CN113009127A

CN113009127A - Application of biosensor and protein combined immunoreaction in detection of deoxyschizandrin signal channel for treating oligospermia

Info

Publication number: CN113009127A
Application number: CN201911316164.4A
Authority: CN
Inventors: 吴志生; 郑燕飞; 马丽娟; 李博怿
Original assignee: Beijing University of Chinese Medicine
Current assignee: Beijing University of Chinese Medicine
Priority date: 2019-12-19
Filing date: 2019-12-19
Publication date: 2021-06-22
Anticipated expiration: 2039-12-19
Also published as: CN113009127B

Abstract

The invention relates to an application of a biosensor and protein immunoreaction in detecting a signal path for treating oligospermia by deoxyschizandrin, which is characterized by comprising an AlGaAs/GaAs HEMT biosensor and protein immunoreaction; through the constructed AlGaAs/GaAs HEMT biosensor, a schizandrin A in-vitro detection experiment for treating the oligospermia signal pathway is carried out, and further combined with protein immunoreaction, a schizandrin A in-vivo pathway detection experiment for treating the oligospermia signal is carried out, so that the schizandrin A plays a role in treating the oligospermia and the oligospermia by regulating an SCF/c-kit pathway; the detection method has strong specificity, the detection concentration is as low as pg/pM level, the detection range covers five orders of magnitude, and the detection method has high sensitivity and strong specificity.

Description

Application of biosensor and protein combined immunoreaction in detection of deoxyschizandrin signal channel for treating oligospermia

Technical Field

The invention relates to the field of traditional Chinese medicines, in particular to application of a biosensor and protein immunoreaction in detection of a deoxyschizandrin signal pathway for treating oligospermia.

Background

Studies have shown that about 5% of the heterosexual couples worldwide have infertility problems. However, more couples have experienced at least one year of involuntary non-children: estimated between 12% and 28%. The male infertility accounts for 20-30% of infertility cases, and researches show that the incidence rate of the male infertility is remarkably increased in recent years, the most common reason is semen deficiency, wherein the male infertility caused by oligospermia and weak sperm is up to 60%, the family harmony and the life quality of people are seriously influenced, and the male infertility is a key difficult problem to be overcome urgently in the world.

Although a great deal of research is carried out on the modern medicine aiming at oligospermia and asthenospermia, a treatment medicine with exact curative effect is not available at present. The traditional Chinese medicine has a long history of treating male infertility based on the overall view and the theory of treatment based on syndrome differentiation, and has definite curative effect. The key pathogenesis of oligospermia and asthenospermia proposed by one of the Wangchen Qi Yao of the traditional Chinese medicine in the science of andrology is 'kidney deficiency with dampness and heat stasis poisonous insect', and the treatment principle of 'tonifying kidney and replenishing essence, clearing heat and promoting diuresis, and promoting blood circulation and removing blood stasis' is proposed according to the pathogenesis, so that the first spermatogenic prescription medicine 'Huangjingzanyu capsule' specially used for treating male infertility in China is researched. Clinical results show that the medicine can increase the number of sperms and improve the activity of the sperms, and has obvious curative effect on male infertility caused by oligospermia and asthenospermia. Early researches show that the active ingredient deoxyschizandrin has the function of improving sperm motility, is expected to develop a low-toxicity and high-efficiency sperm-producing medicament, and the action mechanism of the deoxyschizandrin is still to be further researched.

The method currently used to detect signaling pathways is the protein immunoreaction method, but this method alone lacks an intuitive characterization of the mechanism of action. High Electron Mobility Transistor (HEMT) is used as a third-generation electrochemical device, and provides a key leading-edge technical support for the identification of an intermolecular interaction mechanism by virtue of higher sensitivity and stronger specificity. However, the research of the mechanism of improving the activity of the sperm of the samples with the hypospermia and the asthenospermia by combining the biosensor technology and the protein immunoreaction method is still blank.

Disclosure of Invention

The invention aims to solve the technical problems that the reliability of an independent in-vitro experiment is lacked, and the intuitive representation problem of the action mechanism of the independent in-vivo experiment is lacked, and provides an in-vitro experiment combined with a high-sensitivity and strong-specificity HEMT biosensor and an in-vivo verification experiment method based on a protein immunoreaction method, so that the action mechanism of schizandrin A for improving the sperm activity of a sample with few and weak sperm diseases is analyzed.

The invention provides an application of an AlGaAs/GaAs HEMT biosensor combined protein immunoreaction method in a path for detecting a deoxyschizandrin-treated oligospermia signal, and firstly provides an application of an AlGaAs/GaAs HEMT biosensor in a path for detecting a deoxyschizandrin-treated oligospermia signal, wherein the specific technical scheme is as follows:

(1) the c-kit-AlGaAs/GaAs HEMT biosensor and the SCF-AlGaAs/GaAs HEMT biosensor are constructed by the following specific preparation steps:

a, placing a clean AlGaAs/GaAs HEMT device in a sulfydryl reagent, soaking for 24 hours at room temperature, and generating an Au-S bond on the surface of the AlGaAs/GaAs HEMT device to generate a self-assembled monolayer;

b. washing a sulfydryl reagent on the surface of the AlGaAs/GaAs HEMT device by using deionized water, adding a carbodiimides hydrochloride and N-hydroxysuccinimide mixed solution, and reacting for 15 minutes to generate a stable amine activated product so as to activate carboxyl;

c. washing the AlGaAs/GaAs HEMT device with PBS, adding tyrosine kinase (c-kit) or recombinant human Stem Cell Factor (SCF), and reacting at 4 deg.C for 2 hr to obtain c-kit-AlGaAs/GaAs HEMT biosensor or SCF-AlGaAs/GaAs HEMT biosensor;

(2) a method for detecting a deoxyschizandrin passage for treating oligospermia based on an AlGaAs/GaAs HEMT biosensor comprises the following specific detection steps:

a. preparing a c-kit-AlGaAs/GaAs HEMT biosensor, sequentially adding PBS solution of the recombinant human stem cell factor SCF in a concentration range from low to high, and recording the current intensity I between a source electrode and a drain electrode by adopting an electrochemical device_DS(ii) a Taking logarithm Lg of the concentration of the SCF protein solution as an abscissa and a relative value (I-I) of current change₀)/I₀Linear fitting was performed as ordinate to determine c-kit-AlGaAs/GaAs HEMT biosensorDetecting the concentration range of the SCF protein solution; taking the concentration of the SCF protein solution as an abscissa, changing the concentration/current within the concentration range determined in the step (2) into an ordinate, and performing linear fitting to obtain a dissociation constant of the interaction of the SCF and the c-kit;

b. preparing c-kit-AlGaAs/GaAs HEMT biosensor, sequentially adding deoxyschizandrin standard solution (concentration range of 0.1pM-1.0mM) with concentration from low to high into the biosensor, and recording current intensity I between source and drain by electrochemical device_DS(ii) a Taking the logarithm of the concentration of the deoxyschizandrin standard substance solution as an abscissa and the relative value of current change as an ordinate, and performing linear fitting to determine the detection range of the concentration of the deoxyschizandrin standard substance solution; taking the concentration of the deoxyschizandrin standard solution as an abscissa, changing the concentration/current in a determined detection range into an ordinate, performing linear fitting, and calculating the interaction strength of deoxyschizandrin and c-kit according to a formula;

c. preparing a SCF-AlGaAs/GaAs HEMT biosensor, and analyzing the interaction condition of deoxyschizandrin and SCF according to the same method b;

d. preparing c-kit-AlGaAs/GaAs HEMT biosensor, sequentially adding mixed solution of recombinant human stem cell factor SCF and deoxyschizandrin with concentration from low to high, and recording current intensity I between source and drain by electrochemical device_DSAnd the influence of deoxyschizandrin on SCF/c-kit interaction is examined.

The invention further provides an application of the protein immunoreaction method in detecting the signal path of deoxyschizandrin for treating oligospermia, which comprises the following steps:

(1) constructing animal models of oligospermia and asthenospermia, dividing a blank control group, three groups of high, medium and low dosage of deoxyschizandrin and a levocarnitine group for administration, taking venous blood after all rats are administered for the last time, killing the animals, and taking testis tissues for freezing storage;

(2) taking testis tissues to prepare a paraffin section, and observing the expression conditions of SCF and c-kit proteins in the testis tissues by combining an immunochemical reaction method;

(3) cutting testis tissue, cutting, adding PBS buffer solution, centrifuging at 4 deg.C, discarding supernatant, adding RIPA lysate containing protease inhibitor to lyse testis tissue, centrifuging at 4 deg.C, and collecting supernatant; taking bovine serum albumin solution as standard solution, adopting an enzyme-labeling instrument to measure the protein concentration of the SCF antibody and the c-kit antibody, adjusting the sample concentration, and heating in a water bath to fully denature the protein;

(4) adopting electrophoresis to separate protein, preparing a PVDF membrane through the steps of preparing glue, electrophoresis, membrane transferring, membrane dyeing, sealing and the like, adding SCF and c-kit secondary antibody solution for reaction, analyzing the protein content by combining a digital gel image analysis system through western blot reaction;

(5) and analyzing the mechanism of treating oligospermia by the schizandrin-mediated SCF/c-kit pathway by combining the tissue section result and the western blot result.

The beneficial results are that:

the invention provides an application of an AlGaAs/GaAs HEMT biosensor combined protein immunoreaction method in detecting a schizandrin A treatment oligospermia signal pathway, firstly, an autonomously developed AlGaAs/GaAs HEMT biosensor is adopted to carry out a detection experiment of the schizandrin A treatment oligospermia signal pathway in vitro, and the discovery that schizandrin A can act on an SCF/c-kit pathway, the detection range reaches pg/pM level, the specificity is strong, the sensitivity is high, and the interaction of schizandrin A and protein in the SCF/c-kit pathway can be intuitively reflected in vitro; furthermore, a rat model of oligospermia is constructed, a protein immunoreaction method is adopted, and an in vivo verification experiment of treating the signal path of oligospermia by deoxyschizandrin is carried out, and the result shows that the deoxyschizandrin can improve the testicular tissue structure of an animal model of oligospermia, improve the content of SCF protein in the testicular tissue, and reduce the content of c-kit protein. An in-vitro detection experiment of the deoxyschizandrin signal pathway for treating the oligospermia based on the AlGaAs/GaAs HEMT biosensor and an in-vivo verification experiment of the deoxyschizandrin signal pathway for treating the oligospermia based on the protein immunoreaction overcome the technical problems that the independent in-vitro experiment lacks reliability and the independent in-vivo experiment lacks visual representation of an action mechanism, and provide a detection method guide for the detection of the deoxyschizandrin signal pathway for treating the oligospermia.

Drawings

FIG. 1 c-kit-AlGaAs/GaAs HEMT biosensor protein modification results.

FIG. 2(a) I binding of different concentrations of SCF to c-kit_DS-V_DSA change in signal; (b) linear fit results for different concentrations of SCF in combination with c-kit.

FIG. 3(a) I binding of schizandrin A standard solutions of different concentrations to c-kit_DS-V_DSA change in signal; (b) linear fitting results of combination of the schizandrin A standard solution with different concentrations and the c-kit.

FIG. 4(a) I of schizandrin A standard solution and SCF combined at different concentrations_DS-V_DSA change in signal; (b) linear fitting results of combination of the schizandrin A standard substance solutions with different concentrations and the SCF.

FIG. 5(a) I binding of mixed solution of SCF and schizandrin A standards with different concentrations to c-kit_DS-V_DSA change in signal; (b) and (3) linear fitting results of the combination of the mixed solution of the SCF and schizandrin A standard substances with different concentrations and the c-kit.

FIG. 6(a) I binding of schizandrin A standard and SCF mixed solution with different concentrations to c-kit_DS-V_DSA change in signal; (b) and (3) linear fitting results of combination of the schizandrin standard substance and the SCF mixed solution with different concentrations and the c-kit.

FIG. 7 shows the expression of SCF and c-kit proteins in rat testis tissues of various groups.

Detailed Description

EXAMPLE 1 preparation of a c-kit-AlGaAs/GaAs HEMT biosensor

(1) Measuring I of each channel of AlGaAs/GaAs HEMT device by using CHI-660E electrochemical workstation_DS-V_DSAnd (5) curve judging and comparing the performance of each channel.

(2) Placing the clean AlGaAs/GaAs HEMT device in a 3-mercaptopropionic acid (3-MPA) aqueous solution, soaking at room temperature to generate Au-S bonds on the surface of the AlGaAs/GaAs HEMT device to form a self-assembled monolayer;

(3) preparing a mixed aqueous solution of 20mM carbodiimide hydrochloride and 50mM N-hydroxysuccinimide in a volume ratio of 1:1 as a carboxyl activating solution for standby, washing off 3-MPA on the surface of the AlGaAs/GaAs HEMT device by deionized water, and adding the carboxyl activating solution to generate a stable amine activating product for activating carboxyl;

(4) and (3) washing the AlGaAs/GaAs HEMT device by using 10mM Phosphate Buffer Solution (PBS), adding 100mg/mL tyrosine kinase c-kit, and reacting at 4 ℃ for 2 hours to obtain the c-kit-AlGaAs/GaAs HEMT biosensor.

(5) Electrochemical workstation I employing CHI-660E_DS-V_DSThe signal and result are shown in FIG. 1, and it can be seen that after c-kit modification, the current changes significantly, indicating that c-kit is successfully modified on AlGaAs/GaAs HEMT devices and that c-kit-AlGaAs/GaAs HEMT biosensors are successfully constructed.

EXAMPLE 2 preparation of a SCF-AlGaAs/GaAs HEMT biosensor

(4) washing the AlGaAs/GaAs HEMT device by using 10mM Phosphate Buffer Solution (PBS), adding a recombinant human stem cell factor SCF with the concentration of 100mg/mL, and reacting for 2 hours at 4 ℃ to obtain the SCF-AlGaAs/GaAs HEMT biosensor;

(5) electrochemical workstation I employing CHI-660E_DS-V_DSThe signal and the result show that after the SCF is modified, the current is obviously changed, which shows that the SCF is successfully modified on the AlGaAs/GaAs HEMT device, and the SCF-AlGaAs/GaAs HEMT biosensor is successfully constructed.

Example 3 application of AlGaAs/GaAs HEMT biosensor to detection of SCF and c-kit interactions

(1) Dissolving SCF in 0.1M PBS buffer solution to 0.1mg/L, and diluting to 0.1pg/L according to a ten-fold gradient; a c-kit-AlGaAs/GaAs HEMT biosensor was prepared as in example 1, and using this as a reaction device, a SCF protein solution was added to the c-kit-AlGaAs/GaAs HEMT device in the order of increasing concentration, and the current intensity (I) between the source and drain was recorded using an electrochemical device_DS) I at different concentrations_DS-V_DSThe signals are shown in FIG. 2 (a); as can be seen from the graph, when the concentration ranges from 0.1pg/L to 0.1mg/L, I_DSThe absolute value of the signal continuously decreases;

(2) in terms of the logarithm of the concentration of the SCF protein solution (Lg [ A ]_g]) As abscissa, with relative value of current change (I-I)₀)/I₀The concentration is a vertical coordinate, linear fitting is carried out, and the result shows that the linear relation is better when the concentration range is 0.1pg/L-1.0 ng/L; the results of linear fitting of five concentration points of 0.1pg/L to 1.0ng/L are shown in fig. 2(b), and it is clear that the linear relationship is good, and the equation is y-0.03460 x-0.5036 (R)²＝0.9973)；

(3) The concentration of the SCF protein solution ([ A ] in accordance with the concentration range of (2)_g]) As abscissa, in concentration ([ A ]_g]) Change in Current (I-I)₀Δ I) is ordinate, and linear fitting is performed to obtain the linear equation y-2170.5 x +8E-09, (R)²0.9998); according to the formula

Wherein [ Ab]Is c-kit protein concentration, [ Ag ]]Concentration of SCF protein solution [ C]K and KA are binding constants, K_DIs dissociation constant,. DELTA.I is current change value,. DELTA.I_maxThe maximum change value of the current.

Calculation of dissociation constant K for SCF and c-kit interaction_DTo obtain K_D＝3.686×10^-12M。

Example 4 application of AlGaAs/GaAs HEMT biosensor in detection of Schizandrin A and c-kit interaction

(1) Accurately weighing the deoxyschizandrin standard substance as the effective component of the jingzanyu capsule respectively, placing the deoxyschizandrin standard substance into volumetric flasks, adding 10mM PBS solution respectively to prepare 1mM deoxyschizandrin standard substance solution as mother solution, diluting the mother solution to 0.1pM according to ten times of gradient, and preparing 11 concentration gradient deoxyschizandrin standard substance gradient concentration sample solutions;

(2) the c-kit-AlGaAs/GaAs HEMT biosensor prepared according to the method of example 1 is used as a reaction device, schizandrin A standard solution is added to the c-kit-AlGaAs/GaAs HEMT device in sequence from low concentration to high concentration, and the current intensity (I) between the source and the drain is recorded by an electrochemical device_DS) I at different concentrations_DS-V_DSThe signals are shown in FIG. 3 (a); as can be seen, I is measured at a concentration ranging from 0.1pM to 1.0. mu.M_DSThe absolute value of the signal continuously decreases; when the concentration exceeds 1.0. mu.M, I_DSThe signal change is irregular;

(3) using logarithm of concentration of deoxyschizandrin standard substance solution (Lg [ A ]_g]) As abscissa, with relative value of current change (I-I)₀)/I₀The concentration is the ordinate, linear fitting is carried out, and the result shows that the linear relation is better when the concentration range is 1.0pM-10 nM; the results of linear fitting of five concentration points of 1.0pM to 10nM are shown in FIG. 3(b), which shows good linearity, and the equation is y-0.09810 x-1.5551 (R)²＝0.9981)；

(4) According to the concentration range of (3), the concentration of the deoxyschizandrin standard solution ([ A ]_g]) As abscissa, in concentration ([ A ]_g]) Change in Current (I-I)₀Δ I) is the ordinate, and linear fitting is performed to give the linear equation y-877.06 x +5E-08, (R)²0.9997); calculating the interaction of deoxyschizandrin and c-kit according to formulas (I), (II) and (III)Dissociation constant K of_DTo obtain K_D＝5.701×10^-11M。

Example 5 application of AlGaAs/GaAs HEMT biosensor in detection of interaction between deoxyschizandrin and SCF

(2) the SCF-AlGaAs/GaAs HEMT biosensor prepared according to the method of example 2 is used as a reaction device, a schizandrin A standard solution is added to the SCF-AlGaAs/GaAs HEMT device in sequence from low concentration to high concentration, and the current intensity (I) between the source and the drain is recorded by an electrochemical device_DS) I at different concentrations_DS-V_DSThe signals are shown in FIG. 4 (a); as can be seen from the graph, when the concentration changes, I_DSThe signal is basically unchanged; using logarithm of concentration of deoxyschizandrin standard substance solution (Lg [ A ]_g]) As abscissa, with relative value of current change (I-I)₀)/I₀As an ordinate, a linear fit was made, substantially a straight line parallel to the X-axis (FIG. 4(b)), indicating that schizandrin A had no interaction with SCF.

Example 6 application of AlGaAs/GaAs HEMT biosensor in detecting Schisandrin A influence on SCF and c-kit interaction

(1) Taking 7 parts of 10pM schizandrin A standard solution, adding equal volume of 0.1pg/L-0.1 mug/L SCF protein solution into each 60 μ L schizandrin A standard solution, and mixing uniformly for later use;

(2) the c-kit-AlGaAs/GaAs HEMT biosensor prepared according to the method of example 1 was used as a reaction device, a mixed solution of a deoxyschizandrin standard solution and a SCF protein solution was added to the c-kit-AlGaAs/GaAs HEMT device in the order of increasing concentration, and the current intensity (I) between the source and the drain was recorded using an electrochemical device_DS) I at different concentrations_DS-V_DSThe signals are shown in FIG. 5 (a); as can be seen from the figure, when the concentration range is 01 pg/L-0.1. mu.g/L, I_DSThe absolute value of the signal continuously decreases;

(3) using logarithm of concentration of deoxyschizandrin standard substance solution (Lg [ A ]_g]) As abscissa, with relative value of current change (I-I)₀)/I₀The concentration is a vertical coordinate, linear fitting is carried out, and the result shows that the linear relation is better when the concentration range is 1.0pg/L-10 ng/L; the results of linear fitting of five concentration points of 1.0pg/L to 10ng/L are shown in fig. 5(b), and it is clear that the linear relationship is good, and the equation is y-0.04880 x-0.6145 (R)²＝0.9994)；

(4) According to the concentration range of (3), the concentration of the deoxyschizandrin standard solution ([ A ]_g]) As abscissa, in concentration ([ A ]_g]) Change in Current (I-I)₀Δ I) is the ordinate, and linear fitting is performed to give the linear equation y-585.04 x +6E-08, (R)²0.9992); calculating dissociation constant K of schizandrin A and c-kit interaction according to formulas (I), (II) and (III)_DTo obtain K_D＝1.026×10^-10M。

Example 7 application of AlGaAs/GaAs HEMT biosensor in detecting Schisandrin A influence on SCF and c-kit interaction

(1) Taking 7 parts of SCF protein solution with the concentration of 10 mu g/L, adding isovolume schizandrin A standard solution with the concentration range of 10pM-10 mu M into each part of SCF protein solution with the concentration of 60 mu L, and uniformly mixing for later use;

(2) the c-kit-AlGaAs/GaAs HEMT biosensor was prepared as described in example 1, and the reaction device was used, a mixed solution of SCF protein solution and schizandrin A standard solution was added to the c-kit-AlGaAs/GaAs HEMT device in the order of increasing concentration, and the current intensity (I) between the source and drain was recorded by an electrochemical device_DS) I at different concentrations_DS-V_DSThe signals are shown in FIG. 6 (a); as can be seen from the figure, when the concentration is in the range of 10pM to 10. mu.M, I_DSThe absolute value of the signal continuously decreases;

(3) using logarithm of concentration of deoxyschizandrin standard substance solution (Lg [ A ]_g]) As abscissa, with relative value of current change (I-I)₀)/I₀For the ordinate, a linear fit was performed, the result beingThe linear relation is better when the concentration range is 10pM-0.1 mu M; the results of linear fitting of five concentration points of 1.0pg/L to 10ng/L are shown in fig. 6(b), and it is clear that the linear relationship is good, and the equation is y-0.04880 x-0.6145 (R)²＝0.9994)；

(4) According to the concentration range of (3), the concentration of the deoxyschizandrin standard solution ([ A ]_g]) As abscissa, in concentration ([ A ]_g]) Change in Current (I-I)₀Δ I) is the ordinate, and linear fitting is performed to give the linear equation y-221.07 x +3E-08, (R)²0.9992); calculating dissociation constant K of schizandrin A and c-kit interaction according to formulas (I), (II) and (III)_DTo obtain K_D＝1.357×10^-10M。

Example 8 construction and characterization of rat model for oligoasthenospermia

(1) Laboratory animal

70 SPF male SD rats with the weight of 170-: SCXK (Jing) 2011-. All animals were raised in Peking university of traditional Chinese medicine laboratory at 22-24 deg.C, 52-56 RH% humidity, 12h light/12 h dark alternately, and were fed with free water. All operations of the experiment are strictly carried out according to ethical related regulations of animals.

(2) Main equipment

A centrifuge: eppendorf company, model: centrifuge 5810R; an electronic balance: company a & D, model: GR-200; vientian sperm quality detection system: beijing Weili company, model: WL-9000; 37 ℃ constant temperature incubator: beijing Kogyo Yongxing instruments Inc., model number: HH-S6A type; a stomach filling needle, a 50mL centrifuge tube, a 1.5mL EP tube, a 5mL freezing tube, a 1mL, 2mL or 5mL disposable syringe, an ophthalmic scissors, a forceps, a surgical blade and the like.

(3) Main reagent and experimental medicine

Tripterygium wilfordii Glycosides (GTW): shanghai Compound Dan Fuhua pharmaceutical Co., Ltd, batch number: 160902; sodium carboxymethylcellulose (CMC): source leaf organism, lot number: L13M8G 35918; m199 medium: HYCLONE corporation; chloral hydrate: beijing chemical company. Schizandrin a (schiscandrin a): BioRuler, batch PS 000928; l-carnitine: dalian Mero pharmaceutical plant, Inc., batch number: 01161202, respectively; the above medicine is suspended in 0.5% carboxymethyl cellulose (CMC) for use before administration.

(4) Calculation of drug dosage

Calculating the gavage dosage of the rat:

taking the dose of human oral deoxyschizandrin as a reference, and carrying out dose conversion according to the ratio of the surface area of a rat to the surface area of a human body: the calculation formula of the human body surface area is as follows: body surface area (m)²) (vii) 0.0061 × height (cm) +0.0128 × weight (kg) -0.1529; calculation formula of body surface area of rat: body surface area (m)²)＝9.1×(W ²3/10000), wherein W is the weight of the rat; rat dose (mg/kg) × human dose × human surface area/rat body surface area

The high dose, the medium dose and the low dose of the gavage of the rat are respectively set according to the dosage of the adult, and are respectively 20 times, 10 times and 5 times of the dosage of the adult. High dose of deoxyschizandrin: 22.5 mg/(kg. d); the dosage of the deoxyschizandrin is as follows: 11.25 mg/(kg. d); and (3) low dose of deoxyschizandrin: 5.625 mg/(kg. d); l-carnitine: 2.1 mL/(kg. d)

Molding: tripterygium glycosides 20 mg/(kg. d), and perfusing stomach continuously for 35d (5w), establishing rat model of oligospermia and asthenospermia.

(5) Experimental methods

And (3) performing intragastric administration on the model-made rats by using GTW 20 mg/(kg. d), wherein the intragastric administration is performed once a day for 35 consecutive days (5 weeks), and a rat model of oligospermia and asthenospermia is established.

70 SD rats are randomly divided into 7 groups, namely a normal group 1 group, a model group 1 group, 1 group of deoxyschizandrin with high, medium and low doses respectively, and a western medicine positive control group (levocarnitine group) 1 group; wherein the normal group and the model group are respectively 15, and the schisandra chinensis high, medium and low dose groups, the levocarnitine group and the polygonatum praise group are respectively 10. Adaptive breeding for 5 days.

Normal group (NS): the stomach was perfused with 2mL of 0.5% CMC for 35 days per day. On day 35, 5 random sacrificed and examined by semen parameters as a GTW panel to determine if molding was successful. After the molding was successful, the remaining rats were further gavaged with 2mL of 0.5% CMC for 35 days.

Model Group (GTW): the stomach was perfused with GTW 20 mg/(kg. d) for 35 days, 5 random sacrificed on day 35, and NS groups were examined and compared by semen parameters to determine whether molding was successful. After successful molding, the remaining rats were further gavaged with 2mL of 0.5% CMC for 35 days.

Schizandrin a group (Schizandrin a): divided into three dose groups of high, medium and low. The stomach was perfused with GTW 20 mg/(kg.d) for 35 days, and from day 36, the stomach was perfused with deoxyschizandrin 22.5 mg/(kg.d), 11.25 mg/(kg.d), and 5.625 mg/(kg.d), and the stomach was perfused continuously for 35 days.

Levocarnitine group (Levocarnitine): the stomach was perfused with GTW 20 mg/(kg. d) for 35 days, and with levocarnitine 2.1 mL/kg. d from day 36, the stomach was perfused continuously for 35 days.

During the gavage period, the rats were weighed once a week and the gavage dose was adjusted according to the latest body weight. The eating condition, mental state, activity performance, hair color, skin, stool and the like of each group of experimental animals are observed and recorded every day.

(6) Drawing experimental rat materials

After all rats are administrated 24 hours for the last time, the abdominal aorta is bled after chloral hydrate anesthesia, experimental animals are killed by bleeding, the testis and the epididymis are rapidly picked up, and the weight of the testis is weighed for later use.

In each group, 7 rats were randomly selected for censorship and epididymal tail sperm were collected by diffusion. Placing the tail of epididymis into 3mL of physiological saline at 37 ℃, cutting into pieces, and standing for 1min to prepare sperm suspension. 50 μ L of the sperm suspension was removed and placed in 1mL M199 medium in a thermostatic water bath at 37 ℃ for 5min, from which 13 μ L of the sperm suspension was removed and spread on a pre-warmed blood cell counting plate for sperm quality analysis.

(7) Evaluation of Molding results

And (3) molding: and (3) NS group: the body weight of the rat gradually increased. The fur color is glossy, the movement is quick, and the mental state is good. The food intake is gradually increased and the body temperature is normal. Defecation is normal, and the excrement is solid and granular. The fighting times are increased along with the experiment and are more active. 1 dead in stomach, and 2 nd day after gavage, the corpses were eaten by the companions. And (3) GTW molding each group: rats gained weight gradually and gained weight slightly faster than the NS group. The color and luster of the hair are reduced during the molding process, the hair occasionally falls off, the hair is favored to be clasped and curled, the activity is slower than that of NS group, and the spirit is poor. The food intake is gradually increased, the body temperature is lower, a small part of rats are cold in body, and the tails are cold. Loose stool appears at the initial molding stage, and the stool is soft and rotten like mud and is light brown; the stool gradually returns to normal in the middle and later period of the molding, but the stool quality is generally soft, and part of the rat stool is soft and rotten according to the condition. The death was 2, and the patients died on the 2 nd and 3 rd days of gastric lavage.

The administration period is as follows: and (3) NS group: the weight of the rat is gradually increased, and the weight growth amplitude is slowed down in the later period of the experiment. The fur color is glossy, the movement is quick, the mental state is good, and the fighting is favored. The food intake tends to be stable and the body temperature is normal. Defecation is normal, and the excrement is solid and granular. GTW group: the weight of the rat is gradually increased, and the weight growth amplitude is slowed down in the later period of the experiment. The hair color gradually recovers the luster, and the mental status is slightly improved; the activity is still slow and is not as active as other groups, and the fighting phenomenon is less than that of other groups. The food intake tends to be stable, the body temperature is normal, the tail is cool, and the food intake tends to be normal in the later period of the experiment. Normal stool, soft stool, which is depressed by pressing. Levocarnitine group: the weight of the rat is gradually increased, and the weight growth amplitude is slowed down in the later period of the experiment. The hair color gradually recovers the luster, the mental status and the activity status are improved, and the fighting is increased. The food intake tends to be stable, the body temperature is normal, the tail is cool, and the food intake tends to be normal in the later period of the experiment. Stool is normal, and stool is solid and granular and hard when pressed. Schizandrin A high dose group: the weight of the rat is gradually increased, and the weight growth amplitude is slowed down in the later period of the experiment. The hair color gradually recovers the luster, the mental status and the activity status are improved, and the fighting is increased. The food intake tends to be stable and the body temperature is normal. Stool is normal, and stool is solid and granular and hard when pressed. The dose group of deoxyschizandrin is as follows: the weight of the rat is gradually increased, and the weight growth amplitude is slowed down in the later period of the experiment. The hair color gradually recovers the luster, the mental status and the activity status are improved, and the fighting is increased. The food intake tends to be stable and the body temperature is normal. Feces are normal and solid and granular, which can be trapped by pressing. Schizandrin A low dose group: the weight of the rat is gradually increased, and the weight growth amplitude is slowed down in the later period of the experiment. The hair color gradually recovers the luster, the mental status is slightly improved, the activity state is not as good as other dosage groups, and the fighting is less. The food intake tends to be stable and the body temperature is normal. Feces are normal and solid and granular, which can be trapped by pressing.

The treated sperm suspension was applied to a counting plate using a WL-9000 sperm quality detection System (CASA) set at 37 deg.C, 5 fields of view were selected, and testing was completed within 2 min. The main test indexes are as follows: sperm density (. times.10)⁶(mL), sperm motility (%), sperm fraction at level a (%), sperm fraction at level b (%). The results show (table 1): after modeling, the sperm density and the b-grade sperm rate of the rats in the GTW group are obviously reduced, wherein the sperm density is obviously different from that of the NS group (P is less than 0.05), the sperm mobility of the rats in the GTW group is obviously reduced, and the sperm mobility is obviously different from that of the NS group (P is less than 0.05), which indicates successful modeling.

TABLE 1 sperm density, sperm rate at a level, and sperm rate at b level

Note: comparison with NS group: p < 0.05; comparison with GTW group:^#P＜0.05。

example 9 application of protein immunoreaction method in vitro signal pathway detection of effective components of Huangjingzanyu capsule for treating oligospermia and asthenospermia

(1) Obtaining the testis of the rat: after all rats are administrated 24 hours for the last time, chloral hydrate is used for anesthetizing abdominal aorta to take blood, experimental animals are killed by bleeding, and testis and epididymis are rapidly picked for standby; fixing ipsilateral testis of each rat in Davidson's fixing solution for 24h, wherein the volume ratio of tissue fluid is 1:20, and changing the fluid once every 12 h; after fixation for 24h, the cells are transferred into neutral formalin fixing solution for fixation for 24-48 h. After tissue is trimmed, flushing for 12h by running water; dehydrating along the gradient alcohol concentration from low to high, after dehydration, carrying out xylene transparency, and carrying out paraffin embedding to prepare testis paraffin sections for later use;

(2) extracting the testis protein of the rat: cutting and weighing 100mg testis tissue, cutting into pieces, adding 0.01mol L PBS buffer solution, centrifuging at 4 deg.C under 2500pm for 1min, and discarding supernatant; adding 0.5mL of RIPA lysate containing protease inhibitor into the tissue, and homogenizing until the tissue mass disappears; placing the tissue serous fluid on a shaking bed, shaking and cracking the tissue serous fluid for 30mnin, centrifuging the tissue serous fluid at 12000rpm at 4 ℃ for 15min, and sucking supernatant for later use; measuring and calculating the concentration of the protein in the sample by using a microplate reader by taking 0.5mg/mL Bovine Serum Albumin (BSA) as a standard solution;

(3) according to the protein concentration determination result, adjusting the sample concentration to 10ug/L, adding 5xSDS buffer solution, mixing, heating in a water bath at 95 ℃ for 10min in a heater to fully denature the protein; separating protein by using 12% SDS-PAGE electrophoresis, preparing gel, loading, performing electrophoresis at 60V for 30min, performing electrophoresis at 120V for 1.5h when a sample enters the separation gel, stopping electrophoresis when bromophenol blue runs to the bottom of the gel, washing and cutting the film; after a PVDF film is subjected to constant current conversion for 1.5h at 300mA, the film is immersed into a ponceau red dye solution which is diluted by 10 times to dye the film; sealing the PVDF membrane for later use;

(4) diluting SCF and c-kit antibodies by adopting goat serum working solution in a volume ratio of 1:100, taking out the sealed PVDF membrane, washing the PBST for 3 times, 10min each time, and putting the membrane into a hybridization bag after washing. The SCF and c-kit primary antibodies were diluted 1:100 by volume using goat serum working solution PBST and placed in hybridization bags overnight at 4 ℃. Taking out the PVDF membrane the next day, washing the PVDF membrane for 3 times by shaking PBST, washing for 10min each time, putting the PVDF membrane into a new hybrid bag, pouring prepared secondary antibody, incubating for 1h by shaking, taking out the membrane, washing for 3 times by PBST, putting the PVDF membrane into a luminometer for 10min each time, dripping ECL (Ecl-color development) solution for incubation for 30s, taking a picture by using a digital gel image analysis system, and calculating the expression conditions of SCF (short-cut fluorescent protein) and c-kit;

(5) the expression conditions of SCF and c-kit in testis tissues are shown in Table 2 and figure 7, and the results show that the SCF expression is remarkably reduced in the asthenospermia model group compared with the normal group; after the model rat is administrated, the SCF protein content of the levocarnitine group, the schizandrin A high-dose group and the SCF protein content of the medium-dose group are obviously increased (P is less than 0.05), wherein the schizandrin A high-dose group is closest to a normal group, positive particles are found in part of seminiferous tubules only in the schizandrin A low-dose group, and the SCF content is increased but has no obvious difference. Compared with the normal group, the expression of c-kit protein of the asthenospermia model group is increased, the content of c-kit protein of each group is reduced after administration, wherein the group with high deoxyschizandrin and medium dosage is reduced most obviously.

TABLE 2 SCF and c-kit protein expression results in various groups of testis tissues

Claims

1. An application of an AlGaAs/GaAs HEMT biosensor combined with a protein immunoreaction method in detecting a signal path for treating oligospermia by deoxyschizandrin is characterized by comprising the AlGaAs/GaAs HEMT biosensor and the protein immunoreaction, wherein the signal path for treatment comprises an SCF/c-kit path.

2. The use of the AlGaAs/GaAs HEMT biosensor in combination with protein immunoreaction method in detecting schizandrin-treated oligospermia signaling pathway according to claim 1, wherein the AlGaAs/GaAs HEMT biosensor is applied in detecting schizandrin-treated oligospermia signaling pathway by the following specific steps:

(1) preparing an AlGaAs/GaAs HEMT biosensor:

a. placing the clean AlGaAs/GaAs HEMT device in a sulfydryl-containing reagent, soaking at room temperature to generate an Au-S bond on the surface of the AlGaAs/GaAs HEMT device and generate a self-assembled monolayer;

b. washing away a sulfydryl reagent on the surface of the AlGaAs/GaAs HEMT device by using deionized water, and adding a carboxyl activating agent to generate a stable amine activating product;

c. washing the AlGaAs/GaAs HEMT device by using a phosphate buffer solution, adding a tyrosine kinase c-kit or a recombinant human stem cell factor SCF, and completing the reaction to obtain a c-kit-AlGaAs/GaAs HEMT biosensor or an SCF-AlGaAs/GaAs HEMT biosensor;

(2) the application of the AlGaAs/GaAs HEMT biosensor in the detection of the signal path of the hypospermia and the oligospermia of the effective components of the Huangjingzanyu capsule is as follows:

a. preparing a c-kit-AlGaAs/GaAs HEMT biosensor according to the method in the step (1), sequentially adding a recombinant human stem cell factor SCF solution with the concentration from low to high, and recording the current intensity between a source electrode and a drain electrode by adopting an electrochemical device; taking the logarithm of the concentration of the SCF protein solution as an abscissa and the relative value of the current change as an ordinate, performing linear fitting, and further calculating the interaction strength of the SCF and the c-kit according to a formula;

b. preparing a c-kit-AlGaAs/GaAs HEMT biosensor and a SCF-AlGaAs/GaAs HEMT biosensor according to the method in the step (1), respectively adding deoxyschizandrin standard solution with the concentration from low to high into the two biosensors in sequence, and recording the current intensity between a source electrode and a drain electrode by adopting an electrochemical device; taking the logarithm of the concentration of the deoxyschizandrin standard substance solution as an abscissa and the relative value of current change as an ordinate, performing linear fitting, and further calculating the interaction strength of the deoxyschizandrin, the c-kit and the SCF according to a formula;

c. preparing the c-kit-AlGaAs/GaAs HEMT biosensor according to the method in the step (1), sequentially adding a mixed solution of a recombinant human stem cell factor SCF and schizandrin A with the concentration from low to high, recording the current intensity between a source electrode and a drain electrode by adopting an electrochemical device, observing the influence of the schizandrin A on the SCF/c-kit interaction intensity, and analyzing the SCF/c-kit action mechanism regulated by the schizandrin A.

3. The use of the AlGaAs/GaAs HEMT biosensor in combination with protein immunoreaction method of any one of claims 1-2 for detecting schizandrin signal pathway for treating oligospermia, wherein the carboxyl activating agent in step (1) b is carbonyldiimine hydrochloride and N-hydroxysuccinimide, the activation time is 15-30min, the reaction temperature for modifying c-kit or SCF is 4 ℃, and the reaction time is not less than 2 hours.

4. The use of the AlGaAs/GaAs HEMT biosensor and protein immunoreaction method in detecting the signal path of deoxyschizandrin for treating oligospermia according to claim 1, wherein the specific steps of the protein immunoreaction method in detecting the signal path of deoxyschizandrin for treating oligospermia are as follows:

(2) cutting testis tissue, cutting, adding phosphate buffer solution, centrifuging at 4 deg.C, discarding supernatant, adding RIPA lysate containing protease inhibitor to lyse testis tissue, centrifuging at 4 deg.C, and collecting supernatant;

(3) preparing testis tissue paraffin sections, and observing the testis tissue protein expression condition of rats in each group by an immunohistochemical experimental method;

(4) measuring the protein concentrations of the SCF antibody and the c-kit antibody by using an enzyme-labeling instrument by using a bovine serum albumin solution as a standard solution; separating proteins by adopting an electrophoresis technology, preparing a PVDF membrane through the steps of gel preparation, electrophoresis, membrane transfer, membrane staining, sealing and the like, adding a second antibody solution of the proteins related to a signal path for reaction, and analyzing the protein expression condition by combining a Western Blot and a digital gel image analysis system;

(5) and analyzing the mechanism of treating the hypospermia and the asthenospermia by the schizandrin-mediated SCF/c-kit pathway by combining the protein expression result based on the testis tissue immunochemical reaction and the Western Blot.