Investigation Of The Protective Effct Of HJB-1on LPS Induced Macrophages Activation And Acute Lung Injury Of Mice

Inflammation is a protective response by the body to ensure removal ofdetrimental stimuli, as well as a healing process for repairing damaged tissue.Appropriate inflammatory response is beneficial for the host to protect againstpathogens and wound healing, but uncontrolled inflammation leads to extensive tissuedamage and manifestation of pathological states. Macrophages play a central roleduring inflammation process via releasing multiple inflammatory mediators includingpro-inflammatory and cytotoxic cytokines,bioactive lipids, growth factors, hydrolyticenzymes. Lipopolysaccharide (LPS) is a vital component of the outer membrane ofgram-negative bacteria that activates the cells of the monocyte/macrophage lineageafter interacting with CD14, Toll-like receptor4(TLR4) and MD-2on the cellssurface. These interactions result in the activation of second messenger and signaltransduction pathways, including the IKB kinase (IKK)-NF-κB pathway andMAPK pathways.The dried root of Euphorbia fischeriana, known as ‘lang-du’ in traditionalChinese medicine, has been used to treat patients with cancer, edema and ascites forlone time. Previous phytochemical investigation have demonstrated that this plantcontains diterpenoids, triterpenoids and steroids including three ent-abietanediterpenoids, jolkinolide A (JA), B(JB) and17-hydroxy-jolkinolide B (HJB). Recentstudies indicated that JA, JB and HJB exhibit significant antitumour activities againstseveral tumour lines such as Sarcoma180and Ehrlich ascites carcinoma in mice.Several in vitro studies further indicated that JB and HJB induce apoptosis of humanleukemic cells HL-60, MDA-MB-231and human breast cancer cells MCF-7, humanchronic myeloid leukemia (K562). The anti-inflammatory effect of JB, HJB and HJA(17-hydroxy-jolkinolide A) was reported by Uto et al. The investigation of Uto et alindicated that JB exerted anti-inflammatory effects and the presence of C-17hydroxy group in JB or JA significantly enhancing the anti-inflammatory potency of thechemical modified molecule.The aim of this study was to investigate the anti-inflammatory effect of the HJBderivatives that were specifically modified at C-17hydroxy group and clarify themechanism of anti-inflammation of the derivatives in LPS-stimulated mouseperitoneal macrophages.In this study, two HJB derviatives HJB-1and HJB-2were obtained by chemicalmodification of17hydroxy of HJB. HJB-1more effectively inhibited TNF-α releasein LPS-stimulated mouse peritoneal macrophages. Consistently, HJB-1reducedLPS-induced mRNA expression of TNF-α, IL-1β, IL-6, COX-2and iNOS in aconcentration-dependent manner, but did not alter IL-10mRNA level. Further,LPS-induced NF-κB activation and MAPK phosphorylation were also effectivelyinhibited by HJB-1.Acute lung injury (ALI) remain devastating disorders of the lung that areassociated with a high mortality. ALI is characterized by the intense inflammatoryparenchymal process that includes diffuse alveolar damage, disruption of epithelialintegrity, lung parenchymal injury, interstitial edema, and neutrophil accumulation,resulting in releasing of proinflammatory cytokines, including tumor necrosis factor(TNF)-α, interleukin (IL)-1β, and IL-6. Current therapeutic strategy includesprotective ventilation and supportive fluid conservative. Despite extensive studiesabout the pathogenesis of ALI have been conducted, the mortality of ALI remainsvery high. Thus, it is critical to explore the innovative therapies and effectivemedications for ALI.Male BALB/c mice with ALI, induced by intranasal instillation of LPS, weretreated or not with HJB-1(10and2mg/kg, intraperitoneally)1h prior to LPSexposure. HJB-1treatment significantly attenuated pulmonary edema of mice withALI after LPS challenge, as it markedly decreased the lung W/D ratio of lung samples,protein concentration and the amounts of inflammatory cells in BALF. Similarly,verproduction of proinflammatory cytokines in BALF of ALI mice, including TNF-α,IL-1β and IL-6, was strongly reduced by HJB-1. Histological studies demonstrated that HJB-1substantially inhibited LPS induced alveolar wall thickness, alveolarhemorrhage and leukocytes infiltration in lung tissue with evidence of reducedmyeloperoxidase (MPO) activity. In addition, Western blot analysis indicated that theactivation of MAPKs and NF-κB signaling pathways stimulated by LPS wassignificantly blocked by HJB-1. These results demonstrate HJB-1exertsanti-inflammatory effect in LPS-activated mouse peritoneal macrophages byinhibiting NF-κB activation and MAPK phosphorylation and chemical modificationof17hydroxy group of HJB may enhance the anti-inflammatory potency of HJBderivatives.Futhermore, our results suggest that HJB-1exhibits a protective effect onLPS-induced ALI via suppression of MAPKs and NF-κB signaling pathways, whichmay involve the inhibition of tissue oxidative injury and pulmonary inflammatoryprocess.So we may provide a new potential treatment for ALI from traditionalChinese herbal medicine.