Protective Effect And Mechanism Of Puerarin On Cognitive Dysfunction In Vascular Dementia Rats Induced By Chronic Ischemia

ObjectiveChronic cerebral hypoperfusion (CCH) can be caused by different kinds of neurological diseases or psychiatric diseases. Studies have shown that CCH is closely related with learning and memory dysfunction within those diseases, especially in vascular dementiathe, characterized by cognitive impairment and bad behavior. The CCH animal models can be prepared by disruption of the bilateral common carotid arteries (2VO rats) as the pathological change occurred in vascular dementia. In the 2VO rats, cerebral blood flow was disrupted and cause cerebral chronic hypoperfusion in the cortex, hippocampus and white matter. Cognitive dysfunction and suboptimal metabolism thus occurred, which lead to final neuronal injury. The rennin-angiotensin system (RAS) was reported to be important regulatory systems in the circulatory homeostasis and was regarded as potential target in treating CCH induced cognitive defects.One of main cause of vascular dementia is reactive oxygen species (ROS) overexpression induced by cerebral blood flow decrease. Reactive oxygen species superexpression take part in the RAS process, it is the symbolled the beginning of oxidative stress, which is defined the symbol of unbalance between active oxygen ROS and anti-oxidative reaction. Excessive ROS and free radicals can cause serious cell damage, including the membrane lipid peroxidation, fracture and damage DNA and protein loss. In the brain excessive production of reactive oxygen species can lead to the death or apoptosis of neurocytes and astrocytesl, which can lead to permanent neurological damage. ROS generation and its harmful effects were found not only in the vascular dementia, but also in the ischemic, hemorrhagic desease and the reperfusion lesion of the central nervous system. In this regard, ROS-removal compounds were widely tested in all kinds of disease models in order to reduce the level of reactive oxygen species.Puerarin, one of isoflavones, can be found in a number of plants and herbs like the root of Pueraria. Puerarin is also commonly used in traditional Chinese medicine for symptoms like fever and diarrhea. It has been proved that puerarin has the ability of scavenging ROS and reducing lipid perioxidation. Puerarin was also proved to be neuroprotective in different kinds of neurological diseases like Alzheimer’s disease, Parkinson’s disease and brain ischemia. The ROS cavenging ability of puerarin was emphasized in most of the studies referring the protective effects of puerarin. Several studies also focused on the anti-apoptosis activity of puerarin on particular cell types. However, no study has yet been done considering the protective effects of puerarin on vascular dementia.In this study, we tried to validate that puerarin play a protective role in vascular dementia caused by chronic ischemia through 2VO rats model. At the same time, we also tried to validate that puerarin could reduce cognitive and behavioral barriers caused by vascular dementia. We made a inference that puerarin protective effect in 2 VO rats model may be achieved through clearing ROS in order to reduce oxidative stress reaction.Methods6 to 8 week old male Wistar rats were adopted as experimental animals. The CCH animal models can be prepared by disruption of the bilateral common carotid arteries (2VO rats) as the pathological change occurred in vascular dementia. The rats were divided into defferent groups who accepted puerarin treatment or not. Morris water maze (MWM) test was adopted to test the learning and memory function of rats. MDA, glutathione peroxidase and total thiol assessment was done to reflect the oxidative stress in the brain tissue. Hydrogen peroxide is administered after SH-SY5Y cell culture-simulation of oxidative stress, give puerarin treatment or not. Cell Counting Kit-8 (CCK8) and flow cytometry (FCM) were performed to examine the cell viability and apoptosis rate. Reactive oxygen species (ROS) generation was determined by the 2’,7’-dichlorofluorescein diacetate (DCFH-DA) assay. qPCR and Western blot (WB) were adopted to test the change of gene expression including Nrf2、FoxO1 FoxO3 和 FoxO4, and investigate the molecular mechanism of puerarin treatment.Results1, In Morris water maze test 2VO animals spent more time on escaping time than control group, the delay is significant (P< 0.05). The escape time of 2VO followed puerarin-treat group is significantly shortened than 2VO group(P< 0.05). In the control with puerarin-treatment group the escape time had no obvious change with the control group. In Probe Trains, it took the average time in the target quadrant area by 2VO group model shorter than the control group, it’s significant difference (P< 0.05). 2VO with puerarin treatment group spent more average time in the target quadrant area than 2VO group, it’s significant difference (P< 0.05). Control with puerarin-treatment group spent almost the same average time in the target quadrant area as control group, there is no significant difference. There was no significant difference about swim speed among four groups of animals.2, The malondialdehyde(MDA) content of hippocampus and frontal cortex in 2VO group increased significantly compared with the control group (P< 0.05); Puerarin intervention group can significantly regulated-down the increased MDA level of hippocampus and frontal cortex (P< 0.05); The malondialdehyde content of control group could not be effected by the intervention of puerarin.The concentration of glutathione peroxidase in 2VO group was lower than the control group with statistical difference (P< 0.05); Puerarin intervention group shows the puerarin’s cure effect on glutathione peroxidase concentration compared with the 2VO group.(P< 0.05); The control with puerarin-treatment group did not show any difference compared with control group.2VO rats showed a significant decease in the level of total thiol compare to the sham group (P< 0.05). Administration of puerarin in 2VO rats could partial y improve the total thiol level decreased by 2VO procedure. Administration of puerarin in the sham+puerarin did not show statistical meaningful changes on the thiol level.3, To simulate the in vivo situation of vascular dementia and 2VO models, we stimulated the SH-SY5Y eel s with hydrogen peroxide for oxidative stress. Cell viability was evaluated with or without treatments of puerarin. Puerarin was treated under three different concentrations:0.1 uM,0.5 uM and 1 uM. Cell viability was measured at 12 h,24 h and 48 h respectively. The results showed that oxidative stress induced by hydrogen peroxide could significantly down-regulate the cell viability at the three time points. The cell viability of SH-SY5Y cells was improved by puerarin treatments in a dose-dependent way.1 uM of puerarin showed the best improving effects that almost reached to the same level of the control groups. We then tested the cell apoptosis rate induced by oxidative stress. The results went an opposite pattern showing that pueararin could decrease the apoptosis rate induced by hydrogen peroxide in a dose-dependent way.1 uM of puerarin showed the best anti-apoptosis effects compare to the other puerarin treatments groups.4, To further explore the ROS scavenging ability of puerarin, we performed ROS scavenging assay by DCFH-DA on SH-SY5Y cells. Intracellular ROS levels were analyzed by DCFH-DA, which is cell permeable and oxidation sensitive within cells. After 24h’s induction of hydrogen peroxide, culture medium was replaced with different dosages of puerarin. After 12 h or 24 h of puerarin treatment, intracelular ROS levels were then analyzed by DCFH-DA. The results remarkably showed that puerarin decrease the intensity of DCF fluorescence within SH-SY5Y cells in a dose-dependent way. Quantitative analysis of DCF fluorescence intensity showed that groups induced with hydrogen peroxide and no treatment of puerarin showed the highest fluorescence level. The ROS positive cell number was significantly down regulated by puerarin treatments at both 12 h and 24 h.1 uM of puerarin showed the best ROS scavenging ability compare to other groups (P< 0.05).5, We previously proved that puerarin could effectively scavenge intracellular ROS generation induced by hydrogen peroxide. To explore the underlying mechanisms, we hypothesized that the antioxidant activity of puerarin might be related with alterations of gene expressions. We then investigated several crucial genes related with antioxidant protein expression namely Nrf2, FoxO1. FoxO3 and FoxO4. Qpcr and western blot were performed to test the expression changes of these genes. From the results, we found that hydrogen peroxide is not affective on the mRNA level of Nrf2, while puerarin treatments could significantly improve the expression of Nrf2. As for the FoxO family, FoxO1, FoxO3 and FoxO4 were improved by hydrogen peroxide induction on the mRNA levels. Puerarin treatments could enhance the improving effects of hydrogen peroxide(P< 0.05). The results were consistent on the protein levels proved by WB. The quantitative analysis of WB images showed that these anti oxidative stress genes xpressions were up regulated in different patterns according to the mRNA level alterations.Conclusions1, Our results demonstrated that puerarin administration both in vivo and in vitro could significantly reverse the oxidative stress induced by ischemic conditions or hydrogen peroxide. Puerarin administration could also improve the learning ability and consolidate the memory in 2VO rats. We found that the protective effects of puerarin on the chronic ischemic condition and oxidative stress induced by hydrogen peroxide in vitro is closely related with the ROS scavenging ability of puerarin, which also explained the anti-apoptosis activity of puerarin in vitro. On the molecular level, we discovered that treatments of puerarin is associated with the up regulation of several antioxidant protein expression. The mRNA level and protein levels of Nrf2, FoxOl, FoxO3 and FoxO4 were significantly up regulated by treatments of puerarin. It was also interesting to find that hydrogen peroxide treatment alone could also up regulate the expression of Nrf2.2,2VO rat model was chosen as the in vivo animal model in our study. The ligation of the bilateral common arteries could cause global ischemia of the brain. Two target areas were mostly affected from this ischemia namely hippocampus and frontal cortex in which hippocampus is in charge of the learning and memory function. We then performed the Morris Water Maze test for the evaluation of learning and memory function within each group. Al the results indicated protective effects of puerarin on the impaired function of learning and memory abilities in 2VO rats.3, It has already been proved that oxidative stress plays a crucial role in the ischemic and hypoxic brain damage. Oxidative stress is also related with over expression of inflammatory cytokines and cognitive dysfunction caused by vessels impairments. ROS is capable of oxidize membranous lipids, cell proteins and nuclear acids within the nuclei that eventually lead to cellular dysfunction. In this consideration, we focused on the ROS over generation and free radicals production in the brain tissue of 2VO rats. We measured the levels of MDA, which is a marker of lipid peroxidation. GSH-Px and thiol levels were measured as enzymatic and non-enzymatic defense of ROS generation in vitro respectively. To further explain the ROS scavenging ability of puerarin in vitro, SH-SY5Y cells were selected to be cultured in vitro and stimulated with hydrogen peroxide as simulation of ischemic induced oxidative stress in vivo. In our results, it was indicated that puerarin treatments were capable of reducing the hydrogen peroxide induced cell apoptosis. We found that puerarin treatments partially reversed the adverse effects of oxidative stress induced by hydrogen peroxide in a dose-dependent way.4, To explore the underlying mechanisms, we focused on the forkhead box O (FoxO) family and Nrf2 expression. The FoxO family reduces ROS production by increasing the expression of several antioxidant enzymes of redox signaling. FoxO family proteins are also transcription factors regulating cel proliferation, differentiation, apoptosis, cel cycle arrest and autophagy. Another gene we focused on is Nrf2, which also regulate antioxidant proteins protective on oxidative damages. Both FoxO family and Nrf2 have important roles in the physiological function and pathological conditions in the CNS. For the first time we reported that puerarin treatments is associated with the up regulation of both these genes in the ischemic brain. In conclusion, we hypothesized that puerarin is protective on the vascular dementia induced chronic brain ischemia by alleviating the oxidative stress and improving the cognitive functions.