Anti-inflammatory effects of Reduning Injection (热毒宁注射液) on lipopolysaccharide-induced acute lung injury of rats

Chin J Integr Med 2014 Aug;20(8):591-599 • 591 • ORIGINAL ARTICLE Anti-Inflammatory Effects of Reduning Injection (热毒宁注射液) on Lipopolysaccharide-Induced Acute Lung Injury of Rats TANG Lu-ping (唐陆平)1, XIAO Wei (萧 伟)2, LI Yi-fang (李怡芳)1, LI Hai-bo (李海波)2, WANG Zhen-zhong (王振中)2, YAO Xin-sheng (姚新生)1, Hiroshi Kurihara1, and HE Rong-rong (何蓉蓉)1 Objective:: To evaluate the protective effects of Reduning Injection (热毒宁注射液, RDN), a ABSTRACT Objective patent Chinese medicine, on lipopolysaccharide (LPS)-induced acute lung injury (ALI) in rats and its underlying Methods:: Sixty male Sprague-Dawley rats were randomly divided into 6 groups, including mechanisms of action. Methods normal control, model, dexamethasone (DEX, 5 mg/kg), RDN-H (720 mg/kg), RDN-M (360 mg/kg) and RDN-L (180 mg/kg) groups, with 10 rats in each group. Rats were challenged with intravenous injection of LPS 1 h after intraperitoneal treatment with RDN or DEX. At 6 h after LPS challenge, lung tissues and bronchoalveolar lavage fluid (BALF) were collected, and the number of inflammatory cells was determined. The right lungs were collected for histopathologic examination, measurement of gene and protein expressions, superoxide dismutase Results:: In vivo pretreatment of RDN (360, 720 mg/kg) significantly (SOD) and myeloperoxidase (MPO) activities. Results reduced the weight of wet to dry (W/D) ratio of lung, protein content in BALF, and led to remarkable attenuation of LPS-induced histopathological changes in the lungs. Meanwhile, RDN enormously decreased BALF total inflammatory cells, especially neutrophil and macrophage cell numbers. Moreover, RDN increased SOD activity, inhibited MPO activity, alleviated LPS-induced tumor neurosis factor-α (TNF-α) and inducible nitric oxide synthase (iNOS) expression in lung tissues. Furthermore, RDN (720 mg/kg) efficiently weakened nuclear factorConclusion:: Anti-inflammatory effects of RDN was demonstrated kappa B (NF-κB) gene and protein expression. Conclusion to be preventing pulmonary neutrophil infiltration, lowering MPO activity, TNF-α and iNOS gene expression by inhibiting NF-κB activity in LPS-induced ALI. KEYWORDS acute lung injury, lipopolysaccharide, neutrophils, chemokine, nuclear factor-kappa B, Reduning Injection, Chinese patent medicine Acute lung injury (ALI) is a clinical complication mainly due to gram-negative bacterial infection and an important cause of morbidity and mortality in critically ill patients.(1) Lipopolysaccharide (LPS), major composition of the outermost membrane of gramnegative bacteria, is recognized as a key factor in the pathogenesis of ALI,(2) which even can cause acute respiratory distress syndrome (ARDS).(3) Intravenous administration of LPS has been widely used as an experimental animal model of ALI/ARDS. (4) LPSinduced ALI is characterized by the infiltration of neutrophils and macrophages into alveoli, (5) the release of chemokines and pro-inflammatory cytokines from activated macrophages and neutrophils, (6,7) as well as the increase of protein content in the bronchoalveolar lavage fluid (BALF).(8) Among these pro-inflammatory cytokines, tumor necrosis factor α (TNF-α), which participates in both early and later inflammatory responses, plays a critical role in the progression of ALI.(9,10) The excessive accumulation of inflammatory mediators, especially cytokines, chemokines, adhesion molecules, and bioactive lipid products(11,12) contribute to the increased pulmonary vascular permeability, interstitial edema, disruption of epithelial integrity, and lung parenchymal injury.(13,14) Numerous studies have suggested that nuclear factorkappa B (NF-κB) is activated after LPS exposure to the experimental animal. Activated NF-κB plays a key role in the regulation of inflammatory mediator ©The Chinese Journal of Integrated Traditional and Western Medicine Press and Springer-Verlag Berlin Heidelberg 2014 Supported by Distinguished Trans-Century Training Program Foundation for the Talents of the State Education Commission, China (No. NCET-12-0678), Natural Science Foundation of Guangdong Province, China (No. S20120011316), Science and Technology Program of Guangzhou, China (No. 2012J22000073), Foundation for Distinguished Young Talents in Higher Education of Guangdong, China (No. LYM11023) 1. Pharmacy College, Jinan University, Guangzhou (510632), China; 2. State Key Laboratory of New-Tech for Chinese Medicine Pharmaceutical Process, Lianyungang, Jiangsu Province (222001), China Correspondence to: Prof. HE Rong-rong, Tel: 86-20-85227791, E-mail: DOI: 10.1007/s11655-014-1758-x Chin J Integr Med 2014 Aug;20(8):591-599 • 592 • transcription, (15) which contributes to the vigorous acquisition of neutrophils in the lung and is crucial to the pathogenesis of ALI.(16) LPS challenge also activates neutrophils and macrophages within the lung vasculature, which exaggerates production of reactive oxygen species (ROS), including hydroxyl radical, hydrogen peroxide and superoxide anion radical.(17,18) They are believed to contribute to the severity of several diseases, including ALI and ARDS. The increased ROS will induce lipid peroxidation and alter antioxidant enzyme activity,(17,19) resulting in increased pulmonary vascular permeability and the release of inflammatory cell in BALF.(20) Thus, ROS is believed to be crucial in the induction of lung damage caused by pneumonia, while therapeutic agents that could effectively scavenge ROS may prevent or reduce the deleterious effects of pneumonia. Reduning Injection (热毒宁注射液, RDN) is a patented Chinese medicine prepared by effective components from the extract of Artemisia annua , Gardenia jasminoides Ellis , and Flos Lonicerae . It has been widely used to treat common cold, cough, acute upper respiratory infection (AURI) and acute bronchitis in clinic. Clinical studies reported that RDN was safe and effective in curing pneumonia in the elderly population(21) and also had good therapeutic effect to AURI in children.(22) At the same time, it was reported to be used as a new drug for prophylactic treatment to Staphylococcus aureus , Klebsiella pneumoniae and influenza virus in clinic.(23) However, there is little experimental data regarding the effects of RDN on inflammation. The present study was undertaken in rats to evaluate the effects of RDN on ALI induced by bacterial LPS through examining cellular infiltration and histological changes in the lungs. METHODS Drugs and Reagents RDN (Batch No. 100906) was provided by Jiangsu Kanion Pharmaceutical Co., Ltd. (Jiangsu, China). Its chemical pattern was obtained by highperformance liquid chromatography (HPLC) analysis. The injection was submitted to Dianon HP-20 Toyopearl HW40 (Mitsubishi, Japan) and preparative HPLC chromatographic columns (Silicycle Canada), yielding compounds of the main peaks in the HPLC fingerprint. The structures of these compounds were identified by spectrophotometer (Waters, USA), mass spectrometer (Bruker, USA), and nuclear ma (...truncated)