Use of luteolin in the preparation of a drug for restoring trem2 in diabetic nephropathy + Use of luteolin in the preparation of a drug for restoring trem2 in diabetic nephropathy

Abstract

This invention discloses a method for restoring TREM2 in the preparation of diabetic nephropathy using luteolin. + This invention relates to the application and research methods of macrophage functional drugs, and pertains to the field of biomedical technology. Through the technical solution of this invention, luteolin is used for the intervention of diabetic nephropathy, achieving the effect of regulating TREM2. + Targeted regulation of the key pathological link of macrophage dysfunction; by increasing TREM2 + The number and functional status of macrophages are improved, enhancing their ability to eliminate apoptotic cells, reducing the level of renal tissue cell apoptosis and immune inflammatory damage; thereby alleviating the progression of kidney damage in diabetic nephropathy.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and more particularly to a luteolin-based method for restoring TREM2 in the treatment of diabetic nephropathy. + Applications of macrophage functional drugs, and the use of luteolin in the preparation of drugs that restore TREM2. + Application of macrophage functional drugs and a luteolin-based drug for restoring TREM2 + Methods for studying the functional mechanisms of macrophages. Background Technology

[0002] Diabetic nephropathy (DKD) is one of the most serious microvascular complications of diabetes and the leading cause of end-stage renal disease (ESRD). Its pathological features include progressive proteinuria, glomerular enlargement, mesangial matrix proliferation, foot rupture, and basement membrane thickening, which eventually lead to glomerular sclerosis and interstitial fibrosis.

[0003] Existing research indicates that immune inflammation is one of the key drivers in the occurrence and development of diabetic kidney disease (DKD). Studies have shown that during the development of DKD, multiple factors, including glucose metabolism disorders, lipid metabolism disorders, and hemodynamic changes, ultimately lead to long-term, chronic, localized renal inflammation. Under a prolonged high-glucose environment, the metabolic reprogramming of intrinsic renal cells and immune cells causes them to secrete damage-associated molecular patterns (DAMPs), pro-inflammatory factors, and chemokines in an autocrine or paracrine manner, activating innate immunity. This process leads to the accumulation of myeloid-derived immune cells in the kidneys and continuously exacerbates the local chronic inflammatory response in the kidneys. The persistent immune inflammation within the kidneys not only promotes renal tissue damage but also further accelerates the progression of diabetic nephropathy.

[0004] In the process of renal immune inflammation, macrophages are the most important effector immune cells. Recent studies have found that TREM2... + Macrophages play a protective role in tissue damage clearance, immune inflammation relief, and cell burial. TREM2 + Decreased macrophage function often leads to a weakened ability to clear apoptotic cells, thereby exacerbating local inflammation and tissue damage. Therefore, restoring or enhancing TREM2... + Macrophage function holds promise as a new approach to intervention in diabetic nephropathy.

[0005] However, current drug research for diabetic nephropathy mainly focuses on lowering blood sugar, lowering blood pressure, reducing proteinuria, and general anti-inflammatory measures, and lacks research on restoring TREM2. + A drug regimen that leverages macrophage function to improve diabetic nephropathy. In other words, how to develop a drug that can restore TREM2 in diabetic nephropathy. +Drugs that enhance macrophage function and thus reduce kidney damage and immune inflammation remain unresolved issues in current technology.

[0006] Honeysuckle, a traditional Chinese medicine, has been used to treat diseases for nearly 1000 years. It has been proven to have good anti-inflammatory, immunomodulatory, and antiviral effects, and no related side effects have been found so far. Luteolin, a monomer extracted from honeysuckle, is a natural flavonoid compound with various pharmacological activities such as anti-inflammatory, antioxidant, and immunomodulatory effects, showing potential application in diseases such as abnormal glucose and lipid metabolism. However, current technology has not yet shown that luteolin can be used to restore TREM2 in diabetic nephropathy. + Technical solutions for improving macrophage function and disease progression. Summary of the Invention

[0007] To address the above problems, this invention provides a method for restoring TREM2 in the preparation of diabetic nephropathy using luteolin. + Application and research methods of macrophage functional drugs: By using luteolin for the intervention of diabetic nephropathy, the effects of TREM2 were achieved. + Targeted regulation of the key pathological link of macrophage dysfunction; by increasing TREM2 + This invention aims to improve the number and functional status of macrophages, enhance their ability to eliminate apoptotic cells, reduce the level of renal tissue cell apoptosis and immune inflammatory damage, thereby alleviating the progression of kidney damage in diabetic nephropathy. Compared to existing drugs that mainly focus on lowering blood sugar, lowering blood pressure, or general anti-inflammatory treatments, this invention provides a method to restore TREM2. + This novel application, centered on macrophage function, has a clearly defined target and promising application prospects.

[0008] To achieve the above objectives, this invention provides a method for restoring TREM2 in the preparation of diabetic nephropathy using luteolin. + Application in drugs that promote macrophage function.

[0009] This invention also proposes a method for preparing luteolin in the restoration of TREM2. + Application in drugs that promote macrophage function.

[0010] This invention also proposes a method for restoring TREM2 with luteolin. + Methods for studying the mechanisms of macrophage function include: An animal model of diabetic nephropathy was established, and a control group, a diabetic nephropathy model group, and a luteolin treatment group were set up. The kidney tissues of each group were examined separately to obtain TREM2. + Macrophage number, TREM2 protein expression level, Rab5a expression level, Rab7a expression level, TREM2 in the eutrophic state+ Macrophage count and apoptosis-related indicators; Based on the test results of each group, if TREM2 is treated with luteolin... + Increased macrophage number, elevated TREM2 protein expression levels, elevated Rab5a and Rab7a expression levels, and TREM2 cells in a necrophore state. + An increase in macrophage numbers and a decrease in apoptosis-related markers confirm that luteolin can restore TREM2 levels. + The role of macrophage function.

[0011] In the above technical solution, preferably, luteolin restores TREM2. + Research methods for understanding the mechanisms of macrophage function also include: An in vitro model of macrophage dysfunction stimulated by high glucose was constructed, and a blank control group, a high glucose model group, and a luteolin intervention group were set up. The luteolin intervention group was treated with luteolin, and the TREM2 level in the cells of each group was detected. + Macrophage ratio.

[0012] In the above technical solution, preferably, luteolin restores TREM2. + Research methods for understanding the mechanisms of macrophage function also include: The level of soluble TREM2 in the cell culture medium of each group was detected; A TREM2 overexpression 293T cell model was constructed, and the full-length TREM2 expression level on the surface of the 293T cell membrane was detected after luteolin treatment. Based on the test results, if the soluble TREM2 level decreased after luteolin intervention compared to the high glucose model group, and the full-length TREM2 expression level on the cell membrane surface of the 293T cell model with TREM2 overexpression that was not treated with luteolin increased, then it is determined that luteolin has the effect of inhibiting TREM2 cleavage and restoring full-length TREM2 expression on the cell membrane surface.

[0013] In the above technical solution, preferably, luteolin restores TREM2. + Research methods for understanding the mechanisms of macrophage function also include: Apoptotic renal tubular epithelial cells were prepared and co-cultured with macrophages from various groups; The phosphorylation levels of DAP12 and SYK, as well as the phagocytic clearance capacity of macrophages on the apoptotic renal tubular epithelial cells, were detected in each group. Based on the test results, if the phosphorylation levels of DAP12 and SYK are enhanced after luteolin intervention, and the phagocytic clearance capacity of macrophages for apoptotic renal tubular epithelial cells is enhanced, then it is further determined that luteolin restores TREM2 by enhancing TREM2-DAP12-SYK signaling. + The cytodegenerative function of macrophages.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) Restoring TREM2 in diabetic nephropathy by using luteolin + Macrophage function enables targeted intervention in the key pathological link of abnormal immune inflammation in diabetic nephropathy. Compared to existing treatment approaches that mainly focus on lowering blood sugar, lowering blood pressure, or general anti-inflammatory treatments, this application utilizes TREM2... + The study of macrophage function recovery offers a new application direction and provides a new technical approach for the intervention of diabetic nephropathy.

[0015] (2) By detecting TREM2 + Macrophage number, TREM2 protein expression level, Rab5a expression level, Rab7a expression level, TREM2 in the eutrophic state + The study investigated the number of macrophages and apoptosis-related indicators, achieving the goal of restoring TREM2 by luteolin. + Functional validation of macrophage function. This indicates that luteolin can enhance TREM2. + Increasing the number of macrophages and TREM2 expression levels enhances burial-related capabilities and reduces apoptosis levels, thereby helping to alleviate persistent inflammatory responses in the context of kidney tissue injury.

[0016] (3) By constructing an in vitro model of high glucose stimulation and detecting the levels of soluble TREM2 and full-length TREM2 expression on the cell membrane surface, the mechanism of luteolin's regulation of TREM2 cleavage was verified. This indicates that luteolin can reduce soluble TREM2 levels and restore full-length TREM2 expression on the cell membrane surface, thereby helping to maintain the functional state of TREM2 and restore its function. + Macrophage function provides a clearer basis for its role.

[0017] (4) By further detecting the phosphorylation levels of DAP12 and SYK, as well as the phagocytic clearance capacity of macrophages against apoptotic renal tubular epithelial cells, the enhancement of TREM2 downstream signal transduction and cell burial function by luteolin was further validated. This not only improved the understanding of luteolin's ability to restore TREM2... + This study elucidates the functional chain of macrophages and provides a relatively complete technical basis for their application in related drug development and mechanism of action research. Attached Figure Description

[0018] Figure 1 This is a graph showing the change in body weight of mice over time in each group, as disclosed in one embodiment of the present invention.

[0019] Figure 2 This is a graph showing the change of blood glucose levels over time in various groups of mice according to an embodiment of the present invention.

[0020] Figure 3 This is a graph showing the change of uACR over time in various groups of mice according to an embodiment of the present invention.

[0021] Figure 4 This is a graph showing the comparison of blood urea nitrogen levels in different groups of mice according to an embodiment of the present invention.

[0022] Figure 5 This is a graph showing the comparison of serum creatinine levels in different groups of mice according to an embodiment of the present invention.

[0023] Figure 6 This is a PAS staining image of mouse kidney tissues from various groups, as disclosed in one embodiment of the present invention.

[0024] Figure 7 This is a graph showing the analysis results of the ratio of the glomerular mesangial matrix area to the glomerular area in each group of mice, as disclosed in an embodiment of the present invention.

[0025] Figure 8 This is a graph showing the results of glomerular volume analysis in various groups of mice according to an embodiment of the present invention.

[0026] Figure 9 The images shown are transmission electron micrographs of the glomeruli of mice in various groups, as disclosed in one embodiment of the present invention.

[0027] Figure 10 This is a graph showing the analysis results of the foot process fusion degree of each group of mice disclosed in one embodiment of the present invention.

[0028] Figure 11 This is a graph showing the analysis results of the basement membrane thickness of various groups of mice disclosed in one embodiment of the present invention.

[0029] Figure 12 This is a graph showing the analysis results of the transcriptional levels of pro-inflammatory factors in mice as disclosed in one embodiment of the present invention.

[0030] Figure 13 This is an immunofluorescence image of mouse F4 / 80 disclosed in one embodiment of the present invention.

[0031] Figure 14 This is a graph showing the analysis results of the average number of positive cells in mouse F4 / 80 cells, as disclosed in one embodiment of the present invention.

[0032] Figure 15This is a WB detection result of mouse TREM2 protein disclosed in one embodiment of the present invention.

[0033] Figure 16 This is a graph showing the analysis results of mouse TREM2 protein expression levels as disclosed in one embodiment of the present invention.

[0034] Figure 17 This is an immunofluorescence image of mouse TREM2 / F4 / 80 disclosed in one embodiment of the present invention.

[0035] Figure 18 This is a graph showing the analysis results of the average number of double-positive cells in mouse TREM2 / F4 / 80 as disclosed in one embodiment of the present invention.

[0036] Figure 19 This is a WB detection result diagram of mouse Rab5a and Rab7a proteins disclosed in one embodiment of the present invention.

[0037] Figure 20 This is a graph showing the analysis results of the expression levels of mouse Rab5a and Rab7a proteins disclosed in one embodiment of the present invention.

[0038] Figure 21 This is an immunofluorescence image of mouse TREM2 / Rab5a disclosed in one embodiment of the present invention.

[0039] Figure 22 This is a graph showing the analysis results of the average number of TREM2 / Rab5a double-positive cells in mice, as disclosed in one embodiment of the present invention.

[0040] Figure 23 This is an immunofluorescence image of mouse TREM2 / Rab7a disclosed in one embodiment of the present invention.

[0041] Figure 24 This is a graph showing the analysis results of the average number of TREM2 / Rab7a double-positive cells in mice, as disclosed in one embodiment of the present invention.

[0042] Figure 25 This is a TUNEL staining image of mouse kidney tissue disclosed in one embodiment of the present invention.

[0043] Figure 26 The mean TUNEL of mouse kidneys as disclosed in one embodiment of the present invention + The results of the cell count analysis are shown in the figure.

[0044] Figure 27 This is a WB test result of mouse Cleaved-caspase 3 disclosed in one embodiment of the present invention.

[0045] Figure 28This is a graph showing the analysis results of mouse Cleaved-caspase3 protein expression level as disclosed in one embodiment of the present invention.

[0046] Figure 29 The in vivo TREM2 disclosed in one embodiment of the present invention + The results of the analysis of changes in the proportion of macrophages.

[0047] Figure 30 This is a graph showing the analysis results of TREM2 protein expression changes disclosed in one embodiment of the present invention.

[0048] Figure 31 and Figure 32 TREM2 in kidney tissue as disclosed in one embodiment of the present invention + Figure showing the analysis results of macrophage population changes.

[0049] Figure 33 and Figure 34 This is a graph showing the analysis results of changes in TUNEL apoptosis burden disclosed in one embodiment of the present invention.

[0050] Figure 35 This is a graph showing the analysis results of changes in Cleaved-caspase 3 disclosed in one embodiment of the present invention.

[0051] Figure 36 This is a graph showing the analysis results of the changes in Rab5a and Rab7a disclosed in one embodiment of the present invention.

[0052] Figure 37 and Figure 38 Rab5a is disclosed in one embodiment of the present invention. + / TREM2 + The results of the analysis of changes in double-positive cells are shown in the figure.

[0053] Figure 39 and Figure 40 Rab7a is disclosed in one embodiment of the present invention. + / TREM2 + The results of the analysis of changes in double-positive cells are shown in the figure.

[0054] Figure 41 TREM2 in a high-glucose in vitro model disclosed in one embodiment of the present invention + Flow cytometry results and quantitative analysis results of macrophage proportion.

[0055] Figure 42 This is a graph showing the analysis results of sTREM2 changes in the supernatant of high-glucose macrophage culture, as disclosed in an embodiment of the present invention.

[0056] Figure 43This is a diagram showing the analysis results of sTREM2 changes in kidney tissue according to an embodiment of the present invention.

[0057] Figure 44 This is a diagram showing the analysis results of full-length TREM2 expression and membrane protein purity verification on the 293T cell membrane, as disclosed in an embodiment of the present invention.

[0058] Figure 45 This is a graph showing the analytical results of quantitative changes in TREM2 along the entire length of the membrane surface, as disclosed in an embodiment of the present invention.

[0059] Figure 46 This is a graph showing the analysis results of dose-dependent changes of sTREM2 in the culture supernatant of different treatment groups in one embodiment of the present invention.

[0060] Figure 47 This is a schematic diagram of pHrodo green fluorescence images of different co-culture groups disclosed in one embodiment of the present invention.

[0061] Figure 48 This is a graph showing the analytical results of the quantitative results of the phagocytic clearance capacity disclosed in one embodiment of the present invention.

[0062] Figure 49 This is a schematic diagram of a representative protein blot image disclosed in one embodiment of the present invention.

[0063] Figure 50 This is a graph showing the analysis results of changes in mRNA of IL-1β, IL-18, TNF-α, and MCP-1 disclosed in one embodiment of the present invention.

[0064] Figure 51 This is a graph showing the analytical results of quantitative changes in Rab5a disclosed in one embodiment of the present invention.

[0065] Figure 52 This is a graph showing the analytical results of quantitative changes in Rab7a disclosed in one embodiment of the present invention.

[0066] Figure 53 This is a graph showing the analytical results of quantitative changes in p-SYK as disclosed in one embodiment of the present invention.

[0067] Figure 54 This is a graph showing the analytical results of quantitative changes in p-DAP12 disclosed in one embodiment of the present invention. Detailed Implementation

[0068] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0069] The present invention will now be described in further detail with reference to the accompanying drawings: According to the present invention, a luteolin is used to restore TREM2 in the preparation of diabetic nephropathy. + In one specific embodiment of the application of macrophage functional drugs, luteolin is prepared into a pharmaceutically acceptable dosage form, and the use of luteolin is verified using an animal model of diabetic nephropathy.

[0070] Preferably, db / db mice were selected as the diabetic nephropathy model animals, with normal mice as controls. The effects of luteolin on renal function, renal tissue pathological damage, local renal immune inflammation, and TREM2 in diabetic nephropathy were evaluated through continuous administration. + The effect on macrophage functional status. According to the experimental design, a normal control group, a diabetic nephropathy model group, and a luteolin-treated group can be set up. The luteolin-treated group is intervened according to a preset dose and administration period. During the administration process, indicators such as body weight, blood glucose, urine albumin-to-creatinine ratio, serum creatinine, and blood urea nitrogen can be monitored simultaneously.

[0071] After drug administration, kidney tissue was obtained from each group for pathological and molecular biological evaluation. Kidney tissue pathological examination can be used to observe glomerular hypertrophy, mesangial matrix proliferation, basement membrane thickening, foot process fusion, and tubulointerstitial injury; it can also assess the levels of inflammatory factors, macrophage infiltration, and TREM2 in the kidney tissue. + Macrophage-related indicators were detected. Combined with the in vivo experimental results in the initial draft, it was found that luteolin treatment improved renal function indicators and reduced renal tissue pathological damage in diabetic nephropathy model animals, and TREM2 was also observed. + Macrophage number and functional status were restored. In db / db mice, luteolin upregulated TREM2 protein expression in kidney tissue and further increased TREM2 levels. + Macrophage populations, thereby restoring TREM2 in diabetic nephropathy scenarios. + This application of macrophage function has more direct experimental support.

[0072] In this embodiment, by using luteolin in an animal model of diabetic nephropathy, the effects of reduced renal function impairment, improved renal tissue pathological damage, and TREM2 were achieved.+ The combined effect of luteolin in restoring macrophage function demonstrates its potential use in preparing TREM2-restoring agents for diabetic nephropathy. + Drugs that enhance macrophage function.

[0073] During implementation, the application and effects of the above-described implementation methods will be explained with reference to the following specific examples.

[0074] Example 1: Select 6-8 week old C57BL / 6KSJ mice and db / db Mice, all male. db / db Mice were inbred C57BL / 6KSJ mice with a homozygous point mutation in the leptin receptor gene; mice with fasting blood glucose ≥16.7 mmol / L were selected for the study. Animals were divided into a normal control group (NC), a diabetic nephropathy solvent group (db / db+Vehicle), a low-dose luteolin group (db / db+LUTL, 40 mg / kg), and a high-dose luteolin group (db / db+LUTH, 80 mg / kg). The molecular formula of the luteolin monomer compound is C57BL / 6KSJ. 15 H 10 O6, CAS number 491-70-3, was prepared with 0.5% sodium carboxymethyl cellulose solution as solvent and administered by gavage once daily for 10 weeks.

[0075] Mice were randomly assigned to groups at 10 weeks of age when elevated urinary ACR and significant proteinuria were observed. Starting at 8 weeks of age, blood glucose and body weight were measured every two weeks, and random urine was collected. After 10 weeks of drug administration, mice were anesthetized with 0.1% sodium pentobarbital, and blood and kidney tissue were collected.

[0076] The detection indicators included: body weight, fasting blood glucose, uACR, serum creatinine, and blood urea nitrogen; the proportion of mesangial matrix area and glomerular volume as shown by PAS staining of kidney tissue; the degree of foot process fusion and basement membrane thickness under transmission electron microscopy; the expression of TNF-α, IL-1β, IL-18 and MCP-1 was detected by qPCR, and the infiltration of macrophages in kidney tissue was detected by F4 / 80 antibody.

[0077] Results analysis: such as Figure 1 and Figure 2 As shown, the body weight and blood glucose levels of diabetic mice treated for 10 weeks were slightly lower than those in the model group, but the differences were not statistically significant. This suggests that luteolin does not improve diabetic kidney disease (DKD) by improving diabetes itself, but rather acts directly on the kidneys. Figure 3 As shown, after 10 weeks of luteolin treatment, the uACR of mice decreased significantly, with a more pronounced decrease in the high-dose group, exhibiting a certain dose-response relationship. Figure 4 , 5 As shown, serum creatinine and blood urea nitrogen were significantly elevated in diabetic nephropathy mice, while luteolin could reduce these two indicators, exhibiting a clear dose-response relationship. Figure 6 As shown, compared with the model group, the treatment group showed a significant reduction in basement membrane hyperplasia and mesangial area. Figure 7 , 8 As shown, the volume of the glomerulus decreases, manifested as a reduction in the mesangial coefficient and glomerular volume. For example... Figure 9 As shown in the figure, electron microscopy revealed that, compared to the model group, luteolin increased the density of foot processes and decreased the thickness of the basement membrane. Figure 10 , 11 As shown, this manifests as a significant decrease in foot process fusion and basement membrane thickness. (As...) Figure 12 As shown, compared to the control group (NC), the solvent group showed significantly increased levels of pro-inflammatory factors (TNF-α, IL-1β, IL-18, MCP-1) in the kidneys, while luteolin significantly reduced their transcription. Figure 13 , 14 As shown, immunofluorescence revealed that, compared to the model group, the luteolin treatment group significantly reduced macrophage infiltration.

[0078] The results showed that luteolin had no significant effect on blood glucose and body weight, but significantly reduced uACR, serum creatinine, and blood urea nitrogen, improved PAS and pathological changes under electron microscopy, and reduced the expression of renal pro-inflammatory factors and the infiltration of F4 / 80 positive macrophages. Combined with subsequent TREM2-related results, it can be determined that luteolin can be used to restore TREM2 in diabetic nephropathy. + Drugs that enhance macrophage function.

[0079] This invention also proposes a method for preparing luteolin in the restoration of TREM2. + In one specific embodiment, the application of macrophage functional drugs still prioritizes diabetic nephropathy as the preferred validation scenario. Compared to the previous embodiment, this embodiment emphasizes the effect of luteolin on TREM2. + The restorative effect on macrophage function itself, not just its improvement on diabetic nephropathy outcome markers. To validate this application, in vivo studies can be conducted around TREM2. + The number of macrophages, TREM2 protein expression, cell burial-related proteins, and apoptosis-related indicators were comprehensively detected; in vitro, TREM2 under high glucose conditions could be observed. + Functional validation was performed on macrophage ratio, sTREM2, full-length TREM2 on membrane surface, and phagocytic clearance capacity.

[0080] The above in vitro and in vivo studies demonstrate that luteolin not only improves DKD in a general way, but also has a higher-level effect on restoring TREM2. +Drug applications for macrophage function. In vivo experiments demonstrate functional recovery in pathological tissue environments, while in vitro experiments demonstrate its direct regulatory effects on macrophage phenotype, cleavage state, and phagocytic capacity under high glucose injury conditions.

[0081] During implementation, the application and effects of the above-described implementation methods will be explained with reference to the following specific examples.

[0082] Example 2: Based on the animal grouping and dosing regimen of Example 1, TREM2 in kidney tissue was further detected after the last dose. + Macrophage number, TREM2 protein expression, Rab5a / Rab7a expression, number of Rab5a / TREM2 and Rab7a / TREM2 double-positive cells, and TUNEL and Cleaved-caspase3 indicators.

[0083] Simultaneously, a high-glucose-stimulated macrophage model was established in vitro to further detect TREM2. + The proportion of macrophages, sTREM2 in cell culture supernatant, and the phagocytic clearance capacity of apoptotic renal tubular epithelial cells were measured to verify the restoration of TREM2 by luteolin at both animal and cellular levels. + Applications of macrophage function.

[0084] Results analysis: such as Figure 15 , 16 As shown, compared to the model group, luteolin significantly increased the expression of TREM2 protein. Figure 17 , 18 As shown, immunofluorescence also indicated similar results, with luteolin increasing TREM2 levels in the kidneys. + The number of macrophages. For example... Figure 19 , 20 As shown, the expression of burial-related proteins Rab5a and Rab7a was significantly increased during burial, and luteolin significantly increased the expression of these two proteins compared to the solvent group. Figure 21 , 22 As shown in Figures 23 and 24, co-localization of Rab5a and TREM2, and Rab7a and TREM2 fluorescence, revealed an increase in the number of double-positive cells in the luteolin treatment group, suggesting that TREM2 cells are undergoing endocytosis. + An increase in the number of macrophages. For example... Figure 25 , 26 As shown, compared with the control group, the number of apoptotic cells in the solvent group increased sharply, while luteolin significantly improved the number of apoptotic cells. Figure 27 , 28As shown, molecular changes also reflect a similar trend. Cleaved-caspase 3, a key molecule reflecting apoptosis, was also elevated in the solvent group, and subsequently significantly decreased by luteolin.

[0085] The results showed that TREM2 was reduced after treatment with luteolin. + Increased macrophage number, upregulated TREM2 protein expression, enhanced Rab5a / Rab7a ratio, increased number of double-positive cells, and decreased apoptosis level; in vitro, this is manifested as TREM2... + The proportion of macrophages increased, sTREM2 decreased, and phagocytic clearance capacity was enhanced. This demonstrates that luteolin has the potential to restore TREM2 levels. + Uses of drugs for macrophage function.

[0086] This invention also proposes a method for restoring TREM2 with luteolin. + In one specific implementation method, the research method for the mechanism of macrophage function includes: An animal model of diabetic nephropathy was established, with a control group, a diabetic nephropathy model group, and a luteolin-treated group. This research method is based on the aforementioned in vivo experiments, using TREM2 in kidney tissue... + Macrophage number, TREM2 protein expression, Rab5a / Rab7a ratio, TREM2 cells in the euthanasia state + Synergistic changes in macrophage number and apoptosis-related markers were used to determine whether luteolin could restore TREM2 levels. + The role of macrophage function.

[0087] Specifically, after the administration was completed, the kidney tissue of each group was tested to obtain TREM2. + Macrophage number, TREM2 protein expression level, Rab5a expression level, Rab7a expression level, TREM2 in the eutrophic state + Macrophage count and apoptosis-related indicators.

[0088] During the procedure, F4 / 80 antibody and TREM2 antibody can be used for immunofluorescence co-staining to detect TREM2 in kidney tissue. + Macrophage number; TREM2 protein expression level in kidney tissue was detected by Western blot; Rab5a and Rab7a protein expression levels were detected by Western blot; Rab5a / TREM2 and Rab7a / TREM2 dual immunofluorescence co-staining was used to detect TREM2 in the eutrophic state. +Macrophage number; the number of apoptotic cells in kidney tissue was detected using the TUNEL kit, and the expression level of Cleaved-caspase3 was detected by Western blot to characterize the degree of apoptosis.

[0089] Based on the above test results, if TREM2 is treated with luteolin... + Increased macrophage number, elevated TREM2 protein expression levels, elevated Rab5a and Rab7a expression levels, and TREM2 cells in a necrophore state. + An increase in macrophage numbers and a decrease in apoptosis-related markers confirm that luteolin can restore TREM2 levels. + The role of macrophage function.

[0090] In this embodiment, TREM2 + Macrophage number and TREM2 protein expression are used to reflect the TREM2-associated macrophage population and its receptor expression status; Rab5a and Rab7a are key molecules in the phagocytosis / burial process and can reflect the strength of burial activity; the number of TREM2-positive and Rab5a- or Rab7a-positive cells can reflect TREM2 in the burial state. + Macrophage number; TUNEL and Cleaved-caspase 3 indirectly reflect the cell burial clearance effect from the perspective of apoptosis burden. If luteolin synergistically changes the above indicators in a favorable direction, it indicates that it enhances TREM2. + The number and function of macrophages reduce kidney tissue damage.

[0091] During implementation, the application and effects of the above-described implementation methods will be explained with reference to the following specific examples.

[0092] Example 3: Kidney tissue from each group of mice in Example 1 was used as samples. Immunofluorescence co-staining with F4 / 80 and TREM2 antibodies was performed first, and the TREM2 content in the kidney tissue was statistically analyzed. + Macrophage count; then, Western blot was used to detect the expression of TREM2 protein, Rab5a, Rab7a, and Cleaved-caspase 3; simultaneously, double immunofluorescence co-staining with Rab5a / TREM2 and Rab7a / TREM2 was used to detect TREM2 cells in the eutrophic state. + Macrophage count; the number of apoptotic cells was also detected using the TUNEL assay kit.

[0093] Among them, the number of TUNEL-positive cells and Cleaved-caspase 3 expression were used to reflect the apoptotic burden of renal tubular cells, while Rab5a and Rab7a, as early and late endosome-related proteins, respectively, were used to reflect the strength of the endosome process; an increase in the number of Rab5a / TREM2 and Rab7a / TREM2 double-positive cells indicated that TREM2 cells were undergoing endosome process. + The number of macrophages increases.

[0094] Results analysis: Figure 29 Explanation of TREM2 + The increase in the number of macrophages was further amplified by the action of luteolin. Figure 30 This indicates that TREM2 protein was slightly increased in db / db mice, while the increase was greater in the Lut-H group. Immunofluorescence detection of kidney sections further confirmed this. Figure 31 and Figure 32 This trend was further confirmed by the results showing that luteolin upregulated TREM2. + A population of macrophages. Figure 33 and Figure 34 This indicates that luteolin can significantly reduce the number of apoptotic renal tubular cells. Figure 35 This indicates that luteolin inhibits the expression of cleaved caspase-3, suggesting that luteolin can improve renal tubular cell apoptosis. Figure 35 as well as Figure 36 This indicates that the protein levels of Rab5a and Rab7a were significantly upregulated in the Lut-H group. Figure 37 and Figure 38 This indicates that the average number of double-positive cells increased in the Lut-H group. Figure 39 and Figure 40 This indicates that similar quantitative increases were also detected in immunohistopathological sections co-stained with Rab7a and TREM2 in the Lut-H group.

[0095] Figures 29 to 32 Further evidence demonstrates that luteolin treatment in db / db mice resulted in TREM2 levels in vivo. + Increased proportion of macrophages, enhanced TREM2 protein expression, and TREM2 in kidney tissue + The macrophage population increased. Figures 33 to 40 Further evidence showed that luteolin treatment reduced renal apoptosis burden, decreased Cleaved-caspase 3 expression, and enhanced Rab5a and Rab7a expression, with Rab5a... + / TREM2 + and Rab7a + / TREM2 +The increased number of double-positive cells indicates that luteolin can enhance TREM2. + Macrophage burial reduces renal tubular cell apoptosis.

[0096] The results showed that TREM2 protein and TREM2 were present in the db / db model group. + Macrophages increased slightly, with a more significant increase after luteolin treatment; Rab5a and Rab7a protein levels were significantly upregulated, with Rab5a... + / TREM2 + and Rab7a + / TREM2 + The number of double-positive cells increased; TUNEL-positive cells and Cleaved-caspase3 expression decreased.

[0097] Functional analysis showed that luteolin does not simply increase TREM2. + Instead of increasing the number of macrophages, it simultaneously enhances the expression of burial-related molecules and burial status, while reducing the apoptotic burden on renal tubules, thereby forming an in vivo process of increasing luteolin, TREM2, and macrophages, enhancing burial, and reducing apoptosis.

[0098] In the above-described embodiments, preferably, luteolin restores TREM2. + Research methods for understanding the mechanisms of macrophage function also include: An in vitro model of macrophage dysfunction induced by high glucose stimulation was constructed, and a blank control group, a high glucose model group, and a luteolin intervention group were set up. Preferably, PMA was used to induce THP-1 cells to differentiate into macrophages. Specifically, under the conditions of PMA concentration of 100 ng / mL and induction time of 48 h, THP-1 cells were induced from a suspension state to an adherent state, and CD68 and CD11b staining was used to confirm that they had differentiated into macrophages. Subsequently, the obtained macrophages were exposed to a high glucose environment for 48 h to establish an in vitro model of macrophage dysfunction induced by high glucose stimulation.

[0099] After the model was established, the luteolin intervention group was treated with luteolin, and the TREM2 level in the cells of each group was detected. + Macrophage ratio.

[0100] During implementation, the application and effects of the above-described implementation methods will be explained with reference to the following specific examples.

[0101] Example 4: THP-1 cells were induced for 48 h under 100 ng / mL PMA to transition them from a suspension state to adherent growth. After PMA addition, THP-1 cells gradually lost their proliferative capacity and began adherent growth. CD68 and CD11b staining confirmed differentiation into macrophages. The differentiated macrophages were divided into a blank control group, a high-glucose model group, and a luteolin intervention group. Both the high-glucose model group and the luteolin intervention group were cultured in a 65 mmol / L high-glucose environment for 48 h, followed by luteolin treatment in the luteolin intervention group.

[0102] After treatment, the cells were triple-labeled with CD11b, CD68, and TREM2, and TREM2 was detected by flow cytometry. + Macrophage ratio.

[0103] Results analysis: Figure 41 This indicates that high glucose (HG) conditions reduced TREM2 levels. + The proportion of macrophages, while luteolin significantly increased TREM2. + The proportion of macrophages. Figure 41 It was demonstrated that TREM2 under high glucose stimulation conditions... + The proportion of macrophages decreased, but this proportion rebounded significantly after luteolin intervention, indicating that luteolin can restore the TREM2-related phenotype in an in vitro model of macrophage dysfunction induced by high glucose.

[0104] The results showed that the high-glucose model group TREM2 + The proportion of macrophages decreased significantly, while it rebounded significantly in the luteolin intervention group. Combined with CD68 and CD11b staining results, it was confirmed that luteolin reversed the decrease in TREM2-related phenotypes of differentiated macrophages under high glucose conditions, rather than simply affecting the THP-1 cell differentiation process.

[0105] In the above-described embodiments, preferably, luteolin restores TREM2. + Research methods for understanding the mechanisms of macrophage function also include: Based on the cell culture media obtained in the above embodiments, the soluble TREM2 level in each cell culture medium was detected. Under high glucose conditions, the soluble TREM2 level increased, indicating that TREM2 underwent more significant extracellular shearing. After intervention with luteolin, the soluble TREM2 level decreased, indicating that luteolin may have inhibited the TREM2 shearing process.

[0106] Furthermore, a TREM2 overexpression 293T cell model was constructed, and the full-length TREM2 expression level on the 293T cell membrane surface after luteolin treatment was detected. Preferably, the TREM2 expression plasmid was transfected into 293T cells, and total membrane proteins were extracted for analysis; ATP1A1 was used as a membrane protein loading control, and β-actin was used as a cytoplasmic protein control to verify the purity of the extracted membrane proteins.

[0107] Based on the test results, if the soluble TREM2 level decreased after luteolin intervention compared to the high glucose model group, and the full-length TREM2 expression level on the cell membrane surface of the 293T cell model with TREM2 overexpression that was not treated with luteolin increased, then it is determined that luteolin has the effect of inhibiting TREM2 cleavage and restoring full-length TREM2 expression on the cell membrane surface.

[0108] During implementation, the application and effects of the above-described implementation methods will be explained with reference to the following specific examples.

[0109] Example 5: In the three groups of macrophages in Example 4, culture supernatants were collected, and sTREM2 concentration was detected using an ELISA kit. The results showed that sTREM2 was significantly increased in the high glucose model group and significantly decreased in the luteolin intervention group. Furthermore, the detection results of sTREM2 in the three groups of kidney tissues showed that sTREM2 increased in vivo under model conditions, while luteolin significantly inhibited this increase, thus corroborating the TREM2 shear changes observed in vitro and in vivo.

[0110] In addition, a TREM2 overexpression model was constructed by transfecting 293T cells with the TREM2 expression plasmid. Untreated groups, a GI254023X positive control group, and luteolin treatment groups at 5 μM, 10 μM, and 20 μM were set up. Total membrane proteins were extracted, and the full-length TREM2 expression level on the membrane surface was detected. ATP1A1 was used as a membrane protein loading control, and β-actin as a cytoplasmic protein loading control to verify the purity of membrane protein separation. The results showed almost no cytoplasmic protein contamination.

[0111] Simultaneously, sTREM2 in the culture supernatant of each treatment group was detected. The results showed that different doses of luteolin increased the expression of full-length TREM2 on the cell surface in a dose-dependent manner; the 20 μM group was close to the GI group, while the corresponding sTREM2 in the supernatant decreased, and the sTREM2 level in the supernatant of the 20 μM group was almost parallel to that of the GI group.

[0112] Results analysis: Figure 42 This study demonstrated that the level of soluble TREM2 in macrophage culture supernatant increased under high glucose conditions, and that luteolin could reverse this change. Figure 43Furthermore, the study demonstrated at the in vivo level the inhibitory effect of luteolin on the increase of sTREM2 in kidney tissue. Figure 44 and Figure 45 It was demonstrated that luteolin can enhance full-length TREM2 expression on the membrane surface in a 293T cell model with TREM2 overexpression. Figure 46 Further evidence showed that sTREM2 in the supernatant decreased after treatment with different doses of luteolin, thus demonstrating that luteolin can inhibit TREM2 cleavage and restore full-length TREM2 expression on the cell membrane surface.

[0113] Functional analysis showed that the decrease in sTREM2 in the high-glucose macrophage system and the increase in full-length TREM2 in the 293T membrane protein system constituted a complementary chain of evidence, jointly demonstrating that luteolin can inhibit TREM2 cleavage and maintain full-length TREM2 on the cell membrane surface.

[0114] In the above-described embodiments, preferably, based on the above-described embodiments which have confirmed that luteolin can inhibit TREM2 cleavage and restore full-length TREM2 expression on the membrane surface, further changes in TREM2 downstream signal transduction and cell burial function are detected.

[0115] Specifically, apoptotic renal tubular epithelial cells were prepared and co-cultured with macrophages from various groups. Preferably, apoptotic HK2 cells were used as target cells for phagocytosis. After co-culturing with macrophages from various groups, apoptotic HK2 cells or surviving HK2 cells were labeled with pHrodo dye.

[0116] Meanwhile, the phosphorylation levels of DAP12 and SYK, as well as the phagocytic clearance capacity of macrophages on apoptotic renal tubular epithelial cells, were detected in each group. After TREM2 binds to its ligand, it can promote downstream signal transduction through DAP12 and SYK, thereby promoting phagocytosis and cell burial-related processes.

[0117] Based on the test results, if the phosphorylation levels of DAP12 and SYK are enhanced after luteolin intervention, and the phagocytic clearance capacity of macrophages for apoptotic renal tubular epithelial cells is enhanced, then it is further determined that luteolin restores TREM2 by enhancing TREM2-DAP12-SYK signaling. + The cytodegenerative function of macrophages.

[0118] During implementation, the application and effects of the above-described implementation methods will be explained with reference to the following specific examples.

[0119] Example 6: Apoptotic HK2 cells were first prepared, and either apoptotic or surviving HK2 cells were labeled with pHrodo dye. These cells were then co-cultured with control macrophages + surviving HK2 cells, control macrophages + apoptotic HK2 cells, high-glucose-treated macrophages + apoptotic HK2 cells, and luteolin-pretreated high-glucose macrophages + apoptotic HK2 cells. The number of green fluorescent positive cells was observed and counted.

[0120] Since pHrodo dye only emits green fluorescence in acidic environments, obvious green fluorescence will only appear after macrophages engulf apoptotic HK2 cells and enter late endosomes or lysosomes; therefore, cells that will show pHrodo green fluorescence are defined as phagocytic clearance positive cells.

[0121] The results showed that there was almost no green fluorescence when co-cultured with live HK2 cells; the green fluorescence was enhanced when co-cultured with apoptotic HK2 cells; and the green fluorescence of macrophages treated with high glucose was significantly weakened, and was significantly restored after intervention with luteolin.

[0122] Cellular proteins were then collected from each group, and Rab5a, Rab7a, p-DAP12, and p-SYK were detected by Western blot. IL-1β, IL-18, TNF-α, and MCP-1 were detected by qPCR. The results showed that in the high glucose model group, Rab5a, Rab7a, p-DAP12, and p-SYK decreased, while inflammatory factors increased. After luteolin intervention, Rab5a, Rab7a, p-DAP12, and p-SYK increased, while inflammatory factors decreased.

[0123] Results analysis: Figures 47 to 49 This study demonstrated that macrophages' ability to phagocytose and clear apoptotic renal tubular epithelial cells decreased under high glucose conditions, while luteolin could significantly restore this ability. Figure 50 Further evidence showed that the expression of IL-1β, IL-18, TNF-α and MCP-1 decreased after luteolin treatment, indicating that it can reduce the inflammatory response while restoring cell burial function; Figure 49 and Figures 51 to 54 The results showed that luteolin treatment enhanced the expression of Rab5a and Rab7a, as well as the levels of p-SYK and p-DAP12, indicating that luteolin can enhance TREM2-DAP12-SYK signaling and promote cell death-related processes.

[0124] Functional analysis showed that luteolin not only enhanced the phagocytic clearance capacity of macrophages against apoptotic renal tubular epithelial cells, but also simultaneously enhanced TREM2-DAP12-SYK signaling and Rab5a / Rab7a-related cell burial processes, and reduced the expression of inflammatory factors. Thus, it achieved the complete logic of luteolin, TREM2 cleavage inhibition, full-length TREM2 restoration on the membrane surface, DAP12 / SYK enhancement, enhanced cell burial and phagocytic clearance, and decreased inflammation.

[0125] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A luteolin for restoring TREM2 in the preparation of diabetic nephropathy + Application in drugs that promote macrophage function.

2. A luteolin in the preparation of TREM2 + Application in drugs that promote macrophage function.

3. A luteolin-based method for restoring TREM2 + The research method for studying the mechanism of macrophage function is characterized by, include: An animal model of diabetic nephropathy was established, and a control group, a diabetic nephropathy model group, and a luteolin treatment group were set up. The kidney tissues of each group were examined separately to obtain TREM2. + Macrophage number, TREM2 protein expression level, Rab5a expression level, Rab7a expression level, TREM2 in the eutrophic state + Macrophage count and apoptosis-related indicators; Based on the test results of each group, if TREM2 is treated with luteolin... + Increased macrophage number, elevated TREM2 protein expression levels, elevated Rab5a and Rab7a expression levels, and TREM2 cells in a necrophore state. + An increase in macrophage numbers and a decrease in apoptosis-related markers confirm that luteolin can restore TREM2 levels. + The role of macrophage function.

4. The luteolin-based TREM2 recovery method according to claim 3 + The research method for studying the mechanism of macrophage function is characterized by, Also includes: An in vitro model of macrophage dysfunction stimulated by high glucose was constructed, and a blank control group, a high glucose model group, and a luteolin intervention group were set up. The luteolin intervention group was treated with luteolin, and the TREM2 level in the cells of each group was detected. + Macrophage ratio.

5. The luteolin-based TREM2 recovery method according to claim 4 + The research method for studying the mechanism of macrophage function is characterized by, Also includes: The level of soluble TREM2 in the cell culture medium of each group was detected; A TREM2 overexpression 293T cell model was constructed, and the full-length TREM2 expression level on the surface of the 293T cell membrane was detected after luteolin treatment. Based on the test results, if the soluble TREM2 level decreased after luteolin intervention compared to the high glucose model group, and the full-length TREM2 expression level on the cell membrane surface of the 293T cell model with TREM2 overexpression that was not treated with luteolin increased, then it is determined that luteolin has the effect of inhibiting TREM2 cleavage and restoring full-length TREM2 expression on the cell membrane surface.

6. The luteolin-based TREM2 recovery method according to claim 5 + The research method for studying the mechanism of macrophage function is characterized by, Also includes: Apoptotic renal tubular epithelial cells were prepared and co-cultured with macrophages from various groups; The phosphorylation levels of DAP12 and SYK, as well as the phagocytic clearance capacity of macrophages on the apoptotic renal tubular epithelial cells, were detected in each group. Based on the test results, if the phosphorylation levels of DAP12 and SYK are enhanced after luteolin intervention, and the phagocytic clearance capacity of macrophages for apoptotic renal tubular epithelial cells is enhanced, then it is further determined that luteolin restores TREM2 by enhancing TREM2-DAP12-SYK signaling. + The cytodegenerative function of macrophages.

Images