Method for researching effect of Chemerin in kidney aging process

Abstract

The invention discloses a method for researching the effect of Chemerin in the kidney aging process, and belongs to the technical field of pharmacological research, male mice are selected, mice blood and kidney tissue samples are collected in groups, kidney tissues are dyed, pathological changes of kidney tubules are observed under an optical microscope, and the kidney aging degree is evaluated; the method comprises the following steps: determining the level of Chemerin in serum by using an ELISA (Enzyme-Linked Immunosorbent Assay) kit, and evaluating the correlation between Chemerin and kidney aging; carrying out RT-qPCR (Reverse Transcription-Quantitative Polymerase Chain Reaction) detection on mRNA (Messenger Ribonucleic Acid) levels of the mouse A sample is prepared by extracting tissue protein, SDS-PAGE gel is prepared, and then electrophoresis, membrane transfer, sealing, primary antibody incubation, washing, secondary antibody incubation, washing and developing are carried out to detect the protein level. According to the detection method provided by the invention, the occurrence of kidney aging can be detected, and intervention measures can be taken in time to delay kidney aging as a new target.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of pharmacological research, and particularly relates to a method for studying the role of Chemerin in the aging process of kidneys. BACKGROUND

[0002] In the complex process of human aging, the kidney is one of the organs that is most significantly affected. From the perspective of tissue structure, with age, the weight of the kidney gradually decreases, the number of nephrons decreases significantly, glomerular sclerosis, tubular atrophy and other degenerative changes become increasingly obvious, and these morphological changes directly lead to the gradual decline of kidney function, including decreased filtration function, reduced metabolic waste discharge efficiency, and weakened ability to regulate water, electrolyte and acid-base balance, thereby affecting the stability of the internal environment of the entire body.

[0003] In addition, the impact of kidney aging is not limited to the kidney itself, and it is closely related to the occurrence and development of various chronic diseases. For example, in diabetic nephropathy, the decreased metabolic capacity of the kidney caused by aging exacerbates the damage of high blood glucose to the kidney microvessels, accelerates the pathological process of glomerular basement membrane thickening and mesangial matrix proliferation, and the occurrence of hypertensive nephropathy is closely related to the decreased elasticity of kidney blood vessels and the imbalance of the renin-angiotensin system caused by aging. Kidney aging reduces the tolerance of the kidney to blood pressure fluctuations, making it more susceptible to damage from high blood pressure, forming a vicious cycle of "high blood pressure-kidney damage-blood pressure" further increasing.

[0004] In recent years, Chemerin has gradually become the focus of medical research as a new type of adipocyte factor. With further research, more and more evidence shows that Chemerin is closely related to kidney disease. In various kidney disease models and clinical samples, the expression level of Chemerin abnormally increases, and the change in its concentration is positively correlated with proteinuria and the degree of kidney function damage, suggesting that it may participate in the occurrence and development of kidney disease through pathways such as activation of inflammatory signaling pathways and promotion of kidney fibrosis.

[0005] However, compared to the research of Chemerin in other disease fields, its role and specific mechanism in the aging process of the kidney have not been fully elucidated. Current researches are mostly focused on the pathological state of kidney disease, and there is still a lack of systematic and in-depth exploration of how Chemerin regulates the degenerative changes of kidney tissue structure and function in the normal aging process. SUMMARY

[0006] Therefore, the purpose of the present application is to provide a method for studying the role of Chemerin in the aging process of the kidney.

[0007] To achieve the above-mentioned purpose, the present application provides the following technical solutions:

[0008] The application provides a method for studying the role of Chemerin in the aging process of a kidney, comprising the following steps,

[0009] S1, collecting samples: C57 / BL6J male mice are selected and divided into a young group (about 3 months old) and an old group (about 24 months old) and are raised in a 22 DEG C condition and a 12h day-night alternating light environment; after adaptive feeding for one week, the blood and kidney tissue samples of the mice are collected;

[0010] S2, histological observation: the kidney tissue is dyed, and pathological changes such as glomeruli and renal tubules in the kidney are observed under an optical microscope, and the degree of kidney aging is evaluated;

[0011] S3, serum detection: the level of Chemerin in the serum is determined by using an ELISA kit, and the correlation between Chemerin and kidney aging is evaluated;

[0012] S4, RT-qPCR detection of the mRNA levels of mouse Chemerin and its receptor CMKLR1: after extracting the RNA of the kidney tissue, the RNA is reversely transcribed into cDNA by a two-step method, finally, the cDNA is subjected to RT-qPCR detection according to the reaction program of 1.50 DEG C for 2 min, 2.95 DEG C for 10 min, pre-denaturation, 3.95 DEG C for 15 s, denaturation, 4.60 DEG C for 1 min, annealing and extension, and steps 3 and 4 are 40 cycles.

[0013] S5, Western blot quantitative detection of the protein level of the kidney tissue: first, the tissue protein is extracted for sample preparation, and the SDS-PAGE gel is prepared, then electrophoresis, membrane transfer, blocking, primary antibody incubation, washing, secondary antibody incubation, washing and development are carried out.

[0014] Further, in the step S2, when the kidney tissue is dyed, HE staining, Masson staining and PAS staining are used to observe the pathological changes of kidney aging from different points.

[0015] Further, in the step S3, when the correlation is evaluated, the optical density of each hole is measured by using an enzyme marker at a wavelength of 450 nm.

[0016] Further, in the step S4, when RT-qPCR is detected, a general PCR instrument and a real-time fluorescent quantitative PCR instrument are used for determination.

[0017] Further, in the step S5, the SDS-PAGE gel concentration, electrophoresis, membrane transfer time and the proportion of the primary antibody are adjusted according to the molecular weight of the target protein.

[0018] The application has the following beneficial effects:

[0019] 1. In the present invention, by measuring the Chemerin level in the blood, it is found that the Chemerin level is significantly positively correlated with age, which can realize the quantitative assessment of the aging process.

[0020] 2. In the present invention, HE, Masson, and PAS staining can visually display changes in kidney structure and provide direct evidence for assessing the severity of kidney damage.

[0021] 3. The detection method provided by the present invention can accurately evaluate the impact of Chemerin levels on renal aging through multiple aspects such as serum testing, histological observation and RT-qPCR.

[0022] 4. The detection method provided by the present invention can detect the occurrence of renal aging, which helps to take timely intervention measures and serves as a new target to delay renal aging.

[0023] Other advantages, objectives and features of the present invention will be described in the following description and will be apparent to those skilled in the art to some extent, or those skilled in the art can be taught from the practice of the present invention. The objectives and other advantages of the present invention can be realized and obtained through the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to make the purpose, technical solutions and beneficial effects of the invention clearer, the present invention is described with the following drawings:

[0025] Figure 1 This is a flow chart of the experimental design in the embodiment of the present invention;

[0026] Figure 2 This is a diagram showing the results of renal histological pathological changes in an embodiment of the present invention;

[0027] Figure 3 This is a graph showing a positive correlation between serum Chemerin levels and renal aging in an embodiment of the present invention;

[0028] Figure 4 This is a graph showing the high expression of Chemerin and its related receptor RNA levels in kidney tissue in an embodiment of the present invention;

[0029] Figure 5 This is a graph showing the high expression of aging markers and Chemerin and its related receptor proteins in kidney tissue in the present invention;

[0030] The following are marked in the accompanying drawings: DETAILED DESCRIPTION

[0031] like Figure 1-5 As shown, the present invention provides a method for studying the effect of Chemerin in the aging process of the kidney.

[0032] S1, adopt natural aging model to divide mice into young group, old group, in 22 ℃ condition 12 h day and night alternation light environment feeding, adaptive feeding a week after collecting mouse blood, kidney tissue sample, extract mouse eyeball to collect into EP tube, put EP tube into 4 ℃ centrifuge, 3000g centrifugal 15 min after taking supernatant liquid, with 200 μL EP tube is divided, and is stored under the condition of -80 ℃;Peel off kidney, a part is immersed in 4% paraformaldehyde for histological detection, another part is frozen in -80 ℃ condition for WB analysis;

[0033] S2, the kidney tissue immersed in 4% paraformaldehyde, after dehydration, transparency, embedding, section, carries out HE, Masson, PAS dyeing treatment respectively, and the pathological change of kidney tubule of kidney is observed under optical microscope, and the degree of kidney aging is evaluated;

[0034] Wherein HE dyeing is dewaxing, rehydration, hematoxylin staining, eosin staining, dehydration, transparency, mounting;

[0035] Masson dyeing is dewaxing, rehydration, Weight iron hematoxylin dyeing nucleus, 1% hydrochloric acid ethanol differentiation, 0.1-1% lithium carbonate counterbluing, acidic magenta dyeing, 2% glacial acetic acid aqueous solution washes 1 min, 1% phosphomolybdic acid aqueous solution differentiation, 2% glacial acetic acid aqueous solution washes 1 min, benzamine blue aqueous solution or 1% light green aqueous solution dyes 1 min, 0.2% glacial acetic acid aqueous solution washing, dehydration, transparency, mounting;

[0036] PAS dyeing is periodic acid solution oxidation, Schiff reagent dyeing, hematoxylin staining, differentiation, dehydration, transparency, mounting;

[0037] S3, take out reagent kit and balance to room temperature, configure good washing liquid, standard working solution, biotinylated antibody working solution, HRP enzyme conjugate working solution, set standard hole, blank hole and sample hole, 100 μL times dilution standard is added to standard hole, 100 μL standard sample diluent is added to blank hole, the rest of the holes add 100 μL sample to be measured, give enzyme-labeled plate film, 37 ℃ incubation 90 min, after discarding the liquid in the plate, 100 μL biotinylated antibody working solution is added to the hole, after film covering, 37 ℃ incubation 1 h, remove the liquid in the plate, wash the plate 3 times, add HRP enzyme conjugate working solution 100 μL per hole, after film covering, 37 ℃ incubation 30 min, remove the liquid in the plate, wash 5 times, add substrate solution (TMB) 90 μL per hole, after film covering, 37 ℃ avoid light incubation 15 min, add 50 μL stopping solution per hole, stop the reaction, then the optical density (OD) value of each hole is measured by enzyme-labeled instrument at 450 nm wavelength, that is, the Chemerin level in mouse serum can be detected;

[0038] S4, kidney tissue samples stored at -80℃ were taken out, rinsed in PBS, and then cut into 20 mg in a grinding tube, and sterilized grinding beads and 1 mL of Trizol were added to the tube, and then the tube was placed in a grinder at a frequency of 70 Hz for 90 seconds to fully lyse the tissue. After lysis, 200 μL of chloroform was added for extraction, and then 500 μL of supernatant was aspirated into a new enzyme-free sterile 1.5 mL EP tube, and an equal volume of isopropanol was added. After mixing, centrifugation was performed, and the supernatant was discarded. Then, 1 mL of 75% anhydrous ethanol prepared with DEPC water was added for washing and centrifugation, and the supernatant was discarded. DEPC water was added to the EP tube to dissolve the precipitated RNA. The concentration of the RNA was measured using a spectrophotometer, and 1.5 μg of total RNA was used for reverse transcription into cDNA. The total volume of the sample required was calculated, and the remaining RNA was stored in a -80℃ refrigerator.

[0039] After reverse transcription, RT-qPCR was performed using a two-step method. The reaction system was 2x SYBR Green mix 10 μL, cDNA template 0.5 μL, upstream primer 5 pM, downstream primer 5 pM, and deionized water 10 μL. The reaction system was mixed and then centrifuged, and then added to the reaction well. The reaction program was 1.50℃ for 2 min, 2.95℃ for 10 min for pre-denaturation, 3.95℃ for 15 s for denaturation, 4.60℃ for 1 min for annealing and extension, and steps 3 and 4 were 40 cycles.

[0040] S5. Weigh and cut 20 mg of kidney tissue into a grinding tube, add 1 mL of cell lysis buffer and grinding beads to the tube, grind at a frequency of 70 Hz for 90 seconds, ultrasonically lyse after grinding, and then rinse with clean water; centrifuge at 12000 rpm for 15 minutes in a 4°C centrifuge, remove tissue fragments, and aspirate the supernatant into an empty new 1.5 mL EP tube. Take 1 μL of protein supernatant and measure the concentration on a spectrophotometer. Then calculate the total amount for 20 times based on the sample load of 25 μg / time and the sample volume of 15 μL, and add the corresponding amount of 5× loading Buffer and ddH2O were diluted, and after thorough shaking and mixing, the samples were placed in a metal bath at 100℃ for 10 minutes and sealed at -20℃. Then, SDS-PAGE gel was prepared and electrophoresis was performed. After shaking and mixing, the samples were loaded into the SDS-PAGE gel wells and electrophoresed at 80V constant voltage for 30 minutes. After the protein markers appeared and began to separate, the voltage was adjusted to 100V and electrophoresis was continued for 1 hour until the loading reached the bottom layer of the glass plate. 1× transfer buffer was prepared and PVDF was sheared. The membrane was activated in methanol. According to the molecular weight of the target protein and the position and size of the protein marker band, the SDS-PAGE gel was cut and placed in 1× transfer buffer for later use. The transfer clip was prepared, i.e., sponge pad (white positive electrode side) - double-layer filter paper - PVDF membrane - PAGE gel - filter paper - sponge pad (black negative electrode side). After buckling the transfer clip, it was placed steadily in the transfer tank, ice bath at low temperature, constant voltage transfer 100V, 1.5h; after the transfer was completed, the membrane was blocked with 5% skim milk for 1.5h.

[0041] Incubate the membrane with diluted primary antibody in blocking buffer at 4°C overnight. After incubation, wash the membrane three times with TBST for 10 minutes each time. Then incubate the membrane with conjugated secondary antibody in blocking buffer at room temperature for 1 hour, wash the membrane three times with TBST for 10 minutes each time, and finally prepare ECL chemiluminescent solution in a 1:1 ratio. Add the chemiluminescent solution to the membrane and incubate for 1 minute in the dark. Place the strips in a luminometer for development.

[0042] Test results:

[0043] Depend on Figure 2 It can be seen that the present invention detected that with aging, the kidneys of aging mice showed increased glomerular fibrosis, decreased number of renal tubular epithelial cells, obvious expansion of the tubular lumen, and cell debris, indicating impaired renal function.

[0044] Depend on Figure 3 It can be seen that with aging, the serum Chemerin level of mice is positively correlated with age and is highly expressed in aged mice. Chemerin is very likely to be involved in the occurrence and development of renal aging.

[0045] Depend on Figure 4It can be seen that the expression of Chemerin and its receptor CMKLR1 in the kidney tissues of the mice is significantly increased in the aging mice, and Chemerin and its receptor are positively correlated with the age.

[0046] It can be seen that the expression of Chemerin and its receptor CMKLR1 in the kidney tissues of the mice is significantly increased in the aging mice, and Chemerin and its receptor are positively correlated with the age. Figure 5 It can be seen that the expression of Chemerin and its receptor CMKLR1 in the kidney tissues of the mice is significantly increased in the aging mice, and Chemerin and its receptor are positively correlated with the age.

[0047] Finally, it should be pointed out that the above preferred embodiments are only used to illustrate the technical solutions of the present application and are not limiting. Although the present application has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in form and details without departing from the scope defined by the claims of the present application.

Claims

1. A method for studying the effect of Chemerin on kidney aging, characterized by: The following steps are included: S1. Sample collection: Male mice were selected and divided into groups. They were housed at 22°C in a 12-hour day / night light environment. Blood and kidney tissue samples were collected after one week of adaptive feeding. S2. Histological observation: The kidney tissue was stained and the pathological changes of renal tubules were observed under an optical microscope to assess the degree of renal aging. S3. Serum testing: ELISA kit was used to measure the level of Chemerin in serum and evaluate the correlation between Chemerin and renal aging; S4. RT-qPCR detection of mouse Chemerin and its receptor CMKLR1 mRNA levels: After extracting RNA from kidney tissue, the RNA was reverse transcribed into cDNA using a two-step method. Finally, the cDNA was subjected to RT-qPCR detection according to the instructions according to the reaction program of 1.50°C for 2 min, 2.95°C for 10 min, pre-denaturation, 3.95°C for 15 s, denaturation, 4.60°C for 1 min, annealing and extension, and steps 3 and 4 for 40 cycles; S5. Quantitative detection of renal tissue protein levels by Western blot: First, extract tissue protein for sample preparation and prepare SDS-PAGE gel, followed by electrophoresis, transfer, blocking, incubation with primary antibody, washing, incubation with secondary antibody, washing, and development.

2. The method for studying the effect of Chemerin on kidney aging according to claim 1, characterized in that: In step S2, HE staining, Masson staining, and PAS staining are used to stain the kidney tissue to observe the pathological changes of kidney aging from different points.

3. The method for studying the effect of Chemerin on kidney aging according to claim 2, characterized in that: In step S3, when evaluating the correlation, the optical density of each well is measured at a wavelength of 450 nm using a microplate reader.

4. The method for studying the effect of Chemerin on kidney aging according to claim 3, characterized in that: In step S4, RT-qPCR is detected using a common PCR instrument and a real-time fluorescence quantitative PCR instrument.

5. The method for studying the effect of Chemerin on kidney aging according to claim 4, characterized in that: In step S5, the SDS-PAGE gel concentration, electrophoresis, transfer time, and the ratio of primary antibodies are adjusted according to the molecular weight of the target protein.

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