for the treatment of coronary heart disease, angina pectoris, blood stasis resistance. Our previous studies have displayed that orientin has strongly anti-inflammatory and anti-oxidative effects on D-galactose induced aging mice. In addition, the protective effect of orientin is positive on the hippocampal nerve cells in mice and no cumulative toxication is found.These results show orientin can be used for further study because of its clinical application value. In this study, we built the middle cerebral artery occlusion(MCAO) model, with edaravone as a positive control, and explored the protective effects and the mechnisms of orientin on cerebral ischemia-reperfusion(I/R) injury, which provides experimental basis for the choice of new neural protectant.1 The model establishment of the MCAO in rats According to Longa's suture method, all rats were anesthetized with7% chloral hydrate, then were reperfused after ischemia for 2 h at the right side of the brain. Neurological deficit scores were assayed when the rats revived in order to verify the success of the model.2 Grouping and drug delivery The rats were randomly divided into six groups: sham-operated group,model(I/R) group, edaravone group, low- medium- and high-dose of orientin(1.62 ?mol·kg-1, 3.24 ?mol·kg-1, 6.48 ?mol·kg-1, respectively). The edaravone and orientin groups were treated with drugs, and sham and model groups were given the same volume of saline and solvent. All groups were injected intraperitoneally(ip) after 1 h of reperfusion for 1d or 3d,respectively.3 Effects of orientin on the infarct volume in rats after MCAO After 2 h ischemia followed by 1 d reperfusion, rats were sacrificed after deep anesthesia and the brains were carefully removed from skull.Then we cut each brain into 5 slices of 2 mm thickness and measured the infarct volume using the TTC staining.4 Effects of orientin on blood brain barrier and antioxidation in rats after MCAO After 2 h ischemia followed by 1 d or 3 d reperfusion, rats were sacrificed after deep anesthesia and the brains were carefully removed from skull. The brain water content was measured by wet-dry method; the aquaporin-4(AQP-4) positive cell levels in ischemic side were evaluated by the immunohistochemical staining method. The tissue homogenate from the ischemic regions of brain was prepared for the measurement of superoxide dismutase(SOD) activity and the malondialdehyde(MDA)content using the corresponding kits.5 Effects of orientin on the anti-inflammatory in rats after MCAO After 2 h ischemia followed by 1 d or 3 d reperfusion, rats were sacrificed after deep anesthesia and the brains were carefully removed from skull. The positive cell levels of toll like receptor 4(TLR4) and NF-?B(P65)were measured by the immunohistochemical staining method and the m RNA expression of TLR4 and NF-?B(P65) were measured by the real-time quantitative polymerase chain reaction(RT-q PCR) method. The tissue homogenate from the ischemic regions of brain was prepared for determination of the tumor necrosis factor-?(TNF-?) content by the enzyme-linked immuno sorbent assay(ELISA) method.6 Effects of orientin on the non- antioxidant in rats after MCAO After 2 h ischemia followed by 1 d or 3 d reperfusion, rats were sacrificed after deep anesthesia and the brains were carefully removed from skull. The tissue homogenate from the ischemic regions of brain was prepared to detect nitric oxide(NO) content and the activities of nitric oxide(NOS) and Na+-K+-ATPs. The content of glutamate(Glu), aspartate(Asp) and glycine(Gly) were also measured by the automatic analyzer for amino acids.7 Effects of orientin on the cerebral histomorphology in rats after MCAO After 2 h ischemia followed by 1 d or 3 d reperfusion, rats were sacrificed after deep anesthesia and the brains were carefully removed from skull. The cell morphology including cell shape, quantity, integrity,deformation and ultrastructure of neurons in the ischemic regions of brain were also observed with the light microscope and the transmission electron microscope, respectively.Results:1 Preparation of MCAO model The rats were evaluated for neurological deficits using a five-point scoring standard. Compared with sham group, the rats of MCAO fell down or circle on the contralateral side and couldn't fully extend his left forepaw,suggesting that the MCAO model was successfully constructed.2 Reduction on the infarct volume of MCAO rats after the treatment with orientin Compared with sham group, the model group rats were observed the pale infarcts in the ischemic regions of brain and showed a significant difference(P < 0.01) in infarction volume. After treatment with drugs for 1d on MCAO rats, all treated groups reduced the cerebral infarction volume in a dose- and time-dependent manner(P < 0.05, P < 0.01). The infarction volume with orientin at doses of 1.62 and 3.24 ?mol·kg-1 were larger than that with edaravone at 3.24 ?mol·kg-1(P < 0.05, P < 0.01). However, at6.48 ?mol·kg-1 orientin, infarction volume was almost same to that in edaravone group.3 Effects of orientin on blood brain barrier and anti-oxidation in rats after MCAO Compared with sham group, the model group significantly induced edema, reduced the SOD activity, increased the MDA content and AQP-4 positive cells(P < 0.01) in the right brain of rats. After treatment with drugs for 1 d or 3 d on MCAO rats, all treated groups alleviated edema, reduced MDA content and AQP-4 positive cells, up-regulated the activity of SOD in a dose- and time- dependent manner(P < 0.05, P < 0.01).Compared with edaravone at 3.24 ?mol·kg-1, these effects were weaker than that in the low and middle doses(1.62 and 3.24 ?mol·kg-1,respectively) of orientin(P < 0.05, P < 0.01), but almost at the same level of 6.48 ?mol·kg-1 orientin.4 Effects of orientin on the anti-inflammatory in rats after MCAO Compared with sham group, the level of TNF-? was significantly improved in the model group. Also, there were significant increases in the expression of positive cells and m RNA of TLR4 and NF-?B(P65)(P < 0.01).After treatment with drugs for 1 d or 3 d on MCAO rats, all treated groups reduced the TNF-? content, TLR4 and NF-?B(P65) positive cells, and the m RNA expression of TLR4 and NF-?B(P65) in a dose- and time- dependent manner(P < 0.05, P < 0.01). These effects with edaravone at 3.24?mol·kg-1 were stronger than that in the group of orientin at the concentrations of 1.62 and 3.24 ?mol·kg-1(P < 0.05, P < 0.01), but almost at the same level of 6.48 ?mol·kg-1 orientin.5 Effects of orientin on the non- antioxidant in rats after MCAO Compared with sham group, the content of NO, Asp and Glu, and the activity of NOS were greatly increased in the model group(P < 0.01).Meanwhile, the model group reduced the Gly content and Na+-K+-ATPs activity and had a significant difference(P < 0.01). After treatment with drugs for 1 d or 3 d on MCAO rats, orientin reduced the content of NO,Asp and Glu, decreased the activity of NOS, and improved the Gly content and Na+-K+-ATPs activity in a dose- and time-dependent manner(P < 0.05,P < 0.01). Edaravone at 3.24 ?mol·kg-1 showed better activity than orientin at the concentrations of 1.62 and 3.24 ?mol·kg-1(P < 0.05, P < 0.01), but almost had no significant difference with orientin at 6.48 ?mol·kg-1. These effects with edaravone at 3.24 ?mol·kg-1 were stronger than the low and middle doses(1.62 and 3.24 ?mol·kg-1, respectively) of orientin(P < 0.05,P < 0.01), but almost at the same level of 6.48 ?mol·kg-1 orientin.6 Effects of orientin on the cerebral histomorphology in rats after MCAO From the light microscopy, the cell morphology was clear and there was no nucleolus pyknosis or cellular infiltration in the sham group.However, when rats were subjected to I/R injury, the cell shrinkage, edema,infiltration, and necrosis appeared to some extent. After treated with different doses of orientin, the cell morphology and edema were gradually recovered in a dose- and time-dependent manner. And the treatment with orientin at 6.48 ?mol·kg-1 and edaravone at 3.24 ?mol·kg-1 for 3 d had the same effect on the recovery of cells.In addition, from the SEM, the neuron ultrastructure was complete, the organelles were clear and visible, and there was almost no edema. However,when rats were subjected to I/R injury, severe cell edema and mitochondria damage with swell and vacuole appeared and almost no intact organelles existed. After treated with different doses of orientin, the organelle integrity was recovered and cell edema was gradually reduced in a dose- and time-dependent manner. Moreover, when the treatment lasted for 3 d, the protective effect of orientin at 6.48 ?mol·kg-1 was almost same to edaravone at 3.24 ?mol·kg-1.Conclusions To sum up, orientin is able to protect rats from I/R injury in a doseand time-dependent and the mechanisms are mainly through three pathways in the following.(1) The protection of blood-brain barrier and the antioxidant pathway are through improving the SOD activity and reducing the MDA content and cerebral infarction volume, as well as up-regulating AQP-4 expression,reducing brain edema and improving the function of the blood-brain barrier.(2) The anti-inflammatory effect is through down-regulation in the expression of TLR4, inhibiting the tranfer and release of NF-?B and reducing TNF-? content, suggesting the relationship with the pathway of TLR4 /NF-?B/TNF-?.(3) The amino acid pathway is through the inhibition of TNF-?,promoting the expression of glutamate transporter on astrocyte membrane,reducing the Glu accumulation in synaptic cleft and protecting the neurons of post-synaptic membrane.The protective effects on MCAO rats with edaravone at 3.24 ?mol·kg-1were stronger than that in the groups of orientin at the doses of 1.62 and3.24 ?mol·kg-1. However, there was no significant difference between 6.48?mol·kg-1 orientin and edaravone. In addition, the treatment with orientin for 3 d showed better effects than that for 1 d. |
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