Ginkgolide B functions as a determinant constituent of Ginkgolides in alleviating lipopolysaccharide-induced lung injury

Abstract

Ginkgolides are the major bioactive components of Ginkgo biloba extracts, however, the exact constituents of Ginkgolides contributing to their pharmacological effects remain unknown. Herein, we have determined the anti-inflammatory effects of Ginkgolide B (GB) and Ginkgolides mixture (GM) at equivalent dosages against lipopolysaccharide (LPS)-induced inflammation. RAW 264.7 cell culture model and mouse model of LPS-induced lung injury were used to evaluate in vitro and in vivo effects of GB and GM, respectively. In RAW 264.7 cells, GB and GM at equivalent dosages exhibit an identical capacity to attenuate LPS-induced inducible nitric oxide synthase mRNA and protein expression and subsequent NO production. Likewise, GB and GM possess almost the same potency in attenuating LPS-induced expression and activation of nuclear factor kappa B (p65) and subsequent increases in tumor necrosis factor-α mRNA levels. In LPS-induced pulmonary injury, GB and GM at the equivalent dosages have equal efficiency in attenuating the accumulation of inflammatory cells, including neutrophils, lymphocytes, and macrophages, and in improving the histological damage of lungs. Moreover, GB and GM at equivalent dosages decrease the exudation of plasma protein to the same degree, whereas GM is superior to GB in alleviating myeloperoxidase activities. Finally, though GB and GM at equivalent dosages appear to reduce LPS-induced IL-1β mRNA and protein levels and IL-10 protein levels to the same degree, GM is more potent than GB to attenuate the IL-10 mRNA levels. Taken together, this study demonstrates that GB functions as the determinant constituent of Ginkgolides in alleviating LPS-induced lung injury.

Introduction

Gingko biloba extracts (EGB) prepared from Gingko biloba leaves contain a variety of medicinal ingredients, and have been used in traditional medicines for centuries [1]. Since Ginkgo biloba leaves were registered for a medication (named EGB761) by the mid-1960s, EGB761 has been widely used throughout Europe, North America and Asia for dementia, macular degeneration, tinnitus, and winter depression [2]. In modern China, EGB is mainly used for the treatment of pulmonary and cardiac diseases [3]. The standard EGB761 contains 6% terpene lactones, 24% flavonol glycosides and less than 5 ppm of ginkgolic acids [4]. Ginkgolides predominantly composed of Ginkgolide A (GA), Ginkgolide B (GB), and Ginkgolide C (GC), are the biologically active terpenic lactones, and have been considered as the major active component of EGB [4], [5]. Ginkgolides have been recognized as an antagonist of platelet activating factor (PAF) receptor, and are able to inhibit PAF-induced cascade effect in platelet aggregation and inflammatory reactions [6], [7]. Moreover, Ginkgolides have been shown to possess antioxidant activity and function as potent peroxy radical scavengers [8]. Finally, Ginkgolides appear to reduce the portal vein pressure of liver cirrhosis, exert an anti-shock effect, and protect the lung injury from asthma and infections [9], [10], [11].

Airway inflammation is critical for various respiratory diseases, including chronic obstructive pulmonary disease (COPD), acute respiratory distress syndrome (ARDS), and asthma. It is believed that inflammatory process is responsible for the initiation, progression, and prognosis of these diseases [12]. Among various inflammatory cells, neutrophils play the pivotal roles in airway inflammation [13]. In response to the inflammatory conditions, neutrophils fulfill chemotaxis and respiratory burst, two essentially biological responses. In chemotaxis, neutrophils cross the blood vessel and migrate into inflammatory tissues through cytoskeleton rearrangement [14]. In respiratory burst, neutrophils generate reactive oxygen species (ROS) in a nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-dependent manner to kill the microbes [15]. Lipopolysaccharide (LPS), a bacterial cell wall component, induces the pulmonary inflammation characterized by neutrophil infiltration. Binding of LPS to Toll-like receptors (TLR) triggers MyD88-dependent and MyD88-independent TLR signaling cascades, consequently leading to the expression of pro-inflammatory mediators, such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), through activation of nuclear factor-κB (NF-κB) [16]. Neutrophils activated by LPS not only release pro-inflammatory mediators but also aggravate inflammation.

Despite the fact that mounting evidence has demonstrated the potently anti-inflammatory effects of Ginkgolides in a variety of inflammatory diseases [17], [18], [19], [20], which constituents predominantly function as a biologically active component is not fully understood. Hence, we attempt to compare the pharmacological effects of GB and Ginkgolides mixture (GM) at equivalent dosages on LPS-induced inflammatory lung injury. In the present study, we have shown that GB functions as a determinant constituent of Ginkgolides in alleviating the LPS-induced inflammatory responses both in vivo and in vitro.