Sepsis is a serious medical condition that arises when the body's responses to an infection creates widespread inflammation, leading to organ malfunction and failure. Numerous illnesses, such as bacterial, viral, fungal, and parasitic diseases, can cause it. The lungs (pneumonia), urinary system, digestive system, bloodstream, catheter sites, wounds, and burns are common sources of infection that can result in sepsis [1]. Because of its high pathophysiological, molecular, genetic and clinical complexity; sepsis and septic shock pose a significant and increasing worldwide burden to emergency doctors [2], [3]. Since the initial consensus definition (Sepsis-1) was established in 1991, the prevalence of sepsis and septic shock has steadily climbed, reaching about 49 million cases of sepsis and 11 million sepsis-related fatalities globally in 2017 [4]. Sepsis is an inflammatory condition that helps notify the immune system of an infection, but if left unchecked, it can cause organ failure and a hyperinflammatory state [5]. Multiple organ dysfunction syndrome (MODS) drives morbidity and mortality in sepsis. The utility of supervised machine learning driven identification of the genes associated with persistent MODS in paediatric septic shock was demonstrated [6]. The balance between pro and anti-inflammatory processes is essential for the patient's recovery, and the regulation of inflammatory responses is defective in sepsis, leading to a cascade of harmful effects [7].

Macrophages are key cells in the innate immune system, performing various functions [8]. Phenotypic polarization of macrophages is essential for the initiation and resolution of inflammation in all tissues. Using machine learning algorithms on human macrophages a recent finding provided a formal and universally accepted definition of macrophage typing and a predictive framework (https://hegemon.ucsd.edu/SMaRT) was proposed for the scientific community who are engaged in developing macrophage-targeted diagnostics and therapeutics [9]. The challenges and prospects associated with monitoring immune microenvironment and disease progression through imaging of macrophage phenotypes have been discussed [10].

They exhibit functional diversity through a process called polarization, which involves differentiating into distinct subtypes, such as M1 and M2, in response to micro-environmental stimuli. M1 macrophages are involved in antimicrobial activities, while M2 macrophages are associated with wound healing, tissue repair, and immunoregulation [11]. Recent study demonstrated that the specific activation of nuclear factor erythroid 2–related factor 2 (Nrf2) in macrophages at infection sites is important for management of sepsis. In this aspect, it was shown that inhibition of Kelch-like ECH-associated protein (Keap1) − Nrf2 protein–protein interaction pathway by an heptamethine dye- IR-61 may be promising for the treatment of sepsis [12]. Another group of macrophages namely the tumor-associated macrophages (TAMs) normally displayed an immunosuppressive phenotype, in promoting the tumor growth and invasiveness directly and via angiogenesis, tissue remodelling, production of specific cytokines/ chemokines, and suppression of adaptive immunity [13].

LPS, present on the outer wall of Gram-negative bacteria, binds to Toll-like receptor 4 (TLR4), triggering a cascade of pro-inflammatory mechanisms. TLR4 acts as a receptor for LPS, promptly inducing the production of several pro-inflammatory, anti-viral, and anti-bacterial cytokines [14]. LPS induces inflammatory cytokines primarily through the TLR4 pathway [15]. The LPS molecule is toxic and classified as an endotoxin that elicits a strong immune response. The TLR4 signaling pathway plays an important role in initiating the innate immune response and its activation by bacterial endotoxins. The TLR4 signaling pathway is a potential target for modulators in the treatment of inflammation and sepsis [16]. Additionally, various studies have reported that LPS induces the production and release of inflammatory mediators, including cytokines, which play a crucial role in the pathogenesis of sepsis.

Furthermore, several compounds, such as Corylin and Toddalolactone, have been shown to protect against LPS-induced sepsis and attenuate the LPS-induced inflammatory response by modulating the expression of pro-inflammatory cytokines [17]. Protopine (PTP), an alkaloid with anti-inflammatory and antioxidant properties recently shown to prevent excessive mitophagy and acute lung injury in LPS sepsis [18]. Cytokines are important pleiotropic regulators of the immune response, which have a crucial role in the complex pathophysiology underlying sepsis [19]. As integral components of the innate immune system, interferon gamma (IFN-γ) also provide an early defense against infections and contribute to the overall regulation of the LPS induced immune response [20]. TNF-α, IL-1β, IL-10, IL-17 and IFN-γ reported o play important roles in the immune response and inflammation. TNF-α, IL-1β, and IFN-gamma are considered as prominent mediators of the “cytokine storm” in sepsis, and their circulating concentrations may correlate with disease severity [21]. Recent study highlighted the understandings of the biological functions of IL-34 in macrophage activation and polarization which might provide as new therapeutic approaches in the prevention of heart failure [22]. It was shown that IL-12α expression was upregulated in LPS sepsis mice where macrophages were the main sources of IL-12α. Moreover, deletion of IL-12α increased the LPS-induced macrophage infiltration and drives macrophages toward the M1 phenotype in LPS-treated mice [23].

Efflux pumps may influence the immune response by modulating the production of pro-inflammatory cytokines, such as IFN-γ and TNF-α, which are increased in the peripheral circulation of patients with conditions like chronic obstructive pulmonary disease (COPD) [24]. Macrophages play a crucial role in regulating the host's immune balance and inflammatory response in sepsis [25]. Polarization refers to the ubiquitous process by which macrophages can be directed towards different functional profiles, such as M1 or M2 macrophages. M1 macrophage polarization is related to the activation of the TLR4/NF-κB pathway [26]. Macrophage polarization and ROS production are interconnected in the context of LPS-induced sepsis. M1 macrophages rely on ROS production for their bactericidal function, while M2 macrophages may be protected by increased anti-oxidant levels. Dysregulation of macrophage polarization and reactive oxygen species (ROS) production can contribute to the pathogenesis of sepsis-induced immunosuppression. Therefore, further research is needed to fully understand the complex relationship between macrophage polarization, ROS production, and LPS-induced sepsis.

Additionally, most of the cytokines and chemokines signal through mechanisms dependent of JAK-STAT [27]. The JAKs and STATs are differentially expressed in various cell types, and their activation can lead to distinct downstream effects [28]. The JAK/STAT3 pathway plays a critical role in the immune response, mediating the cellular inflammation response, and regulating the development and function of various immune cells. It is involved in the transduction of extracellular signals and is essential for the regulation of immune responses, including the induction of pro-inflammatory and anti-inflammatory cytokines. Certain primary immunodeficiencies result from genetic mutations affecting JAK-STAT signaling components, leading to immunological abnormalities and increased susceptibility to infections [29].

PGP is a transmembrane transporter responsible for the efflux of a number of drugs [30]. It is also expressed on murine macrophages [31]. Differences in transporter expression between M1 and M2 macrophages may result in differences in intracellular concentrations of many drugs, leading to sub-therapeutic intracellular concentrations, and may cause in the inability for the elimination of bacteria from these cells. Based on the provided search results, there are natural products that have been shown to decrease inflammation and efflux transporter expression. For example, curcumin-loaded noisome have been shown to decrease immune cell influx and inflammatory mediators in an in vivo study [32]. Additionally, natural products such as quercetin, curcumin, honokiol, magnolol, caffeic acid phenetyl ester and xanthohumol have been investigated for their ability to modulate the function of ATP-binding cassette (ABC) drug transporters, which are efflux transporters [33]. Furthermore, dietary polyphenols such as quercetin, curcumin, and others have been shown to affect the expression of drug efflux transporters. Fruits and vegetables also contain various natural polyphenols that can provide substantial health benefits beyond basic nutrition [34]. These natural products have been researched for their potentiality to reduce inflammation and may offer alternative or complementary approaches for managing inflammatory conditions.

Verapamil improves survival of mice receiving a lethal dose of LPS and significantly potentiates the protective effect against LPS-induced sepsis, probably due to both P-gp inhibition and a reduced level of cytokines [35]. Effects of verapamil on different parameters in LPS Induced inflammatory disease has been reported in mice model [36], [37]. Verapamil has shown positive therapeutic effects via suppressing uncontrolled inflammatory response, oxidative stress and NF-κB signalling [38]. It has been reported that verapamil provided protection against ROS in diabetic nephropathy [39], suppressed LPS-induced production of pro-inflammatory and anti-inflammatory cytokines in sepsis [40]. It also reduced the acute liver injury via suppressing the production of pro-inflammatory cytokines by inhibiting the NF-κB activation [41], decreased in vitro pro-inflammatory gene signaling and macrophage responsiveness [42]. Verapamil also down-regulated the production of pro-inflammatory cytokines (TNF-α and IL-6) and increased IL-10 in vivo [43].

Tangeretin, a flavonoid found in citrus fruit peels, has demonstrated anti-inflammatory and antioxidant properties [44]. Tangeretin has been found to inhibit LPS-induced production of nitric oxide, TNF-α, IL-6 and IL-1β, as well as LPS-induced mRNA expression of inducible nitric oxide synthases and cytokines [44]. In various studies, tangeretin has shown its potential in inhibiting oxidative stress and inflammation through upregulating the Nrf-2 signaling pathway [45]. Tangeretin (50 mg/kg) given orally once daily for 14 days showed therapeutic effects on rheumatoid arthritis by decreasing oxidative stress damage, regulating inflammatory cytokine levels (IL-1β, TNF-α, IFN-γ), and enhancing antioxidant enzyme activity [45].

Several potential therapeutic targets have been identified for LPS-induced sepsis. These include intracellular LPS sensing, which has been suggested as a potential therapeutic target for sepsis [46]. Additionally, natural compounds have been shown to protect against LPS-induced sepsis and attenuate the LPS-induced inflammatory response by modulating the expression of pro-inflammatory cytokines [47]. Notoginsenoside R1 (NG-R1) which is a unique bioactive component of Panax notoginseng could also protect against sepsis-induced cardiomyopathy (SIC) by regulating the expression of TNF-α inflammatory factors providing a new concept for drug development in sepsis [48]. Each step involved in the development of LPS-induced sepsis, including microbial invasion, recognition of bacterial products, immune response and immune dysregulation, endothelial and organ damage, and organ crosstalk and multiple organ dysfunction [49], can be a potential target for a specific therapeutic approach in favour of usage of such natural products. Therefore, a combination of targeted treatments and precision medicine may be needed to effectively treat LPS-induced sepsis using components obtained from natural sources.

A recent study demonstrated that tangeretin protects against LPS-induced acute lung injury in male C57BL/6 mice by suppressing Th17 response via a Notch-dependent mechanism [50]. Sepsis is associated with T-cell exhaustion. The role of Sialic acid-binding Ig-like lectin 5 (SIGLEC5) as an immune checkpoint ligand (IC ligand) and its potential as a biomarker for sepsis explored has been explored [51].Tangeretin has been reported to inhibit inflammation by inhibiting NF-κB activation and proinflammatory cytokines such as IL-1β and TNF-α [52]. However, the effects of verapamil or tangeretin on lipopolysaccharide (LPS)-induced sepsis in Swiss albino mice and its anti-inflammatory mechanism have not been reported. It is well known than LPS induces NFκB activation after binding to TLR4 [53]. LPS also affects the cytokine network and efflux drug transporters [54]. Inflammation and oxidative damage driven by proinflammatory macrophages (i.e. M1) are the major contributors to the development septic shock and therefore inhibition of macrophage-driven inflammation is an important target in the search of compounds (here Verapamil and tangeretin) capable of combating LPS sepsis. Verapamil or Tangeretin by downregulating the PGP expression and subsequent modulation of efflux pump that synergize with innate immunity to kill Gram negative bacteria especially the MDRs and might have potential for development of alternative therapy along with antibiotics; when P-gp is inhibited anticipating retaining of the drugs [for example DEX or the antibiotics] in the macrophages. However, the role of either verapamil or tangeretin in mediating the effects of LPS on P-gp expression at the murine macrophage functional and downstream signalling level has not been fully elucidated. In this study, we have determined that verapamil and tangeretin protects against LPS-induced sepsis by suppressing M1 macrophages in the circulation and also through the inhibition of P-glycoprotein expression via downregulating STAT1/STAT3 and upregulating SOCS3 expression in peritoneal macrophages.