| Vascular dementia(VD), a heterogeneous group of brain disorders in which cognitive impairment is attributable to cerebrovascular pathologies, being the second most common dementia in elderly, after Alzheimer’s disease(AD). Epidemiological studies have shown that the prevalence of vascular dementia is increased in accompany with the dramatic increase in aged population. This not only seriously affects patients’ quality of life, but also add a heavy burden to family and society. Thus, there is an urgent need to develop an effective anti-dementia drug, while improving cerebral pathological change with less adverse effects.Taohong Siwu Decoction(TSD), a famous Chinese compound prescription, first recorded in ‘Yi Zong Jin Jian’(a famous ancient Chinese medicine book written by Wu Qian). TSD can not only promote blood circulation and nourish blood, but also eliminate blood stasis to promote new blood. Previous studies also confirm that TSD exhibited many pharmacological activities, including inhibiting platelet aggregation, improving microcirculation, antioxidant and enhancing immune function. Furthermore, recent studies have shown that TSD possesses effective neuroprotective activity against MCAO-induced neuronal injury and consequent neurological deficits, and it has been reported for treatment of VD. However, its still have not been subjected to intensive studies. In this study, we investigated the protective effects of TSD on neuronal cells from ischemic insult and improvement on ability of learning and memory from VD, as well as its potential mechanisms in two levels: systemic level and cellular level. This research provides scientific basis for the exploration of new promising alternative for the treatment of vascular dementia.Objective: The current study was performed to investigate the neuroprotective effect and partial mechanism of Taohong Siwu decoction(TSD) on memory impairment and brain damage in VD model rats, meanwhile to investigate the protective effects of serum with Taohong Siwu decection(S-TSD) on the injury of PC12 cells induced by oxygen glucose deprivation(OGD) and its relative mechanisms. From two aspects of animals and cells to provide experimental basis for TSD in treatment of vascular dementia.Method: In the vivo experiment, VD injury was induced through middle cerebral artery occlusion reperfusion(MCAO/R). Adult male SD rats were randomly divided into six groups: sham-operated group, VD model group, TSD-treated group at doses of 18 g/kg, 9 g/kg and 4.5 g/kg and Nimodipine group(20 mg·kg-1). Their spatial learning and memory abilities were assessed using the Morris water maze and step-down test. The level of cerebral hippocampus tissue Acetylcholinesterase(ACh E), 5-hydroxytryptamine(5-HT) level, endothelin-1(ET-1) and vascular endothelial growth factor(VEGF) as well as the concentration of serum Thromboxane B2(TXB2), 6-keto-prostagladin F1α(6-keto-PGF1α) were detected by enzyme-linked immunosorbent assay(ELISA). Pathological changes in hippocampal CA1 field were observed by HE staining. Blood plasma were collected by abdominal aortic method to detect the hemorheological index. The expression of microvascular density(CD34) of all groups was detected using immunohistochemical assay. Moreover, the expressions of Bax and Bci-2 protein in hippocampal CA1 were detected by immunohistochemistry and western blot. In the vitro experiment, We used the sodium dithionite(Na2S2O4)+DMEM(Low Glucose) in PC12 cells to build OGD injury model, cell viability was detected by MTT method, to establish the appropriate concentration of and Na2S2O4 serum. After treatment with different concentrations of TSD medicated serum, the cell viability was assessed by the MTT assay. Morphological changes of PC12 cells were observed under reversal phase microscope. Oxidative stress parameters such as superoxide dismutase(SOD), malondial dehyde(MDA) and lactate dehydrogenase(LDH) were observed. Cell apoptosis morphological changes were detected by Hoechst33258 staining under fluorescence microscope. Western blot to analyze expression of Caspase-3 protein.Results:In vivo experiment,In the Morris water maze test: the average escape latency was significantly longer in model rats compared to sham-operated rats(P<0.01); the high-dosage and medium dosage treatment group significantly shortened the escape latency by comparison with model group(P<0.05 or P<0.01). During probe trials, the percent of traveled distance in target quadrant and the number of times through virtual platform of VD model rats were significantly less than sham-operated rats(P<0.01). The results showed that rats administered high-dosage and medium dosage of TSD exhibited significantly increased distance in the target quadrant(P<0.05 or P<0.01). However, in all dosage of TSD treated rats, the number of crossing platform location were no significant difference when compared with VD model rats(P>0.05). In the jump platform experiments, although the response time and the number of errors in the model group had no significant difference(P>0.05), but showed an increasing trend; compared with model group, every-dosage group of TSD had the trend of decrease. Compare to the sham operation group, the latency had the tend of increasing in the model group(P>0.05)and the error times enhanced significantly(P<0.05); however, the high-dosage treatment group significantly shortened the error times by comparison with model group(P<0.05 or P<0.01).Histological observation of hippocampal CA1 region on VD rats: In the sham-operated group, no histopathological abnormalities were observed in the hippocampal area. The hippocampal CA1 region remain intact and normal cell organelles, neurons kept arranged well and the nuclei were centered with clear staining and the cytoplasm was abundant. In contrast, most neurons in CA1 region in the model group appeared pycnotic shape and condensed nuclear, and their arrangement was disordered. After treatment with high-dosage and medium dosage of TSD for 14 days, the necrotic neurons markedly decreased and most neurons have normal morphology.The effects of TSD on the content of TXB2, 6-Keto-PGF1α in serum of VD rats: Compared with the sham-operated group, the content of 6-keto-PGF1α in serum of VD model group was obviously decreased(P<0.01). After treatment with TSD at the doses of 18 mg/kg showed a significant increase in activities of 6-Keto-PGF1α compared with the model group(P<0.01). Compared with the sham-operated group, the content of TXB2 in serum of VD model group was obviously increased(P<0.01). Compared with the model group, the content of TXB2 was decreased in high and medium dosage treatment groups, with statistical significance(P<0.05 or P<0.01).Effect of TSD on blood rheology in VD model rats: Compared with the sham-operated group, whole blood viscosity and plasma viscosity of rats in VD model group were significantly increased(P<0.01). Compared with the VD model group, the high and medium dosage of TSD treatment groups significantly decreased the whole blood viscosity and plasma viscosity by comparison with model group(P<0.01 or 0.05). Meanwhile the low dosage of TSD treatment group significantly decreased the plasma viscosity by comparison with model group(P<0.05).Compared with the sham-operated rats, ACh E and ET-1 activities significantly increased in model group, whereas a significantly decrease of the 5-HT content was found. Compared with the VD model group, ET-1 and ACh E activity in the TSD group(9 and 4.5 mg/kg/day) and the Nimodipine group were significantly decreased(P<0.01 for both). Surprisingly, rats administered to all doses of TSD and significantly increase the 5-HT content(P<0.01 or 0.05). TSD treatment caused dose-dependent reduction of ET-1 and ACh E activities, meanwhile an increase of 5-HT content. The effects of TSD on numbers of MVD in the hippocampus CA1 area: The MVD was markedly increased in the VD model group from 20.50±2.26 to 26.17±3.06(P<0.01) when compared with the sham-operated group. In the treatment groups with high dose of TSD, MVD markedly increased to 33.83±5.95(P<0.05), in comparison to the model group.The effects of TSD on the expression of Bax and Bcl-2 in hippocampus on VD model rats:The results of immunohistochemical staining: The immunohistochemistry showed that there was a little expression of Bax and Bcl-2 could be seen on hippocampal CA1 area in sham operation group. The mean optical density of Bax and Bcl-2 was significantly higher and the positive cells was deeply stained in the VD model group than in sham operation group(P<0.01 or P<0.05). After treated with drugs, every-dosage group of TSD could significantly decrease the mean optical density of Bax(P<0.01 or P<0.05). Meanwhile, the high and medium dosage of TSD groups could significantly increase the mean optical density of Bcl-2(P<0.01). The results of Western blot: The expression of Bax protein in the models were significantly higher than those in the sham operation group(P<0.01). The expression of Bcl-2 protein in the models were significantly lower than those in the sham operation group(P<0.01). Compared to sham operation group,the ratio of Bcl-2/Bax decreased significantly in model group(P<0.01). While the expression Bax protein in the high and medium dosage groups were significantly lower than those in the model group(P<0.01), the expression of Bcl-2 proteins in the high dosage treatment group were significantly higher than those in the model group(P<0.01). The ratio of Bcl-2/Bax increased obviously in the high and low dosage of TSD treatment group(P<0.01 or P<0.05).In vitro experiment:Selected the optimal conditions of OGD damage PC12 cell: MTT assay results showed that growth inhibition rate of the Na2S2O4 20 m M + DMEM(Low Glucose) acted on PC12 cells for 24 h nearly 50%.Morphological monitoring of S-TSD on the OGD-induced PC12 cells: The morphology observation results showed that the model group cells had lower cell density, rounded cells with varying degrees of shrinkage, synapse shorten; while 10% medicated serum group of cells form adherent cell number is more, number of exfoliated cells decreased, cell refractivity increased, much closer to the normal cells.S-TSD protected PC12 cells against OGD-induced cytotoxicity: MTT assay results showed that, in the range of 5% ~ 15%, cell viability increased gradually with the concentration of blank serum. Compared with the model group, TSD medicated serum with 5%, 10%, 15% concentrations on injured PC12 cells induced by OGD showed a certain degree of protection.Effects of S-TSD on LDH levels in PC12 cells: The LDH leakage in PC12 cells increased remarkably after treatment of OGD compared with that of blank serum group(P<0.01). 10% S-TSD significantly stopped OGD-induced increase of the level of LDH leakage(P<0.05).Effect of S-TSD on OGD-induced intracellular accumulation of SOD and MDA: When PC12 cells pretreated with 10% and 15% S-TSD, followed by exposure to OGD, intracellular MDA level was significantly decreased and SOD activity was significant increased as compared with the OGD group(P<0.01 or P<0.05).Effect of S-TSD on OGD-induced PC12 cells apoptosis by Hoechst 33258 staining: Hoechst 33258 staining showed that cells of model group were stained to bright blue fragment, which was a typical feature of apoptosis of cell nucleus, 10% S-TSD treated group presented less of morphology of apoptosis, compared with the model group.Effect of S-TSD on caspase-3 activity in OGD-induced PC12 cells: Western blotting results indicated that, after the treatment of OGD injury, the expression of caspase-3 significantly increased(P<0.01). Comparing with control group, 10% S-TSD treated group can significantly drop the expression of caspase-3 protein induced by OGD(P<0.05).Conclusion:(1) TSD possesses remarkable anti-dementia properties probably involve its adjusting neurotransmitter content, dilating blood vessels, promoting cerebrovascular growth, improving blood circulation and decrease apoptosis.(2) S-TSD has protective effect on in vitro cultured PC12 cells injured by OGD. The mechanism may be related to the antioxidative and anti-apoptotic effects. |
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