Study On Ameliorative Effects Of Semen Pruni Persicae And Carthamus Tinctorius L. Carbon Dots On The Blood-Brain Barrier Permeability In Traumatic Brain Injury

OBJECTIVE: To examine the role of carbon dots(CDs)derived from Semen pruni persicae and Carthamus tinctorius L.(Taoren-Honghua carbon dots,TH-CDs)in protecting the blood-brain barrier(BBB)in the acute phase of traumatic brain injury(TBI).METHOD: The TH-CDs with maximum quantum yield(QY)were prepared and screened by a green hydrothermal synthesis method.The structural features of TH-CDs were comprehensively detected and identified using various characterization methodologies.A mouse model of TBI was established through a controlled cortical impact(CCI)instrument.After doses-dependent of TH-CDs were injected intravenously,the therapeutic effects were verified using modified neurological severity score(m NSS),cerebral water content detection,Nissl staining and brain tissue pathological observation.To discover whether TH-CDs protect the BBB,we evaluated the effects of TH-CDs in the acute stages of TBI models through the content of Evans blue and Ig G immunohistochemical staining.Furthermore,the expressions of tight junction(TJ)proteins including Claudin-5 and ZO-1 were measured by immunofluorescence staining.Molecular docking was utilized to verify the binding ability and interaction force of the hypothetical TH-CDs models with Claudin-5protein.Finally,the biological safety of TH-CDs was validated in vitro and in vivo using CCK-8 cytotoxicity test,blood routine detection and histopathological staining.RESULT: TH-CDs with maximum QY were prepared at 240 °C for16 h.Physicochemical characterizations showed that TH-CDs were uniformly dispersed and quasi-circular spherical dots with negative charges on their surface.The average particle size was about 3.12 ± 0.95 nm.The maximum excitation wavelength was 361.6 nm and the maximum emission wavelength was 434.6 nm.TH-CDs were mainly composed of carbon,nitrogen and oxygen,and the surface contains abundant groups from Taoren and Honghua.In the acute phase of TBI mice models,we found that all TH-CDs at high,medium,and low doses improved neurological functions,neuronal damage and pathological changes in brain tissues after intravenous injection of TH-CDs solution.However,only high-dose group of TH-CDs were observed to significantly reduce brain water content.We further showed that the high-dose group of TH-CDs could significantly reduce the BBB permeability and upregulated the expressions of Claudin-5 and ZO-1 proteins.There were electrostatic interactions between three hypothetical TH-CDs models and Claudin-5protein,including Van der Waals forces,hydrogen bond and π-π stacking.The results of cytotoxicity experiments,routine blood tests and pathological staining demonstrated that TH-CDs had good biological safety in vitro and in vivo.CONCLUSION: In this study,a kind of nano-scale CDs with negative surface charges,intravenous injection and good biosafety was synthesized derived from green Taoren-Honghua.TH-CDs might bind to the Claudin-5 through electrostatic interactions,and exerted a pharmacodynamic role in reducing the BBB permeability in the acute phase of TBI.