The Study On Protective Effect And Mechanism Of Combination Therapy With Ginsenoside Rg1 And Bone Marrow Mesenchymal Stem Cells On Dementia Model Rats

Alzheimer's disease (AD) is an age-related neuro-degenerative disease, characterized by progressive disorder with cognitive and memory decline. Many approaches have been undertaken to understand AD, but the heterogeneity of the etiologic factors makes it difficult to define the most important factor determining the onset and progression of the disease. Until now, AD is considered to be multi-etiopathogenisis disease. It is difficult to find a good way for cure of AD. But the change of cholinergic nerve system is one of the important pathology. How to retrieve the decline of cholinergic nerve system and promote the recovery of disease, which has become the hot spot of AD research.BMSCs is a kind of ideal seeding cells, because of being easily obtained, poor immunogenicity, dismissing any ethical concern, phenotypic plasticity (Adopting neural phenotypes under a certain circumstances) and so on. Cell therapies with BMSCs are promising approaches for the treatment of degenerative neurological diseases such as AD, for breaking the traditional ideas that adult stem cell can't different by spaning blastodermic layer. But the research of grafting BMSCs on AD treament is still in the primary stage, doubt has being emerged that whether the same result will be happened in vivo as in vitro, considering the effects that low rate of survival and differentiation of bone marrow BMSCs in vivo, although, BMSCs can survive and express neural differentiation well in vitro in many research before. In order to raise rate of survival and differentiation, strengthen the therapeutic efficacy. We use ginsenoside Rg1, which can induce BMSCs into neuron-like cells in vitro to combine mith BMSCs transplantion on AD model rats treatment, to observe effects on Central cholinergic nerve system by col-contribution and simple contribution. In addition to the change level of NGF and TrkA, we observed the survival and differentiation of BMSCs, to investigate mechanisms of col-contribution in AD. By which we hope to explore a more effective method for AD treatment.1. Isolating, culturing, and inducing diffentiation of rat bone marrow BMSCs.Pure BMSCs were acquired by this way: isolated by wall sticking method, then cultured and amplified in condition of L-DMEM with 20% fetal calf serum , 37℃,5%CO2, saturated humidity, passaged with 0.25% trypsinization in vitro. The third generation BMSCs were induced to differentiate into neuron-like cells with ginsenoside Rg1, then differentiated cells were identified by immunohistochemistry of NSE and GFAP after 72 hours, 10μmol/L, 20μmol/L final concentration groups have higher expression rates of NSE than others, but there was no significant difference between them. Positive expression rates of GFAP was low, and there was no significant difference between each group. The results suggested that, After being isolated, cultured and passaged, BMSCs could be induced by ginsenoside Rg1 to express NSE mostly but GFAP a few.2.The establishment of the dementia model rats and the effect of combining treament on ethology and histological anatomy of rats2.1 Transecting hippocampal fimbria-fornix of rat to set up AD model ratsMale Wistar rats were divided into five groups randomly(each 15 rats): sham operation control group (SOC group), ambi-hippocampal fimbria-fornix transected model group(FF lesioned group).simple ginsenoside Rg1 treatment group(ginsenoside Rg1 group), simple BMSCs treatment group(BMSCs group),and ginsenoside Rg1 combining BMSCs treatment group (combining treatment group).Rats were anesthetized by an peritoneal infusion of 10% chloral hydrate (3~3.5ml/kg) and then put on the stereotaxis instrument, to be transected in ambihippocampal fimbria-fornix,and SOC group received the same process but ambihippocampal fimbria-fornix not cut off .After 2 weeks , ginsenoside Rg1 group and combining group received peritoneal infusion of ginsenoside Rg1 (5mg/kg),one day a time, untill a month. Rats in the other groups received peritoneal infusion of saline isodose. BMSCs group and combining group received transplantation of BMSCs (10μl , 1×106cells) into hippocampus in both sides of rats by Hamilton microinjector with stereotaxis. Rats in the other groups received infusion of PBS (0.01 M, PH 7.4). After 1 month, they received experiment below.2.2 Determination of learning and memory ability of ratsLearning and memory ability of each group rats were determined by Morris water maze and the escape latency were recorded. The shorter of escape latency, the better of learning and memory ability they had. Experimental results showed that, having been compared the escape latency of FF transected group with SOC group revealed significant difference, the former is longer than the latter. The result suggests that operation resulted in the damage of central cholinergic system and caused the decrease of learning and memory ability of rats. Every treatment group compared with FF transected group, showing escape latency was diminished, with significant difference, which indicates that the learning and memory ability of rats increased significantly after being treated with ginsenoside Rg1 or BMSCs or the both. The combining group compared with Rg1 or BMSCs treatment group, showing the escape latency decreased with significant difference, which demonstrated that the combining treament was better than the simple treament.2.3 HE staining and electron microscope results in hippocampus of ratsHE staining of SOC group showed that hippocampal pyramidal cell lined up in order, uniformity, and cellular structure was integrity with normal morphous; Electron microscope showed that cellular membrane was integrity, neuron nucleole was clear, chromatin distribution was uniformity, much rough endoplasmic reticulum and few lipofuscin was in cytoplasm, neuropil was normal. HE staining of FF transected group showed that hippocampal pyramidal cell was lost and derangement with unclear construction, Some nucleus were pyknosis and anachromasisand ; Electron microscope showed that most of neurons were intumesce with cavitation, and neuropil was the same, chondriosome were intumesce were accumulation in cytoplasm. HE staining of every treatment group showed that the number of cellular layer in hippocampal was more than FF transected group, cells lined up nearly in order, and cellular structure was almost clear. But morphous of combining group was approach to SOC group. The experimental results confirmed that transecting hippocampal fimbria-fornix could induce the damage of neuron in rat hippocampal and result in the learning and memory ability of rats decreased. Simple ginsenoside Rg1 treatment, simple BMSCs treatment and ginsenoside Rg1 combining BMSCs treatment all have protective effects on hippocampal pyramidal cell of FF transected rats, but more effective in combining treatment group.3.The changes of central cholinergic nerve system in FF transected rats and the effect of treatment3.1 The effect of combining treatment on ChAT positive cholinergic fibers in dementia model rats hippocampusBasic on choline energy hypothesis of cognitive handicap in AD, this experiment The expression of ChAT in rats hippocampal was measured with immunohistochemistry and image-analysis, to develop effect of treament on dementia model rats, results showed:ChAT positive cholinergic fibers could be found in rats hippocampus of each group; The IOD value of ChAT positive cholinergic fibers in hippocampus of FF transected group was lower than those of SOC group obviously. IOD of ChAT in each treatment group was higher than those of FF transected group, difference was significant. And also the same difference was found when comparing combining treatment group with simple group. The results suggested that, simple ginsenoside Rg1 treatment, simple BMSCs treatment and ginsenoside Rg1 combining BMSCs treatment could elevate level of ChAT activity in hippocampus of FF transected rats, result in increasing the function of cholinergic CNS and improving memory decline, combining treatment was more obviously than BMSCs treatment.3.2 The changes of basal forebrain TrkA positive cholinergic neuron in FF transected rats and the effect on it of treatmentThe TrkA positive cholinergic neuron s in basal forebrain of FF transected rats were observed by using immunohistochemistry staining. The result showed: the number of TrkA positive cholinergic neurons in basal forebrain of FF transected group was lessen than those in SOC group, difference was significant. Those of each treatment group was more than those of FF transected group, difference was significant. And those of combining treatment group was more than those of simple treatment group, difference was significant. The results suggested that FF lesion could evoke the lost of TrkA positive cholinergic neurons in basal forebrain of rats, which resulted in decline of learning and memory ability in rats. Simple ginsenoside Rg1 treatment, simple BMSCs treatment and ginsenoside Rg1 combining BMSCs treatment could decrease the loss of cholinergic neurons in basal forebrain of rats, then improved cognition function, but combining treatment was superior to BMSCs treatment.3.3 The changes of NGFmRNA expression in basal forebrain of rats and the effect of combining treatmentTo explore the protective mechanism of combining treatment on center cholinergic nerve system , RT–PCR technique was used to detect the change of NGFmRNA expression in basal forebrain of FF transected rats and the effect of each treament on that. The result showed: The level of NGFmRNA expression in basal forebrain of FF transected rats was decrease apparently, difference was significant comparing to SOC group. The level of NGF mRNA expression raised in each treament group than FF transected group, difference was significant. The result suggested that: After being transected in FF, NGF mRNA expression in basal forebrain of rats diminished, Which resulted in cholinergic system cannot obtaining enough nutrition and happening to be cataplasis, to induce ChAT activity to descend, then composition and release of Ach to decrease, leading to learn and memory ability declined. Simple ginsenoside Rg1 treatment, simple BMSCs treatment and ginsenoside Rg1 combining BMSCs treatment could raise learn and memory ability by up-regulating NGF mRNA expression in basal forebrain of FF transected rats. NGF mRNA expression in basal forebrain in combining treatment group increased obviously than in simple treatment group, with significant difference, It suggested that ginsenoside Rg1 combining with BMSCs have synergia effect on up-regulating NGF mRNA expression in basal forebrain to play the role of trophism in center cholinergic nerve system.4 To investigate mechanism and effect of survival and differentiation of ginsenoside Rg1 on implanted BMSCs4.1 status of survival and differentiation of implanted BMSCs Survival and migration was observed by using immunohistochemistry staining. Result showed that: BrdU positive cells were alive in both combining treatment group and simple BMSCs treatment group in rats brain, distributing along pin hole of hippocampus region, A few of BrdU positive cells migrate to stratum radiatum as well. The number of BrdU positive cells in hippocampus of combining treatment group was more than those of simple BMSCs treatment group, with significant difference. It suggested that ginsenoside Rg1 could change the environment in FF transected rats brain, which is benefit to the Survival of implanted BMSCs. Differentiation of cells was observed by using immunohistochemistry double staining. Result showed that: Few BrdU and NSE double positive cells in hippocampus. We can not draw a conclusion that ginsenoside Rg1 promote the differentiation of implanted BMSCs.4.2 To detect the change of NGF mRNA expression in hippocampus by RT –PCR techniqueTo investigate ginsenoside Rg1 changed environment in brain and increased the number of implanted BMSCs by which means, we used RT–PCR technique to detect the change of NGF mRNA expression in hippocampus of combining treatment group and BMSCs treatment group, Results showed that: level of NGFmRNA expression in hippocampus of combining treatment group was higher than that of BMSCs treatment group, difference was significant. It suggested that ginsenoside Rg1 might facilitate the survival of implanted BMSCs by up-regulating content of NGFmRNA in hippocampus.In conclusion, ginsenoside Rg1 combining BMSCs can protect central cholinergic nerve system by enhancing NGF and TrkA there, to play the trophic action on central cholinergic nerve system, to delay degeneration and death of cells there and improve cognition founction. Rg1 might facilitate the survival of implanted BMSCs in FF transected rats. We hypothesize that ginsenoside Rg1 combining BMSCs would be used to improve cognition founction of AD patient.