The Study Of Correlation Between Nrf2/ARE Upregulation Of Polydatin In Promoting Neural Differentiation And BMSC Transplantation For Spinal Cord Injury

Objective1.To isolate and culture mouse bone-marrow mesenchymal stem cells(BMSCs),and to investigate the effect of polydatin on the differentiation potential of BMSCs into neuron-like cells,and to screen out the working concentration of polydatin(PD)in vitro to lay a foundation for further experiments.2.A mouse spinal cord injury(SCI)model was established to investigate the effect of PD on neural differentiation of transplanted BMSCs in vivo by grouping intervention.3.The regulatory mechanism of the PD-Nrf2/ARE axis in neural differentiation of BMSCs was investigated using the neural differentiation system of BMSCs and contusive SCI animal model.4.The therapeutic effects of PD gavage,BMSC transplantation,and the combination of PD gavage and BMSC transplantation in SCI animal model were compared through behavioral assessment,neurological functional recovery,and histopathological examination.Methods1.Effects of PD on neural differentiation of BMSCs in vitro(1)Isolation,culture,passage and identification of BMSCsPrimary BMSCs were obtained from humeri,femurs and tibiae of C57BL/6 mice,and cultured and passaged.Osteogenic and chondrogenic differentiation potential was detected by their specific staining.The surface markers of BMSCs were detected by flow cytometry.(2)Effects of PD on the activity and neural differentiation potential of BMSCsThe Cell Counting Kit-8(CCK-8)method was used to detect the effects of PD at different concentrations on BMSCs activity.The working concentration of PD was screened out for further neural differentiation experiments,and the morphological changes of BMSCs were dynamically observed under a microscope.(3)Effects of PD on the expression of neural markers in neurally differentiated BMSCs?Western blot(WB),RT-qPCR analysis and immunofluorescence(IF)staining were used to detect the expression and distribution of MAP-2,NeuN,NF-M and NSE in different groups.?The protein levels of DBH,ChAT,GAD65 and TH in the control(CT)group and the 30?mol/1 PD group were detected by WB.2.Effects of PD on neural differentiation of transplanted BMSCs in vivo(1)Labeling of BMSCsThe frozen BMSCs were resuscitated,and 10?mol/1 BrdU was added to the culture medium for 48 hours when the cell confluence reached 70%.The cell labeling rate was detected by IF assay.(2)Animal grouping,modeling,intervention and sampling?125 male C57BL/6 mice aged 8 weeks were randomly divided into 5 groups(N=25 each):sham-operated group(Sham),model group(SCI),polydatin-treated group(PD),BMSC transplantation group(BMSCs),and polydatin combined with BMSC transplantation group(PD+BMSCs).?The contusive SCI model at T10 segment was established and treated according to grouping.?Fresh spinal cord tissues containing the compression epicenter were extracted at 7,10 and 28 days post-injury(dpi)and stored at-80? for subsequent experiments.For IF assays,spinal cords was removed at 28 dpi,and fixed for 24 hours,then sent for frozen sections following gradient dehydration.(3)Spinal cord tissue detection?IF staining was used to detect BrdU/Nestin,BrdU/NeuN and BrdU/NSE double-positive transplanted cells in the injured spinal cords.WB was used to assess the protein expression of Nestin,NeuN and NSE in spinal cord tissues at 28 dpi.?The protein expression of iNOS and NF-?B and the contents of I1-6,I1-1? and TNF-? of tissues were detected by WB and ELISA at 7 dpi.?The contents of SOD and MDA in the injured spinal cords were detected at 7 and 10 dpi.3.The regulatory mechanism of the PD-Nrf2/ARE axis in neural differentiation of BMSCs(1)Cell experiments?Cell grouping and interventionExperiment 1(5 groups):CT group,positive group(NIM),and 3,10 and 30?mol/1 PD groups.Experiment 2(5 groups):CT group,NIM group,inhibitor group(Bru),3?mol/1 PD group,and PD+Bru group.?To explore the potential regulatory pathways of PD-promoted neuronal differentiation of BMSCs in vitroThe protein expression of PI3K-AKT,MAPK and Nrf2/ARE signaling pathways of cells in the experiment 1 was detected by WB.And the mRNA expression of the Notch,Wnt,BMP/Smad and Nrf2/ARE signaling pathways-related genes were detected by RT-qPCR.?To investigate the effect of PD-Nrf2/ARE axis in neural differentiation of BMSCs in vitroIF staining,WB,and RT-qPCR analysis were used to detect the expression and distribution of MAP-2,NeuN,NF-M and NSE of different groups in the experiment 2.(2)Animal experiments?Animal grouping and interventionExperiment 1(5 groups,N=7 each):as mentioned above.Experiment 2(4 groups,N=7 each):Sham group,SCI group,PD+BMSCs group,PD+BMSCs+Bru group.After modeling,intervention was conducted according to grouping,and samples were collected at 28 dpi.?To explore the effects of different treatments on the expression of Nrf2/ARE pathway in vivoIF assay was used to detect the expression of Nrf2 in the injured spinal cords of each group in the experiment 1,and WB was used to assess the protein expression of Nrf2/ARE pathway.?To investigate the effect of PD-Nrf2/ARE axis in neural differentiation of BMSCs in vivoIF staining was used to detect BrdU/Nestin,BrdU/NeuN and BrdU/NSE double-positive transplanted cells in the injured spinal cords of PD+BMSCs group and PD+BMSCs+Bru group.WB was used to assess the protein expression of Nest in,NeuN and NSE in the experiment 2.4.Effects of PD gavage combined with BMSC transplantation in SCI(1)Animal grouping and interventionAnimal grouping,modeling,and intervention were carried out according to the section 2,and samples were collected at 28 dpi.(2)Behavioral assessmentThe recovery of hindlimb motor function was evaluated using the BBB locomotor rating scale and inclined plane test at different time points post-injury.(3)Nerve electrophysiological testSpinal cord evoked potentials(SCEP)were recorded at 28 dpi,and the changes in the amplitudes and latencies of SCEP was measured.(4)Histopathological examination?The lesion area of spinal cord surface was quantified and the damage degree of spinal cords was assessed by HE staining and Nissl staining.?IF staining,WB,and RT-qPCR analysis were used to detect the expression and distribution of MAP-2.The ultrastructural changes of spinal axons and myelin sheaths were observed by transmission electron microscope(TEM).?The IF double labeling staining for GAP43 and Tujl was carried to assess the axonal regeneration in spinal cords.?IF staining was used to observe the formation of glial scar in the injured spinal cords,and GFAP protein expression was detected by WB.The IF double labeling staining for GFAP and NF-200 was carried to observe the relationship between glial scar and nerve filaments.Results1.Effects of PD on neural differentiation of BMSCs in vitroThe cultured BMSCs have multi-directional differentiation potency and high purity.BMSCs incubated with PD at concentrations of 3,10,and 30 ?mol/1 showed better cell viability,so this concentration range was chosen for further experiments.It was found that the number of neural-like cells in the PD group was significantly higher than that in the NIM group at the middle and later stages of induction.Compared with the NIM group,PD upregulated the protein and mRNA expression of MAP-2,NeuN,NF-M,and NSE in a concentration-dependent manner in vitro(P<0.05).Moreover,PD also increased the fluorescence intensity of NF-M and ChAT,and the number of MAP-2-and NeuN-positive cells(P<0.05),which was most significant in the 30?mol/l PD group(P<0.05).2.Effects of PD on neural differentiation of transplanted BMSCs in vivoThe SCI animal model showed high stability and reproducibility.After 4 weeks of modeling,Brdu-labeled exogenous BMSCs could be detected in vivo,and some cells expressed Nestin,NeuN or NSE simultaneously.Compared with the BMSCs group,more BrdU/neural marker positive cells were detected in the injured spinal cords of the PD+BMSCs group(P<0.05).The results of WB showed that the expression of Nestin,NeuN and NSE could be up-regulated by different treatment methods,but the expression level of PD+BMSCs group was higher(P<0.05).There was no statistical difference between all treatment groups in the expression of inflammation-related factors and the contents of oxidative stress indicators(P>0.05).3.The regulatory mechanism of the PD-Nrf2/ARE axis in neural differentiation of BMSCs(1)Cell experimentsWhen compared with the NIM group,the protein expression of the PI3K-AKT and MAPK signaling pathways-related biomarkers and the mRNA expression of the Notch,Wnt and BMP/Smad signaling pathways-related genes in the three PD groups were not significantly different(P>0.05).However,the protein and mRNA expressions of Nrf2,NQO-1 and HO-1 in the PD groups were significantly up-regulated in a concentration-dependent manner than those in the NIM group(P<0.05).After blocking the Nrf2/ARE signaling pathway with brusatol,the effect of PD on up-regulating the protein and mRNA expression of neural markers in cells was suppressed,and the expression level was significantly lower than that of the PD group(P<0.05),which was similar to the trend of IF results.(2)Animal experimentsCompared with the SCI group,the fluorescence intensity of Nrf2 and the protein expression of Nrf2/ARE signaling pathway-related biomarkers in each treatment group were significantly increased(P<0.05),and the expression level in the PD+BMSCs group was higher than that of the other treatment groups(P<0.05).After blocking the Nrf2/ARE signaling pathway,compared with the PD+BMSCs group,the efficiency of neural differentiation of transplanted BMSCs in the PD+BMSCs+Bru group was dramatically decreased(P<0.05),and the protein expression of Nestin,NeuN and NSE were also significantly down-regulated(P<0.05).4.Effects of PD gavage combined with BMSC transplantation in SCIWithin 14 to 28 dpi,compared with other treatment groups,the BBB score of the PD+BMSCs group significantly recovered(P<M.05),and the critical angle of the inclined plane test was higher(P<0.05).Results of nerve electrophysiological test showed that SCEP of mice recovered significantly after treatment(P<0.05).And the combination of PD gavage and BMSC transplantation significantly increased the SCEP amplitudes and shortened the SCEP latencies compared to that in the mice under monotherapy(P<0.05).In the histopathological examination,the lesion area of spinal cord surface in the PD+BMSCs group was significantly lower than that in the other treatment groups and SCI group(P<0.05).Besides,the results of HE staining and Nissl staining also indicated that the degree of SCI-induced histopathological damage and neuronal damage was attenuated more obviously compared to the other treatment groups(P<.05).When compared with the other mice subjected to contusive SCI,the levels of indicators associated with sheaths myelin repair and axonal regeneration in the PD+BMSCs group increased(P<0.05),while the levels of glial scar formation-related indicators decreased(P<0.05).Conclusion1.On the basis of neuronal induction medium,PD could promote the differentiation of BMSCs into(cholinergic)neuron-like cells in vitro in a concentration-dependent manner,which is most significant at a concentration of 30 ?xmol/1.Besides,PD could also promote the differentiation of transplanted BMSCs into neuron-like cells in vivo.All of the above effects might be achieved through the activation of the Nrf2/ARE signaling pathway.2.Combination therapy with PD gavage and BMSC transplantation can effectively improve hindlimb locomotor performance of SCI mice and promote the recovery of spinal nerve function,which may be based on its important role in helping the sheaths myelin repair in the injured spinal cords,accelerating axonal regeneration,and weakening the blocking effect of glial scar on nerve filaments growth.