| AIM:In this study,we observed the effect of berberine on the secretion of BMSCs in vitro,and explored the protective effect,anti-apoptotic and anti-oxidant effects of the secretion of BMSCs pretreated by berberine on neurons.And further clarify its mechanism,so as to explore a new effective treatment method and way for the prevention and treatment of AD.METHODS:(1)Primary BMSCs from C57BL/6 mice were cultured in vitro and identified by flow cytometry,osteogenesis and adipogenic differentiation.(2)CCK-8 method was used to determine the optimal concentration of BBR without damaging BMSCs.(3)The conditioned medium of P4 BMSCs serum-free D/F12 cultured at different time points(6 h,12 h,24 h,48 h)was collected.The ELISA method was used to determine the highest concentration of NGF in secretion at different time points to select the optimal collection time.(4)After BBR was applied to BMSCs for 24 h,the culture medium which was continuously cultured for 12 h with D/F12 was collected and divided into D/F group,BMSC-CM group and BBR-BMSC-CM group.The ELISA method was used to detect the three groups' NGF and BDNF neurotrophic factors.The secretion was collected and cultured.(5)Primary mouse cerebral cortical neurons were cultured on six-well plates for 7 days,and identified on day 7 by immunofluorescence(MAP2).(6)The experimental concentrations of t-BHP(0 ?M,2.5 ?M,5 ?M,10 ?M,20 ?M,and 40 ?M),the optimal volume percentage of BBR-BMSC-CM to improve t-BHP-induced neuronal damage and the effect of BMSC-CM and BBR-BMSC-CM on protecting oxidative were selected by MTT method.(7)To determine the neuroprotective effect of secretion against t-BHP–induced cell death,damaged neurons,The experiment is divided into 5 groups: Control group(neuron+Neurobasal),t-BHP group(neuron+t-BHP+Neurobasal),D/F group(neuron +D/F12+t-BHP+Neurobasal),BMSC-CM group(neuron+BMSC-CM+t-BHP+Neurobasal)and BBR-BMSC-CM group(neuron+BBR-BMSC-CM+t-BHP+Neurobasal).MTT method was used to detect the activity of neurons in different groups after treatment in different groups.(8)ROS production in neurons was measured with the DCFH-DA assay kit according to the manufacturer's instructions.(9)The changes in MMP were measured with an MMP assay kit with JC-1 according to the manufacturer's instructions.(10)The changes apoptosis of neurons were measured with an Annexin V-FITC and PI assay kit according to the manufacturer's instructions.(11)Western blots were used to detect expression of apoptotic proteins(Bax/Bcl-2,Cytochrome c,Cleaved-Caspase 3/Caspase 3),synaptic function(SYP,PSD 95),and antioxidant proteins(Keap1,Nrf2,HO-1)in each group.RESULTS:(1)Flow cytometry detection of P4 BMSCs was performed in a cellular immunophenotype.The results showed that Sca-1,CD 105,CD 29 were expressed,and CD 45 was not expressed.After osteogenic and adipogenic induction,alizarin red and oil red "O" staining were performed respectively,and a large number of calcified nodules and lipid droplets were formed under the microscope.(2)When BBR is 10 ?M,the cell viability of BMSCs is the best,so 10 ?M is the optimal concentration.(3)The NGF and BDNF contents in the culture medium collected at 12 h were the highest.(4)BBR-BMSC-CM contained more NGF and BDNF than conditioned medium from BMSCs(BMSC-CM)after the cells were incubated with BBR for 24 h.(5)Mouse primary neocortical neurons labeled with MAP2 green fluorescence were observed under fluorescence microscope.(6)MTT assay showed that when the t-BHP was 10 ?M,the degree of neuronal damage reached to 50%,and 10% BBR-BMSC-CM increased the viability of surviving neurons to 71% under oxidative stress.(7)The activity of neurons in the t-BHP group significantly decreased.Compared with the BMSC-CM group,the neuron cell viability increased in the BBR-BMSC-CM group.(8)The ROS fluorescence level of the t-BHP group was significantly increased.The green fluorescence intensity of the BMSC-CM and BBR-BMSC-CM group was significantly lower than the t-BHP group,and the ROS fluorescence level of the BBR-BMSC-CM group was lower than that of the BMSC-CM group.(9)Oxidative damage reduced the mitochondrial membrane potential in the neurons of the t-BHP group.Compared with the t-BHP group,the mitochondrial membrane potential in the BMSC-CM group and the BBR-BMSC-CM group increased,and the membrane potential of the BBR-BMSC-CM group was higher than the BMSC-CM group.(10)T-BHP caused neuronal apoptosis.Compared with the t-BHP group,the number of neuronal apoptosis in the BBR-BMSC-CM group was significantly reduced,while the decrease in the neuronal apoptosis in the BMSC-CM group was not obvious.(11)Western blot results showed that compared with t-BHP group,common apoptotic proteins Bax / Bcl-2,Cytochrome c,and Cleaved-Caspase 3 / Caspase 3 were down-regulated in both BMSC-CM group and BBR-BMSC-CM group.BBR-BMSC-CM significantly increased SYP and PSD 95.Moreover,BBR-BMSC-CM down regulated Keap1,Cytosolic Nrf2 levels and up-regulated Nuclear Nrf2,HO-1 proteins.SUMMARY:(1)Berberine(BBR)can promote BMSCs secrete NGF and BDNF neurotrophic factors;(2)Compared with the BMSC-CM,the BBR-BMSC-CM group can significantly reverse injury induced by t-BHP;(3)The BBR-BMSC-CM can reduce ROS production,reverse MMP in neurons,and attenuate apoptosis caused by oxidative damage to neurons;(4)The BBR-BMSC-CM can down-regulate Bax/Bcl-2,Cytochrome c,Cleaved-Caspase3/Caspase 3 apoptotic proteins to alleviate neuronal apoptosis;(5)The anti-apoptotic effect of BBR-BMSC-CM restored the synaptic protein of SYP and PSD95 and saved the synaptic function of neurons;(6)The mechanism of BBR-BMSC-CM against oxidative damage of neurons may be through activation of the Keap1-Nrf2-HO-1 signaling pathway.CONCLUSION:The experimental results confirmed that berberine can significantly promote the secretion of neurotrophic factors NGF and BDNF by BMSCs.Moreover,the BMSCs secretion pretreated with berberine can more effectively restore the vitality of oxidative damaged neurons,improve mitochondrial dysfunction,and reduce neuronal apoptosis and oxidative damage.The experimental results further indicate that this effect against oxidative damage may be achieved by Keap1-Nrf2-HO-1 anti-oxidation signaling pathway inhibiting the generation of ROS. |
PDF Full Text Request