Mechanisms Of Melanin Nanoparticles Enhanced The Protection Of Mesenchymal Stem Cells On Ischemic Stroke Model In Vitro

Background and objectiveIschemic stroke is one of the leading causes of disability even death.Ischaemic hypoxia and reperfusion injury,especially the ensuing intense oxidative stress（OS）response,is an important pathogenesis of neuronal death and neurological deficits in ischaemic stroke.However,at present,the methods that can effectively treat ischemic stroke in clinic are limited,and affected by time window,which are restricted from a wide range of applications.A number of preclinical studies have shown that transplantation of mesenchymal stem cells（MSCs）can lead to improved neurological function and reduced neuronal death in ischaemic stroke models in vivo or in vitro,regardless of the treatment time window.However,the poor microenvironment in the host leads to a low survival rate after MSCs transplantation,which affects the therapeutic efficacy of MSCs.Therefore,it is particularly important to explore new ways to improve the survival rate of MSCs after transplantation,and to increase the efficiency of stem cell therapy for ischaemic stroke.Polydopamine nanoparticles are produced by oxidising dopamine hydrochloride under alkaline conditions and polymerising it through a series of reactions.They are also known as melanin nanoparticles（MNPs）because their physicochemical properties are similar to those of true melanin.It has been shown that MNPs have potent antioxidant and anti-inflammatory activities,scavenging reactive oxygen species（ROS）from OS-injured cells and significantly reducing the size of brain infarcts in ischaemic stroke models.Thus,we propose the hypothesis that MNPs may improve the survival rate of transplanted stem cells in an ischemic-hypoxic environment and enhance the neuroprotective effects of stem cells.Therefore,in this study,the oxygen-glucose deprivation（OGD）model of primary cortical neuronal and the hydrogen peroxide（H₂O₂）/serum deprivation（SD）model of MSCs were established to investigate whether MNPs could enhance the neuroprotective effects of rat bone marrow MSCs on neurons damaged by OGD and the underlying mechanism,so as to laid the foundation for the clinical application of MNPs combined with MSCs transplantation in the treatment of ischemic stroke.Method1.Construction of melanin nanoparticle pretreated rat bone marrow MSCs.（1）MNPs were synthesised with dopamine hydrochloride（DA）solution and Na OH after a series of reactions,and then the MNPs were characterised and identified.（2）To assay the free radical scavenging ability of MNPs.（3）Isolation,culture and purification of rat bone marrow MSCs in vitro,for morphological observation and detection of cell surface markers.（4）The cellular activity of MSCs after pretreatment with different concentrations of MNPs for different times were measured by CCK-8 assay.（5）Morphological observation of MSCs pretreated with MNPs by light microscopy and electron microscopy.2.To investigate whether pretreatment with MNPs can enhance the neuroprotective effect of MSCs and the underlying mechanism.（1）Experimental grouping and construction of an in vitro model of ischaemic stroke treated with bone marrow MSCs transplantation.Primary cortical neurons cultured in vitro were identified.Cortical neurons on day 7 of culture were randomly divided into normal control group,ischemic stroke model（OGD-vehicle）group,MNPs treated（OGD-MNPs）group,stem cells treated（OGD-MSCs）group,MNPs pretreated stem cells treated（OGD-MNPs-MSCs）group,ROS inhibitor（OGD-ROS Inhibitor）group,poly（adenosine diphosphate（ADP）-ribose）polymerase 1（PARP-1）-dependent cell death（parthanatos）inhibitor（OGD-DPQ）group.Except for the normal control group,an in vitro model of cerebral ischaemic injury was established by the method of OGD-injury.The OGD-MSCs group and the OGD-MNPs-MSCs group applied a culture plate with a polycarbonate membrane with 0.4μm pores as a septum to establish a cross-compartmental co-culture system to establish an in vitro model of bone marrow MSCs transplantation for ischemic stroke by co-culture of bone marrow MSCs with OGD-injured neurons for 48 hours.（2）Propidium Iodide（PI）/Hoechst 33342staining method to determine neuronal mortality.（3）The ROS/Superoxide kit measures the fluorescence intensity of intracellular ROS and superoxide respectively.Biochemical assay kits are used to measure cellular superoxide dismutase（SOD）and catalase（CAT）activity.（4）Assay of apoptosis-related indicators:cleaved caspase-3 in various groups of neuronal cells was detected by immunofluorescence staining;relative expression of cleaved caspase-3,Bcl-2 and Bax were measured by western blot（WB）.（5）Assay of parthanatos-related markers:apoptosis-inducing factor（AIF）and Iduna were detected by immunofluorescence staining;relative expression of AIF,Iduna and PARP-1 were measured by WB.3.To investigate whether pretreatment with MNPs can promote the survival of MSCs in OS and SD conditions.Rats bone marrow MSCs with good growth in the 3rd-5th generation were randomly divided into normal control group,H₂O₂/SD injury model（H₂O₂）group,MNPs pretreatment（MNPs-H₂O₂）group,MNPs culture（MNPs）group,ROS inhibitor（H₂O₂-ROS Inhibitor）group and apoptosis inhibitor（H₂O₂-Z-VAD-FMK）group,according to the specific experiment after 24-48 hours of affixed culture.In vitro models of oxidative stress and ischemia after MSCs transplantation were established by applying H₂O₂damage and SD,except for the normal control group and the MNPs culture（MNPs）group.（1）MSCs activity after treatment with serum-free medium at different H₂O₂ concentrations were detected by CCK-8 assay.（2）Mortality of MSCs was determined by PI/Hoechst 33342 staining.（3）The ROS/Superoxide Assay Kit detects the fluorescence intensity of intracellular ROS and superoxide,respectively.（4）The expression of apoptosis marker cleaved caspase-3 in each group of MSCs was measured by immunofluorescence staining,and the relative expression of apoptosis-related proteins cleaved caspase-3,Bcl-2 and Bax was detected by WB.Result1.Characterization of MNPs and construction of MSCs pretreated with MNPs.（1）The synthesised MNPs had an average diameter of 115.13±8.02 nm,an average hydrated particle size of 106.5 nm and a zeta potential of-21.3 m V.The FTIR spectra of the MNPs showed a broad peak in the range of 3700-2800cm-1 with peaks observed at 1621 cm-1,1401 cm-1 and 1141 cm-1.Compared to dopamine monomer,the synthesised MNPs exhibit a wide range of absorption from UV to NIR wavelengths.The Raman spectra of the MNPs show characteristic peaks at 1594 cm-1 and 1360 cm-1.（2）The DPPH radical scavenging rate of MNPs increased from 2.04%to 38.58%as the concentration of MNPs increased（5-200μg/m L）.（3）Bone marrow MSCs cultured in vitro were identified by flow cytometry as cells positive for CD29,CD44 and CD90expression,and negative for CD34 and CD45 expression.（4）MSCs co-incubated with MNPs at concentrations of 10,20 and 40μg/m L showed significantly higher cell viability than the control（P<0.05）,and a significant decrease in stem cell viability was first observed at a MNPs concentration of 200μg/m L（P<0.05）.After 48 and 72 hours of co-incubation,cell viability began to decrease significantly at MNPs concentrations of 10μg/m L and 20μg/m L,significantly lower than that after 24 hours（P<0.05）.（5）MSCs were observed by inverted phase contrast microscopy after co-incubation with MNPs at a concentration of 120μg/m L for 24 h.The morphology of MSCs did not change and remained shuttle-shaped,with abundant cytoplasm and large nuclei.When MSCs were observed by TEM,the MNPs were dispersed in the cytoplasm and vesicles in a single or clustered form after uptake into the cells,and no abnormal changes in the ultrastructure were observed.2.Neuronal mortality,OS response,apoptosis and parthanatos-related indicators in each group.（1）Rat primary cortical neurons in vitro cultured were identified as cells positive for MAP2 labelling>95%.（2）Compared with the OGD-vehicle group,neuronal mortality was reduced by 79.32%,59.97%and 91.24%in the OGD-MSCs,OGD-MNPs and OGD-MNPs-MSCs groups,respectively.Neuronal mortality in the OGD-MNPs-MSCs group was significantly less than that in the OGD-MSCs or OGD-MNPs groups（n=6,P<0.01）.（3）Antioxidant-related indexes:ROS:compared with the OGD-vehicle group,the mean fluorescence intensity of neuronal ROS was reduced by 45.13%,24.72%and61.80%in the OGD-MSCs,OGD-MNPs and OGD-MNPs-MSCs groups,respectively and the effect of MNPs-MSCs co-culture in reducing intracellular ROS was better that of MSCs or MNPs（n=6,P<0.01）.Superoxide:compared to the OGD-vehicle group,the mean fluorescence intensity of neuronal superoxide was reduced by 46.63%,32.37%and 52.26%in the OGD-MSCs,OGD-MNPs and OGD-MNPs-MSCs groups,respectively,and the effect of MNPs-MSCs co-culture in reducing intracellular superoxide was better than that of MSCs（n=6,P<0.05）or MNPs（n=6,P<0.01）.SOD:compared to the OGD-vehicle group,neuronal SOD activity was significantly higher in the OGD-MNPs-MSCs,OGD-MSCs and OGD-MNPs groups（n=3,P<0.05）.SOD activity was higher in the OGD-MNPs-MSCs group than in the OGD-MSCs,OGD-MNPs and normal control groups（n=3,P<0.01）.CAT:Neuronal CAT activity was significantly higher in the OGD-MNPs-MSCs,OGD-MSCs and OGD-MNPs groups compared to the normal control and OGD-vehicle groups（n=3,P<0.01）.CAT activity was higher in the OGD-MNPs-MSCs group than in the OGD-MNPs group（n=3,P<0.01）and comparable to that of the OGD-MSCs group（n=3,P>0.05）.（4）Apoptosis-related indexes:immunofluorescence staining:the expression of neuronal cleaved caspase-3 in the OGD-MNPs-MSCs group,OGD-MSCs group and OGD-MNPs group decreased by 84.66%,71.53%and 56.49%,when compared with the OGD-vehicle group,respectively.Cleaved caspase-3expression was significantly lower in the OGD-MNPs-MSCs group than in the OGD-MSCs group or OGD-MNPs group（n=6,P<0.01）.WB:compared to the OGD-vehicle group,the expression of cleaved caspase-3 and Bax proteins were significantly decreased（n=6,P<0.01）and Bcl-2 protein expression was significantly increased（n=6,P<0.01）after treatment with MNPs-MSCs,MSCs or MNPs.The effect of MNPs-MSCs in up-regulation of cleaved caspase-3 and Bax,or up-regulation of Bcl-2 was better than that of MSCs or MNPs（n=6,P<0.05）.（5）Parthanatos-related indicators:Immunofluorescence staining:AIF nucleation rate of neurons in the OGD-MNPs-MSCs group,OGD-MSCs group and OGD-MNPs group was reduced by 74.44%,56.94%and 23.30%compared to the OGD-vehicle group,respectively.The rate of AIF nucleation in neurons in the OGD-MNPs-MSCs group was significantly lower than in the OGD-MSCs or OGD-MNPs groups（n=6,P<0.01）,and the effect of MNPs-MSCs in reducing AIF nucleation in OGD-injured neurons was comparable to that of DPQ（n=6,P>0.05）.Compared to normal controls and the OGD-vehicle group,Iduna was significantly increased in neurons in the OGD-MNPs-MSCs group,the OGD-MSCs group and the OGD-MNPs group（n=6,P<0.05）,while the expression rate of Iduan in neurons in the OGD-MNPs-MSCs group was significantly higher than that in the OGD-MSCs group or the OGD-MNPs group（n=6,P<0.01）.WB:AIF levels were significantly reduced in nuclear extracted proteins in the OGD-MNPs-MSCs,OGD-MSCs and OGD-MNPs groups compared to the OGD-vehicle group（n=6,P<0.05）.AIF levels of nuclear protein were significantly lower in the OGD-MNPs-MSCs group than in the OGD-MSCs or OGD-MNPs groups（n=6,P<0.05）.The inhibition of AIF entering the nucleus by MNPs-MSCs was comparable to that of DPQ（n=6,P>0.05）.MNPs-MSCs,MSCs and MNPs treatments all resulted in a significant decrease in intra-neuronal PARP-1 levels（n=6,P<0.01）.In contrast,MNPs-MSCs caused a better decrease in PARP-1 than MSCs and even better than DPQ（n=6,P<0.05）.The expression of neuronal Iduna was significantly elevated in both the OGD-MNPs-MSCs group and the OGD-MSCs group compared to the normal control group and the OGD-vehicle group（n=6,P<0.05）,while the level of Iduna in the OGD-MNPs-MSCs group was significant higher than that in OGD-MSCs group（n=6,P<0.05）,which was consistent with the Iduna immunofluorescence staining results.3.Viability,mortality,OS response and apoptosis-related indicators of MSCs in each group.（1）The viability of MSCs decreased significantly when the H₂O₂concentration was 50μmol/L.The cell viability decreased more significantly when the H₂O₂ concentration was 200μmol/L（n=3,P<0.01）.When the H₂O₂concentration was greater than 300μmol/L,the cell viability was basically the same and remained at a very low level（n=3,P>0.05）.（2）The mortality of MSCs increased gradually with H₂O₂concentration increasing,and the cell mortality was significantly higher in the 200 and 300μmol/L groups with no statistically significant difference（n=3,P>0.05）.Compared with the H₂O₂-injured group,MNPs pretreatment at the concentrations of 10,20 and 40μg/m L reduced the death of MSCs by 57.24%,70.00%and 72.00%,respectively.MNPs of 20μg/m L pretreatment had a better protective effect on MSCs than 10μg/m L（n=6,P<0.05）,and the difference with 40μg/m L was not statistically significant（n=6,P>0.05）.（3）Pretreatment with MNPs reduced ROS in H₂O₂-induced MSCs by 70.95%（n=6,P<0.01）;and reduced superoxide in H₂O₂-induced MSCs by 23.13%（n=6,P<0.01）compared with the H₂O₂-injured group.（4）Apoptosis-related indicators:immunofluorescence staining:compared with the H₂O₂-injured group,the expression of cleaved caspase-3 was reduced by 52.20%and 57.53%in the H₂O₂-Z-VAD-FMK and MNPs-H₂O₂ groups of MSCs,respectively;the effect of MNPs pretreatment in reducing caspase-3 cleavage was comparable to that of Z-VAD-FMK（n=6,P>0.05）.WB:MNPs pretreatment reduced caspase-3 cleavage by40.20%compared with the H₂O₂-injury group,and the effect in reducing caspase-3 cleavage was comparable to that of Z-VAD-FMK（n=6,P>0.05）.Bcl-2expression was significantly higher in the MNPs-H₂O₂ group compared with the H₂O₂-injury group.Bax expression was significantly reduced in the MNPs-H₂O₂group compared with the H₂O₂-injured group（n=6,P<0.01）and was comparable to that in the H₂O₂-Z-VAD-FMK group（n=6,P>0.05）.ConclusionMNPs can be taken up by MSCs through incubation and have no adverse effect on the biological properties of MSCs at a range of concentrations.Pretreatment with MNPs promotes the survival of MSCs under oxidative stress and serum deprivation by reducing intracellular ROS and inhibiting apoptosis,and enhances the protection of MSCs against OGD-injured neurons by improving the antioxidant defense,inhibiting apoptosis and parthanatos effects of MSCs.This study suggests that the combination of MNPs and MSCs may be a potential means to improve the efficacy of stem cells in ischaemic stroke treatment,providing new ideas for effective clinical use of stem cell therapy.