Intervention Study Of Catalpol On Oxygen And Glucose Deprivation/reperfusion Injury In A Microfluidic-based Approach Of Neurovascular Unit Model

Background:Cerebrovascular disease is one of the three most deadly diseases in human beings.It has the characteristics of high morbidity,high mortality rate,high disability rate,etc.,which seriously endangers human health and imposes a heavy burden on families and society.Among them,hypoxic ischemic encephalopathy(HIE)accounts for about 85%of all cerebrovascular diseases,and its onset begins with local blood flow and sudden interruption of glucose and energy supply in the infarct area,which leads to serious brain dysfunction.However,the nerve damage continues even while the blood supply to the brain has been restored.The pathological process is not only cerebral ischemia and neuronal death,but also reperfusion injury can aggravate the degree of brain damage,which leads to further worsening of neurological dysfunction,and the pathological process is cerebral ischemia reperfusion injury(CIRI).CIRI includes a series of multi-link,multi-factor,multi-pathological pathological cascades.Previous studies had focused on specific structures such as specific neurons or blood-brain barrier(BBB)without considering the interaction among multiple factors.As the intensive study on brain structure and brain function,the research in single structures has gradually turned to the study of structural complexes,which was the research in neurovascular unit(NVU).As the basic unit of brain structure and function,NVU is mainly composed of neurons,glial cells and BBB.Researches had shown that the steady state relationship between nerves and blood vessels was destroyed after CIRI,which lead to the release of inflammatory factors,vascular endothelial cells and glial cells were activated,BBB permeability changes,neuronal damage was intensified,and NVU componented different degrees of damage.Taking NVU as the research object,the whole structure of neuron-glial-vascular endothelial cells was controlled.Neuroprotection and injury repair are of great significance for the prevention and treatment of hypoxic ischemic encephalopathy.Traditional Chinese medicine has unique advantages in the treatment of CIRI because of its overall regulation and syndrome differentiation.As one of the "four great medicines" in China,Rehmannia glutinosa is a commonly used medicine in traditional Chinese medicine.In recent years,the separation,identification and pharmacological research on the active ingredients of Rehmannia glutinosa have been researched deeply,and the most representative one is the iridoid steroid catalpol.Modern research have confirmed that catalpol have a wide range of pharmacological effects such as neuroprotection,cardiovascular protection,hypoglycemia,and anti-tumor.Catalpol have good therapeutic effect on multiple neurological diseases such as cerebral ischemic injury,Parkinson’s disease,Alzheimer’s and neurodegeneration through anti-inflammatory,anti-oxidation,anti-apoptosis,neurological pathways and promotion of nerve repair and remodeling.However,the protective effect of catalpol on CIRI at the cellular level and its mechanism have not been elucidated.It is clear that the protective effect of catalpol on CIRI will provide new ideas for the study of neuroprotective mechanisms and drug screening.Objectives:The purpose of this study was divided into three aspects:Firstly,using the multi-directional differentiation potential of neural stem cells and the high-throughput and integration of microfluidic chip technology,the functionalized NVU model was constructed in vitro,which provided base for the research of nervous system-related diseases,the screening of potential therapeutic targets and the development of new drugs.Secondly,based on the NVU model,explored the appropriate oxygen sugar deprivation/reoxygenation sugar(OGD/R)damage conditions,and established a reliable and simple OGD/R injury model in vitro brain function unit at the cellular level,which provided base for research of the intervention effect of Chinese medicine catalpol on CIRI.Finally,the intervention effect of catalpol on OGD/R injury in NVU vitro model was discussed,which provided new ideas for the research of neuroprotective mechanism and drug screening in vitro.Materials and methods:1.Design,preparation and functional detection of brain NVU in vitro model based on microfluidic technologyThe aborted embryos were collected for 6-12 weeks of pregnancy,and human neural stem cells(hNSCs)were isolated and cultured in vitro to identify their dryness,specificity and growth characteristics.Human microvascular endothelial cells(hBMECs)were cultured in vitro to identify their specificity and growth characteristics.CAD software was employed to draw microfluidic chip design and print film mask;soft lithography was employed to prepare SU-8 male mold;PDMS casting into mold and punching was employed;lower layer chip and glass substrate plasma bonding was employed;hBMSCs polycarbonate membrane was placed in the middle of the upper and lower chips and then bonded by plasma;assembly,integrated and microchannel NVU in vitro model sealing was manufactured.The hNSCs inoculated in the chip model were subjected to neurogenic differentiation using the serum-promoting method.The proportion of differentiated neurons(Map2 positive),astrocytes(GFAP positive)and oligodendrocytes(MBP positive)was identified by real-time quantitative PCR and immunofluorescence staining.Immunofluorescence staining was employed to detect the expression of vWF and tight junction protein ZO-1 in hBMSCs in NVU model,and to investigate the integrity of BBB;to detect the pass rate of FITC-labeled low molecular dextran,and to investigate the permeability of BBB.2.Establishment of OGD/R damage modelOn the basis of the NVU model,a normal control group and an OGD/R group were established.Especially,the OGD/R group need to use sterile PBS to perfuse the upper and lower cell cultures of the model,and replaced the perfusate with an anaerobic,sugar-free,serum-free medium,then,placed in a three-gas culture chamber which volume fractions was 1%O2,5%CO2,and 94%N,respectively,and cultured for 3 hours under hypoxia.After the end of OGD,the perfusate was replaced with a conventional complete culture solution,and placed in a resuscitation sugar culture in an incubator with a volume fraction of 5%CO2 at 37 C,and detected at 0,6,12,24,and 48 h,respectively:LDH detection kit was employed to detect LDH activity in model perfusate;Calcein-PI double staining method was employed to detect the survival rate of neuro-glial cells and microvascular endothelial cells(BMECs)in OGD/R injury model;and FITC Labeled low molecular dextran was employed to detect changes in BBB permeability in the OGD/R injury model.3.Intervention of catalpol on OGD/R injury in NVU vitro modelThe effects of different concentrations of catalpol(0.05 mg/ml,0.1 mg/ml,0.25 mg/ml,0.5 mg/ml,1.0 mg/ml)on the growth activities of hNSCs and hBMECs were examined by CCK-8 method,and whether the dose of catalpol has any toxic effects on each cell component was investigate.High-performance liquid chromatography(HPLC)was used to determine the permeability of catalpol in the NVU model,and the efficiency of the application of catalpol to nerve-glial cells via BBB was investigated.Based on the successfully constructed OGD/R injury model,the catalpol immediate administration group(oxycatalpol-containing reoxygenation solution perfusion immediately after OGD injury)and the pretreatment administration group(catalpol was perfused for 6 hours before OGD injury,and the catalpol effect was maintained throughout the OGD/R process)were established,then,each group was divided into three doses of low,medium and high,and OGD 3 h/R 24 h was employed except for the normal control group.The LDH assay kit was used to detect the LDH activity in the perfusate of each treatment group,and the degree of injury was evaluated to determine the optimal administration mode and dosage of catalpol.The cell viability of hNSCs and hBMECs in each treatment group was further measured by Calcein-PI double staining method;The permeability of BBB in each model was detected by FITC-labeled low molecular weight dextran(4000 Da),and the intervention effect of catalpol on OGD/R injury in NVU vitro model was evaluated.Results:1.Constructing a functionalized NVU model in vitro based on neural stem cells and microfluidic chip technologyThe extracted hNSCs that can be differentiated into percentage of neurons(Map2 positive),percentage of astrocytes(GFAP positive),and percentage of oligodendrocytes(MBP positive),which was identified that NSCs-specific proteins(Nestin,SOX2)were stably expressed,and inoculated in a chip model after 7 days of induction by FBS.hBMECs cultured in vitro were identified to stably express endothelial cell-specific proteins(CD31,vWF),and after 5-7 days inoculation on polycarbonate membrane,tight junctions were formed between cells(ZO-1 positive)and stable expression of vWF.The microfluidic biochip with PDMS as the cell attachment material has good biocompatibility,and the survival rates of nerve-glial cells and hBMECs are above 95%.The model consisted of a multi-layered vertical neurological module and a vascular module which were connected to a syringe pump,provided controlled cell inoculation and material transport conditions,and achieved the distinctive features of NVU,such as spatial three-dimensional structure(neural module+vascular module),shear stress(continuous rate-controlled perfusion),cell diversity(neurons derived from hNSCs,astrocytes,and oligodendrocytes).Compared with the pure polycarbonate membrane,the BBB in the NVU model can significantly inhibit the FITC-labeled dextran permeability,indicating that the integrity and permeability were good.2.Construction of OGD/R damage model based on NVU in vitroAfter the NVU in vitro model oxygen oxidative deprivation(OGD)was damaged for 3 hours,the reoxygenated sugar(R)treatment was performed,and the results shown that:at R 6 h,the activity of LDH in the perfusate began to increase,and reached the maximum at R 24 h;the survival rate of nerve-glial cells began to decrease,and reached the minimum at R 24 h.Meanwhile,the permeability of the endothelial barrier began to increase in the model,and the cell survival rate reached the minimum after 24 h.When R reaches 48 h,there was no significant difference between the detection indexes and R 24 h,and the modeling need a long time,which was not conducive to obtaining stable experimental results.Therefore,OGD 3 h/R 24 h was selected as the molding condition for the subsequent research.3.Investigate the effect of catalpol on OGD/R injury in NVU in vitro modelAfter different concentrations of catalpol(0.05 mg/ml,0.1 mg/ml,0.25 mg/ml,0.5 mg/ml,1.0 mg/ml)were applied at different time points(24,48,72 hours),none of them adversely affected the growth activities of hNSCs and hBMECs.The reversed-phase HPLC method was used to determine the catalpol content with good precision and reliable stability,and the detection concentration of catalpol in the range of 7.8125-1000 μg/mL showed a good linear relationship with the peak area integral value.The permeation of catalpol in the NVU model increased with the perfusion time,until it was stable after 4 hours,and the BBB permeation efficiency was 58.18±1.52%.Compared with the OGD 3h/R 24h group,the medium(0.25 mg/mL)and high(1.0 mg/mL)concentrations of the catalpol pretreatment group significantly reduced the activity of LDH in the model perfusate;there was no significant difference in LDH activity in the perfusate compared with the OGD 3h/R 24h group.lmg/ml catalpol by pretreatment can significantly improve the survival rate of neuro-glial cells and hBMECs in the OGD/R injury model,and effectively alleviate cells denaturation and necrosis which induced by OGD/R injury;protected the endothelial barrier function and maintain its integrity and permeability;reduced LDH activity in model perfusate,and reduce cell damage.Conclusions:1.The NVU in vitro model was successfully constructed by combining human neural stem cells and microfluidic chip technology.The chip model was easy to assemble,and spatial structure(three-dimensional culture),physiological stimulation(continuous perfusion,shear stress)and cell diversity(such as interactive co-culture)can be integrated into one.NSCs had stable differentiation efficiency,and each cell component survival rate on the chip was high.2.The OGD/R injury model was successfully constructed,after the human NVU in vitro model was treated with oxygen sugar deprivation for 3 hours and reoxygen sugar for 24 hours.The release level of LDH in perfusate can reflect the degree of damage sensitively and conveniently;the increase of BBB permeability can be used as a marker to judge the success of OGD/R damage modeling.3.The therapeutic dose of catalpol had no toxic effect on each cellular component.Catalpol can alleviate cell degeneration and necrosis caused by OGD/R injury,reduced cell damage,inhibited glial cell activation,protected BBB integrity and permeability,and protected OGD/R injury in human NVU in vitro model.Moreover,the protective effect of pretreatment administration on OGD/R injury was better than immediate administration.