CN113009127B - Application of biosensor combined protein immune response method in detecting schizandrin A treating oligospermia signal path - Google Patents
Application of biosensor combined protein immune response method in detecting schizandrin A treating oligospermia signal path Download PDFInfo
- Publication number
- CN113009127B CN113009127B CN201911316164.4A CN201911316164A CN113009127B CN 113009127 B CN113009127 B CN 113009127B CN 201911316164 A CN201911316164 A CN 201911316164A CN 113009127 B CN113009127 B CN 113009127B
- Authority
- CN
- China
- Prior art keywords
- scf
- algaas
- kit
- schizandrin
- concentration
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/34—Genitourinary disorders
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention relates to an application of a biosensor combined with protein immune response in detecting a signal path of schisandrin for treating oligospermia, which is characterized by comprising an AlGaAs/GaAs HEMT biosensor and protein immune response; through the constructed AlGaAs/GaAs HEMT biosensor, an in-vitro detection experiment of the signal path of the schizandrin for treating oligospermia is carried out, the protein immune reaction is further combined, an in-vivo detection experiment of the signal path of the schizandrin for treating oligospermia is carried out, and the effect of the schizandrin for treating oligospermia and oligospermia is found by adjusting the SCF/c-kit path; the detection method has strong specificity, the detection concentration is as low as pg/pM, the detection range covers five orders of magnitude, and the detection method has high sensitivity and strong specificity.
Description
Technical Field
The invention relates to the field of traditional Chinese medicines, in particular to an application of a biosensor combined protein immune response method in detecting a signal path of schizandrin A for treating oligospermia.
Background
Studies have shown that about 5% of all couples worldwide have infertility problems. However, more couples have experienced at least one year of involuntary non-children: estimated to be between 12% and 28%. The male infertility accounts for 20% -30% of infertility cases, and researches show that the occurrence rate of male infertility is remarkably increased in recent years, the most common cause is semen deficiency, wherein the male infertility caused by oligospermia and asthenozoospermia is up to 60%, the family harmony and the life quality of people are seriously affected, and the male infertility is a key difficult problem to be overcome in the world.
Aiming at oligospermia and asthenozoospermia, a great deal of research is carried out in modern medicine, but currently, no therapeutic drug with definite curative effect is available. The traditional Chinese medicine has long history of treating male infertility based on overall view and dialectical treatment ideas and has definite curative effect. One Wang Qi yard of the science foundation of the traditional Chinese medicine brings forward the core pathogenesis of oligospermia and weak sperm disease as kidney deficiency with damp-heat stasis and poison insect, and according to the pathogenesis, the treatment principle of tonifying kidney and replenishing essence, clearing heat and promoting diuresis, and activating blood circulation and removing blood stasis is provided, and based on the principle, the first sperm producing prescription medicine 'Polygonatum sibiricum in the China specially for treating male infertility' is developed. Clinical results show that the medicine can increase the number of sperms and improve the sperm motility, and has obvious curative effect on male infertility caused by oligospermia and asthenozoospermia. Early researches show that the effective component schisandrin A has the effect of improving sperm activity, is hopeful to develop low-toxicity and high-efficiency spermatic medicine, but the action mechanism of the schisandrin A is still to be further researched.
The method currently used to detect signal pathways is the protein immune response method, but this method alone lacks visual characterization of the mechanism of action. High electron mobility transistors (High Electron Mobility Transistor, HEMT) as third generation electrochemical devices provide a key technological support for the identification of intermolecular interaction mechanisms with their higher sensitivity and higher specificity. However, the research on the action mechanism of combining the biosensor technology and the protein immune reaction method for detecting the improvement of the sperm motility of the sample with oligospermia and asthenospermia of schisandrin is still blank.
Disclosure of Invention
Aiming at the problem that the independent in-vitro experiment lacks reliability and the independent in-vivo experiment lacks visual characterization of action mechanism, the invention provides an in-vivo verification experimental method combining the in-vitro experiment of a HEMT biosensor with high sensitivity and strong specificity and based on a protein immune reaction method, and the action mechanism of analyzing schizandrin to improve sperm motility of samples with less and weak sperm.
The invention provides an application of a biosensor combined protein immune response method in detecting a signal path of schisandrin for treating oligospermia, firstly provides an application of an AlGaAs/GaAs HEMT biosensor in detecting the signal path of schisandrin for treating oligospermia, and specifically adopts the following technical scheme:
(1) The c-kit-AlGaAs/GaAs HEMT biosensor and the SCF-AlGaAs/GaAs HEMT biosensor are constructed, and the specific preparation steps are as follows:
a, placing a clean AlGaAs/GaAs HEMT device in a sulfhydryl 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 off sulfhydryl reagent on the surface of AlGaAs/GaAs HEMT device by deionized water, adding mixed solution of carbodiimide hydrochloride and N-hydroxysuccinimide, and reacting for 15min to generate stable amine activation product to activate carboxyl;
c. cleaning an AlGaAs/GaAs HEMT device by using PBS, adding a tyrosine kinase (c-kit) or a recombinant human Stem Cell Factor (SCF), and reacting for 2 hours at 4 ℃ to obtain a c-kit-AlGaAs/GaAs HEMT biosensor or a SCF-AlGaAs/GaAs HEMT biosensor;
(2) The method for detecting the schizandrin A treatment oligospermia pathway based on AlGaAs/GaAs HEMT biosensor comprises the following specific detection steps:
a. preparing a c-kit-AlGaAs/GaAs HEMT biosensor, sequentially adding PBS solution of recombinant human stem cell factor SCF in a concentration range from low to high, and recording current intensity I between a source electrode and a drain electrode by adopting an electrochemical device DS The method comprises the steps of carrying out a first treatment on the surface of the The log Lg of the concentration of SCF protein solution is taken as the abscissa, and the relative value of current change (I-I 0 )/I 0 Performing linear fitting on the ordinate, and determining the concentration range of the c-kit-AlGaAs/GaAs HEMT biosensor for detecting the SCF protein solution; taking the concentration of the SCF protein solution as an abscissa and the concentration/current change of the concentration range determined in the step (2) as an ordinate, performing linear fitting to obtain the dissociation constant of the interaction of the SCF and the c-kit;
b. preparing a c-kit-AlGaAs/GaAs HEMT biosensor, sequentially adding a schisandrin A standard substance solution with concentration ranging from low to high (concentration range is 0.1 pmol/L-1.0 mmol/L) into the sensor, and recording the current intensity I between a source electrode and a drain electrode by adopting an electrochemical device DS The method comprises the steps of carrying out a first treatment on the surface of the Logarithmic concentration of schisandrin A standard substance solutionTaking the current change relative value as an ordinate as an abscissa, performing linear fitting, and determining the detection range of the concentration of the schisandrin A standard substance solution; taking the concentration of the schizandrin A standard substance solution as an abscissa, determining the concentration/current change of a detection range as an ordinate, performing linear fitting, and calculating the interaction strength of schizandrin A and c-kit according to formulas (1), (2) and (3);
wherein [ A ] b ]For protein receptor concentration, [ A ] g ]Concentration of ligand [ C]K, K A For binding constant, K D For dissociation constant, ΔI is the current change value, ΔI max Is the maximum change value of the current.
c. Preparing an SCF-AlGaAs/GaAs HEMT biosensor, and analyzing the interaction condition of schisandrin A and SCF according to the b same method;
d. preparing a c-kit-AlGaAs/GaAs HEMT biosensor, sequentially adding mixed solution of recombinant human stem cell factor SCF and schizandrin A with low-to-high concentration, and recording current intensity I between source electrode and drain electrode by adopting an electrochemical device DS The effect of schizandrin A on SCF/c-kit interaction was examined.
The invention further provides an application of the protein immune response method in detecting the signal path of schisandrin for treating oligospermia, which comprises the following specific steps:
(1) Constructing animal models of oligospermia and asthenozoospermia, dividing a blank control group, taking three high, medium and low doses of schizandrin A and a levocarnitine group, taking venous blood from all rats after the last administration, and then killing the animals and taking testis tissue for freezing storage;
(2) Preparing testis tissue into paraffin sections, and observing the expression of SCF and c-kit protein in the testis tissue by combining an immunochemistry reaction method;
(3) Cutting testis tissue, shearing, adding PBS buffer solution, centrifuging at 4deg.C, discarding supernatant, adding RIPA lysate containing protease inhibitor to lyse testis tissue, centrifuging at 4deg.C, and collecting supernatant; using bovine serum albumin solution as standard solution, measuring protein concentration of SCF antibody and c-kit antibody by using an enzyme-labeled instrument, adjusting sample concentration, and heating in water bath to enable protein to be fully denatured;
(4) Separating protein by electrophoresis, preparing PVDF film by steps of gel preparation, electrophoresis, film transfer, film dyeing, sealing and the like, adding SCF and c-kit secondary antibody solution for reaction, and analyzing the protein content by western blot reaction and combining a digital gel image analysis system;
(5) Combining the tissue slice result and the western blot result, analyzing the mechanism of the schisandrin A mediated SCF/c-kit path for treating oligospermia.
Beneficial results:
the application of the biosensor combined protein immune response method in detecting the signal path of schizandrin for treating oligospermia, provided by the invention, firstly, an AlGaAs/GaAs HEMT biosensor which is independently researched and developed is adopted to develop a detection experiment of the signal path of schizandrin for treating oligospermia in vitro, and the detection range of schizandrin can reach pg/pM level by acting on SCF/c-kit path, so that the specificity is strong, the sensitivity is high, and the interaction of schizandrin and protein in the SCF/c-kit path is intuitively reflected in vitro; further, a oligospermia rat model is constructed, and an in-vivo verification experiment of the oligospermia signal path of the schisandrin treatment is carried out by adopting a protein immune reaction method, and the result shows that the schisandrin can indeed improve the testis tissue structure of the oligospermia animal model, improve the content of SCF protein in testis tissues and reduce the content of c-kit protein. The in-vitro detection experiment of the schizandrin-treated oligospermia signal path based on the AlGaAs/GaAs HEMT biosensor and the in-vivo verification experiment of the schizandrin-treated oligospermia signal path based on the protein immune response overcome the technical problems of lack of reliability of the in-vitro experiment and lack of visual representation of the action mechanism of the in-vivo experiment, and provide detection method guidance for the detection of the schizandrin-treated oligospermia signal path.
Drawings
FIG. 1 c-kit-AlGaAs/GaAs HEMT biosensor protein modification results.
FIG. 2 (a) I binding to c-kit at various concentrations of SCF DS -V DS A signal change; (b) Linear fitting results of binding of different concentrations of SCF to c-kit.
FIG. 3 (a) Schisandrin A standard solution at different concentrations I combined with c-kit DS -V DS A signal change; (b) Linear fitting results of binding of different concentrations of schizandrin a standard solution to c-kit.
FIG. 4 (a) shizandra berry A standard solution of different concentrations I combined with SCF DS -V DS A signal change; (b) Linear fitting results of binding of schizandrin a standard solutions of different concentrations to SCF.
FIG. 5 (a) I binding to c-kit of mixed solutions of SCF and schizandrin A standards at different concentrations DS -V DS A signal change; (b) Linear fitting results of c-kit binding to mixed solutions of different concentrations of SCF and schizandrin a standard.
FIG. 6 (a) shizandra berry A standard and SCF mixed solution at various concentrations I combined with c-kit DS -V DS A signal change; (b) Linear fitting results of binding of mixed solutions of schizandrin standard and SCF at different concentrations to c-kit.
FIG. 7 shows the expression of SCF and c-kit proteins in rat testis tissue of each group.
Detailed Description
Example 1 preparation of c-kit-AlGaAs/GaAs HEMT biosensor
(1) Measuring I of each channel of AlGaAs/GaAs HEMT device by adopting CHI-660E electrochemical workstation DS -V DS And (5) curves, judging and comparing the performances of the channels.
(2) Placing the clean AlGaAs/GaAs HEMT device in 3-mercaptopropionic acid (3-MPA) aqueous solution, soaking at room temperature, and generating 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 20mmol/L of carbodiimide hydrochloride and 50mM of N-hydroxysuccinimide with a volume ratio of 1:1, using the mixed aqueous solution as a carboxyl activating solution for standby, washing off 3-MPA on the surface of the AlGaAs/GaAs HEMT device by using deionized water, and adding the carboxyl activating solution to generate a stable amine activating product for activating carboxyl;
(4) And (3) cleaning the AlGaAs/GaAs HEMT device by using 10mmol/L Phosphate Buffer Solution (PBS), adding the amino acid kinase c-kit with the concentration of 100mg/mL, and reacting for 2 hours at the temperature of 4 ℃ to obtain the c-kit-AlGaAs/GaAs HEMT biosensor.
(5) Electrochemical workstation using CHI-660E DS -V DS The signal and the result are shown in figure 1, and the current is obviously changed after c-kit modification, which shows that the c-kit is successfully modified on the AlGaAs/GaAs HEMT device, and the c-kit-AlGaAs/GaAs HEMT biosensor is successfully constructed.
Example 2 preparation of SCF-AlGaAs/GaAs HEMT biosensor
(1) Measuring I of each channel of AlGaAs/GaAs HEMT device by adopting CHI-660E electrochemical workstation DS -V DS And (5) curves, judging and comparing the performances of the channels.
(2) Placing the clean AlGaAs/GaAs HEMT device in 3-mercaptopropionic acid (3-MPA) aqueous solution, soaking at room temperature, and generating 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 20mmol/L of carbodiimide hydrochloride and 50mM of N-hydroxysuccinimide with a volume ratio of 1:1, using the mixed aqueous solution as a carboxyl activating solution for standby, washing off 3-MPA on the surface of the AlGaAs/GaAs HEMT device by using deionized water, and adding the carboxyl activating solution to generate a stable amine activating product for activating carboxyl;
(4) Cleaning an AlGaAs/GaAs HEMT device by using 10mmol/L Phosphate Buffer Solution (PBS), adding recombinant human stem cell factor SCF with the concentration of 100mg/mL, and reacting for 2 hours at the temperature of 4 ℃ to obtain the SCF-AlGaAs/GaAs HEMT biosensor;
(5) Electrochemical workstation using CHI-660E DS -V DS Signal, result tableAfter 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 in detecting interaction of SCF and c-kit
(1) Dissolving SCF to 0.1mg/L by using 0.1mol/L PBS buffer solution, and diluting to 0.1pg/L by ten times of gradient; preparing a c-kit-AlGaAs/GaAs HEMT biosensor according to the method of the embodiment 1, taking the biosensor as a reaction device, sequentially adding SCF protein solution to the c-kit-AlGaAs/GaAs HEMT device from low concentration to high concentration, and recording the current intensity (I) between a source electrode and a drain electrode by adopting an electrochemical device DS ) I at different concentrations DS -V DS The signals are shown in fig. 2 (a); as can be seen from the graph, when the concentration is in the range of 0.1pg/L to 0.1mg/L, I DS The absolute value of the signal continuously drops;
(2) Logarithmic concentration of SCF protein solution (Lg [ A ] g ]) On the abscissa, in terms of the relative value of the current change (I-I 0 )/I 0 As the ordinate, the linear fitting is carried out, and the result shows that the linear relation is better when the concentration range is 0.1pg/L to 1.0 ng/L; as shown in FIG. 2 (b), the result of linear fitting is obtained by taking five concentration points of 0.1pg/L to 1.0ng/L, and the linear relationship is good, and the equation is y= -0.03460x-0.5036 (R) 2 ＝0.9973)；
(3) According to the concentration range of (2), the concentration of the SCF protein solution ([ A ] g ]) On the abscissa, in concentration ([ A ] g ]) Current variation (I-I) 0 Δi) is the ordinate, and linear fitting is performed to obtain a linear equation of y=2170.5x+8e-09, (R 2 =0.9998); according to the formula
Wherein [ A ] b ]For protein receptor concentration, [ A ] g ]Concentration of ligand [ C]K, K A For binding constant, K D For dissociation constant, ΔI is the current change value, ΔI max Is the maximum change value of the current.
Calculation of dissociation constant K for interaction of SCF with c-kit D Obtaining K D ＝3.686×10 -12 mol/L。
Example 4 application of AlGaAs/GaAs HEMT biosensor in detecting interaction of schizandrin A and c-kit
(1) Respectively precisely weighing schisandrin A standard substances which are active components of the Polygonatum sibiricum Zanyu capsules, placing the standard substances in a volumetric flask, respectively adding 10mmol/L PBS solution to prepare 1mmol/L schisandrin A standard substance solution as mother solution, and diluting the mother solution to 0.1pmol/L according to ten times gradient to prepare 11 gradient schisandrin A standard substance gradient concentration sample solutions;
(2) Preparing a c-kit-AlGaAs/GaAs HEMT biosensor according to the method of the embodiment 1, taking the c-kit-AlGaAs/GaAs HEMT biosensor as a reaction device, sequentially adding a schizandrin A standard substance solution to the c-kit-AlGaAs/GaAs HEMT device from low concentration to high concentration, and recording the current intensity (I) between a source electrode and a drain electrode by adopting an electrochemical device DS ) I at different concentrations DS -V DS The signals are shown in fig. 3 (a); as can be seen from the graph, when the concentration is in the range of 0.1pmol/L to 1.0. Mu. Mol/L, I DS The absolute value of the signal continuously drops; when the concentration exceeds 1.0. Mu. Mol/L, I DS The signal change is irregular;
(3) The concentration of schisandrin A standard solution is logarithmic (Lg [ A ] g ]) On the abscissa, in terms of the relative value of the current change (I-I 0 )/I 0 As the ordinate, the linear fitting is carried out, and the result shows that the linear relation is better when the concentration range is 1.0pM-10 nM; as shown in FIG. 3 (b), the result of linear fitting was performed by taking five concentration points of 1.0pmol/L to 10nmol/L, and the linear relationship was found to be good, and the equation was y= -0.09810x-1.5551 (R) 2 ＝0.9981)；
(4) According to the concentration range of (3), the concentration of the schisandrin A standard solution ([ A) g ]) On the abscissa, in concentration ([ A ] g ]) Current variation (I-I) 0 Δi) is the ordinate, and linear fitting is performed to obtain a linear equation of y=877.06x+5e-08, (R) 2 =0.9997); calculating dissociation constant K of the interaction of schizandrin A and c-kit according to formulas (1), (2) and (3) D Obtaining K D ＝5.701×10 -11 mol/L。
Example 5 application of AlGaAs/GaAs HEMT biosensor in detecting interaction of schizandrin A and SCF
(1) Respectively precisely weighing schisandrin A standard substances which are active components of the Polygonatum sibiricum Zanyu capsules, placing the standard substances in a volumetric flask, respectively adding 10mmol/L PBS solution to prepare 1mmol/L schisandrin A standard substance solution as mother solution, and diluting the mother solution to 0.1pmol/L according to ten times gradient to prepare 11 gradient schisandrin A standard substance gradient concentration sample solutions;
(2) An SCF-AlGaAs/GaAs HEMT biosensor was prepared according to the method of example 2, and the SCF-AlGaAs/GaAs HEMT device was sequentially added with a schisandrin standard solution from low concentration to high concentration, and the current intensity (I) between source electrode and drain electrode was recorded by using an electrochemical device DS ) I at different concentrations DS -V DS The signal is shown in fig. 4 (a); as can be seen from the graph, when the concentration is changed, I DS The signal is substantially unchanged; the concentration of schisandrin A standard solution is logarithmic (Lg [ A ] g ]) On the abscissa, in terms of the relative value of the current change (I-I 0 )/I 0 On the ordinate, a linear fit was performed, essentially a straight line parallel to the X-axis (fig. 4 (b)), and the results indicated that schizandrin a did not interact with SCF.
Example 6 application of AlGaAs/GaAs HEMT biosensor in detecting influence of Schisandrin A on interaction of SCF and c-kit
(1) 7 parts of schisandrin A standard solution with the concentration of 10pmol/L is taken, 60 mu L of each part is added with SCF protein solution with the concentration range of 0.1pg/L to 0.1 mu g/L in equal volume, and the mixture is uniformly mixed for standby;
(2) A c-kit-AlGaAs/GaAs HEMT biosensor was prepared as in example 1, and was used as a reaction device, followed by a concentration step down to c-kiAdding mixed solution of schisandrin A standard substance solution and SCF protein solution to t-AlGaAs/GaAs HEMT device, and recording current intensity (I) between source electrode and drain electrode by using electrochemical device DS ) I at different concentrations DS -V DS The signals are shown in fig. 5 (a); as can be seen from the graph, the concentration range is 0.1pg/L to 0.1. Mu.g/L, I DS The absolute value of the signal continuously drops;
(3) The concentration of schisandrin A standard solution is logarithmic (Lg [ A ] g ]) On the abscissa, in terms of the relative value of the current change (I-I 0 )/I 0 As the ordinate, the 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; as shown in FIG. 5 (b), the result of linear fitting with five concentration points of 1.0pg/L to 10ng/L shows that the linear relationship is good, and the equation is y= -0.04880x-0.6145 (R) 2 ＝0.9994)；
(4) According to the concentration range of (3), the concentration of the schisandrin A standard solution ([ A) g ]) On the abscissa, in concentration ([ A ] g ]) Current variation (I-I) 0 Δi) is the ordinate, and linear fitting is performed to obtain a linear equation of y=585.04x+6×10 -8 ,(R 2 = 0.9992); calculating dissociation constant K of the interaction of schizandrin A and c-kit according to formulas (1), (2) and (3) D Obtaining K D ＝1.026×10 -10 mol/L。
Example 7 application of AlGaAs/GaAs HEMT biosensor in detecting influence of Schisandrin A on interaction of SCF and c-kit
(1) 7 parts of SCF protein solution with the concentration of 10 mu g/L and 60 mu L of each part are taken, and the schisandrin A standard substance solution with the concentration range of 10pmol/L to 10 mu mol/L with the same volume is respectively added into the SCF protein solution and is uniformly mixed for standby;
(2) Preparing a c-kit-AlGaAs/GaAs HEMT biosensor according to the method of example 1, taking the c-kit-AlGaAs/GaAs HEMT biosensor as a reaction device, sequentially adding a mixed solution of an SCF protein solution and a schizandrin A standard substance solution to the c-kit-AlGaAs/GaAs HEMT device from low concentration to high concentration, and recording the current intensity (I DS ) I at different concentrations DS -V DS The signal is shown in fig. 6 (a); from the graphAs is known, when the concentration is in the range of 10pmol/L to 10. Mu. Mol/L, I DS The absolute value of the signal continuously drops;
(3) The concentration of schisandrin A standard solution is logarithmic (Lg [ A ] g ]) On the abscissa, in terms of the relative value of the current change (I-I 0 )/I 0 As the ordinate, the linear fitting is carried out, and the result shows that the linear relation is better when the concentration range is 10pmol/L to 0.1 mu mol/L; as shown in FIG. 6 (b), the result of linear fitting with five concentration points of 1.0pg/L to 10ng/L shows that the linear relationship is good, and the equation is y= -0.04880x-0.6145 (R) 2 ＝0.9994)；
(4) According to the concentration range of (3), the concentration of the schisandrin A standard solution ([ A) g ]) On the abscissa, in concentration ([ A ] g ]) Current variation (I-I) 0 Δi) is the ordinate, and a linear fit is performed to obtain a linear equation of y=221.07x+3×10 -8 ,(R 2 = 0.9992); calculating dissociation constant K of the interaction of schizandrin A and c-kit according to formulas (1), (2) and (3) D Obtaining K D ＝1.357×10 -10 mol/L。
Example 8 construction and characterization of a rat model with oligospermia and Weak sperm disease
(1) Experimental animal
SPF-class male SD rats 70, weight 170-190 g, from St Bei Fu (Beijing) laboratory animal science and technology Co., ltd., license number: SCXK (jing) 2011-0004. All animals are raised in the laboratory of Beijing university of Chinese medicine and laboratory at 22-24 deg.c and humidity of 52-56RH alternately in 12 hr light and 12 hr dark for free drinking and eating. All operations of the experiment are strictly carried out according to the relevant regulations of animal ethics.
(2) Main equipment
Centrifuge: eppendorf corporation, model: centrifuge 5810R; an electronic balance: a & D company, model: GR-200; the Weili sperm quality detection system comprises: beijing Weili company, model: WL-9000; constant temperature incubator at 37℃: beijing family Yongxing instruments Co., ltd., model: HH-S6A type; stomach needle, 50mL centrifuge tube, 1.5mL EP tube, 5mL cryopreservation tube, 1mL, 2mL, 5mL disposable syringe, ophthalmic scissors, forceps, surgical blade, etc.
(3) Main reagent and experimental medicine
Tripterygium glycosides (glucoside tripterygium wilfordine, GTW): shanghai double denier re-bloom pharmaceutical Co., ltd., lot number: 160902; sodium carboxymethyl cellulose (CMC): source leaf organisms, lot number: L13M8G35918; m199 medium: HYCLONE Co; hydration of chloral: beijing chemical reagent Co. Schisandrin A (Schisandrin A): bioRuler company, lot number PS000928; levocarnitine: dalian Meiro pharmaceutical Co., ltd., lot number: 01161202; the medicine is suspended in 0.5% of carboxymethyl cellulose (CMC) for standby before use.
(4) Calculation of drug dosage
Calculation of the gastric lavage dosage of the rat:
based on the dosage of human oral schizandrin A, the dosage conversion is carried out according to the ratio of the surface area of the rat to the surface area of the human body: the calculation formula of the human body surface area is as follows: body surface area (m) 2 ) =0.0061×height (cm) +0.0128×weight (kg) -0.1529; rat body surface area calculation formula: body surface area (m) 2 )＝9.1×(W 2 3/10000), wherein W is the body weight of the rat; rat dose (mg/kg) =human dose×human body surface area/rat body surface area
The high dose, the medium dose and the low dose of the stomach irrigation 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 schisandrin: 22.5 mg/(kg.d); the dosage of schisandrin A: 11.25 mg/(kg.d); low dose of schizandrin a: 5.625 mg/(kg.d); levocarnitine: 2.1 mL/(kg. D)
And (3) molding: tripterygium glycosides 20 mg/(kg.d), continuous gastric lavage 35d (5 w), and establishment of rat model with oligospermia and asthenozoospermia.
(5) Experimental method
The model rats were gavaged with GTW 20 mg/(kg.d), once daily, for 35 consecutive days (5 weeks), and a oligospermia rat model was established.
The SD rats are randomly divided into 7 groups, namely a normal group 1 group, a model group 1 group, and 1 group of schisandrin A with high, medium and low doses respectively, and a western medicine positive control group (left carnitine 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 rhizoma polygonati praise group are respectively 10. And (5) adaptively feeding for 5 days.
Normal group (NS): 2mL of 0.5% CMC was gavaged daily for 35 days. 5 animals were randomly sacrificed on day 35 and tested as a control for the GTW group by semen parameters to determine if molding was successful. After successful molding, the remaining rats continued to be 2ml of 0.5% cmc for 35 days.
Model Group (GTW): the GTW was infused with 20 mg/(kg.d) for 35 days, 5 animals were sacrificed at random on day 35, and the NS group was compared by semen parameter detection to determine if modeling was successful. After successful molding, the remaining rats were continuously gavaged with 2ml of 0.5% cmc for 35 days.
Schisandrin A group (Schizandrin A): the three dosage groups are high, medium and low. The stomach was irrigated with GTW 20 mg/(kg.d) for 35 days, and 22.5 mg/(kg.d), 11.25 mg/(kg.d) and 5.625 mg/(kg.d) of schizandrin A was irrigated with each of the above materials from day 36, followed by continuous gastric irrigation for 35 days.
Left carnitine group (Levocarnitine): the cells were filled with GTW 20 mg/(kg.d) for 35 days, and with L-carnitine 2.1 mL/kg.d from day 36, the cells were filled for 35 days.
During the gavage period, rats were weighed once a week and the gavage dose was adjusted based on the latest body weight. Eating, mental state, activity, hair color, skin, stool, etc. of each group of experimental animals were observed and recorded daily.
(6) Laboratory rat material
After the last administration of all rats for 24 hours, the abdominal aorta is bled after the chloral hydrate is anesthetized, the experimental animal is sacrificed by bleeding, testis and epididymis are rapidly picked up, and the testis weight is weighed for standby.
7 rats were randomly selected for each group and epididymal tail sperm was collected using a diffusion method. Placing the epididymal tail into 3mL of 37 ℃ physiological saline, shearing, standing for 1min, and preparing sperm suspension. mu.L of the sperm suspension was removed and placed in 1mL M199 medium, incubated at 37℃for 5min, and 13. Mu.L of the sperm suspension was removed therefrom and smeared on a pre-warmed cytometer for sperm quality analysis.
(7) Evaluation of Molding results
And (3) a molding period: NS group: the body weight of the rats gradually increased. The hair color is glossy, the activity is agile, and the mental state is good. The food intake is gradually increased and the body temperature is normal. Normal defecation, and solid and granular stool. The number of fighting times is increased along with the experiment, so that the method is more active. 1 dead, dead on 2 nd day of gastric lavage, and cadavers were eaten by peers. GTW modeling groups: rats gain weight gradually, slightly faster than NS group. The glossiness of the hair is reduced during the molding process, the hair is sometimes lost, the hair is curled with a preference for the hair, the activity is slower than that of an NS group, and the hair is poor in spirit. The food intake is gradually increased, the body temperature is lower, and a small part of rats cool in body and the tails cool. Loose stool occurs in the initial stage of molding, and the stool is soft and rotten like mud and is light brown; the feces gradually return to normal in the middle and later stages of molding, but the feces are generally softer, and part of the rat feces are soft and rotten according to the feces. 2 deaths occurred on the 2 nd and 3 rd day of gastric lavage, respectively.
Dosing period: NS group: the weight of the rat gradually increases, and the weight increase amplitude is slowed down in the later period of the experiment. The hair color is glossy, the activity is agile, the mental state is good, and the fight is favored. The food intake tends to be stable and the body temperature is normal. Normal defecation, and solid and granular stool. GTW group: the weight of the rat gradually increases, and the weight increase amplitude is slowed down in the later period of the experiment. The hair color gradually recovers the luster, and the mental condition is slightly improved; the activity is still slower, and the fighting phenomenon is less than that of other groups, which are not active. The food intake tends to be stable, the body temperature is normal, the tail is cool, and the later period of the experiment tends to be normal. Normal stool, soft stool, and sinking in the press. Left carnitine group: the weight of the rat gradually increases, and the weight increase amplitude is slowed down in the later period of the experiment. The hair color gradually recovers the luster, the mental condition and the activity state are improved, and the fight is increased. The food intake tends to be stable, the body temperature is normal, the tail is cool, and the later period of the experiment tends to be normal. The feces are normal, the feces are solid particles and are hard. Schisandrin a high dose group: the weight of the rat gradually increases, and the weight increase amplitude is slowed down in the later period of the experiment. The hair color gradually recovers the luster, the mental condition and the activity state are improved, and the fight is increased. The food intake tends to be stable and the body temperature is normal. The feces are normal, the feces are solid particles and are hard. Schizandrin a dose group: the weight of the rat gradually increases, and the weight increase amplitude is slowed down in the later period of the experiment. The hair color gradually recovers the luster, the mental condition and the activity state are improved, and the fight is increased. The food intake tends to be stable and the body temperature is normal. The feces are normal, solid and granular, and can be trapped. Low dose group of schizandrin a: the weight of the rat gradually increases, and the weight increase amplitude is slowed down in the later period of the experiment. The hair color gradually recovers the luster, the mental condition is slightly improved, the activity state is inferior to other dosage groups, and the fight is less. The food intake tends to be stable and the body temperature is normal. The feces are normal, solid and granular, and can be trapped.
The WL-9000 type sperm quality test system (CASA) was used, the test temperature was set at 37℃and the treated sperm suspension was applied to a counter plate, 5 fields of view were selected and the test was completed within 2 min. The main test indexes are as follows: sperm density (×10) 6 Per mL), sperm motility (%), a-stage sperm rate (%), b-stage sperm rate (%). The results showed (table 1): after molding, the sperm density and b-level sperm rate of rats in the GTW group are obviously reduced, wherein the sperm density is obviously different from that of rats in the NS group (P is less than 0.05), the sperm motility of rats in the GTW group is obviously reduced, and the sperm motility of rats in the GTW group is obviously different from that of rats in the NS group (P is less than 0.05), so that the molding is successful.
TABLE 1 sperm density, a-stage sperm count, b-stage sperm count test values for each group
Note that: comparison to NS group: * P is less than 0.05; comparison to the GTW group: # P＜0.05。
example 9 application of protein immune response method in detecting in vitro Signal paths of Polygonatum sibiricum and Zanyu Capsule effective ingredient treatment less sperm weakness
(1) Rat testis material selection: after the last administration of all rats for 24 hours, the chloral hydrate is used for anesthetizing the abdominal aorta to take blood, the experimental animals are sacrificed by bleeding, and testes and epididymis are rapidly picked up for later use; fixing testes on the same side of each rat in Davidson's fixing solution for 24 hours, wherein the volume ratio of tissue liquid is 1:20, and replacing the liquid once every 12 hours; after 24h fixation, the samples were transferred into neutral formalin for fixation for 24-48h. Flushing the tissue after repairing the blocks for 12 hours by running water; dehydrating the graded alcohol concentration from low to high, and embedding the dehydrated alcohol concentration in paraffin through xylene transparency after dehydration, so as to prepare testicular paraffin sections for later use;
(2) Extraction of rat testis protein: cutting and weighing 100mg testis tissue, cutting, adding 0.01mol/LPBS buffer, centrifuging at 2500pm at 4deg.C for 1min, and discarding supernatant; adding 0.5mL of RIPA lysate containing protease inhibitor into the tissue, homogenizing until the tissue mass disappears; placing the tissue slurry on a shaking table, vibrating and cracking for 30min, centrifuging at 12000rpm at 4deg.C for 15min, and sucking the supernatant for use; measuring and calculating the concentration of a sample protein by using 0.5mg/mL Bovine Serum Albumin (BSA) as a standard solution and adopting an enzyme-labeled instrument;
(3) According to the protein concentration measurement result, the concentration of the sample is adjusted to 10 mug/L, 5xSDS buffer solution is added for uniform mixing, and the mixture is heated in a water bath at 95 ℃ in a heater for 10min to enable the protein to be fully denatured; separating protein by 12% SDS-PAGE electrophoresis, preparing gel, loading sample, performing electrophoresis at 60V voltage for 30min, performing electrophoresis at 120V voltage for 1.5h when sample enters into the separation gel, stopping electrophoresis when bromophenol blue runs to gel bottom, and washing and cutting film; 300mA constant current is converted into PVDF film for 1.5h, and then the PVDF film is immersed into ponceau dye liquor after 10 times dilution for dyeing; sealing the PVDF film for standby;
(4) Diluting the SCF and the c-kit antibody by adopting goat serum working solution according to the volume ratio of 1:100, taking out the sealed PVDF membrane, washing by PBST for 3 times for 10min each time, and putting the membrane into a hybridization bag after washing. SCF and c-kit primary antibody were diluted 1:100 by volume using goat serum working solution PBST and placed in hybridization bags overnight at 4 ℃. Taking out PVDF membrane the next day, washing with PBST shaking for 3 times, 10min each time, placing PVDF membrane into new hybridization bag after washing, pouring prepared secondary antibody, shaking for 1h incubation, taking out membrane, washing PBST for 3 times, 10min each time, placing PVDF membrane into luminometer, dripping ECL color development liquid for 30s incubation, photographing with digital gel image analysis system, calculating SCF and c-kit expression;
(5) The expression of SCF and c-kit in testis tissue is shown in Table 2 and FIG. 7, and the results show that the expression of SCF is significantly reduced in the oligospermia model group compared with the normal group; after the administration of the model rats, the SCF protein content of the levocarnitine group and the schizandrin A high-dose group and the medium-dose group is obviously increased (P is less than 0.05), wherein the schizandrin A high-dose group is closest to the normal group, and the schizandrin A low-dose group only finds positive particles in part of seminiferous tubules, and the SCF content is increased but the difference is not obvious. The c-kit protein expression of the weak sperm model group is increased compared with that of the normal group, and the c-kit protein content of each group is reduced after administration, wherein the reduction of the schisandrin A high and medium dose groups is most obvious.
TABLE 2 results of expression of SCF and c-kit proteins in testis tissue of each group
Claims (2)
1. The application of the biosensor combined protein immune response method in detecting the signal path of schisandrin for treating oligospermia is characterized by comprising the following specific steps:
(1) Preparation of AlGaAs/GaAs HEMT biosensor:
a. placing the clean AlGaAs/GaAs HEMT device in a sulfhydryl-containing reagent, soaking 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 off sulfhydryl reagent on the surface of AlGaAs/GaAs HEMT device by deionized water, and adding carboxyl activating agent to generate stable amine activating product;
c. cleaning an AlGaAs/GaAs HEMT device by using 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 a SCF-AlGaAs/GaAs HEMT biosensor;
(2) The application of AlGaAs/GaAs HEMT biosensor in detecting the signal path of the weak sperm disease of the effective component treatment of the Siberian solomonseal rhizome Zanyu capsule:
a. preparing a c-kit-AlGaAs/GaAs HEMT biosensor according to the method in the step (1), sequentially adding recombinant human stem cell factor SCF solution with low concentration to high concentration, 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 current change as an ordinate, performing linear fitting, and further calculating the interaction strength of the SCF and the c-kit according to formulas (1), (2) and (3);
b. preparing c-kit-AlGaAs/GaAs HEMT and SCF-AlGaAs/GaAs HEMT biosensors according to the method in the step (1), sequentially adding schisandrin A standard substance solutions with low concentration to high concentration into the two biosensors respectively, 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 schizandrin A 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 schizandrin A, c-kit and SCF according to formulas (1), (2) and (3);
the formulas described in a and b in step (2) are as follows:
wherein [ A ] b ]For the concentration of c-kit, [ A ] g ]Concentration of SCF/schizandrin A [ C ]]K, K A For binding constant, K D For dissociation constant, ΔI is the current change value, ΔI max Is the maximum variation value of the current;
c. preparing a c-kit-AlGaAs/GaAs HEMT biosensor according to the method in the step (1), sequentially adding mixed solution of recombinant human stem cell factor SCF and schizandrin A with low concentration from low concentration to high concentration, recording current intensity between a source electrode and a drain electrode by adopting an electrochemical device, examining influence of schizandrin A on SCF/c-kit interaction intensity, and analyzing action mechanism of schizandrin A for adjusting SCF/c-kit;
(3) The specific steps of the protein immune response method applied to detecting the oligospermia signal path of schisandrin A treatment are as follows:
a. constructing animal models of oligospermia and asthenozoospermia, dividing a blank control group, taking three high, medium and low doses of schizandrin A and a levocarnitine group, taking venous blood from all rats after the last administration, and then killing the animals and taking testis tissue for freezing storage;
b. cutting testis tissue, shearing, adding phosphate buffer, centrifuging at 4deg.C, discarding supernatant, adding RIPA lysate containing protease inhibitor to lyse testis tissue, centrifuging at 4deg.C, and collecting supernatant;
c. making testis tissue paraffin sections, and observing the expression condition of the testis tissue proteins of each group of rats by an immunohistochemical experiment method;
d. using bovine serum albumin solution as standard solution, and adopting an enzyme-labeled instrument to measure protein concentration of SCF antibody and c-kit antibody; separating proteins by adopting an electrophoresis technology, preparing a PVDF membrane through the steps of gel preparation, electrophoresis, membrane transfer, membrane dyeing and sealing, adding a secondary antibody solution of the proteins related to a signal path for reaction, and analyzing the protein expression condition by combining a Western Blot with a digital gel image analysis system;
e. combining protein expression results based on testis tissue immunochemistry reaction and Western Blot, analyzing the mechanism of schisandrin A mediated SCF/c-kit pathway for treating oligospermia and oligospermia.
2. The application of the AlGaAs/GaAs HEMT biosensor combined protein immune reaction method in detecting the signal path of schisandrin for treating oligospermia, which is characterized in that the carboxyl activating agent in the step (1) b is carbodiimide hydrochloride and N-hydroxysuccinimide, the activating time is 15-30min, the reaction temperature of modifying c-kit or SCF is 4 ℃, and the reaction time is not less than 2 hours.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911316164.4A CN113009127B (en) | 2019-12-19 | 2019-12-19 | Application of biosensor combined protein immune response method in detecting schizandrin A treating oligospermia signal path |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911316164.4A CN113009127B (en) | 2019-12-19 | 2019-12-19 | Application of biosensor combined protein immune response method in detecting schizandrin A treating oligospermia signal path |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113009127A CN113009127A (en) | 2021-06-22 |
| CN113009127B true CN113009127B (en) | 2023-07-07 |
Family
ID=76381413
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201911316164.4A Active CN113009127B (en) | 2019-12-19 | 2019-12-19 | Application of biosensor combined protein immune response method in detecting schizandrin A treating oligospermia signal path |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113009127B (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102313765A (en) * | 2011-07-17 | 2012-01-11 | 北京科技大学 | Biosensor base on zinc oxide and high electron mobility transistor and its preparation method |
| CN206270282U (en) * | 2016-12-23 | 2017-06-20 | 成都海威华芯科技有限公司 | A kind of HEMT biology sensors |
| CN107121544A (en) * | 2017-06-02 | 2017-09-01 | 中国科学院半导体研究所 | Biology sensor of diagnosing acute heart infarction and preparation method thereof |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8836351B2 (en) * | 2008-06-10 | 2014-09-16 | University Of Florida Research Foundation, Inc. | Chloride detection |
| US20110117669A1 (en) * | 2008-11-06 | 2011-05-19 | Fan Ren | Materials and methods for detecting toxins, pathogens and other biological materials |
-
2019
- 2019-12-19 CN CN201911316164.4A patent/CN113009127B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102313765A (en) * | 2011-07-17 | 2012-01-11 | 北京科技大学 | Biosensor base on zinc oxide and high electron mobility transistor and its preparation method |
| CN206270282U (en) * | 2016-12-23 | 2017-06-20 | 成都海威华芯科技有限公司 | A kind of HEMT biology sensors |
| CN107121544A (en) * | 2017-06-02 | 2017-09-01 | 中国科学院半导体研究所 | Biology sensor of diagnosing acute heart infarction and preparation method thereof |
Non-Patent Citations (2)
| Title |
|---|
| AlGaN/GaN high electron mobility transistors for protein–peptide binding affinity study;Chih-Cheng Huang等;《Biosensors and Bioelectronics》;20121008;第41卷;摘要，第719-720页 * |
| 五味子甲素通过SCF/c-kit信号通路调控少、弱精子症大鼠模型精子凋亡作用机制研究;李博怿;《中国优秀硕士学位论文全文数据库 医药卫生辑》;20180815;第2018年卷(第08期);摘要，正文第98-122页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113009127A (en) | 2021-06-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Bennett et al. | Breast cancer, prostaglandins, and bone metastases | |
| Tang et al. | Protective effect of Ginkgo biloba leaves extract, EGb761, on myocardium injury in ischemia reperfusion rats via regulation of TLR-4/NF-κB signaling pathway | |
| CN116173112B (en) | Traditional Chinese medicine formula probiotic composition, preparation method and application thereof in improving male sexual function | |
| CN104435306A (en) | Application of blood-nourishing and brain-clearing preparation in preparation of medicament for treating Alzheimer disease | |
| CN105434894A (en) | Method for preparing traditional Chinese medicine composition for treating systemic lupus erythematosus | |
| Takei et al. | The herbal medicine Toki-shakuyaku-san improves the hypertension and intrauterine growth retardation in preeclampsia rats induced by Nω-nitro-L-arginine methyl ester | |
| CN113009127B (en) | Application of biosensor combined protein immune response method in detecting schizandrin A treating oligospermia signal path | |
| CN1569884B (en) | Method for preparing astragaloside and its use in preparation of drug for preventing and treating diabetic nephropathy | |
| Pippard et al. | Prolactin stimulates Na+-K+-ATPase activity located in the outer renal medulla of the rat | |
| CN107041924A (en) | It is a kind of prevent and treat diabetic nephropathy towards medicine compound extract and preparation method thereof | |
| Chang et al. | Interaction of estrogen and progesterone in the regulation of sodium channels in collecting tubular cells | |
| CN109125331A (en) | GABABApplication of the receptor antagonist in terms of blocking the channel TREK-1 | |
| Stephenson et al. | The effect of monensin on the chemotactic function of bovine neutrophils | |
| CN105456481B (en) | Application of the Fructus Schisandrae Sphenantherae extract in preparation liver regeneration drug | |
| CN103417629B (en) | Traditional Chinese medicine for treating hepatitis B liver cirrhosis | |
| CN111514209A (en) | Application of traditional Chinese medicine composition in preparation of medicine for preventing and/or treating myocardial ischemia-reperfusion injury | |
| CN104922700A (en) | Experimental method for applying Sirt1-Nrf2 novel oxidation resisting pathway in oxidative damage treatment | |
| CN108888652A (en) | A kind of renal lobe beggarweed alcohol extract and its application | |
| CN109170914A (en) | A kind of anti-fatigue health-caring composition and preparation method thereof | |
| CN114982713B (en) | Construction method, evaluation method and application of model combining yin deficiency syndrome type 2 diabetes with animal | |
| CN110141634A (en) | A kind of qi-regulating dissipating bind particle and preparation method thereof | |
| Nuñez et al. | α-MSH potentiates the responsiveness of mammotropes by increasing Ca2+ entry | |
| CN107385029A (en) | LBP-X suppresses the experimental method of glioma growth by adjusting blood-brain barrier | |
| Lam et al. | Chlorpromazine-induced histamine release from guinea-pig skin in vitro—A photosensitive reaction | |
| Weatherall et al. | Urinary porphyrins in experimental lead poisoning |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |