| Background:Vascular dementia(VD)refers to cognitive dysfunction syndrome caused by cerebral vascular abnormalities caused by ischemia,hypoxia or bleeding,and is a common type of senile dementia.With the aging of population,the incidence of VD is increasing,but its complex pathogenesis makes clinical treatment difficult.At present,cognitive,learning and memory dysfunction caused by VD has brought serious harm and heavy economic burden to patients and their families.Therefore,it is of great social and economic significance to explore VD intervention drugs and their mechanism of action.Phytoestrogens have a weak estrogen-like effect,but can reduce a series of adverse reactions caused by estrogen.This effect can be realized by activating GPER,and its protective effect on neural function has been widely concerned.Among them,genistein-3’-sodium sulfonate(GSS)is a sulfonated structural modification of phytoestrogens,genistein-3’-sodium sulfonate(GSS).Previous studies have confirmed that GSS has a better protective effect on brain,but the protective mechanism is still being further explored.It is known that the regulation of mitochondrial dynamic balance is crucial for the survival of mitochondria and even neurons.In particular,there are many factors that can break the mitochondrial dynamic balance during the evolution from cerebral ischemia to VD.Therefore,this study aimed to explore the protective effect and mechanism of sodium genistein sulfonate on cognition,learning and memory function of VD rats from the pathway of regulating mitochondrial dynamic balance.Objective:In vivo and in vitro experiments were conducted to investigate the improvement effect of GSS on cognitive and learning and memory dysfunction induced by chronic cerebral ischemia in 2-VO rats.From the perspective of mitochondrial dynamic balance,we explored the molecular mechanism of Rictor-MTFP1-m PTP axis protecting hippocampal neurons in 2-VO rats by GSS,providing experimental basis for further clarifying the prevention mechanism of GSS on 2-VO rats,and also indirectly providing a new target for VD treatment or the development of new brain protective drugs.Finally,it provides reference for the prevention and treatment of VD in clinic.Methods:Healthy male Sprague-Dawley rats,weighing 180-210 g.VD rat model was induced by 2-vessel Occlusion(2-VO)in vivo.In vitro mouse hippocampal neuron line(HT-22)and mouse microglial cell line(BV2)were co-cultured,and cell damage model was prepared by OGD/R.Morris water maze and new object recognition test were used to detect spatial learning and memory of 2-VO rats.The morphological changes of neurons and mitochondria in hippocampal CA1 region of 2-VO rats were observed by transmission electron microscopy.The functional changes of mitochondrial MMP and m PTP were observed by JC-1 and Calein AM probes,respectively.The expression of relevant functional proteins in the hippocampus of rats was detected by i TRAQ technique.The differential proteins related to mitochondrial dynamics were screened by bioinformatics method,and the differential expressions of related molecules were further verified by Q-PCR and Western Bolt.Meanwhile,HT-22 cells were transfected with lentivirus for intervention experiments,and the intervention mechanism of GSS on mitochondrial dynamic balance and key regulatory molecules was preliminarily clarified.Results:1.In vivo experiment:(1)GSS can improve learning and memory and new object recognition ability of 2-VO rats;(2)GSS can significantly reduce the damage of hippocampal neurons and mitochondria in 2-VO rats;(3)GSS significantly increased the expression of GPER in hippocampal neurons of 2-VO rats;(4)i TRAQ technique and bioinformatics analysis showed that the expression of MTFP1 protein in hippocampal CA1 neurons of 2-VO rats increased significantly,but decreased significantly after GSS treatment.(5)WB and Q-PCR results showed that GSS increased the expression of mitochondrial fusion signaling molecules Opa1 and Mfn1 and decreased the expression of fission signaling molecule Drp1 in hippocampal neurons of 2-VO rats.(6)GSS can increase the m RNA and/or protein expression levels of m TORC2,Rictor and decrease VDAC1 in hippocampal neurons of 2-VO rats.2.In vitro experiment:(1)The viability of cocultured cells decreased significantly after OGD/R treatment,and increased significantly after GSS treatment;(2)MMP and m PTP opening in OGD/R group were significantly decreased and increased,which were improved after GSS treatment.(3)The expression of fusion proteins Opa1 and Mfn1 in mitochondria of OGD/R cells decreased,while the expression of fission protein Drp1 increased,which was alleviated after GSS treatment.(4)The protein expression levels of GPER,m TORC2 and Rictor decreased in OGD/R group,while the protein expression levels of MTFP1 and VDAC1 increased significantly,which was reversed after GSS treatment.(5)After Rictor knockdown in HT-22 cells,the protective effect of GSS on OGD/R injury in direct co-culture cells was reversed,indicating that the regulation of GSS on mitochondrial dynamics related molecules was reversed.Conclusion:(1)GSS can improve the learning function and memory function of 2-VO rats;(2)GSS may act on hippocampal neurons through GPER;(3)GSS can effectively correct the mitochondrial dynamic imbalance of in 2-VO rats and OGD/R direct co-culture cells;(4)The Rictor-MTFP1-m PTP signal axis may be involved in the regulation of GSS on mitochondrial dynamic balance,of which Rictor is a key target. |
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