An in vitro candidate screening method based on the LAPTM5 / USP10 / PTEN pathway

Abstract

This invention discloses an in vitro candidate screening method based on the LAPTM5 / USP10 / PTEN pathway. This method is based on the mechanism by which LAPTM5 weakens PTEN stability by promoting lysosomal degradation of USP10, thereby activating the PI3K / AKT / mTOR pathway, inhibiting autophagy, and promoting epithelial-mesenchymal transition. The screening method includes: establishing senescence-induced or LAPTM5-overexpressing cell models; after candidate treatment, detecting multi-level indicators such as LAPTM5 / USP10 / PTEN expression and modification, PI3K / AKT / mTOR phosphorylation, autophagy markers LC3 and p62, EMT markers, cell morphology, and migration ability; and comprehensively evaluating and ranking candidates based on their regulatory effects on each indicator. The screening system integrates data acquisition, processing, analysis, evaluation, storage, and visualization output modules to automate the screening process. This invention establishes a candidate screening platform based on a clear molecular mechanism, a comprehensive indicator system, and quantifiable evaluation, which can be used to screen candidates from compound libraries or natural products that have regulatory effects on the aforementioned signaling axis, and provides a technical basis for subsequent research.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an in vitro candidate screening method based on the LAPTM5 / USP10 / PTEN pathway. Background Technology

[0002] Age-related renal fibrosis is a crucial pathological process in the progression of chronic kidney disease, characterized by renal tubular epithelial cell senescence, epithelial-mesenchymal transition (EMT), and excessive extracellular matrix deposition. Senescent renal tubular epithelial cells secrete pro-inflammatory and pro-fibrotic factors through the senescence-associated secretory phenotype (SASP), creating a vicious cycle. Autophagy, a highly conserved lysosomal degradation pathway, plays a vital role in maintaining cellular homeostasis and resisting stress. Autophagy defects are closely associated with renal tubular epithelial cell senescence, EMT, and renal fibrosis.

[0003] The PI3K / AKT / mTOR signaling pathway is a key negative regulatory pathway for autophagy; its activation inhibits autophagy initiation. Phosphatases and tensin homologs (PTENs), as negative regulators of PI3K, promote autophagy by inhibiting PI3K / AKT / mTOR pathway activation. Lysosome-associated transmembrane protein 5 (LAPTM5) is significantly upregulated in aging kidneys and weakens the deubiquitination of PTEN by promoting the lysosomal degradation of the deubiquitinase USP10. This leads to the proteasomal degradation of PTEN via the K48-linked polyubiquitination pathway, thereby activating the PI3K / AKT / mTOR pathway, inhibiting autophagy, and promoting EMT and renal fibrosis.

[0004] Currently, there is a lack of systematic methods based on clearly defined molecular mechanisms for screening candidate drugs for age-related renal fibrosis. Existing screening methods mostly focus on single targets or pathways, making it difficult to comprehensively evaluate the regulatory effects of candidates on the intact signaling axis and their biological effects. Therefore, there is an urgent need to establish candidate screening methods and systems based on the LAPTM5 / USP10 / PTEN / PI3K / AKT / mTOR / autophagy intact signaling axis to identify, evaluate, and screen candidates that have regulatory effects on renal tubular epithelial cell phenotypic transformation and autophagy-related indicators. Summary of the Invention

[0005] 1. Technical problems to be solved This invention aims to solve the following technical problems: (1) Establish an in-situ screening method for candidates based on the LAPTM5 / USP10 / PTEN signal axis to identify candidates that can regulate the signal axis and its downstream PI3K / AKT / mTOR-autophagy pathway; (2) Provide a multi-level, quantifiable candidate evaluation index system, including molecular level indicators (protein expression and modification), cellular level indicators (autophagy activity, EMT markers) and functional level indicators (cell phenotype and migration ability). (3) Construct a candidate screening system that integrates data collection, indicator analysis and candidate evaluation functions to improve standardization and comparability; (4) To provide technical basis for further research and evaluation of relevant candidates.

[0006] 2. Technical Solution This invention provides a candidate screening method based on the LAPTM5 / USP10 / PTEN axis regulation of the PI3K / AKT / mTOR autophagy pathway, comprising the following steps: (1) Cell model construction Establish cell models reflecting LAPTM5 / USP10 / PTEN signaling axis dysregulation, including but not limited to: a) Senescence-induced model: D-galactose (20 g / L, treatment for 5 days) was used to induce senescence in renal tubular epithelial cells (such as HK-2 cells). In this model, LAPTM5 expression was upregulated, PTEN expression was downregulated, and autophagy activity was reduced. b) Senescent conditioned medium (CM) model: Senescent cells were collected and treated with conditioned medium to treat non-senescent cells. In this model, LAPTM5 expression was increased, inducing EMT and inhibiting autophagy. c) LAPTM5 overexpression model: LAPTM5 was overexpressed by plasmid transfection. In this model, USP10 degradation increased, PTEN level decreased, PI3K / AKT / mTOR was activated, autophagy was inhibited, and EMT was enhanced.

[0007] (2) Candidate processing The candidate substance to be tested was added to the cell model at different concentrations, and a solvent control group and a positive control group (such as the known PTEN agonist matrine) were set up. After an appropriate culture time (e.g., 24-48 hours), cells and / or culture medium were collected for subsequent detection.

[0008] (3) Detection of molecular level indicators The expression and modification status of key molecules in the LAPTM5 / USP10 / PTEN signaling axis were detected after candidate treatment, including: a) LAPTM5 protein expression level (Western blot, immunofluorescence); b) USP10 protein expression level and stability (half-life determined by Western blot and CHX tracing assay). c) PTEN protein expression level and stability (half-life determined by Western blot and CHX tracing assay); d) PTEN ubiquitination levels, especially K48-linked polyubiquitination (immunoprecipitation combined with Western blot). e) PI3K / AKT / mTOR pathway activation status: p-PI3K, p-AKT, p-mTOR and their total protein levels (Western blot).

[0009] (4) Autophagy level index detection The changes in autophagy activity of cells after candidate treatment were detected, including: a) LC3 protein expression and LC3II / LC3I ratio (Western blot, immunofluorescence); b) p62 protein accumulation level (Western blot, immunofluorescence); c) Other autophagy markers (optional): Beclin1, ATG5, ATG7, etc.

[0010] (5) Detection of EMT-related indicators Detect changes in cellular EMT markers after candidate treatment, including: a) Epithelial markers: E-cadherin expression level; b) Mesenchymal markers: expression levels of N-cadherin, α-smooth muscle actin (α-SMA), fibronectin, and type I collagen (COL1A1); c) Detection methods: Western blot, immunofluorescence, qRT-PCR.

[0011] (6) Detection of cell phenotype and functional indicators Detecting changes in cell morphology and function after candidate treatment, including: a) Cell morphology observation: The transformation of epithelial cell morphology into mesenchymal-like morphology; b) Cell migration ability: Transwell migration assay; c) Age-related phenotypes (optional): SA-β-galactosidase staining, expression of aging markers p16 / p21 / p53, and Lamin B1 expression; d) SASP factor secretion (optional): TNF-α, IL-6, TGF-β1, etc.

[0012] (7) Evaluation and judgment of candidates The candidates are comprehensively evaluated based on the changes in the above indicators: a) Preferred candidates should exhibit one or more of the following effects: - Reduce LAPTM5 expression or inhibit its function in promoting USP10 degradation; - Stabilizes or increases USP10 protein levels, prolonging the USP10 half-life; - Stabilizes or increases PTEN protein levels, prolongs PTEN half-life, and reduces PTEN K48-linked polyubiquitination; - Reduces the phosphorylation levels of PI3K, AKT, and mTOR; - Increasing the LC3II / LC3I ratio and reducing p62 accumulation suggests autophagy activation; - Increased expression of E-cadherin and decreased expression of N-cadherin, α-SMA, fibronectin, and COL1A1 suggest EMT inhibition; - Maintains epithelial cell morphology and reduces cell migration ability.

[0013] b) Quantitative scoring system (optional): A scoring system is established based on the degree to which the candidates regulate each indicator, such as: - Core metrics (USP10 stability, PTEN stability, autophagy level) have high weighting; Downstream indicators (EMT biomarkers, cell migration) have the second highest weighting; - Calculate the overall score and select the candidate with the highest score to proceed to the next stage of verification.

[0014] c) Positive control verification: The reliability of the screening method was verified by comparing it with matrine, a known PTEN agonist.

[0015] (8) Optional further verification Perform one or more of the following further validations on the selected candidates: a) Mechanism validation: Verify whether the candidate affects the interaction between LAPTM5-USP10 and USP10-PTEN by co-immunoprecipitation; verify whether the candidate affects the lysosomal localization of USP10 by immunofluorescence. b) Animal model validation: The effect of the candidate on renal fibrosis was evaluated in a D-galactose-induced aging mouse model. The indicators included serum creatinine (Cr), blood urea nitrogen (BUN), histological staining (Masson, PAS, HE), PTEN expression, p-PI3K / p-AKT / p-mTOR levels, autophagy markers, EMT markers, etc.

[0016] Furthermore, the core of the screening method of this invention does not lie in the individual detection of a single endpoint indicator, but in establishing a candidate evaluation pathway around the following continuous mechanism: LAPTM5 promotes the degradation of USP10 via the lysosomal pathway; the decrease in USP10 leads to a weakening of its deubiquitination stabilizing effect on PTEN, thereby reducing PTEN stability and causing activation of the downstream PI3K / AKT / mTOR pathway, inhibition of autophagy, and enhancement of EMT. Therefore, this invention preferably evaluates candidates through a combination of changes in USP10 protein levels, PTEN protein levels, K48-linked polyubiquitination of PTEN, autophagy levels, and EMT markers.

[0017] 3. Beneficial effects The beneficial effects of this invention include: (1) Mechanism clarity: The screening method is based on the well-defined LAPTM5 / USP10 / PTEN / PI3K / AKT / mTOR / autophagy signal axis, which can systematically evaluate the role of candidates in the complete regulatory pathway, rather than just targeting a single target, thus improving the rationality of the screening mechanism.

[0018] (2) Comprehensiveness of the indicator system: A multi-dimensional evaluation indicator system covering the molecular level (protein expression, post-translational modification), pathway level (phosphorylation status), organelle function level (autophagy activity), cell phenotype level (EMT markers, morphology) and cell function level (migration ability) has been established, which can comprehensively evaluate the biological effects of candidates.

[0019] (3) Quantifiability: All indicators are quantifiable molecular or cellular level parameters, which facilitates objective evaluation and comparison of the effects of different candidates.

[0020] (4) Model diversity: It provides a variety of cell models such as senescence induction model, conditioned medium model, and LAPTM5 overexpression model. Appropriate models can be selected according to the screening purpose and candidate characteristics, thereby improving screening flexibility.

[0021] (5) Positive control availability: Matrine, as a validated PTEN agonist, can be used as a positive control, providing a reference standard for the establishment and validation of screening methods.

[0022] (6) Adequacy of experimental verification: - Signal axis integrity: The interaction and regulatory relationship between LAPTM5-USP10-PTEN have been verified by various methods such as Co-IP, immunofluorescence colocalization, CHX half-life assay, and ubiquitination assay. - Observable downstream effects: It has been confirmed that changes in PTEN levels can affect PI3K / AKT / mTOR phosphorylation, autophagy markers LC3 and p62, and EMT markers E-cadherin / N-cadherin / α-SMA / fibronectin, etc. - Positive control efficacy: Matrine has been shown to upregulate PTEN, inhibit PI3K / AKT / mTOR phosphorylation, restore autophagy, and affect related indicators in a D-galactose-induced aging mouse model. Attached Figure Description

[0023] Figure 1 Identification of key biomarkers in an aging-related kidney model. (A) Heatmap of differentially expressed genes in the merged GEO gene sets (GSE181797, GSE233718, and GSE155407); (B) Volcano plot showing all genes; (CF) Weighted co-expression network analysis (WGCNA); (GH) LASSO regression analysis; (I) Overlapping genes among LASSO, differentially expressed genes, and MEgrey modules; (J) Gene expression patterns in the merged dataset samples; (K) Gene expression patterns in GSE261354.

[0024] Figure 2 LAPTM5 expression is upregulated in D-galactose-induced aging kidneys. (A) qRT-PCR analysis of LAPTM5 and CFI mRNA expression in kidneys of different mouse models; (B, C) Western blot analysis of LAPTM5 protein levels; (D, E) SA-β-gal staining of kidney tissue sections; (F, G) Western blot analysis of aging markers p16 and p21; (H) qRT-PCR analysis of SASP-related gene mRNA; (I, J) Protein levels of TNF-α, IL-6, and TGF-β1; (K, L) Representative images and quantification of Masson staining, PAS staining, and LAPTM5 immunohistochemistry; (M, N) Western blot analysis of fibrosis-related proteins.

[0025] Figure 3EMT is associated with senescence in HK-2 cells. (A) Viability of HK-2 cells treated with different concentrations of D-galactose; (B, C) SA-β-gal staining; (D, E) Western blot analysis of senescence-related proteins p21, p16, and p53; (F, G) LaminB1 immunofluorescence; (H) qRT-PCR analysis of SASP-related factors; (I, J) Protein levels of TNF-α, IL-6, and TGF-β1; (K) Schematic diagram of experimental design; (L) Cell morphological changes; (M, N) Transwell migration assay; (P, Q) Western blot analysis of EMT-related proteins and LAPTM5.

[0026] Figure 4 LAPTM5-mediated EMT induced in senescent cells by conditioned medium. (A, B) LAPTM5 overexpression verification; (C) Cell morphology; (D, E) Transwell migration assay; (F) Immunofluorescence of EMT markers; (G, H) LAPTM5 knockdown verification; (I) Cell morphology; (J, K) Transwell migration assay; (L) Immunofluorescence of EMT markers; (M, N) Western blot analysis of EMT-related proteins.

[0027] Figure 5 The PI3K / AKT / mTOR pathway is involved in CM-induced EMT in HK-2 cells and is regulated by PTEN. (A) GO functional enrichment analysis; (B) KEGG pathway enrichment analysis; (C, D) Western blot analysis of PI3K and AKT protein expression; (EG) Western blot analysis of mTOR, p62, and LC3 protein expression; (H, I) PTEN protein expression analysis; (JK) PTEN overexpression inhibits senescent CM-induced EMT; (L) AKT, mTOR, p62, and LC3 protein expression levels.

[0028] Figure 6LAPTM5 accelerates K48-linked polyubiquitination of PTEN via the lysosomal pathway of USP10 degradation. (A) IP-MS identification of the interaction between USP10 and LAPTM5 and PTEN; (B, C) Immunoprecipitation verification of the interaction; (DE) PTEN protein expression analysis; (F) USP10 degradation inhibited by chloroquine; (G) Co-localization of LAPTM5 and USP10 in lysosomes; (H, I) CHX tracking assay to detect the half-life of USP10 and PTEN; (J, K) Immunofluorescence analysis of PTEN and USP10 co-localization; (L) PTEN degradation inhibited by MG132; (M, N) PTEN ubiquitination and K48-linked ubiquitination analysis.

[0029] Figure 7 Matrine alleviates D-gal-induced renal fibrosis pathological damage through the PTEN / PI3K-AKT-mTOR / autophagy pathway. (A) Schematic diagram of animal model establishment and sample collection; (B, C) Serum creatinine (Cr) and blood urea nitrogen (BUN) levels; (D, E) Histological staining (HE, PAS, Masson); (F, G) Western blot analysis of EMT markers; (H, I) Immunohistochemical staining and quantification of PTEN; (J, K) Western blot analysis of the PTEN-PI3K-AKT-mTOR signaling pathway; (L, M) Immunofluorescence staining and quantification of LC3B; (N, O) Western blot analysis of P62, Beclin1, and LC3B. Detailed Implementation

[0030] The present invention will be described in detail below through specific embodiments, but the scope of protection of the present invention is not limited to these embodiments.

[0031] Example 1: Establishment of an aging-induced cell model The human renal tubular epithelial cell line HK-2 was used as the selection model cell. HK-2 cells were cultured in DMEM / F12 medium supplemented with 10% fetal bovine serum and penicillin / streptomycin, and incubated at 37°C with 5% [missing information - likely a specific temperature range]. Cultured under specific conditions.

[0032] To establish an aging-induced model, HK-2 cells were seeded in culture plates and treated with medium containing 20 g / L D-galactose for 5 days after cell attachment. The medium was changed daily. Control group cells were cultured in normal medium without D-galactose.

[0033] Validation of aging models: (1) SA-β-galactosidase staining: Cells were fixed with 4% formaldehyde, washed with PBS, and incubated overnight at 37°C with SA-β-gal staining solution (pH 6.0). The results showed that the number of SA-β-gal positive cells in the D-galactose treatment group increased significantly.

[0034] (2) Detection of aging markers: Western blot analysis showed that the expression of p16, p21 and p53 proteins was upregulated in the D-galactose treatment group, while the expression of Lamin B1 was downregulated.

[0035] (3) SASP factor detection: qRT-PCR showed increased expression of IL-6, IL-1β, MMP3, CCL2, and CXCL10 mRNA; Western blot showed increased protein levels of TNF-α, IL-6, and TGF-β1.

[0036] (4) LAPTM5 expression: Both Western blot and qRT-PCR showed that LAPTM5 expression was significantly upregulated in the D-galactose treatment group.

[0037] Example 2: Verification of signal axis misalignment of LAPTM5 / USP10 / PTEN In the aging model established in Example 1, the misalignment state of the LAPTM5 / USP10 / PTEN signal axes was further verified.

[0038] (1) Detection of USP10 and PTEN protein levels: Western blot showed that the levels of USP10 and PTEN proteins were reduced in the D-galactose treatment group.

[0039] (2) Detection of PI3K / AKT / mTOR pathway activation: Western blot showed that the levels of p-PI3K, p-AKT and p-mTOR increased in the D-galactose treatment group, while the levels of total PI3K, AKT and mTOR did not change significantly.

[0040] (3) Autophagy level detection: Western blot showed that the LC3II / LC3I ratio was decreased and p62 protein accumulation was increased in the D-galactose treatment group. Immunofluorescence showed a decrease in punctate aggregation of LC3.

[0041] (4) Detection of EMT markers: Western blot showed that the expression of E-cadherin was decreased and the expression of N-cadherin, α-SMA, and fibronectin was increased in the D-galactose treatment group. Immunofluorescence showed that the cell morphology changed from polygonal to spindle-shaped.

[0042] (5) Cell migration ability test: Transwell migration assay showed that the number of migrating cells in the D-galactose treatment group increased significantly.

[0043] The above results confirm that in the D-galactose-induced aging model, LAPTM5 upregulation is accompanied by a decrease in USP10 / PTEN, activation of PI3K / AKT / mTOR, inhibition of autophagy, and enhancement of EMT, making it suitable for candidate screening.

[0044] The above results indicate that, in the established model, the upregulation of LAPTM5 is not an isolated phenomenon, but rather occurs sequentially with the decrease of USP10 and PTEN, activation of the PI3K / AKT / mTOR pathway, inhibition of autophagy, and enhancement of EMT. Therefore, the model used in this invention can simultaneously provide a multi-level evaluation basis encompassing upstream molecule stability, midstream pathway activity, and downstream phenotypic changes.

[0045] Example 3: Verification of the positive control matrine To establish a reference standard for screening methods, the effects of the known PTEN agonist matrine were first verified in a cell model.

[0046] HK-2 cells treated with D-galactose (5 days after modeling) were treated with different concentrations of matrine (0, 1, 5, 10, 20 μM) for 48 hours (matrine was dissolved in DMSO, and the final DMSO concentration was <0.1%).

[0047] Test results: (1) Cell viability: CCK-8 assay showed that matrine at concentrations of 20 μM and below had no significant effect on cell viability.

[0048] (2) PTEN expression: Western blot showed that matrine upregulated PTEN protein levels in a dose-dependent manner, with significant effects at concentrations of 10 μM and 20 μM.

[0049] (3) PI3K / AKT / mTOR pathway: Matrine treatment reduced the levels of p-PI3K, p-AKT and p-mTOR, with 10 μM and 20 μM concentrations showing significant effects.

[0050] (4) Autophagy recovery: Matrine treatment increased the LC3II / LC3I ratio and reduced p62 accumulation, with significant effects at 10 μM and 20 μM concentrations.

[0051] (5) EMT inhibition: Matrine treatment increased the expression of E-cadherin and decreased the expression of N-cadherin, α-SMA and fibronectin. The effects were significant at concentrations of 10 μM and 20 μM.

[0052] (6) Cell phenotype: cells treated with matrine maintained more epithelial-like morphology and had reduced migration ability.

[0053] Based on the above results, 10-20 μM matrine was selected as the positive control concentration for subsequent screening.

[0054] Example 4: Candidate Screening Process Step 1: Candidate Preparation Dissolve the candidate sample in a suitable solvent (such as DMSO, water, ethanol, etc.) to prepare a stock solution (e.g., 10 mM). Based on the preliminary experimental results, select an appropriate screening concentration range (recommended initial screening concentrations: 1, 5, 10, 20 μM).

[0055] Step 2: Cell seeding and modeling HK-2 cells were seeded in 96-well plates (for CCK-8 assay), 24-well plates (for immunofluorescence), or 6-well plates (for Western blot) and treated with 20 g / L D-galactose for 5 days to establish an aging model.

[0056] Step 3: Candidate Processing After the aging model was established, the culture medium was replaced with fresh medium containing the candidate, and the following control group was set up: - Blank control (normal cells, without D-galactose and candidate substances) - Model control (D-galactose treatment, no candidate added) - Solvent control (D-galactose treatment, with an equal volume of solvent added) - Positive control (D-galactose treatment, with the addition of 10-20 μM matrine) Candidate processing time: 24-48 hours.

[0057] Step 4: Cell viability assay Cell viability in each group was assessed using a CCK-8 assay kit to exclude candidates with significant cytotoxicity.

[0058] Step 5: Molecular index detection Cells were collected for Western blot analysis. - LAPTM5, USP10, PTEN expression - p-PI3K, PI3K, p-AKT, AKT, p-mTOR, mTOR - LC3I, LC3II, p62 - E-cadherin, N-cadherin, α-SMA, fibronectin, COL1A1 - Internal reference: GAPDH or β-actin Immunofluorescence assay (optional): - USP10 and PTEN co-positioning - LC3 point-like aggregation - EMT biomarker expression and cell morphology Step 6: Cell function index detection Transwell migration assay: After 12-24 hours of candidate treatment, cells were digested and seeded into Transwell chambers. After 24 hours, the cells were stained and counted to determine the number of migrating cells.

[0059] Step 7: Data Collection and Analysis (1) Acquire Western blot strip images and perform grayscale analysis using ImageJ software.

[0060] (2) Calculate the relative expression levels of each protein (standardized with internal reference and based on the model control group).

[0061] (3) Calculate the phosphorylated protein ratio (p-protein / total protein).

[0062] (4) Calculate the LC3II / LC3I ratio.

[0063] (5) Count the number of Transwell cells that migrated.

[0064] Step 8: Candidate Evaluation The candidates were comprehensively evaluated based on the changes in various indicators after treatment relative to the model control group, solvent control group, and positive control group. Preferably, the candidates exhibited one or more of the following changes after treatment: increased USP10 protein levels, increased PTEN protein levels, decreased K48-linked polyubiquitination of PTEN, decreased PI3K / AKT / mTOR pathway activity, increased LC3II / LC3I ratio, decreased p62 protein accumulation, and restoration of EMT markers to the epithelial phenotype. Candidates meeting the above trends and not exhibiting significant cytotoxicity can be used for further validation.

[0065] Example 5: Further Validation of the Screened Candidates The further verification in this embodiment is not a prerequisite for the success of the screening method of this invention, but rather serves to further confirm, based on the preliminary screening results, whether the candidate exerts a regulatory effect along the LAPTM5—USP10—PTEN action chain. Verification of interactions, protein stability, ubiquitination, and localization changes can enhance the reliability of determining the candidate's action direction.

[0066] The candidates selected in Example 4 were further verified as follows: (1) Validation of dose-response relationship: Test finer concentration gradients (e.g., 0.1, 0.5, 1, 5, 10, 20, 50 μM), plot dose-response curves, and calculate EC50 values.

[0067] (2) Verification of mechanism of action: a) Immunoprecipitation: to detect whether the candidate affects the interaction between LAPTM5-USP10 and USP10-PTEN.

[0068] b) Protein stability assay: CHX tracking assay to detect whether the candidate extends the half-life of USP10 and PTEN.

[0069] c) Ubiquitination experiment: to detect whether the candidate reduces K48-linked polyubiquitination of PTEN.

[0070] d) Lysosomal localization: Immunofluorescence assay to determine whether the candidate affects the colocalization of USP10 and LAMP1.

[0071] (3) Validation using other cell models: The efficacy consistency of the candidate was validated in the LAPTM5 overexpression model or the aging conditioned medium model.

[0072] (4) Animal model validation (optional): In a D-galactose-induced aging mouse model, the effects of candidate compounds on related indicators (Cr, BUN), histopathology (HE, PAS, Masson staining), PTEN expression, autophagy, and EMT markers were evaluated, referencing the matrine dosing regimen (20 mg / kg, intraperitoneal injection daily for 4 weeks). Summary of Implementation Examples

[0073] The above examples demonstrate that: 1. A cell model based on the dysregulation of the LAPTM5 / USP10 / PTEN signaling axis (D-galactose-induced aging model) was established. 2. The effect of the positive control matrine in this model was verified; 3. It provides a complete candidate screening process, including candidate processing, multi-level indicator detection, data analysis, and candidate evaluation; 4. A quantitative scoring system has been established, which can objectively rank the candidates; 5. An integrated screening system was built, which automated and standardized the screening process.

[0074] The screening method and system provided by this invention have advantages such as clear mechanism, comprehensive indicators, quantifiable evaluation, and process scalability. They can be used to screen candidates from compound libraries or natural products that have the potential to regulate the LAPTM5 / USP10 / PTEN signal axis and related pathological processes.

Claims

1. An in vitro candidate screening method based on the LAPTM5 / USP10 / PTEN pathway, characterized in that, The method for screening candidates that can promote the degradation of USP10 lysosomes by regulating LAPTM5 and thereby affect the stability of PTEN protein includes the following steps: (1) Provide a model of renal tubular epithelial cells; (2) Apply the test candidate to the cell model; (3) Detect the changes in USP10 and PTEN protein levels after candidate treatment; (4) Evaluate the regulatory role of the candidate based on the increase in USP10 protein level and / or PTEN protein level.

2. The in vitro candidate screening method based on the LAPTM5 / USP10 / PTEN pathway according to claim 1, characterized in that, The renal tubular epithelial cell model is a D-galactose-induced senescent renal tubular epithelial cell model.

3. The in vitro candidate screening method based on the LAPTM5 / USP10 / PTEN pathway according to claim 2, characterized in that, The renal tubular epithelial cells were HK-2 cells.

4. A method for screening in vitro candidates based on the LAPTM5 / USP10 / PTEN pathway according to claim 2 or 3, characterized in that, The D-galactose-induced senescent renal tubular epithelial cell model was constructed by treating the cells with 20 g / L D-galactose for 5 days.

5. The in vitro candidate screening method based on the LAPTM5 / USP10 / PTEN pathway according to claim 1, characterized in that, Step (3) also includes detecting changes in the stability of the USP10 protein and / or the PTEN protein after candidate treatment.

6. The in vitro candidate screening method based on the LAPTM5 / USP10 / PTEN pathway according to claim 5, characterized in that, The stability of the USP10 protein and / or PTEN protein was evaluated by determining the half-life using a CHX tracking assay.

7. The in vitro candidate screening method based on the LAPTM5 / USP10 / PTEN pathway according to claim 1, characterized in that, Step (3) also includes detecting the K48-linked polyubiquitination level of PTEN after candidate treatment.

8. The in vitro candidate screening method based on the LAPTM5 / USP10 / PTEN pathway according to claim 7, characterized in that, If a candidate reduces the level of K48-linked polyubiquitination of PTEN, then the candidate is evaluated as having a regulatory effect.

9. The in vitro candidate screening method based on the LAPTM5 / USP10 / PTEN pathway according to claim 1, characterized in that, Step (3) also includes detecting changes in the colocalization of USP10 and the lysosomal marker LAMP1 after candidate treatment.

10. The in vitro candidate screening method based on the LAPTM5 / USP10 / PTEN pathway according to claim 9, characterized in that, If a candidate reduces the colocalization of USP10 and LAMP1, then the candidate is evaluated as having a regulatory effect.

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