Effects Of Dendrobium Officinale Polysaccharides On The Differentiation Of Bone Marrow Mesenchymal Stem Cells Into Neural-like Cells And The Prognosis Of MCAO Model Rats

Objective:The purpose of this study was to investigate the effects of a traditional Chinese medicine-Dendrobium officinale,its main component-Dendrobium officinale polysaccharides on the differentiation of bone marrow mesenchymal stem cells(BMSCs)into neural-like cells and its effects on neural functional recovery in rats with cerebral ischemia-reperfusion(MCAO),which provide new strategies for the treatment of stroke.Methods:Experiment 1: After the fresh bulb of Dendrobium officinale was dried and pulverized,its main component-Dendrobium officinale polysaccharide(DOP)was extracted by water extraction and alcohol precipitation method,and the content of Dendrobium officinale polysaccharide was detected by phenol-sulfuric acid method.Experiment 2: The bone marrow mesenchymal stem cells was extracted and purified by the method of density gradient centrifugation.Flow cytometry(the detection markers were CD90,CD44,CD34)was used to identify the P3-P4 generation of bone marrow mesenchymal stem cells.Then the polysaccharides extracted in Experiment 1 were configured into Dendrobium officinale polysaccharide solutions of different concentrations(10,25,50,75,100 ?g / ml),and co-cultured with the P3-P4 generation of bone marrow mesenchymal stem cells for 7 days.RT-PCR(real time-polymerase chain reaction)was used to detect the expression levels of neural-related markers nestin,MAP2,and VEGF-A m RNA in each group and to screen the optimal concentration;Western blot was used to detect the expression of neural differentiation-related markers Enolase,Thy-1,?-tubullin ? at the protein level,and immunofluorescence staining technology was used to detect the expression of neuraldifferentiation-related markers CD24,Thy-1 in each group to evaluate the effect of Dendrobium officinale polysaccharides on the differentiation of bone marrow mesenchymal stem cells into neural-like cells.Experiment 3: 80 male SD rats were randomly divided into the sham operation group,the MCAO model group,the BMSCs group,and the neural-like cells group.24 hours after model was founded,the neurnal-like cells induced by Dendrobium officinale polysaccharide were injected into the focal lateral ventricle of rats in the Neural-like cells group,The BMSCs group rats were received the same amount of BMSCs,and the same amount of normal saline was injected in the MCAO group and the sham operation group.The modified neurologic severity scores(m NSS)of rats in each group were compared on day 1,4,7,and 14 after injection.On the 14 th day,the cerebral infarction volume and serum inflammatory factors(IL-1?,TNF-?,IL-6)of each group were compared by TTC staining and ELISA.Results:Experiment 1:The extraction rate of Dendrobium officinale was 17.04%,and the polysaccharide content in the extraction was 89%.Experiment 2:(1)The expression rate of CD90 on the surface of BMSCs was 96.79%,the expression rate of CD44 was98.27%,and the expression rate of CD34 was 2.64%,which confirm that the collected cells were BMSCs.(2)Nestin m RNA expression levels in different concentrations of Dendrobium officinale polysaccharides(10,25,50,75,100 ?g / ml)groups were significantly higher than those in the control groups(negative control group,positive control group).Comparing the different concentration groups with each other,the nestin m RNA expression level in the 25 ?g / ml concentration group was significantly higher than that in the 10,50,75 and 100 ?g / ml concentration groups.The expression of other two m RNAs(MAP2,VEGF-A)was consistent with the expression of nestin m RNA.All these data indicate that Dendrobium officinale polysaccharide could enhance the differentiation of BMSCs into neural-like cells,and 25 ?g / ml is the optimal concentration.Subsequent experiment were carried out at the concentration of 25?g /ml.The results of Western blot and immunofluorescence staining showed that Enolase,Thy-1,?-tubullin ? and CD24 were all gradually increased in the negative control group,positive control group and Dendrobium candidum polysaccharide group.Experiment 3: The m NSS results showed that the Neural-like cell group was significantly less than the MCAO group at 4,7,and 14 days after injection.And on the14 th day,the m NSS in the Neural-like cell group was significantly less than that in the BMSCs group.TTC staining of brain tissue showed that the volume of cerebral infarction was significantly increased after the MCAO model was established;After target injection,the volume of cerebral infarction in the BMSCs and Neural-like cell groups were significantly lower than that in the MCAO model group,and the Neural-like cell group was lower than that in the BMSCs group.ELISA results showed that the expression levels of serum inflammatory factors(TNF-?,IL-1?,IL-6)were increased after the MCAO model was established.After injection,the expressions of the three inflammatory factors in the BMSCs group and the Neural-like cell group were significantly lower than those in the MCAO model group,and the levels of three inflammatory factors in the Neural-like cell group were significantly lower than those in the BMSCs group.Conclusions: 1.Dendrobium officinale polysaccharide could promote the neural differentiation of bone mesenchymal stem cells into neural-like cells.2.Neural-like cells induced by Dendrobium officinale polysaccharides could promote the neural functional recovery of rats with cerebral ischemia-reperfusion injury,and the mechanism may related to reducing the expression of inflammatory factors.