# Chitoheptaose Promotes Heart Rehabilitation in a Rat Myocarditis Model by Improving Antioxidant, Anti-Inflammatory, and Antiapoptotic Properties

Authors: Qini Zhao; Liquan Yin; Lirong Zhang; Dongli Jiang; Long Liu; Hong Ji
Journal: Oxidative Medicine and Cellular Longevity
 (2020)

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## Abstract

Background. Myocarditis is one of the important causes of dilated cardiomyopathy, cardiac morbidity, and mortality worldwide. Chitosan oligosaccharides (COS) may have anti-inflammatory and cardioprotective effects on myocarditis. However, the exact molecular mechanism for the effects of functional COS on myocarditis remains unclear. Methods. Anti-inflammatory activities of COS (chitobiose, chitotriose, chitotetraose, chitopentaose, chitohexaose, chitoheptaose, and chitooctaose) were measured in lipopolysaccharide- (LPS-) stimulated RAW264.7 cells. A rat model with myocarditis was established and treated with chitopentaose, chitohexaose, chitoheptaose, and chitooctaose. Serum COS were measured by using high-performance liquid chromatography (HPLC) in all rats. Myocarditis injury, the levels of reactive oxygen species (ROS), reactive nitrogen species (RNS), inflammatory factors, and apoptotic factors were also measured. Pearson’s correlation coefficient test was used to explore the relationship between the levels of ROS/RNS and cardiac parameters. Results. Among all chitosan oligosaccharides, the COS>degreesofpolymerizationDP4 showed anti-inflammatory activities (the activity order was chitopentaose<chitohexaose<chitoheptaose<chitooctaose) by reducing the levels of interleukin- (IL-) 1β, IL-17A, and interferon- (IFN-) γ and increasing the level of IL-10. However, the serum level of chitooctaose was low whereas it showed significant therapeutic effects on myocarditis by improving cardiac parameters (left ventricular internal dimension, both end-systolic and end-diastolic, ejection fraction, and fractional shortening), inflammatory cytokines (IL-1β, IL-10, IL-17A, and IFN-γ), oxidative factors (ROS and RNS), and apoptotic factors (caspase 3, BAX, and BCL-2) when compared with chitopentaose, chitohexaose, and chitooctaose (COSDP>4). The levels of ROS/RNS had a strong relationship with cardiac parameters. Conclusions. Chitoheptaose plays a myriad of cardioprotective roles in the myocarditis model via its antioxidant, anti-inflammatory, and antiapoptotic activities.


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## Body

## 1. Introduction

Myocarditis is an inflammation of the myocardium, which is one of the important causes of dilated cardiomyopathy [1] and cardiac morbidity and mortality worldwide [2]. The proinflammatory factors include the following: high-level interleukin- (IL-) 1β is associated with high myocarditis risk [3] and IL-1 receptor antagonist (IL-1ra) can prevent cardiac dysfunction [4]. Myocarditis involves many myocardial inflammatory diseases, such as acute myocarditis, chronic myocarditis, and inflammatory cardiomyopathies, and inflammatory diseases with cardiomyopathies [5]. Oxidative stress is also an important risk in the pathogenesis of myocarditis [6]. Inflammatory and oxidative stress can affect the left ventricular internal diameter in end diastole (LVIDd) and left ventricular internal diameter in end systole (LVIDs) [7]. The volume of the ejection fraction (EF) is often measured clinically to assess cardiac mechanics and function and significantly reduced in the progression of myocarditis [8]. The value of fractional shortening (FS) is widely used to assess left ventricular dysfunction and often decreased after myocarditis [9]. On the other hand, preserved EF [10] is associated with predominant inflammation and oxidant stress.IL-17A is a CD4+ T cell-derived proinflammatory cytokine and plays an important role in the pathogenesis of myocarditis [11]. Interferon- (IFN-) γ is an important immune regulator in normal immunity and involved in the regulation of most immune and inflammatory responses. IL-10 is a type 2 cytokine, which inhibits proinflammatory cytokine formation. Lowering the IFN-γ level and increasing the IL-10 level will reduce the progression of myocarditis [12].Reactive oxygen species (ROS) [13] and reactive nitrogen species (RNS) [14] are normally produced highly in myocarditis. Antioxidants can ameliorate cardiac apoptosis and dysfunction in an animal model with autoimmune myocarditis [15, 16]. From the above information, antioxidant and anti-inflammatory therapy has become a very important approach in preventing myocarditis progress.Chitosan oligosaccharides (COS) are homo- or heterooligomers ofN-acetylglucosamine and D-glucosamine and active and have more physiological functions than chitosan with solubility in water. COS exert cardiac protective effects on the patients with coronary heart disease (CHD) by improving antioxidant capacity [17]. COS also possess anti-inflammatory properties by activating mitogen-activated protein kinase (MAPK) signaling [18]. However, the effects of COS on myocarditis and which oligosaccharide of COS plays a more important functional role still remain unclear. Normally, long-chain oligosaccharides (degreesofpolymerizationDP>5) may have an excellent function than short-chain ones [19]. The effects of COS with different DP on myocarditis were explored in a rat model.


## 2. Materials and Methods

### 2.1. Reagents

The standards of COS (chitobiose, chitotriose, chitotetraose, chitopentaose, chitohexaose, chitoheptaose, and chitooctaose) were purchased from Qingdao BZ Oligo Biotech Co., Ltd. (Qingdao, China). Lipopolysaccharide (LPS, #L2880) was from Sigma (Sigma, Louis, MO, USA). RAW264.7 cells (an appropriate model of macrophages) were from Shanghai Institute of Cell Biology, CAS (Shanghai, China), and cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (Hyclone, Logan, UT, USA), 100 U/mL penicillin, and 100 mg/mL streptomycin at 37°C and 5% CO2.


### 2.2. Measurement of COS Anti-Inflammatory Activity

According to previous reports, RAW264.7 cells were seeded in 96-well plates at a density of1×104 cells/well and incubated in the above medium with 100 μM different DP of COS (chitobiose, chitotriose, chitotetraose, chitopentaose, chitohexaose, chitoheptaose, and chitooctaose) for 24 h and stimulated with LPS (200 ng/mL, 6 h). RAW264.7 cells were washed 3 times with cold PBS and centrifuged at 1,000 g for 10 min at 4°C. The cell pellet thus obtained was resuspended in 0.5 mL Tris buffer (20 mM, pH 7.5, 1 μg/mL chymostatin, 2 μg/mL of pepstatin A, 100 μM phenylmethylsulfonyl fluoride, and 5 μg/mL aprotinin). The cells were lysed by two freeze-thaw cycles [20]. The supernatants were collected via centrifugation at 15,000 g for 10 min at 4°C. The inflammatory production was measured by using the kits (mouse IL-1β ELISA Kit, ab100704; mouse IL-10 ELISA Kit, ab108870; mouse IL-17A ELISA Kit, ab100702; and mouse IFN-γ ELISA Kit, ab100689; Abcam, USA).


### 2.3. Establishment of the Experimental Autoimmune Myocarditis (EAM) Model

Before the present experiment, all procedures were approved by the animal research ethics committee of China-Japan Union Hospital of Jilin University (approval no. 2017CCZYY03017). Forty-eight 8-week-old specific pathogen-free (SPF) rats (200-220 g) were purchased from the animal center of Jilin University. Porcine cardiac myosin (PCM, Sigma Aldrich, M0531) was dissolved in 0.10 mol/L PBS solution (10 g/L). The solution was mixed with complete Freund’s adjuvant (CFA, Sigma Aldrich, F5881) in an equal volume of 1 : 1 to make it a homogeneous emulsion. On the first day and the eighth day, 200μL of myosin (1 mg) was injected subcutaneously into the bilateral inguinal and axillary sites of the 40 rats. The control group (n=8) was immunized with myosin-free PBS and CFA emulsion. From day 1, all rats were housed at a temperature of 23±1°C in a 12 h/12 h daylight/dark cycle and allowed indicated food and water ad libitum.


### 2.4. Animal Grouping

After the establishment of an EAM model, the mice were orally treated with 100μM different DP of COS (chitopentaose, chitohexaose, chitoheptaose, and chitooctaose in saline solution) daily for one month. Forty-eight rats were evenly divided into the CG group (control), MG group (EAM model), PG group (chitopentaose-treated EAM model), HexG group (chitohexaose-treated EAM model), HepG group (chitoheptaose-treated EAM model), and OG group (chitooctaose-treated EAM model) (Figure S1). The mice were treated with equal-volume saline solution in the CG and MG groups.


### 2.5. Echocardiography Analysis of Cardiac Parameters

After one-month intervention, all rats were anesthetized by intraperitoneal injection of 10% chloral (300 mg/kg). After depilation of the chest, echocardiography was performed on the left side of the supine position, and the experiments were performed in triplicate. Cardiac parameters were measured by echocardiography, including the left ventricular internal diameter in end diastole (LVIDd), left ventricular internal diameter in end systole (LVIDs), and ejection fraction (EF). Fractional shortening (FS) was calculated asLVIDd−LVIDs/LVIDd×100.


### 2.6. HPLC Analysis of Serum COS

Theoretically, the larger size oligosaccharide will be more difficult to be absorbed into blood vessels. Therefore, it is necessary to measure the serum level of different DP of COS. After one-month COS intervention, serum levels of COS were measured in all rats (FigureS1). One mL of blood was sampled from each rat tail in anticoagulant-free tubes and kept at room temperature for 1 h before the serum was isolated (centrifugation at 1,500 g for 10 min at 22°C). Serum levels of COS were analyzed by using HPLC (Varian 920-LC, HPLC-DAD/UV system, Varian, Inc., Palo Alto, CA, USA) [21] according to COS standards (chitopentaose, chitohexaose, chitoheptaose, and chitooctaose). The column was Metacarb 87H (300×7.8mm, Varian, USA) with an RID detector; the flow rate was set at 0.6 mL/min, and the temperature was 80°C. A sample of 20 μL was injected into the HPLC.


### 2.7. Measurement of Serum Inflammatory Cytokines

After one-month COS intervention, serum inflammatory cytokines were measured (FigureS1) by using the kits (rat IL-1β ELISA Kit, ab100768; rat IL-10 ELISA Kit, ab214566; rat IL-17A ELISA Kit, ab119536; and rat IFN-γ ELISA Kit, ab239425; Abcam, USA).


### 2.8. Oxidative Stress Measurement

After one-month COS intervention, the oxidative stress levels were measured (FigureS1) by using reactive oxygen species (ROS) and reactive nitrogen species (RNS) via DCF DA and/or DAF-FM DA fluorescence. Cardiac tissues were digested into a single cell by trypsin. The single cells were resuspended in RPMI 1640 medium (Gibco) and adjusted to 1×106 and were incubated using DCF DA with DAF-FM DA for 15 min at 37°C in the dark. The cells were washed with fresh medium twice and transferred to PBS buffer (20 mM, pH 7.0). The fluorescence intensity was measured using Synergy H1 Hybrid Reader (BioTek Instruments, Winooski, VT, USA).


### 2.9. Analysis of Myocardial Histopathology

After one-month COS intervention, myocardial histopathology was analyzed (FigureS1). The rat chest was opened to expose the anterior cardiac region, and the heart was separated and removed. After being repeatedly washed with cold PBS on ice, the heart was cut longitudinally into two parts along the coronal plane, half of which was fixed in 10% formaldehyde solution for HE staining and immunohistochemistry, and the other part was stored at -80°C for subsequent analysis of the mRNA and protein expression of caspase 3, BAX, and BCL-2 (Figure S1). The myocardial tissue fixed in 10% formaldehyde solution was dehydrated with gradient ethanol and treated with xylene I/II, respectively, and then dipped in benzene/wax I/wax II and embedded in solid paraffin at 65~70°C. After cooling, the paraffin was cut into 2-3 μm slices and treated in warm water at about 56°C for 30 min at 70°C. The heart tissue of each group of rats was subjected to hematoxylin-eosin (HE) staining and myocarditis injury, and inflammatory status was observed. H&E stain was scored to assess infiltration based on the following scale: 0 = normal myocardium, 1 = mild myocarditis (<5% cross section of infiltration), 2 = moderate myocarditis (5-10% cross section of infiltration), 3 = marked myocarditis (10-25% cross section of infiltration), and 4 = severe myocarditis (>25% cross section of infiltration).Paraffin sections were baked for 2 h at 65°C; after dewaxing to water, sections were repaired, incubated, and sealed; primary antibodies (anti-BAX 1 : 1000, Abcam, ab32503; anti-BCL-2 1 : 1000, Abcam, ab59348; and 1 : 500 monoclonal rat anti-caspase 3 (sc-7148) and anti-β-actin (sc-47778), Santa Cruz Biotechnology) were incubated at 4°C overnight, and secondary antibodies (1 : 50,000 goat anti-rabbit HRP (ab6702, Abcam) in 3% BSA in TBS) were incubated at 37°C for 50 min. Meanwhile, the NC group (negative control group without primary antibody) and the PC group (positive control group with β-actin as a primary antibody) were detected in intestinal tissues. Freshly prepared DAB solution was added to develop the color. After the color development was complete and after counterstaining, differentiation, blue return, dehydration, and drying, the xylene was transparent and the slides were sealed. Immunohistochemistry (IHC) was scored as follows: 0 (negative), 1 (weak brown), 2 (moderate brown), and 3 (strong brown).


### 2.10. Real-Time Quantitative RT-PCR

Ten mg heart tissue was ground into powder in liquid nitrogen, and RNA was extracted using RNeasy Mini Kit (Qiagen, Valencia, CA, USA). The RNA was treated with RQ1 RNase-Free DNase and reverse transcribed using the M-MLV reverse transcriptase (TaKaRa, Dalian, China). The following primers were used: caspase 3 (forward primer 5′-ATGCTTACTCTACCGCACCCG-3′ and reverse primer 5′-GGTTAACACGAGTGAGGATGTGC-3′), BAX (forward primer 5′-TGAACTGGACAACAACATGGAG-3′ and reverse primer 5′-AGCAAAGTAGAAAAGGGCAACC-3′), BCL-2 (forward primer 5′-TTGTGGCCTTCTTTGAGTTCG-3′ and reverse primer 5′-TTCAGAGACAGCCAGGAGAAATC-3′), and β-actin (forward primer 5′-AGGTCATCACTATCGGCAAT-3′ and reverse primer 5′-ACTCATCGTACTCCTGCTTG-3′) as an internal control. qRT-PCR was conducted in 96-well plates using the following parameters: 10 μL 2x SYBR Green PCR master mix, 0.5 μL forward primer (10 μM), 0.5 μL reverse primer (10 μM), 1 μL (10 ng) of diluted cDNA, and 8 μL ddH2O. The qRT-PCR program was performed on a LightCycler 480 Real-Time PCR system (Roche, Basel, Switzerland): 94°C 20 s; 40 amplification cycles of 94°C 10 s and 60°C 45 s. qRT-PCR data were normalized to β-actin.


### 2.11. Western Blot

Ten mg heart tissues were ground into a fine powder using a mortar and pestle in liquid nitrogen. The ground powder was placed into the Qiagen TissueLyser II® for lysing (MD, USA). The mixture was centrifuged at 12,000 g for 10 min, and the supernatants were obtained for Western blot analysis. Total proteins were separated by 10% SDS-PAGE and transferred to the polyvinylidene difluoride (PVDF) membrane. The PVDF membrane was blocked with 5% nonfat milk in TBS-T buffer for 2 h, incubated with specific primary antibodies overnight at 4°C, washed with TBS-T, and incubated with peroxidase-conjugated antibodies. Protein bands were observed by using chemiluminescence, and density was analyzed using ImageJ software and normalized withβ-actin.


### 2.12. Statistical Analysis

The data were processed by SPSS17.0 software (SPSS Statistics, IBM, Armonk, NY, USA). The measurement data were expressed asmean±S.D. and analyzed by using the t-test, and the count data were analyzed by the χ2 test. The Wilcoxon rank-sum test was used to evaluate grade data. One-way (with 1 independent variable) analysis of covariance (ANCOVA) was used to explore the response of the dependent variable among all groups. Pearson’s correlation coefficient test was used to explore the relationship between the levels of ROS/RNS and the values of cardiac parameters. The difference was statistically significant if p<0.05.


## 2.1. Reagents

The standards of COS (chitobiose, chitotriose, chitotetraose, chitopentaose, chitohexaose, chitoheptaose, and chitooctaose) were purchased from Qingdao BZ Oligo Biotech Co., Ltd. (Qingdao, China). Lipopolysaccharide (LPS, #L2880) was from Sigma (Sigma, Louis, MO, USA). RAW264.7 cells (an appropriate model of macrophages) were from Shanghai Institute of Cell Biology, CAS (Shanghai, China), and cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (Hyclone, Logan, UT, USA), 100 U/mL penicillin, and 100 mg/mL streptomycin at 37°C and 5% CO2.


## 2.2. Measurement of COS Anti-Inflammatory Activity

According to previous reports, RAW264.7 cells were seeded in 96-well plates at a density of1×104 cells/well and incubated in the above medium with 100 μM different DP of COS (chitobiose, chitotriose, chitotetraose, chitopentaose, chitohexaose, chitoheptaose, and chitooctaose) for 24 h and stimulated with LPS (200 ng/mL, 6 h). RAW264.7 cells were washed 3 times with cold PBS and centrifuged at 1,000 g for 10 min at 4°C. The cell pellet thus obtained was resuspended in 0.5 mL Tris buffer (20 mM, pH 7.5, 1 μg/mL chymostatin, 2 μg/mL of pepstatin A, 100 μM phenylmethylsulfonyl fluoride, and 5 μg/mL aprotinin). The cells were lysed by two freeze-thaw cycles [20]. The supernatants were collected via centrifugation at 15,000 g for 10 min at 4°C. The inflammatory production was measured by using the kits (mouse IL-1β ELISA Kit, ab100704; mouse IL-10 ELISA Kit, ab108870; mouse IL-17A ELISA Kit, ab100702; and mouse IFN-γ ELISA Kit, ab100689; Abcam, USA).


## 2.3. Establishment of the Experimental Autoimmune Myocarditis (EAM) Model

Before the present experiment, all procedures were approved by the animal research ethics committee of China-Japan Union Hospital of Jilin University (approval no. 2017CCZYY03017). Forty-eight 8-week-old specific pathogen-free (SPF) rats (200-220 g) were purchased from the animal center of Jilin University. Porcine cardiac myosin (PCM, Sigma Aldrich, M0531) was dissolved in 0.10 mol/L PBS solution (10 g/L). The solution was mixed with complete Freund’s adjuvant (CFA, Sigma Aldrich, F5881) in an equal volume of 1 : 1 to make it a homogeneous emulsion. On the first day and the eighth day, 200μL of myosin (1 mg) was injected subcutaneously into the bilateral inguinal and axillary sites of the 40 rats. The control group (n=8) was immunized with myosin-free PBS and CFA emulsion. From day 1, all rats were housed at a temperature of 23±1°C in a 12 h/12 h daylight/dark cycle and allowed indicated food and water ad libitum.


## 2.4. Animal Grouping

After the establishment of an EAM model, the mice were orally treated with 100μM different DP of COS (chitopentaose, chitohexaose, chitoheptaose, and chitooctaose in saline solution) daily for one month. Forty-eight rats were evenly divided into the CG group (control), MG group (EAM model), PG group (chitopentaose-treated EAM model), HexG group (chitohexaose-treated EAM model), HepG group (chitoheptaose-treated EAM model), and OG group (chitooctaose-treated EAM model) (Figure S1). The mice were treated with equal-volume saline solution in the CG and MG groups.


## 2.5. Echocardiography Analysis of Cardiac Parameters

After one-month intervention, all rats were anesthetized by intraperitoneal injection of 10% chloral (300 mg/kg). After depilation of the chest, echocardiography was performed on the left side of the supine position, and the experiments were performed in triplicate. Cardiac parameters were measured by echocardiography, including the left ventricular internal diameter in end diastole (LVIDd), left ventricular internal diameter in end systole (LVIDs), and ejection fraction (EF). Fractional shortening (FS) was calculated asLVIDd−LVIDs/LVIDd×100.


## 2.6. HPLC Analysis of Serum COS

Theoretically, the larger size oligosaccharide will be more difficult to be absorbed into blood vessels. Therefore, it is necessary to measure the serum level of different DP of COS. After one-month COS intervention, serum levels of COS were measured in all rats (FigureS1). One mL of blood was sampled from each rat tail in anticoagulant-free tubes and kept at room temperature for 1 h before the serum was isolated (centrifugation at 1,500 g for 10 min at 22°C). Serum levels of COS were analyzed by using HPLC (Varian 920-LC, HPLC-DAD/UV system, Varian, Inc., Palo Alto, CA, USA) [21] according to COS standards (chitopentaose, chitohexaose, chitoheptaose, and chitooctaose). The column was Metacarb 87H (300×7.8mm, Varian, USA) with an RID detector; the flow rate was set at 0.6 mL/min, and the temperature was 80°C. A sample of 20 μL was injected into the HPLC.


## 2.7. Measurement of Serum Inflammatory Cytokines

After one-month COS intervention, serum inflammatory cytokines were measured (FigureS1) by using the kits (rat IL-1β ELISA Kit, ab100768; rat IL-10 ELISA Kit, ab214566; rat IL-17A ELISA Kit, ab119536; and rat IFN-γ ELISA Kit, ab239425; Abcam, USA).


## 2.8. Oxidative Stress Measurement

After one-month COS intervention, the oxidative stress levels were measured (FigureS1) by using reactive oxygen species (ROS) and reactive nitrogen species (RNS) via DCF DA and/or DAF-FM DA fluorescence. Cardiac tissues were digested into a single cell by trypsin. The single cells were resuspended in RPMI 1640 medium (Gibco) and adjusted to 1×106 and were incubated using DCF DA with DAF-FM DA for 15 min at 37°C in the dark. The cells were washed with fresh medium twice and transferred to PBS buffer (20 mM, pH 7.0). The fluorescence intensity was measured using Synergy H1 Hybrid Reader (BioTek Instruments, Winooski, VT, USA).


## 2.9. Analysis of Myocardial Histopathology

After one-month COS intervention, myocardial histopathology was analyzed (FigureS1). The rat chest was opened to expose the anterior cardiac region, and the heart was separated and removed. After being repeatedly washed with cold PBS on ice, the heart was cut longitudinally into two parts along the coronal plane, half of which was fixed in 10% formaldehyde solution for HE staining and immunohistochemistry, and the other part was stored at -80°C for subsequent analysis of the mRNA and protein expression of caspase 3, BAX, and BCL-2 (Figure S1). The myocardial tissue fixed in 10% formaldehyde solution was dehydrated with gradient ethanol and treated with xylene I/II, respectively, and then dipped in benzene/wax I/wax II and embedded in solid paraffin at 65~70°C. After cooling, the paraffin was cut into 2-3 μm slices and treated in warm water at about 56°C for 30 min at 70°C. The heart tissue of each group of rats was subjected to hematoxylin-eosin (HE) staining and myocarditis injury, and inflammatory status was observed. H&E stain was scored to assess infiltration based on the following scale: 0 = normal myocardium, 1 = mild myocarditis (<5% cross section of infiltration), 2 = moderate myocarditis (5-10% cross section of infiltration), 3 = marked myocarditis (10-25% cross section of infiltration), and 4 = severe myocarditis (>25% cross section of infiltration).Paraffin sections were baked for 2 h at 65°C; after dewaxing to water, sections were repaired, incubated, and sealed; primary antibodies (anti-BAX 1 : 1000, Abcam, ab32503; anti-BCL-2 1 : 1000, Abcam, ab59348; and 1 : 500 monoclonal rat anti-caspase 3 (sc-7148) and anti-β-actin (sc-47778), Santa Cruz Biotechnology) were incubated at 4°C overnight, and secondary antibodies (1 : 50,000 goat anti-rabbit HRP (ab6702, Abcam) in 3% BSA in TBS) were incubated at 37°C for 50 min. Meanwhile, the NC group (negative control group without primary antibody) and the PC group (positive control group with β-actin as a primary antibody) were detected in intestinal tissues. Freshly prepared DAB solution was added to develop the color. After the color development was complete and after counterstaining, differentiation, blue return, dehydration, and drying, the xylene was transparent and the slides were sealed. Immunohistochemistry (IHC) was scored as follows: 0 (negative), 1 (weak brown), 2 (moderate brown), and 3 (strong brown).


## 2.10. Real-Time Quantitative RT-PCR

Ten mg heart tissue was ground into powder in liquid nitrogen, and RNA was extracted using RNeasy Mini Kit (Qiagen, Valencia, CA, USA). The RNA was treated with RQ1 RNase-Free DNase and reverse transcribed using the M-MLV reverse transcriptase (TaKaRa, Dalian, China). The following primers were used: caspase 3 (forward primer 5′-ATGCTTACTCTACCGCACCCG-3′ and reverse primer 5′-GGTTAACACGAGTGAGGATGTGC-3′), BAX (forward primer 5′-TGAACTGGACAACAACATGGAG-3′ and reverse primer 5′-AGCAAAGTAGAAAAGGGCAACC-3′), BCL-2 (forward primer 5′-TTGTGGCCTTCTTTGAGTTCG-3′ and reverse primer 5′-TTCAGAGACAGCCAGGAGAAATC-3′), and β-actin (forward primer 5′-AGGTCATCACTATCGGCAAT-3′ and reverse primer 5′-ACTCATCGTACTCCTGCTTG-3′) as an internal control. qRT-PCR was conducted in 96-well plates using the following parameters: 10 μL 2x SYBR Green PCR master mix, 0.5 μL forward primer (10 μM), 0.5 μL reverse primer (10 μM), 1 μL (10 ng) of diluted cDNA, and 8 μL ddH2O. The qRT-PCR program was performed on a LightCycler 480 Real-Time PCR system (Roche, Basel, Switzerland): 94°C 20 s; 40 amplification cycles of 94°C 10 s and 60°C 45 s. qRT-PCR data were normalized to β-actin.


## 2.11. Western Blot

Ten mg heart tissues were ground into a fine powder using a mortar and pestle in liquid nitrogen. The ground powder was placed into the Qiagen TissueLyser II® for lysing (MD, USA). The mixture was centrifuged at 12,000 g for 10 min, and the supernatants were obtained for Western blot analysis. Total proteins were separated by 10% SDS-PAGE and transferred to the polyvinylidene difluoride (PVDF) membrane. The PVDF membrane was blocked with 5% nonfat milk in TBS-T buffer for 2 h, incubated with specific primary antibodies overnight at 4°C, washed with TBS-T, and incubated with peroxidase-conjugated antibodies. Protein bands were observed by using chemiluminescence, and density was analyzed using ImageJ software and normalized withβ-actin.


## 2.12. Statistical Analysis

The data were processed by SPSS17.0 software (SPSS Statistics, IBM, Armonk, NY, USA). The measurement data were expressed asmean±S.D. and analyzed by using the t-test, and the count data were analyzed by the χ2 test. The Wilcoxon rank-sum test was used to evaluate grade data. One-way (with 1 independent variable) analysis of covariance (ANCOVA) was used to explore the response of the dependent variable among all groups. Pearson’s correlation coefficient test was used to explore the relationship between the levels of ROS/RNS and the values of cardiac parameters. The difference was statistically significant if p<0.05.


## 3. Results

### 3.1. Anti-Inflammatory Effects of COS

The inflammatory analysis showed that chitobiose, chitotriose, and chitotetraose had almost no effects on the levels of inflammatory cytokines IL-1β (Figure 1(a)), IL-10 (Figure 1(b)), IL-17A (Figure 1(c)), and IFN-γ (Figure 1(d), p>0.05) in LPS-stimulated RAW264.7 cells when compared with the control group. Among all chitosan oligosaccharides, the COS with more than degrees of polymerization (DP) 4 (a qualitative anti-inflammatory activity order, chitopentaose<chitohexaose<chitoheptaose<chitooctaose) showed anti-inflammatory activities by reducing the levels of interleukin- (IL-) 1β (Figure 1(a)), IL-17A (Figure 1(c)), and interferon- (IFN-) γ (Figure 1(d)) and increasing the level of IL-10 (Figure 1(b), p<0.05). The results suggest that the anti-inflammatory activity is improved with the increase in the degrees of polymerization of COS.Figure 1
Anti-inflammatory effects of chitosan oligosaccharides (chitobiose, chitotriose, chitotetraose, chitopentaose, chitohexaose, chitoheptaose, and chitooctaose) in LPS inducing the release of inflammatory cytokines in RAW264.7 cells. (a) Interleukin- (IL-) 1β. (b) IL-10. (c) IL-17A. (d) Interferon- (IFN-) γ. Ap<0.05 vs. the control, Bp<0.05 vs. the chitobiose group, Cp<0.05 vs. the chitotriose group, Dp<0.05 vs. the chitotetraose group, Ep<0.05 vs. the chitopentaose group, Fp<0.05 vs. the chitohexaose group, Gp<0.05 vs. the chitoheptaose group, and Hp<0.05 vs. the chitooctaose group.
(a)
(b)
(c)
(d)


### 3.2. HPLC Analysis of Serum COS Levels

HPLC analysis showed that the elution time of chitopentaose, chitohexaose, chitoheptaose, and chitooctaose was 9.3, 10.3, 12.1, and 23.1 min, respectively (Figure2(a)). No serum COS were detected in the rats from the CG group (Figure 2(b)) and the MG group (Figure 2(c)), respectively. The serum level of chitopentaose was highest and chitooctaose was lowest, and the serum contents of chitopentaose (Figure 2(d)), chitohexaose (Figure 2(e)), chitoheptaose (Figure 2(f)), and chitooctaose (Figure 2(g)) were 230, 110, 45, and 18 μM in the PG, HexG, HepG, and OG groups, respectively. The results suggest that the COS with high degrees of polymerization become more difficult to be absorbed into blood vessels with the increase in the size of COS.Figure 2
HPLC analysis of blood chitosan oligosaccharides. (a) Standard of chitosan oligosaccharides. (b) Control group. (c) Model group. (d) Chitopentaose. (e) Chitohexaose. (f) Chitoheptaose. (g) Chitooctaose.
(a)
(b)
(c)
(d)
(e)
(f)
(g)


### 3.3. Chitoheptaose Improved Cardiac Function Better Than Other COS

After the model establishment, the values of LVIDd (Figure3(a)) and LVIDs (Figure 3(b)) in the MG group were higher than those in the CG group whereas the percent of EF (Figure 3(c)) and FS (Figure 3(d)) in the MG group was lower than that in the CG group. The results suggest the model establishment causes cardiac injury. Meanwhile, chitoheptaose showed significant therapeutic effects by reducing the values of LVIDd (Figure 3(a)) and LVIDs (Figure 3(b)) and increasing the percent of EF (Figure 3(c)) and FS (Figure 3(d)) when compared with other COS (p<0.05).Figure 3
Cardiac function analysis. (a) LVIDd: left ventricular internal diameter in end diastole; (b) LVIDs: left ventricular internal diameter in end systole; EF: ejection fraction; (d) FS: fractional shortening.Ap<0.05 vs. the CG group, Bp<0.05 vs. the MG group, Cp<0.05 vs. the PG group, Dp<0.05 vs. the HexG group, Ep<0.05 vs. the HepG group, and Fp<0.05 vs. the OG group. n=8 for each group.
(a)
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### 3.4. Chitoheptaose Prevented Myocarditis Injury Better Than Other COS

H&E staining of the whole heart showed that the myocardium was thickest and the cells with light blue may be necrotic in the MG group when compared with other groups, and chitoheptaose showed significant inhibitory effects on the thickness of the myocardium and necrosis situation (FigureS2). H&E analysis showed that myocarditis injury was observed with damaged myomuscular fiber and abundant inflammatory cells in the MG group after the model establishment (Figure 4). COS intervention repaired the damage, prevented the injury in the HepG group, and reduced the amounts of inflammatory cells. Chitoheptaose showed significant therapeutic effects by reducing more pathological scores and inflammatory situation when compared with other COS (Figure 4, p<0.05).Figure 4
H&E analysis of myocarditis injury. (a) Control group (CG). (b) Model group (MG). (c) Chitopentaose-treated group (PG). (d) Chitohexaose-treated group (HexG). (e) Chitoheptaose-treated group (HepG). (f) Chitooctaose-treated group (OG).Ap<0.05 vs. the CG group, Bp<0.05 vs. the MG group, Cp<0.05 vs. the PG group, Dp<0.05 vs. the HexG group, Ep<0.05 vs. the HepG group, and Fp<0.05 vs. the OG group. n=8 for each group.


### 3.5. Chitoheptaose Showed Higher Anti-Inflammatory Effects Than Other COS

After the model establishment, the serum levels of IL-1β (Figure 5(a)), IL-17A (Figure 5(b)), and IFN-γ (Figure 5(c)) in the MG group were more than those in the CG group whereas the level of IL-10 (Figure 5(d)) in the MG group was lower than that in the CG group. The results suggest the model establishment causes inflammatory responses. Meanwhile, chitoheptaose showed significant anti-inflammatory properties by reducing the serum levels of IL-1β (Figure 5(a)), IL-17A (Figure 5(b)), and IFN-γ (Figure 5(c)) and increasing the level of EF (Figure 5(d)) when compared with other COS (p<0.05).Figure 5
ELISA analysis of serum inflammatory factors. (a) Interleukin- (IL-) 1β. (b) IL-17A. (c) Interferon- (IFN-) γ. (d) IL-10. Ap<0.05 vs. the CG group, Bp<0.05 vs. the MG group, Cp<0.05 vs. the PG group, Dp<0.05 vs. the HexG group, Ep<0.05 vs. the HepG group, and Fp<0.05 vs. the OG group. n=8 for each group.
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### 3.6. Chitoheptaose Showed Higher Antioxidant Effects Than Other COS

After the model establishment, the levels of ROS (Figure6(a)) and RNS (Figure 6(b)) in the MG group were higher than those in the CG group. The results suggest the model establishment increases the oxidative stress. Meanwhile, chitoheptaose showed significant antioxidant properties by reducing the levels of ROS (Figure 6(a)) and RNS (Figure 6(b)) when compared with other COS (p<0.05).Figure 6
Oxidative stress analysis. (a) The levels of reactive oxygen species (ROS). (b) The levels of reactive nitrogen species (RNS).Ap<0.05 vs. the CG group, Bp<0.05 vs. the MG group, Cp<0.05 vs. the PG group, Dp<0.05 vs. the HexG group, Ep<0.05 vs. the HepG group, and Fp<0.05 vs. the OG group. n=8 for each group.
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### 3.7. Chitoheptaose Intervention Reduced More the Relative mRNA Level of Apoptotic Factors Than Other COS

After the model establishment, the relative mRNA levels of caspase 3 (Figure7(a)) and BAX (Figure 7(b)) in the MG group were higher than those in the CG group whereas the level of BCL-2 (Figure 7(c)) in the MG group was lower than that in the CG group. The results suggest the model establishment causes the apoptotic responses. Meanwhile, chitoheptaose showed higher antiapoptotic properties by reducing the relative mRNA levels of caspase 3 (Figure 7(a)) and BAX (Figure 7(b)) and increasing the level of BCL-2 (Figure 7(c)) when compared with other COS (p<0.05).Figure 7
Real-time qPCR analysis of the relative mRNA levels of apoptotic factors. (a) Caspase 3. (b) BCL-2-associated X protein (BAX). (c) B cell lymphoma 2 (BCL-2). CG: control group; MG: model group; PG: chitopentaose-treated group; HexG: chitohexaose-treated group; HepG: chitoheptaose-treated group; OG: chitooctaose-treated group.n=3 for each group. Ap<0.05 vs. the CG group, Bp<0.05 vs. the MG group, Cp<0.05 vs. the PG group, Dp<0.05 vs. the HexG group, Ep<0.05 vs. the HepG group, and Fp<0.05 vs. the OG group.
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### 3.8. Chitoheptaose Intervention Reduced More the Relative Protein Level of Apoptotic Factors Than Other COS

For Western blot analysis, three samples were measured from each group. Figure8 shows the Western blot analysis for sample 1 from each group. Supplementary Figure S3 shows the Western blot analysis of samples 2 and 3 from each group. After the model establishment, the relative protein levels of caspase 3 (Figure 8(a)) and BAX (Figure 8(b)) in the MG group were more than those in the CG group whereas the level of BCL-2 (Figure 8(c)) in the MG group was lower than that in the CG group. The results suggest the model establishment causes apoptotic responses. Meanwhile, chitoheptaose showed higher antiapoptotic properties by reducing the relative protein levels of caspase 3 (Figure 8(a)) and BAX (Figure 8(b)) and increasing the level of BCL-2 (Figure 8(c)) when compared with other COS (p<0.05).Figure 8
Relative protein levels of apoptotic factors among different groups. (a) Caspase 3. (b) BCL-2-associated X protein (BAX). (c) B cell lymphoma 2 (BCL-2). CG: control group: MG: model group: PG: chitopentaose-treated group: HexG: chitohexaose-treated group: HepG: chitoheptaose-treated group: OG: chitooctaose-treated group.n=3 for each group. Ap<0.05 vs. the CG group, Bp<0.05 vs. the MG group, Cp<0.05 vs. the PG group, Dp<0.05 vs. the HexG group, Ep<0.05 vs. the HepG group, and Fp<0.05 vs. the OG group. Three samples were measured from each group with the numbers 1, 2, and 3. Supplementary Figure S2 showed Western blot analysis of the samples 2 and 3 from each group.
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### 3.9. Chitoheptaose Intervention Reduced the Expression of Apoptotic Factors More Than Other COS

IHC analysis showed that the IHC scores were lowest in the NC group and highest in the PC group, suggesting that the method was conducted successfully (Figure9(a)). After the model establishment, the relative expression of caspase 3 (Figure 9(a)) and BAX (Figure 9(b)) in the MG group was higher than that in the CG group whereas the expression of BCL-2 (Figure 9(c)) in the MG group was lower than that in the CG group. The results suggest the model establishment causes apoptotic responses. Meanwhile, chitoheptaose showed significant antiapoptotic properties by reducing the relative expression of caspase 3 (Figure 9(a)) and BAX (Figure 9(b)) and increasing the level of BCL-2 (Figure 9(c)) when compared with other COS (p<0.05). Whole heart staining of IHC also showed that the caspase 3 (Figure S4A) and BAX (Figure S4B) expression was highest in the MG group and significantly inhibited in the HepG group. In contrast, the BCL-2 expression was lowest in the MG group and significantly improved in the HepG group (Figure S4C).Figure 9
Histoimmunity analysis of apoptotic factors. (a) Caspase 3. (b) BCL-2-associated X protein (BAX). (c) B cell lymphoma 2 (BCL-2). CG: control group; MG: model group; PG: chitopentaose-treated group; HexG: chitohexaose-treated group; HepG: chitoheptaose-treated group; OG: chitooctaose-treated group; NC: negative control group without primary antibody; PC: positive control group with the primary antibody forβ-actin. n=8 for each group. Ap<0.05 vs. the CG group, Bp<0.05 vs. the MG group, Cp<0.05 vs. the PG group, Dp<0.05 vs. the HexG group, Ep<0.05 vs. the HepG group, Fp<0.05 vs. the OG group, Gp<0.05 vs. the NC group (negative control group without primary antibody), and Hp<0.05 vs. the PC group (positive control group with primary antibody for β-actin).
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### 3.10. Oxidative Stress Levels Had a Strong Relationship with Cardiac Parameters

The correlation test showed that with the increase in the level of ROS, the levels of LVIDd (Figure10(a)) and LVIDs (Figure 10(b)) were increased whereas the percent of EF (Figure 10(c)) and FS (Figure 10(d)) was reduced (p<0.001). In a similar situation, with the increase in the level of RNS, the levels of LVIDd (Figure 10(e)) and LVIDs (Figure 10(f)) were increased whereas the percent of EF (Figure 10(g)) and FS (Figure 10(h)) was reduced (p<0.001). The results suggest that oxidative stress levels have a strong relationship with cardiac parameters.Figure 10
The relationship between ROS factors and cardiac function. (a) The relationship between ROS levels and LVIDd. (b) The relationship between ROS levels and LVIDs. (c) The relationship between ROS levels and EF. (d) The relationship between ROS levels and FS. (e) The relationship between RNS levels and LVIDd. (f) The relationship between RNS levels and LVIDs. (g) The relationship between RNS levels and EF. (h) The relationship between RNS levels and FS.
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## 3.1. Anti-Inflammatory Effects of COS

The inflammatory analysis showed that chitobiose, chitotriose, and chitotetraose had almost no effects on the levels of inflammatory cytokines IL-1β (Figure 1(a)), IL-10 (Figure 1(b)), IL-17A (Figure 1(c)), and IFN-γ (Figure 1(d), p>0.05) in LPS-stimulated RAW264.7 cells when compared with the control group. Among all chitosan oligosaccharides, the COS with more than degrees of polymerization (DP) 4 (a qualitative anti-inflammatory activity order, chitopentaose<chitohexaose<chitoheptaose<chitooctaose) showed anti-inflammatory activities by reducing the levels of interleukin- (IL-) 1β (Figure 1(a)), IL-17A (Figure 1(c)), and interferon- (IFN-) γ (Figure 1(d)) and increasing the level of IL-10 (Figure 1(b), p<0.05). The results suggest that the anti-inflammatory activity is improved with the increase in the degrees of polymerization of COS.Figure 1
Anti-inflammatory effects of chitosan oligosaccharides (chitobiose, chitotriose, chitotetraose, chitopentaose, chitohexaose, chitoheptaose, and chitooctaose) in LPS inducing the release of inflammatory cytokines in RAW264.7 cells. (a) Interleukin- (IL-) 1β. (b) IL-10. (c) IL-17A. (d) Interferon- (IFN-) γ. Ap<0.05 vs. the control, Bp<0.05 vs. the chitobiose group, Cp<0.05 vs. the chitotriose group, Dp<0.05 vs. the chitotetraose group, Ep<0.05 vs. the chitopentaose group, Fp<0.05 vs. the chitohexaose group, Gp<0.05 vs. the chitoheptaose group, and Hp<0.05 vs. the chitooctaose group.
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## 3.2. HPLC Analysis of Serum COS Levels

HPLC analysis showed that the elution time of chitopentaose, chitohexaose, chitoheptaose, and chitooctaose was 9.3, 10.3, 12.1, and 23.1 min, respectively (Figure2(a)). No serum COS were detected in the rats from the CG group (Figure 2(b)) and the MG group (Figure 2(c)), respectively. The serum level of chitopentaose was highest and chitooctaose was lowest, and the serum contents of chitopentaose (Figure 2(d)), chitohexaose (Figure 2(e)), chitoheptaose (Figure 2(f)), and chitooctaose (Figure 2(g)) were 230, 110, 45, and 18 μM in the PG, HexG, HepG, and OG groups, respectively. The results suggest that the COS with high degrees of polymerization become more difficult to be absorbed into blood vessels with the increase in the size of COS.Figure 2
HPLC analysis of blood chitosan oligosaccharides. (a) Standard of chitosan oligosaccharides. (b) Control group. (c) Model group. (d) Chitopentaose. (e) Chitohexaose. (f) Chitoheptaose. (g) Chitooctaose.
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## 3.3. Chitoheptaose Improved Cardiac Function Better Than Other COS

After the model establishment, the values of LVIDd (Figure3(a)) and LVIDs (Figure 3(b)) in the MG group were higher than those in the CG group whereas the percent of EF (Figure 3(c)) and FS (Figure 3(d)) in the MG group was lower than that in the CG group. The results suggest the model establishment causes cardiac injury. Meanwhile, chitoheptaose showed significant therapeutic effects by reducing the values of LVIDd (Figure 3(a)) and LVIDs (Figure 3(b)) and increasing the percent of EF (Figure 3(c)) and FS (Figure 3(d)) when compared with other COS (p<0.05).Figure 3
Cardiac function analysis. (a) LVIDd: left ventricular internal diameter in end diastole; (b) LVIDs: left ventricular internal diameter in end systole; EF: ejection fraction; (d) FS: fractional shortening.Ap<0.05 vs. the CG group, Bp<0.05 vs. the MG group, Cp<0.05 vs. the PG group, Dp<0.05 vs. the HexG group, Ep<0.05 vs. the HepG group, and Fp<0.05 vs. the OG group. n=8 for each group.
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## 3.4. Chitoheptaose Prevented Myocarditis Injury Better Than Other COS

H&E staining of the whole heart showed that the myocardium was thickest and the cells with light blue may be necrotic in the MG group when compared with other groups, and chitoheptaose showed significant inhibitory effects on the thickness of the myocardium and necrosis situation (FigureS2). H&E analysis showed that myocarditis injury was observed with damaged myomuscular fiber and abundant inflammatory cells in the MG group after the model establishment (Figure 4). COS intervention repaired the damage, prevented the injury in the HepG group, and reduced the amounts of inflammatory cells. Chitoheptaose showed significant therapeutic effects by reducing more pathological scores and inflammatory situation when compared with other COS (Figure 4, p<0.05).Figure 4
H&E analysis of myocarditis injury. (a) Control group (CG). (b) Model group (MG). (c) Chitopentaose-treated group (PG). (d) Chitohexaose-treated group (HexG). (e) Chitoheptaose-treated group (HepG). (f) Chitooctaose-treated group (OG).Ap<0.05 vs. the CG group, Bp<0.05 vs. the MG group, Cp<0.05 vs. the PG group, Dp<0.05 vs. the HexG group, Ep<0.05 vs. the HepG group, and Fp<0.05 vs. the OG group. n=8 for each group.


## 3.5. Chitoheptaose Showed Higher Anti-Inflammatory Effects Than Other COS

After the model establishment, the serum levels of IL-1β (Figure 5(a)), IL-17A (Figure 5(b)), and IFN-γ (Figure 5(c)) in the MG group were more than those in the CG group whereas the level of IL-10 (Figure 5(d)) in the MG group was lower than that in the CG group. The results suggest the model establishment causes inflammatory responses. Meanwhile, chitoheptaose showed significant anti-inflammatory properties by reducing the serum levels of IL-1β (Figure 5(a)), IL-17A (Figure 5(b)), and IFN-γ (Figure 5(c)) and increasing the level of EF (Figure 5(d)) when compared with other COS (p<0.05).Figure 5
ELISA analysis of serum inflammatory factors. (a) Interleukin- (IL-) 1β. (b) IL-17A. (c) Interferon- (IFN-) γ. (d) IL-10. Ap<0.05 vs. the CG group, Bp<0.05 vs. the MG group, Cp<0.05 vs. the PG group, Dp<0.05 vs. the HexG group, Ep<0.05 vs. the HepG group, and Fp<0.05 vs. the OG group. n=8 for each group.
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## 3.6. Chitoheptaose Showed Higher Antioxidant Effects Than Other COS

After the model establishment, the levels of ROS (Figure6(a)) and RNS (Figure 6(b)) in the MG group were higher than those in the CG group. The results suggest the model establishment increases the oxidative stress. Meanwhile, chitoheptaose showed significant antioxidant properties by reducing the levels of ROS (Figure 6(a)) and RNS (Figure 6(b)) when compared with other COS (p<0.05).Figure 6
Oxidative stress analysis. (a) The levels of reactive oxygen species (ROS). (b) The levels of reactive nitrogen species (RNS).Ap<0.05 vs. the CG group, Bp<0.05 vs. the MG group, Cp<0.05 vs. the PG group, Dp<0.05 vs. the HexG group, Ep<0.05 vs. the HepG group, and Fp<0.05 vs. the OG group. n=8 for each group.
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## 3.7. Chitoheptaose Intervention Reduced More the Relative mRNA Level of Apoptotic Factors Than Other COS

After the model establishment, the relative mRNA levels of caspase 3 (Figure7(a)) and BAX (Figure 7(b)) in the MG group were higher than those in the CG group whereas the level of BCL-2 (Figure 7(c)) in the MG group was lower than that in the CG group. The results suggest the model establishment causes the apoptotic responses. Meanwhile, chitoheptaose showed higher antiapoptotic properties by reducing the relative mRNA levels of caspase 3 (Figure 7(a)) and BAX (Figure 7(b)) and increasing the level of BCL-2 (Figure 7(c)) when compared with other COS (p<0.05).Figure 7
Real-time qPCR analysis of the relative mRNA levels of apoptotic factors. (a) Caspase 3. (b) BCL-2-associated X protein (BAX). (c) B cell lymphoma 2 (BCL-2). CG: control group; MG: model group; PG: chitopentaose-treated group; HexG: chitohexaose-treated group; HepG: chitoheptaose-treated group; OG: chitooctaose-treated group.n=3 for each group. Ap<0.05 vs. the CG group, Bp<0.05 vs. the MG group, Cp<0.05 vs. the PG group, Dp<0.05 vs. the HexG group, Ep<0.05 vs. the HepG group, and Fp<0.05 vs. the OG group.
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## 3.8. Chitoheptaose Intervention Reduced More the Relative Protein Level of Apoptotic Factors Than Other COS

For Western blot analysis, three samples were measured from each group. Figure8 shows the Western blot analysis for sample 1 from each group. Supplementary Figure S3 shows the Western blot analysis of samples 2 and 3 from each group. After the model establishment, the relative protein levels of caspase 3 (Figure 8(a)) and BAX (Figure 8(b)) in the MG group were more than those in the CG group whereas the level of BCL-2 (Figure 8(c)) in the MG group was lower than that in the CG group. The results suggest the model establishment causes apoptotic responses. Meanwhile, chitoheptaose showed higher antiapoptotic properties by reducing the relative protein levels of caspase 3 (Figure 8(a)) and BAX (Figure 8(b)) and increasing the level of BCL-2 (Figure 8(c)) when compared with other COS (p<0.05).Figure 8
Relative protein levels of apoptotic factors among different groups. (a) Caspase 3. (b) BCL-2-associated X protein (BAX). (c) B cell lymphoma 2 (BCL-2). CG: control group: MG: model group: PG: chitopentaose-treated group: HexG: chitohexaose-treated group: HepG: chitoheptaose-treated group: OG: chitooctaose-treated group.n=3 for each group. Ap<0.05 vs. the CG group, Bp<0.05 vs. the MG group, Cp<0.05 vs. the PG group, Dp<0.05 vs. the HexG group, Ep<0.05 vs. the HepG group, and Fp<0.05 vs. the OG group. Three samples were measured from each group with the numbers 1, 2, and 3. Supplementary Figure S2 showed Western blot analysis of the samples 2 and 3 from each group.
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## 3.9. Chitoheptaose Intervention Reduced the Expression of Apoptotic Factors More Than Other COS

IHC analysis showed that the IHC scores were lowest in the NC group and highest in the PC group, suggesting that the method was conducted successfully (Figure9(a)). After the model establishment, the relative expression of caspase 3 (Figure 9(a)) and BAX (Figure 9(b)) in the MG group was higher than that in the CG group whereas the expression of BCL-2 (Figure 9(c)) in the MG group was lower than that in the CG group. The results suggest the model establishment causes apoptotic responses. Meanwhile, chitoheptaose showed significant antiapoptotic properties by reducing the relative expression of caspase 3 (Figure 9(a)) and BAX (Figure 9(b)) and increasing the level of BCL-2 (Figure 9(c)) when compared with other COS (p<0.05). Whole heart staining of IHC also showed that the caspase 3 (Figure S4A) and BAX (Figure S4B) expression was highest in the MG group and significantly inhibited in the HepG group. In contrast, the BCL-2 expression was lowest in the MG group and significantly improved in the HepG group (Figure S4C).Figure 9
Histoimmunity analysis of apoptotic factors. (a) Caspase 3. (b) BCL-2-associated X protein (BAX). (c) B cell lymphoma 2 (BCL-2). CG: control group; MG: model group; PG: chitopentaose-treated group; HexG: chitohexaose-treated group; HepG: chitoheptaose-treated group; OG: chitooctaose-treated group; NC: negative control group without primary antibody; PC: positive control group with the primary antibody forβ-actin. n=8 for each group. Ap<0.05 vs. the CG group, Bp<0.05 vs. the MG group, Cp<0.05 vs. the PG group, Dp<0.05 vs. the HexG group, Ep<0.05 vs. the HepG group, Fp<0.05 vs. the OG group, Gp<0.05 vs. the NC group (negative control group without primary antibody), and Hp<0.05 vs. the PC group (positive control group with primary antibody for β-actin).
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## 3.10. Oxidative Stress Levels Had a Strong Relationship with Cardiac Parameters

The correlation test showed that with the increase in the level of ROS, the levels of LVIDd (Figure10(a)) and LVIDs (Figure 10(b)) were increased whereas the percent of EF (Figure 10(c)) and FS (Figure 10(d)) was reduced (p<0.001). In a similar situation, with the increase in the level of RNS, the levels of LVIDd (Figure 10(e)) and LVIDs (Figure 10(f)) were increased whereas the percent of EF (Figure 10(g)) and FS (Figure 10(h)) was reduced (p<0.001). The results suggest that oxidative stress levels have a strong relationship with cardiac parameters.Figure 10
The relationship between ROS factors and cardiac function. (a) The relationship between ROS levels and LVIDd. (b) The relationship between ROS levels and LVIDs. (c) The relationship between ROS levels and EF. (d) The relationship between ROS levels and FS. (e) The relationship between RNS levels and LVIDd. (f) The relationship between RNS levels and LVIDs. (g) The relationship between RNS levels and EF. (h) The relationship between RNS levels and FS.
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## 4. Discussion

The present findings demonstrated that COS improved cardiac function by increasing the antioxidant and anti-inflammatory capacities and reducing the apoptotic risk in a rat model with myocarditis. COS can exert better physiological function than chitosan with excellent water solubility. Theoretically, the COS with high degrees of polymerization will be with high bioactivity than the low-degree ones. Thus, different COS (chitobiose, chitotriose, chitotetraose, chitopentaose, chitohexaose, chitoheptaose, and chitooctaose) were explored by using the rat model with myocarditis. Just as we supposed, chitobiose, chitotriose, and chitotetraose almost showed no anti-inflammatory activities on LPS inducing the release of inflammatory cytokines in RAW264.7 cells when compared with the control group. The COS exhibited anti-inflammatory effects when the DP were more than 4. Meanwhile, the anti-inflammatory activity reached the highest level in the chitooctaose group (Figure1, p<0.05). The results also suggest that the COS with low degrees of polymerization may have no functional activity with simple structure.This study showed that cardiac parameters (LVIDd, LVIDs, EF, and FS) were improved significantly in the HepG group compared with the other groups (Figure3). The results suggest that chitoheptaose shows higher therapeutic effects than chitooctaose according to the improvement of cardiac parameters although chitooctaose has higher anti-inflammatory activity than chitoheptaose. The reason may be that chitooctaose is more difficult than chitoheptaose to be absorbed into the blood vessels in the small intestine in the rat models because the former has the longer chain. Just as we proposed, the short-chain COS in serum reached the highest level in the PG group and the long-chain COS in serum reached the lowest level in the OG group (Figure 2).On the other hand, apoptotic activity is generally following oxidative [22, 23] and inflammatory responses [24, 25]. Therefore, the effects of COS on the apoptotic activity were also investigated here. Just as the antioxidant and anti-inflammatory properties, the COS showed similar antiapoptotic activity in different COS. Chitoheptaose showed higher antiapoptotic properties than other COS (Figures 7–9). The results suggest that oxidative stress, inflammation, and apoptosis may affect each other.The correlation test showed that the oxidative stress levels (ROS and RNS) had a strong relationship with the cardiac parameters. The results suggest that chitoheptaose may exert its cardioprotective function by affecting oxidative stress. Further work is highly needed to approve the central role for the correction among inflammatory, oxidative stress, and apoptotic activities in the pathogenesis of myocarditis. The results indicated that the COS with DP 7 exerted the highest antioxidant and anti-inflammatory activity in the animal model, which suggested that their activity had a close relationship with the degree of polymerization of COS. Larger oligosaccharides (chitohexaose and chitoheptaose) have the highest capability for scavenging DPPH [26]. Chitoheptaose has higher capability for scavenging ROS and RNS when compared with other COS and effectively scavenges ROS and RNS generated by electron leakage and protects cells against apoptosis induced by ROS and RNS. Chitoheptaose treatment showed the highest protective effects on the EAM model among the COS chitopentaose, chitohexaose, chitoheptaose, and chitooctaose. The effects of chitopentaose and chitooctaose were minimal when compared with the EAM model only treated with saline solution (Figure 9). Larger COS become more potent, and the biological activity of COS is size-dependent. Chitoheptaose was most active compound as the COS with the DP>4. However, much work is needed to explore the exact molecular mechanism.There were some limitations in the present work. Only 4 cardiac parameters were investigated here, and the interaction of these parameters was too complex to be explored. The relationship between the levels of ROS and RNS and the values of the parameters of cardiac parameters was only analyzed by using Pearson’s correlation coefficient test and not demonstrated by the experiments. Thus, the mechanisms were examined by using an animal model with CHD via related gene overexpression or silence in future work. The effects of the interaction among different COS on heart therapy are very complex, and it is difficult to understand its exact functional molecular mechanism. The deep molecular mechanism should be explored in the subsequent experiment.



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*Source: 2394704-2020-04-11.xml*