Studies On Design,synthesis Of Novel Fluorescent Probes And Its Application In Dynamic Evaluation Of The Effect Of Dendrobium Officinale Polysaccharide In Improving Acute Alcoholic Liver Injury

Acute alcoholic liver injury（AALI）,as the early stage of alcoholic liver disease,is often overlooked or misdiagnosed due to the lack of typical clinical symptoms.Without appropriate intervention,it is likely to develop into severe liver disease.Dendrobium officinale polysaccharide（DOP）,a natural polysaccharide extracted from Dendrobium officinale,has the potential to protect the liver by enhancing the antioxidant system to maintain the relative balance of ROS（reactive oxygen species）and antioxidants in AALI.However,the real-time improvement effect of DOP on AALI is still unclear and there is no accurate medication guidance,which limits the clinical application of DOP.The stable metabolism of physiological indicators in the liver is the basis for the response of liver cells to liver diseases,and the abnormal changes of related indicators are closely related to liver injury.In particular,physiological indicators such as glutathione（GSH）,viscosity,blood flow in the liver play crucial roles in many metabolic processes,for instance cell signaling and transport.Due to its advantages of high sensitivity,non-invasiveness,and visualization,near-infrared（NIR）fluorescence imaging has been widely studied in the fields of biochemistry and biomedicine.To better understand the detailed physiological functions of the above index in the liver and the relationship between them and liver injury,it is necessary to use near-infrared fluorescent probes to track and monitor the abnormal changes of the above index in the liver,so as to comprehensively analyze and diagnose the progression of liver injury.In this paper,to diagnose AALI more comprehensively and to conduct multi-dimensional evaluation of the dynamic process of DOP improve AALI through various index,three near-infrared fluorescent probes were designed and constructed for dynamic evaluation of it by hepatic GSH,viscosity,and blood flow.The specific research contents are as follows:In the first part,based on 2,3,3-trimethylbenzindole,methyl iodide,phosphorus oxychloride and cyclohexanone as raw materials,a"turn-on"near-infrared GSH fluorescent probe HCG was developed.The structure of the intermediate and HCG was characterized by ¹HNMR,¹³CNMR and high-resolution mass spectrometry.The sensitivity,selectivity and response time of HCG for GSH detection were studied.The results showed that HCG could effectively detected GSH with a detection limit of 252 n M and a fast response time of30 s.The contents of GSH in MCF-7,Hep G2 and HT-29 cells was detected and analyzed by flow cytometry and confocal microscopy.The response mechanism of HCG was studied by HPLC,HRMS,and DFT methods.The animal in vivo imaging system of HCG in GSH group and non-GSH group mice were studied,which examined its fluorescence imaging ability at the animal level.The results showed that HCG had potential application in biological science research,and laid the foundation for the subsequent evaluation of the efficacy of DOP in improving AALI mice.In the second part of this paper,high-purity DOP was prepared and characterized by using Dendrobium officinale stems as raw materials.An AALI mouse model was established to simulate the human AALI model and DOP was used to improve AALI mice.HCG was applied to analyze the fluorescence imaging of different groups of mice,the relationship between hepatic GSH and AALI was studied.Near-infrared fluorescence analysis technology was used to dynamically evaluate the efficacy of DOP in improving AALI mice,which proved that DOP can maintain the relative balance of GSH content in the liver and protect the liver from damage.In the third part,a fluorescent probe HOTPy with aggregation-induced emission properties was developed,which using tetraphenylethylene as the fluorophore.The stability,sensitivity and specificity of HOTPy response to viscosity in different solutions were investigated.Confocal microscopy was used to conduct mitochondria-targeted localization experiments in LO2 cells and the co-localization ability of HOTPy and mitochondrial commercial probes was compared.The biocompatibility of the probe in mice was investigated,and the diagnosis ability of HOTPy in AALI mice was further studied.When DOP was used to alleviate liver injury in AALI mice,HOTPy could dynamically track the liver improvement of process in situ.The results showed that HOTPy could diagnose AALI with microviscosity changes in the liver and evaluate the improvement effect of DOP on AALI in real time through hepatic viscosity changes.In the fourth part,a high-resolution NIR-II fluorescent probe NTPB-NPs was prepared by using TPB and DSPE-PEG-2000 as raw materials.The structure and composition of NTPB-NPs were characterized by TEM,DLS and other methods.The biocompatibility and metabolic process of NTPB-NPs in mice were studied with blood biochemical analysis and in vivo dynamic NIR-II imaging.The high-resolution fluorescence imaging capabilities of NTPB-NPs was compared with clinical contrast agent ICG.The relationship between changes in blood flow velocity and liver fluorescence was investigated.The imaging ability of NTPB-NPs in situ tracking of the improvement effect of DOP on AALI was studied.It was comfirmed that the decreased blood flow velocity of the liver caused by alcohol could lead to the lower fluorescence intensity in the liver of AALI mice.The results showed that NTPB-NPs could diagnose AALI by NIR-II fluorescence imaging technology and dynamically evaluate the improvement effect of DOP on AALI.