Protective Effect of Deer Heart Peptide on Cardiac Injury in Mice

Peptides are widely used as natural bio-small molecules because of their various pharmacological activities such as enhancing immunity, promoting wound healing, and improving inflammation. Alcoholic heart injury has become one of the major health problems worldwide, and alcohol consumption is now the main cause of alcoholic cardiomyopathy. In this study, deer heart peptides were extracted from deer hearts by enzymatic digestion and the antioxidant activity of deer heart peptides extracted at different times was evaluated by three in vitro antioxidant methods, and the active peptide with the best enzymatic effect has been selected for in vivo animal experiments. The anti-inflammatory and antioxidant properties of deer heart enzymatic extracts were evaluated in in vivo experiments in mice. In this study, mice were orally gavaged with white wine (12 mL/kg body weight) to induce a mouse model of cardiac injury, while mice were orally administered a single dose of 100 mg/kg/bw and 200 mg/kg/bw of deer heart enzyme digest and were examined for body weight, dietary intake, water intake, and coat gloss, as well as for general behaviors, adverse effects, and mortality. Histology, serum, anti-inflammatory factors, and oxidative stress parameters were subsequently assessed. In all modeled mice, no four-way or any significant behavioral changes were observed in all groups, but in the modeled group, mice showed weight loss, decreased diet and water intake, and decreased cardiac index. For in vivo tests, the extract inhibited the anti-inflammatory activity with a significant decrease in inflammatory factors of TNF-α, IL-6, and IL-1β in cardiac tissues, a significant increase in serum levels of both CAT and SOD, an increase in MDA content, and a remarkable increase in the level of the marker CK in the cardiac myocardial enzyme profile. Significant improvement in myocardial disorders by deer heart peptide could be observed from heart tissue sections. The present study emphasizes the anti-inflammatory and antioxidant activity of deer heart peptide, an enzymatic digest of deer heart, which provides empirical as well as supportive role for the anti-inflammatory properties of traditional medicine.


Introduction
Cervus nippon is a valuable medicinal and animal-based food in China, and its body is full of treasures and extremely rich in bio actives [1].China is the frst country in the world to breed and produce C. nippon, and with the development and progress of the times, many countries in the world, such as Japan, Korea, and the United Kingdom, have also started to breed C. nippon on a large scale and develop and utilize its related functional products [2,3].Products recovered from C. nippon are well known for its health promising properties, thus identifed as valuable medicinal herbs at home and abroad.Its deer heart, deer blood, and antler blood are the hot spots for the development of plum deer resources in China in recent years [4].Deer hearts mostly refers to the heart of plum deer or horse deer [5], which is rich in a variety of amino acids, vitamins, peptide proteins, and other substances and is a good medicine to enhance the metabolic function of human body and improve cardiovascular diseases and nervous system functions [6].As far as our traditional Chinese medicine theory is concerned, the tissues and organs of animals have protective, preventive, and therapeutic efects on the corresponding tissues and organs of the human body [7].In recent years, it has been shown that small molecule active peptides from deer heart can regulate cardiac dysfunction, promote blood circulation, and replenish myocardial prime mover, thus achieving a protective efect on the heart [8].
Peptides are protein fragments formed by peptide bonds connecting two or more amino acid molecules and are biologically active substances with multiple cellular functions.Peptides are classifed into dipeptides, tripeptides, and polypeptides according to amino acid residues, and they are a class of protein precursors or degradable products between proteins and amino acids [9,10].In recent years, peptides have become popular among domestic and foreign researchers owing to their small molecular weight, easy absorption by the body, high activity, and low side efects [11].According to the available studies, peptides have various functional characteristics such as immunomodulation, antioxidant, hypotensive, antibacterial, and antiviral [12][13][14][15].
Dong [16] et al. have pointed out that the chemical composition of plum deer heart is complex and has revealed that it contains a variety of physiologically active substances, and its products can signifcantly increase coronary blood fow in rats under specifc experimental conditions.It showed that the heart of the plum deer has a signifcant protective efect on the cardiovascular system.Zhao [17] et al. showed by UPLC analysis that the enzymatic digest of deer heart contained 17 amino acids, the content of which was higher than that of deer heart extract without enzymatic treatment, and the nutritional value was higher.Chen [18] et al. demonstrated that deer heart small molecule active peptides signifcantly reduced the release of creatine kinase (CK), aspartate transaminase(AST), and LDH-L after H/R injury; decreased the content of lipid peroxide metabolite malondialdehyde (MDA); increased the SOD activity of cardiomyocytes; inhibited the expression of infammatory factors tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β) in cardiomyocyte cultures; and had a protective efect on cultured cardiomyocytes in vitro.
Te proper functioning of the heart depends on a timely and adequate supply of blood from the coronary arteries.When the blood supply to the heart is inadequate, cardiac ischemia or myocardial hypoxia can occur.Myocardial ischemia or hypoxia can lead to cellular metabolic disorders, cellular damage, and thus heart dysfunction, which can lead to many heart diseases [19].Cardiovascular disease is the main disease that endangers human life, and its incidence is increasing year by year [20].Currently, alcohol consumption is the main cause of dilated cardiomyopathy in China, and dilated cardiomyopathy caused by alcohol consumption, also known as alcoholic cardiomyopathy, is also common in clinical practice.Te heart is damaged, causing ischemia and hypoxia in the myocardial cells of the blood supply area, resulting in myocardial cell necrosis.Cardiomyocytes are the basic units that make up the heart and are contractile and diastolic.Alcohol consumption leads to fbrosis of cardiomyocytes and interstitial myocardium, making the myocardium less systolic and diastolic.Studies have shown that excessive alcohol consumption can cause heart failure and irreversible damage to the heart muscle.Once alcoholic cardiomyopathy occurs, besides causing severe heart failure, it can cause serious arrhythmias, which pose a serious threat to human health and have become urgent concerns [21].
At present, most of the research reports on peptide extraction from deer products focus on antler, deer blood, and deer whip.In spite of this, few studies have been published on the extraction of peptides from deer hearts.Trough the search of corresponding literature, it was found that there are few systematic data reported about the antioxidant, antiinfammatory, and preventive treatment of deer heart in heart diseases.In order to investigate the efects of deer heart peptide on populations with potential risk factors for diseases such as heart injury and potential therapeutic targets, we established a mouse model of alcoholic heart injury by using a high concentration of edible alcohol and then administered diferent concentrations of deer heart peptide to the mice by transoral gavage for subsequent testing.In this study, we investigated the efects of deer heart peptide on alcoholinduced cardiac injury in mice and explored the potential mechanisms and relationships between the antioxidant activity, anti-infammatory properties, and cardiac injury of deer heart peptide in vivo and in vivo.Terefore, the study of using deer heart peptide on mouse heart injury model not only provides theoretical basis for the development and utilization of bioactive peptides but also lays the theoretical foundation for the development of heart nutritional supplements and treatment of cardiovascular diseases using deer heart peptide as raw material.

Methodology
2.1.Drugs and Chemicals.Te deer heart was originally purchased from Jilin Dong'ao Deer Technology Development Co. Ltd. and extracted and purifed to deer heart peptide by Jilin China-Korea Institute of Animal Science.Chemicals used in this study were purchased from local commercial stores from the corresponding suppliers.

Sample Preparation.
We precisely weighed 1g of deer heart tissue and pepsin was added at 1% with a solid-liquid ratio = 1 : 100 and enzymatically digested for 3 h, 5 h, 7 h, and 9 h at 37 °C.Subsequently, the samples were centrifuged and fltered, evaporated, and concentrated; after freezing, the product is lyophilized.Trough the results of in vitro antioxidant, the 7 h digested deer heart peptide was fnally selected for the subsequent in vivo experiments in mice, taking into account the economic factors and the smooth running of the experiments.

Scavenging of Radicals by DPPH.
A slightly modifed version of the Blois method was used to measure each deer heart peptide's DPPH scavenging activity [22].Te DPPH 2 International Journal of Infammation solution (1.5 × 10 −4 M, 100 μL) was mixed with and without each extract (100 μL) and incubated at room temperature for 30 minutes.After 30 minutes of standing, with the aid of an enzyme marker, the absorbance was measured at 540 nm.Using the following equation, clearance activity was calculated as a percentage.
An absorbance measurement of the reaction mixture without deer heart peptide is used as A control and deer heart peptide absorbance A sample is determined by the reaction mixture containing the sample.

Hydrogen Peroxide Radical Scavenging Activity.
As determined by Muller's method, hydrogen peroxide scavenging activity was calculated [23].In 96-microtiter plates, 100 μL of 0.1 M phosphate bufer (pH 5.0) was added to each extract.After 5 minutes of incubation at 37 °C, 20 μL of hydrogen peroxide was added to the mixture.A mixture of 30 μL of ABTS containing 1.25 mM and 30 μL of peroxidase containing 1 unit/mL is then added to the mixture and incubated for 10 minutes at 37 °C.In order to calculate the percentage of scavenging activity, a 405 nm enzyme marker was used to measure absorbance and a scavenging activity percentage was determined by using (1).

ABTS Free Radical Scavenging
Activity.ABTS scavenging activity of deer heart peptides enzymatically digested at diferent times was assessed according to the method of Chung et al. [24].A solution of ABTS-+ and a solution of potassium persulfate were used as stock solutions.Mixing equal amounts of the 2 stock solutions and allowing them to react for 12 hours prepared the working solutions.Te working solution was diluted with fresh ABTS-+ solution and mixed with or without extracts.Two-hour incubation was followed by the measurement of each solution's absorbance at 735 nm.As a result of (1), a scavenging activity percentage was calculated.

Animals and Diets.
Eight-week-old male C57BL/6 mice weighing 23 ± 03 g were purchased from an approved laboratory animal supplier.In a 12-hour light/12-hour dark cycle, the animals were housed with thermoregulation (22 ± 1 °C) and humidity (40 ± 10%).Mice are acclimated to the laboratory environment for one week prior to the test.Te animals are fed a standard rodent diet and received free access to purifed water (reverse osmosis autoclaved water).

Alcoholic Cardiac Injury Induction.
Treating mice with alcohol resulted in alcohol-induced heart disease with 60 °white wine for 1 week (12 mL/kg body weight).During the establishment of the alcoholic heart injury model, there was a 90% success rate and no animals died during the experiment, which can be used for further studies.

Experimental Design
Animals were randomly divided into four groups of nine mice each.Following is a list of the groups: Group I: control group-normal diet with water; Group II: alcoholic heart injury model group-received white wine by oral gavage; Group III: model mice received 100 mg/kg of deer heart peptide; Group IV: model mice received 200 mg/kg of deer heart peptide for 21 days.Te deer heart peptide was dissolved in distilled water.Te mice in the model group and the administered group were given an equal amount of distilled water along with transoral gavage in the control group (Figure 1).Subsequently, they were intervened with daily oral administration of deer heart peptide (DH; 100 and 200 mg/mL) for 21 days.White wine (ALC; 12 mL/kg) was administered orally daily for 6 days starting from day 15 to induce alcoholic heart injury.
All samples were dissolved in deionized water and administered orally once a day for 21 days.Mice were fasted and executed after 21 days.
Blood was collected for the next step of analysis.Mice were weighed, and mouse hearts were collected, frozen in liquid nitrogen, and stored at −80 °C until further study, and heart tissues were also fxed in 10% formalin for the next histological analysis.
Ethical approval for this study was obtained from Changchun Sci-Tech University (CKARI202302).
3.1.Weight, Food Intake, Water Intake, and Cardiac Weight Index Are Assessed Each Week.Food intake, water intake, and body weight of the animals were recorded every other day until the end of the experimental period.Te absolute weight of the heart was recorded on the day of dissection, and its relative weight was calculated using the formula below [25].
(2)  1).Te target gene expression was normalized to that of glyceraldehyde 3-phosphate dehydrogenase using GAPDH as an internal reference gene, and the relative mRNA expression of the target gene was determined using the 2 −ΔΔCt method.

Statistical Analysis.
One-way ANOVA was used to determine diferences between groups, and data were expressed as mean and standard error.A post hoc test for Turkey was performed using GraphPad Prism software (v.8.0; GraphPad Software, La Jolla, CA, USA); P < 0.05 was considered signifcant.

Antioxidant Activity.
Te antioxidant activity of deer heart peptides may not be attributed to a single mechanism.Terefore, in this study, three methods were chosen to assess diferent aspects of the antioxidant activity of deer heart peptides.
In Vitro Antioxidant Assay.Te antioxidant activity of the enzymatic extracts of deer heart was assessed by ABTS, DPPH, and H 2 O 2 assay and is shown in Figure 2. Overall, the antioxidant activity of the 9 h enzymatic digestate of deer heart peptide was markedly better than that of deer heart   depicts the weight changes of mice in the control and experimental groups every two days.During the frst 15 days of oral administration of deer heart peptide, the body weight of mice in both the normal diet group and the deer heart peptide-fed group stabilized.However, after the 15th day, the white wine modeling phase was performed, and the mice fed orally with white wine lost the most signifcant weight compared to the mice fed with normal diet.In contrast, after deer heart peptide administration (100 and 200 mg/mL body weight), body weight decreased but the decrease was alleviated compared to the model group, and the decrease was slower in the deer heart peptide at high dosage (Figure 3(b)).Figures 3(c) and 3(d) depict the changes of diet and water consumption of mice during the experimental period, and the trends of both were largely the same, with a signifcant decrease in diet and water consumption after oral gavage of white wine.Te heart weight/body weight of the experimental animals did not show any signifcant changes compared with the control animals (Figure 3(e)), but the heart weight index of the mice in the deer heart peptide tended to approach that of the control group.

Discussion
With the growing concern of consumers towards the adverse efects associated with synthetic compounds, researchers focused on the exploration of natural remedies for many diseases.Cervus nippon has played a central role in traditional Chinese medicine owing to the promising bioactive compounds present.Terefore, in the current study, we examined the protective efect of deer heart peptide recovered with enzymatic digestion on alcoholic heart-injured mice.Our fndings revealed the promising efect of deer International Journal of Infammation  6 International Journal of Infammation heart peptide on alleviating the clinical features of alcoholic heart-injured mice [26][27][28].In the present study, we created an alcoholic heart-injured mice model through the administration of white wine and increased levels of serum SOD, MDA, and CK confrmed that alcoholic heart-injured mice model was successfully created.In addition, clinical characteristics such as weekly body weight, food intake, heart weight, and heart weight/body weight demonstrated that the creation of a mouse model of alcoholic cardiac injury was successful.Similar results were reported with the previous study where reduced heart weight and serum cardiac markers were the prominent features of heart-injured mice [29].Cardiac injury is a common clinical cardiovascular disease, and its incidence has been increasing year by year in recent years.Terefore, it is urgent to solve this problem.Xu [30] et al. used velvet polypeptide as a pharmacodynamic substance to observe its protective efect on mouse heart injury and found that velvet has various functions such as antioxidation and promoting protein and nucleic acid synthesis in vivo.Cardiomyocyte-specifc transcription factors (Nkx2.5,GATA4, ATF-2, and MEF-2C) have a signifcant inhibitory efect on cardiac injury.Terefore, it can be speculated that deer heart peptide has good antioxidant activity, can activate various proteins that regulate the heart muscle, and has a protective efect on the heart.Modern pharmacological studies have shown that the protein content in velvet antler is as high as 55.26%.Zhao et al. [31] confrmed that velvet antler protein can signifcantly reduce the damage changes of the electrocardiogram, reduce the area of myocardial ischemia, and reduce myocardial fbrosis.In summary, we can guess that the protein in deer heart peptide can reduce heart injury in mice.In addition, Chen et al. [32] further confrmed the protective efect of pilose antler polypeptide on rat heart injury and observed its efect on SOD activity in ischemic myocardial  Deer heart peptide is highly antioxidant, which means that it activates the body's antioxidant capacity, increases SOD activity, and reduces MDA production in the body.Te degree of cardiomyocyte damage was measured by CK and H&E staining, which is a marker enzyme for the degree of cardiac damage, while H&E staining allows visualization of the degree of myocardial disorder in mice to count the size of the cells.Apparently, deer heart peptide can successfully reduce the level of CK in vivo and can reduce cardiac fber disorders and inhibit cardiomyocyte swelling in mice, which confrms that it has a wide therapeutic potential and has a series of prospects for development in the near future.However, this study was conducted to induce acute heart injury in mice by white wine, so it was more impaired to the body function and afected the organ index of mice.Terefore, it is necessary to pay attention to the loss of body function in mice in future studies, and it is needed to maintain the body function as much as possible while conducting efective studies.Terefore, further studies are needed in order to explore the possible mechanism of action of deer heart peptide and make it a useful clinical treatment.
Te ancient literary works provide evidence of the favorable impact of deer heart on cardiac well-being, thereby highlighting its potential therapeutic signifcance.In contrast, there exist a plethora of scholarly investigations into the diverse medicinal properties of deer antler velvet, leaving deer heart as a relatively neglected subject of scientifc inquiry [33,34].Consequently, this valuable organ, considered a byproduct of the deer industry, frequently falls victim to wastage.Compared to deer antler, deer heart exhibits a more modest price point in the market.Compared to antler, deer heart exhibits a smaller price in the market.Deer heart has great potential as a functional food in the health care industry, and in future studies, small molecule active peptides from deer heart can be used to explore related heart diseases such as myocarditis, myocardial infarction, coronary artery disease, and angina pectoris to investigate the efcacy of deer heart peptide and the related mechanism and to further confrm the ameliorative efect of deer heart peptide on heart injury.However, its intrinsic value as a functional food surpasses mere monetary considerations.Rich in bioactive compounds and potentially possessing unique physiological efects, deer heart holds promise as a natural source of health-promoting substances [18].Te integration of deer heart into the realm of functional foods can not only diversify the agricultural landscape but also enhance the economic prospects of farmers.By recognizing and utilizing the untapped potential of deer hearts, rural communities engaged in deer farming can expand their sources of income.In turn, it ofers a compelling incentive for the growth and development of the deer industry, bolstering employment opportunities and local economies.Moreover, the sustainable utilization of deer heart aligns with the principles of resource conservation, promoting ecological balance within the agricultural sector.
In conclusion, the underappreciated deer heart, in light of its documented health benefts and comparative afordability, possesses immense potential as a value-added agricultural product.By elevating its status through scientifc research, appropriate marketing channels, and consumer education, deer heart can emerge as a sought-after ingredient in the realm of functional foods, providing a tangible pathway for farmers to enhance their income and invigorate the burgeoning deer industry.

C. nippon: Cervus nippon DH:
Deer heart peptide ALC: Alcoholic heart injury A + DH_L: Mice with alcoholic heart injury treated with 100 mg/kg/bw of deer heart peptide A + DH_H: Mice with alcoholic heart injury treated with 200 mg/kg/bw of deer heart peptide.International Journal of Infammation 9

Figure 1 :
Figure 1: Adaptive feeding for one week prior to oral administration.Subsequently, they were intervened with daily oral administration of deer heart peptide (DH: 100 and 200 mg/mL) for 21 days.White wine (ALC: 12 mL/kg) was administered orally daily for 6 days starting from day 15 to induce alcoholic heart injury.

Figure 3 (
a) illustrates the morphological map of the heart of each group of mice.Figure3(b)

Figure 2 :Figure 3 :
Figure 2: A comparison of the antioxidant activity of deer heart peptides.(a) ABTS radical scavenging activity of deer heart peptides enzymatically digested at diferent times.(b) DPPH radical scavenging activity of deer heart peptides enzymatically digested at diferent times.(c) H 2 O 2 radical scavenging activity of deer heart peptides enzymatically digested at diferent times.Data represent mean ± standard error of the mean.* * p < 0.05; * p < 0.01 according to Dunnett's multiple range test.

Figure 3 :Figure 5 :
Figure 3: Efects of deer heart peptide on body weight, food intake, water intake, and heart weight.(a) Heart morphology in mice with alcoholic heart injury.(b) Food intake.(c) Water intake.(d) Heart weight/body weight (HW/BW) of mice with alcoholic heart injury.(e) Cardiac index in mice.CON: normal control mice; ALC: alcoholic heart injury; A + DH_L: mice with alcoholic heart injury treated with 100 mg/kg/bw of deer heart peptide; A + DH_H: mice with alcoholic heart injury treated with 200 mg/kg/bw of deer heart peptide.
μL of SYBR Premix Ex Taq (Takarabio Inc.), and 9.5 μL of dH 2 O were mixed to obtain 25 μL of solution.PCR primers used for gene expression analysis are listed in Table 1.Te amplifcation conditions were as follows: 95 °C for 10 s, 95 °C for 5 s, 60 °C for 30 s, 95 °C for 15 s, 60 °C for 30 s, 95 °C for 15 s, and 40 cycles of C. Primers for qPCR were synthesized by Enotech Co.
Cardiomyocyte Injury Markers andMarkers of Oxidative Stress.Blood samples were obtained by cardiac puncture and serum was separated by centrifugation (3000 rpm, 20 min) and stored at −80 °C until assay.Creatine kinase isoenzyme (CK) concentrations were assessed as indicators of cardiomyocyte injury.Superoxide dismutase (SOD) activity, catalase (CAT), and malondialdehyde (MDA) levels were used as indicators of oxidative stress.CK levels in serum cardiac tissues and markers of oxidative stress in cardiac tissues were measured by commercially prepared kits (Nanjing Jincheng Institute of Biological Engineering, Nanjing, China).International Journal of Infammation 3.3.Histological Analysis.An aqueous solution was applied to the heart tissue to fx it in a phosphate bufer solution that contained 10% formalin.Te dehydrated tissue was dipped in wax, embedded and cut into 5 μm thick, thin pieces, and stained with hematoxylin and eosin (H&E).Te heart samples were observed and photographed (×100 original magnifcation) under a microscope.3.4.Evaluation of the Transcript-Level Expression of TargetGenes Using Quantitative Real-Time PCR (qPCR).Total RNA was isolated from heart tissues of mice belonging to each treatment using TRIzol according to the manufacturer's protocol.cDNA was obtained using Superscript II switch transcriptase (Invitrogen).cDNA was analyzed by RT-qPCR on Warm Cycler Shakers TP850 (Takarabio Inc., Shigatse, Japan) according to the manufacturer's protocol.Briefy, 2 μL of cDNA (100 ng), 1 μL of sense and antisense primers (0.4 µM), 12.5 (Daejeon, Korea) (Table

Table 1 :
Primers used for qPCR., 5 h, and 7 h enzymatic digestate and appeared to be dose dependent.From the ABTS results, the antioxidant activity of 7 h deer heart enzyme digestate (65.1505 μM TE/mg, Figure2(a)) was higher than that of 9 h deer heart peptide (62.336 μM TE/mg, Figure2(a)), but there was no signifcant diference between them (P < 0.01).
most obvious in the high-dose group.Te results indicate that deer heart peptide can reduce the degree of cellular disorder in heart tissue and has a protective efect on the heart.It can be seen from the CAT activity (Figure5(c)) that the deer heart peptide supplement can improve the antioxidant activity in the body, and the value of the high-dose group is close to that of the CON group.And compared with the model group, the deer heart peptide supplement group had A + DH_L and A + DH_H groups compared with the ALC group.Meanwhile, it is more visualized from the fgure that the cardiomyocytes in the ALC group had obvious swelling and gradual disappearance of transverse lines.However, after deer heart peptide prophylaxis, the cardiomyocytes gradually decreased in size and appeared cross-striations, which was a signifcant increase (P < 0.05).Serum levels of CK were signifcantly (P < 0.05) increased in alcoholic heart-injured mice compared to that in control group (Figure5(d)).Treatment of mice with 100 mg/kg (P < 0.05) or 200 mg/kg (P < 0.05) of deer heart peptide resulted in a signifcant decrease in CK levels compared to mice with alcoholic heart injury, with a signifcant diference.
Terefore, we can speculate that peptides have good antioxidant activity, which can reduce the generation of free radicals in the body and increase SOD activity.
* * p < 0.05; * p < 0.01 according to Dunnett's multiple range test.CON: control group; ALC: ALC-treated group; A + DH_L; 100 mg/ mL deer heart peptide; A + DH_H; 200 mg/mL deer heart peptide.CON ALC A+DH_L A+DH_HFigure 4: Efect of deer heart peptide on the pathological pattern of alcoholic heart injury.Tissue sections were photographed under a microscope (original magnifcation ×100).CON, control group; ALC, alcoholic heart injury model group; A + DH_L, supplemented with low-dose deer heart peptide 100 mg/kg body weight/day; A + DH_H, supplemented with high-dose deer heart peptide 200 mg/kg body weight/day.8 International Journal of Infammation tissue.