| Taste,as an important physiological sensation,helps organisms to perceive the taste,safety and nutritional information of different substances in the external environment.The transient receptor potential vanilloid 1?TRPV1?is a noxious stimulus molecular integrator that can be activated by a variety of factors such as capsaicin,allicin,and arachidonic acid metabolites.TRPV1 not only participates in the development of spicy taste,but also plays an important role in the development of pain perception and signal transmission.In this paper,two types of biosensors were constructed using tissue and cell membranes expressing TRPV1 channel proteins as biosensory elements.The activation of TRPV1 channel by capsaicin,allicin and sanshool was determined by time-current statutory quantification.Then,the TRPV1 channel activation/inhibition model was established using capsaicin as an activator,and the inhibitory effects of capsazepine,loureirin B,aconitine,tetrahydropalmatine,AMG517 and anandamide on the activated TRPV1 channel were determined.The kinetic parameters of each substance were analyzed by analogy enzymatic reaction kinetics and enzyme inhibition theory,and the activation constants and inhibition constants of each compound were calculated.The main research results are as follows:?1?Using the sodium alginate-starch gel as a fixing agent,taste-bud tissues of SD rats were fixed between two nuclear microporous membranes.The sodium alginate was gelated by a CaCl2 solution to form a sandwich type sensing film to prepare a taste bud tissue biosensor.The inhibition constants of the six compounds against the activated TRPV1channel were determined using capsaicin as an activator.It turned out that capsazepine,AMG 517,loureirin B and tetrahydropalmatine were competitive allosteric regulatory ligands for capsaicin,while aconitine and anandamide were non-competitive and competitive hybrid allosteric regulatory ligand.The suppression constants are:Ki capsazepine=3.9796×10-18 mol/L,Ki AMG517=2.0743×10-18 mol/L,Ki loureirin B=1.6196×10-18mol/L,Ki tetrahydropalmatine=4.4440×10-17 mol/L,Ki'aconitine=4.7931×10-17 mol/L,Ki'anandamide=9.2206×10-17 mol/L.This indicates that the order of inhibition of the six compounds is:loureirin B>AMG517>capsazepine>tetrahydropalmatine>aconitine>anandamide.?2?The pcDNA3.1?+?eukaryotic expression vector containing the human TRPV1channel protein was synthesized by whole-gene synthesis method,and then the vector was transfected into HEK293T cells by lipofection.HEK293T cell line stably expressing human TRPV1 channel protein was screened by G418 selective medium.The successful construction of pcDNA3.1?+?vector and the expression of TRPV1 channel protein on the cell membrane of TRPV1-HEK293T cells were confirmed by agarose gel electrophoresis and Western Blotting experiments.The constructed TRPV1-HEK293T cells can be used for subsequent studies of TRPV1 channel proteins.?3?A collagen/cell membrane/intermediate filaments protein/cysteamine/AuNPs/thionin-chitosan/reduced graphene oxide biosensor was constructed by reducing graphene oxide and gold nanoparticles as signal amplification materials.The time-current method was used for the determination.The activation constants were Ka capsaicin=3.5206×10-16mol/L,Ka allicin=5.0227×10-15 mol/L,Ka sanshool=1.7832×10-15 mol/L.This indicates that the stimulating intensity of the three substances is:capsaicin>sanshool>allicin.The use of cell membranes as sensing elements maintains the six-transmembrane domain of the TRPV1 channel and improves sensor stability and shelf life.The biosensor can be used for the screening of TRPV1 channel activators.In this paper,the activation/inhibition mechanism of TRPV1 channel was studied at the tissue and molecular levels,respectively,through the construction and application of the above two different electrochemical biosensors.On this basis,various TRPV1 channel protein activators and inhibitors were subjected to intensity grading,and the antagonistic types of different inhibitors were defined.In addition,by kinetic law and molecular structure analysis,it was first discovered that capsaicin and TRPV1 antagonist can regulate TRPV1 channel status through cannabinoid?CB?receptor/G protein/PI3K/PIP2signaling pathway.This provides an important theoretical basis for revealing the activation and inhibition mechanism of TRPV1 protein.Furthermore,it will lay the theoretical foundation and new ideas for investigating nociceptive signal?pain?and screening potential analgesic drugs. |
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