CN108853082B - Application of hematoxylin in preparation of medicine for treating immune glomerulonephritis - Google Patents
Application of hematoxylin in preparation of medicine for treating immune glomerulonephritis Download PDFInfo
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- CN108853082B CN108853082B CN201810673027.5A CN201810673027A CN108853082B CN 108853082 B CN108853082 B CN 108853082B CN 201810673027 A CN201810673027 A CN 201810673027A CN 108853082 B CN108853082 B CN 108853082B
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- membranous nephropathy
- immune glomerulonephritis
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Abstract
The invention discloses an application of hematoxylin in preparation of a medicine for treating immune glomerulonephritis. In the research of immune glomerulonephritis of a membranous nephropathy animal model, hematoxylin is found to achieve the aim of treating membranous nephropathy through at least the following three aspects: 1) the hematoxylin has effects of reducing urine protein, improving lipid metabolism disorder, and increasing plasma albumin level. 2) Hematoxylin has the function of improving the pathological morphological damage of kidney tissues. 3) Hematoxylin has the effects of reducing the level of Vascular Endothelial Growth Factor (VEGF) in serum, regulating the permeability of glomerular filtration membrane by inhibiting VEGF secretion, reducing the excretion rate of urine protein, and protecting renal function. The invention defines that the hematoxylin has the regulating and treating functions on the immune glomerulonephritis, provides a new technical means for treating the immune glomerulonephritis, and has wide application prospect in the field of treating the immune glomerulonephritis.
Description
Technical Field
The invention relates to a new application of hematoxylin, in particular to an application of hematoxylin in treating immune glomerulonephritis, and belongs to the technical field of traditional Chinese medicine development.
Background
Immune glomerulonephritis is a chronic glomerular disease that is primarily initiated in the kidney due to various etiologies. The etiology of most immune nephritis is not clear, and researches prove that various microorganisms including bacteria, viruses, rickettsia, protozoa and the like can cause the disease through immune pathogenesis. The treatment effect and prognosis of the immune nephritis are closely related to the pathological change type, and the immune nephritis is internationally classified according to the histopathological changes: 1. minor pathologic change. 2. Membranous nephropathy. 3. Mesangial proliferative nephritis (including IgA nephritis). 4. Endothelial-mesangial proliferative nephritis. 5. Mesangial capillary nephritis, also known as membranous proliferative nephritis. 6. Focal segmental glomerulosclerosis. 7. Crescentic nephritis, and the like.
Among them, Membranous Nephropathy (MN) is one of the common pathological types of immune glomerulonephritis in adults, is a kidney disease seriously harming human health, and its pathological changes are slowly progressive, often manifested as nephrotic syndrome, which is a common clinical nephrotic syndrome, and the main clinical manifestations are: a large amount of proteinuria, hypoalbuminemia, edema and hyperlipidemia are mainly characterized in that glomerular basement membrane is thickened, deposits are formed under epithelial cells of glomerular capillary walls, a spiky structure is seen, and about 25% of patients with membranous nephropathy can gradually develop renal failure and uremia after the onset of the nephropathy for several years. At present, clinically, glucocorticoid and immunosuppressant are applied for treatment, a certain curative effect can be achieved in a short time, but the long-term application has great toxic and side effects and low disease remission rate, and the disease is easy to relapse after the medicine is stopped. With the research and development of traditional Chinese medicines in China, a plurality of traditional Chinese medicines with immunosuppressive action are widely applied to clinical treatment and obtain considerable curative effects, for example, tripterygium glycosides have immunosuppressive action, so that the tripterygium glycosides are widely applied to clinical treatment, and tacrolimus is taken as a powerful novel immunosuppressant, is also widely applied to clinical treatment of nephrotic syndrome at present, and plays an active role.
Sappan wood, also known as sappan wood, red firewood, palm wood, Mongolian medicine name broom-muea all is the dry heartwood of sappan wood (Caesalpinia sappan L.) of leguminous plants, produced in Burma, Vietnam, India, Malaya and Sri Lanka, is mainly distributed in Guangxi Bai color and heaviness, Yun nan Jingdong, Yunjiang, Maguan, Lijiang, Taiwan, Hainan and the like at home, mainly contains flavonoid compounds and sterol compounds including sappan flavin, sappan chalcone, brazilein, protosappan and the like, and is considered by traditional Chinese medicine to have the effects of activating blood, removing stasis, reducing swelling, relieving pain and the like. Can be used for treating blood, qi, heart and abdominal pain, amenorrhea, puerperal blood stasis, distending pain, asthma, dysentery, tetanus, carbuncle, swelling, liver blood exuberance, and pain due to blood stasis. In addition, sapanwood is used as a natural pigment and widely applied to daily chemicals, food, leather, fabric dyeing and the like, and is used as a staining agent for cell tissue slices and applied to pathological experiments. Since the beginning of research on the pharmacological action of sapanwood marked by research on the in-vitro tumor inhibition effect of the sapanwood water extract by Japanese scholars in the 70 th 20 th century, through continuous exploration and research at home and abroad for decades, the sapanwood is found to have the effects of immunoregulation, tumor resistance, oxidation resistance, bacteria resistance, inflammation resistance, complement resistance, sugar reduction and the like besides the traditional effects. In clinical practice, sapanwood and other Chinese medicinal herbs are formulated to treat autoimmune diseases (such as scleroderma). The traditional Mongolian medicine Gurigumu-Zhu Cao idion can also relieve liver injury caused by anaphylactic reaction by inhibiting cellular immune reaction of organism, thereby treating hepatitis. Studies of Yang Feng and the like find that the sappan wood water extract has obvious inhibition effect on the functions of T cells and B cells of mice, the effect of the sappan wood water extract is enhanced along with the increase of the dosage, and the immunosuppressive effect of the sappan wood is proved to be stronger than that of tripterygium wilfordii. The comparison research with the cyclosporine A (CSA) shows that the composition has the effect of resisting the immune rejection and has no obvious toxic or side effect. Although clinical application of immunosuppressive drugs such as CSA creates a new era for organ transplantation and treatment of immune diseases, most of the drugs lack of selectivity and specificity at present, and long-term application of the drugs can easily cause the body resistance to be reduced to induce severe infection, malignant tumor and the like; on the other hand, the immunosuppressant itself has a high toxicity to the body and a large number of side effects, and serious cases may cause dysfunction or even loss of function of internal organs or transplanted organs. Therefore, researchers urgently expect to find out a novel immunosuppressant with high efficiency, low toxicity and strong selectivity from Chinese herbal medicines, so that the immunosuppressant can replace or partially replace western medicine immunosuppressants currently applied and can be better applied to clinic.
The inventor of the invention has used the water extract of the sappan wood as the animal experiment research of a rat nephrotic syndrome model caused by adriamycin and a cationized bovine serum albumin induced membranous nephropathy model, and also clinically observes the treatment of the patient with the immune glomerulonephritis, and the result further proves that the sappan wood has the immunoregulation treatment effect. Therefore, the inventor and the team perform detailed analysis, take the most main possible effective component hematoxylin in the sappan wood as a breakthrough, perform related animal experiments and in-vitro experiments, discuss the action mechanism of the hematoxylin on the immune glomerulonephritis, take a membranous nephropathy rat model as a research object, observe the influence of the hematoxylin on biochemical indexes, pathological tissues and cytokines of the hematoxylin, compare the influence with tacrolimus, and provide experimental basis for the clinical treatment of the immune glomerulonephritis.
Disclosure of Invention
The invention aims to provide a new application of hematoxylin in preparation of a medicine for treating immune glomerulonephritis.
The molecular formula of hematoxylin is: c16H14O6Molecular weight: 302.28, the molecular structural formula is:
in order to achieve the purpose, the invention adopts the following technical means:
the method selects 50 adult male SD rats, randomly divides the adult male SD rats into a normal control group (group A, N ═ 10), a model group (group B, N ═ 10), a hematoxylin group (group C, N ═ 10), a tacrolimus group (group D, N ═ 10), a prednisone group (group E, N ═ 10) and a B, C, D, E group, respectively establishes a cationized bovine serum albumin (C-BSA) induced rat membranous nephropathy model, namely C-BSA 1mg, adds the cationized bovine serum albumin (C-BSA) and rat membranous nephropathy model into 0.5ml of Phosphate Buffer Solution (PBS), uniformly mixes the cationized bovine serum albumin (C-BSA) and the cationized bovine serum albumin (C-BSA) with an equal amount of Freund incomplete adjuvant to form milk white suspension, performs subcutaneous injection on the axilla and the inguinal multipoint of the rat, performs preimmunization for one week, performs intravenous injection of C-BSA2.5mg (dissolved in 1. Hematoxylin (refined extract, purity 95%) 0.1mg/D is administered to group C, tacrolimus 0.1mg/D is administered to group D, prednisone 1mg/D is administered to group E, and the gavage treatment is continued for 4 weeks. Urine was left for 24 hours before treatment and 24 hours urine protein quantification was assayed. All groups of rats are killed at the end of the fourth week after treatment, kidney tissues are kept, slices are stained by HE, PAS and the like, pathological changes of all groups of rats under a nephroscope and a renal electron microscope of the rats are observed, quantitative enzyme detection of Vascular Endothelial Growth Factor (VEGF) of the rats is carried out in parallel, and relevant data obtained by experiments are compared and statistically analyzed. The results show that the urine protein of the model group is quantified within 24 hours, the blood fat is obviously increased, and the plasma albumin is obviously reduced; the indexes related to the hematoxylin group, the tacrolimus group and the prednisone group are improved. Histological examination can find diffuse proliferation of glomerular cells and matrix of kidney tissue of the model group under the optical microscope, the basement membrane is obviously thickened, and a spiky-like structure can be seen; under an electron microscope, a plurality of electron compact matters are deposited under a basement membrane and epithelium; the basement membrane of each treatment group is not obviously thickened, the damage is relatively reduced, and a small amount of electron compact deposition or exact electron compact deposition is not seen under an electron microscope. The expression level of VEGF in each treatment group was reduced compared to that in the model group. The hematoxylin is proved to have an improvement trend in blood and urine tests after being applied to a rat membranous nephropathy model induced by C-BSA, the survival performance of rats is improved, and pathological and biochemical detection prompts that the hematoxylin has a certain treatment effect on the rat membranous nephropathy. VEGF detection suggests that hematoxylin plays a certain role in inhibiting VEGF expression, reducing kidney tissue damage and the like.
According to the conclusion, the invention provides the application of hematoxylin in preparing the medicine for treating the immune glomerulonephritis.
Preferably, the immune glomerulonephritis is membranous nephropathy.
Wherein the hematoxylin has effects of reducing urine protein of patients with membranous nephropathy, improving lipid metabolism disorder, and increasing plasma albumin level.
Wherein the hematoxylin has the function of improving the pathological morphological damage of the kidney tissue of the patient with membranous nephropathy.
Wherein the hematoxylin has the effects of reducing the level of VEGF in the serum of a patient with membranous nephropathy, regulating the permeability of a glomerular filtration membrane by inhibiting VEGF secretion, reducing the excretion rate of urine protein and protecting the kidney function.
Drawings
FIG. 1 shows the variation of the excretion amount of urinary protein in each group of rats at 24 hours;
note: Δ represents P <0.05 compared to model group; it means that P <0.01 compared to week 4 (before treatment).
FIG. 2 shows the change in VEGF levels in rats of various groups;
note: Δ means P <0.01 compared to normal group, it means P <0.05 compared to normal group; # denotes P <0.05 compared to model group;
FIG. 3 is HE staining (X10) of normal control group;
FIG. 4 shows PAS staining (X40) of normal control group;
FIG. 5 is model group HE staining (x 100);
figure 6 is model set PAS staining (× 100);
FIG. 7 is HE staining (X100) for hematoxylin group;
fig. 8 is a PAS staining of tacrolimus group (× 100);
figure 9 is HE staining (x 100) in the hormone group;
FIG. 10 is a pattern of nail projections (× 100) from the model set;
FIG. 11 shows the changes in the normal control group under an electron microscope;
FIG. 12 shows the change in model set electron microscopy;
FIG. 13 shows the change in hematoxylin group under electron microscope;
FIG. 14 shows the change in Tacrolimus group electron microscopy;
FIG. 15 shows the change under a Podocarpon electron microscope.
Detailed Description
The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.
EXAMPLE 1 test of the efficacy of hematoxylin in the treatment of membranous nephropathy
1. Materials and methods
1.1 materials
1.1.1 Experimental animals
50 healthy male clean SD rats (weight 200-. Animals were acclimatized for one week prior to the experiment, with free access to food and water during the experiment.
1.1.2 Main Experimental drugs
And (3) hematoxylin: the finished product of hematoxylin (purity, 95%) was purchased from Harbin Hongbo GmbH.
Tacrolimus capsule (kangtian): approval document No.: the national drug standard H20084522. Production unit: zhejiang hong Sheng pharmaceutical Co Ltd.
Prednisone (prednisone acetate tablets): approval document No.: H41020283. production unit: henan Tian Fang pharmaceutical industry Co., Ltd.
1.1.3 Main test reagents
Natural bovine serum albumin (N-BSA), production unit: hebei Bohai bioengineering, Inc.
Carbodiimide (EDC), production unit: sigma Co (USA).
Anhydrous Ethylenediamine (EDA), production unit: shanghai Meter and chemical reagent factory.
Freund's incomplete adjuvant, production unit: sigma Co (USA).
10% chloral hydrate solution: the university of Harbin medical is affiliated with the pharmacy department of the second hospital.
Rat VEGF quantitative analysis enzyme-linked immunoassay kit, production unit: beijing Boling Ke is a Biotech Co.
1.1.4 Main Experimental instruments
Electronic analytical balance: resolution 0.1mg, manufactured by Siderris, Germany.
Full-automatic biochemical analyzer: hitachi model 7600 full-automatic biochemical analyzer.
Tungsten filament scanning electron microscope: resolution 3.0nm @30KV (SE and W); 4.0nm @30kv (vp with bsd), magnification: 5-1000000x, carl zeiss, germany.
OLYMPUS BX51M light microscope: ocular magnification: 10, objective magnification: 5-100, instrument magnification: 50-1000, olympus (china) limited.
1.2 test methods
1.2.1 preparation before experiment
(1) Preparation of cationized bovine serum albumin (C-BSA): bovine serum albumin is cationized according to the literature (Broder WA, Ward HJ, Kamil ES, et al.indication of membrane chromatography in situ by administration of an exogenously cationic antigen [ J ]. J Clin Invest, 1982; 69: 451-. EDA67ml was first dissolved in 500ml of distilled water, 350ml of 6mol/L HCl solution was added, the pH was adjusted to 4.75, the temperature was controlled at 25 ℃, 5g BSA and 1.8g EDA were stirred uniformly for 2 hours, the pH was adjusted to 4.75 with 4mol/L acetic acid solution, these were dialyzed against distilled water at 4 ℃ for 48 hours, freeze-dried and the isoelectric Point (PI) was determined with polyacrylamide to be > 8.5.
(2) Preparation of Phosphate Buffered Saline (PBS):
weighing the reagents with an electronic balance: 8.0007 g of sodium chloride, 0.20015 g of potassium chloride, 0.2410 g of potassium dihydrogen carbonate and 3.5289 g of disodium hydrogen phosphate. The above four reagents were dissolved in distilled water to 800ml, the pH was adjusted to about 7.4, and distilled water was added to 1000 ml. Subpackaging in 2 500ml rubber plug glass bottles, sealing, and sterilizing for later use.
(3) Preparing pre-immune liquid
Weighing 50mg of C-BSA by using an electronic balance, putting the C-BSA into a sterilized mortar, adding a small amount of PBS, carefully grinding until C-BSA particles cannot be seen by naked eyes, adding PBS, totaling 25ml, and uniformly mixing; then 25ml Freund's incomplete adjuvant is added and ground until the solution turns milky white (the solution is easy to separate after standing, so when pre-immune injection is carried out, injection is carried out after stirring).
(4) Preparing C-BSA solution
Weighing C-BSA1.848g by an electronic balance, placing into a sterilized mortar, adding a small amount of PBS, carefully grinding until C-BSA particles cannot be seen by naked eyes, pouring the suspension into a 1000ml measuring cylinder, washing the mortar by the PBS for a plurality of times, pouring a cleaning solution into the measuring cylinder, supplementing the solution to 1000ml by the PBS, subpackaging into 10 1000ml rubber plug glass bottles, and storing at 4 ℃ for later use (the whole preparation process is aseptic operation, all glassware is sterilized at 200 ℃ for 4 hours, and the rubber plugs are boiled for 15 minutes for sterilization).
1.2.2 Experimental animal grouping and model replication
After 50 SD rats were acclimatized for one week, they were randomly divided into a normal control group (group a, N ═ 10), a model group (group B, N ═ 10), a hematoxylin group (group C, N ═ 10), a tacrolimus group (group D, N ═ 10), a prednisone group (group E, N ═ 10), and B, C, D, E groups to establish a C-BSA induced rat membranous nephropathy model, respectively.
When the model is replicated, 1mg of C-BSA is added into 0.5ml of PBS for each rat, the mixture is mixed with an equal amount of Freund incomplete adjuvant to form a milk white suspension, the milk white suspension is injected into bilateral armpits and groin of the rat at multiple points, and after one week of preimmunization, 2.5mg of C-BSA2 (dissolved in 1ml of PH7.4PBS) is injected into rat tail veins three times a week for three weeks.
1.2.3 administration mode for Experimental animals
Group a and B rats: gavage was performed with 1ml of physiological saline for 4 weeks.
Group C: 1mg of hematoxylin (95% pure) was dissolved in 10ml of physiological saline, and 1ml of the solution was administered to each rat by gavage (i.e., 0.1mg of hematoxylin per rat per day) for 4 weeks.
Group D: 1mg of tacrolimus was dissolved in 10ml of physiological saline and 1ml of tacrolimus was administered per rat by gavage (i.e. 0.1mg of tacrolimus per day per rat) for 4 weeks.
Group E: 10mg prednisone was dissolved in 10ml physiological saline and 1ml intragastric administered to each rat (i.e. 1mg prednisone per day per rat) for 4 weeks.
1.2.4 taking materials
Urine is collected for 24 hours before administration of the drug for quantitative detection of urine protein, all rats are subjected to abdominal opening after anesthesia and fixation of a left lower abdominal cavity by chloral hydrate (300mg/Kg) at the end of 4 weeks of drug treatment, blood is collected by abdominal aorta after blood stasis, and biochemical index and VEGF detection are performed. After sacrifice, the kidneys were cannulated through the abdominal aorta, and renal tissue was repeatedly lavaged with normal saline, blood was cleared from the vessels, the kidneys were removed, divided into several pieces, fixed in 4% paraformaldehyde and 2% glutaraldehyde, respectively, and examined under light and electron microscopy (each group of rats was left with urine for 24 hours a day before sacrifice).
1.2.5 pathological examination
(1) Hematoxylin-eosin staining (HE staining) of kidney tissue specimen
The method comprises the following operation steps:
1) kidney tissue was fixed with 4% paraformaldehyde overnight;
2) tissue dehydration: soaking in 75% alcohol for several hours, transferring to 80% alcohol for several hours, soaking in 95% alcohol I and 95% alcohol II for 1 hour and 2 hours, transferring to 100% alcohol I and 100% alcohol II for 1 hour, and soaking in chloroform for several hours;
3) embedding conventional tissue, slicing with a slicer to about 5 μm, and oven drying;
4) paraffin sections were dewaxed using xylene and washed with various grades of alcohol to water: sequentially soaking in xylene I, xylene II, 100% ethanol, 95% ethanol, 80% ethanol and 75% ethanol for 5min, 2min, 1min and 1min, and washing with distilled water for 2 min;
5) staining with hematoxylin for 5min, washing with tap water, differentiating with hydrochloric acid ethanol for 30sec, soaking in tap water for 15min, and re-staining with eosin for 2 min;
6) dehydrating, transparent and sealing: dehydrating in 95% ethanol, 100% ethanol, and xylene for 2 times, each for 1min, sealing with neutral resin, and taking pictures under microscope.
(2) PAS staining of kidney tissue specimen
The method comprises the following operation steps:
1) fixing kidney tissue with 4% paraformaldehyde for 10 min;
2) tissue dehydration: soaking in 75% alcohol for several hours, transferring to 80% alcohol for several hours, soaking in 95% alcohol I and 95% alcohol II for 1 hour and 2 hours, transferring to 100% alcohol I and 100% alcohol II for 1 hour, and soaking in chloroform for several hours;
3) embedding conventional tissue, slicing with a slicer to about 5 μm, and oven drying;
4) after the slices are subjected to conventional dewaxing hydration, reducing for 10min by using 1% periodic acid aqueous solution;
5) washing with tap water for 2 times, and washing with double distilled water for 1 time;
6) adding schiff reagent, and incubating for 20min in a greenhouse in a dark place;
7) 0.5% sodium metabisulfite is washed 3 times for about 5min each time;
8) cleaning with double distilled water for 5min for 3 times;
9) performing light hematoxylin staining for 10 min;
10) conventional dewatering, transparency and mounting: dehydrating in 95% ethanol, 100% ethanol, and xylene for 2 times, each for 1min, sealing with neutral resin, and taking pictures under microscope.
(3) Examination of renal tissue specimens under electron microscope
The method comprises the following operation steps:
1) fixing in 2% glutaraldehyde buffer solution;
2) then putting the mixture in osmate fixed solution with l percent for post-fixing at room temperature;
3) dehydrating with gradient ethanol (50% -70% -95% -100% ethanol), clearing kidney tissue with propylene oxide, soaking tissue in mixed solution of propylene oxide and EPON812 resin, embedding tissue with fresh EPON812 resin, heating and condensing: the embedded tissue block can be preserved for a long time.
4) Firstly, cutting 1um semi-thin slices by a semi-thin slicer;
5) after dyeing with Melanin solution, finding out glomerulus with obvious pathological changes under a light microscope, cutting 50nm ultrathin section with an ultrathin microtome at the position where the glomerulus with pathological changes is found in the tissue block, and loading on a copper mesh collodion film. Double dyeing is carried out by uranium acetate and lead citrate. The stained sections were photographed under an electron microscope.
1.2.6 quantitative enzyme assay for rat VEGF
The principle is as follows: the concentration of rat VEGF in the sample is detected by a double-antibody sandwich ELISA method in the experiment. The rat VEGF trap antibody is pre-coated on the microplate, and when the sample or reference is added, the rat VEGF will be bound to the trap antibody, and other free components will be removed by washing. When biotinylated anti-rat VEGF antibody is added, the anti-rat VEGF antibody binds to rat VEGF to form a sandwich immune complex, and the other free components are removed by washing. Followed by the addition of horseradish peroxidase-labeled avidin. Biotin binds specifically to avidin, which binds the avidin-linked enzyme to the sandwich immune complex; other free components are removed by the process of washing. And finally adding a color developing agent, wherein if VEGF exists in the sample, an immune complex is formed, and the horseradish peroxidase catalyzes the colorless color developing agent to be oxidized into a blue substance which is yellow after the stop solution is added. Detecting by an enzyme-labeling instrument, reading the OD value at 450nm, drawing a standard curve by a reference substance in a manner that the rat VEGF concentration is in direct proportion to the OD450 value, and calculating the VEGF concentration in the sample by contrasting the OD value in an unknown sample. The operation procedure is as follows:
(1) the kit is taken out from the refrigerator and then is placed at room temperature for balancing for 20 minutes; the remaining reagents after each assay were immediately stored at 4 ℃.
(2) The concentrated washings were diluted with double distilled or deionized water (1 part with 19 parts water).
(3) And (3) adding 0.25ml of standard product diluent into a freeze-dried standard product bottle to enable the final concentration of VEGF to reach 1000pg/ml, standing for 15 minutes, then gently suspending for complete decomposition, diluting by using the standard product diluent in a multiple ratio gradient manner, and then sequentially adding the diluted standard product diluent into a detection hole (seven points are taken on a standard curve, the highest concentration is 1000pg/ml, and the standard product diluent is directly added to serve as 0 concentration).
(4) The number of slats required for a single experiment was calculated and determined, the required slats were removed and placed in a frame, temporarily sealed with a foil bag until the slats were replaced, and stored at 4 ℃. Respectively arranging a blank hole, a sample hole to be detected and a standard hole, marking the positions of the holes, respectively adding a specimen or a standard substance with different concentrations (50 mu l/hole) into the corresponding holes, adding 10 mu l of a sample to be detected into the sample hole to be detected, and adding 40 mu l of sample diluent (namely the sample is diluted by 5 times); and (4) adding only TMB developing solution and stopping solution into blank control holes. The reaction wells were sealed with a sealing plate of gummed paper and incubated at room temperature for 120 minutes.
(5) The plate was washed 5 times and finally patted dry on thick absorbent paper.
(6) Biotinylated antibody working solution (50. mu.l/well) was added. The reaction wells were sealed with a sealing plate of gummed paper and incubated at room temperature for 60 minutes.
(7) The plate was washed 5 times and finally patted dry on thick absorbent paper.
(8) Add the enzyme conjugate working solution (50. mu.l/well). The reaction wells were sealed with a sealing plate of gummed paper and incubated for 20 minutes at room temperature in the dark.
(9) The plate was washed 5 times and finally patted dry on thick absorbent paper.
(10) Color reagent TMB 50. mu.l/well was added and incubated for 20 minutes at room temperature in the dark.
(11) Add stop solution 50. mu.l/well and measure OD450 value immediately after mixing.
(12) And (3) calculating: drawing a standard curve according to the concentration of the standard substance and the corresponding OD value, calculating the corresponding sample concentration according to the OD value of the sample, and finally, the final concentration is the actual measured concentration multiplied by the dilution multiple.
1.3 statistical methods
The obtained data all adopt the mean value plus or minus standard deviation
It shows that SPSS13.0 statistical software is used for analysis, and the variance analysis and t test are adopted for the comparison of the mean values among groups, and the statistical significance is obvious when p is less than 0.05.
2. Results
2.1 general conditions of the experimental rats
The rats in the normal group have obvious weight gain, good mental status, free movement, glossy fur and sensitive reaction. B. C, D, E rats are modeled for about 10 days, and gradually have the phenomena of lassitude, slow activity, yellow and lusterless fur, slow reaction, anorexia, slow weight increase, rolling, dislike activity, urine volume reduction and the like, and some rats have diarrhea and loose stool with different degrees. In the second week or so, some rats showed mild edema of feet and abdomen, and in the 4 th week, the rats in the model group showed subcutaneous edema of different degrees, such as feet and scrotum. In the 4 th to 6 th weeks, subcutaneous and scrotal edema of the rats in the group B is aggravated, 1 rat dies, in the 7 th to 8 th weeks, C, D, E rats have better states than before, the appetite is recovered, the food intake is gradually increased, subcutaneous edema is obviously relieved, the states of the model groups are worse, 1 rat dies, and after the rats are sacrificed in the 8 th week, the rats in the model control group have ascites with different degrees. The death causes of the rats consider that the plasma albumin is continuously reduced, the viscera are edematous, the resistance is reduced, the infection is caused, and finally the death is caused.
2.2 Change in urinary protein excretion in rats in Each group in 24 hours
After molding, a large amount of proteinuria appears in each group of rats, which prompts successful molding, and the proteinuria level between molding groups (B, C, D, E) has no obvious difference; the quantitative amount of urine protein in each treatment group at week 8 was significantly reduced compared to that before treatment at week 4 (P < 0.01); two-by-two comparisons between the model group and each treatment group at week 8 showed that the urinary protein levels were significantly reduced (P <0.05) in each treatment group compared to the model group, and no significant difference was observed between the treatment groups. See table 1, fig. 1 for details.
TABLE 1 variation of urinary protein excretion in each group of rats in 24 hours
Note: # denotes P <0.05 compared to model group; p <0.01 compared to week 4 (before treatment).
2.3 Change of Biochemical index in groups of rats
The results in table 2 show that TG and TC in the model group are obviously increased compared with the normal group, and the ALB level is obviously reduced compared with the normal group (P < 0.05); after 8 weeks of treatment, TG is increased compared with a normal group in each treatment group, and compared with a model group, the TG level is reduced in each treatment group after medicament treatment; the TC level of each treatment group is basically recovered to the normal level after treatment and is obviously lower than that of the model group (P < 0.01); the ALB level of the treatment group is obviously increased compared with that of the model group, and the hypoproteinemia is improved after the treatment (P < 0.01); the levels of Cr and BUN between 5 groups have no significant difference, and the difference of each index between treatment groups has no statistical significance. See table 2 for details.
TABLE 2 Biochemical index changes in rats of each group
Note: p <0.05 compared to normal group; p <0.01 compared to normal group; # denotes P <0.01 compared to model group.
2.4 changes in VEGF levels in groups of rats
The results in table 3 show that the levels of peripheral blood VEGF of rats in the model group, the hematoxylin group, the tacrolimus group and the prednisone group are higher than those of the normal control group, and the P is less than 0.01, so that the obvious difference exists; the peripheral blood VEGF level of rats in the treatment group is reduced to a certain extent compared with that of a model group, and the statistical difference exists, wherein P is less than 0.05, which shows that the drug has a certain reduction effect on the VEGF level; there were no statistical differences between treatment groups. See table 3, fig. 2 for details.
TABLE 3 change in VEGF levels in groups of rats
Note: p <0.01, P <0.05, compared to normal; # denotes P <0.05 compared to model group.
2.5 Change of Kidney tissue in groups of rats
(1) Change under the light mirror:
the kidney tissues of the normal control group stained by HE and PAS are observed under a light microscope, the structure of the kidney tissues is normal, the structures of the renal cortex and medulla are clear, the glomerulus is not hardened and swollen, the capillary plexus is not proliferated, the wall of the arteriole is not thickened, the lumen is not narrowed, the renal tubule is not expanded and shrunk, the renal stroma is not obviously infiltrated by lymphocytes, and the fibrotic lesion is not seen. See figures 3-4 for details.
The model group has obvious glomerulus swelling, diffuse proliferation of mesangial cells and stroma, spike-like structure, diffuse thickening of basement membrane, vacuole and granule degeneration of renal tubule epithelial cells, infiltration of a large amount of inflammatory cells of renal interstitium, thickening of arteriole wall and stenosis of lumen. See figures 5-6, 10 for details.
The glomerular swelling is not obvious in the treatment group, mesangial cells and stroma are slightly hyperplastic, and inflammatory cells are correspondingly reduced to different degrees, wherein the basement membrane is not obviously thickened in the hematoxylin group and the tacrolimus group, and the interstitial small-focus inflammatory cells infiltrate into the kidney. See figures 7-9 for details.
(2) Change under electron microscope:
the extent of thickening of the glomerular basement membrane and the deposition of electron dense matter within the visceral epithelial cells, the epithelium, the basement membrane and the mesenteric region were mainly observed under an electron microscope.
The basement membrane of the normal control group under an electron microscope is not thickened, and obvious electron compact deposition and foot process cell fusion are not seen in each area. See figure 11 for details.
The irregular thickening of the basement membrane can be seen under the electron microscope of the model group, the deposition of a plurality of electron compact matters can be seen in the basement membrane, the foot processes of epithelial cells of the visceral layer are diffused and fused, and the deposition of a plurality of electron compact matters can be seen under the epithelium. See figure 12 for details.
The hematoxylin group and tacrolimus group have similar performance under an electron microscope, the basement membrane has no obvious thickening, the podophyllum is segmentally fused, and exact electron compact substance deposition is not seen under the epithelium and the mesentery region. See figures 13, 14 for details.
The hormone group shows that the basement membrane is not obviously thickened under an electron microscope, foot processes are widely fused, and a small amount of electron compact substance deposition can be seen under the epithelium and the mesentery. See figure 15 for details.
3. Conclusion
1) Hematoxylin can reduce urine protein of rat model of C-BSA membranous nephropathy, improve lipid metabolism disorder, and increase plasma albumin level.
2) Hematoxylin has the function of improving the pathological morphological damage of the kidney of the rat model with the C-BSA membranous nephropathy.
3) Through the detection of VEGF, the level of VEGF in the serum of a rat model with C-BSA membranous nephropathy is increased, and the level of VEGF in the serum of the rat after hematoxylin treatment is reduced, so that hematoxylin is suggested to inhibit the secretion of VEGF, regulate the permeability of a glomerular filtration membrane, reduce the excretion rate of urine protein and protect the kidney function.
4) The experimental study provides part of experimental basis for the application of hematoxylin serving as a novel traditional Chinese medicine immunosuppression in the treatment of clinical nephrotic syndrome in the future.
Claims (5)
1. The application of hematoxylin in preparing medicines for treating immune glomerulonephritis is characterized in that the molecular formula of the hematoxylin is as follows: c16H14O6Molecular weight: 302.28, the molecular structural formula is:
2. the use according to claim 1, wherein the immune glomerulonephritis is membranous nephropathy.
3. The use according to claim 2, wherein hematoxylin has the effects of reducing urine protein, improving lipid metabolism disorder, and increasing plasma albumin level in patients with membranous nephropathy.
4. The use according to claim 2, wherein hematoxylin has the effect of ameliorating the histopathological morphological damage of the kidney of a patient with membranous nephropathy.
5. The use according to claim 2, wherein hematoxylin has the effect of reducing the level of VEGF in the serum of patients with membranous nephropathy, and the renal function is protected by inhibiting VEGF secretion, regulating glomerular filtration membrane permeability, reducing the rate of excretion of urinary protein.
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| JP2009242247A (en) * | 2008-03-28 | 2009-10-22 | Institute Of Physical & Chemical Research | Human immunodeficiency virus infection inhibitor and aids-treating or preventing drug |
| CN105748461A (en) * | 2016-04-13 | 2016-07-13 | 天津大学 | Application of haematoxylin to inhibition of amyloid beta-protein aggregation and related toxicity |
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| JP2009242247A (en) * | 2008-03-28 | 2009-10-22 | Institute Of Physical & Chemical Research | Human immunodeficiency virus infection inhibitor and aids-treating or preventing drug |
| CN105748461A (en) * | 2016-04-13 | 2016-07-13 | 天津大学 | Application of haematoxylin to inhibition of amyloid beta-protein aggregation and related toxicity |
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| Title |
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| Brazilin Suppresses Inflammation via the Down-regulation of IRAK4 in LPS-stimulated Raw264.7 Macrophage;Kye-Yoon Yoon等;《Journal of Food and Nutrition Research》;20151231;第3卷(第9期);第575-580页 * |
| 小分子IRAK-4 抑制剂的研究进展;陈钊 等;《中南药学》;20151031;第13卷(第10期);第1017-1024页 * |
| 苏木治疗大鼠膜性肾病的实验研究;胡克杰 等;《中医药信息》;20121231;第29卷(第1期);第108-111页 * |
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