Application of clopidogrel in preparation of medicine for preventing renal ischemia reperfusion injury

Abstract

The invention relates to an application of clopidogrel in preparation of a medicine for preventing renal ischemia-reperfusion injury. The medicine is administrated 2 hours ahead of time, so that mouse kidney IRI can be prevented, and cell ferroptosis is inhibited through an FSP1 way. In-vitro cell experiments show that in various cell lines such as HT1080, HK2 and the like, clopidogrel can inhibit ferroptosis induced by ferroptosis inducers (such as RSL3, IKE, FIN56 or JKE-1674), and cell lipid peroxidation and intracellular Fe < 2 + > content increase caused by ferroptosis can be partially recovered. The clopidogrel disclosed by the invention has an obvious effect of inhibiting cell ferroptosis and is beneficial to prevention of renal ischemia reperfusion injury.

Description

Technical Field

[0001] The invention belongs to the field of medicine, and particularly relates to the application of clopidogrel as a ferroptosis inhibitor to prevent renal ischemia-reperfusion injury. Background Art

[0002] Renal ischemia-reperfusion injury (IRI) is the main cause of acute kidney injury and an important clinical problem often encountered during surgery, organ transplantation, and severe hypotension. About 30% to 50% of kidney transplant recipients experience IRI. Renal IRI is characterized by inflammation, oxidative stress, and cell death. There are currently no effective preventive measures. Therefore, new strategies are needed, such as small molecule drugs and the addition of a third drug to improve the remission rate. Discovering new uses for preventing renal IRI from existing clinically used drugs can shorten the drug promotion cycle and is of great significance.

[0003] Recent studies have found that ferroptosis (an iron-dependent, oxidative form of regulated cell death) is an important pathological mechanism of renal IRI. During renal IRI, excess iron accumulates in renal cells, catalyzing the production of reactive oxygen species (ROS), leading to lipid peroxidation and cell damage, which is a hallmark event of ferroptosis. Therefore, targeting the ferroptosis pathway may be a powerful therapeutic strategy to reduce renal injury and enhance recovery after IRI, providing ideas for more effective kidney protection interventions. Ferroptosis is intricately controlled by a variety of mechanisms that protect cells from oxidative damage. The core of this regulation is key antioxidant proteins, such as FSP1. Research on molecular mechanisms provides potential therapeutic targets for addressing ferroptosis-related diseases.

[0004] Clopidogrel (CPD) is an antiplatelet drug that belongs to the thienopyridine class and is primarily used to prevent thrombotic events in patients with cardiovascular disease. It works by irreversibly inhibiting the P2Y12 receptor on platelets, thereby reducing platelet activation and aggregation. Clopidogrel is commonly used in patients with acute coronary syndrome, patients undergoing percutaneous coronary intervention, and patients with peripheral artery disease. Recent studies have also highlighted its antioxidant properties, indicating that clopidogrel can reduce oxidative stress by reducing ROS levels and increasing antioxidant enzyme activity, which may help improve vascular function, and its antioxidant function has potential value in preventing renal IRI.

[0005] Clopidogrel is a second-generation thienopyridine drug. It was approved for marketing in 1998 after strict placebo-controlled clinical trials (DIG) evaluation and was launched in my country in 2001. Due to its good performance in pharmacodynamics and pharmacokinetics, clopidogrel has become a classic antithrombotic drug that is still used today. The molecular formula of clopidogrel is C16H16 ClNO2S, and the molecular weight is 321.82. Current studies have found that in addition to preventing thrombosis, other pharmacological effects and related molecular mechanisms of clopidogrel are still being explored in depth. We found that clopidogrel can prevent renal ischemia-reperfusion injury by inhibiting cell ferroptosis, and this result has not been reported at home or abroad. It has good application prospects. Summary of the invention

[0006] The purpose of the present invention is to provide an application of clopidogrel in the preparation of a drug for preventing renal ischemia-reperfusion injury. As a ferroptosis inhibitor, clopidogrel can inhibit ferroptosis through the FSP1 pathway and alleviate renal ischemia-reperfusion injury.

[0007] The technical solution of the present invention is:

[0008] Application of clopidogrel in the preparation of drugs for preventing renal ischemia-reperfusion injury.

[0009] The damage is ferroptosis caused by lipid peroxidation and cell damage.

[0010] The drug is an inhibitor.

[0011] The inhibitor inhibits ferroptosis in multiple cell lines.

[0012] The plurality of cells are HT1080, HK2, 786O, U937, BXPC3, CAOV3, RL95-2 or PANC-1.

[0013] The drug inhibits cell ferroptosis via the FSP1 pathway.

[0014] The drug of the present invention can prevent renal IRI in mice by administering it 2 hours in advance, and inhibit cell ferroptosis through the FSP1 pathway. In vitro cell experiments show that clopidogrel can inhibit ferroptosis induced by ferroptosis inducers (such as RSL3, IKE, FIN56 or JKE-1674) in various cell lines such as HT1080 and HK2, and can partially restore cell lipid peroxidation and intracellular Fe caused by ferroptosis. 2+ The clopidogrel of the present invention has a significant effect in inhibiting cell ferroptosis and is beneficial for preventing renal ischemia-reperfusion injury.

[0015] The applicant's experiment verified that clopidogrel administered 2h in advance can prevent renal ischemia-reperfusion injury in C57BL / 6J mice, and clopidogrel can inhibit cell ferroptosis induced by a variety of ferroptosis inducers (RSL3, IKE, FIN56, BSO, Dihydroartemisinin, Artesunate). Clopidogrel has the effect of inhibiting ferroptosis in multiple cell lines (HT1080, HK2, 786O, U937, BXPC3, CAOV3, RL95-2, PANC-1, etc.). Clopidogrel inhibits ferroptosis in RSL3-induced ferroptosis model cell line HT1080 and liver cell line HK2 through FSP1.

[0016] Clopidogrel, Chinese alias: Α-(2-chlorophenyl)-2-(4,5,6,7-tetrahydrothiophene 3,2-C and pyridine-5 (4H) acetate methyl ester hydrogen sulfate; Clopidogrel; Clopidogrel base, English name: Clopidogrel, chemical structure:

[0017]

[0018] Physical and chemical properties: density: 1.317g / cm3, boiling point: 423.7℃ at 760mmHg, flash point: 210℃, commonly used as disulfate, white powder, insoluble in water, but easily soluble in water with a pH of 1. Soluble in methanol.

[0019] Production method: racemic 2-(2-chlorophenyl)glycine (I) is esterified with methanol under the action of hydrogen chloride to obtain racemic 2-(2-chlorophenyl)glycine methyl ester (II), which is then split with (+)-tartaric acid to obtain (+)-(II). It is then alkylated with 2-(2-bromoethyl)thiophene in methyl acetate in the presence of dipotassium hydrogen phosphate; or alkylated with 2-[2-(phenylsulfonyloxy)ethyl]thiophene in methyl acetate in the presence of sodium bicarbonate to obtain compound (III). Finally, it is cyclized with formic acid and formaldehyde to obtain clopidogrel.

[0020] Pharmacological action: It is a platelet aggregation inhibitor, which selectively inhibits the binding of ADP to platelet receptors and the activation of ADP-mediated glycoprotein GPⅡb / Ⅲa complex, thereby inhibiting platelet aggregation. It can also inhibit platelet aggregation caused by non-ADP. The effect on platelet ADP receptors is irreversible. It is rapidly absorbed orally, with a plasma protein binding rate of 98%. It is metabolized in the liver, and its main metabolites have no antiplatelet aggregation effect. See Baidu Encyclopedia.

[0021] Clopidogrel is an FDA-approved drug with a bioavailability of more than 50% through oral absorption. It has a protein binding rate of 95% to 98% in plasma. The half-life of the prototype drug, t1 / 2, is 7 to 8 hours, and the elimination half-life of the active metabolite, t1 / 2, is 0.5 to 1 hour. It is a well-performing bioprecursor prodrug with important value in preventing renal IRI. In addition, it is easily available, has low cost, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Clopidogrel inhibits renal ischemia-reperfusion injury in C57BL / 6J mice. Figure 1 A Clopidogrel molecular formula; Figure 1 B Illustration of animal experiments; Figure 1 C H&E staining; Figure 1 D PAS staining; Figure 1 EMasson staining; Figure 1 F Collagen volume analysis; Figure 1 G renal tubular injury score; Figure 1 H Glomerular injury score; Figure 1 I. Creatinine CRE content; Figure 1 J Urea nitrogen BUN content;

[0023] Figure 2 Clopidogrel inhibits renal IRI in C57BL / 6J mice by inhibiting ferroptosis. Figure 2 A Prussian blue staining; Figures BC Western blot detection and quantification;

[0024] Figure 3 Clopidogrel inhibits ferroptosis in vitro; Figure 3 Effects of clopidogrel (10-50 μM) on ferroptosis induced by RSL3, IKE, FIN56, BSO, Dihydroartemisinin, and Artesunate in AF HT1080 cells; Figure 3 G. Flow cytometry was used to detect the effect of clopidogrel (20 μM) on RSL3-induced HT1080 cell death; Figure 3 Effects of clopidogrel (10-50 μM) on ferroptosis induced by RSL3, JKE-1674, and FIN56 in HJ HK2 cells; Figure 3 Effects of clopidogrel (10-50 μM) on RSL3-induced ferroptosis in KP 786O, U937, BXPC3, CAOV3, RL95-2, and PANC-1 cells;

[0025] Figure 4Clopidogrel inhibited ferroptosis-induced lipid peroxidation and the increase of intracellular Fe2+ in HT1080 and HK2 cells. Figure 4 AB flow cytometry was used to detect the effect of clopidogrel treatment on lipid peroxidation in HK2 and HT1080 cells; Figure 4 C Flow cytometry was used to detect the effect of clopidogrel treatment on intracellular Fe2+ content in HK2 cells; Figure 4 D. Immunofluorescence detection of the effect of clopidogrel treatment on intracellular Fe2+ content in HT1080 cells;

[0026] Figure 5 Clopidogrel affects cell ferroptosis through the FSP1 pathway, Figure 5 Effects of clopidogrel on cell viability in combination with FSP1 inhibitor (iFSP1), CoQ inhibitor (4-CBA) and RSL3 in AD HT1080 and HK2 cells; Figure 5 Effects of EG clopidogrel on the viability of FSP1 gene knockout HT1080 cells (sgFSP1); Figure 5 HJ Effect of clopidogrel on the viability of FSP1 gene knockout HK2 cells (sgFSP1); Figure 5 Effects of clopidogrel on cell viability in combination with DHODH inhibitor (TEF), SCD1 inhibitor (CAY1056), and GCH1 / BH4 inhibitor (DAHP) and RSL3 in KP HT1080 and HK2 cells. DETAILED DESCRIPTION

[0027] Clopidogrel, RSL3, FIN56, Imidazole ketone erastin (IKE), DL-Buthionine-(S, R)-sulfoximine (BSO), iFSP1, Teriflunomide (TEF), CAY10566, Liproxstatin-1 (Lip1), Artesunate, and Dihydroartemisinin used in the experiment of this example were purchased from MCE Company;

[0028] JKE-1674 and 2,4-Diamino-6-hydroxypyrimidine (DAHP) were purchased from TargetMol;

[0029] 4-Chlorobenzoic (4-CBA) was purchased from Sangon Biotech;

[0030] Cell lines HK2, HT1080, 786-O, U937, BXPC3, CAOV3, RL95-2, and PANC-1 cells were from the Hematological Disease Center of the Army Medical University, and all cell lines were authenticated by short tandem repeat analysis (STR);

[0031] Experiments in this Example All CCK8 detection kits in this experiment were purchased from Bio-Tech, and mouse CRE and BUN detection kits were purchased from Elab science;

[0032] The flow cytometry antibodies PI and APC Annexin V were purchased from Vazyme;

[0033] Western blot reagents were purchased from Bio-Tech Company;

[0034] MDA, NGAL, FSP1, and GAPDH antibodies were purchased from Abclonal;

[0035] 4-HNE was purchased from Invitrogen;

[0036] KIM-1 was purchased from Huabio;

[0037] Fluorescent dye C11 BODIPY 581 / 591 was purchased from Abclonal;

[0038] FeRhoNox-1 was purchased from Maokang Company;

[0039] Hoechst 33342 was purchased from MCE;

[0040] The DMDM ​​and RPMI1640 culture media used were purchased from Gibco;

[0041] Fetal bovine serum was purchased from Excell;

[0042] Trypsin and 1% penicillin-streptomycin were purchased from Bio-Tech Company;

[0043] Other reagents were commercially available analytical grade reagents.

[0044] Example 1: Preventive effect of clopidogrel on renal ischemia-reperfusion injury in C57BL / 6J mice

[0045] 1.1 Experimental methods

[0046] Experimental subjects and groups: C57BL / 6J mice (6-8 weeks old) were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. 24 male mice were randomly divided into sham operation group: clopidogrel (40 mg / kg) (sham-CPD group, n=6) or corn oil (sham group, n=6) were injected intraperitoneally 2 hours before ischemia; ischemia reperfusion operation group: clopidogrel (40 mg / kg) (IRI-CPD, n=6) or corn oil (IRI group, n=6) were injected intraperitoneally 2 hours before ischemia. All mice were anesthetized with avertin and depilated before surgery.

[0047] Sham operation group: mice in the sham operation group were not clamped on both sides of the kidney pedicles.

[0048] Ischemia-reperfusion surgery: After bilateral back incision, the mouse renal pedicles were clamped (the time interval between clamping the left and right renal pedicles was controlled at 1 to 1.5 minutes). When the color of the kidneys changed from bright red to purple-black, the renal ischemia model was successfully established. The clamps were removed after 35 minutes. After blood supply was restored, the color of the kidneys changed from purple-black to bright red, which indicated that renal ischemia-reperfusion was successful. The mice were humanely killed after 48 hours.

[0049] Tissue processing and detection: The bilateral renal tissues of mice were collected and divided into two parts. One part was fixed with 4% paraformaldehyde, embedded in paraffin and sectioned. Hematoxylin-eosin (H&E) staining (Servicebio), periodic acid-Schiff reagent (PAS, Servicebio), Masson's trichrome staining (Servicebio) and Prussian blue staining (Servicebio) were performed according to standard protocols; one part was lysed by RIPA protein for Western blot detection; mouse serum was collected, separated and serum creatinine (CRE) level and urea nitrogen (BUN) level were detected.

[0050] 1.2 Experimental Results

[0051] Compared with the sham or sham-CPD groups, the renal ischemia-reperfusion group (IRI) showed mild glomerular lesions, mild mesangial proliferation, and severe damage to tubular and interstitial cells ( Figure 1 C, 1D, black arrows). The renal tubular epithelial cells are detached, and the tubules are dilated and notched. In addition, the renal tubular basement membrane is exposed ( Figure 1 C, 1D, green arrows). The IRI group showed interstitial edema accompanied by inflammatory cell infiltration ( Figure 1 C, 1D, blue arrows). Clopidogrel pretreatment significantly alleviated the damage caused by IRI. Masson trichrome staining showed that clopidogrel pretreatment (IRI-CPD group) reduced the degree of renal fibrosis caused by IRI ( Figure 1 E, blue area, 1F). The tubular and glomerular injury scores clearly showed the protective effect of clopidogrel ( Figure 1 G, 1H). In addition, the renal histological lesions in the IRI group were increased ( Figure 1 H), elevated serum creatinine (CRE) and blood urea nitrogen (BUN) levels ( Figure 1 I, 1J). IRI mice pretreated with clopidogrel (IRI-CPD group) showed significantly reduced necrotic areas. Prussian blue staining was used to detect the iron content in the renal tissue of IRI model mice ( Figure 2 A). Clopidogrel treatment (IRI-CPD group) significantly reduced the elevated intracellular iron level caused by IRI. Western blot analysis was used to detect the protein expression levels of Kim-1 and NGAL, two markers of renal tubular injury ( Figure 2 B-2C). After IRI, the protein levels of Kim-1 and NGAL increased significantly, while those of the clopidogrel pretreatment group (IRI-CPD) decreased significantly. In addition, clopidogrel pretreatment (IRI-CPD) significantly reduced the protein level of ferroptosis-related signal FSP1 and lipid peroxidation index 4-HNE, but MDA did not. Figure 2 B-2C).

[0052] Conclusion: Clopidogrel pretreatment has a protective effect on renal injury and fibrosis induced by renal IRI; the inhibitory effect of clopidogrel on ferroptosis may be one of the mechanisms to improve acute kidney injury caused by IRI; clopidogrel may inhibit ferroptosis through the FSP1 pathway.

[0053] Example 2: Clopidogrel inhibits cell ferroptosis in vitro

[0054] 2.1 Experimental methods

[0055] The CCK-8 method was used to detect the cell viability of clopidogrel (10-50 μM)-treated and control cells in HT1080, HK2, 786O, U937, BXPC3, CAOV3, RL95-2, and PANC-1 cell lines. Flow cytometry was used to detect the effect of clopidogrel intervention on apoptosis of HT1080 cells treated with RSL3. Cells were plated at 1.5*10 5 The cells were inoculated in 6-well plates with the dose of clopidogrel (50 μM) after cell attachment, and RSL3 (0.5 μM) was added 3 h later. The cells were harvested 12 h after the addition of RSL3, and double-stained with 5 μL PI and APC Annexin V (Vazyme). Flow cytometry was used to detect cell death 15 min later.

[0056] 2.2 Experimental Results

[0057] CCK8 experiments showed that clopidogrel inhibited cell death induced by RSL3, IKE, FIN56, BSO, dihydroartemisinin and artesunate in HT1080 cells in a dose-dependent manner ( Figure 3 A-3F). Flow cytometry further confirmed that clopidogrel rescued RSL3-induced cell death ( Figure 3 G). Clopidogrel also inhibited the death of HK2 cells induced by RSL3, JKE-1674 and FIN56 in a dose-dependent manner ( Figure 3 H-3J). Clopidogrel also dose-dependently protected these cancer cells from RSL3-induced ferroptosis in multiple tumor cell lines (786-O, U937, BXPC3, CAOV3, RL95-2, and PANC-1) (Fig. 3K-3P).

[0058] Example 3: Clopidogrel reduces ferroptosis-induced cellular lipid peroxidation and Fe 2+ Increase

[0059] 3.1 Experimental methods

[0060] Flow cytometry was used to detect the effects of clopidogrel on lipid peroxidation and intracellular Fe in HT1080 cells treated with RSL3. 2+ Content. 2*10 per hole 5 The cells were seeded in a 6-well culture dish. After 3 h of clopidogrel pretreatment, RSL3 (1.0 μM for HK2 cells and 0.5 μM for HT1080 cells) was added. The culture medium was discarded 3 h or 12 h after RSL3 was added. The cells were washed with PBS, 1.0 μM C11 BODIPY 581 / 591 (Abclonal) or 2 μM FeRhoNox-1 (Maokang Biotech) staining solution was added, and incubated at 37 °C for 40 min. Subsequently, the cells were trypsinized and resuspended in 200 μL Hanks' balanced salt solution (HBSS, Gibco) and analyzed by flow cytometry (FACS Canto II System, BD). The free iron content in HT1080 cells was detected by immunofluorescence. 4*10 4 Cells were seeded on 24-well plates, covered with pre-treatment, and treated with RSL3 (1.0 μM). Clopidogrel was added 3 h before RSL3 was added. Cells were incubated with 2 μM FeRhoNox-1 staining solution at 37 °C for 1 h. Hoechst 33342 (MCE) staining solution was added for 10 minutes and then washed with PBS. 4% paraformaldehyde was added for fixation for 15 minutes, and the lid was removed and sealed. Images were detected using a fluorescence microscope (Leica).

[0061] 3.2 Experimental Results

[0062] In HK2 and HT1080 cells, clopidogrel partially reversed the significant increase in lipid peroxidation and ferrous iron levels caused by ferroptosis ( Figure 4 A-4C). Fe 2 + fluorescence imaging also showed that in clopidogrel-treated HT1080 cells exposed to RSL3, cellular iron content was significantly reduced ( Figure 4 D).

[0063] Conclusion: Clopidogrel reduces ferroptosis-induced cellular lipid peroxidation and Fe 2+ increase.

[0064] Example 4: Clopidogrel-mediated inhibition is partially dependent on the canonical ferroptosis FSP1 pathway

[0065] 4.1 Experimental methods

[0066] The FSP1 gene knockout HT1080 cell line (sgFSP1) was constructed, and the knockout efficiency was detected by Western blot. CCK8 was used to detect the protective effect of clopidogrel against ferroptosis under the action of multiple inhibitors and in knockout cells.

[0067] 4.2 Experimental Results

[0068] We used specific inhibitors to target the FSP1 / CoQ (iFSP1, 4-CBA) pathway. The FSP1 inhibitor iFSP1 and the CoQ synthesis inhibitor 4-CBA significantly reduced the anti-ferroptotic activity of clopidogrel in HT1080 and HK2 cells. Knockout of FSP1 in HT1080 and HK2 cells significantly reduced the anti-ferroptotic activity of clopidogrel, and clopidogrel could reverse the cell viability of FSP1 knockout cells. ( Figure 5 )Conclusion: Clopidogrel can inhibit ferroptosis through the FSP1 pathway.

Claims

1. Application of clopidogrel in the preparation of drugs for preventing renal ischemia-reperfusion injury.

2. The use according to claim 1, characterized in that: The damage is ferroptosis caused by lipid peroxidation and cell damage.

3. The use according to claim 1, characterized in that: The drug is an inhibitor.

4. The use according to claim 3, characterized in that: The inhibitor inhibits ferroptosis in multiple cell lines.

5. The use according to claim 4, characterized in that: The plurality of cells are HT1080, HK2, 786O, U937, BXPC3, CAOV3, RL95-2 or PANC-1.

6. The use according to claim 2, characterized in that: The drug inhibits cell ferroptosis via the FSP1 pathway.

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