Research method of influence of S100A9 on AQP4 and influence mechanism thereof

Abstract

The invention discloses a method for researching the influence of S100A9 on AQP4 and an influence mechanism of the S100A9, and relates to the technical field of biomedicine. Comprising the following steps: cell culture; the method comprises the following steps: inoculating U251 astroglioma cells or SD (Sprague Dawley) rat primary astroglioma cells into a culture dish, after the cells are completely adhered to the wall, replacing a culture medium containing S100A9 proteins with different concentrations, and continuously culturing for 12 hours; the cell activity is detected through CCK8; and adding a CCK8 reagent into the culture dish, incubating for a period of time, and detecting the activity of each group of cells. The invention provides a systematized and detailed research method, and the influence of the S100A9 on the AQP4 can be clarified; through the experimental steps of cell culture, CCK8 detection, Western Blot, RT-qPCR and the like, the change of the S100A9 on the expression and positioning of the AQP4 under different concentrations can be comprehensively known.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a research method for the influence of S100A9 on AQP4 and its influence mechanism. Background Art

[0002] Alzheimer's disease is a common progressive neurodegenerative disease characterized by the extracellular deposition of amyloid-β (Aβ) and the intracellular deposition of neurofibrillary tau tangles (NFTs). The pathological changes of the extracellular deposition of amyloid-β (Aβ) and the intracellular deposition of neurofibrillary tau tangles (NFTs) lead to synaptic loss and neuronal death, ultimately impairing the patient's cognitive function.

[0003] Current studies have shown that Aβ in the interstitium is mainly cleared locally by cellular uptake and degradation, efflux through the blood-brain barrier, and by the glial lymphatic system that exchanges cerebrospinal fluid (CSF) and interstitial fluid (ISF) along the perivascular space pathway. Among them, impaired polarization of aquaporin 4 (AQP4) may be an important factor in the pathological development and disease progression of Alzheimer's disease. AQP4 is mainly located on the end feet of perivascular astrocytes. AQP4 molecules are densely aggregated to form orthogonal arrays of particles (OAPs), which are anchored to the end feet of perivascular astrocytes by the dystrophin-associated complex (DAC), including dystrophin (DAG), dystrophin (DMD), scissor peptide (DTNA), and α-syntrophin (SNTA1), providing a low-resistance path for water diffusion and facilitating the efficient flow of CSF and ISF. Changes in the localization of AQP4 can be found in a variety of central nervous system diseases. AQP4 localization is known to show changes in AQP4 localization in postmortem frontal cortex tissue from patients diagnosed with AD, including loss of AQP4 from perivascular end-feet, and similar changes have been observed in aged mice and AD mouse models. Therefore, targeting the clearance function of the glymphatic system may be a potential therapeutic approach for the treatment of AD.

[0004] The pro-inflammatory factor S100A9 is a small molecular weight protein with a molecular weight of 13KD. It was initially found to be highly expressed in macrophages infiltrating inflammatory tissues. This protein is secreted in large quantities by microglia in the injured nervous system, but is not produced in the physiological state. It is widely expressed in the AD brain. The S100A9 protein is known as a damage-related molecule or alarm molecule, and is widely involved in infection, cell stress, tissue damage, and cancer. In recent years, the role of the S100A9 protein as a pro-inflammatory protein in central nervous system lesions has been valued, including studies on its role in acute traumatic brain injury (TBI), multiple sclerosis (MS), and AD.

[0005] In AD, a large number of studies have shown that S100A9 has complex effects on the central nervous system. The research team's previous studies have found that S100A9 can co-aggregate with Aβ42 or Aβ40 to reduce the overall cytotoxicity of amyloid protein. Low concentrations of S100A9 can promote microglia to phagocytose Aβ, promote the differentiation of neural stem cells, and remedy neuronal damage.

[0006] S100A9 inhibits astrocyte proliferation and promotes the release of inflammatory factors by microglia.

[0007] Therefore, in-depth research on whether S100A9 is involved in the polarization change of AQP4 and whether S100A9 can affect the expression and localization of AQP4 on the astrocyte membrane is of great significance for studying the flow of endogenous proinflammatory factors in cerebrospinal fluid and solute exchange in the brain after injury. Summary of the invention

[0008] The purpose of the present invention is to solve the shortcomings of the prior art and to propose a research method for the effect of S100A9 on AQP4 and its effect mechanism.

[0009] In order to achieve the above object, the present invention adopts the following technical solutions:

[0010] A method for studying the effect of S100A9 on AQP4, comprising:

[0011] S1: Cell culture;

[0012] U251 astroglioma cells or primary SD rat astrocytes were seeded in culture dishes, and after the cells were completely attached to the wall, the culture medium containing different concentrations of S100A9 protein was replaced and cultured for another 12 hours;

[0013] S2: CCK8 detection of cell viability;

[0014] CCK8 reagent was added to the culture dish, and after incubation for a period of time, the viability of cells in each group was detected;

[0015] S3: Western Blot detection of protein levels of AQP4 and anchoring proteins;

[0016] The cells in each group were collected, total protein was extracted, and the protein levels of AQP4 and anchoring proteins were detected by Western Blot;

[0017] S4: RT-qPCR detection of mRNA levels of AQP4 and anchoring proteins;

[0018] Cells from each group were collected, total RNA was extracted, and the mRNA levels of AQP4 and anchoring proteins were detected by RT-qPCR;

[0019] S5: AQP4 cell immunofluorescence assay.

[0020] Preferably, in S1, cell culture and treatment specifically include the following steps:

[0021] Prepare U251 astroglioma cells or primary SD rat astrocytes and inoculate them into a culture dish containing an appropriate amount of culture medium under sterile conditions;

[0022] After the cells reached 70-80% adhesion rate, the culture medium containing different concentrations of S100A9 protein was replaced;

[0023] The treated cells were placed in a constant temperature incubator at 5% CO2 and 37°C for further culture for 12 hours to simulate the action environment of S100A9.

[0024] Preferably, in S2, CCK8 is used to detect cell viability, which specifically includes the following steps:

[0025] At the end of the culture, CCK8 reagent was added to each group of cells and incubated for a certain period of time according to the standard protocol;

[0026] The absorbance was measured using a microplate reader to evaluate the effects of different concentrations of S100A9 on cell viability;

[0027] The data were statistically analyzed and the IC50 value was calculated to determine the S100A9 concentration threshold for inhibiting cell proliferation.

[0028] Preferably, in S3, Western Blot is used to detect protein levels, which specifically comprises the following steps:

[0029] The cell samples treated with S100A9 were collected and the culture medium residues were removed by centrifugation and washing;

[0030] Total protein was extracted using RIPA buffer, and protein concentration was determined by BCA method;

[0031] The proteins were separated by SDS-PAGE electrophoresis and transferred to PVDF membrane;

[0032] Incubate with specific primary antibodies to label AQP4 and its anchoring proteins;

[0033] HRP-labeled secondary antibodies were applied, and protein bands were visualized by enhanced chemiluminescence (ECL).

[0034] Preferably, in S4, RT-qPCR detection of mRNA levels specifically includes:

[0035] The total RNA of each group of cells was extracted by TRIzol method, and the RNA purity and concentration were determined by nanopipette;

[0036] cDNA was synthesized using reverse transcriptase using the extracted RNA as a template;

[0037] Specific primers were designed to measure the mRNA expression levels of AQP4 and its anchoring proteins by qPCR reaction;

[0038] The CT values ​​were analyzed, the relative gene expression levels were calculated, and statistical comparisons were performed.

[0039] Preferably: in said S5, the immunofluorescence staining of AQP4 cells specifically comprises:

[0040] Treated cells were plated in culture plates with glass slides;

[0041] Use permeable cell membranes to increase antibody permeability;

[0042] Nonspecific binding sites were blocked with 5% BSA solution;

[0043] The primary antibody against AQP4 was diluted and incubated with cells overnight to ensure adequate binding;

[0044] Wash to remove excess primary antibody, add fluorescently labeled secondary antibody, and incubate in a dark room for a certain period of time;

[0045] Fluorescence microscopy was used to observe and capture fluorescence images, and the expression and distribution of AQP4 under different treatment conditions were compared and analyzed.

[0046] Preferably, in S5, the cells are seeded on a glass slide, fixed for 20 minutes, and permeabilized for 10 minutes; blocked with 5% BSA at room temperature for 1.5 hours; incubated with AQP4 primary antibody at 4°C overnight; recovered primary antibody, and incubated with goat anti-rabbit fluorescent secondary antibody at 37°C for 1.5 hours.

[0047] Preferably: in S1, after the cells reach an adhesion rate of 70-80%, the culture medium containing different concentrations of S100A9 protein is replaced, including: 0 μg / ml, 5 μg / ml, 10 μg / ml, and 30 μg / ml.

[0048] Preferably, in S5, the treated cells are inoculated in a culture plate with a glass slide, and then the cells are fixed with 4% paraformaldehyde.

[0049] Preferably: in S5, 0.1% TritonX-100 is used to permeabilize the cell membrane.

[0050] The beneficial effects of the present invention are:

[0051] 1. The present invention provides a systematic and detailed research method, which can clarify the effect of S100A9 on AQP4. Through experimental steps such as cell culture, CCK8 detection, Western Blot and RT-qPCR, the changes of S100A9 on AQP4 expression and localization at different concentrations can be fully understood. This provides a solid foundation for further studying the mechanism of action of S100A9 in Alzheimer's disease.

[0052] 2. The present invention reveals the specific interaction between S100A9 and AQP4. Experimental results show that S100A9 can promote the expression of AQP4 in astrocytes and change the level of anchoring proteins, thereby affecting the localization of AQP4 on the astrocyte membrane; this finding helps to understand the potential pathological role of S100A9 in neurodegenerative diseases.

[0053] 3. This invention provides new research directions and ideas. Through this study, we can further explore the specific molecular mechanisms of the interaction between S100A9 and AQP4, such as signal transduction pathways and regulatory networks. These studies will help to reveal the complex pathological process of AD and provide a theoretical basis for the development of new treatment strategies.

[0054] 4. The present invention has important clinical application potential; since S100A9 exhibits pro-inflammatory effects in a variety of inflammatory diseases, the study of its relationship with AQP4 may provide new ideas for the development of new anti-inflammatory drugs for AD; in addition, understanding how S100A9 affects the function of aquaporins may also help improve cerebrospinal fluid circulation and waste removal, thereby delaying disease progression. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 This is a schematic diagram of the effect of different drug concentrations of S100A9 on U251 cell viability detected by CCK8 in a research method for the effect of S100A9 on AQP4 proposed by the present invention;

[0056] Figure 2 This is the first schematic diagram of a method for studying the effect of S100A9 on AQP4 proposed by the present invention, in which S100A9 in vitro intervenes to promote the protein expression of AQP4 in U251 astrocytes;

[0057] Figure 3 The second schematic diagram of the method for studying the effect of S100A9 on AQP4 proposed by the present invention, wherein S100A9 in vitro intervenes to promote the protein expression of AQP4 in U251 astrocytes;

[0058] Figure 4This is a schematic diagram of 5 μg / mL S100A9 promoting the proliferation of primary astrocytes in a research method for the effect of S100A9 on AQP4 proposed in the present invention;

[0059] Figure 5 A schematic diagram of using Western Blot to verify the expression of S100A9 stimulating primary astrocytes in a research method for the effect of S100A9 on AQP4 proposed by the present invention;

[0060] Figure 6 This is a schematic diagram of using RT-qPCR to verify the expression of S100A9-stimulated primary astrocytes in a research method for the effect of S100A9 on AQP4 proposed in the present invention. DETAILED DESCRIPTION

[0061] The technical solution of the present invention is further described in detail below in conjunction with specific implementation methods.

[0062] Embodiment 1:

[0063] A method for studying the effect of S100A9 on AQP4, comprising:

[0064] (1) S100A912h promotes the expression of AQP4 in U251 astroglioma cells:

[0065] CCK8 detected the effects of different drug concentrations of S100A9 on U251 cell viability:

[0066] 50,000 U251 astrocytes were seeded per well, and after the cells were completely attached to the wall, different concentrations of S100A9 protein were added for intervention for 12 hours; then 100 μl of CCK8 reagent was added to detect cell viability.

[0067] Results: After 12 h of in vitro intervention with S100A9, the proliferation inhibition of U251 astrocytes increased with increasing concentrations. S100A9 at a concentration of 30 μg / ml could inhibit proliferation, with significant differences (p<0.05) (e.g. Figure 1 ).

[0068] (2) S100A9 promotes the expression of AQP4 and its anchoring proteins in U251 cells:

[0069] U251 cells were intervened in vitro with S100A9 at concentrations of 0 μg / ml, 5 μg / ml, 10 μg / ml, and 30 μg / ml and LPS at 1 μg / ml for 12 h, and with a hypotonic solution of 78 mOsm / kg for 10 min. The protein levels of AQP4 and anchoring proteins were then detected by Western Blot.

[0070] Results: S100A9 intervention for 12 h in vitro promoted the expression of AQP4 in U251 astrocytes in a concentration-dependent manner, with a significant change at 5 μg / ml (p<0.01) (e.g. Figure 2 , Figure 3 ). S100A9 also promoted the expression of anchoring proteins SNTA1, DTNA, and dystrophin, but the protein level of β-DG was not affected. The results showed that the effect of pro-inflammatory protein S100A9 on AQP4 was similar to that of lipopolysaccharide LPS, and there was no statistical difference between the two.

[0071] Embodiment 2:

[0072] A method for studying the effect of S100A9 on AQP4. This embodiment, based on embodiment 1, further includes:

[0073] (1) Extract primary astrocytes and identify the purity of primary astrocytes using GFAP immunofluorescence:

[0074] Take the brain tissue of newborn SD rats, prepare the cell suspension, and replace the culture medium to remove the suspended cells after overnight inoculation. Continue to culture for 6-7 days. Under the microscope, the cells can be seen to be layered and spread all over the bottom of the dish. The upper layer of cells is in the shape of a "hammer" with multiple protrusions; the lower layer of cells is flat and polygonal, with larger cell bodies. Gently shake to remove the upper layer of oligodendrocytes, digest and passage the lower layer of cells, and after 3 days of culture, flat polygonal cells of basically similar size are formed, and many short and thick branching protrusions can be seen. GFAP immunofluorescence staining is positive, and the positive rate reaches about 95% and can be used for subsequent experiments.

[0075] (2) CCK detection of the effects of different drug concentrations of S100A9 on the viability of primary astrocytes of SD rats:

[0076] 8000 SD rat primary astrocytes were seeded per well. After the cells were completely attached to the wall, different concentrations of S100A9 protein were added for intervention for 12 h, and then 100 μl of CCK8 reagent was added to detect cell viability.

[0077] Results: 5 μg / mL S100A9 promoted the proliferation of astrocytes (e.g. Figure 4 ), with significant differences (p<0.01). As the concentration of S100A9 further increased, S100A9 inhibited astrocyte proliferation. The inhibitory effect of the 40-80μg / mL concentration group was significantly different (p<0.01), and was concentration-dependent. Since the pathological characteristics of astrocyte activation and glial cell proliferation are often observed in a variety of neurodegenerative diseases, 5μg / mL of S100A9 was selected as the optimal stimulating concentration for astrocytes in subsequent experiments.

[0078] (3) S100A9 promotes the expression of AQP4 and its anchoring proteins in primary astrocytes:

[0079] S100A9 at concentrations of 0 μg / ml and 5 μg / ml was used to intervene in primary astrocytes in vitro for 8 h, 12 h and 24 h, respectively. The protein and mRNA levels of AQP4 and its anchoring proteins were detected by Western Blot and cell RT-qPCR, and the expression of AQP4 was detected by cell immunofluorescence.

[0080] In addition, Western Blot and RT-qPCR were used to verify that S100A9 stimulated astrocytes to express AQP4, β-DG, α-DG, SNTA1, DTNA, and dystrophin protein and mRNA levels ( Figure 5-Figure 6 ), the expression of AQP4, SNTA1, DTNA and dystrophin proteins in astrocytes increased, and the transcription levels of AQP4, SNTA1, DTNA and dystrophin increased at 8h and 12h, which was statistically significant compared with the control group (p<0.05). There was no significant change in the protein levels of β-DG and α-DG and the transcription level of gene DAG1 (p>0.05). There was no significant difference between the 24h group and the control group (p>0.05). The experimental results suggest that S100A9 protein can promote the expression of AQP4 in astrocytes, and also change the level of anchoring protein and its localization on the membrane.

[0081] For AQP4 cell immunofluorescence:

[0082] 12500 cells / well were inoculated and cultured for 12h, and then treated with 0μg / ml S100A9 and 5μg / ml S100A9 for 12h. The cells were removed and fixed with 4% paraformaldehyde for 20min at room temperature, and permeabilized with 0.1% Triton-X 100 for 10min at room temperature. They were blocked with 5% BSA for 1.5h at room temperature. AQP4 primary antibody was incubated at 4℃ overnight. The primary antibody was recovered and the goat anti-rabbit fluorescent secondary antibody was incubated at 37℃ for 1.5h.

[0083] Results: After 5 μg / ml S100A9 was used to intervene in primary astrocytes in vitro for 12 hours, the immunofluorescence results of AQP4 showed that 5 μg / ml S100A9 could promote the expression of AQP4 protein (p<0.01), which was consistent with the results of Western Blot.

[0084] In summary: The pro-inflammatory protein S100A9 can promote the expression of AQP4, and the results are consistent in U251 astroglioma cells and primary astrocytes. At the same time, S100A9 also affects the level of the anchoring-related complex - the dystrophin-associated complex (DAC), including DMD, DTNA, and SNTA1, but the transcription of DAG1 and the encoded proteins α-DG and β-DG are not affected. Therefore, the above experimental results show that S100A9 affects the expression of AQP4, and it is possible to affect the localization of AQP4 on the astrocyte membrane by changing the level of anchoring proteins.

[0085] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A method for studying the effect of S100A9 on AQP4, characterized in that: include: S1: Cell culture; U251 astroglioma cells or primary SD rat astrocytes were seeded in culture dishes, and after the cells were completely attached to the wall, the culture medium containing different concentrations of S100A9 protein was replaced and cultured for another 12 hours; S2: CCK8 detection of cell viability; CCK8 reagent was added to the culture dish, and after incubation for a period of time, the viability of cells in each group was detected; S3: Western Blot detection of protein levels of AQP4 and anchoring proteins; The cells in each group were collected, total protein was extracted, and the protein levels of AQP4 and anchoring proteins were detected by Western Blot; S4: RT-qPCR detection of mRNA levels of AQP4 and anchoring proteins; Cells from each group were collected, total RNA was extracted, and the mRNA levels of AQP4 and anchoring proteins were detected by RT-qPCR; S5: AQP4 cell immunofluorescence assay.

2. The method for studying the effect of S100A9 on AQP4 according to claim 1, characterized in that: In S1, cell culture and treatment specifically include the following steps: Prepare U251 astroglioma cells or primary SD rat astrocytes and inoculate them into a culture dish containing an appropriate amount of culture medium under sterile conditions; After the cells reached 70-80% adhesion rate, the culture medium containing different concentrations of S100A9 protein was replaced; The treated cells were placed in a constant temperature incubator at 5% CO2 and 37°C for further culture for 12 hours to simulate the action environment of S100A9.

3. The method for studying the effect of S100A9 on AQP4 according to claim 1, characterized in that: In S2, CCK8 is used to detect cell viability, which specifically includes the following steps: At the end of the culture, CCK8 reagent was added to each group of cells and incubated; The absorbance was measured using a microplate reader to evaluate the effects of different concentrations of S100A9 on cell viability; The data were statistically analyzed and the IC50 value was calculated to determine the S100A9 concentration threshold for inhibiting cell proliferation.

4. The method for studying the effect of S100A9 on AQP4 according to claim 1, characterized in that: In S3, Western Blot is used to detect protein levels, which specifically includes the following steps: The cell samples treated with S100A9 were collected and the culture medium residues were removed by centrifugation and washing; Total protein was extracted using RIPA buffer, and protein concentration was determined by BCA method; The proteins were separated by SDS-PAGE electrophoresis and transferred to PVDF membrane; Incubate with specific primary antibodies to label AQP4 and its anchoring proteins; HRP-labeled secondary antibodies were applied, and protein bands were visualized by enhanced chemiluminescence.

5. The method for studying the effect of S100A9 on AQP4 according to claim 1, characterized in that: In S4, RT-qPCR detection of mRNA levels specifically includes: The total RNA of each group of cells was extracted by TRIzol method, and the RNA purity and concentration were determined by nanopipette; cDNA was synthesized using reverse transcriptase using the extracted RNA as a template; Specific primers were designed to measure the mRNA expression levels of AQP4 and its anchoring proteins by qPCR reaction; The CT values ​​were analyzed, the relative gene expression levels were calculated, and statistical comparisons were performed.

6. The method for studying the effect of S100A9 on AQP4 according to claim 1, characterized in that: In S5, AQP4 cell immunofluorescence staining specifically includes: Treated cells were plated in culture plates with glass slides; Use permeable cell membranes to increase antibody permeability; Nonspecific binding sites were blocked with 5% BSA solution; The primary antibody against AQP4 was diluted and incubated with cells overnight to ensure adequate binding; Wash to remove excess primary antibody, add fluorescently labeled secondary antibody, and incubate in a dark room for a certain period of time; Fluorescence microscopy was used to observe and capture fluorescence images, and the expression and distribution of AQP4 under different treatment conditions were compared and analyzed.

7. The method for studying the effect of S100A9 on AQP4 according to claim 6, characterized in that: In the S5, the cells were seeded on a glass slide, fixed for 20 minutes, and permeabilized for 10 minutes; blocked with 5% BSA at room temperature for 1.5 hours; incubated with AQP4 primary antibody at 4°C overnight; recovered primary antibody, and incubated with goat anti-rabbit fluorescent secondary antibody at 37°C for 1.5 hours.

8. The method for studying the effect of S100A9 on AQP4 according to claim 2, characterized in that: In S1, after the cells reach an adhesion rate of 70-80%, the culture medium containing different concentrations of S100A9 protein is replaced, including: 0 μg / ml, 5 μg / ml, 10 μg / ml, and 30 μg / ml.

9. The method for studying the effect of S100A9 on AQP4 according to claim 7, characterized in that: In the above-mentioned S5, the treated cells were seeded on a culture plate with a glass slide, and then the cells were fixed with 4% paraformaldehyde.

10. The method for studying the effect of S100A9 on AQP4 according to claim 7, characterized in that: In S5, 0.1% TritonX-100 was used to permeabilize the cell membrane.

Images