Substance P Enhances Doxorubicin (Adriamycin)-associated Autophagy and Reduces Cardiomyocyte Apoptosis.

Background Anthracyclines, including doxorubicin, are some of the most potent anticancer drugs available. However, the use of doxorubicin as a chemotherapeutic agent is severely hindered by dose-limiting toxicity, particularly cardiotoxicity, while degrading other organ systems. Despite years of use and the number of details published on this drug, the understanding of its cellular mechanisms remains limited. Method Cardiomyocyte grouping was carried out, where H9C2 cardiomyocytes were randomly divided into a control group, a myocardial model group, an group, and an SP antagonist group. For replication in animal models, twelve rats were similarly randomly assigned into a control group, a myocardial model group, an SP group, and an SP antagonist group. Except for the control group, the rats in the other groups were modelled: the rats were injected with adriamycin solution. Flow cytometry was used to detect apoptosis, while HE staining, TUNEL stain, Western Blot detection, and transmission electron microscopy were performed to detect autophagy levels accordingly. All the results were analysed and carefully interpreted.

containing nerve bres are often found around coronary vessels, making them ideally situated to detect changes in the myocardial environment. Apoptosis and autophagy are genetically regulated, evolutionaryconserved processes that regulate cell fate and are important for development, normal physiology, and a wide range of diseases. Recent studies show that despite the signi cant differences between these two processes, their regulation is closely connected and certain regulators are able to control both apoptosis and autophagy.

Conclusions
In this study, the in uence of substance P was discussed, providing possible molecular mechanisms for crosstalk between apoptosis and autophagy mediating heart failure due to doxorubicin therapy. Background 1. In physiological conditions, autophagy and apoptosis play essential roles in cardiac health and integrity, with the structure and function of cardiac myocytes closely related to autophagic ux.

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Cardiac myocytes retain a limited ability to re-enter the cell cycle, leading to a limited regeneration capacity in the adult heart. As a result, a continuous process of cell renovation including the removal and replacement of damaged tissue is present. In addition, autophagy is necessary for continual heart contraction and large cytoplasmic calcium transients without disrupting cardiac function [1].
During heart development, apoptosis is involved in the development of the embryonic out ow tract, cardiac valves, conducting system, and coronary vasculature [2]. In pathological conditions, the interplay between autophagy and apoptosis is closely linked to certain cardiac diseases including ischaemic heart disease, pressure overload-induced cardiac disease, and diabetic cardiomyopathy.
2. Substance P (SP) belongs to the tachykinin family of sensory nerve neuropeptides and other classical members of this family include neurokinin A (NKA) and neurokinin B (NKB). Although NKB is encoded by its gene (TAC2) and is restricted to the central nervous system, SP and NKA are both encoded by the TAC1 gene and are located in the central nervous system and peripheral afferent sensory neurons [3].
3. There has been a focus on SP due to its good and bad sides in myocardial remodelling and heart failure. It offers protection following ischaemia-reperfusion [4], however, it also causes long-term nonischaemic myocardial remodelling and heart failure [5].  Figure 1). Studies have found that treating rats with heart failure using drugs can effectively reduce cardiomyocyte autophagy and inhibit cardiomyocyte apoptosis, thereby improving the heart function of the rats [10]. Although the apoptosis and autophagy systems can perform their functions independently, they are also clearly related to each other, with both systems jointly regulating cell biological processes through crosstalk, thus participating in the occurrence and development of heart failure. It is currently believed that apoptosis-autophagy crosstalk can provide new ideas and directions for the study of heart failure pathogenesis and therapeutic targets.
5. Substance P (SP) is released in large quantities when the heart is noxiously stimulated, and plays a dual role in the occurrence and development of heart failure: During the acute ischaemic phase of the heart, SP exerts myocardial protection through strong vasodilation, especially in the diastolic coronary arteries. Both non-ischaemic-induced myocardial remodelling and chronic heart failure are detrimental [12]. Several studies, including Valdemarsson [13], con rmed that the expression of SP in the plasma of patients with chronic heart failure is signi cantly higher than that of healthy people.
Moreover, the SP level in plasma of patients with heart failure and angina symptoms is higher than patients without angina [14].
. The studies mentioned above suggested that SP plays a pathogenic role in the pathogenesis of heart failure. However, the molecular mechanism of SP-mediated heart failure is not yet clear. In recent years, studies have shown that the increase of SP expression, whether in hair tissue or bladder tissue, signi cantly improves autophagy activity [15,16] and that SP has a clear induction effect on autophagy. In this study, doxorubicin was used to induce cardiomyocyte damage in vitro and exogenous SP was used for intervention during the preliminary test. Findings showed that the expression of cardiomyocyte autophagy marker Beclin-1 signi cantly and the apoptosis rate of cardiomyocytes signi cantly increased. These preliminary experimental results indicated that the mechanism of SP-mediated heart failure could be related to the regulation of autophagy/apoptosis [17], but remained unclear.

Methods
The study was submitted to and approved by the Flow cytometry to detect apoptosis 1 × 10 6 -3 × 10 6 cells were collected, then added with 1 ml PBS, centrifuged at 1500 rpm for 3 minutes, and washed twice. A 5 × Binding Buffer was diluted to 1 × with double distilled water and 300 ul of prechilled 1 × Binding Buffer was used to resuspend the cells. 3 ul Annexin V-FITC and 5 ul PI-PE were added to each tube. After mixing slightly, each tube was incubated for 10 minutes at room temperature in the dark, then 200 ul of pre-cooled 1 × Binding Buffer was added to each tube. After mixing, the mixtures were tested using a ow meter.

Animal model replication and drug treatment
Twelve rats were randomly divided into a control group, a myocardial model group, an SP group, and an SP antagonist group. Except for the control group, the rats in the other groups were modelled: the rats were injected with the prepared adriamycin solution at a dose of 3 mg/kg (the adriamycin concentration was 1 mg/ml, so the injection volume was 3 ml/kg). The injection was carried out once every 3 days, 5 times in a row. In the same period, the control group was intraperitoneally injected with the same amount (3ml/kg) of normal saline once every 3 days for 5 consecutive times. Every 4 days, the SP group was injected with exogenous SP through the tail vein at a dose of 6.7 μg/kg (exogenous SP concentration was 1 μg/ml, so the injection volume was 6.7 ml/kg), the SP antagonist group injected with the SP antagonist at a dose of 6.7 μg/kg (SP antagonist concentration was 1 μg/ml, so the injection volume was 6.7 ml/kg), and both the control and myocardial model group were injected with the same amount (6.7 ml /kg) normal saline.

HE staining
The tissues were taken out and rinsed with running water for several hours, dehydrated by 70%, 80%, and 90% ethanol solutions, mixed with pure alcohol and xylene for 15 minutes, xylene I for 15 minutes, and xylene II for 15 minutes (until transparent). Then, the tissues were placed in a mixture of xylene and para n in half for 15 minutes, followed by placement in para n I and para n II for 50-60 minutes each before embedded in para n and sectioned. The para n slices were baked, dewaxed, and hydrated then put in a hematoxylin aqueous solution, with distilled water added, and stained for 3 minutes.
Subsequently, the slices were placed into a hydrochloric acid ethanol differentiation solution for 15 seconds and washed with water. The solution was allowed to turn back to blue for 15 seconds then rinsed with running water and stained with eosin for 3 minutes. Finally, the samples were rinsed with running water, dehydrated, and covered with a transparent lm for microscopic examination.

TUNEL stain
The tissue sections were placed in xylene for 10 minutes. After that, xylene was removed and the samples were allowed to depara nise for 10 minutes, before being put in gradient ethanol for hydration. The slices were then placed into a wet box, with 50 μg/ml Proteinase K working solution added dropwise to each sample, and allowed to react at 37 °C for 30 minutes for antigen and antibody retrieval. Following that, the slices were washed thoroughly with PBS 3 times for 5 minutes each time. An absorbent paper was used to absorb the PBS around the tissue, with a su cient amount of TUNEL detection solution added to each slide, and the samples were incubated for 2 hours at 45 °C in the dark. Then, the samples were washed with PBS 3 times for 5 minutes each time and the liquid was blotted on a glass slide with absorbent paper. Finally, the slides were mounted with anti-uorescence quenching and observed under a uorescence microscope.

Western Blot detection
The tissue was taken and ground into a powder with liquid nitrogen, then the lysis solution was added and the tissue was ground again. Following this, the tissue homogenate was sucked into an Eppendorf (EP) tube, lysed on ice for 30 minutes, centrifuged at 12,000 rpm for 15 minutes and the supernatant was carefully aspirated to obtain the total protein. The protein concentration was determined according to the BCA kit, then the protein was denatured, loaded, and subjected to sodium dodecylbenzene sulfonate gel electrophoresis (SDS-PAGE) for 2 hours. It was then transferred to the membrane with a constant current of 300 mA for 80 minutes. The primary antibody solution was incubated overnight at 4 °C before incubation with the secondary antibody solution for 2 hours at room temperature. The Chemiluminescence (ECL) luminescent liquid was dropped onto the lm and exposed in the gel imaging system. The "Quantityone" software was used to analyse the grey value of each antibody band.
Transmission electron microscopy to detect autophagy levels After the embedded para n sections were depara nised and dehydrated, the sections were cut into 80 nm thickness using an ultra-thin microtome, and then stained with 3% uranyl acetate-lead citrate, washed with distilled water, and the autophagosomes were observed under a transmission electron microscope.

Results
The effect of SP on the viability of H9C2 cells induced by Adriamycin As shown in Figure 2, the cell proliferation rate of the myocardial model group was signi cantly reduced compared to the control group (P < 0.05), while a comparison between the myocardial model and SP group showed that there was a signi cant increase in the cell proliferation rate of the SP group (P < 0.05). For the SP antagonist group, there was a signi cantly decreased cell proliferation rate compared to the SP group (P < 0.05).
The effect of SP on H9C2 cell apoptosis induced by Adriamycin Figure 3 demonstrates that the apoptosis rate of the myocardial model group was signi cantly increased compared to the control group (P < 0.05), while the apoptosis rate of the SP group was signi cantly decreased compared to the myocardial model group (P < 0.05), and the SP antagonist group mortality rate was signi cantly increased compared to the SP group (P < 0.05).
The effect of SP on Bax, Bcl-2, Beclin-1, LC / protein expressions in H9C2 cells induced by Adriamycin Figure 4 shows that the expressions of Bcl-2, Beclin-1, and LC / protein in the myocardial model group were signi cantly down-regulated (P < 0.05), and the expression of Bax protein was signi cantly upregulated (P < 0.05) compared to the control group. A comparison between the myocardial model and SP groups revealed that Bcl-2, Beclin-1, LC / protein expressions were signi cantly down-regulated (P < 0.05) but Bax protein expression was signi cantly up-regulated (P < 0.05) in the myocardial model group.
The effect of SP on the pathological changes of the myocardium in rats with doxorubicin Based on Figure 5, the cardiomyocytes of the control group were neatly arranged without any in ammatory factor in ltration, the myocardial cell structure of the myocardial model group was disordered with myocardial hypertrophy and a large number of in ammatory factors in ltrated, the cardiomyocytes of the SP group were more neatly arranged and had fewer in ammatory cells, while the cardiomyocytes of the SP antagonist group were arranged disorderly and a large number of in ammatory cells that in ltrated.
The effect of SP on cardiomyocyte apoptosis induced by Adriamycin According to Figure 6, the apoptosis rate of the myocardial model group was signi cantly increased compared to the control group (P < 0.05) while the apoptosis rate of the SP group was signi cantly decreased compared to the myocardial model group (P < 0.05) and the SP antagonist group mortality rate was signi cantly increased compared to the SP group (P < 0.05).
The effect of SP on the level of myocardial autophagy in rats with myocardial model induced by Adriamycin As observed in Figure 7, the autophagy level of the myocardial model group was signi cantly reduced compared to the control group, (P < 0.05), while the autophagy level of the SP group was signi cantly increased compared to the myocardial model group (P < 0.05) and the autophagy level of the SP antagonist group signi cantly decreased compared to the SP group. (P < 0.05).
The effect of SP on the expressions of Bax, Bcl-2, Beclin-1 and LC / protein in the myocardial tissue of rats with a doxorubicin-induced myocardial model As shown in Figure 8, the expressions of Bcl-2, Beclin-1, and LC / protein in the myocardial model group were signi cantly down-regulated (P < 0.05), and the expression of Bax protein was signi cantly upregulated (P < 0.05) compared to the control group. A comparison of the myocardial model group to the SP group revealed that the Bcl-2, Beclin-1, and LC / protein expressions were signi cantly downregulated (P < 0.05) while Bax protein expression was signi cantly up-regulated (P < 0.05). The SP antagonist group's Bcl-2, Beclin-1, LC / protein expression were signi cantly down-regulated (P < 0.05), and Bax protein expression was signi cantly up-regulated (P 0.05) compared to the SP group.

Discussion And Conclusion
In summary, doxorubicin-induced H9C2 myocardial cell viability decreased and apoptosis rate increased, while rats with doxorubicin-induced heart failure had an increased myocardial apoptosis rate and decreased autophagy. After SP intervention, the viability of H9C2 cardiomyocytes induced by doxorubicin was signi cantly increased. The rate of apoptosis was reduced, and the rate of myocardial apoptosis in rats with heart failure decreased, with an increase in autophagy level. This process could be related to the up-regulation of Bcl-2, Beclin-1, and LC / protein expressions and the down-regulation of Bax expression.

Abbreviations
Substance P: SP