Application of N-acetylcysteine in preparation of medicine for relieving toxic and side effects of PD-1 inhibitor

Abstract

The invention relates to the technical field of medicines, in particular to application of N-acetylcysteine (NAC) in preparation of a medicine for relieving toxic and side effects of a PD-1 inhibitor. The invention provides application of NAC in preparation of a medicine for relieving toxic and side effects of a PD-1 inhibitor. The mechanism of the PD-1 inhibitor on cardiac collagen remodeling is reported for the first time. Researches show that the PD-1 inhibitor causes increase of mouse cardiac perivascular collagen deposition and reduction of interstitial collagen deposition, and after NAC is supplemented, cardiac collagen redistribution caused by the PD-1 inhibitor is effectively improved, so that the cardiac function is improved. The invention aims to find reasons and key targets of cardiac collagen remodeling caused by the PD-1 inhibitor, and provides support for clinically and reasonably using anti-tumor drugs.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to application of N-acetylcysteine ​​(NAC) in the preparation of drugs for reducing the toxic and side effects of PD-1 inhibitors. Background Art

[0002] Cardiac fibrosis is still an unresolved problem in heart disease. It is a cardiac remodeling state in the late stage of various cardiovascular diseases such as myocardial infarction, hypertension, and cardiac hypertrophy. When fibroblasts in the myocardium over-synthesize extracellular matrix, especially when collagen is deposited in the interstitium, the density of fibrous connective tissue increases, the type I / type III collagen ratio is seriously imbalanced, and the collagen arrangement is disordered, which leads to increased myocardial stiffness, decreased compliance, and decreased contractility, and eventually develops into heart failure. TGF-β1 is currently recognized as a regulatory factor closely related to the occurrence of fibrosis. Its downstream target molecule Smads can induce the upregulation of extracellular matrix-related protein expression. The TGF-β1 / Smads signaling pathway plays an important regulatory role in the process of tissue fibrosis. However, due to the large number of triggers of myocardial fibrosis and the complex pathogenesis, effective preventive and therapeutic drugs have not yet been found.

[0003] Endothelial-to-mesenchymal transition (EndMT) refers to the biological process in which endothelial cells lose their endothelial cell characteristics and acquire mesenchymal cell characteristics under certain specific physiological or pathological conditions. EndMT is a complex cell differentiation process that is crucial for the development of heart valves on the one hand, and may lead to pathological processes such as cardiac fibrosis on the other. Its main characteristics are that endothelial cells lose their specific markers such as CD31 and VE-cadherin, and weaken their original endothelial cell functions such as tube formation, and then appear mesenchymal cell-specific markers such as α-SMA and FSP1 and have mesenchymal cell functions such as collagen secretion. Studies have shown that EndMT occurs in a pressure overload-induced cardiac fibrosis model, and endothelial cell lineage tracing technology has found that myofibroblasts derived from EndMT account for 27% to 35% of all fibroblasts. In a study of doxorubicin-induced cardiotoxicity in mice, EndMT was found to be a potential mechanism for the occurrence of perivascular fibrosis. Many studies have shown that reactive oxygen species (ROS) can induce the occurrence of EndMT. N-acetylcysteine ​​(NAC) is a precursor of glutathione (GSH), and NAC neutralizes ROS by increasing the level of GSH. NAC can inhibit cell damage caused by oxidative stress by reducing ROS. This makes NAC play an important role in the prevention and treatment of many diseases. Summary of the invention

[0004] The purpose of the present invention is to provide the use of N-acetylcysteine ​​(NAC) in the preparation of drugs for reducing the toxic side effects of PD-1 inhibitors. The present invention aims to find the causes and key targets of cardiac collagen remodeling caused by PD-1 inhibitors, and intervene in key targets for the auxiliary use of clinical PD-1 inhibitors in the treatment of tumors, so as to reduce the clinical cardiovascular toxicity of PD-1 inhibitors.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides the use of NAC in preparing a drug for alleviating the toxic and side effects of a PD-1 inhibitor.

[0007] Preferably, the toxic side effect refers to reducing the cardiovascular toxicity of PD-1 inhibitors.

[0008] Preferably, the PD-1 inhibitor is InVivoMAb anti-mouse PD-1.

[0009] Preferably, the cardiovascular toxicity is cardiac collagen remodeling, manifested by increased perivascular collagen deposition and decreased interstitial collagen deposition.

[0010] The present invention also provides a pharmaceutical composition for treating cancer, comprising NAC and a PD-1 inhibitor.

[0011] Preferably, the cancer is melanoma, renal cancer, bladder cancer, liver cancer, gastric cancer or head and neck squamous cell carcinoma.

[0012] Preferably, the mass ratio of NAC to PD-1 inhibitor is 5000-6000:5.

[0013] Compared with the prior art, the present invention has the following beneficial effects: the present invention constructs a mouse model treated with PD-1 inhibitors and finds that PD-1 inhibitors significantly reduce the cardiac function of mice. Through Masson trichrome staining, we found that PD-1 inhibitors lead to the redistribution of cardiac collagen, which is manifested as a decrease in cardiac interstitial collagen and an increase in perivascular collagen deposition. Based on the results of PD-1 immunofluorescence staining of cardiac tissue, we speculate that PD-1 inhibitors are likely to act on endothelial cells to cause cardiac toxicity. Cell communication analysis was performed based on single-cell RNA-seq data, and the results showed that compared with normal mice, three cardiac endothelial-fibroblast interaction pairs, TGFβ1_EGFR, TGFβ1_TGFβR3, and TGFβ1_TGFβR1, which are closely related to fibrosis, disappeared in the hearts treated with PD-1 inhibitors. Further experiments found that PD-1 inhibitors downregulated the expression level of TGF-β1 in cardiac endothelial cells, thereby leading to a decrease in their secretion of TGF-β1. Fibroblasts were cultured with conditioned medium containing endothelial cell secretions, and it was found that the secretions of endothelial cells treated with PD-1 inhibitors could induce a decrease in fibroblast collagen secretion, while direct treatment of fibroblasts with PD-1 inhibitors did not affect their collagen secretion.

[0014] It is speculated that the reduction of TGF-β1 secreted by endothelial cells and then acting on fibroblasts is the main reason for the reduction of cardiac interstitial collagen induced by PD-1 inhibitors. Finally, it was found through detection that PD-1 inhibitors can induce endothelial to mesenchymal cell transformation (EndMT) in cardiac endothelium in vitro and in vivo. KEGG analysis of single-cell RNA-seq data of endothelial cells found that in mouse cardiac endothelial cells treated with PD-1 inhibitors, upregulated differential genes were significantly enriched in the oxidative phosphorylation pathway. Overactivation of oxidative phosphorylation can induce increased production of reactive oxygen species (ROS), and ROS is an important inducing factor of EndMT. The present invention found through in vitro experiments that PD-1 inhibitors induced a significant increase in ROS levels in endothelial cells, and the ROS inhibitor NAC can reverse the EndMT induced by PD-1 inhibitors. Therefore, we speculate that the increase in ROS in endothelial cells leads to EndMT, which is the reason why PD-1 inhibitors induce perivascular collagen deposition in the heart. Supplementation of NAC in animals can effectively improve the redistribution of cardiac collagen caused by PD-1 inhibitors and improve cardiac function.

[0015] The present invention reports the mechanism of PD-1 inhibitors on cardiac collagen remodeling. It shows that PD-1 inhibitors lead to increased perivascular collagen deposition and reduced interstitial collagen deposition in mice, and NAC supplementation can effectively improve the cardiac collagen redistribution caused by PD-1 inhibitors and improve cardiac function. The present invention aims to find the causes and key targets of PD-1 inhibitors leading to cardiac collagen remodeling, and provide support for the rational use of anti-tumor drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The dosage and time of PD-1 inhibitors.

[0017] Figure 2 Representative images of cardiac function and evaluation of EF and FS levels in mice were collected for echocardiography. n = 5-6; Note: **p < 0.01.

[0018] Figure 3 Expression levels of mRNA of related fibrosis genes and determination of soluble collagen content in the hearts of mice treated with PD-1 inhibitors; (AD) qRT-PCR detection of mRNA levels of Fn1, CTGF, Col1a1, and Col3a1 in the hearts of mice. n = 5-6; (E) Determination of soluble collagen content in the heart (collagen content per 10 mg of heart tissue). n = 5-6. Note: *p<0.05, **p<0.01, ****p<0.0001.

[0019] Figure 4 Masson trichrome staining; (A) Masson staining sections of cardiac interstitium and statistics of interstitial collagen content. n = 5-6. Scale bar: 100 μm (B) Masson staining sections of cardiac perivascular tissue and statistics of perivascular collagen content. n = 5-6. Scale bar: 100 μm. Note: **p<0.01, ****p<0.0001.

[0020] Figure 5 These are the related fibrosis indicators after HCFs were treated with PD-1 inhibitors for 24 hours; (AD) mRNA expression of Fn1, CTGF, Col1a1, and Col3a1 in HCFs after treatment with PD-1 inhibitors. n = 6. (E) Soluble collagen production in the cell supernatant of HCFs after treatment with PD-1 inhibitors. n = 3.

[0021] Figure 6 The expression of PD-1 in four major cardiac cells (CD31 marks endothelial cells, FSP1 marks fibroblasts, F4 / 80 marks macrophages, and cTnT marks cardiomyocytes). Scale bar: 100 μm.

[0022] Figure 7 This is the change in the interaction between endothelial cells and fibroblasts treated with PD-1 inhibitors, green is the ligand; yellow is the receptor.

[0023] Figure 8The expression of TGF-β1 in HCEACs after 24h of PD-1 inhibitor treatment; (A) qRT-PCR detected the mRNA expression of TGF-β1 in HCEACs after PD-1 inhibitor treatment. n = 4. (B) Western blot detected the protein expression of TGF-β1 in HCEACs after PD-1 inhibitor treatment. n = 3. (C) ELISA detected the secretion level of TGF-β1 in the supernatant of HCEACs after PD-1 inhibitor treatment. n = 6. Note: *p<0.05, **p<0.01.

[0024] Fig. 9 To detect the related fibrosis indicators after HCFs were cultured with conditioned medium; (A) Schematic diagram of co-culture with conditioned medium. (B) qRT-PCR detection of mRNA expression of related fibrosis genes Fn1, CTGF, Col1a1, and Col3a1 after HCFs were cultured with conditioned medium for 24 hours. n = 5. (C) Determination of soluble collagen production in cell supernatant medium after HCFs were cultured with conditioned medium for 24 hours. n = 3. Note: **p<0.01, ***p<0.001.

[0025] Fig.10 PD-1 inhibitors induce EndMT in endothelial cells; (A) Immunofluorescence detection of the expression of CD31 and α-SMA in the heart vessels of mice after treatment with PD-1 inhibitors and the expression of CD31 + α-SMA + CD31 + The percentage of cells in the HCAECs was counted. n=3. Scale bars: 50μm and 10μm. (B) The mRNA expression levels of CD31, VE-cadherin, Vimentin, and α-SMA in the hearts of mice in the normal group and the PD-1 inhibitor group. n=3. The mRNA expression levels of CD31, VE-cadherin, Vimentin, and α-SMA in HCAECs after 24h of PD-1 inhibitor treatment. n=4. Note: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0026] Fig.11 KEGG analysis was performed to identify the enrichment results of upregulated differentially expressed genes in cardiac endothelial cells of normal mice and mice treated with PD-1 inhibitors.

[0027] Fig.12Effects of PD-1 inhibitors on oxidative stress in HCAECs; (A) DHE was used to detect the generation of ROS in HCAECs after treatment with PD-1 inhibitors. n = 3. Scale bar: 50 μm. (B) MDA levels were detected in HCAECs after treatment with PD-1 inhibitors. n = 3. (C) SOD activity was detected in HCAECs after treatment with PD-1 inhibitors. n = 3. Note: *p<0.05, **p<0.01, ***p<0.001.

[0028] Fig.13 Effects of NAC on oxidative stress in HCAECs treated with PD-1 inhibitors; (A) DHE detected the expression of ROS after NAC was added to HCAECs treated with PD-1 inhibitors. n=3. Scale bar: 50μm. (B) Detection of MDA level after NAC was added to HCAECs treated with PD-1 inhibitors. n=3. (C) Detection of SOD activity after NAC was added to HCAECs treated with PD-1 inhibitors. n=3. (D) qRT-PCR detected the mRNA expression of CD31, VE-cadherin, Vimentin and α-SMA after NAC was added to HCAECs treated with PD-1 inhibitors. n=4. Note: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0029] Fig.14 Echocardiography: representative images of cardiac function and EF and FS levels of mice collected by echocardiography. n = 6. Note: *p < 0.05, **p < 0.01.

[0030] Fig.15 Masson trichrome staining; mouse cardiac interstitial and perivascular staining sections and collagen content statistics. n = 6. Scale bar: 100 μm. Note: *p < 0.05, **p < 0.01, ***p < 0.001. DETAILED DESCRIPTION

[0031] Example 1

[0032] PCR Primers Sangon Biotech (Shanghai) Co., Ltd.

[0033] D-PBS Beijing Solable Technology Co., Ltd.

[0034] InVivoMAb anti-mouse PD-1 Bioxcell, USA (BP0146)

[0035] 4% paraformaldehyde solution Wuhan Saiweier Biotechnology Co., Ltd.

[0036] Ready-to-use PBS powder Sangon Biotech (Shanghai) Co., Ltd.

[0037] 4×gDNAwiper Mix solution Nanjing Novogene Biotech Co., Ltd.

[0038] 4×HiScript III qRT SuperMix Nanjing Novogene Biotech Co., Ltd.

[0039] 4×ChamQ SYBR qPCR Master MixNanjing Novogene Biotech Co., Ltd.

[0040] Masson trichrome staining kit Wuhan Saiweier Biotechnology Co., Ltd.

[0041] Sircol TM Soluble Collagen AssayBiocolor, UK

[0042] Ultrasonic coupling agent Shandong Yuanke Biological

[0043] SOD Kit Biotech

[0044] MDA Kit Biotech

[0045] Superoxide Anion Fluorescent Probe Bio-Technology Company

[0046] Vevo 3100LT Small Animal Ultrasound Cardiography System FUJIFILM Visual Sonics

[0047] All mice in this study were 8-week-old male C57BL / 6 mice (20 g-25 g), purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd., animal production license number: SCXK (Lu) 20190003; animal husbandry and related experimental operations were in accordance with the theoretical guidelines for animal experiments. The cells used in this study were human coronary artery endothelial cells (HCAECs) and human fibroblasts (HCFs) purchased from ScienCell Research Laboratories, USA.

[0048] 1 Experimental methods

[0049] 1.1 Cardiac ultrasound examination

[0050] Mice were placed on a 40°C thermostatic heating table and anesthetized with 1.5% to 2% isoflurane. After hair removal, two-dimensional M-mode echocardiography was measured using a Vevo3100LT high-resolution imaging system and a 30MHz center frequency scan head. EF and FS were recorded for evaluation of cardiac function. Three consecutive cardiac cycles were selected for data measurement and analysis using Vevo LAB 5.6.0 software.

[0051] 1.2 Histomorphological analysis

[0052] 1.2.1 Preparation of cardiac tissue paraffin sections

[0053] (1) Sample fixation: The heart tissue was fixed in 4% paraformaldehyde for more than 24 h.

[0054] (2) Take out the heart tissue soaked in 4% paraformaldehyde and dehydrate it according to the sequence in Table 1.

[0055] Table 1 Dehydration sequence and dehydration schedule of cardiac tissue

[0056]

[0057]

[0058] (3) Paraffin embedding: After embedding, place it in the freezer.

[0059] (4) Paraffin tissue sections

[0060] 1.2.2 HE staining

[0061] (1) Dewaxing of paraffin sections: Place the paraffin sections in xylene I for 3 min, xylene II for 3 min, anhydrous ethanol I for 3 min, anhydrous ethanol II for 3 min, 95% ethanol for 3 min, 85% ethanol for 3 min, and 75% ethanol for 3 min, and then rinse with tap water for 1 min.

[0062] (2) Hematoxylin staining of cell nuclei: stain with hematoxylin for 4 min and rinse with tap water for 10 s.

[0063] (3) Differentiation and blueing: Differentiate in 1% saline-alcohol differentiation solution for 1 to 2 seconds, rinse in tap water for 20 to 30 seconds to blue.

[0064] (4) Eosin staining of cytoplasm: Stain with eosin for 15 to 30 seconds, remove excess stain and perform rapid dehydration.

[0065] (5) Dehydrate, make transparent, and seal the slides for observation under a microscope.

[0066] 1.2.3 Masson staining

[0067] (1) Dewaxing of paraffin sections: Place the paraffin sections in xylene I for 3 min, xylene II for 3 min, anhydrous ethanol I for 3 min, anhydrous ethanol II for 3 min, 95% ethanol for 3 min, 85% ethanol for 3 min, and 75% ethanol for 3 min, and then rinse with tap water for 1 min.

[0068] (2) Weigert iron hematoxylin staining of cell nuclei: Take equal amounts of Weigert stain solution A and Weigert stain solution B and mix them to make Weigert iron hematoxylin staining for 10 min. Wash with distilled water for 30 s.

[0069] (3) Differentiation: Differentiate in acidic differentiation solution for 10 s and wash with distilled water for 30 s.

[0070] (4) Bluing: Use Masson bluing solution for 3 min and rinse with distilled water for 30 s.

[0071] (5) Ponceau staining: stain with Ponceau fuchsin solution for 5 min.

[0072] (6) Prepare weak acid working solution: prepare weak acid working solution in the ratio of distilled water to weak acid dye solution = 2:1, and clean with weak acid working solution for 1 min.

[0073] (7) Phosphomolybdic acid differentiation: Differentiate with phosphomolybdic acid dye solution for 1 min, and wash with weak acid working solution for 1 min.

[0074] (8) Aniline blue staining of collagen fibers: staining with aniline blue staining solution for 1 min and washing with weak acid for 1 min.

[0075] (9) Dehydration, transparency, and sealing: 95% ethanol for 10 seconds, 100% ethanol I for 10 seconds, and 100% ethanol II for 10 seconds. Transparentize with xylene I for 1 minute, and transparentize with xylene II for 1 minute. Air-dry and seal with neutral gum, and observe under a microscope.

[0076] 1.2.4 Soluble collagen production assay

[0077] (1) First, draw a standard curve: take 0, 5, 10, and 15 μg of collagen reference standard and add it to cell culture medium to make up to 1 mL; take 1 mL of HCFs supernatant after treatment with PD-1 inhibitor.

[0078] (2) Add 200 μL of Isolation & Concentration Reagent to each EP tube containing the protein reference standard and test sample and incubate at 4°C overnight.

[0079] (3) The next day, centrifuge at 12,000 rpm for 10 min, and remove 1 mL of supernatant using a pipette and discard.

[0080] (4) Then, add 1 mL of Sircol staining reagent to each centrifuge tube and place it on a shaker for 30 min to form a collagen-dye complex at the bottom.

[0081] (5) After washing with ice-cold saline washing reagent, add 250 μL of Alkali reagent to dissolve collagen, and then transfer 200 μL of the sample into a 96-well plate.

[0082] (6) Measure the absorbance at 555 nm using an ELISA reader.

[0083] (7) Draw a standard curve

[0084] (8) Take the corresponding heart tissue or cell supernatant and measure the absorbance value according to the above steps, and calculate the collagen content of the sample to be tested according to the standard curve.

[0085] 1.2.5 Real-time fluorescence quantitative PCR

[0086] 1.2.5.1 RNA extraction

[0087] (I) Sample preparation

[0088] (1) Tissue samples

[0089] Take 10 mg of tissue and put it into a 1.5 mL EP tube; after cutting it with scissors, add 200 μL Trizol first, grind it into a homogenate with a handheld grinder, then add 800 μL Trizol to make it up to 1 mL, and let it stand at room temperature for 5 minutes to lyse.

[0090] (2) Cell samples

[0091] After taking out the six-well plate, shake the well plate first to aspirate out all the old culture medium and dead cells in the well plate, wash it twice with pre-cooled PBS, add 1mL Trizol to each well, scrape the cells vigorously with a cell scraper to fully lyse the cells, let it stand for 5 minutes, and then transfer it to a 1.5mL EP tube.

[0092] (ii) Liquid phase separation

[0093] Add 200 μL (1 / 5 of Trizol) chloroform, shake vigorously for 5 minutes, let stand for 5 minutes; centrifuge at 12000g, 4°C for 15 minutes. After centrifugation, take out the 1.5mL EP tube. The sample is divided into three layers, and the RNA is distributed in the colorless supernatant aqueous phase. Carefully pipette the colorless supernatant into another new 1.5mL EP tube.

[0094] 3. RNA precipitation

[0095] Add 500 μL (1 / 2 of Trizol) isopropanol, shake vigorously, incubate on ice for 10 min; centrifuge at 12000 g, 4°C for 10 min, discard the supernatant, invert the EP tube on filter paper, and drain the isopropanol.

[0096] (IV) Washing RNA

[0097] Add 1 mL of 75% ethanol and pipette to mix well. Vortex and gently invert. Centrifuge at 12000g, 4°C for 5 min, discard the supernatant, invert the EP tube on filter paper, and dry the RNA at room temperature.

[0098] (V) Dissolving RNA

[0099] Add 10 μL of DEPC water to resuspend the RNA precipitate. After the white precipitate is completely dissolved, transfer it into a 0.2 mL EP tube and place it in a -80°C refrigerator for later use.

[0100] 1.2.5.2 PCR amplification

[0101] (1) Removal of genomic DNA:

[0102] Table 2 Amplification system

[0103]

[0104] Mix well by gently pipetting. Incubate at 42℃ for 2 min.

[0105] (2) Prepare the reverse transcription reaction system:

[0106] Add 5×HiScript III qRT SuperMix directly to the reaction tube in step 1.

[0107] Table 3 Transcription reaction system

[0108]

[0109] Use a pipette to gently blow and mix. After mixing, put it into the PCR instrument and set the following program: 37℃, 15min; 85℃, 5s; 4℃, ∞.

[0110] (3) Real Time PCR:

[0111] 1. Prepare the following mixture in a qPCR tube:

[0112] Table 4 Reaction system

[0113]

[0114] 2. Perform qRT-PCR reaction according to the following conditions

[0115] Table 5 Reaction procedure

[0116]

[0117] (4) Real-time PCR primer sequences

[0118] Table 6 Primer sequences

[0119]

[0120]

[0121] As shown in SEQ ID NOs: 1-36.

[0122] 1.2.5.3 Analyze the data:

[0123] According to the threshold cycle (Ct) values ​​of each gene, GAPDH was used as the internal reference gene and 2 -△△Ct The expression levels of target genes in different samples were calculated.

[0124] 1.2.6 Cell culture

[0125] 1.2.6.1 Culture conditions and cell morphology

[0126] HCFs were cultured in Fibroblast Medium-2 (FM-2) containing 5% fetal bovine serum (FBS), 1% fibroblast growth factor-2 (FGS-2) and 1% penicillin / streptomycin. The conditions of the cell culture incubator were 37°C, 5% CO2 and 95% air. Normally grown HCFs were mainly star-shaped or spindle-shaped under an optical microscope.

[0127] HCAECs were cultured in endothelial cell culture medium (ECM) containing 5% FBS, 1% endothelial cell growth factor (ECGS), and 1% penicillin / streptomycin. The conditions of the cell culture incubator were 37°C, 5% CO2, and 95% air. Normally grown HCAECs were round / oval in shape.

[0128] 1.2.6.2 Cell recovery

[0129] (1) Prepare a fibronectin (FN)-coated T-25 cell culture flask and place the flask in an incubator overnight (at least 2 h at 37°C).

[0130] (2) The next day, FN was recovered, the culture flask was washed twice with DPBS, and 4 mL of complete culture medium was added to the flask.

[0131] (3) Take out the HCAECs cryopreservation tube from liquid nitrogen and place it in a 37°C water bath. Gently hold the bottle and rotate it until it dissolves into the size of a soybean. Immediately take the bottle out of the water bath, dry it, wash it with 75% ethanol, place it on a sterilized operating table, and remove the cap.

[0132] (4) Gently aspirate the cell suspension in the cryopreservation tube with a 1 mL pipette and add the cell suspension to a T-25 cell culture flask containing culture medium. Replace the culture medium the next day and continue culturing.

[0133] 1.2.6.3 Cell passaging

[0134] (1) Observe the growth status of cells under an optical microscope and subculture them when the cell confluence reaches 90% to 95%.

[0135] (2) Prepare FN-coated cell culture flasks one day before subculturing.

[0136] (3) Place the tube in a clean bench, discard the old culture medium, and rinse the cells twice with DPBS to clean the remaining blood in the bottle.

[0137] (4) Add 2.4 mL of DPBS and 1 mL of 0.05% trypsin, and observe under a microscope. When the cells become round and shrink, transfer the trypsin solution to a new 15 mL conical centrifuge tube containing 2 mL of DPBS, and then gently tap the culture flask until the cells fall off.

[0138] (5) Observe under a microscope to ensure that all cells have been detached.

[0139] (6) Add 2 mL of ECM complete medium to the cell culture flask to terminate digestion. Use a pipette to gently pipette and transfer the free cells to a 15 mL centrifuge tube.

[0140] (7) Centrifuge a 15 mL centrifuge tube at 1000 rpm / min for 5 min.

[0141] (8) After centrifugation, discard the supernatant, add 2 mL of ECM complete medium, pipette evenly, inoculate the cell suspension into a new coated cell culture flask at a ratio of 1:2, then add 4 mL of ECM complete medium and place in a cell culture incubator for culture.

[0142] 1.2.6.4 Cell cryopreservation

[0143] Steps (1)-(7) are the same as those in “1.2.6.3 Cell Passaging”.

[0144] (8) Preparation of cell freezing solution: Prepare 1 mL of cell freezing solution according to DMSO: FBS = 1:9.

[0145] (9) Discard the supernatant, resuspend the cells in 1 mL of cell freezing solution, pipette repeatedly to mix well, and transfer the cell suspension to a 2 mL cell freezing tube.

[0146] (10) Gradient freezing: Place at 4°C for 30 min, -20°C for 2-4 h, and -80°C overnight. The next day, transfer the cells frozen at -80°C to liquid nitrogen for long-term storage.

[0147] 1.2.6.5 Cellular administration

[0148] Dissolve the PD-1 inhibitor in ECM culture medium to control its concentration at 5 μg / mL. When the cell confluence reaches 80%, replace the culture medium containing PD-1 inhibitor and culture for 24 hours to detect indicators.

[0149] 1.2.7 Immunofluorescence

[0150] 1.2.7.1 Immunofluorescence on frozen sections

[0151] (1) The heart was fixed in 4% paraformaldehyde.

[0152] (2) Preparation of cardiac frozen sections: The heart in 4% paraformaldehyde was dehydrated with 10%, 20%, and 30% sucrose aqueous solutions for about 10 h, and then embedded in OCT embedding medium. The embedding box mold was frozen at a gradient temperature of -20°C and -40°C. The temperature of the cryostat was set to -20°C, the thickness of the cryostat was adjusted to 7 μm, and the slices were stored at -40°C.

[0153] (3) Remove the slide and allow to warm to room temperature for 30 min.

[0154] (4) Wash the slides three times with PBS and add 4% Triton for 1.5 h to prevent the slides from drying out.

[0155] (5) Wash the slides three times with PBS and add 3% BSA to block for 1.5 h to prevent the slides from drying out.

[0156] (6) Primary antibody incubation: After the sections were dried, a circle was drawn around the tissue with an immunohistochemistry pen. Different tissues were co-incubated with PD-1 primary antibody (1:1000) and CD31 primary antibody (1:500), PD-1 primary antibody (1:1000) and FSP1 primary antibody (1:500), PD-1 primary antibody (1:1000) and cTnT primary antibody (1:1000), and PD-1 primary antibody (1:1000) and F4 / 80 primary antibody (1:1000) at 4°C overnight.

[0157] (7) The next day, the slides were washed three times with PBS. After the sections were slightly dried, donkey anti-rabbit IgG and Alexa Fluor 488 goat anti-mouse IgG fluorescent secondary antibodies were added dropwise into the circle and incubated at room temperature in the dark for 1 h.

[0158] (8) The slides were washed three times with PBS, dried, and then DAPI stain was added to the circle and incubated at room temperature for 10 min to stain the cell nuclei.

[0159] (9) Collect images using a laser confocal microscope.

[0160] 1.2.7.2 Immunofluorescence on paraffin sections

[0161] (1) Dewax the paraffin sections until they are hydrated. Place the sections in environmentally friendly dewaxing solution for 30 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, anhydrous ethanol III for 5 min, and then wash with distilled water.

[0162] (2) Antigen retrieval: The tissue sections were placed in a retrieval box containing EDTA antigen retrieval buffer and subjected to antigen retrieval in a microwave oven at 180°C for 16 min, then at 160°C for 16 min.

[0163] (3) Circle blocking: After the slices are dried, use an immunohistochemistry pen to draw a circle around the tissue and add 3% BSA to block for 30 minutes.

[0164] (4) Add CD31 primary antibody (1:500) and α-SMA primary antibody (1:1000) and incubate at 4°C overnight.

[0165] (5) The next day, the slides were washed three times with PBS. After the sections were slightly dried, donkey anti-rabbit IgG and Alexa Fluor 488 goat anti-mouse IgG fluorescent secondary antibodies were added dropwise into the circle and incubated at room temperature in the dark for 1 h.

[0166] (6) Wash the slides three times with PBS, spin dry, and then add DAPI stain to the circle and incubate at room temperature for 10 min to stain the cell nuclei.

[0167] (7) Collect images using a laser confocal microscope.

[0168] 1.2.8 Western blot

[0169] 1.2.8.1 Total cell protein extraction

[0170] (1) Preparation of lysis buffer: Protease inhibitor and RIPA lysis buffer were prepared at a ratio of 1:50, that is, 20 μL of protease inhibitor was added to 1 mL of RIPA lysis buffer, mixed and shaken before use.

[0171] (2) Discard the supernatant: Pour out the cell culture medium or use a pipette to absorb it cleanly.

[0172] (3) Rinse: Wash three times with PBS, 2 mL each time, and blot dry with filter paper.

[0173] (4) Lysis: Add 300 μL of RIPA lysis buffer (containing protease inhibitors) to each cell culture flask, let it stand for 5 seconds, and blow it several times with a gun to allow the lysis buffer to fully contact with the cells for 2 minutes.

[0174] (5) Scraping cells: Scrape the cells attached to the wall, aspirate them into an EP tube, and lyse them on ice for 10 minutes.

[0175] (6) Vortex: 30 s each time, once every 3 min, for a total of 3 times to ensure more complete cell lysis.

[0176] (7) Place the lysed cells into a 1.5 mL EP tube and centrifuge at 4°C, 13,000 × g for 5 min. Pipette the supernatant into the EP tube and mark it.

[0177] (8) Measure protein concentration.

[0178] (9) Protein denaturation: Mix the remaining protein with 5× loading buffer in a ratio of 4:1, heat in a 100°C metal bath for 10 min to denature it, cool to room temperature, and store at -20°C for later use.

[0179] (10) Storage: If the protein is not to be used for a long time, store it at -80°C and label it.

[0180] 1.2.8.2 Glue preparation

[0181] (1) Lower layer glue: Prepare glue of corresponding concentration according to the instructions. Use a pipette to pour glue from one side without any bubbles. Add glue to 2 cm from the upper edge, and add ddH2O to flatten the liquid surface. Let it stand at room temperature for 30 to 40 minutes. After the lower layer of glue is completely solidified, pour out the ddH2O on the top and absorb the moisture.

[0182] (2) Upper layer of glue: Prepare according to the instructions. When adding TEMED, quickly vortex, pour the glue, insert the comb, and let it stand at room temperature for 20 to 40 minutes.

[0183] 1.2.8.3 Liquid preparation

[0184] (1) Prepare 1× electrophoresis buffer: Weigh 3 g of Tris, 14.4 g of glycine, and 1 g of SDS, add ddH2O to make the total volume 1 L, and stir evenly with a stirring bar to fully dissolve. This is the preparation of 1× electrophoresis buffer.

[0185] (2) Prepare 1× transfer buffer: Weigh 3 g of Tris and 14.4 g of glycine, add 200 mL of methanol, and add ddH2O to make the total volume 1 L. Stir evenly with a stirring bar to fully dissolve. This is the preparation of 1× transfer buffer.

[0186] (3) Preparation of 1×PBST solution: Add 95.7 g of ready-to-use PBS powder to ddH2O to a total volume of 1 L, stir with a stirring rod to make it fully dissolved, and then prepare 10×PBS solution. Take 100 mL of 10×PBS solution, add ddH2O to 1 L, and then add 500 μL of Tween-20 and mix thoroughly to prepare 1×PBST solution.

[0187] 1.2.8.4 Electrophoresis

[0188] (1) Fixation: Use electrophoresis clamps to fix the gel plate.

[0189] (2) Leakage detection: Fill the electrophoresis tank with electrophoresis liquid and check for leakage.

[0190] (3) Loading: Use a pipette to add samples close to the gel plate. Load samples uniformly in each lane according to the amount of protein expression.

[0191] (4) Gel running: During the upper gel stage, set the voltage to a constant voltage of 80 V. When the Maker moves to the lower gel, adjust the voltage to a constant voltage of 120 V. Stop electrophoresis when the bromophenol blue indicator band reaches the bottom edge of the gel plate.

[0192] 1.2.8.5 Transfer

[0193] (1) Place the cut PVDF membrane in a container containing methanol solution and soak it for 1 min to activate it, then wash it with ddH2O, and finally place it in a container containing transfer solution for later use.

[0194] (2) Open the transfer clip and place it in the transfer solution. Place the sponge, filter paper, PVDF membrane, and glue in order from the negative electrode to the positive electrode. Finally, place the filter paper and sponge. Use a plastic rod to remove excess bubbles and tighten the transfer clip. Finally, place the transfer clip in the transfer tank and transfer the membrane at a constant current of 200 mA for about 2 hours. Pour a lot of ice around the transfer tank to cool it down.

[0195] 1.2.8.6 Closure

[0196] (1) Prepare blocking solution: Use 1× PBS to prepare 10% skimmed milk powder blocking solution, shake gently on a horizontal shaker at room temperature, and block for 2 h.

[0197] (2) After blocking, wash the membrane three times with 1× PBST solution, each time for 10 min.

[0198] 1.2.8.7 Incubation with primary antibody

[0199] (1) Determine the primary antibody concentration based on the protein expression level. Use 1× PBST to dilute the antibody to an appropriate concentration, completely place the PVDF membrane in the antibody, and incubate overnight at 4°C.

[0200] (2) The next day, the primary antibody was recovered and labeled. The PVDF membrane was washed three times with 1× PBST, each time for 10 min.

[0201] 1.2.8.8 Incubation with secondary antibody

[0202] (1) Dilute the secondary antibody to an appropriate concentration using 1× PBST according to the instructions and incubate at room temperature for 2 h.

[0203] (2) Recover the secondary antibody, label it, and store it at 4°C. Wash it three times with 1× PBST, each time for 10 min.

[0204] 1.2.8.9 Development

[0205] (1) Prepare the developer: Mix the ECL reagent A and B in a ratio of 1:1 and keep away from light.

[0206] (2) Place the developer on the plastic wrap and place the film on top so that the developer is in full contact with the strips. Turn off the light and incubate for 2 minutes. Then, develop and fix the strips.

[0207] (3) Use Image J software to measure the gray value of the band.

[0208] Table 7 Primary antibody related information

[0209]

[0210]

[0211] Table 8 Secondary antibody related information

[0212]

[0213] 1.2.9 Co-culture with conditioned medium 1.2.9.1 Collection of conditioned medium

[0214] Prepare a 5 mL EP tube and plate the donor HCAECs in a cell bottle. When the density of HCAECs is about 60%, add PD-1 inhibitor for 24 hours, replace with fresh ECM, and continue incubation for 24 hours. Collect the obtained cell supernatant in the EP tube prepared in advance.

[0215] 1.2.9.2 Mixed culture medium

[0216] According to the growth rates of HCAECs and HCFs, the appropriate time point was selected to culture HCFs, and the culture medium of HCAECs was collected and mixed with the culture medium of HCFs in a ratio of 1:1 to form a conditioned medium.

[0217] 1.2.10 TGF-β1 ELISA

[0218] 1) Sample collection method

[0219] After HCACEs were treated with PD-1 inhibitors, the supernatant was collected and centrifuged at 1000×g for 20 min at 2-8°C, and the supernatant was detected.

[0220] 2) Sample activation method

[0221] TGF-β1 is inactive in the body and can only be detected after activation. Cell culture supernatant: Add 20μL of standard and sample diluent to 100μL of sample, mix, add 40μL of activation reagent 1, and incubate at room temperature for 10 minutes. Further add 40μL of activation reagent 2, mix and measure.

[0222] 3) Operation steps

[0223] 1. Set up standard wells, blank wells and sample wells, add 100 μL of standard to the standard wells, add 100 μL of standard and sample diluent to the blank wells, and add 100 μL of the sample to be tested to the remaining wells. Cover the ELISA plate with a film and incubate at 37°C for 90 minutes.

[0224] 2. Pour out the liquid in the wells without washing. Add 100 μL of biotinylated antibody working solution to each well, cover the ELISA plate with a film, and incubate at 37°C for 1 hour.

[0225] 3. Pour out the liquid in the small wells and pat dry on clean absorbent paper. Add 350 μL of washing solution to each well, soak for 1 minute, and then pat dry and discard the liquid in the well. Repeat three times.

[0226] 4. Add 100 μL of HRP enzyme conjugate working solution to each well, cover the ELISA plate with a film and incubate at 37°C for 30 minutes.

[0227] 5. Shake off all liquid in the wells and wash the plate 5 times using the same method as step 3.

[0228] 6. Add 90 μL of substrate solution to each well, cover the plate with film, and incubate at 37°C in the dark for about 15 minutes. The time can be shortened or extended as appropriate according to the color development of the reagent, but should not exceed 30 minutes.

[0229] 7. Add 50 μL of stop solution to each well to end the reaction.

[0230] 8. Use a microplate reader to measure the absorbance (OD) of each well at a wavelength of 450 nm.

[0231] 1.2.11 Determination of MDA content

[0232] For tissue and cell samples, the protein concentration can be determined by BCA protein concentration after preparation, thereby facilitating the calculation of MDA content per unit protein weight in tissues or cells.

[0233] 1) Preliminary preparation

[0234] (i) Preparation of TBA stock solution: Weigh an appropriate amount of TBA and prepare 0.37% TBA stock solution using TBA preparation solution.

[0235] (ii) Preparation of MDA working solution: Refer to the following table and prepare appropriate MDA working solution according to the number of samples to be measured.

[0236] Table 9 Preparation of MDA detection working solution

[0237]

[0238] (III) Dilution of standard: Take an appropriate amount of standard and dilute it with distilled water to 1, 2, 5, 10, 20, 50 μM to prepare the standard curve.

[0239] 2) Sample determination: Add 0.1 mL of PBS to each well of a six-well plate for grinding, and set up the detection reaction system according to the following table:

[0240] Table 10 Reaction detection system

[0241]

[0242] (i) After mixing, heat at 100℃ or in boiling water for 15 minutes. Do not allow the liquid to splash during the heating process.

[0243] (ii) Cool to room temperature in a water bath, then centrifuge at 1000 g for 10 min at room temperature. After centrifugation, take 200 μL of the supernatant and add it to a 96-well plate. Set the absorbance of the microplate reader to 532 nm.

[0244] (iii) MDA calculation: The molar concentration of MDA is calculated based on the standard curve, and the MDA content in the initial sample is expressed as unit weight protein content or tissue weight, such as μmol / mg protein or μmol / mg tissue.

[0245] 1.2.12 SOD activity assay

[0246] 1) Sample preparation

[0247] Remove the treated cells from the incubator, aspirate the culture medium, wash with PBS that has been pre-bathed on ice, add 200 μL of the SOD sample preparation solution provided by the kit for every 1 million cells, pipette appropriately to fully lyse the cells, centrifuge at 12,000 g for 3 to 5 minutes at 4°C, and extract the supernatant as the sample to be tested.

[0248] 2) Preliminary preparation

[0249] (I) Preparation of WST-8 / enzyme working solution: Prepare an appropriate amount of WST-8 / enzyme working solution according to the number of samples to be tested. The specific preparation method can be referred to Table 11.

[0250] Table 11 Preparation of WST-8 enzyme working solution

[0251]

[0252]

[0253] (ii) Preparation of reaction start working solution: Dissolve the reaction start solution (40×) in the kit at room temperature in advance, then mix evenly, and dilute it at a ratio of adding 39 μL SOD detection buffer to every 1 μL reaction start solution (40×). After mixing evenly, it is the reaction start working solution.

[0254] 3) Sample determination

[0255] (i) Add the sample to be tested and other different solutions according to Table 12. Add the reaction starter working solution and mix thoroughly.

[0256] Table 12 Sample measurement system

[0257]

[0258] (ii) Incubate at 37°C for 30 min.

[0259] (iii) Measure the absorbance at 450 nm.

[0260] 4) Calculation of total SOD activity in samples

[0261] (I) Calculation of inhibitor percentage: refer to the following calculation formula:

[0262] Inhibition percentage = (A 空白对照1 -A 样品 ) / (A 空白对照1 -A 空白对照2 )×100%;

[0263] (ii) SOD enzyme activity unit formula: SOD enzyme activity units in the sample to be tested = SOD enzyme activity units in the detection system = inhibition percentage / (1-inhibition percentage) units.

[0264] 1.2.13 Determination of cellular superoxide anion levels

[0265] Weigh DHE powder and dissolve it in DMSO to prepare a mother solution (0.5-5 μM). When the cell confluence reaches about 80%, slowly add the prepared solution to the cell culture medium to ensure that each drop is evenly distributed in the entire culture system. Then, incubate at 37°C for about 30 minutes. After incubation, wash the cells with PBS and then collect images using a laser confocal microscope.

[0266] 1.2.14 Pathway enrichment analysis

[0267] In order to investigate the potential functions of differentially expressed genes, the Kyoto Encyclopedia of Genes and Genomes (KEGG) was used to analyze the enrichment of up-regulated differentially expressed genes in mouse cardiac endothelial cells.

[0268] 1.2.15 Data Statistical Analysis

[0269] All data were analyzed and plotted by GraphPad Prism 9.0.0. The data between two groups were analyzed by Student's t-test, and one-way ANOVA was used for three or more groups. All data are presented as mean ± SD. P values ​​less than 0.05 were considered statistically significant. Results:

[0270] 2.1PD-1 inhibitors reduce cardiac function in mice

[0271] In order to study the toxic effects of PD-1 inhibitors on the heart, the present invention used 8-week-old C57BL / 6 mice to intraperitoneally inject PD-1 inhibitors, i.e., PD-1 monoclonal antibodies, to establish an animal model treated with PD-1 inhibitors ( Figure 1 ). On the day of sampling, echocardiograms of mouse heart function were collected. The results of mouse heart echocardiograms showed that compared with the control group, the cardiac contractile function of mice treated with PD-1 inhibitors was significantly decreased, as shown by a significant decrease in EF and FS ( Figure 2 The above results indicate that PD-1 inhibitors cause significant impairment of cardiac contractile function in mice.

[0272] 2.2 Effects of PD-1 inhibitors on cardiac collagen in mice

[0273] To investigate the effect of PD-1 inhibitors on cardiac fibrosis, qRT-PCR was used to detect the expression of fibrosis-related genes in the hearts of mice treated with PD-1 inhibitors. The mRNA levels of Fn1, CTGF, Col1a1, and Col3a1 were significantly lower than those in the control group ( Figure 3AD). The soluble collagen production kit was used to detect the level of collagen production in the mouse heart, showing that compared with the control group, the soluble collagen production in the mouse heart treated with PD-1 inhibitors was significantly reduced ( Figure 3 E).

[0274] 2.3PD-1 inhibitor induces collagen redistribution in mouse hearts

[0275] In order to further detect the fibrosis changes inside the mouse heart, the present invention observed the interstitial and perivascular fibrosis of the mouse heart by Masson trichrome staining. The results showed that PD-1 inhibitors led to the redistribution of cardiac collagen. Compared with the control group, the cardiac interstitial collagen was significantly reduced, while the perivascular collagen deposition was significantly increased ( Figure 4 The above results indicate that PD-1 inhibitor treatment leads to the redistribution of cardiac collagen, which is manifested by a significant decrease in cardiac interstitial collagen and a significant increase in perivascular collagen deposition, which may be the cause of the impaired cardiac function in mice.

[0276] 2.4 Effects of PD-1 inhibitors on HCFs

[0277] Fibroblasts are key cells for collagen synthesis. In order to study whether PD-1 inhibitors act on fibroblasts to affect collagen secretion, the present invention detected the changes of fibrosis-related genes in HCFs treated with PD-1 inhibitors by qRT-PCR. The results showed that the mRNA expression of Fn1, CTGF, Col1a1, and Col3a1 did not change significantly compared with the control group ( Figure 5 AD). The soluble collagen content in the HCFs supernatant was detected by a soluble collagen production kit, and the results showed that PD-1 inhibitors had no significant effect on HCFs collagen production ( Figure 5 E) The above results confirm that PD-1 inhibitors do not directly act on fibroblasts to exert biological effects.

[0278] 2.5PD-1 is mainly expressed in endothelial cells, cardiomyocytes, and macrophages

[0279] Since PD-1 inhibitors are monoclonal antibodies against PD-1 that function through PD-1, the expression of PD-1 in various cardiac cells was studied next. Through immunofluorescence experiments, cTnT for cardiomyocytes, CD31 for endothelial cells, F4 / 80 for macrophages, and FSP1 for fibroblasts were detected. Figure 6 ), the results showed that PD-1 is not expressed in fibroblasts, so PD-1 inhibitors do not directly act on fibroblasts to exert biological effects.

[0280] 2.6 Changes in the interaction between endothelial cells and fibroblasts in the heart after PD-1 inhibitors

[0281] Since PD-1 is not expressed in fibroblasts and PD-1 inhibitors do not directly act on fibroblasts, do PD-1 inhibitors reduce cardiac interstitial fibrosis by regulating the interaction between other cardiac cells and fibroblasts? The present invention first uses Cellphone DB to study the interaction between cells and found that the three interaction pairs between endothelial cells and fibroblasts, TGFβ1_EGFR, TGFβ1_TGFβR3, and TGFβ1_TGFβR1, which are closely related to fibrosis, disappeared in the hearts of mice treated with PD-1 inhibitors ( Figure 7 ). Therefore, it is speculated that the decrease in cardiac interstitial collagen secretion may be related to the interaction between endothelial cells and fibroblasts.

[0282] 2.7PD-1 inhibitors downregulate TGF-β1 in endothelial cells

[0283] Since the three endothelial-fibroblast interaction pairs of TGFβ1_EGFR, TGFβ1_TGFβR3, and TGFβ1_TGFβR1 disappeared in the heart treated with PD-1 inhibitors, we considered whether PD-1 inhibitors reduced the expression of TGF-β1 in endothelial cells. Therefore, a cell model treated with PD-1 inhibitors was constructed by treating HCAECs with PD-1 inhibitors. The expression of TGF-β1 in HCAECs treated with PD-1 inhibitors was detected by qRT-PCR and Western blot, showing that the expression of TGF-β1 in HCAECs was significantly lower than that in the control group ( Figure 8 AB). The results of the ELISA kit showed that after HCAECs were treated with PD-1 inhibitors, the secretion of TGF-β1 in the supernatant was significantly lower than that in the control group ( Figure 8 C).

[0284] 2.8PD-1 inhibitors downregulate TGF-β1 in endothelial cells, affecting endothelial-fibroblast interaction and leading to reduced fibroblast collagen secretion

[0285] In order to further explore whether PD-1 inhibitors affect collagen secretion of fibroblasts by regulating endothelial-fibroblast interaction through acting on endothelial cells, the present invention uses HCAECs treated with PD-1 inhibitors for 24 hours, replaces with fresh culture medium and continues to culture for 24 hours, then collects and mixes with FM-2 at a ratio of 1:1 to form conditioned medium, and cultures HCFs with conditioned medium for 24 hours ( Fig. 9 A). qRT-PCR results showed that after HCFs were cultured with conditioned medium, the mRNA expression of fibrosis-related genes Fn1, CTGF, Col1a1, and Col3a1 decreased significantly ( Fig. 9 B). The results of the soluble collagen production kit showed that the collagen production of HCFs decreased after being cultured with conditioned medium ( Fig. 9C).

[0286] Based on the above results, when PD-1 inhibitors act directly on HCFs, the collagen synthesis capacity of HCFs does not change, but when HCFs are cultured in conditioned medium, fibrosis-related Fn1, CTGF, Col1a1 and Col3a1 are significantly decreased. Therefore, PD-1 inhibitors affect endothelial-fibroblast interaction by downregulating TGF-β1 in endothelial cells, resulting in reduced collagen secretion by fibroblasts.

[0287] 2.9PD-1 inhibitors induce increased perivascular collagen deposition in the heart through EndMT

[0288] In order to study the main reason for the increase in perivascular collagen deposition caused by PD-1 inhibitors, we performed immunofluorescence staining, and the results showed that in normal mouse hearts, red-stained CD31-positive endothelial cells were continuously and completely laid on the inner wall of the lumen, while in mice treated with PD-1 inhibitors, CD31-positive endothelial cells became intermittent and appeared in the green smooth muscle layer marked by α-SMA. The results confirmed that PD-1 inhibitors caused EndMT ( Fig.10 A). qRT-PCR results confirmed that the mRNA expressions of endothelial markers CD31 and VE-cadherin were decreased, and the mRNA expressions of fibroblast markers Vimentin and α-SMA were increased in cardiac tissues and HCAECs after treatment with PD-1 inhibitors ( Fig.10 B). The above results suggest that PD-1 inhibitors may lead to increased perivascular collagen deposition by inducing EndMT.

[0289] 2.10PD-1 inhibitors significantly increase reactive oxygen species production in endothelial cells

[0290] In order to study how PD-1 inhibitors lead to the EndMT process, KEGG data analysis found that after PD-1 inhibitor treatment, the upregulated differentially expressed genes in mouse cardiac endothelial cells were mainly enriched in the oxidative phosphorylation pathway ( Fig.11 ), and the overactivation of oxidative phosphorylation can induce the increase of reactive oxygen species (ROS), which is an important inducing factor of EndMT. HCAECs were stained with superoxide anion probe (DHE). DHE staining is a fluorescent probe for detecting intracellular reactive oxygen species. It can freely enter cells and dehydrogenate to form ethidium bromide under the action of superoxide anions in cells. Ethidium bromide can combine with RNA or DNA to produce red fluorescence, which can determine the amount and changes of cellular ROS content. The results showed that HCAECs produced more ROS after being treated with PD-1 inhibitors ( Fig.12 A). The results of malondialdehyde (MDA) kit test showed that the MDA content of HCAECs increased after treatment with PD-1 inhibitors ( Fig.12 B). The results of superoxide dismutase (SOD) activity detection kit showed that the SOD activity of HCAECs decreased after treatment with PD-1 inhibitors ( Fig.12 C). The above results all confirmed that PD-1 inhibitors significantly activated the oxidative stress response of endothelial cells.

[0291] 2.11PD-1 inhibitors induce EndMT by increasing ROS generation in HCAECs

[0292] To investigate whether activation of oxidative stress leads to EndMT, we added NAC, a commonly used antioxidant, to HCAECs after treatment with PD-1 inhibitors. DHE staining results showed that the addition of NAC significantly reversed the increase in ROS caused by PD-1 inhibitors ( Fig.13 A). The results of the MDA test kit showed that the addition of NAC could significantly reverse the increase in MDA production caused by PD-1 inhibitors ( Fig.13 B). The results of the SOD test kit showed that the addition of NAC could significantly reverse the decrease in SOD activity caused by PD-1 inhibitors ( Fig.13 C). The above results all confirmed that the addition of NAC to HCAECs can significantly reverse the increased ROS generation caused by PD-1 inhibitors. Can the addition of NAC reverse EndMT caused by PD-1 inhibitors? The present invention detected the expression of EndMT-related genes by qRT-PCR, and the results showed that NAC can significantly reverse the down-regulation of mRNA expression of endothelial cell markers CD31 and VE-cadherin in HCAECs caused by PD-1 inhibitors, and the up-regulation of mRNA expression of fibroblast markers Vimentin and α-SMA ( Fig.13 D) The above results all confirm that PD-1 inhibitors cause EndMT by increasing ROS in endothelial cells.

[0293] 2.12 NAC supplementation in vivo can reverse cardiac collagen remodeling induced by PD-1 inhibitors

[0294] We used C57 / BL6 male mice and added NAC (400 mg / kg / d) to the drinking water. The use of PD-1 inhibitors was as described above. Echocardiography of mouse cardiac function was collected on the day of sampling. The results showed that compared with the control group, the cardiac contractile function of mice treated with PD-1 inhibitors was significantly decreased, and daily oral administration of NAC could significantly restore the decreased cardiac contractile function of mice caused by PD-1 inhibitors ( Fig.14 Masson trichrome staining showed that oral administration of NAC significantly increased interstitial collagen and decreased perivascular collagen secretion ( Fig.15). The above results confirm that the addition of NAC significantly reverses the redistribution of cardiac collagen, keeps collagen secretion in a relatively balanced state, and significantly restores the impaired cardiac contractile function caused by PD-1 inhibitors. The above description is only a preferred embodiment of the present invention.

Claims

1. Application of N-acetylcysteine ​​in the preparation of drugs that reduce the toxic side effects of PD-1 inhibitors.

2. The use according to claim 1, characterized in that: The toxic side effects refer to reducing the cardiovascular toxicity of PD-1 inhibitors.

3. The use according to claim 1, characterized in that: The PD-1 inhibitor is InVivoMAb anti-mousePD-1.

4. The use according to claim 2, characterized in that: The cardiovascular toxicity is increased perivascular collagen deposition and decreased interstitial collagen deposition.

5. A pharmaceutical composition for treating cancer, characterized in that: The pharmaceutical composition comprises N-acetylcysteine ​​and a PD-1 inhibitor.

6. The composition according to claim 5, characterized in that The cancer is melanoma, kidney cancer, bladder cancer, liver cancer, stomach cancer or head and neck squamous cell carcinoma.

7. The composition according to claim 5, characterized in that The mass ratio of the N-acetylcysteine ​​to the PD-1 inhibitor is 5000-6000:5.

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