Elsevier

Phytomedicine

Volume 21, Issue 4, 15 March 2014, Pages 461-469
Phytomedicine

Shuang-Huang-Lian exerts anti-inflammatory and anti-oxidative activities in lipopolysaccharide-stimulated murine alveolar macrophages

https://doi.org/10.1016/j.phymed.2013.09.022Get rights and content

Abstract

Shuang-Huang-Lian (SHL) is a traditional Chinese compound formula prepared from Lonicerae Japonicae Flos, Scutellariae Radix and Fructus Forsythiae. In this study, we demonstrate the anti-inflammatory and anti-oxidative activities of SHL in lipopolysaccharide (LPS)-stimulated murine alveolar macrophages (MH-S). SHL significantly reduces the transcriptional and translational levels of iNOS and COX-2 as well as the production of NO and prostaglandin E2 (PGE2). It also suppresses the transcription and translation of inflammatory cytokines production, such as TNF-α, IL-1β and IL-6. These inhibitory effects mainly act via ERK1/2- and p38-mediated AP-1 rather than the NF-κB pathway. In parallel with the anti-inflammatory activity, SHL suppresses LPS-induced intracellular total ROS levels by weakening NADPH oxidase activity, enhancing SOD activity and increasing GSH content. In addition, SHL directly scavenges radical dotOH and O2radical dot. Thus, our study elucidates the anti-inflammatory and anti-oxidative mechanisms of SHL in LPS-stimulated MH-S.

Introduction

SHL is a traditional Chinese medicine preparation composed of three herbs: Lonicerae Japonicae Flos, Scutellariae Radix and Fructus Forsythiae. It is officially recorded in the Chinese Pharmacopeia and has the ability to remove toxic heat and induce diaphoresis (State Pharmacopoeia Committee, 2010). Clinically, SHL products are delivered by different routes of administration (e.g. oral, injectable and pulmonary routes) (Sun et al., 2006) and has been widely used to treat various infectious diseases caused by bacteria or viruses in the respiratory tract. Although the pharmacological effects are studied extensively (Zhou et al., 2011), the potential protection mechanisms of SHL against infection-induced lung injury remain uncertain.

During the process of gaseous exchange, the lung is exposed to various foreign particles, pathogens and atmospheric oxygen. The LPS present in the outer membrane of bacteria is a major contributor to lung injury. LPS-induced lung injury is characterized by a series of responses, including the infiltration of macrophages into the alveoli and the release of chemokines, pro-inflammatory cytokines, etc. The excessive accumulation of inflammatory mediators and oxidants, especially cytokines, reactive oxygen species (ROS) and bioactive lipid products, may contribute to various pulmonary diseases. These include acute lung injury, adult respiratory distress syndrome, asthma, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis and others (Tiwari et al., 2010). Therefore, compounds with anti-oxidative and anti-inflammatory functions are useful therapeutic agents for the treatment of respiratory tract and lung infections (Reddy et al., 2012).

Alveolar macrophages (AMs), which play an important role in innate and adaptive immunity, can protect the lungs as a first line of defense AMs absorb foreign particles by phagocytosis, which recruits and activates inflammatory cells. When AMs are stimulated by LPS, there is over-production of ROS and pro-inflammatory mediators such as iNOS, COX-2, TNF-α, IL-1β, IL-6, which in turn cause inflammation and damage to neighboring tissues and the macrophages themselves (Tiwari et al., 2010). In the present study, we explore the anti-inflammatory and anti-oxidative effects of SHL based on LPS-stimulated MH-S. Furthermore, we investigate the underlying mechanisms of these effects.

Section snippets

Materials and reagents

Considering the advantages of injection for in vitro assay, we used SHL injection from Duoduo Pharmaceutical Co., Ltd. (Jiamusi, Heilongjiang, China) in this study. PGE2 high sensitivity ELISA kit was purchased from Enzo Life Sciences (Geneva, Switzerland). Mouse TNF-α, IL-1β and IL-6 ELISA kits were obtained from Excell Technology Co. (Shanghai, China). Antibodies for iNOS, COX-2 and β-actin were from Santa Cruz Biotechnology, Inc. (Santa Cruz, California, USA). Antibodies against

HPLC analysis of SHL

The main component profile of SHL was analyzed via HPLC–UV. The representative chromatogram is shown in Fig. 1. The identification of SHL constituents was based on the retention times and the UV spectrum in comparison with authentic standards at a wavelength of 350 nm. Ten components in SHL assigned to three herbal drugs (Lonicerae Japonicae Flos, Scutellariae Radix and Fructus Forsythiae) were identified and quantified with the indicated conditions (Table 3).

Inhibition of NO and PGE2 production

Cell viability analysis showed that

Discussion

In the present study, we identified ten main components in SHL using HPLC–UV analysis. Of these components, chlorogenic acid (Francisco et al., 2013, Kwon et al., 2010), caffeic acid (Huang et al., 2013), forsythiaside (Kim et al., 2011), rutin (Lee et al., 2013), baicalin (Guo et al., 2013, Cao et al., 2011) and luteolin (Nishitani et al., 2013, Park et al., 2012) were reported to have anti-inflammatory and anti-oxidative activities. These components mainly contributed to the effects of SHL in

Acknowledgments

This work was supported by the National Natural Science Foundation of China (Nos. 81274163 and 81173645) and National S&T Major Project and Scientific Researchers Aiding Enterprise Item (No. 2012ZX09301-002-001) from the Ministry of Science and Technology of the People's Republic of China.

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