Puerarin protects against sepsis-induced myocardial injury through AMPK-mediated ferroptosis signaling

Introduction

Sepsis is a chronic systemic inflammatory response syndrome (SIRS) that can lead to septic shock and multiple organ dysfunction syndrome (MODS) [1, 2]. According to statistics, sepsis accounts for 11% of all acute and critical illnesses in ICUs, and its incidence increases by 8% to 13% annually [3]. Over 18 million patients with severe sepsis each year, with a 30-70% mortality rate. In critically ill patients, sepsis is one of the leading causes of death [4]. Sepsis is most frequently caused by Gram-negative bacteria [5]. The Gram-negative bacterial cell wall is primarily composed of lipopolysaccharides composed of lipid A, core polysaccharides, and O-antigens [1]. The elicitor functions after the invasion, triggering the inflammatory response and resulting in multiple organ dysfunction, ultimately leading to MODS [5]. The heart is one of the vulnerable target organs of sepsis. Research has demonstrated that 40%-50% of patients with sepsis have a myocardial injury, manifesting as arrhythmia, hypotension, and heart failure, and 20% of patients have latent myocardial injury [6, 7]. In addition, sepsis-mediated myocardial dysfunction has been proposed as a contributing factor to hospital-acquired mortality [8]. Studies have confirmed that various drugs effectively improve sepsis-induced myocardial injuries, such as metformin and ulinastatin [9, 10]. In recent years, using antibiotics has been rampant, leading to the emergence of resistant bacteria, implying the urgent need for novel drugs and innovative treatments for sepsis.

Sepsis patients have long been treated with traditional Chinese herbal medicine, which has had good therapeutic outcomes. Research has revealed that traditional Chinese medicine monomers and gene knockout technology can improve LPS-induced multiple organ dysfunction, including heart, lung, brain, kidney, and other essential organs [11]. Both berberine and artesunate can protect against acute lung injury caused by LPS [12, 13]. Concurrently, curcumin can effectively improve LPS-induced acute kidney injury [14]. In addition, studies have indicated that emodin can reduce damage to brain tissue caused by LPS [15]. Puerarin is the main active ingredient in the traditional Chinese medicine Pueraria. With its anti-inflammatory, anti-oxidation, and other effects, Puerarin has been deployed to prevent and treat cardiovascular disease [16]. Recent research reveals that Puerarin can reduce the risk of myocardial injury caused by LPS, inhibit inflammation, and reduce oxidative stress [17–19]. Puerarin inhibits the inflammatory response by inhibiting TLR4/NF-κB/JNK signaling pathway, improving LPS-induced multiple organ injury [17]. Puerarin reduced the myocardial injury induced by LPS by inhibiting the expression of inflammatory cytokines IL-1β, TNF-α and apoptosis-related protein Bax in an in vitro experiment [18].

Additionally, Puerarin can improve LPS-induced myocardial contractile dysfunction, increasing LVESP in septic rats [19]. Further research is required to determine the specific mechanism Puerarin uses to reduce sepsis-induced myocardial injury. Several factors have been implicated in the etiology of septic myocardial damage, including inflammation, oxidative stress, myocardial energy metabolism disorders, and excessive activation of the renin-angiotensin system (RAS) [20, 21]. Several scholars have proposed that ferroptosis plays an essential role in LPS-induced myocardial damage [22]. Iron-dependent ferroptosis is a brand new cell death model regulated by lipid oxidation and depends on iron [23, 24]. Ferroptosis occurs predominantly because Erastin inhibits the entry of cystine into a cell, resulting in glutathione depletion and inactivation of GPX4.

As a result, a large amount of Fe²⁺ enters mitochondria, causing a large volume of ROS to be produced, resulting in cell death [22, 23]. Dexmedetomidine inhibits ferroptosis by reducing HO-1 expression and increasing GPX4 expression, thereby reducing LPS-induced myocardial damage [25]. Ferroptosis is believed to be regulated by an AMPK signaling pathway, and activation of this pathway inhibits ferroptosis [26]. It should be observed that Puerarin stimulates AMPK and Akt signaling pathways in myocardial tissues, improving various myocardial diseases, including pathological myocardial hypertrophy and myocardial hypoxia/reoxygenation injury [27, 28]. Puerarin can also prevent heart failure caused by pressure overload by reducing ferroptosis [29].

We hypothesize that Puerarin prevents ferroptosis by acting on AMPK signaling pathway, thus protecting myocardial damage and cardiac dysfunction caused by LPS. Several basic and clinical studies have demonstrated that extracts from traditional Chinese herbal medicine can help protect against various cardiovascular diseases. Once Puerarin enters the body, it can be absorbed and utilized by the human myocardium, protecting myocardial cells and improving the ability to contact them. To further elucidate Puerarin’s protective mechanisms against LPS-induced myocardial injury, we used an in vivo sepsis model with LPS administration and corresponding medications to investigate the protective mechanisms of Puerarin.

Results

Discussion

Gram-negative bacteria produce LPS in their cell walls, one of the sepsis’s most critical pathogenic factors [5]. LPS-induced sepsis can cause the body to produce many inflammatory mediators (IL-1, IL-6, and TNF-α), resulting in a cascade of inflammatory responses that can cause extensive tissue and cell damage and damage to multiple organ structures. Injury and dysfunction eventually result in the patient’s death [20, 30, 31]. Experiments revealed that Puerarin, through its anti-inflammatory and antioxidant effects, can effectively improve LPS-induced myocardial injury, reduce the release of myocardial enzyme release, and maintain normal heart function. Western blot demonstrated that Puerarin inhibited the expressions of cleaved caspase-3, Bax, Bcl-2, ACSL4, and TFR, meanwhile up-regulating AMPK phosphorylation in heart tissues. Furthermore, we added compound C, an AMPK inhibitor, to LPS model rat, and subsequent experimental data suggested that Puerarin’s cardioprotective effect was possibly related to AMPK-mediated ferroptosis signaling pathway (Figure 7).

Sepsis is a systemic inflammatory response syndrome caused by infection [2] and is characterized by sepsis-induced cardiotoxicity [32]. Sepsis can cause myocardial injury and increase the concentration of myocardial damage markers (CK-MB and LDH) in mouse plasma [31]. In addition, it can lead to cardiac dysfunction manifested as a decline in ejection fraction (EF%) and fractional shortening (FS%) [31]. Studies have demonstrated that oxidative stress and inflammation caused by LPS through various signaling pathways are the main pathophysiological mechanisms of sepsis leading to myocardial damage and cardiac dysfunction [21]. LPS can up-regulate the expression of inflammatory cytokines through TLR4/NFκB signaling pathway to promote inflammation [33]. LPS can also enhance the oxidative stress of mouse myocardial tissue by inhibiting AMPK signaling pathway [34]. Researchers used many anti-inflammatory and antioxidant drugs to conduct experiments to determine the mechanism of LPS-induced myocardial injury. Leonurine inhibits oxidative stress induced by LPS by inhibiting NF-κB signaling pathway, thereby protecting the heart [35]. Salidroside inhibits the inflammatory response induced by LPS by inhibiting ROS-mediated PI3K/Akt/mTOR pathways, thus improving sepsis-induced cardiotoxicity [36]. Numerous studies have indicated that many traditional Chinese herbs have potent anti-inflammatory and antioxidant properties [12–15]. Due to various diseases, Puerarin can effectively reduce oxidative stress and inflammation in the myocardium [37, 38]. In addition, through our research, we demonstrate that Puerarin significantly reduced oxidative stress indicators (MDA, GSH, and SOD) and inflammatory cytokines (TNF-α, IL-6, and IL-10) in the serum of LPS model rats.

Puerarin is a monomer of traditional Chinese medicine derived from the Radix Pueraria, one of the most effective sources of isoflavones. The chemical name is 4, 7-dihydroxy-8-β-D-glucosyl isoflavone [16]. Puerarin is used to treat various diseases, and the concentration used for each disease varies [39, 40]. Puerarin has been revealed to successfully improve myocardial damage caused by coronary heart disease, diabetes, ischemia-reperfusion, hypoxia, and severe burns. Puerarin is notable for protecting against myocardial damage caused by various heart diseases through multiple mechanisms. Puerarin inhibits cardiomyocyte apoptosis by down-regulating the expression of apoptosis-related proteins cleaved-caspase 3 and Bax, increasing Bcl-2 expression, and increasing Bcl-2/Bax ratio [41].

Furthermore, we also confirmed this conclusion in our research. In a study of myocardial infarction, researchers found that Puerarin can protect against myocardial infarction by regulating PPAR-Υ/NF-κβ signaling pathway [42]. Puerarin inhibits inflammation by activating SIRT1/NF-κβ signaling pathway, improving myocardial ischemia-reperfusion injury [37].

Moreover, Puerarin can protect against myocardial injury caused by various diseases by modulating AMPK signaling pathway. In a study of myocardial ischemia-reperfusion injury, researchers found that Puerarin can reduce the expression of inflammatory cytokines IL-6, IL-1β, and TNF-α through AMPK/Akt/GSK-3β /Nrf2 signaling pathway to prevent myocardial ischemia-reperfusion injury [43]. Interestingly, researchers also conducted experiments with other drugs and discovered that AMPK could inhibit oxidative stress and inflammation induced by LPS after activation [44].

AMPK, an AMP-dependent protein kinase, is a key molecule that regulates bioenergy metabolism and a potential therapeutic target for various metabolic diseases [45]. AMPK is a heterotrimeric protein kinase composed of one catalytic subunit α and two regulatory subunits β and γ [46]. AMPK signaling pathway plays a vital role in the human body, and several substances influence the body through this signaling pathway [46]. Studies have found that luteolin can protect against LPS-induced myocardial injury by activating the AMPK signaling pathway [47], suggesting that activating the AMPK signaling pathway can effectively improve LPS-induced myocardial injury. In addition, other studies have indicated that activation of the AMPK signaling pathway can inhibit ferroptosis [26]. Ferroptosis is a non-apoptotic cell death that depends on iron and is regulated by lipid oxidation [23, 24].

Additionally, ferroptosis is implicated in organ damage and dysfunction caused by various diseases. Several scholars have suggested that GPX4, acyl-CoA synthase long-chain family member 4 (ACSL4), transferrin receptor (TFR), and ferritin (Ferritin) play a critical role in ferroptosis and can be used as ferroptosis occurrence detection metrics [48, 49]. As a result, our study also chose these four as ferroptosis monitoring indicators. Based on recent research, Puerarin can protect organs by directly inhibiting ferroptosis [29]. More importantly, ferroptosis is important in myocardial injury resulting from various heart diseases [50]. Ferroptosis inhibitors can effectively improve heart damage caused by various heart diseases. Researchers used ferrostatin-1 to treat H9c2 cells and found that ferrostatin-1 can inhibit the damage to cardiomyocytes caused by ischemia-reperfusion [51]. More interestingly, it has been recently confirmed that dexmedetomidine improves tissue and organ damage by inhibiting LPS-induced ferroptosis [25]. In addition, we discovered that ferroptosis activator Erastin could effectively abolish Puerarin’s protective effect on LPS-induced myocardial injury, confirming ferroptosis’s key role in myocardial injury. In echocardiography and HE stained sections, we found that Erastin combined with Puerarin exacerbated myocardial injury, the release of myocardial enzymes increased (CK-MB, LDH, and cTnI), while myocardial contractility decreased. By measuring the heart weights of the rats in each group, we discovered that compared to LPS group, HW/BW and HW/TL ratios of LPS+Pue group were significantly reduced (p < 0.05), demonstrating that Puerarin can alleviate LPS-induced myocardial edema (Figure 1B).

Furthermore, ELISA results show that Erastin in conjunction with Puerarin group increases the oxidation index MDA in cardiomyocytes while decreasing the antioxidant index GSH and SOD. We also observed that Erastin effectively abolished the anti-inflammatory effect of Puerarin. After adding Erastin, the expressions of IL-1, IL-6, and TNF-α in myocardial cells increased (Figure 3A–3C). In addition, Erastin can directly abolish the anti-apoptotic effect of Puerarin. The expression level of cleaved caspase-3 and Bax/Bcl-2 ratio in Erastin combined with Puerarin group were significantly higher than those of LPS+Pue group. Employing tissue-iron quantitation and Western blotting, we discovered that Puerarin could effectively reduce the iron content in the myocardium while promoting AMPK phosphorylation. Puerarin can effectively inhibit the expression of ferroptosis-promoting protein ferritin and ACSL4 and increase the expression of various anti-ferroptosis-related proteins (Figure 5). AMPK phosphorylation level in LPS+Pue group increased compared to that in LPS group. The expression of ferroptosis promoting proteins Ferritin and ACSL4 decreased, while the anti-ferroptosis proteins GPX4 and TFR increased (Figures 5, 6).

Furthermore, after AMPK inhibitor compound C inhibited AMPK activation, the regulatory effect of Puerarin on ferroptosis-related proteins was inhibited. Similarly, the iron content of CC-added group was significantly higher than that of LPS+Pue group (Figure 6). Our results indicate that Puerarin has an anti-ferroptosis effect, but this effect was blocked by pretreatment with AMPK inhibitor CC.

Conclusions

The present study demonstrated that Puerarin exerts cardioprotective effects against sepsis-induced cardiotoxicity. Puerarin exerts cardioprotective effects by attenuating inflammation and oxidative damage in myocardial tissues and suppressing apoptosis and ferroptosis in cardiomyocytes. The cardioprotective effects of Puerarin can be inhibited by inhibiting AMPK phosphorylation or promoting ferroptosis. Our results suggest that targeting AMPK or ferroptosis key proteins could be vital for future prevention and treatment of LPS-induced myocardial injury. More research is required to investigate the clinical application of Puerarin.

Materials and Methods