Exosomes miRNA-499a-5p targeted CD38 to alleviate anthraquinone induced cardiotoxicity: experimental research

Background: The purpose of this study was to investigate the effects of cardiac homing peptide (CHP) engineered bone marrow mesenchymal stem cells (BMMSc) derived exosomes (B-exo) loaded miRNA-499a-5p on doxorubicin (DOX) induced cardiotoxicity. Methods: miRNA chip analysis was used to analyze the differences between DOX induced H9c2 cells and control group. CHP engineering was performed on BMMSc derived exosomes to obtain C-B-exo. miRNA-499a-5p mimic was introduced into C-B-exo by electroporation technology to obtain C-B-exo-miRNA-499a-5p. DOX was used to establish a model of cardiotoxicity to evaluate the effects of C-B-exo- miRNA-499a-5p in vivo and in vitro. Western blot, immunohistochemistry, immunofluorescence, and other molecular biology methods were used to evaluate the role and mechanism of C-B-exo-miRNA-499a-5p on DOX induced cardiotoxicity. Results: miRNA chip analysis revealed that miRNA-499a-5p was one of the most differentially expressed miRNAs and significantly decreased in DOX induced H9c2 cells as compared to the control group. Exo-and B-exo have a double-layer membrane structure in the shape of a saucer. After engineering the CHP of B-exo, the results showed that the delivery of miRNA-499a-5p significantly increased and significantly reached the target organ (heart). The experimental results showed that C-B-exo-miRNA-499a-5p significantly improved electrocardiogram, decreased myocardial enzyme, serum and cardiac cytokines, improved cardiac pathological changes, inhibited CD38/MAPK/NF-κB signal pathway. Conclusions: In this study, C-B-exo-miRNA-499a-5p significantly improved DOX-induced cardiotoxicity via CD38/MAPK/NF-κB signal pathway, providing a new idea and method for the treatment of DOX induced cardiotoxicity.


Introduction
Doxorubicin (DOX), an anthracycline antibiotic found in mutant streptomyces, is one of the most effective chemotherapeutic drugs.It is commonly used to treat many solid tumors and malignant hematological diseases, including breast cancer, lung cancer, lymphoma, ovarian cancer, soft tissue sarcoma, and leukemia [1] .However, in clinical, it has been found that the use of DOX produced many toxic and side effects, such as hematopoietic suppression, nausea, vomiting, extravasation, and alopecia, and the most serious was cumulative and dose-dependent cardiotoxicity [2,3] .Clinically, the cardiotoxicity of DOX is diverse [4] .Although DOX has serious toxic and side effects, it is still one of the main drugs for treating cancer.However, its cardiotoxicity needs further elaboration.
Bone marrow mesenchymal stem cells (BMSCs) are mammalian cells, which can be differentiated into bone, muscle tissue, and connective tissue.At present, BMSCs, as a kind of stem cells with multidifferentiation function, are widely used in the treatment of cardiovascular diseases [5] .Exosomes are endogenous vesicular particles that have attracted attention in recent years.They exist in almost all biological fluids, including blood, urine, saliva, cerebrospinal fluid, and cell pretreatment media [6] .Exosomes from different cell sources can carry different bioactive substances, such as lipids, proteins, nucleic acids, etc., which act as mediators of information exchange between cells and participate in the regulation of various cell biological functions.Therefore, exosomes play an important role in many diseases [7] .The aim of this study was to investigate the effects of BMSCs derived exosomes carrying miRNA-499a-5p on DOX induced cardiotoxicity.

Reagent
Creatine kinase MB (CK-MB) and lactate dehydrogenase (LDH) assay kits (Wuhan Huamei Bioengineering Co., Ltd.).Fetal bovine serum, phosphate buffer (PBS), and DMEM medium were purchased from America National Hyclone Company.Transfection reagent Transit-X2 was purchased from America Minas Company.Endotoxin-free plasmid small amount extraction kit was purchased from Qiagen Company of Germany.BCA protein quantitative kit was purchased from Beijing Pulilai Company.DOX was purchased from Sigma Company.Diamino polyethylene glycol was purchased from Yarebio Company.All antibodies were purchased from Cell Signaling Technology Company.

DOX stimulated H9c2 cells
H9c2 cells were cultured in DMEM medium containing 10% fetal bovine serum, 1% penicillin, and streptomycin in a 5% CO2 saturated humidity incubator at 371C.The experiment was divided into two groups: control group and model (M) group (1 μmol/L DOX) for 24 h.

miRNA chip analysis
The miRNA-microarray expression profile chip of Shanghai Bohao biological company was used for the experiment.The brief process was as follows: the cells were labeled with fluorescence and then was subjected to chip hybridization and subjected to rolling hybridization at 551C, 20 r/min, for 20 h in a rolling hybridization furnace.After hybridization was completed, the plates were washed in a washing tank.The chip results were scanned using Agilent Microarray Scanner and data were read using Feature Extraction software 10.7.1.1,and finally normalized using R language package and the algorithm used was Quantile.All miRNAs primer sequences are shown in Table 1.

Isolation and identification of BMSC and extraction of exosomes
Isolation and identification of BMSC Mice were killed with isoflurane (50 mg/kg).After being soaked and disinfected with 75% ethanol for 5 min, bilateral femurs and tibias were aseptically taken out and brought back to the cell chamber.The muscle, fascia, and other connective tissues attached to the bone surface in the ultra-clean table were removed, the bone marrow cavity along the longitudinal axis of the long bond was cut, repeatedly.The bone marrow cavity was rinsed with 3 ml of low sugar DMEM complete medium containing 10% fetal bovine serum until the bone marrow cavity became white, the rinsing solution was sucked into a 50 ml plastic culture flask for use.The remaining bone tissue in the glass dish was cut into about 1 mm 3 bone fragments, soaked in 0.1% type II collagenase, shaken and digested for 90 min.Finally, the digested bone fragments were inoculated into the culture flask with bone marrow liquid, and placed horizontally in the culture box for static culture.After 48 h, the first full amount of liquid shall be changed, and then half amount of liquid shall be changed once every other day.

Gens
Primer sequence (5'-3') Data can be requested through the corresponding author.

Observation of cell morphology
The morphology, adherence, density, and fusion degree of primary and subculture cells were observed under a microscope.

Molecular detection of surface marker
The BMSCs were cultured to about 60% confluence, and then fluorescent CD90, CD73 and CD29, antibodies were added in turn.After incubation at 4°C for 40 min, CDs was added for flow cytometry detection.

Extraction of exosomes from BMSC
After 48 h of cell culture, the culture medium was collected and transferred to an ultrafiltration centrifuge tube.The culture medium was centrifuged at 41C at 12736 r/min for 20 min to further remove cells and cell debris and then passed through 0.2 μM to remove particles > 200 nm.Exosomes were collected by centrifugation at 34 288 r/min for 80 min at 41C, and the resulting precipitates were exosomes (B-exo).

Transmission electron microscope (TEM) examination
The B-exo was dropped on the wax tray, the surface with supporting film was contacted with the surface of the B-exo solution by the copper mesh, and it was taken out after standing for 3-5 min.The copper mesh was taken out, and the excess droplets were sucked out with filter paper strips and dried slightly.Three percent phosphotungstic acid solution was dropped on the wax dish.The copper mesh was adsorbed with the sample on the surface of the dye solution (B-exo) was in contact with the dye solution) for 3-5 min.The copper mesh was taken out, the excess droplets were sucked out with filter paper strips, and dried under incandescent lamps.TEM photographs were taken to observe the morphology of B-exo.

Nanoparticle tracking analysis (NTA)
The B-exo with 1 × PBS buffer was diluted appropriately and particle size was analyzed (Zeta view system was calibrated with 110 nm polystyrene particles, and the temperature was maintained at 23 and 371C).

Determination of exosomes surface markers
The expression of exosome marker proteins CD63, CD9, and TSG101 were detected by Western blot.

Localization of C-B-exo in vitro
DiR-labeled C-B-exo and B-exo C-B-exo or B-exo was incubated with 1 mol fluorescent lipophilic tracer DiR at room temperature for 15 min and then DiR-C-Bexo and DiR-B-exo were obtained.
Small animal imaging analysis 60 μl of DiR-C-B-exo and 60 μl of DiR-B-exo were injected into the mice via tail vein respectively.After 2 h injection, the mice were put into a small animal living imager (IVIS SPECTRUM) for back and abdomen living imaging.

Relationship between miRNA-499a-5p and DOX-induced cardiotoxicity
Search for miRNA-499a-5p target gene The target gene of miRNA-499a-5p was calculated by using bioinformatics analysis method and miRbase and Targets-can software database.

Analysis of double luciferase reporter gene
The construction of plasmid was completed by Guangdong Ruibo biological Co., Ltd.Plasmid and miRNA-499a-5p were co-transfected as follows: 25 μl dilute miRNA-499a-5p NC or miRNA-499a-5p mimics with serum-free opti-MEM, and gently mixed with the gun head.25 µl serum free opti-MEM was diluted with 1 µl reporter plasmid.Plasmids were divided into two types: wild-type (WT) and mutant plasmids (Mutant) were mixed.25 μl serum free opti-MEM was diluted with 1.5 μl lipofectamine 3000.The final reaction system was 100 μl, and the remaining liquid was replaced by serum-free medium.100 μl of fish phospholipase B was added to cells/each well, the suspension was added to 96 well plate (50 μl/per well), then 100 μl LAR-II was added, the fluorescein reaction intensity value was recorded at the same time, then 100 μl of reaction termination solution was added and finally the fluorescein reaction intensity value of fluorescein was recorded.The results were expressed by firefly fluorescein reaction intensity value/firefly fluorescein reaction intensity value.

Detection of target gene-related gene and protein expression by PCR and Western Blot
Total RNA of transfected cells was extracted by Trioal.After the concentration was determined, reverse transcription was performed by reverse transcription kit, and then fluorescent PCR was performed by AeCQ PCR SYBR Green Master Mix kit.The transfected cells were obtained, and the protein expression of related genes was detected by Western blot.

Relevant index detection and relevant methods
Myocardial enzyme (CK), Phosphocreatine kinase isoenzyme and lactate dehydrogenase (LDH) test The levels of CK, CK-MB, and LDH in serum were detected by related kits.The operation process strictly followed the instructions of the kits.

Cytokine measurement
The concentrations of IL-6, IL-1β, and TNF-α in serum, heart tissues and cell supernatant were analyzed by enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer's instructions.

Hematoxylin-eosin (H&E) staining
The heart was fixed in 4% formalin solution, embedded in paraffin, cut into 4 μm thick sections, and placed on a glass slide for hematoxylin-eosin staining.

Echocardiographic determination
The mice in each group were examined by transthoracic color Doppler echocardiography.Left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), left ventricular internal dimension-diastole (LVIDD), and left ventricular internal dimension-systole (LVIDS) were measured by ultrasound through the sternal left heart long axis section.
Cell viability was detected by CCK-8 method.

Immunohistochemistry
CD38 protein in heart tissue was detected by immunohistochemistry, specific operation methods according to previous report [3] .Heart tissue blank slices were baked at 601C for 1 h, then paraffin was removed by xylene, dehydrated by gradient ethanol and heated by sodium citrate buffer for antigen repair.
After natural cooling to room temperature, it was cultured with 3% hydrogen peroxide for 10 min.Each section was sealed with 3% BSA at room temperature.After removing the blocking solution, the section was incubated with the primary antibody at 41C overnight, the secondary antibody was incubated for 10 min, and PBS was washed three times, each time for 3 min, the third antibody was incubated for 10 min and washed with PBS for 3 min each time.Samples were stained with DAB and hematoxylin, dehydrated by gradient ethanol and xylene, dried, sealed with neutral resin, and the expression of related proteins in lung tissue was observed under light microscope.

Western blot analysis
CD38/MAPKP38/NF-κB signaling pathway related proteins in heart tissue and H9c2 cells were detected by Western blot analysis, specific operation methods according to previous report [3] .SDS-PAGE electrophoresis was performed on protein samples and transferred to PVDF membrane.The 5% skimmed milk powder was sealed at room temperature for 2 h, and incubated with related equal primary antibodies, respectively.The membrane was rinsed with TBST and then reacted with horseradish peroxidase coupled secondary antibody.The membrane was rinsed with TBST and then developed with enhanced chemiluminescence (ECL) luminescent reagent.The optical density of the main band was measured by gray-scale imaging software (UVP, UK) to calculate the expression level of the above proteins in lung tissue.

Immunofluorescence
The levels of CD38 in H9c2 cells were evaluated by immunofluorescence, specific operation methods according to previous report [3] .H9c2 cells were exposed to 5% Triton X-100 room temperature for 20 min, washed three times with PBS, and sealed at room temperature with sheep serum for 1 h and corresponding primary antibodies (all at 1:1000) were added and incubated overnight at 41C.The next day, it was incubated at room temperature for 1 h, washed three times in PBS, labeled with fluorescent secondary antibody, incubated in dark at 371C for 30 min, washed three times in PBS, and observed under a fluorescence microscope

Statistical analysis
All data were expressed as mean SD and analyzed by one-way analysis of variance (ANOVA) followed by Tukey multiple comparison test using GraphPad prism 8 (GraphPad Software, USA).A value of P < 0.05 was considered statistically significant.

Analysis of differential miRNAs between control H9c2 cells and DOX-induced H9c2 cells
Since miRNA plays a role in regulating DOX-induced cardiotoxicity, this study used miRNA chip to detect the difference of differential miRNAs between control H9c2 cells and DOXinduced H9c2 cells (Fig. 1A).There were eight differentially expressed miRNAs between control H9c2 cells and DOXinduced H9c2 cells were detected.Among them, miRNA-499a-5p, miR-146a, miR-143 were significantly down-regulated.And miRNA-499a-5p was most down-regulated most obviously.The other five miRNAs were significantly up-regulated.Therefore, miRNA-499a-5p attracted our attention (Fig. 1B).In order to further confirm the down-regulation of miRNA-499a-5p expression in DOX-induced cardiotoxicity in vivo and in vitro, PCR was used to detect the expression of miRNA-499a-5p in DOX-induced cardiotoxicity in vivo and in vitro.The results showed that the expression of miRNA-499a-5p in DOXinduced models was significantly lower than that of control (Fig. 1C-D).
In order to verify the changes of CD38, we investigated the expression of CD38 gene in DOX-induced cardiotoxicity in vivo and in vitro, and in vitro models, and found that in in vivo and in vitro M groups, CD38 mRNA significantly increased (Fig. 2B-C).Compared with the wild-type 3'-URT, the double luciferase activity of CD38-wt + miRNA-499a-5p group significantly decreased, and the double luciferase activity of mutant group did not significantly change (Fig. 2D-F).
The expression of CD38 protein was further detected, and the expression of CD38 proteins in DOX-induced cardiotoxicity in vivo and in vitro were investigated.The results showed that M groups in vivo and in vitro, CD38 protein was significantly increased (Fig. 2 G-H).These results suggested that the expression of miRNA-499a-5p was negatively correlated with the expression of CD38 (Fig. 2 I-J).
Other literatures indicated that the expression of CD38 was MAPK pathway dependent.In LPS stimulated dendritic cells, p38 inhibitor effectively inhibited the upregulation of CD38 expression, ERK inhibitor also has a small inhibitory effect on CD38 expression, while JNK inhibitor has no obvious effect on CD38 expression [9] .Therefore, the interaction between CD38 and P38 protein was detected by immunoprecipitation.The result showed that CD and P38 could be endogenous bound into protein complexes (Fig. 3 A-B).These results suggest that there was an interaction between CD38 and P38.
To further confirm the relationship between CD38 and P38, in this experiment, CD38 inhibitor luteolinidin (Lut, 0, 2.5, 5, 10 µM) was used to treat H9c2 cells to detect the expression of P38.The results showed that Lut significantly inhibited the expression of P38 (Fig. 3 C).Conversely, in this experiment, P38 inhibitor SB203580 (0, 1, 2, 4 µM) was used to treat H9c2 cells to detect the expression of CD38, the results showed that SB203580 significantly inhibited the expression of CD38 in a dose-dependent manner (Fig. 3 C).The above results showed that CD38 was positively correlated with p38 and there was an interaction between CD38 and P38 (Fig. 3 D).

TME observation of B-exo
TEM results clearly show that the BMSC derived exosomes are in the shape of saucers, with double-layer membrane structure and complete membrane structure (Fig. 4A).

Nanoparticle tracking analysis of B-exo
Through NTA particle size analysis, the B-exo with an average particle size of 79.6 nm (Fig. 4B).The particle size distribution conforms to the exosome particle size.

B-exo marker protein test results
As shown in Figure 4C, the expressions of marker proteins of B-exo were detected by Western blot.The results showed that CD63, CD9, and TSG101 were highly expressed, which was consistent with the characteristics of exosome surface markers.

Identification of BMSC
As shown in Figure 4D, under light microscope, it was found that BMMCs gradually generated spindle cells from day 3 to day 7.The results of flow cytometry showed that, CD29, CD90, and CD73 were positive (Fig. 4E).

C-B-exo-miRNA-499a-5p can reach the target organ-heart tissue of mice and cardiac release of miRNA-499a-5p
DiR-C-B-exo were injected into the tail vein of the mice.After in vivo imaging, it was found that fluorescence showed in the heart of mice.The results showed that C-B-exo could reach the target organ-heart tissue (Fig. 5A).It was suggested that CHP contributed to the targeted delivery of miRNA-499a-5p to the heart via B-exo.
As shown in Figure 5B-C, the levels of miRNA-499a-5p were significantly rapidly increased in heart of C-B-exo-miRNA-499a-5p group, and the decrease rate of miRNA-499a-5p content was very slow in heart of C-B-exo-miRNA-499a-5p group.While, the levels of miRNA-499a-5p were increased slowly in in heart of B-exo-miRNA-499a-5p group and the decrease rate of miRNA-499a-5p content was fast heart of B-exo-miRNA-499a-5p group.

The effects of C-B-miRNA-499a-5p on DOX-induced cardiotoxicity in vivo and in vitro
The effects of C-B-miRNA-499a-5p on CK, CK-MB, and LDH in DOX-induced mice As shown in Figure 6 A-F, the levels of CK, CK-MB, and LDH in DOX-induced mice were markedly increased in DOX group than control group.Meanwhile, B-exo-miRNA-499a-5p and C-B-exo-miRNA-499a-5p markedly reduced the levels of CK, CK-MB, and LDH in DOX-induced mice.And the effect of C-B-exo-miRNA-499a-5p was better than that of B-exo-miRNA-499a-5p, suggesting that CHP was helpful for targeted delivery of miRNA-499a-5p.

The effects of C-B-miRNA-499a-5p on heart histopathology and echocardiography in DOX-induced mice
The cardiomyocytes in the control group were evenly arranged, with normal morphology and no abnormal morphological cells.In the M group, myocardial cell edema, vacuolar degeneration, disordered arrangement, cell proliferation, and even interstitial fibrosis appeared.In B-exo-miRNA-499a-5p and C-B-exo-miRNA-499a-5p groups mice, cardiomyocytes were arranged evenly, edema was reduced, vacuolar degeneration was reduced, and cell proliferation was not obvious.
Echocardiography is a common method to evaluate cardiac function.Compared with the control group, LVEF and LVFS of mice in DOX (M) group were significantly decreased, and LVIDS were significantly increased.Compared with DOX group, LVEF and LVFS were significantly increased and LVIDS were significantly decreased in B-exo-miRNA-499a-5p and C-B-exo-miRNA-499a-5p groups (Fig. 7).

The effects of C-B-miRNA-499a-5p on cell viability and cytokine in DOX-induced H9c2 cells
As shown in Figure 10, DOX significantly increased levels of TNF-α, IL-1β, IL-6 in cell supernatant of H9c2 cells and the decrease of cell viability.Compared with DOX group, B-exo-miRNA-499a-5p and C-B-exo-miRNA-499a-5p significantly decreased the levels of TNF-α, IL-1β, and IL-6 and increased cell viability.
The effects of C-B-miRNA-499a-5p on CD38/MAPKP38/NF-κB pathway in DOX-induced H9c2 cells As shown in Figure 11A-H, compared with control group, the levels of CD38, p-P38, and p-NF-κBp65 were significantly increased in DOX group.Compared with DOX group, B-exo-miRNA-499a-5p and C-B-exo-miRNA-499a-5p significantly decreased the levels of CD38, p-P38, and p-NF-κBp65.In  As shown in Figure 12 A-C, the levels of CK, CK-MB, and LDH in DOX-induced mice were significantly increased than control group.B-exo-miRNA-499a-5p and C-B-exo-miRNA-499a-5p significantly reduced the levels of CK, CK-MB, and LDH.
Heart histopathology: The cardiomyocytes in the control group were evenly arranged, with normal morphology and no abnormal morphological cells.In the M group, myocardial cell edema, vacuolar degeneration, disordered arrangement, cell proliferation, and even interstitial fibrosis appeared.In B-exo-miRNA-499a-5p and C-B-exo-miRNA-499a-5p groups mice, cardiomyocytes were arranged evenly, edema was reduced, vacuolar degeneration was reduced, and cell proliferation was not obvious (Fig. 12H-K).

Discussion
DOX is a widely used anticancer drug.However, DOX induced cardiomyopathy is a major obstacle to its use in cancer chemotherapy.Although many mechanisms have been demonstrated to be related to DOX induced cardiotoxicity, the exact and complete mechanism is unclear [10] , and the current treatment is not very satisfactory.In this study, a new therapeutic method was proposed, which is engineered exosomes loaded miRNA to the heart to treat DOX induced cardiotoxicity.
MicroRNAs (microRNAs, miRNAs) are a class of endogenous small molecule non coding RNAs with a length of  about 20-25 nucleotides, which mediate and regulate a variety of pathophysiological processes in vivo [11,12] .In this study, the DOX induced H9c2 cell injury model was established and eight different miRNAs were identified by miRNA chip technology of which miRNA-499a-5p was the most different miRNA.It was further found that the expression of miRNA-499a-5p was significantly decreased in the DOX induced cardiotoxicity model.It was suggested that miRNA-499a-5p may be a potential target for intervention of DOX induced cardiotoxicity.Extracellular vesicles are receiving increasing attention in heart related diseases.The cardioprotective effect of exosomes originally came from stem cell transplantation after myocardial infarction [13,14] and exosomes are nano vesicles secreted by cells.It has been proved that exosomes are a natural carrier for drug delivery [15,16] .In addition, exosomes have high affinity and natural targeting ability.For example, exosomes can cross the bloodbrain barrier and be selectively taken up by glial cells [17] .Exosomes have the intrinsic ability to carry and protect miRNA, cross biological barriers, and transport miRNA to rake cells for a long distance, and are nonimmunogenic to the host, exosomes can be used as a new delivery vehicle for the therapeutic use of miRNA.In this study, miRNA-499a-5p mimic was electrotransferred to exosomes.In addition, in order to deliver miRNA-499a-5p to the heart, the exosomes were engineered with the cardiac homing peptide to deliver miRNA-499a-5p to the heart.
In this study, we found that the target gene of miRNA-499a-5p was CD38 via bioinformatics.Previous studies have shown that miRNA-499a-5p was differentially expressed in cardiomyocytes and is closely related to a variety of heart diseases [17] .It has been reported that miRNA-499a-5p reduced cardiomyocyte apoptosis and lactate dehydrogenase (LDH) activity induced by hypoxia/reoxygenation by downregulating CD38 protein, thereby alleviating cardiomyocyte injury [18] .This study found that miRNA-499a-5p was negatively correlated with CD38 and MAPKp38 was positively correlated with CD38 expression.B-exo-miRNA-499a-5p and C-B-exo-miRNA-499a-5p significantly inhibited CD38/MAPK/NF-κB pathway to alleviate DOX-induced cardiotoxicity.

Conclusions
In conclusion, this study found that miRNA-499a-5p is a potential marker of DOX induced cardiotoxicity, and the targeted delivery of miRNA-499a-5p via engineered exosomes effectively improved DOX induced cardiotoxicity.This study provides a new treatment for DOX induced cardiotoxicity.

Limitations and prospect
Limitations: (1) The discovery of miRNA-499a-5p is based on the establishment of a DOX induced H9c2 cell model in vitro.Next, we will conduct validation of miRNA-499a-5p for clinical DOX induced cardiotoxicity patients.(2) This study only focused on the role of miRNA-499a-5p, and did not investigate other changed miRNAs, which has certain limitations.Therefore, in the future, we will conduct in-depth research on other miRNAs in DOX induced cardiotoxicity.
Prospects: (1) The discovered miRNA-499a-5p in this study can serve as a potential clinical diagnostic criterion.In clinical practice, a decrease in miRNA-499a-5p was found after DOX chemotherapy, which can provide a warning for DOX induced cardiac toxicity.(2) This study provides a rapid detection method for DOX induced cardiac toxicity in clinical practice.In clinical practice, DOX induced cardiac toxicity can be quickly warned.

Figure 3 .
Figure 3. Immunoprecipitation of CD38 and P38.(A-B) The total protein cleaved by H9c2 cells was co precipitated with IgG, P38 antibody, or CD38 antibody of the control group, and then western blot was performed with P38 antibody or CD38 antibody, respectively, to confirm the binding of P38 to CD38.(C) CD38 inhibitor luteolinidin (Lut, 0, 2.5, 5, 10 µM) was used to treat H9c2 cells to detect the expression of P38, P38 inhibitor SB203580 was used to treat H9c2 cells to detect the expression of CD38.(D) Correlation between CD38 and P38.