Zeaxanthin Exhibits Protective Effects in Myocardial Injury by Inhibiting TGF-β/Smad2/3 and p38MAPK/NF-κB Signaling Pathways

Background: Zeaxanthin is a newly discovered natural product in β-carotenoid family with multiple bioactivities. Recently, it has been shown that zeaxanthin may have cardioprotective effects in several studies, but its mechanisms have not been fully investigated. Herein, we explored the role and mechanism of zeaxanthin in myocardial injury. Methods and Results: In this study, three different models were used to investigate the mechanism by which zeaxanthin alleviates myocardial injury. H9C2 Cardiomyocyte injury models were induced by H 2 O 2 . TUNEL assay, Flow cytometry, and Western blot analysis showed that treatment with zeaxanthin signicantly decreased cardiomyocyte apoptosis and apoptosis-related protein expression. And reactive oxygen species (ROS) measurement analysis and Western blot analysis showed that treatment with zeaxanthin also could reduce the production of ROS and affect the expression of p38-Mitogen activated protein kinase/nuclear factor-κ gene bindin (p38MAPK/NF-κB) signaling pathway. Transforming Growth Factor-β1 (TGF-β1) was used to establish the brosis model in cardiac broblasts (CFs). QRT-PCR and Western blot analysis showed that treatment with zeaxanthin signicantly decreased the expression of brosis markers in CFs. Myocardial injury animal models were induced by high-fat diet (HFD). Our results demonstrated that zeaxanthin improved brosis damage and cardiomyocyte apoptosis in HFD mice. Furthermore, Western blot analysis showed that TGF-β/Drosophila mothers against decapentaplegic2/3 (TGF-β/Smad2/3) signaling pathway related protein p-Smad2/3, Smad2/3, and TGF-β1 were signicantly downregulated by zeaxanthin treatment. Conclusions: Zeaxanthin may alleviate HFD and H 2 O 2 -induced heart injury by regulating TGF-β/Smad2/3 and p38MAPK/NF-κB signaling pathways, which is immense clinical signicance in the


Background
Myocardial brosis and cardiomyocyte apoptosis are the predominant manifestations of pathological changes associated with myocardial injury. When myocardial injury occurs, it induces cardiomyocyte apoptosis by affecting the expression of apoptosis-related signaling pathways and genes. And it also leads to the activation of CFs, followed by their transdifferentiation into myo broblasts. Subsequently, scar tissue formed by extracellular matrix (ECM) deposition replaces normal myocardial tissue, increases ventricular wall stiffness, resulting in decreased cardiac compliance, cardiac dysfunction, and eventually heart failure [1]. Myocardial injury is closely related to the occurrence and development of a range of cardiovascular diseases, such as hypertension, heart failure, myocardial hypertrophy, and arrhythmia [2][3][4]. However, insights into the mechanism underlying myocardial injury remain scarce, although such mechanistic would be helpful in preventing cardiac dysfunction and identifying therapeutic targets for clinical treatment [5,6]. Therefore, identifying targets and effective therapeutic drugs for myocardial injury have gained substantial clinical signi cance. cultured in a standard humidi ed incubator at 37 °C with 5% CO 2 . After starvation in serum-free medium for 12 h, cardiomyocytes were treated with zeaxanthin (60 µM) for 24 h and stimulated with H 2 O 2 (200 µM) for 12 h. And in the H9C2 cardiomyocyte experiments, the ctrl groups were untreated as the blank control. At the end of the experimental period, all used materials were treated innocuously.

CF culture
Primary CFs were isolated from the heart tissue of Kunming mice. CFs were cultured in DMEM with 10% FBS and 1% penicillin-streptomycin and then placed in an incubator maintained at 37 °C with 5% CO2 and 95% air. Before TGF-β1 treatment, CFs were cultured in a serum-free medium for 6 h. The concentration of TGF-β1 used was 10 ng/mL. CFs were stimulated for 24 h with TGF-β1 and treated with zeaxanthin for 24 h, followed by extraction of RNA or protein for further analysis. In the CF experiments, the ctrl groups were untreated as the blank control. At the end of the experimental period, all used materials were treated innocuously.

Flow cytometry
An Annexin V-FITC/PI apoptosis detection kit (Solarbio, Beijing, China) was used to check for apoptosis in H9C2 cells. After the cells were grown and subjected to the appropriate treatment, an appropriate amount of cells were collected in the logarithmic growth phase and washed twice with PBS. The harvested cells were then incubated with a buffer solution containing annexin V-FITC for 10 min, followed by treatment with PI for another 10 min at room temperature. Flow cytometry was used to measure cellular apoptosis levels.

ROS measurement
A DCFH-DA (10 µM) probe was used to detect ROS formation in cardiomyocytes. Cells were incubated with a probe for 30 min at 37 ℃. The culture medium was discarded, and cells were xed with 4% paraformaldehyde, followed by three steps of washing with PBS. Images were acquired using a uorescence microscope and analyzed using ImageJ.

H&E and Masson staining
The ventricular tissues of 3 mice in each group were taken out for experiment. Myocardial tissues were xed with 4% paraformaldehyde at 4 °C for 24 h. Samples were dehydrated to transparency, and para n embedding was performed. The embedded tissue was cut into 5-μm thick slices using a para n slicer and xed on an adhesive slide. Sections were then stained using a Hematoxylin-Eosin (H&E) staining kit (Solarbio, Shanghai, China). Masson's trichrome staining kit (Solarbio, Shanghai, China) was used to examine collagen deposition in myocardial tissues.

Immuno uorescence assay
The ventricular tissues of 3 mice in each group were taken out for experiment. The para nembedded myocardial tissue was sectioned into slices of 5-μm thickness. Sections were dewaxed and rehydrated, and permeabilized by treatment with 0.1% Triton X-100 (4 μL Triton X-100 and 0.1 g BSA in 1 mL PBS) for 1 h. Subsequently, the permeabilized sections were blocked with 50 % normal goat serum at 37 ℃ for 1 h. Then, 50 μL of rabbit anti-Bax primary antibody (1:100, Abcam) was added to each tissue section and incubated overnight at 4 ℃. The primary antibody was recovered the next day. Tissue sections were washed with PBS, followed by incubation with a goat anti-rabbit secondary antibody labeled with FITC (1:500, Alexa Fluor 594, Life Technology) at room temperature for 1 h. Nuclei were stained with DAPI for 5 min. Immuno uorescence images were acquired in a uorescence microscope.
MTT assay H9C2 cells were plated in 96-well plates, and 200 μL of culture medium was added to each well. After the appropriate treatment, 20 μL of 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazoliumbromide (MTT) solution (5 mg/ml, Beyotime Biotechnology, China) was added to each well and cells were incubated at 37 °C for 4 h. Then, 150 μL of dimethyl sulfoxide was added to each well and incubated for 15 min. Absorbance was measured at a wavelength of 490 nm using a uorescence microplate reader.

CCK-8 viability assay
Cell Counting Kit-8 (CCK-8) was used to assess the optimal time point for zeaxanthin treatment in H9C2 cells. H9C2 cells were seeded into 96-well plates. After pretreatment with 60 µM zeaxanthin for 0, 12, 24, and 48 h, H 2 O 2 (200 μM) was added for 4 h to induce cardiomyocyte hypoxia. Then, the culture medium was replaced with CCK-8 solution. Absorbance was measured at 450 nm using a uorescence microplate reader.
TUNEL apoptosis assay H9C2 cells were grown to con uence after treatment and washed thrice with PBS. Cells were then treated with TUNEL solution, according to the manufacturer's instructions. TUNEL apoptosis images were obtained using an Olympus microscope. The ratio of apoptosis-positive cell nuclei/total cell nuclei represents the rate of cardiomyocyte apoptosis. Cellular apoptosis events were quanti ed using the Image J software. with HRP-conjugated secondary antibodies at room temperature for 50 min. The Image Lab software was used to detect protein expression levels.

RNA extraction and qRT-PCR assay
Total RNA was extracted from cells using the TRIzol reagent. After TRIzol was used to lyze the cells, chloroform was added and cells were thoroughly vortexed to ensure strati cation of the aqueous and organic phases. The upper aqueous phase was separated after centrifugation for 10 min and mixed gently with the same amount of isopropanol for 10 min. Afterwards, the samples were again centrifuged for 10 min at 4 ℃ at 12000 rpm. The supernatant was discarded and the extracted RNA was washed with ethanol. Finally, 20 μL of DEPC-treated water was added to dissolve the RNA.

Statistical analysis
All data were analyzed using GraphPad Prism 8.0 and expressed as mean ± SEM. The signi cance level was calculated by Student's t-test between two groups, and one-way analysis of variance (ANOVA) was used for comparisons across multiple conditions. Bonferroni correction was used to evaluate whether there were differences in each group. A value of p<0.05 was considered as statistically signi cant.

Results
Zeaxanthin suppresses cardiomyocyte apoptosis First, we used H 2 O 2 to induce H9C2 cardiomyocyte injury. The CCK-8 assay was used to determine the optimal time for zeaxanthin treatment in H9C2 cells by estimating the viability of H9C2 cells after 0, 12, 24, and 48 h of exposure to zeaxanthin. Results from the CCK-8 assay showed that treatment with zeaxanthin for 24 h had the strongest effect on restoring H9C2 cell viability (*p<0.05, **p<0.01). At the 48h time point, zeaxanthin had an inhibitory effect on cell viability (Fig.1a). In addition, the MTT assay was used to detect the effect of different concentrations of zeaxanthin on cell activity. Zeaxanthin was found to recover cell activity reduced by H 2 O 2 treatment(**p<0.01) (Fig.1b) . Through the detection of apoptosis-related markers including Bax, Bcl2, and cleaved caspase-3, western blotting con rmed that zeaxanthin at a concentration of 60 μM signi cantly decreased the expression of apoptosis-related markers (**p<0.01) (Fig.1c).

Zeaxanthin inhibits ROS production
We measured the intracellular ROS level in H9C2 cells treated with H2O2 using DCFH-DA probe. As shown in Fig.2d, we found that H 2 O 2 promoted ROS production in H9C2 cardiomyocytes, whereas zeaxanthin inhibited ROS production (**p<0.01). In addition, Western blotting was used to detect changes in p38MAPK/NF-κB levels in ROS-related signaling pathways. Oxidative stress injury increased the expression of p-p38MAPK, T-p38MAPK, and NF-κB, which were signi cantly decreased by zeaxanthin (*p<0.05, **p<0.01) (Fig.2e).

Zeaxanthin alleviates myocardial brosis in vitro and in vivo
Zeaxanthin has been reported to reduce liver brosis [9]. However, the role of zeaxanthin in myocardial brosis remains unclear. Therefore, we explored the role of zeaxanthin in myocardial brosis. First, we used TGF-β1 to induce brogenesis in primary CFs. qRT-PCR results showed that TGF-β1 could increase the mRNA levels of FN1, Col-I, and CTGF, compared with the control (Fig.3a). On the other hand, zeaxanthin signi cantly decreased the mRNA levels of these brosis markers (*p<0.05, **p<0.01).
To further explore the function and mechanism of zeaxanthin in myocardial brosis, we established an ND group that served as a control and a HFD group where myocardial brosis was induced in a C57BL/6 mice background. The myocardial tissue of mice was stained with H&E and Masson's trichrome stain for examining collagen deposition. As shown in Fig.4a, these results proved the successful establishment of the myocardial brosis animal model. HFD caused excessive collagen deposition. Zeaxanthin restored the orderly arrangement of cardiomyocytes and reduced collagen deposition, thereby reverting the deleterious effects of HFD. The results of blood lipid determination showed that zeaxanthin could reduce the levels of TG, TC, and LDL in serum (*p<0.05, **p<0.01), which were increased by HFD (Fig.4b). qRT-PCR and western blot results illustrated that zeaxanthin could decrease FN1, Col-I, MMP9, and CTGF expression both at the mRNA and protein levels (*p<0.05, **p<0.01) (Fig.4c, Fig.4d). In addition, HFD activated the expression of TGF-β/Smad2/3 signaling pathway. And after zeaxanthin treatment, the expression levels of TGF-β1 and p-Smad2/3 decreased (*p<0.05, **p<0.01) (Fig.4d).

Zeaxanthin inhibits myocardial apoptosis in HFD mice
We further veri ed the inhibitory effect of zeaxanthin on apoptosis in vivo. Immuno uorescence analysis of ventricular tissue showed that the expression of Bax in the HFD group was signi cantly increased.

Discussion
Zeaxanthin is an oxygen-containing carotenoid that is commonly found in medicinal plants, vegetables, and fruits [14]. The easy availability and inexpensive nature of zeaxanthin makes it feasible for use alone or in combination with other drugs for the treatment of diseases. Zeaxanthin has many protective effects in the human body, such as reverting the effects of blue light damage to the retina [15], liver brosis [8], and synergistic skin antioxidant activity [16]. It also has a protective effect on cardiovascular diseases, including cardiac dysfunction [17], atherosclerosis [18], and coronary heart disease [19]. In the arterial system and blood, zeaxanthin exerts its vascular protective effect in three ways: decreased oxidation of LDL [20], reduced arterial stiffness [21], and prevention of atherosclerosis [22]. However, the speci c mechanism and role of zeaxanthin in myocardial injury remains unclear. In this study, we illustrated the protective effect of zeaxanthin on myocardial injury for the rst time.
Oxidative stress is accompanied by the excessive production of ROS, leading to myocardial injury and in ammation. Antioxidants can effectively inhibit ROS production. And antioxidants combined with antiin ammatory drugs have a bene cial effect on the treatment of patients with heart failure [23]. Zeaxanthin has strong antioxidant activity and plays an important role in protecting cell membranes and lipoproteins from oxidative stress induced by ROS [24]. In addition, it has been reported that oxidative stress accompanied by cardiomyocyte apoptosis hinders the recovery of cardiac function after myocardial ischemia-reperfusion injury, which is reversed by inhibition of the ROS-related p38MAPK/NF-κB pathway [25]. Our results showed that zeaxanthin inhibited cardiomyocyte apoptosis, decreased the excessive production of ROS, and reduced the expression of p38MAPK/NF-κB pathway. Thus, we consider that zeaxanthin may inhibit ROS production and reduce cardiomyocyte apoptosis by affecting the expression of p38MAPK/NF-κB signaling pathway (Fig. 2).
Over the last decades. increasing studies described the direct effect of HFD and its related metabolites such as Triglycerides and Free Fat Acids (FFAs), on cardiovascular disease [26]. The HFD and its related metabolites can cause the chronic accumulation of excess fat in the myocardium. It will further lead to various metabolic changes in myocardial cells, activate systems modulating oxidative stress and in ammation, and induce myocardial injury [27]. And HFD is known to promote collagen deposition and brosis in heart tissue [28]. These processes are collectively termed "Myocardial lipotoxicity" [29]. Here, we con rmed that HFD could induce myocardial brosis and cardiomyocyte apoptosis in the heart tissue of C57BL/6 mice, and the treatment of zeaxanthin could alleviate these pathological changes (Fig. 4,  Fig. 5).
TGF-β/Smad2/3 signaling pathway is a key regulator of myocardial brosis [30]. The heart tissue is rich in interstitial and perivascular broblasts. TGF-β1 can regulate the phenotype and function of CFs by promoting the phosphorylation of intracellular Smad2/3, then stimulate broblast activation, induce ECM protein synthesis, promote brosis, and diastolic dysfunction [31]. In our study, through experiments in vitro and in vivo, the results showed that zeaxanthin could inhibit the activation of the TGF-β/Smad2/3 signaling pathway. And it also reduced the ECM protein synthesis and myocardial brosis at the same time (Fig. 3, Fig. 4). Thus, we consider that the inhibitory effect of zeaxanthin on myocardial brosis may be closely related to its in uence on TGF-β/Smad2/3 signaling pathway.
In conclusion, our study has con rmed the protective effect of zeaxanthin on myocardial injury. First, we found that zeaxanthin could inhibit cardiomyocyte apoptosis and the production of ROS induced in H9C2 cells by H2O2. And we also found that this protective effect of zeaxanthin was closely related to the inhibition of ROS-related p38MAPK/NF-κB signaling pathway. Next, we used TGF-β1 to induce myocardial brosis in CFs. Zeaxanthin was again found to signi cantly reduce the expression of brosisrelated markers. Furthermore, we established an animal model of myocardial injury induced by HFD to verify the effects of zeaxanthin on myocardial brosis and apoptosis in vivo. In vivo experiments showed that zeaxanthin could reduce apoptosis in heart tissue. In addition, zeaxanthin also could signi cantly reduce the excessive deposition of collagen in myocardial tissue and reduce the protein expression levels of TGF-β1 and p-Smad2/3. These results show that zeaxanthin has a protective effect against myocardial brosis and this protective effect may be closely related to the TGF-β/Smad2/3 signaling pathway. Cardiovascular disease remains a major global health problem, and further research is needed to develop effective treatments and drugs. In this context, the present work assumes immense clinical signi cance by providing insights into the protective mechanism of zeaxanthin against myocardial injury.

Conclusion
In our study, we have found that zeaxanthin may alleviate HFD and H 2 O 2 -induced heart injury by regulating TGF-β/Smad2/3 and p38MAPK/NF-κB signaling pathways. Thus, based on our research, we consider that zeaxanthin may play an important role in cardiovascular diseases. It offers a potential treatment for myocardial injury.

Declarations
Ethics approval All methods including animal experimental procedures were adopted in the study were performed in accordance with ARRIVE guidelines. Animal experimental protocols used in the present study were approved by the Harbin Medical University Research Ethics Committee (2021-SCILLSC-87). The further methods and procedures adopted in the study are in agreement with appropriate guidelines and regulations.

Consent for publication
Not applicable.

Availability of data and materials
Corresponding author will provide the data used in the present work upon request.

Declaration of competing interest
All authors declare that they have no competing interests.  Western blotting analysis of Bcl2, Bax, and cleaved-caspase-3. GAPDH was taken as an internal control gene. All data were expressed as mean ± SEM. Differences in expression were analyzed by Student's ttest and one-way ANOVA (n = 3. * p<0.05, ** p<0.01). Magni cation, 400x. (e) Western blotting analysis of p-p38MAPK and p-NF-kB. GAPDH was taken as an internal control gene. All data were expressed as mean ± SEM. Differences in expression were analyzed by Student's t-test and one-way ANOVA (n = 3. * p<0.05, ** p<0.01).

Figure 3
Zeaxanthin reduces CFs activation induced by TGF-β1 (a) qRT-PCR analysis of FN1, Col-I, and CTGF expression. (b) Western blotting analysis of FN1 and Col-I. GAPDH was taken as an internal control gene.
All data were expressed as mean ± SEM. Differences in expression were analyzed by Student's t-test and one-way ANOVA (n = 3. * p<0.05, ** p<0.01). was taken as an internal control gene. All data were expressed as mean ± SEM. Differences in expression were analyzed by Student's t-test and one-way ANOVA (n = 3. * p<0.05, ** p<0.01).