CN115364088A - Application of TrkB agonist in preparation of medicine for treating visual cortex damage - Google Patents

Abstract

The invention belongs to the technical field of biological medicines, and discloses an application of a TrkB agonist in preparing a medicine for treating visual cortex damage, which comprises the following steps: application of TrkB agonist in preparing medicine for relieving, preventing and treating visual cortex damage is provided. The TrkB agonist is a small molecule TrkB agonist; the small molecule TrkB agonist is 7,8,3' -THF. Visual cortex damage is visual cortex damage caused or contributed to by diabetes. TrkB agonists act by improving the azimuthal selectivity, response characteristics, and synaptic function of neurons and/or by improving impaired BDNF/TrkB signaling pathways. The invention defines that 7,8,3' -THF acts on BDNF/TrkB signal channel damaged by diabetes, effectively improves disturbed BDNF/TrkB channel function, can effectively activate TrkB receptor, prevents synapse loss and improves synapse function.

Description

Application of TrkB agonist in preparation of medicine for treating visual cortex damage

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to application of a TrkB agonist in preparation of a medicine for treating visual cortex damage.

Background

Currently, the primary selective agonists of TrkB include monoclonal antibodies and peptidomimetics. The tricyclic dimer peptide originally designed as a partial agonist of TrkB can promote the survival of the sensory neurons of the cultured chicken embryos. Subsequently, BDNF/NT-4/5 mimetics were designed using the tandem repeat peptide agonist approach, but none of these compounds had a satisfactory in vivo agonistic effect on TrkB. Adenosine and pituitary adenylate cyclase activating peptide have also been reported to transactivate TrkB in cultured hippocampal neurons, however, this G protein-coupled receptor ligand does not act as a TrkB agonist, as it activates the immature form of TrkB only after 1 hour of treatment. These attempts to "manufacture" potent and selective in vivo agonists of TrkB have all ended up with disappointing results.

The step of exploring has never stopped, and finding small molecules that mimic the biological function of BDNF has become the direction of researchers' efforts. Prior art 1 developed a cell-based TrkB receptor-dependent survival assay system and used it to screen chemical libraries to identify a series of compounds, and finally identified lead compounds: 7,8-dihydroxyflavone, which specifically binds to TrkB receptor, promotes receptor dimerization and autophosphorylation, and activates downstream signaling cascades, thereby mimicking the physiological effects of BDNF. The compound can cross blood brain barrier, induces TrkB activation in the brain of a mouse in a BDNF independent mode, has relatively low cell EC50 in the aspect of promoting neuron survival, and shows strong neuroprotective effect under 5mg/kg dosage, so that the compound becomes an ideal candidate drug for subsequent research in animal or clinical research. 7,8-DHF has been widely explored in various cell types and disease models since its first report due to its commercial availability and favorable chemical and physical properties.

Then, prior art 2 carries out extensive structure-activity relationship (SAR) studies, and reports that a hydrogen bond receptor on the 4' position of the B ring of flavone is important for the TrkB agonism of 7,8-DHF, and the 7,8-DHF derivative 4' -dimethylamino-7,8-dihydroxyflavone (4 ' -DMA-7,8-DHF) synthesized based on the hydrogen bond receptor has stronger agonism on TrkB than that of the maternal parent, and the stimulatory effect of the derivative lasts for a longer time in animals. By orally administering the wild-type mice and TrkB F616A knock-out mice for 21 days, it was confirmed that 5mg/kg of 4' -DMA-7,8-DHF showed potent antidepressant effect in a TrkB-dependent manner, and the effect was stronger at 1mg/kg than the parent. Subsequently, prior art 3 demonstrated that chronic administration of 7,8-DHF (5 mg/kg) or 4' -DMA-7,8-DHF (1 mg/kg) significantly ameliorated the motor deficits, improved brain atrophy and prolonged survival in N171-82Q HD mice. In addition, 4' -DMA-7,8-DHF retained DARPP32 levels in the striatum and rescued the neurogenesis injury induced in N171-82Q HD mice by the mutant Huntington protein. These data underscore the consideration of TrkB as a therapeutic target for HD and suggest that small molecule TrkB agonists that penetrate the brain have high potential for further testing in HD clinical trials. Next, prior art 4 found that prodrug R13 exhibited good absorption capacity by modifying the catechol group and was readily hydrolyzed in the liver to 7,8-DHF. R13 has very long half-life, and can improve the oral bioavailability of 7,8-DHF from 4.6% to 10.5%. Plasma concentrations and brain exposure were also significantly enhanced. In the 5XFAD mouse strain, chronic oral administration of R13 ameliorated Α β deposition, reduced loss of hippocampal synapses, and improved memory deficits in a dose-dependent manner. These exciting data provide the basis for pushing such prodrugs to clinical trials.

In fact, 7,8-dihydroxyflavone (7,8-DHF) is a naturally occurring plant flavonoid. Flavonoids are a diverse group of plant secondary metabolites found in fruits and vegetables, capable of exerting diverse biological effects, including as antioxidants and cancer preventative agents. Flavonoids can improve cognitive performance by protecting fragile neurons, enhancing existing neuronal function, and stimulating neuronal regeneration. In addition, flavonoids have an effect on the long-term potentiation of learning, memory and cognition through interaction with signaling pathways, including PI3K/Akt and MAPK.

The small molecular substance 7,8,3 '-trihydroxyflavone (7,8,3' -THF) is a derivative of 7,8-DHF, and has better in-vivo pharmacokinetic characteristics to trigger TrkB phosphorylation activation. The structure-activity relationship (SAR) research finds that 7,8,3'-THF is a more powerful compound for stimulating TrkB phosphorylation, the efficacy of the compound is 2-3 times higher than that of a parent 7,8-DHF, and the compound is mainly derived from that the 3' -hydroxyl in a B ring can effectively improve the agonism.

Currently, the application research of 7,8,3' -THF is still in the exploration stage. In an in vitro study, THF was shown to induce neurite outgrowth in mouse cochlear Spiral Ganglion (SGC) explants, reproducing BDNF-like effects. Prior art 5 discloses that the survival of SGC after treatment with small molecule TrkB agonists 7,8-DHF and 7,8,3' -THF is similar to that after treatment with BDNF in cochlear organic type cultures. In vivo experiments in the same group of mice even showed SGC survival similar to normal hearing control groups, further underscoring the potential of this small molecule TrkB agonist. Since the results of prior art 5 in mice are very promising, prior art 6 continued to compare the protective effects of THF and BDNF on SGC, and confirmed the protective effect of BDNF, but the previously reported protective effects of THF have not been confirmed by clinically used administration methods, and this contradictory finding suggests that the effect of THF on TrkB is not fully understood and deserves further investigation.

Through the above analysis, the problems and defects of the prior art are as follows: the prior art has not been researched or reported for 7,8,3' -THF to treat or improve visual cortex damage caused by diabetes.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides an application of TrkB agonist in preparing a medicine for treating visual cortex damage.

The invention is realized by the application of TrkB agonist in preparing medicine for treating visual cortex damage.

Further, the application further comprises: application of TrkB agonist in preparing medicine for relieving and preventing visual cortex damage is provided.

Further, the TrkB agonist is a small molecule TrkB agonist; the small molecule TrkB agonist is 7,8,3' -THF.

Further, the visual cortex damage is visual cortex damage caused or caused by diabetes.

Further, the TrkB agonist acts by improving the azimuthal selectivity, response characteristics, and synaptic function of neurons.

Further, the TrkB agonists act by ameliorating the impaired BDNF/TrkB signaling pathway.

Further, the applying further comprises: the TrkB agonist is administered according to a dosing regimen repeated once weekly for 4 weeks.

Further, said administering a TrkB agonist comprises: 7,8,3' -THF was dissolved in 17% DMSO for TrkB agonist administration.

Further, the amount of the TrkB agonist to be administered is 5mg/kg.

Further, the administration mode comprises: the administration is carried out in the abdominal cavity.

In combination with the technical solutions and the technical problems to be solved, please analyze the advantages and positive effects of the technical solutions to be protected in the present invention from the following aspects:

first, aiming at the technical problems existing in the prior art and the difficulty in solving the problems, the technical problems to be solved by the technical scheme of the present invention are closely combined with results, data and the like in the research and development process, and some creative technical effects are brought after the problems are solved. The specific description is as follows:

the invention defines that 7,8,3' -THF acts on BDNF/TrkB signal channel damaged by diabetes, can effectively activate TrkB receptor, prevent synapse loss and improve synapse function.

The invention discovers that receptor agonist 7,8,3' -THF applied externally can up-regulate TrkB phosphorylation level and activate TrkB receptor, thereby effectively improving disturbed BDNF/TrkB channel function.

According to the invention, a Western Blot experiment (Western Blot) finds that the level of a p-TrkB receptor of a visual cortex of a diabetic mouse is obviously lower than that of a healthy mouse of the same age group, and shows that the activation level of the TrkB receptor of the visual cortex of the diabetic mouse is low and the TrkB/BDNF signal channel is damaged. The TrkB/BDNF signaling pathway is involved in excitatory synaptic transmission and maintenance of neuronal excitatory firing levels. Meanwhile, a postsynaptic compact substance (PSD-95) and Synaptophysin (SYP) of synaptophysin are detected by Western Blot, and the level of the two synaptophysins in the visual cortex of a diabetic mouse is obviously lower than that of a healthy animal, which indicates that the synaptophysin in the visual cortex is damaged by diabetes. Using extracellular electrophysiological recording, we found that diabetic mouse visual cortical neurons elicited responses significantly less intense than healthy animals to visual stimuli, and at the same time, evaluation of synaptic function revealed that diabetic mouse visual cortical synaptic function was significantly less attenuated than healthy animals, as reflected in the efficiency of information transfer between neurons, consistent with Western Blot results. Furthermore, the most important receptor field characteristic azimuth direction selectivity of the visual cortex neuron is evaluated by using an electrophysiological extracellular recording means, and the azimuth direction selectivity of the diabetic mouse is found to be obviously inferior to that of a healthy mouse, and the azimuth direction selectivity of the visual cortex neuron determines the perception of a visual system on the boundary and contour of an object, and is the basis of visual perception.

These results indicate that TrkB/BDNF signaling pathway impairment severely affects the function of visual cortical neurons. Aiming at a key damage target TrkB/BDNF signal channel, a small molecular substance 7,8,3' -THF is selected to activate a TrkB receptor and enhance the TrkB/BDNF signal channel. Traditional BDNF and other polypeptide substances have large molecular weight, are difficult to penetrate through a blood brain barrier, have short metabolic cycle and are difficult to enter the brain to generate effects. 7,8,3' -THF has small molecular weight, can penetrate blood brain barrier, and can specifically activate TrkB receptor, thus being an ideal medicine for pre-TrkB/BDNF signal channel in brain.

After continuous peripheral abdominal injection of 7,8,3' -THF, the levels of p-TrkB receptors in the visual cortex of diabetic mice increased, indicating that the TrkB/BDNF signaling pathway was activated. At the same time, the level of the synaptic markers PSD-95 and SYP is obviously improved, which indicates that the synaptic density is improved. Electrophysiological recording shows that the visual evoked response intensity of the neuron is obviously improved and is close to that of healthy animals. At the same time, improvements in synaptic connection strength and information transfer also occur.

To further observe the improved effect of 7,8,3'-THF, we compared the ability to distinguish orientation of diabetic mice after 4 weeks treatment with 7,8,3' -THF. We used go/nogo detection method to test the discrimination ability of mice to 90 DEG and 80 DEG/100 DEG black-and-white grating stimulation (angle difference is 10 DEG), and found that the orientation discrimination ability of diabetic mice after 7,8,3' -THF is 4 weeks of treatment is obviously better than that of diabetic mice without treatment.

Secondly, considering the technical scheme as a whole or from the perspective of products, the technical effect and advantages of the technical scheme to be protected by the invention are specifically described as follows:

the invention determines the intrinsic mechanism of DM damage visual cortex neuron function and determines a new way of improving visual function under DM background by carrying out intraperitoneal administration of 4-week TrkB micromolecule stimulant 7,8,3' -THF to DM mice, detecting the functional characteristics of V1 neurons and measuring the changes of BDNF/TrkB channel and synapse-related protein.

Third, as an inventive supplementary proof of the claims of the present invention, there are also presented several important aspects:

(1) The expected income and commercial value after the technical scheme of the invention is converted are as follows:

treatment of the retina is difficult to completely improve the visual disorder of a diabetic patient. Our studies found that visual cortex function impairment caused by visual cortex BDNF/TrkB signaling pathway impairment is also one of the important causes of diabetic visual disturbance. Calculated according to the cardinality of the diabetic patients with visual disturbance in China, the medicine market scale is about 160 to 240 hundred million RMB.

(2) The technical scheme of the invention overcomes the technical prejudice that:

1. the action position overcomes the previously thought technical prejudice that the diabetic visual disturbance exists only in the retina. Diabetes is usually accompanied with retinopathy, which seriously affects the visual function of patients, and about 3200 to 4800 million patients suffering from visual function damage in the current diabetes patients. The mechanism research and the improvement approach research aiming at the visual function impairment of the diabetic patients focus on the retina, however, the retina is only the front part of the visual system, and little attention is paid to how other parts of the visual system, such as the visual center, change. Diabetes causes the possibility that impaired function of cortical neurons of the brain suggests impaired function of the visual cortex. Through extracellular electrophysiological recording, the most important receptive field characteristic of visual cortical neurons, namely impaired azimuth direction tuning ability, is found, and a key injury target, namely a BDNF/TrkB receptor signal channel, is also found. This finding complements the understanding of the mechanism of impaired visual function in diabetes. Aiming at a target spot of the diabetes visual cortex damage, the TrkB receptor micromolecule agonist-7,8,3' -THF is used for intervening the diabetes visual dysfunction, and the result shows that the activation level of the TrkB is increased, the visual cortex neuron function is also normal, and the visual cortex neuron function is expressed by increased reaction strength and improved neuron direction tuning capability.

2. Aiming at improving the function of the nervous system and overcoming the technical prejudice that the prior art only aims at reducing the blood sugar. Currently, all drugs for diabetes treatment do not work to protect or improve neuronal and synaptic function, however, in diabetic population, central nervous system damage is a considerable problem, and the damage affects cognition and perception. The 7,8,3' -THF used by the invention is a TrkB receptor specific agonist, can activate a BDNF/TrkB receptor signal pathway, and can improve synaptic function damage, neuron function and visual perception ability damage caused by diabetes.

Drawings

FIG. 1 is a schematic diagram of metabolic profile changes in diabetic mice provided by an embodiment of the invention;

FIG. 1 (A) is a schematic diagram showing the time-dependent blood glucose changes of an NC group and a DM group of mice after STZ injection;

FIG. 1 (B) is a schematic diagram showing the comparison of blood sugar levels between mice in NC group and mice in DM group provided by the embodiment of the present invention;

FIG. 1 (C) is a schematic diagram showing the change of body weight of diabetic mice in NC and DM groups with time after STZ injection according to the embodiment of the present invention;

FIG. 1 (D) is a schematic diagram showing the comparison of the body weights of mice in NC group and DM group provided by the embodiment of the present invention;

FIG. 1 (E) is a schematic diagram showing the comparison of serum insulin of mice in NC group and DM group provided by the embodiment of the present invention;

FIG. 1 (F) is a schematic diagram showing the change of blood glucose during OGTT of NC group and DM group mice provided by the embodiment of the invention;

FIG. 1 (G) is a graph showing the comparison of the AUC of OGTT of mice in NC group and DM group provided by the embodiment of the invention;

FIG. 2 is a schematic diagram of the azimuthally selective damage of V1 neurons in diabetic mice provided by an embodiment of the invention;

FIG. 2 (A) is a schematic diagram of an embodiment of an electrophysiological recording in vitro;

FIG. 2 (B) is an exemplary plot of single neuron orientations for the NC and DM group of mice V1 provided by an embodiment of the present invention;

FIG. 2 (C) is a graph showing the comparison of the OB values of V1 neurons in mice of NC and DM groups according to the present invention;

FIG. 2 (D) is a graphical representation of a comparison of OSI values of V1 neurons from NC and DM groups provided by an embodiment of the invention;

FIG. 3 is a schematic diagram of impaired V1 neuronal response characteristics in diabetic mice according to an embodiment of the present invention;

FIG. 3 (A) is an exemplary graph of V1 neuronal reactivity in mice of the NC and DM groups provided by an embodiment of the invention;

FIG. 3 (B) is a graph showing the comparison of V1 neuron response activity in NC and DM groups provided by the present invention;

FIG. 3 (C) is a schematic diagram of the frequency distribution of the V1 neuron spike response in NC and DM mice provided by the embodiments of the present invention;

FIG. 3 (D) is a schematic diagram comparing SNR of NC and DM rat group V1 neurons provided by an embodiment of the present invention;

FIG. 3 (E) is a schematic diagram of the frequency distribution of the SNR of the V1 neurons in NC and DM mice provided by the embodiment of the invention;

fig. 3 (F) is a schematic diagram comparing the V1 neuron Fano fans of NC and DM group mice provided by the embodiment of the present invention;

FIG. 3 (G) is a schematic diagram of the frequency distribution of the V1 neuron Fano Fator in the NC and DM group mice provided by the embodiment of the invention;

FIG. 4 is a schematic diagram of the impairment of synaptic function of V1 neurons in diabetic mice according to an embodiment of the present invention;

FIG. 4 (A) is a schematic diagram of a synaptic connection and synaptic transmission analysis process provided by an embodiment of the invention;

FIG. 4 (B) is an exemplary graph of synaptic connection strength of V1 neurons of mice in NC and DM groups, provided by embodiments of the invention;

FIG. 4 (C) is a graph showing the comparison of synaptic connection strength between V1 neurons in NC and DM groups according to the present invention;

FIG. 4 (D) is a schematic diagram illustrating the comparison of synaptic transmission efficiency of V1 neurons in NC and DM groups provided by the embodiment of the present invention;

FIG. 5 is a schematic representation of the functional properties of LGN neurons in diabetic mice provided by embodiments of the invention unchanged;

FIG. 5 (A) is a schematic diagram comparing LGN neuron OB values of NC and DM mice provided by the embodiments of the present invention;

FIG. 5 (B) is a graphical representation of a comparison of LGN neuron OSI values for NC and DM mice provided by an embodiment of the invention;

FIG. 5 (C) is a schematic diagram comparing the LGN neuron evoked response peaks of NC and DM mice provided by the present example;

FIG. 5 (D) is a schematic diagram of the frequency distribution of the LGN neuron evoked response peaks of NC and DM mice provided by the examples of the present invention;

FIG. 5 (E) is a schematic diagram showing the comparison of LGN neurons SNR of NC and DM mice provided by an embodiment of the present invention;

FIG. 5 (F) is a schematic diagram of the frequency distribution of LGN neurons SNR in NC and DM mice provided by an embodiment of the invention;

FIG. 5 (G) is a schematic diagram comparing LGN neurons of NC and DM mice provided by the embodiments of the present invention, fano Factor;

FIG. 5 (H) is a schematic frequency distribution diagram of LGN neurons Fano Factor of NC and DM mice provided by the embodiment of the invention;

FIG. 6 is a schematic diagram showing the effect of 8,3-THF in improving the metabolic profile of DM mice;

FIG. 6 (A) is a schematic diagram showing the comparison of blood glucose levels of mice in DM and THF groups provided by the present invention;

FIG. 6 (B) is a graph showing the body weight comparison between the DM and THF groups of mice according to the present invention;

FIG. 6 (C) is a schematic diagram showing the comparison between the serum insulin of DM group and THF mouse provided by the embodiment of the present invention;

FIG. 6 (D) is a schematic graph showing the time course of blood glucose in mice in DM and THF groups during administration provided by an embodiment of the present invention;

FIG. 6 (E) is a graph showing the body weight of mice in DM and THF groups as a function of time during the administration of THF as provided in the example of the present invention;

FIG. 6 (F) is a schematic graph showing the change in blood glucose during the OGTT after the end of 4-week THF administration provided by an embodiment of the present invention;

FIG. 6 (G) is a schematic graph showing the change in blood glucose during OGTT after the end of 4-week THF administration, provided by an embodiment of the present invention;

FIG. 7 is a schematic diagram of the improvement of the azimuthal selectivity of V1 neurons in diabetic mice by 8,3-THF, provided by an embodiment of the present invention; (A) Is a schematic diagram of the position selectivity of a single neuron of a DM and THF group V1 provided by the embodiment of the invention; (B) Is a schematic diagram comparing the DM and THF group neuron OB values provided by the embodiment of the invention; (C) Is a frequency distribution diagram of OB values of neurons in DM and THF groups provided by the embodiment of the invention;

FIG. 8 is a schematic diagram of the response characteristics of 8,3-THF in improving V1 neurons in diabetic mice, as provided by an embodiment of the present invention; (A) Is an exemplary graph of V1 single neuron reactivity for mice in DM and THF groups provided by the examples of the invention; (B) Is a comparative schematic diagram of V1 neuron evoked response peaks of mice in DM and THF groups provided by the embodiment of the invention; (C) Is a schematic diagram of the frequency distribution of mouse V1 neuron evoked response peaks in DM and THF groups provided by the embodiment of the invention; (D) Is a comparison schematic diagram of SNR of mouse V1 neurons in DM and THF groups provided by the embodiment of the invention; (E) Is a schematic diagram of the frequency distribution of SNR of mouse V1 neurons in DM and THF groups provided by the embodiment of the invention; (F) Is a comparative schematic diagram of mouse V1 neuron Fano Factor for DM and THF groups provided by the embodiment of the invention; (G) Is a frequency distribution schematic diagram of mouse V1 neuron Fano Factor in DM and THF groups provided by the embodiment of the invention;

FIG. 9 shows that 7,8,3' -THF provided by embodiments of the present invention improves synaptic function in V1 neurons in diabetic mice; (A) Is an exemplary graph of synaptic connection strength between DM group and THF group provided by the embodiment of the invention; (B) Is a comparative schematic diagram of synaptic connection strength between DM group and THF group provided by the embodiment of the invention; (C) Is a diagram of examples of synaptic transmission efficiency in DM and THF sets provided by embodiments of the present invention; (D) Is a comparative schematic diagram of synaptic transmission efficiency between DM group and THF group provided by the embodiment of the invention;

FIG. 10 is a schematic diagram showing the variation of V1 protein expression in different groups of mice provided by the examples of the present invention; (A) Is a comparative schematic diagram of BDNF protein expression in different groups of mice V1 provided by the embodiment of the invention; (B) Is a schematic diagram for comparing the activation of TrkB protein in V1 of different groups of mice provided by the embodiment of the invention; (C) Is a comparison schematic diagram of SYP protein expression in different groups of mice V1 provided by the embodiment of the invention; (D) Is a schematic diagram for comparing the expression of PSD95 protein in V1 of different groups of mice provided by the embodiment of the invention.

FIG. 11 is a graph showing the results of the positional discrimination between Diabetic Mice (DM) and 7,8,3' -THF treatment in the diabetic group according to the present invention.

FIG. 12 is a graph of the results of metformin enhanced excitatory synaptic transmission provided by other literature in evidence of the relevant effects provided by the practice of the present invention; (A) Representative example diagrams of hippocampal CA1 pyramidal neurons tiny excitatory postsynaptic currents meppsc; (B) metformin increases the frequency of mepscs in vertebral neurons; (C) a cumulative probability distribution curve for the mepscs event interval period; (D) Metformin does not affect the amplitude of mepscs of vertebral neurons; (E) cumulative probability distribution curve of mepscs amplitude; (F) Metformin does not affect the membrane potential, membrane capacitance and input impedance of the vertebral neurons.

FIG. 13 is a graph comparing metformin provided by other references to increase glutamate release from hippocampal pyramidal neurons in evidence of relevant effects provided by practice of the present invention; (A) schematic representation of the Paired-pulse reaction (PPRs); (B) PPRs vs. stimulation interval time plot. Met represents metformin.

FIG. 14 is a graph of the inability of metformin provided by the practice of the present invention to affect hippocampal pyramidal neuron excitability; (A) A plot of representative traces of action potentials induced by 200pA depolarisation current injection; (B) a graph of emission rate versus injected current intensity; (C) The slope of the curve of the emission rate versus the intensity of the injected current is compared.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

1. Illustrative embodiments are explained. This section is an explanatory embodiment expanding on the claims so as to fully understand how the present invention is embodied by those skilled in the art.

1.1 methods

1.1.1 construction of diabetic mouse model

In the present invention, all experiments were approved by the committee on animal protection and use of the university of science and technology in china, and all drugs and agents for inducing and maintaining the anesthetic state of animals were in compliance with safety regulations.

The present invention constructs a diabetes model using STZ-induced male Wild Type (WT) mice. The present invention purchased about seven weeks old C57BL/6J mice from shanghai si Bei Fu, randomly divided the mice into a Normal Control (NC) group and a Diabetes (Diabetes mellitis, DM) group after one week of SPF acclimation, and fasted overnight. After a 12h fast, the DM group was given five consecutive days of intraperitoneal injection of 50mg/kg streptozotocin (STZ; sigma, st. Louis, mo, USA). STZ was dissolved in citrate buffer at pH 4.5 for injection. NC group mice were injected with an equal volume of citrate buffer. 7-10 days after completion of STZ injection, accu-Check Active was used

The blood glucose meter measures the fasting blood glucose level of the mouse. When the fasting blood sugar is more than or equal to 250mg/dl, the model of the diabetes is successfully built. Blood glucose and body weight of the mice were measured and recorded weekly throughout the experiment.

1.1.2 glucose tolerance test (OGTT)

An Oral Glucose Tolerance Test (OGTT) was performed. The day before the experiment, the fasting body weight of the mouse is weighed, and the required 20% glucose (1 mg (glucose)/g (body weight)) solution amount is calculated and prepared for standby. After 16h without water deprivation in an overnight fast, the tail was clipped and the blood glucose levels at 0min (before gavage), 15min, 30min, 60min, 90min and 120min of gavage 20% glucose were measured using a glucometer. Upon completion, sufficient food was given immediately.

1.1.3 insulin concentration detection

After fasting, the mice were kept at rest and blood was taken through the tail vein. After the whole blood was allowed to stand at room temperature for 2 hours, the supernatant was centrifuged at 2000rpm for 10 min. Serum insulin concentrations were measured using the insulin Elisa kit.

1.1.4 in vivo Multi-channel electrophysiological recording

(1) Mice were anesthetized with a mixture of isoflurane and oxygen in an in vivo electrophysiological surgery. And the eyes of mice were protected with erythromycin ointment. Subsequently, the scalp is disinfected and cut and the skull surface connective tissue is removed. Craniotomy procedures exposing brain tissue at above V1 (about 3.5mm posterior to bregma and about 2mm lateral to midline) or dorsolateral geniculate (dLGN, about 2.5mm posterior to bregma and about 2.0mm lateral to midline) with an aperture of about 0.5X 0.5cm ² 。

(2) Visual stimuli in the present invention, all stimuli are displayed on a CRT display 30cm from the animal's eye (640X 480 pixels, 75Hz, 45.2cd/m) ² Average brightness; sony G220, japan). The visual stimulus in the experiment was a moving sinusoidal grating, written using MATLAB (MathWorks, USA) and its extension PsychCooolBox-3 (Brainerd, 1997). During stimulation, the optimal spatial frequency and temporal frequency are firstly determined, and then visual stimulation with 10-power is performed by taking the obtained optimal spatial frequency and temporal frequency as fixed parameters. At each stimulation, 12 differently oriented rasters (0 °,30 °,60 °,90 °,120 °,150 °,180 °,210 °, 240 °,270 °,300 °,330 ° and 360 °) and one blank stimulus (gray screen of average brightness) were presented on the screen in a pseudo-random sequence, each stimulus time being 1s. The contrast of the stimulus was set to 95%.

(3) Data recording for neuron signal recording, linear array electrodes (A1X 32, poly2, 177 μm) were implanted by a hydraulic thruster in the present invention ² ；NeuroNexus，USA) to acquire neural signals. All neuron signals were digitized by a front-end signal amplifier (cut-off frequency 10kHz, 1000 × Blackrock, usa salt lake city) and by a neural signal processing system (sampling frequency 30khz,16 bits, blackrock), and finally saved in a hard disk for further processing.

(4) After the electrophysiological experiment, the mouse is perfused with normal saline, the brain tissue is frozen in liquid nitrogen and stored in a refrigerator at-80 deg.c for subsequent experiment.

1.1.5 data analysis

All neuron signals were analyzed using Offline data analysis Offline Sorter software (Plexon, version 3.3.5, USA) for neuron-fired waveforms, separating individual neurons (single unit) from background noise and other neuron waveforms based on 2D or 3D features and spatial distribution, thus obtaining single unit signals. And finally, carrying out data analysis on the separated single unit signal by using a self-written Matlab script.

In order to explore the influence of diabetes on the orientation selectivity of the primary visual cortex of the mouse, the orientation preference and the orientation specificity index of the single unit are detected and analyzed.

Orientation Bias (OB) is one of the important indicators in neuro-optics to measure the selectivity of neuron orientation. The orientation selectivity is described by the ratio of the vector sum of the response of the neuron to each orientation to the sum of the response of the neuron in all orientations, and the calculation method comprises the response in all stimulation orientations and is a relatively comprehensive characterization method.

In this formula, R _K Is theta to the azimuth after removing the baseline _K (radius) neuronal response values for visual stimuli. OB values range from 0 to 1, where OB equal to 0 indicates that the neuron is not selective for any orientation, and OB equal to 1 indicates that the neuron only reacts to a particular orientation. OSI universal serial busBy comparing the reaction intensities for the optimal orientation and the vertical orientation, the following is calculated:

here, R _vertical Is the response of the neuron to a vertical orientation, R _optical Is its reaction to the optimal orientation. OSI values range from 0 to 1, where OSI equal to 1 represents the strongest azimuth selectivity and 0 represents the weakest azimuth selectivity.

Fano Factor (FF) is used to characterize the variability of responses of neuronal responses in the visual system, usually quantified by the ratio of variance to mean firing count,

signal-to-noise ratio (SNR) is an important parameter for measuring the signal extraction capability and fidelity of a single neuron, and is calculated as follows:

in both functions, σ is the standard deviation of the dispensing count, R _means Is the average response of the neuron to each test.

Synaptic connection strength

The present invention uses cross-correlation to describe the strength of synaptic connections. Cross-correlation is calculated using the open source toolkit fieldtip. A cross-correlation histogram (CCH) is constructed for the firing sequences of synchronously recorded neuron pairs. The bin width of the CCH is 2ms and extends around zero time lag by 200ms. Before characterizing the CCH shape, the present invention corrects the original CCH by subtracting a shift predictor (shift predictor) and smoothes the shift-corrected CCH using a 5ms kernel ([ 0.005 0.25.40.25.0.005 ]). The peak is determined within a zero time lag of 10 ms. At time lags from-200 ms to-150 ms and from 150ms to 200ms, only the peak of 5SDs above the noise level is considered significant, that is, this has a theoretical functional connection to the neuron. For neuron pairs recorded from the same electrode, the present invention ignores the-2 to +2ms interval because the CCH cannot detect simultaneous or near-simultaneous firing, which can lead to artificial peaks in the CCH. The peak height is defined as the amplitude of the peak and is used to characterize the connection strength of the neuron pair.

Efficiency of synaptic transmission

Based on CCH analysis, the present invention calculates synaptic transmission efficiency for pairs of neurons with functional connections. The peak position is defined as the time lag of the peak in the CCH. Based on this, the present invention moves and aligns firing sequences of neuron pairs such that peak positions of a reference neuron (pre-synaptic neuron) and a target neuron (post-synaptic neuron) are the same. The invention then uses a sliding window with bin width of 5ms to slide along their firing sequence. If both produce firing in a sliding window, the firing of the post-synaptic neuron is considered to be caused by the pre-synaptic neuron, denoted as a synaptic transmission. The present invention calculates the ratio of the number of transmissions occurring in each trial to the total number of firing, with the average representing the efficiency of transmission between neurons.

1.1.6 administration treatment of diabetic mice

In the present invention, all experiments were approved by the committee on animal protection and use of the university of science and technology in china, and all drugs and agents for inducing and maintaining the anesthetic state of animals were in compliance with safety regulations.

The successfully modeled diabetic mice were randomly divided into DM and THF groups. The mice in the THF group were injected intraperitoneally with 5mg/kg 7,8,3' -THF (dissolved in 17% DMSO) for 4 weeks, while the mice in the NC and DM groups were injected with an equal volume of a PBS solution dissolved in 17% DMSO. Blood glucose and body weight of each group of mice were measured and recorded weekly throughout the experiment.

1.1.7 glucose tolerance test (OGTT)

OGTT experiments were performed after 4 weeks of dosing, and it was examined whether 7,8,3' -THF affected metabolic characteristics of diabetes. After an overnight fast of 16h, blood glucose levels were measured in tail-tip bleeding using a glucometer before gavage of 1mg/g 20% glucose (0 min) and after 15, 30, 60, 90 and 120 min.

1.1.8 serum insulin concentration detection

After 4 weeks of administration, tail vein blood sampling was performed to detect serum insulin concentration, and it was examined whether 7,8,3' -THF affected the diabetic serum insulin content. Tail vein bleeds were performed after fasting mice. After the blood was allowed to stand at room temperature for 2 hours, the supernatant was collected by centrifugation at 2000rpm for 10 min. Serum insulin concentrations were measured using an insulin kit.

1.1.9 protein immunoblotting (Western blot)

(1) Extracting tissue protein, taking frozen V1 tissue (n = 6-8/group) from a refrigerator at-80 ℃ after the electrophysiological experiment is finished, placing the tissue in an EP tube, adding a proper amount of RIPA lysate, fully grinding and crushing to obtain homogenate, and then continuously cracking on ice for 1h. The lysed homogenate was centrifuged at 12000 rpm at 4 ℃ for 15min and the supernatant collected.

(2) BCA assay protein concentration the total protein concentration extracted was determined by means of a bicinchoninic acid (BCA) assay quantification kit. After the absorbance value is measured, a standard curve is drawn and the concentration of the extracted sample is calculated. Leveling the protein concentration of each sample according to the Loading concentration, adding an equal volume of Loading buffer, boiling for 10min in boiling water, and storing at-20 ℃ for later use.

(3) Electrophoresis was carried out on 12% separation gel and 4.5% concentrated gel to separate the target protein by SDS-PAGE gel electrophoresis. And (3) performing constant-pressure 80V electrophoresis, adjusting the voltage to 120V after the sample is transferred to the separation gel until the protein band reaches the bottom end, and finishing the electrophoresis.

(4) After membrane conversion methanol activates polyvinylidene fluoride (PVDF) membrane, a sandwich clamp consisting of three layers of filter paper, PVDF membrane and gel is placed in a groove in which membrane conversion solution is added, and wet membrane conversion is carried out. Constant current 250mA, and film turning on ice for 2h.

(5) Blocking the PVDF membrane containing the target protein is placed in a 5% bovine serum albumin solution and is blocked for 1h in a shaking table at room temperature.

(6) After blocking of antibody incubation, primary antibodies, including anti- β -actin (Cell Signaling Technology,1: 1000), BDNF (Sigma-Aldrich, 1: 1000), trkB (Abcam, ab178847,1: 1000), p-TrkB (Millipore, #5541, 1. The following day, protein bands were washed and incubated with horseradish peroxidase (HRP) conjugated anti-rabbit or anti-mouse secondary antibodies (Cell Signaling Technology, 1.

(7) Visualization western blots were detected using Electrochemiluminescence (ECL). The optical density of the strips was quantitatively analyzed using NIH Image J software to obtain results.

1.1.10 statistical analysis

Statistical analysis was performed using GraphPad Prism software (Version 9. The distribution of all data was first analyzed and for normal distributions the data are expressed as mean ± SEM and compared using the unoiled T test. For non-normal distributions, a comparison was made using Mann Whitney test and the data is shown as Median. P <0.05 was considered statistically significant.

1.2 results of the experiment

1.2.1 Metabolic Profile alterations in diabetic mice

Before injection of STZ, the mice are active and agile, the hair color is bright, the diet, drinking water and urine volume are normal, the body weight is normally increased along with time, and the inside of the cage is dry; after injection, the mice are dull and dull, the hair color is dull, the food and water consumption is obviously increased, the weight is slowly increased and even reduced, and the urine volume is increased. No mice died during the experiment.

Typical metabolic features of STZ-induced DM mice are elevated fasting glucose (BG) and reduced Body Weight (BW). Therefore, the blood glucose and body weight of the mice were measured and recorded before (0 w) and after (1 w, 2w, 3w, 4w, 5w, 6 w) SZT injection. As shown in a in fig. 1, there was no significant difference in blood glucose after fasting between Two groups of mice before injection (0 w), blood glucose began to rise gradually in the DM group given SZT injections of 5d consecutively (a, two-way ANOVA, F (1,49) =776.9, p <0.0001 in fig. 1), which had exceeded 13.9mg/dL at week 2, and blood glucose was maintained at a high level, significantly higher than that in the NC group, during the subsequent experiments. For body weight, mice started to lose lean after STZ injection, decreased body weight and were significantly lower than normal mice (B in fig. 1, two-way ANOVA, F (1,49) =199.6, P < 0.0001). Before electrophysiological experiments, the DM group mice exhibited significantly elevated blood glucose (C in fig. 1, unaired T test, mean =6.075 ± 0.1398, 21.31 ± 1.425, p-straw 0.0001) and reduced body weight (D in fig. 1, unaired T test, mean =28.37 ± 1.488, 21.17 ± 1.331, p-straw 0.0001), typical metabolic characteristics of these DMs indicated that DM mice were successfully modelled. Meanwhile, SZT-induced diabetic mice are based on the principle that destruction of islet β cells leads to a decrease in insulin release, i.e., the formation of T1DM, which is absolutely deficient in insulin in the general sense. Thus, the present invention verifies serum insulin levels. As shown by E in fig. 1, the DM group mice did have significant reduction in serum insulin (E in fig. 1, unaired T test, mean =0.2586 ± 0.06218,0.08939 ± 0.03228, p-herd 0.0001).

In addition, the Oral Glucose Tolerance Test (OGTT) is used to measure glucose tolerance, i.e. to assess the body's ability to catabolize glucose. After an overnight fast, blood glucose levels were measured 15, 30, 45, 60, 90, 120min after gavage of glucose (1 g/kg). In F in FIG. 1, the present inventors can see that the blood glucose in DM group rapidly rises and has a large amplitude, reaches a peak at around 15min, and the decline is relatively slow, indicating that impaired glucose tolerance of DM group mice has occurred, while the blood glucose level in NC group reaches a peak and returns to the basal level immediately after about 60min, indicating normal glucose consumption (F, two-way ANOVA, F (1,42) =379.3, P < (0.0001) in FIG. 1). Meanwhile, the area under the OGTT curve AUC was calculated, as shown in G in fig. 1, the DM group was higher than the NC group, and again the DM group mice were verified to have reduced glucose metabolism ability (G in fig. 1, unaired T test, mean =1130 ± 67.94, 2588 ± 166.8, p = 0.0028). In summary, after STZ injection, DM mice were successfully modeled, and metabolic characteristics such as blood glucose, body weight, insulin and glucose metabolism were changed in DM group mice.

1.2.2 Azimuth Selective decline of diabetic mouse V1 neurons

Azimuthal orientation selectivity is one of the most important receptive field properties of primary visual cortical (V1) neurons, determining perception of object edges and contours. To explore the impact of diabetes on the visual cortex, the present invention first examined the azimuthal selectivity of individual neurons in V1, as shown at a in fig. 2. B in fig. 2 shows exemplary diagrams of single neuron orientations from the NC group and the DM group, respectively. In C in fig. 2, the present invention statistically counts OB values from different groups of mice, and finds that the DM group neurons OB value is decreased, indicating that there is an orientation selective defect in the DM mouse cortical neurons (C in fig. 2, man Whitney test, media =0.1011,0.08245, n =191, 164, p = 0.0009). OSI is also used to describe the directionally selective variation of neurons. D in fig. 2 counts and analyzes OSI values for neurons in different groups. Similar to OB results, OSI values for diabetic mouse opsortical neurons decreased (D in fig. 2, mann Whitney test, median =0.2089, 0.1828, n =191, 164, p = 0.0096). The above results indicate that in the visual cortex in diabetic mice, the azimuthal selectivity of V1 neurons is impaired.

1.2.3 diabetic mice have impaired response characteristics of V1 neurons

The present invention measures the peak response of neurons in the presence of visual stimuli to characterize neuronal activity. A in fig. 3 is an exemplary diagram of a typical firing of a single neuron from the NC and DM group visual cortex. The upper is the Raster plot of the example neuron firing in each Trial, and the lower is the line plot of the neuron firing intensity variation over 2 s. The peak firing rate of the DM group-induced responses was significantly lower than that of the NC group (B in fig. 3, mann Whitney test, media =14.6,9.4, n =245,250, p-straw 0.0001), indicating that diabetes impaired the activity of visual cortical neurons.

In the process of information processing, neurons must distinguish the desired signal from background noise to ensure the accuracy of information encoding, which is measured by the signal-to-noise ratio (SNR). The SNR reflects the fidelity of the neuron to the stimulus coding. Fano Factor is an important parameter for characterizing the response variability of neurons, which is often regarded as "noise" reflecting the degree of instability in information processing of the visual system. In the nervous system, large neuron response variability may limit information encoding capabilities. Therefore, the invention discusses the influence of diabetes on the information processing capability of cortical neurons by analyzing SNR and Fano Factor. The results show that neurons of DM group exhibited lower SNR (D in fig. 3, mann Whitney test, media =1.517,1.058, n =245,250, p <0.0001) and higher Fano far (F in fig. 3, mann Whitney test, media =1.55,1.701, n =245,250, p = 0.01108) than NC group, indicating that diabetes increased the response variability of neurons and decreased the coding fidelity, leading to impaired cortical neuron information processing ability.

1.2.4 synaptic function impairment in diabetic mouse V1

The above results indicate that in the context of diabetes, the orientation selection and information processing functions of visual cortical neurons decline. Neurons in cortical networks form functional connections through synapses, collectively encoding neural information. Then is this functional decline associated with an impaired functional connection between the interconnected neuron pair? Thus, the present invention quantifies and analyzes synaptic function of the cortex in an attempt to decode the neural mechanisms by which DM contributes to the impairment of neuronal function. The strength of cross-correlation is first used to measure synaptic connections between pairs of neurons. A in FIG. 4 is a schematic diagram of a computational analysis of synaptic connections and synaptic transmission of pairs of functionally connected neurons. On the left is a pair of neurons with functional connections. Yellow represents pre-synaptic neurons and green is post-synaptic neurons. If the two have synaptic connections, as shown on the right side, then the time window of the sliding post-synaptic neuron has a certain time point, so that the firing of the two neurons before and after the synapse has a certain synchronicity in time sequence, in other words, the firing correlation is the highest, and the firing cross-correlation strength at this time is the synaptic connection strength of the defined neuron pair. With respect to the efficiency of synaptic transmission, after a pre-synaptic neuron produces a firing, a post-synaptic neuron also produces a firing, as indicated by the red box on the right, indicating that the pre-synaptic neuron causes a firing of the post-synaptic neuron, i.e., produces a defined one-time synaptic transmission, whereas, no synaptic transmission is produced, as indicated by the blue box. And calculating the ratio of the synaptic transmission times of the postsynaptic neurons to the total number of the postsynaptic neurons, wherein the ratio is the defined synaptic transmission efficiency. The correlation plot (CCH) of B in fig. 4 is an example plot of synaptic connection strength, with higher peaks indicating stronger synaptic connection strength. The magnitude (peak height) of CCH in the DM group V1 neuron pairs was significantly reduced compared to the NC group. A decrease in the peak indicates that diabetes impairs the synaptic connection strength of the local neural network. 359 pairs and 203 pairs of neurons with synaptic connections are obtained in the NC and DM groups respectively. C in fig. 4 is a statistical result of the synaptic connection strength of each group of neurons, and the neuron pair connection strength of the DM group was found to be significantly decreased (C in fig. 4, mann Whitney test, media =0.01568,0.01323, n =359, 203, p = 0.0009). Synaptic transmission efficiency is used to characterize the efficiency of information transmission of neurons around synapses, and is also an important parameter describing synaptic function. The results show that the synaptic transmission efficiency of the neurons in the DM group also decreased significantly (D in fig. 4, mann Whitney test, median =0.256,0.1763, n =359, 203, p = 0.0021). Thus, these results indicate that the functional connections between V1 neurons in diabetic mice are impaired, i.e. synaptic dysfunction.

1.2.5 diabetic mice were not altered in the functional properties of LGN neurons

Although the results of the present study indicate that diabetes impairs the directionally selective intensity of V1 neurons, the impact of LGN is not negligible. As known in the present invention, in the classical visual pathway, visual information is projected through the LGN to the primary visual cortex and then passed on to higher-level cortex for further processing. And studies showed that the directional preference of the mouse primary visual cortex is in part from LGN. Therefore, it is necessary to determine whether primary cortical retinal neuronal changes are from the primary visual cortex or the subcortical pathways (LGN and more primary visual systems such as the retina, etc.). The present invention records and analyzes the change in receptor field characteristics and firing of dLGN neurons. The data show that diabetic mice OB (a in fig. 5, mann Whitney test, media =0.0977,0.1093, n =218,202, p > 0.9999) and OSI values (B in fig. 5, man Whitney test, media =0.2085, 0.2213, n =218,202, p = 0.8297) are not significantly different from the control group, indicating that the neuron-azimuthally-selective lesions in diabetic mice do not originate from the subcortical pathways but the primary visual cortex itself. Evidence of neuron firing and information transmission fidelity also supports this view. Since between diabetic mice and the control group, neuron firing (D in fig. 5, mann Whitney test, media =3.8,3.7, n =245,250, p > 0.9999), signal-to-noise ratio SNR (E in fig. 5 and F in fig. 5, mann Whitney test, media =0.7958,0.7443, n =245,250, p = 0.9397) and Fano factor (G in fig. 5 and H in fig. 5, mann Whitney test, media =1.292,1.248, n =245,250, p = 0.1755) also did not differ.

1.2.6 7,8,3' -THF did not improve the metabolic profile of diabetic mice

The metabolic profile of the mice remains a first concern of the present invention (fig. 6). The present inventors found that after the end of the 4-week period of THF administration, the metabolic characteristics of the DM group mice did not improve and still exhibited the characteristics of diabetes, such as sustained hyperglycemia (a in fig. 6, unapiared T test, mean =21.31 ± 1.425, 20.188 ± 1.444, p = 0.5838) and significantly reduced body weight (B in fig. 6, unapiared T test, mean =21.17 ± 0.4706, 21.83 ± 0.3846, p = 0.2906) and plasma insulin (C in fig. 6, unapiared T test, mean =0.08938 ± 0.01141,0.1038 ± 0.01478, p = 0.4523). And no significant difference was observed between blood glucose (D in fig. 6, two-way ANOVA, F (1,49) =3.077E-031, p > -0.9999) and body weight (E in fig. 6, two-way ANOVA, F (1,49) =3.212, p = 0.0817) in the Two groups of DM mice given 7,8,3' -THF and solvent control throughout the dosing period. Similarly, the present inventors performed OGTT to investigate whether 7,8,3' -THF affects the blood glucose metabolic capability of mice. The trend lines for blood glucose changes during OGTT were nearly overlapping in mice given 7,8,3' -THF compared to DM mice given solvent control, meaning that both had similar glucose metabolism ability (F, two-way ANOVA, F (1,49) =0.002695, P =0.9591 in figure 6), and the AUC of the second Two groups was also not different (G, unaired T test, mean =2588 ± 160.5, 2562 ± 139.2, P =0.902 in figure 6). Taken together, these data indicate that 7,8,3' -THF does not have any improving effect on metabolic indicators of DM mouse disorders, including blood glucose, body weight, insulin, etc.

1.2.7 7,8,3' -THF improved the regioselectivity of V1 neurons in diabetic mice

To explore the effect of 7,8,3'-THF on the visual cortex of diabetes, after 4 weeks of 7,8,3' -THF administration was completed, the present invention recorded neuronal responses at V1 by multichannel electrophysiology and analyzed the azimuthal selectivity of individual neurons. Shown as a in fig. 7 is a map of the azimuthal preference of typical neurons of the visual cortex in the DM and THF groups. Statistically, it was found that OB values were significantly increased in mice treated with 7,8,3' -THF compared to solvent-controlled DM mice (D in fig. 7, mann Whitney test, median =0.0825,0.1046, n =164, 244, p =, 0.00094). Similarly, the present invention analyzes OSI values for both groups of mouse neurons. Similarly to this result, OSI values of THF group mice increased (D in fig. 7, mann Whitney test, median =0.1828,0.2211, n =164, 244, p = 0.0152). The results of the above studies indicate that chronic THF administration reverses the reduced azimuthal selectivity of neurons in diabetic mice, thereby improving the azimuthal discrimination of the visual cortex.

1.2.8 7,8,3' -THF improves the response characteristics of V1 neurons in diabetic mice

To further investigate whether THF can improve the response characteristics of visual cortical neurons, the present invention examined evoked responses to analyze neuronal activity. A in fig. 8 is a typical example of the neuron activity of the mice in the DM group and the THF group. Data from the THF group showed that 7,8,3' -THF treatment significantly increased the firing rate of diabetic mouse optic cortical neurons, effectively restoring their activity (B in fig. 8 and C in fig. 8, mann Whitney test, median =9.4, 13,n =250, 337,p <0.0001). Further, the invention discusses whether 7,8,3' -THF has a promoting effect on information processing damage of the cortical neurons induced by diabetes by analyzing SNR and Fano Factor. The results show that 7,8,3' -THF administration resulted in a significant improvement in the neuronal response characteristics of the diabetic visual cortex, exhibiting higher SNR (D in fig. 8 and E in fig. 8, mann Whitney test, median =1.058,1.345, n =250, 337, p <0.0001) and lower Fano Factor (F in fig. 8 and G in fig. 8, mann Whitney test, median =1.701,1.526, n =250, 337, p = 0.0131), indicating that long-term 7,8,3' -THF administration reversed the increased response variability and decreased coding fidelity induced by diabetes, that is 7,8,3' -THF administration improved the impaired information handling capacity of diabetic mice.

1.2.9 7,8,3' -THF improved synaptic function in diabetic mouse V1

Electrophysiological results showed that 7,8,3' -THF improved the azimuthal selectivity and information processing ability of DM mouse V1, and then, did it have an improving effect on the synaptic function of the cortex? Thus, the present invention also analyzed the synaptic connection strength of neuronal pairs in the mouse visual cortex after THF administration. The present invention pairs 203 and 349 pairs of neurons in the DM and THF groups, respectively. As shown by the CCH plot of a in fig. 9, the peaks in the DM group were significantly lower than those in the THF group, indicating that the synaptic connection strength of V1 in diabetic mice was reversed after dosing. Statistical analysis confirmed that 7,8,3' -THF was beneficial in enhancing the functional connectivity of paired neurons of the diabetic visual cortex (B, DM: mean =0.01323,0.01618, n =203, 349, p =0.002 in fig. 9). By calculating synaptic transmission efficiency, the present invention observes similar changes to synaptic connection strength: the transmission efficiency in the THF group administered was higher than that in the solvent-controlled DM group, indicating that the decreased synaptic transmission efficiency between neurons in the DM group was reversed by chronic THF administration (D, DM: mean =0.1763,0.2755, n =203, 349, p =0.0009 in fig. 9). Thus, these results indicate that 7,8,3' -THF improves the impaired functional properties of synapses for V1 neuronal interactions in diabetic mice.

1.2.10 7,8,3' -THF improves BDNF/TrkB pathway and synapsin expression in diabetic mice

The BDNF/TrkB signal channel can regulate excitatory synaptic transmission, increase cortical excitability and play an important role in visual perception. Impairment of the brain hippocampal BDNF/TrkB signaling pathway has been reported multiple times in diabetes. Electrophysiological experiments of the present invention indicate that TrkB receptor agonist 7,8,3' -THF can improve the functional properties of the retinal neurons in DM mice, and then whether such improvement is associated with the regulation of the BDNF/TrkB signaling pathway in the visual cortex? Thus, to further elucidate the underlying mechanism of the beneficial effects of 7,8,3' -THF on diabetic neuronal function, the present invention measures the expression of BDNF/TrkB signaling pathway protein in the primary visual cortex using Western Blot. DM mice had lower levels of p-TrkB/TrkB ratios compared to normal mice (B, one-way ANOVA, F (2,21) =4.705, p =0.0205 in fig. 10). The expression levels of BDNF (a, one-way ANOVA, F (2,21) =4.705, p =0.0205 in fig. 10) and TrkB (B, one-way ANOVA, F (2,21) =0.9185, p =0.4146 in fig. 10) were not different between the groups. While the diabetic group treated with 7,8,3'-THF up-regulated TrkB phosphorylation, indicating that in the DM primary visual cortex, trkB receptor activation (p-TrkB) was disturbed so as to impair the normal physiological function of this pathway, while external application of the receptor agonist 7,8,3' -THF can up-regulate TrkB phosphorylation levels, thereby effectively improving disturbed BDNF/TrkB pathway function.

At the same time, the expression of synapse-associated proteins supports the electrophysiological consequences of synaptic injury previously observed in the present invention. SYP and PSD95 are synapse-specific expression proteins, often used as molecular markers of synaptic function, and the present invention selects detection of changes in both to evaluate synaptic damage. The results showed that SYP (C, one-way ANOVA, F (2,18) =11.80, p =0.0005 in fig. 10) and PSD95 protein (D, one-way ANOVA, F (2,15) =7.27, p = 0.0062) expression was significantly reduced in the primary visual cortex in the mice of the diabetic group compared to the normal control group, while treatment with 7,8,3' -THF reversed the reduction in SYP and PSD95 protein in the mice of the diabetic group. These results indicate that diabetes damages the BDNF/TrkB signaling pathway and synaptic function, and 7,8,3' -THF acts on the pathway, can effectively activate TrkB receptors, prevent synaptic loss and improve synaptic function.

2. Application examples. In order to prove the creativity and the technical value of the technical scheme of the invention, the part is the application example of the technical scheme of the claims on specific products or related technologies.

Example 1

1. Construction of diabetic mouse model

Mice were divided into Normal Control group (Normal Control, NC) and diabetic group (Diabetes mellitis, DM). The mice used for modeling were seven week old male C57BL/6J mice, fasted overnight prior to modeling. After fasting for 12h, the DM group was given a five-day continuous intraperitoneal injection of 50mg/kg streptozotocin (STZ; sigma, st. Louis, mo., USA) dissolved in citrate buffer (pH 4.5). NC mice were injected with only an equal volume of citrate buffer. 7-10 days after completion of injection, accu-Check Active was used

The blood glucose meter measures the fasting blood glucose level of the mouse. When the fasting blood sugar is more than or equal to 250mg/dl, the model of the diabetes is successfully built. Blood glucose and body weight of the mice were measured and recorded weekly throughout the experiment.

2.7,8,3' -THF in STZ-induced improvement of visual perception in diabetic mice

Mice were divided into a Normal Control group (Normal Control, NC), a diabetic group (Diabetes mellitis, DM), a Diabetes-imparting 7,8,3' -THF group (THF), seven-week-old male C57BL/6J mice, and a diabetic mouse model was constructed using Streptozotocin (STZ).

Normal Control group (Normal Control, NC): the fasting state is 12h before the injection of citrate buffer. Visual perception was measured 4 weeks after injection of 17% dmso in PBS.

Diabetes group (Diabetes mellitis, DM): after fasting for 12 hours, streptozotocin dissolved in a citric acid buffer solution is injected on an empty stomach, and when the fasting blood sugar is more than or equal to 250mg/dl, the diabetes model is successfully constructed. The visual perception assay was performed 4 weeks after the DM group mice were injected with 17% DMSO in PBS.

Diabetes administration 7,8,3' -THF group (DM + THF): the DM group mice were injected intraperitoneally with 5mg/kg 7,8,3' -THF (dissolved in 17% DMSO) for 4 weeks before visual perception was examined.

Go nogo visual behavior detection device: the mouse head was placed in an acrylic tube. The display was placed 30cm in front of the mouse. When the visual stimulus (go stimulus) that the mouse is required to respond to appears, the mouse responds by licking the tap. A set of infrared emitting and receiving devices is used for detecting whether the tongue of the mouse touches the water nozzle. When the mouse responds correctly to the go stimulus, the water nozzle pumps out water droplets to reward the mouse. When the disturbing stimulus (no go stimulus) occurs, the mouse cannot lick the water tap, otherwise it will be punished to blow on its face, while the next visual stimulus appears 7 seconds later.

Visual behavior training: 1) Habitual training without visual stimulation, and the mouse can get rewarding of water drops when licking the water nozzle each time; 2) Conditioned reflex training, training mice to lick in response to visual stimuli, we set the vertically appearing grating to go stimuli, which is only go stimuli. There is a cue tone one second before the stimulus appears and go stimulus appears for 4 seconds. If licking was detected within the last 2 seconds of the visual stimulus appearing (reaction window), the mouse was rewarded with 4 μ l of water. If licking is not detected during the reaction window, a reward is given to the water at the end of the conditioned reflex phase visual stimulus. Once the hit frequency exceeds 150 in 30 minutes, the mouse enters the next stage; 3) Go-nogo stimulation training, 5-10 days, this section contains Go and nogo stimuli (horizontally oriented to the grating), both of which occur randomly, licking in the response window of Go stimuli will result in a water drop reward, licking in the response window of no Go stimuli will result in a 100ms insufflation penalty and delayed appearance of the next visual stimulus. Visual behavior detection: after the mouse learns to distinguish between go and no go stimuli, the mouse needs to learn to distinguish between no go visual stimuli which are different from go stimuli by a certain angle (delta theta =30 degrees, 60 degrees and 90 degrees), and the smaller the delta theta, the more difficult the task is, and the more difficult the mouse is to distinguish between the difference and the go stimuli. We used d to distinguish the performance of mice in different Δ θ visual stimulus discrimination, d representing the degree of difference between the distribution of mouse discrimination correctness for go stimulation and wrong discrimination ratio distribution for no go stimulation, with larger indicating higher degree of difference and better mouse discrimination for visual stimulus, calculated as d = norm (hit rate) -norm (false report rate). norm is the inverse of the cumulative normal function. Hit rate = number of go-stimulated hits/(number of go-stimulated hits + number of go-stimulated non-hits). No go stimulation false report rate = No go stimulation false report number/(No go stimulation false report number + No go stimulation correct rejection number). When the mice exhibited d >0.5 on visual stimulus discrimination of Δ θ =30 °, the mice were initially tested for visual stimulus discrimination of Δ θ =10 °.

Visual behavior detection results: as shown in fig. 11, 7 mice in DM group, 5 mice in DM + THF group, go nogo behavioral training, and go nogo behavioral testing after the mice had achieved d >0.5 for respective ability of Δ θ =30 °, test angle Δ θ =10 °, it can be seen that the mice in DM + THF group with 7,8,3'-THF intervention had significantly better orientation discrimination on visual stimuli than the mice in DM group without 7,8,3' -THF intervention, the average d of DM group was 0.0444 ± 0.09242 (mean ± SEM), the average d of DM + THF group was 0.608 ± 0.1634 (mean ± SEM), p =0.0303, man Whitney test.

3. Evidence of the relevant effects of the examples. The embodiment of the invention achieves some positive effects in the process of research and development or use, and has great advantages compared with the prior art, and the following contents are described by combining data, diagrams and the like in the test process.

The research result shows that the diabetic has the function damage of the central nervous system, the diabetic dementia is a typical representative, and the recent research of Zhou Yifeng and the like shows that the function of the visual cortex neurons of the diabetic also has the serious damage, which is represented by the reduced neuron reaction strength, the reduced neuron orientation direction tuning and the reduced neuron reaction signal-to-noise ratio. However, no diabetes treatment medicine specially aiming at improvement of neuronal function damage exists at present, and the diabetes treatment medicine specially aiming at improvement of visual center is a blank. Metformin is a first-line drug for treating type 2 diabetes, and has been reported to have an effect of improving central nervous system functions in addition to controlling blood glucose, and metformin can increase the release of glutamate from hippocampal pyramidal neurons (fig. 12) and increase the transmission of excitatory synapses (fig. 13), but does not change the firing strength of excitatory neurons (fig. 14), so that metformin is difficult to improve functional impairments such as down-regulation of response strength of visual cortical neurons in diabetic animals.

The invention specifically activates a BDNF/TrkB receptor signal channel by using 7,8,3' -THF, obviously improves the synaptic connection strength and information transmission efficiency between neurons (figure 9), obviously improves the release strength of the neurons (figure 8), and enhances the orientation discrimination capability of diabetic mice (figure 7), and the improvements are not reported to exist in the curative effects of metformin and other drugs.

The above description is only for the purpose of illustrating the embodiments of the present invention, and the scope of the present invention should not be limited thereto, and any modifications, equivalents and improvements made by those skilled in the art within the technical scope of the present invention as disclosed in the present invention should be covered by the scope of the present invention.

Claims (10)

1. Application of TrkB agonist in preparing medicine for treating visual cortex damage is provided.

2. The use of a TrkB agonist according to claim 1 in the preparation of a medicament for the treatment of visual cortex damage, further comprising: application of TrkB agonist in preparing medicine for relieving and preventing visual cortex damage is provided.

3. Use of a TrkB agonist according to any one of claims 1-2 in the manufacture of a medicament for the treatment of visual cortex damage, wherein the TrkB agonist is a small molecule TrkB agonist; the small molecule TrkB agonist is 7,8,3' -THF.

4. Use of a TrkB agonist according to any one of claims 1 to 3 in the manufacture of a medicament for the treatment of visual cortex damage, wherein the visual cortex damage is caused or caused by diabetes.

5. Use of a TrkB agonist according to any one of claims 1 to 4 in the manufacture of a medicament for the treatment of visual cortex damage, wherein the TrkB agonist acts by improving the azimuthal selectivity, response characteristics and synaptic function of neurons.

6. Use of a TrkB agonist according to any one of claims 1 to 4 in the manufacture of a medicament for the treatment of visual cortex damage, wherein the TrkB agonist acts by modifying the impaired BDNF/TrkB signalling pathway.

7. Use of a TrkB agonist according to any one of claims 1-2 in the manufacture of a medicament for the treatment of visual cortex damage, further comprising: the TrkB agonist is administered according to a dosing regimen repeated once weekly for 4 weeks.

8. Use of a TrkB agonist according to claim 7 in the preparation of a medicament for the treatment of visual cortex damage, wherein said administration of the TrkB agonist comprises: 7,8,3' -THF was dissolved in 17% DMSO for TrkB agonist administration.

9. Use of a TrkB agonist according to claim 7 in the manufacture of a medicament for the treatment of visual cortex damage wherein the TrkB agonist is administered in an amount of 5mg/kg.

10. Use of a TrkB agonist according to claim 7 in the preparation of a medicament for the treatment of visual cortex damage, wherein said administration comprises: the administration is carried out in the abdominal cavity.

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