Application of angiotensin II receptor antagonist in preparation of medicine for preventing and treating renal tubular injury caused by triptolide

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of an angiotensin II receptor antagonist in preparation of a medicine for preventing and treating renal tubular injury caused by triptolide. According to the invention, the upstream key target protein of the TPL-induced renal tubule injury is determined to be HSPA1 through experiments for the first time, and the problem that the key target spot of TPL toxicity is not determined in the prior art is solved. Based on a key target spot of TPL toxicity, through molecular screening and parallel pharmacodynamic verification on various sartan drugs such as irbesartan, losartan, olmesartan and valsartan, it is determined that the angiotensin II receptor antagonist has a general protection effect on TPL toxicity. Wherein the protective efficacy of irbesartan as a preferable embodiment is obviously superior to that of losartan of the same kind (the efficiency slope is 2.451 to 1.584).

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to application of an angiotensin II receptor antagonist in preparation of a medicine for preventing and treating tubular injury caused by triptolide. BACKGROUND

[0002] Tripterygium wilfordii Hook.f. and its preparations such as Tripterygium wilfordii Hook.f. polyglycoside tablets (TWPT) are widely used in the treatment of rheumatoid arthritis, nephrotic syndrome and other immune-related diseases in clinic due to their significant anti-inflammatory and immunosuppressive effects. However, its clinical application is greatly limited by serious toxic side effects, and nephrotoxicity is one of the most common and most serious adverse reactions, with a high incidence and clinical manifestations that can cause acute renal failure. Among them, triptolide (TPL) is the main active and toxic component.

[0003] Although previous studies have extensively explored the nephrotoxicity of Tripterygium wilfordii, there are still the following key deficiencies, which fail to provide a feasible intervention program:

[0004] (1) Lack of upstream targets: Existing studies (Chen, P, Zhong, X, Song, Y, et al. Triptolide induces apoptosis and cytoprotective autophagy by ROS accumulation via directly targeting peroxiredoxin 2 in gastric cancer cells. Cancer Lett. 2024; 587 216622. doi: 10.1016 / j.canlet.2024.216622; Liu, X, Zhao, P, Wang, X, et al. Triptolide Induces Glioma Cell Autophagy and Apoptosis via Upregulating the ROS / JNK and Downregulating the Akt / mTOR Signaling Pathways. Front Oncol. 2019; 9 387. doi: 10.3389 / fonc.2019.00387; Zhou, J, Li, S, Yang, Y, et al. Triptolide alleviates acute lung injury by reducing mitochondrial dysfunction mediated ferroptosis through the STAT3 / p53 pathway. Free Radic Biol Med. 2025; 230 79-94. doi: 10.1016 / j.freeradbiomed.2025.02.001; Wu, H, Cao, P, Wang, H, et al. Postoperative Injection of a Triptolide-Preloaded Hydrogel Prevents the Recurrence of Glioblastoma by Dual-Pathway Activation of Ferroptosis. Small. 2024; 20 (50): e2406036. doi: 10.1002 / smll.202406036) Although involving multiple cell injury mechanisms such as oxidative stress, autophagy, ferroptosis, etc., these mechanisms are common to various toxicants, and no tissue and cell specificity of TPL nephrotoxicity has been found, and no "upstream key molecular target protein" or starting pathway specific to TPL-mediated cell injury has been found.

[0005] (2) Limited clinical drug guidance: Existing studies (Zhang, Zhao, Sun. Leigongteng induces acute renal injury in rats. Heat shock protein 70 expression in the kidney [J]. Chinese disability medicine, 2007, (02): 4-6. Pablo Kizelsztein, Slavko Komarnytsky, Ilya Raskin, Oral administration of triptolide ameliorates the clinical signs of experimental autoimmune encephalomyelitis (EAE) by induction of HSP70 and stabilization of NF-κB / IκBα transcriptional complex, Journal of Neuroimmunology, Volume 217, Issues 1-2, 2009, Pages 28-37.) have found that Leigongteng induces acute renal injury, and the expression of stress proteins such as heat shock protein 70 (HSP70) is up-regulated, but these are non-specific compensatory responses of the body to injury; and it is known that there are many subtypes of HSP70 protein, and previous studies have not been able to determine the specific subtype. Therefore, these existing technologies have not revealed the direct starting mechanism of TPL renal tubular toxicity, and the guidance value for clinical drug intervention is limited.

[0006] In summary, the existing technology has not been able to develop safe and effective protective drugs or combination drug regimens based on the tissue and cell specificity of TPL nephrotoxicity. Clinically, it is still impossible to effectively avoid or reduce the renal damage caused by TPL, which greatly limits the clinical value of Leigongteng preparations.

[0007] The lack of TPL-specific target protein-mediated cell injury mechanisms and clinical drug intervention strategies has resulted in patients always facing a high risk of renal function impairment when receiving Leigongteng treatment, and doctors cannot achieve the purpose of "synergistic and reducing toxicity" through reasonable drug combination. Therefore, there is an urgent need for a safe and effective targeted intervention means to prevent, reduce or treat TPL and related preparations induced specific cell injury. SUMMARY

[0008] The present application finds that the renal toxicity of TPL has high tissue and cell specificity, which mainly targets renal tubular epithelial cells, leading to serious cell damage such as apoptosis and necrosis. The present application further provides a new use of angiotensin II receptor antagonists (ARB) in preventing, reducing or treating the damage to target renal tubular epithelial cells or renal toxicity caused by TPL and related preparations.

[0009] The present application specifically adopts the following technical solutions:

[0010] In a first aspect, the present application provides an application of angiotensin II receptor antagonists in the preparation of a medicine for preventing and treating the renal tubular damage caused by triptolide.

[0011] The cell-level pharmacodynamic verification of the present application establishes the new use of angiotensin II receptor antagonists in resisting the renal tubular toxicity of TPL, and further clarifies that irbesartan has the best protective efficacy and dosage advantage. Therefore, in a further scheme, the angiotensin II receptor antagonist is a sartan drug. Preferably, the sartan drug is at least one of irbesartan, losartan and olmesartan. More preferably, the sartan drug is irbesartan.

[0012] In a second aspect, the present application provides a medicine for preventing and treating the renal tubular damage caused by triptolide, wherein the active ingredient in the medicine is an angiotensin II receptor antagonist.

[0013] In a further scheme, the angiotensin II receptor antagonist is a sartan drug. Preferably, the sartan drug is at least one of irbesartan, losartan and olmesartan. More preferably, the sartan drug is irbesartan.

[0014] The present application has the following beneficial effects:

[0015] (1) The present application first determines through experiments that the upstream key target protein for TPL-induced renal tubular damage is HSPA1, solving the problem that the key target of TPL toxicity is not clear in the prior art.

[0016] (2) The present application establishes the effective protective effect and superiority of sartan drugs: based on the key target of TPL toxicity, the present application establishes the common protective effect of angiotensin II receptor antagonists against TPL toxicity through molecular screening and parallel pharmacodynamic verification of irbesartan, losartan, olmesartan and valsartan and other sartan drugs. Among them, irbesartan as a preferred embodiment has a significantly better protective effect than the similar drug losartan (efficiency slope 2.451 vs. 1.584).

[0017] (3) The present application proves the target point dependence of the protective effect of angiotensin II receptor antagonists: the results of two-factor variance analysis show that the protective effect of irbesartan has clear HSPA1 target point dependence (interaction P = 0.0368). This statistical evidence strongly supports the mechanism innovation and credibility of the present application, that is, angiotensin II receptor antagonists exert protective effects by affecting the HSPA1 target point.

[0018] (4) The present application provides significant clinical application value: the present application provides a mechanism-specific and superior ARB combination strategy for clinical use, which is expected to be developed into an effective prevention and treatment drug for targeting nephrotoxicity of tripterygium, greatly improving the safety of tripterygium preparations, and has great clinical application potential and social value.

[0019] In summary, the present application clearly proves the protective effect of angiotensin II receptor antagonists on triptolide targeting nephrotoxicity and its dependence on the HSPA1 target point; and by using angiotensin II receptor antagonists, the toxic side effects of TPL are effectively reduced, and the safety of its clinical use is improved, so that the therapeutic effect of tripterygium preparations can be more fully exerted.

[0020] Figure 1 : Mouse body weight change graph (A) and mouse kidney index statistics (B).

[0021] Figure 2 : Kidney pathological section results (A) and TNS score results (B, C, D, E).

[0022] Figure 3 : Spatial metabolomics research results (n = 3). Note: A, kidney HE staining graph (upper for CON group; lower for TPL group); B, UMAP clustering cortex medulla graph; C, UMAP obtains PLS-DA score graph between groups in the cortex area; D, UMAP obtains significant difference metabolites volcano graph between groups in the cortex area; E, significant difference enrichment bubble graph of the cortex area, the horizontal coordinate is the Impact value enriched into different metabolic pathways, and the vertical coordinate is the -log10 (P value) value. The color is related to the P value, the darker the color, the smaller the P value, the lighter the color, the larger the P value. The closer the pathway is to the upper right corner, the more significant the enrichment of the differential metabolites in the pathway, and the greater the influence on the pathway.

[0023] Figure 4: Quantification of renal tubular injury proteins SKYLINE (n=10). Note: A, quantification of relative abundance of Lcn2 (NGAL), a marker of renal tubular injury. The results showed that the expression of Lcn2 in TPL group was significantly higher than that in CON group (*P <0.05). B, quantification of relative abundance of Clu (CYTC), a marker of renal tubular injury. The results showed that the expression of Clu in TPL group was significantly higher than that in CON group (*P <0.05). C, quantification of relative abundance of Havcr 1 (KIM-1), a marker of renal tubular injury. The results showed that the expression of Havcr1 in TPL group was significantly higher than that in CON group (*P <0.05).

[0024] Figure 5 : TPL drug concentration-cell inhibition rate (A) and survival rate (B).

[0025] Figure 6 : TPL promotes apoptosis of HK-2. Note: A, flow cytometry assay HK-2 cell apoptosis four quadrant diagram; B, flow cytometry assay HK-2 cell apoptosis quantitative diagram.

[0026] Figure 7 : TPL causes HK-2 cells to increase the level of ROS. Note: A, flow cytometry assay HK-2 cell oxidative stress peak diagram; B, flow cytometry assay HK-2 cell oxidative stress quantitative diagram.

[0027] Figure 8 : TPL causes HK-2 cells (A) and mouse kidney tissue MDA levels to rise (B).

[0028] Figure 9 : TPL causes HK-2 cells to increase the level of ferrous ion. Note: A, flow cytometry assay HK-2 cell ferrous ion content peak diagram; B, flow cytometry assay HK-2 cell ferrous ion quantitative diagram.

[0029] Figure 10 : TPL causes HK-2 cells (A) and mouse kidney tissue GSH levels to decrease (B).

[0030] Figure 11 : Volcano plot of DEPs in kidney tissue of model group and control group (A), biological processes in GO analysis (B), cellular components (C) and molecular functions (D).

[0031] Figure 12 : Cluster heat map of DEPs in kidney tissue of model group and control group.

[0032] Figure 13: Volcano plot (A), clustering heat map (B), GO analysis (C) and KEGG analysis (D) of DEPs of model group and control group cells.

[0033] Figure 14 : Target protein of TPL acting on HK-2 cells based on thermal proteomics. Note: A, thermal shift volcano plot of model group and control group cells; B: temperature-content curve change graph of target protein.

[0034] Figure 15 : Verification of target protein of TPL acting on HK-2 cells. Note: A, CETSA-WB result graph; B: molecular docking result graph; C: surface plasmon resonance response-concentration curve; D: surface plasmon resonance dissociation constant.

[0035] Figure 16 : HSPA1A knockdown shows that triptolide inhibits the proliferation of HK-2 cells. Note: A, PCR results of HSPA1A gene knockdown effect; B: WB results; C: comparison of CCK8 results of TPL toxicity before and after HSPA1A knockdown.

[0036] Figure 17 : Validation of protective effect of sartan drugs on TPL nephrotoxicity.

[0037] Figure 18 : HSPA1 silencing significantly reduces the protective effect of irbesartan on TPL nephrotoxicity. DETAILED DESCRIPTION

[0038] In order to make the objectives, technical solutions and advantages of the present application clearer, the technical solutions in the embodiments of the present application will be clearly described below. Obviously, the described embodiments are a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0039] Some reagents used in the embodiments are as follows:

[0040] Methanol (Thermo Fisher Scientific, USA), mass spectrometry grade acetonitrile (Thermo Fisher Scientific, USA), formic acid (Shanghai Aladdin Bio-Chem Technology Co., Ltd.), triptolide (Med Chem Express, China), chromatographic grade methanol (Thermo Fisher Scientific, USA), chromatographic grade acetonitrile (Thermo Fisher Scientific, USA), mass spectrometry grade acetonitrile (Thermo Fisher Scientific, USA), formic acid (Shanghai Aladdin Bio-Chem Technology Co., Ltd.), mass spectrometry grade water (Thermo Fisher Scientific, USA), iodoacetamide (IAM, Sigma), ammonium bicarbonate (ABC, Sigma), DL-dithiothreitol (DDT, Sigma), sequencing grade modified trypsin (Promega), guanidine hydrochloride (Sigma), Precal Mix (AB Sciex, USA).

[0041] DMEM high-sugar medium (Beijing Solabio Science and Technology Co., Ltd.), GSH detection kit (Beijing Solabio Science and Technology Co., Ltd.), CCK-8 kit (APExBIO Technology LLC, USA), MDA detection kit (Beijing Solabio Science and Technology Co., Ltd.), DMSO (Beijing Solabio Science and Technology Co., Ltd.), ROS detection kit (Nanjing Jiancheng Biological Engineering Institute), Annexin V-APC / PI apoptosis detection kit (Jiangsu KeyGen Biotech Co., Ltd.), divalent iron ion detection probe-FerroOrange (TOKYO CHEMICAL INDUSTRY CO., LTD.).

[0042] Example 1

[0043] I. Determination of the targeting and key mechanism of TPL nephrotoxicity

[0044] 1.1. Establishment and functional verification of TPL nephrotoxicity model

[0045] Twenty SPF-grade male C57BL / 6 mice (purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd., production license number: SCXK(Zhe)2019-0001), 6 weeks old. All mice were adaptively raised in a constant temperature and humidity environment for 2 weeks and randomly divided into a control group (CON) and a model group (TPL) according to body weight, with 10 mice in each group. The mice were fed with SPF-grade breeding feed (Huanyu Biology, GB13078), and the drinking water was tap water; each mouse in the model group was intragastrically administered 0.1 mL of TPL solution (0.5 mg / kg, that is, 0.5 mg of TPL was given per kg of mice) every day, and each mouse in the control group was intragastrically administered 0.1 mL of normal saline. The model was established continuously for 4 weeks, and the body weight changes were recorded every two days. At the end of the experiment, blood, urine and kidneys were collected under anesthesia. Some renal tissues were fixed with 4% paraformaldehyde, and the remaining renal tissues were stored in liquid nitrogen. The TPL solution was prepared by dissolving TPL in normal saline.

[0046] The collected kidneys were sectioned and embedded in paraffin. Subsequently, the sections were processed using hematoxylin and eosin (HE) staining and observed and evaluated under an optical microscope. The degree of renal tubular injury was mainly evaluated by the total nephropathy score (TNS).

[0047] The results showed that compared with the control group, the weight of the mice in the model group decreased significantly ( Figure 1 A), and the kidney index increased significantly ( Figure 1 B).

[0048] The pathological sections of the kidneys showed that the renal tissue structure of the mice in the control group was intact, the boundary between the cortex and the medulla was clearly visible, the glomerular capsule cavity was clear, no degeneration or necrosis was observed in the renal tubules, and no inflammatory cell infiltration or other pathological abnormalities were observed in the interstitium. In the mice of the model group, mesangial cell proliferation (P) occurred in the renal tissue. A large number of renal tubular epithelial cell exfoliation (S), renal tubular dilation (D) and significant glomerular atrophy (AG) were visible in the cortical part of the kidney; medullary hemorrhage (H) was visible in the medullary part, and a small amount of inflammatory cell infiltration (I) was present in the interstitium ( Figure 2 A). Through the renal tubular necrosis score, the results showed that TPL caused severe necrosis of the renal tubules ( Figure 2 B, C, D and E).

[0049] 1.2. Spatial metabolomics study of TPL injury points to cortex specificity

[0050] Three kidneys from each of the model group and control group mice were used for spatial metabolomics analysis. Spatial metabolomics data acquisition was performed using AP-MALDI and QE PLUS tandem at a resolution of 50 μm. Various ion spatial imaging maps were generated using Mzkit software, enabling intelligent region selection within the sample spatial area and subsequent functional analysis. Substance identification was performed using databases such as KEGG, HMDB, LipidMaps, and MoNA, excluding non-endogenous or plant-derived data. Ultimately, 689 endogenous metabolites and 389 lipids were identified.

[0051] UMAP was used to perform dimensionality reduction and cluster analysis on high-dimensional mass spectrometry imaging data. Then, the phenograph clustering algorithm was combined to obtain different subtype regions and features on the slices. Regions with similar metabolite expression patterns and features were marked with the same color. Finally, a spatial data filling algorithm was used to obtain segmentation results with high consistency with actual tissue distribution characteristics. Ultimately, spatial clustering identification revealed differential metabolic characteristics in the cortical and medullary regions of mouse kidneys; TPL induced significant metabolic changes in the cortical and medullary regions of mouse kidneys (…). Figure 3 A and 3B). For mice in the control or model groups whose renal cortex and medulla were identified, 32 scanning sites were randomly selected, ion information was extracted, and PLS-DA analysis was performed, indicating that TPL caused significant metabolic changes in the nephrotoxic cortical region. Figure 3 C). The substitution test confirmed that no overfitting was observed, indicating the model's reliability and accuracy. Metabolites showing significant intergroup changes were visualized using a volcano plot. Results showed that spermine, arginine, glutamic acid, histidine, phenylalanine, creatine, and UDP were significantly decreased in the TPL-induced nephrotoxicity group; while isoleucine, S-adenosylmethionine, uridine triphosphate, hydroxyadenine, and sulfoethylglutamine were significantly increased in the nephrotoxicity model group. Figure 3 D). KEGG pathway enrichment analysis was performed on differentially metabolites, and the main metabolic differences were identified as arginine and proline metabolism, protein degradation and absorption, glutamate metabolism, ferroptosis, tryptophan and methionine metabolism, and arachidonic acid metabolism. Figure 3 E). The above results suggest a differential metabolic mechanism between the cortex and medulla.

[0052] 1.3 Skyling quantification of TPL injury is specific to renal tubular injury.

[0053] To further validate the specific damage to renal tubular epithelial cells (RTEC) caused by triptolide (TPL) at the molecular level, this invention integrates quantitative proteomics (SWATH-MS) analysis data to accurately quantify key renal tubular injury biomarkers. Using Skyline software, this invention extracted specific protein marker peptides corresponding to the recognized renal tubular injury biomarkers—Havcr1 (KIM-1), Lcn2 (NGAL), and Clu (CYTC)—from SWATH-MS data, and quantified their relative abundance based on the total intensity of their fragment ions (MS2 conversion).

[0054] Quantitative analysis results showed that the relative abundance of three key renal tubular injury markers, Lcn2, Clu, and Havcr1, was significantly increased in the TPL-treated group compared to the normal control group (CON) (p<0.05). Figure 4 These molecular-level quantitative results strongly confirm that TPL-induced nephrotoxicity is a specific toxicity primarily characterized by renal tubular epithelial damage, providing solid molecular evidence for subsequent identification of TPL targets and screening of targeted protective agents.

[0055] 1.4. Phenotypic Validation of Human Renal Tubular Epithelial Cells with TPL Injury

[0056] (1) Cell proliferation

[0057] TPL was prepared as a stock solution using DMSO and diluted to different concentrations (0, 6.25, 12.5, 25, 50, 100, 200, 400 nM). 10,000 HK-2 cells were seeded into each well of a 96-well plate, and 100 μL of DMEM high-glucose medium containing 10% FBS was added to each well. After 24 h of seeding, 10 μL of different concentrations of TPL were added and incubated for another 24 h. 10 μL of CCK8 reagent was added to each well, and after incubation for 2 h, the absorbance at 450 nm was recorded using a microplate reader to investigate the effect of TPL on HK-2 cell proliferation.

[0058] The results showed that, compared with the control group (0 nM TPL), 6.25-400 nM TPL had an inhibitory effect on HK-2 cells, and the IC50 of HK-2 cells was significantly lower. 50 It is 46.67 nM, see Figure 5 .

[0059] (2) Apoptosis

[0060] TPL was prepared into a stock solution using DMSO and diluted to different concentrations (0, 50, 200 nM). 5 × 10⁶ cells were inoculated into each well of a 6-well plate. 5HK-2 cells were cultured in 2 mL of DMEM high-glucose medium containing 10% FBS in each well. After 24 h of plating, 100 μL of different concentrations of TPL were added and incubated for another 24 h. Then, apoptosis assays were performed.

[0061] First, transfer the old culture medium to two new 5 mL EP tubes using a pipette. Then, wash the cells with 500 μL of pre-chilled PBS, adding the washing solution to the same EP tube. Digest the cells with EDTA-free trypsin, stopping the digestion with the culture medium collected in the EP tube. After rehydration, collect the cells into the EP tubes. Centrifuge and discard the supernatant, then wash with pre-chilled PBS and centrifuge again, discarding the supernatant. Add 500 μL Binding Buffer, 5 μL PI, and 5 μL Annexin-V-APC to each tube, and incubate at room temperature in the dark for 10 min. Flow cytometry was used to detect the effect of TPL on HK-2 cell apoptosis within 1 h. The results showed that under TPL treatment conditions, compared with the control group (0 nM TPL), the apoptosis rate of HK-2 cells in the model groups (50 and 200 nM) was significantly increased. Figure 6 Furthermore, the induction of apoptosis by TPL increases in a dose-dependent manner.

[0062] (3) ROS level

[0063] Cell culture and treatment were the same as in the apoptosis experiment. After 24 h of incubation, cells were digested and collected into centrifuge tubes. After centrifugation and discarding the supernatant, cells were washed twice with pre-cooled PBS. Then, 500 μL of diluted DCFH-DA probe solution (DCFH-DA diluted 1:5000 with serum-free DMEM high-glucose medium) was added to each centrifuge tube. The cells were incubated at 37℃ for 20 min, inverting and mixing every 3-5 min to ensure sufficient contact between the probe and cells. After incubation, the DCFH-DA solution was discarded, and the cells were washed three times with serum-free culture medium to thoroughly remove any DCFH-DA that had not entered the cells. Then, 1 mL of PBS buffer was added, and flow cytometry was used to detect changes in intracellular ROS levels in HK-2 cells under TPL treatment. The results showed that with increasing TPL concentration, compared with the control group (0 nM TPL), the intracellular ROS levels in the model groups (50, 200 nM TPL) of HK-2 cells increased in a dose-dependent manner. Figure 7 ).

[0064] (4) MDA level

[0065] Cell culture and treatment were the same as in the apoptosis experiment. After incubation for 24 h, cells were digested and collected, lysed, and protein concentration was measured. MDA detection solution was mixed with protein and heated in a metal bath at 100℃ for 60 min. The supernatant was collected by centrifugation. 200 μL of the sample was added to each well of a 96-well plate, and the absorbance of each sample was measured at 532 nm and 600 nm. The results showed that in the model groups (50 and 200 nM TPL), the MDA level in HK-2 cells increased in a dose-dependent manner. Figure 8 A).

[0066] In addition, we also measured the MDA level in mouse kidney tissue preserved in liquid nitrogen, as described in the section on "Establishment and Functional Validation of the TPL Nephrotoxicity Model". The results also showed a significant increase in MDA levels in the kidneys of the model group mice. Figure 8 B).

[0067] (5) Ferrous ion level

[0068] Cell culture and treatment were the same as in the apoptosis experiment. After incubation for 24 h, the culture medium was removed, and the cells were washed three times with HBSS. FerroOrange working solution was then added, and the cells were cultured in an incubator for 30 min. Flow cytometry was then used to detect the effect of TPL treatment on the intracellular ferrous ion concentration in HK-2 cells. The results showed that in the model groups (50 and 200 nM), the intracellular ferrous ion level in HK-2 cells increased in a dose-dependent manner. Figure 9 ).

[0069] (6) GSH level

[0070] Cell culture and treatment were the same as in the apoptosis experiment. After 24 h of incubation, the effect of TPL on GSH levels in HK-2 cells was detected using a glutathione content assay kit. The results showed that, compared with the control group (0 nM TPL), the GSH levels in HK-2 cells of the model groups (50 and 200 nM TPL) decreased in a dose-dependent manner. Figure 10 A).

[0071] We also measured GSH levels in mouse kidney tissue preserved in liquid nitrogen, as described in the section on "Establishment and Functional Validation of the TPL Nephrotoxicity Model". The results similarly showed that GSH levels in the kidneys of the model group mice were significantly decreased. Figure 10 B), consistent with the results of cell experiments.

[0072] II. Identifying TPL target proteins and their pathways of action

[0073] 2.1 Proteomics elucidates the mechanism of ferroptosis leading to nephrotoxicity

[0074] (1) Renal proteomics

[0075] Fresh kidney tissue of 50 mg from control group (CON) and model group (TPL) mice was taken, ground and lysed, and the protein concentration was measured. 250 mg of protein solution was taken for enzymatic digestion, and dithiothreitol (DTT) and iodoacetamide (IAM) were added for reduction and alkylation. After digestion with trypsin at 37°C for 18 h, formic acid with a final concentration of 0.1% was added to terminate the enzyme digestion reaction and obtain the sample peptide.

[0076] This embodiment uses an Eksigent MicroLC 400 system in tandem with a TripleTOF 6600 system (AB Sciex, USA) for SWATH proteomics analysis. First, sample peptides were loaded onto a Trap column (5 μm, ChromXP C18CL, 120 Å, 10 × 0.3 mm) (AB Sciex, USA) at a flow rate of 10 μL / min for 5 min. Then, elution was performed on a chromatographic column (3 μm, ChromXP C18CL, 120 Å, 150 × 0.3 mm) (AB Sciex, USA) using two mobile phases: Mobile phase A consisted of 2% acetonitrile, 98% water, and 0.1% formic acid; Mobile phase B consisted of 98% acetonitrile, 2% water, and 0.1% formic acid. The autosampler and column oven temperatures were maintained at 8°C and 40°C, respectively. A microfluidic gradient method was used for 60 min, and the elution gradients are shown in Table 1. The injection volume was 8 μL and the flow rate was 5 μL / min.

[0077] Table 1. Proteomics Elution Gradients

[0078]

[0079] Renal proteomics results identified a total of 3847 proteins. After screening, 111 DEPs were obtained, and the volcano plot showed that 35 DEPs were upregulated and 76 DEPs were downregulated. Figure 11 A). Gene Ontology (GO) analysis showed that 106 of the 111 DEPs were related to metabolic processes. Figure 11 B, C, D).

[0080] Cluster heatmap of DEPs between control and model groups is as follows Figure 12 As shown.

[0081] (2) Cell proteomics

[0082] TPL was prepared into a stock solution using DMSO and diluted to different concentrations (0, 50, 200 nM). 5 × 10⁶ cells were inoculated into each well of a 6-well plate. 5HK-2 cells were cultured in DMEM high-glucose medium containing 10% FBS in each well for 24 hours. After plating, 100 μL of different concentrations of TPL were added and incubated for another 24 hours. The HK-2 cells were then lysed and digested with enzymes, followed by mass spectrometry analysis. The results showed the identification of 3163 proteins. Proteomics analysis identified 200 DEPs, of which 85 were upregulated and 115 were downregulated. (See [link to table]). Figure 13 A. Clustering heatmap of DEPs between the two groups is shown below. Figure 13 B. GO functional annotation and KEGG enrichment analysis were performed on the above DEPs. Among the main pathways identified, the main pathways associated with kidney injury were ferroptosis and necroptosis, etc. (See below) Figure 13 C and 13 D.

[0083] 2.2 Target Screening and Validation of TPL Renal Tubular Toxicity

[0084] (1) Thermal proteomics

[0085] TPL was prepared into a stock solution using DMSO and diluted to different concentrations (0, 50 nM). HK-2 cell lysates were incubated with equal volumes of different concentrations of TPL at room temperature for 30 min. After incubation, the cell lysates were divided into 7 aliquots and subjected to temperature gradients of 37, 42, 47, 52, 57, 62, and 67 °C for 3 min each. The supernatant was then digested enzymatically, and mass spectrometry analysis was performed to screen target proteins and plot thermal curves of the target proteins. The results showed that 605 target proteins were detected, with 18 proteins significantly upregulated and 7 proteins downregulated. (See [link to table]). Figure 14 A. Clinical studies have shown that HSP70 exerts a renal protective effect through regulatory T cells (TREG), therefore, the target proteins HSPA1A / HSPA1B (hereinafter referred to as HSPA1) were identified through screening. They are members of the HSP70 family. Figure 14 B is its hot melt curve.

[0086] (2) CETSA-WB

[0087] Cell lysates were incubated with TPL and then heat-treated using the same method as in "thermal proteomics". Western blot analysis was used to detect and compare the degradation of HSP70 protein after heat treatment following drug incubation. Results showed that, compared to the control group (0 nM TPL), the stability of HSP70 protein in the model groups (50 and 200 nM TPL) increased after binding to TPL. Figure 15 A.

[0088] (3) Molecular docking

[0089] Molecular docking of the HSP70 receptor with ligand TPL was performed using AutoDock Vina, and the results were analyzed. The results showed that triptolide bound to the target protein molecules with HSPA1A at a rate of -10 kcal / mol and with HSPA1B at a rate of -7.7 kcal / mol, demonstrating that HSPA1A has a good binding effect with triptolide. (See attached image) Figure 15 B.

[0090] (4) Surface Plasmon Resonance (SPR) experiment

[0091] Surface plasmon resonance (SPR) analysis revealed that HSP70 immobilized on the CM5 chip can bind to triptolide, with an affinity constant of 2.11 × 10⁻⁶. -4 M, see Figure 15 C, D.

[0092] (5) Target silencing verification

[0093] The effect of silencing HSP70 gene expression on the inhibitory effect of triptolide on HK-2 cells was detected using CCK8 assay. Specifically, HK-2 cells were plated for 24 hours, and then 200 μL of opti-MEM and siRNA (MCE small interfering RNA fragment (catalog number HY-RS06424)) were added as transfection solution. After 24 hours, cells were harvested for PCR experiments to detect the knockdown effect of mRNA levels, and after 48 hours, samples were harvested to detect the knockdown effect of protein levels. The transfected cells were then re-plated and drug-treated according to the previous CCK8 procedure.

[0094] The results showed that, compared with the control group, HSP70 silencing increased the sensitivity of HK-2 cells to TPL and enhanced TPL toxicity. Figure 16 C. Figure 16 A and B represent the knockdown effects of the HSP70 gene at the mRNA and protein levels, respectively.

[0095] III. Target-Based Protectant Screening and Validation: Virtual Screening

[0096] 3.1 Target Screening for TPL Renal Tubular Toxicity

[0097] The preceding content first identified HSPA1A (Heat Shock Protein A1A) as the key upstream target protein for renal tubular injury induced by triptolide (TPL). Based on this, to efficiently and precisely develop targeted protective agents, this embodiment employed structural bioinformatics and virtual screening technology to systematically screen thousands of marketed or potential drug molecules to evaluate their binding affinity to HSPA1A. After initial screening, this embodiment focused on the top 50–100 candidate drugs with the optimal binding energy to HSPA1A for refined molecular docking and energy assessment, ultimately successfully identifying the angiotensin II receptor antagonist, irbesartan. Irbesartan exhibits extremely high affinity for HSPA1A in both binding energy and binding mode. To further verify the advantages of irbesartan as a protective agent, this embodiment also conducted molecular docking comparison experiments with the similar drug, losartan.

[0098] Docking experiments clearly showed that irbesartan's binding energy to HSPA1A was significantly superior to that of losartan (Table 2). This result not only provides a theoretical basis for irbesartan's superiority in combating TPL tubular toxicity at the molecular structure level, but also strongly supports the novel use of irbesartan in this invention as a drug for the prevention, mitigation, or treatment of TPL tubular injury.

[0099] Table 2. Virtual screening results of the protective effects of sartan drugs

[0100]

[0101] 3.2 Verification of the protective effect of sartan drugs against TPL renal tubular injury

[0102] This embodiment also validated the pharmacodynamic effects of angiotensin II receptor antagonists (ARBs) identified through molecular screening in a human renal tubular epithelial cell (HK-2 cell) model. The experiment involved treating four representative angiotensin II receptor antagonists—irbesartan, losartan, olmesartan, and valsartan—with concentration gradients from 0 to 100 μM to evaluate their protective effects against TPL (100 nM)-induced HK-2 cell cytotoxicity. Specifically, HK-2 cells were seeded in 96-well plates, and 5 μL of different concentrations (0, 1, 2.5, 5, 10, 20, 50, 100 μM) of angiotensin II receptor antagonists and 5 μL of TPL (100 nM) were added simultaneously 24 h after drug administration. CCK8 assays were performed 24 h after drug administration.

[0103] Verification results (such as) Figure 17As shown in the figure, this strongly demonstrates that sartans have a common protective effect against TPL tubular toxicity: irbesartan, losartan, and olmesartan all showed a clear dose-dependent protective trend; although valsartan's protective effect was relatively weaker, it still showed a trend of alleviating toxicity, establishing the potential of sartans as a class of drugs in combating TPL tubular damage. Among the tested drugs, irbesartan's protective effect was particularly outstanding (linear fit Slope = 2.451, significance P < 0.0001), and its efficacy was significantly better than losartan (losartan Slope = 1.584).

[0104] In summary, the pharmacodynamic validation at the cellular level not only established a new use for angiotensin II receptor antagonists to combat TPL tubular toxicity, but also clarified that irbesartan has the best protective efficacy and dosage advantage, providing direct experimental support for the clinical selection of this drug or this class of drugs as a protective agent against TPL tubular damage.

[0105] 3.3 Irbesartan's protective effect against TPL tubular injury is dependent on the target protein HSPA1(A).

[0106] The aforementioned pharmacodynamic validation has established that angiotensin II receptor antagonists (ARBs) have a common protective effect against TPL tubular toxicity, with irbesartan exhibiting the best protective efficacy. To further confirm the protective effect of ARBs and their dependence on the target protein HSPA1A at the molecular mechanism level, this embodiment selects irbesartan as the optimal embodiment for in-depth mechanism validation. The study is based on silencing HSPA1 protein at the HK-2 cell level, observing the changes in the protective effect of irbesartan against TPL (100 nM) cytotoxicity before and after target protein silencing. Specifically, HK-2 cells before and after transfection were seeded in 96-well plates. After 24 hours of seeding, 5 μL of different concentrations of irbesartan and 5 μL of TPL (100 nM) were added simultaneously. CCK8 assay was performed 24 hours after drug addition. The transfection method was described in the "Target Silencer Validation" section.

[0107] The results showed that silencing the HSPA1A target protein significantly reduced the protective effect of irbesartan against TPL-induced HK-2 cytotoxicity (two-way ANOVA, P < 0.05). This study found that the protective effect of irbesartan against TPL is dependent on the TPL target protein HSPA1A, suggesting a possible mechanism of target protein competition between the two. Figure 18 ).

[0108] This embodiment utilizes two-way ANOVA to assess the interaction between "target gene silencing status" and "irbesartan concentration" on HK-2 cell survival, statistically confirming the target-dependent protective effect of irbesartan. The ANOVA analysis results (Table 3) show that both the group main effect and the concentration main effect were significant (C(Group) P≈1.6×10⁻⁶). -12 ; C(Concentration) P ≈ 1.8 ×10 -6 This demonstrates that target gene silencing itself has a significant impact on cell state, and that irbesartan, as a treatment factor, can independently exert a protective effect. The core interaction was significant: the most critical interaction term, C(Group):C(Concentration), reached statistical significance (P = 0.0368 < 0.05). Based on the above, it can be concluded that the significant interaction effect strongly confirms that the protective effect of irbesartan is not independent of the target gene state, but significantly depends on the presence of the target gene. Silencing this target gene (HSPA1A) significantly weakened (attenuated) the protective effect of irbesartan against TPL nephrotoxicity. This ANOVA result provides crucial statistical evidence, clearly indicating that HSPA1A plays a key role in the mechanism pathway by which irbesartan exerts its protective effect, and is an important support for the core hypothesis of this invention.

[0109] Table 3. Results of Two-Way Analysis of Variance

[0110]

[0111] Angiotensin II receptor blockers (ARBs), including irbesartan, losartan, olmesartan, and valsartan, are commonly used in clinical practice. These drugs have been reported to have protective effects against chronic kidney diseases such as hypertension and diabetic nephropathy. However, TPL-induced nephrotoxicity is an acute, tubular-targeting drug toxicity, and its pathophysiological mechanism is fundamentally different from that of chronic metabolic kidney injury (such as diabetic nephropathy). Therefore, the protective effect of ARBs against chronic kidney disease in the prior art cannot directly predict their intervention effect on TPL acute nephrotoxicity. This invention, based on parallel studies of various sartan drugs such as irbesartan, olmesartan, valsartan, and losartan, aims to discover and demonstrate for the first time that angiotensin II receptor blockers (ARBs) can specifically counteract TPL-induced tubular damage, thus providing a novel intervention approach to address this acute drug toxicity.

[0112] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of angiotensin II receptor antagonists in the preparation of drugs for preventing and treating renal tubular damage induced by triptolide.

2. The application of the angiotensin II receptor antagonist according to claim 1 in the preparation of a drug for preventing and treating renal tubular injury induced by triptolide, characterized in that, The angiotensin II receptor antagonist is an angiotensin II class of drugs.

3. The application of the angiotensin II receptor antagonist according to claim 2 in the preparation of a drug for preventing and treating renal tubular injury induced by triptolide, characterized in that, The sartan drug is at least one of irbesartan, losartan, and olmesartan.

4. The application of the angiotensin II receptor antagonist according to claim 2 in the preparation of a drug for preventing and treating renal tubular injury induced by triptolide, characterized in that, The sartan drug in question is irbesartan.

5. A drug for preventing and treating renal tubular damage induced by triptolide, characterized in that, The active ingredient in the drug is an angiotensin II receptor antagonist.

6. The drug for preventing renal tubular damage induced by triptolide according to claim 5, characterized in that, The angiotensin II receptor antagonist mentioned is an angiotensin II receptor blocker (ARB).

7. The drug for preventing renal tubular damage induced by triptolide according to claim 6, characterized in that, The sartan drug is at least one of irbesartan, losartan, and olmesartan.

8. A drug for preventing renal tubular damage induced by triptolide according to claim 6, characterized in that, The sartan drug in question is irbesartan.

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