Apigenin Alleviates Endotoxin-Induced Myocardial Toxicity by Modulating Inflammation, Oxidative Stress, and Autophagy

Apigenin, a component in daily diets, demonstrates antioxidant and anti-inflammatory properties. Here, we intended to explore the mechanism of apigenin-mediated endotoxin-induced myocardial injury and its role in the interplay among inflammation, oxidative stress, and autophagy. In our lipopolysaccharide- (LPS-) induced myocardial injury model, apigenin ameliorated cardiac injury (lactate dehydrogenase (LDH) and creatine kinase (CK)), cell death (TUNEL staining, DNA fragmentation, and PARP activity), and tissue damage (cardiac troponin I (cTnI) and cardiac myosin light chain-1 (cMLC1)) and improved cardiac function (ejection fraction (EF) and end diastolic left ventricular inner dimension (LVID)). Apigenin also alleviated endotoxin-induced myocardial injury by modulating oxidative stress (nitrotyrosine and protein carbonyl) and inflammatory cytokines (TNF-α, IL-1β, MIP-1α, and MIP-2) along with their master regulator NFκB. Apigenin modulated redox homeostasis, and its anti-inflammatory role might be associated with its ability to control autophagy. Autophagy (determined by LAMP1, ATG5, and p62), its transcriptional regulator transcription factor EB (TFEB), and downstream target genes including vacuolar protein sorting-associated protein 11 (Vps11) and microtubule-associated proteins 1A/1B light chain 3B (Map1lc3) were modulated by apigenin. Thus, our study demonstrated that apigenin may lead to potential development of new target in sepsis treatment or other myocardial oxidative and/or inflammation-induced injuries.


Introduction
Sepsis affects significant number of people worldwide and has significant cost in emergency care and stunning high mortality rate [1]. Mortality rate is even higher in patients who have sepsis in past several years [2]. Cardiovascular risk is much higher among patients who have survived from severe sepsis [3]. Sepsis is attributed to a component of the cell wall of Gram negative bacteria: lipopolysaccharide (LPS). Cardiac dysfunction is one of the key factors in sepsis-related complications [4,5]. The significant role of inflammation and associated activation of NFκB in LPSinduced sepsis have been reported in the literature [6][7][8].
Oxidative stress in sepsis is well documented [9,10]. The role of autophagy in sepsis is crucial but the relevant research is very limited [11,12].
Incident of cardiovascular disease is rising due to epidemic of obesity, diabetes, and hypertension [13]. Increase of oxidative stress and chronic low-grade inflammation occurs in all these health disorders in addition to aging [14,15]. Low-grade inflammation leads to circulating immune cells and platelets, which generate superoxide as byproduct of mitochondrial respiration or by enzymatic reactions such as lipoxygenases, myeloperoxidase, NADPH oxidases, and xanthine oxidase [16]. Thus, interplay of inflammation and oxidative stress plays critical role in cardiovascular disease.
Flavonoids, present in fruits and vegetables, are polyphenols and have great potential against cardiovascular complications including sepsis [17][18][19]. Apigenin, a flavonoid, is a traditional Chinese medicine isolated from celery Apium graveolens. Apigenin is also present in parsley and chamomile. Various researches have demonstrated that apigenin is anticancer, antioxidant, and anti-inflammatory [20][21][22]. Cardioprotective effects of apigenin have been reported in numerous studies. Apigenin ameliorates myocardial ischemia/reperfusion injury via the inactivation of p38 mitogen-activated protein kinase [23]. Apigenin reduces the blood pressure, heart weight, heart weight index, cardiomyocyte cross-sectional area, and serum angiotensin II in a cardiac hypertrophy model via HIF-1 and PPARα pathways [24]. In an autoimmune myocarditis model of mice, apigenin mediates protection by inhibiting lymphocyte proliferation [25]. Apigenin attenuated proinflammatory cytokine expression by inactivating NF-kappaB through the suppression of p65 phosphorylation in vitro study of human monocytes and reduced LPS-induced mortality in mice [26]. In a LPSinduced endotoxemic rat model, apigenin attenuates heart injury by suppressing sphingosine kinase 1/sphingosine 1phosphate signaling pathway [27]. We have shown earlier the role of autophagy, inflammation, and oxidative stress in sepsis model [28] and the cardioprotective role of resveratrol in LPS-induced myocardial toxicity via NRF2 [29].
In this study, we demonstrated that apigenin protected against LPS-induced cardiac tissue damage, cardiac injury, cardiomyocyte cell death, and cardiac dysfunction. Cardioprotection by apigenin was mediated by its antiinflammatory and antioxidant effect. Autophagy by apigenin also played a role in cardioprotection.

Animal Experiments.
Male C57BL/6 mice that are 4-6 weeks old were obtained from the Experimental Animal Center of Shandong University (Jinan, Shandong, China). LPS was purchased from Sigma (Beijing, China). LPS was dissolved in saline and administered intraperitoneally (i.p.) as described earlier [29]. The mice were given 4 mg/kg dose of LPS and kept for 18 hours for endpoint analyses. Apigenin (>98 purity) was purchased from Shanghai Winherb Medical S&T Development Co. Ltd. (China) and administered at 50 mg/kg of body weight intraperitoneally (i.p.) 1 hour post challenge of LPS. Vehicle for drug was 5% dimethyl sulfoxide (DMSO) in sterile saline. Mice experimental protocols were approved by the Institutional Animal Care and Use Committee of Shandong University and were in compliance with the Health Ministry of the People's Republic of China. Mice were sacrificed under deep anesthesia after completion of echocardiography.

Cardiac Injury and Tissue Damage Markers.
Plasma CK and LDH levels were determined using an automated analyzer (Abbott Architect, Abbot Park, Illinois, USA) as described earlier [11]. Plasma cTnI concentrations were measured by ELISA-based assay according to the manufacturer's protocol (Abnova, Taiwan). Plasma cardiac myosin light chain-1 (cMLC1) was determined by ELISA (Life Diagnostics Inc., USA) according to the manufacturer's protocol. Both were described earlier [28].
2.6. Cardiac Cell Death Markers. DNA fragmentation was measured by ELISA-based kit (Roche) as described earlier [29]. For PARP activity, we used the HT Universal Colorimetric PARP assay kit from Trevigen as described earlier [31]. 2.8. Cardiac Glutathione Level. Cardiac glutathione levels from tissue lysates were determined by GSR-DTNB recycling assay and used in previous publication [33].
2.9. Cardiac Oxidative Stress Markers. Protein nitrotyrosine nitration was determined using OxiSelect™ Nitrotyrosine ELISA Kit (Cell Biolabs) as described earlier [12]. Carbonyl content in protein from tissue lysate was determined by Protein Carbonyl Colorimetric Assay Kit (Cayman Chemical) as described earlier [28].

Statistical
Analysis. Data were expressed as mean ± standard deviation (SD), and statistical analysis was done by using GraphPad Prism software. Paired t-test or one-way analysis of variance followed by Tukey's posttest was performed and considered statistically significant.

Apigenin Attenuates Endotoxin-Induced Myocardial
Injury and Cell Death of Cardiomyocytes in Mice. To examine cardioprotective effect of apigenin, we administered 4 mg/kg of LPS for 18 hours in C57BL/6 mice posttreated with either vehicle (control) or apigenin. LPS-induced myocardial injury was evident by increase in LDH and CK ( Figure 1). Aginenin treatment significantly reduced LPSinduced myocardial injury. Cell death including apoptotic cell death markers were determined by TUNEL staining (Figure 2(a)). Quantitative cell death markers DNA fragmentation and PARP activity were determined and both were induced at 398% and 2.8-fold in response to LPS, respectively. Both markers, DNA fragmentation and PARP activity, were reduced to 55% and 57%, respectively, by apigenin treatment (Figure 2(b)). Cardiac troponin I (cTnI) and cardiac myosin light chain-1 (cMLC1) are proficient markers of acute heart disorders specifically for heart muscle cell death. cTnI and cMLC1 were induced by LPS administration to 6.3 ng/ml and 0.89 ng/ml in mice serum from 0.34 ng/ml and 0.14 ng/ml in the control group, respectively ( Figure 3). Treatment with apigenin significantly attenuated LPS-induced serum cTnI and cMLC1 to 4.22 and 0.42, respectively. Cardiac function parameters such as left ventricular (LV) structure and function were assessed by echocardiography. LPS administration cause a decrease in ejection fraction (EF) and an increase of end diastolic left ventricular inner dimension (LVID), which was significantly attenuated by apigenin administration (Figure 4). The heart rates of the mice among all the four treatment groups were not statistically different. Treatment with apigenin alone did not alter any cardiac injury markers or function in the above experiments.
Multiple factors have been demonstrated to be involved in the endotoxin-mediated myocardial injury and cardiac dysfunction [5,[34][35][36]. Consistent with previous findings, our in vivo experiments indicated that LPS dramatically increased the plasma level of LDH, CK, cMLC1, cTnI, and cell death markers TUNEL staining, DNA fragmentation, and PARP activity. Apigenin treatment significantly reduced all the above markers. Primary mechanism of cell death in sepsis is by both apoptosis and necrosis with overlapping signaling pathways. In apoptosis, cell shrinkage and associated loss of myocardial structure leads to cardiac dysfunction [37]. In necrosis, an inflammatory response occurs, which also cause cardiac dysfunction [37]. We observed both types of cell death and apigenin-reduced cell death and cardiac dysfunction.
Pharmacokinetics study of apigenin in a rat model demonstrated that apigenin can be available in the system up to 10 days [38]. Apigenin can be found in human red blood cells after parsley consumption and thus increase its distribution and bioavailability [39]. We also observed that apigenin treatment improved LPS-induced cardiac dysfunction. In a long-term study of diabetic cardiomyopathy in mice, apigenin administration improves left ventricular functions in the heart [40]. Apigenin also improves the recovery of cardiac function during ischemia/reperfusion injury of isolated rat heart using Langedorff system [23]. Thus, our data on apigenin-mediated recovery of myocardial injury and cardiac dysfunction may lead to potential therapeutic development in sepsis.  Cardiac damage was measured by plasma cTnI and cMLC1, which were secreted by damaged cardiomyocytes from the heart. Both of them were significantly increased in LPS-treated mice and were significantly attenuated by apigenin treatment. Values represented as means ± SD; * P < 0 05 and n = 6/group.

Apigenin Suppresses Endotoxin-Induced
Real-time PCR analyses of proinflammatory cytokines demonstrated induction of TNF-α, IL-1β, MIP-1α, and MIP-2 by LPS administration up to 3.9, 3.7, 3.8, and 4.1, respectively. Apigenin treatment reduced LPS-induced cytokine gene expression by 43%, 45%, 51%, and 48% for TNF-α, IL-1β, MIP-1α, and MIP-2, respectively ( Figure 5(a)). Two proinflammatory cytokines were verified at protein level, and it was consistent with mRNA level (Figure 5(b)). Western blot analyses demonstrated that NFκB p65 was increased in nuclear fraction in heart lysate from LPS-treated mice and such increase was significantly reduced with apigenin treatment (Figure 6(a)). Similar analyses with cytoplasmic fraction also demonstrated that the cytoplasmic level of p65 was decreased by endotoxin and apigenin treatment restored to normal. NFκB pathway is considered a conventional proinflammatory signaling pathway [41]. NFκB is implicated in the expression of proinflammatory sources including cytokines, chemokines, and adhesion molecules. In sepsis, canonical pathways of NFκB activation occurs [42]. Role of NFκB activation is critical in inhibition of lipopolysaccharide-induced shock [43]. Thus, inhibition of p65 nuclear localization by apigenin in endotoxin-induced sepsis was beneficial. Quantitative determination of oxidative stress markers protein nitrotyrosine and protein carbonyl by ELISA demonstrated significant increase 3.6 and 5.1, respectively. Apigenin attenuated LPS-induced protein nitrotyrosine and protein carbonyl by 61% and 515, respectively (Figure 6(b)). We also determined glutathione level in both oxidized (GSSG) and reduced (GSH) form. Endotoxin induced oxidized glutathione level and decreased reduced glutathione level, which were reversed by apigenin treatment (Figure 7). Endotoxin-induced myocardial injury is associated with defects in redox balance and antioxidant enzymes, which leads to inflammation [44]. We have shown earlier the role of NRF2 in the sepsis and the protective effect by resveratrol [29]. In consistent with earlier literatures, we found increase in oxidative stress markers nitrotyrosine and protein carbonyl by LPS. LPS also induced inflammatory cytokines. Apigenin treatment significantly reduced both oxidative stress and inflammatory response. In a rat model of sepsis, cecal ligation and puncture induces oxidative stress and inflammatory response in the spleen and is attenuated by apigenin [45]. In another rat model of LPS-induced sepsis, apigenin ameliorates inflammatory response by suppressing sphingosine kinase 1 pathway, known as modulator of transcription factor NFκB [27]. We also observed in this study that LPS-induced nuclear translocation of NFκB was attenuated by apigenin. Apigenin inhibits the release of cytokines and suppresses NFκB activity in LPS-stimulated  Figure 6: Apigenin attenuates LPS-induced cardiac NFκB nuclear translocation and oxidative damage. (a) Western blot analyses of NFκB (p65) in nuclear and cytoplasmic fractions from heart lysates. Histone H3 was used as a nuclear control whereas tubulin was used as a cytoplasmic control. (b) Cardiac oxidative markers protein nitration and carbonyl content measured by quantitative ELISA. All markers were significantly increased in LPS-treated mice. Apigenin significantly reduced LPS-induced oxidative stress markers. Values represented as means ± SD; * P < 0 05 and n = 6/group. monocytes isolated from human donor [26]. In a recent report, apigenin reduces NFκB-associated neuroinflammation in a diet-induced obesity model of rat [46]. Thus, our study demonstrating apigenin as antioxidant and antiinflammatory modulator via NFκB in endotoxin-induced myocardial injury may be effective to other inflammatory heart injury models such as myocarditis.

Apigenin Modulates Autophagy in Endotoxin-Induced
Cardiac Injury. In animal model of sepsis and in clinical setting of sepsis, cardiac autophagy is increased [47]. Autophagy is also beneficial for cardioprotection during sepsis [48]. We examined autophagy by Western blot analyses of LAMP1, ATG5, and p62 ( Figure 8). All three autophagy markers were increased in endotoxin-induced cardiac injury. Apigenin enhanced LAMP1 and ATG5 protein levels whereas p62 level was decreased by apigenin. These findings were in consistent with the fact that apigenin induced autophagy as p62 level was decreased and is a marker of autophagy induction in addition to be degraded by autophagy linking ubiquitinated proteins to the autophagic machinery in the lysosome [49]. Western blot analyses of nuclear fraction demonstrated that TFEB, a major modulator of autophagy and associated CLEAR pathway, was increased by both LPS and apigenin alone. Apigenin treatment along with LPS significantly enhanced nuclear localization (Figure 9(a)).
To understand the functional role of nuclear translocation of TFEB, we determined two of its target genes Vps11 and Map1lc3 by real-time PCR (Figure 9(b)). LPS induced Vps11 and Map1lc3 gene expression by 1.53-and 1.83-fold, respectively. Apigenin treatment enhanced further LPSinduced gene expression to 2.6-and 2.9-fold for Vps11 and Map1lc3, respectively. Flavonoids are known to modulate autophagy in a variety of pathological condition or physiological processes in animal models [50][51][52][53]. We have shown earlier that TFEBmediated autophagy is modulated during aging [28]. In this study, we found that apigenin modulated autophagy pathway during sepsis. Curcumin, a flavonoid, also targets TFEB for induction of autophagy [54]. Recent literatures demonstrate the link of flavonoids with TFEB and/or autophagy [55][56][57]. Autophagy is also regulator for cardiovascular redox homeostasis and thus interplay of oxidative stress, autophagy, and inflammation is crucial in apigeninmediated cardioprotection [58,59]. We observed the protective role of TFEB and its downstream targets in LPS-induced myocardial injury and its modulation by apigenin. In doxorubicin-induced cardiomyopathy, TFEB is also modulated [60]. However, activation of autophagy has been identified as cardioprotective in some settings, but in other cases, sustained autophagy has been linked with cardiopathology [61]. Autophagy is thus known for its function as a double-edged sword [62,63]. In this study, autophagy may work as redox homeostasis, enhancing the lysomal degradation of LPS-induced myocardial injury. Both are important for apigenin-mediated cardioprotection.
Our study on apigenin-mediated protection of endotoxin-induced myocardial injury demonstrated interplay of oxidative stress, inflammation, and autophagy. Increased cell death lead to increased inflammation, which is linked to increased oxidative stress. This process goes in cycle and thus enhances the damaging effect of endotoxin. Autophagy and antioxidant defense are also linked [64]. Autophagy regulates both redox balance and ROS formation, and autophagy plays a major role in the degradation of oxidized proteins [65].

Conclusion
We demonstrated that apigenin protects against LPSinduced cardiac injury, tissue damage, and cardiomyocyte cell death. Apigenin improves LPS-induced cardiac dysfunction. Apigenin-mediated protection against LPS-induced myocardial toxicity is mediated by multiple mechanisms where inflammation and oxidative stress were modulated by apigenin along with their master regulator NFκB. In addition to that, autophagic pathway regulator TFEB was enhanced by apigenin. Due to autophagy enhancement, apigenin reduced LPS-induced inflammation and oxidative stress ( Figure 10). Thus, the interplay of autophagy, inflammation, and oxidative stress and its modulation by apigenin played important role in cardioprotection.

Conflicts of Interest
The authors declare that they have no competing interests.  Figure 10: Schematic diagram of apigenin-mediated protection in LPS-induced myocardial toxicity and heart failure. Interplay of autophagy with oxidative damage and inflammation plays a critical role in pathophysiology of sepsis-induced cardiac dysfunction which results in heart failure. By enhancing autophagy, apigenin reduces oxidative damage, which also leads to inflammation and cell death.