Apigenin C-glycosides of Microcos paniculata protects lipopolysaccharide induced apoptosis and inflammation in acute lung injury through TLR4 signaling pathway

Highlights

• Apigenin C-glycosides (ACGs) are separated from the medicinal plant M. paniculata.

• M. paniculata ACGs target MAPKs pathway to exert their anti-inflammatory activity.

• M. paniculata ACGs inhibit apoptosis through mitochondrial-dependent pathways.

• Activation of TLR4-TRPC6 pathway is involved in LPS-induced acute lung injury (ALI).

• M. paniculata ACGs can attenuate inflammation and apoptosis in LPS-induced ALI.

Abstract

Acute lung injury (ALI) and its more severe form acute respiratory distress syndrome (ARDS) are life-threatening conditions with high morbility and mortality, underscoring the urgent need for novel treatments. Leaves of the medicinal herb Microcos paniculata have been traditionally used for treating upper airway infections, by virtue of its content of flavonoids such as apigenin C-glycosides (ACGs). C-glycosides have been shown to exert strong anti-inflammatory properties, although their mechanism of action remains unknown. Herein, hypothesizing that ACGs from M. paniculata inhibit progression of ALI, we used the experimental model of lipopolysaccharide (LPS)-induced ALI in BALB/c mice to evaluate the therapeutic potential of purified ACGs. Our results showed that M. paniculata ACGs inhibited lung inflammation in animals undergoing ALI. The protective effects of ACGs were assessed by determination of cytokine levels and in situ analysis of lung inflammation. ACGs reduced the pulmonary edema and microvascular permeability, demonstrating a dose-dependent down-regulation of LPS-induced TNF-α, IL-6 and IL-1β expression in lung tissue and bronchoalveolar lavage fluid, along with reduced apoptosis. Moreover, metabolic profiling of mice serum and subsequent Ingenuity Pathway Analysis suggested that ACGs activated protective protein networks and pathways involving inflammatory regulators and apoptosis-related factors, such as JNK, ERK1/2 and caspase-3/7, suggesting that ACGs-dependent effects were related to MAPKs and mitochondrial apoptosis pathways. These results were further supported by evaluation of protein expression, showing that ACGs blocked LPS-activated phosphorylation of p38, ERK1/2 and JNK on the MAPKs signaling, and significantly upregulated the expression of Bcl-2 whilst down-regulated Bax and cleaved caspase-3. Remarkably, ACGs inhibited the LPS-dependent TLR4 and TRPC6 upregulation observed during ALI. Our study shows for the first time that ACGs inhibit acute inflammation and apoptosis by suppressing activation of TLR4/TRPC6 signaling pathway in a murine model of ALI. Our findings provide new evidence for better understanding the anti-inflammatory effects of ACGs. In this regard, ACGs could be exploited in the development of novel therapeutics for ALI and ARDS.

Introduction

Acute lung injury (ALI) and its more severe form, acute respiratory distress syndrome (ARDS), are life-threatening medical conditions triggered by common pathologies such as trauma, pneumonia or sepsis. As a consequence, patients develop hypoxemic respiratory failure with high morbidity and mortality rate. ALI pathophysiology is characterized by increased permeability of the alveolar-capillary barrier and pulmonary edema as a result of macrophage activation and extensive neutrophil influx into the lungs. Infiltrating cells release cytokines that activate local pro-inflammatory networks, leading to irreversible damage of the lung epithelium and endothelial cells [1], [2].

Lipopolysaccharide (LPS), a main component of the out membrane of gram-negative bacteria, has been identified as a potent inducer of bacterial sepsis-induced ALI [3], [4], [5]. Upon stimulation by LPS, Toll-like receptor 4 (TLR4) expressed in macrophages and endothelial cells is recruited to lipid rafts and interacts with different adaptor molecules [6], [7], resulting in activation of downstream signaling pathways responsible for the production of pro-inflammatory cytokines and subsequent infiltration of inflammatory cells, such as neutrophils, into the lungs [8]. This uncontrolled inflammatory responses finally induce loss of normal alveolar capillary barrier function and pulmonary edema [6], [7]. In this regard, transient receptor potential channel 6 (TRPC6), a permeable non-selective cation channel, has been shown to modulate Ca²⁺ entry into endothelial cells to further enhance TLR4-dependent vascular permeability and local inflammation in the lung [9], [10]. Thus, targeting the TLR4/TRPC6 axis in endothelial cells might offer novel therapeutic targets to inhibit aberrant pro-inflammatory cytokine networks in the lungs of ALI patients. However, despite our increasing understanding of the pathophysiology of ALI and ARDS [11], [12], there is still an alarming lack of therapeutic agents to treat these conditions and the mortality rate remains high, reflecting that key events regulating pathogenesis remain elusive. Indeed, the only effective therapy against ALI consists of mechanical ventilation and there is no pharmacological approach available [13]. Thus, developing novel therapeutics for ALI is an urgent and unmet clinical need.

Microcos paniculata, also known as shiral (India, Bengal), is an edible and medicinal plant, whose leaves (Microctis folium) have been used in traditional medicine for hundreds of years to relieve diseases and infections affecting the upper respiratory tract involving fever, throat irritation, cough or phlegm [14]. It has been proved that M. paniculata total flavonoids are responsible for its main therapeutic effects, which is coincided with its anti-oxidative and anti-inflammatory abilities [15]. However, little is known about the bioactivity of the apigenin C-glycosides (ACGs), the main sub-fraction found in M. paniculata total flavonoids. Recent work suggests that ACGs may act as strong anti-inflammatory compounds [16], [17], [18], [19], although the molecular mechanism of the anti-inflammatory actions elicited by ACGs is still unknown.

In this study, we isolated and characterized M. paniculata ACGs fraction to test its anti-inflammatory potential in the LPS-induced model of ALI in Balb/c mice. Our results show that ACGs are effective at inhibiting disease progression and this was associated with a clear reduction in cellular infiltration and local inflammation as shown by histological analysis and cytokine production, such as IL-6, IL-1β and TNF-α. This provides proof of concept in the in vivo model that such compounds can be active against ALI. Furthermore, we elucidated the underlying mechanism using metabolomics analysis combined with Ingenuity Pathway Analysis, IPA. Metabolomics, which focus on a comprehensive analysis of small molecule metabolites (< 1 kDa), is highly propitious to the discovery of novel biomarkers and therapeutic targets in biomedicine [20], [21]. Combined with IPA, metabolomics is acting as one cutting-edge tool for pharmacological research [22], [23], [24], especially for illustrating the molecular mechanism of bioactive natural products. This approach revealed that ACGs inhibited the MAPKs pathway and apoptosis pathway. Moreover, we also identified the TLR4/TRPC6 axis as a critical pathway down-regulated by ACGs to mediate protection in ALI mice.

Collectively, our data contribute to the identification of the TLR4/MAPKs/TRPC6 axis as therapeutic target in ALI and suggest that ACGs extracted from M. paniculata could offer new molecules to therapeutically intervene these inflammatory and apoptosis pathways.