Shikonin Attenuates Lipopolysaccharide-induced Acute Lung Injury In Mice

Background and AimsAcute lung injury (ALI), a common clinical severe disorder, presents a high mortalityrate of approximately40%. To data, those available therapies for ALI are not satisfactory.Shikonin is a natural naphthoquinone pigment extracted from the roots of Lithospermumerythrorhizon, which has been used to treat inflammatory diseases such as erysipelas andcarbuncles in traditional Chinese medicine for thousands years. Shikonin has shownanti-inflammatory effect in some in vivo and in vitro studies. In the current study, weaimed to analyze the role of shikonin in acute lung injury (ALI), in order to find out thepotential beneficial agent for ALI.Methods1. Peripheral blood mononuclear cells (PBMCs) were isolated from citrated bloodwhich was obtained from consenting healthy donors.3μM shikonin was added into the culture media of PBMC before lipopolysacchride (LPS) stimulation, and24hours later thesupernatants were collected. The concentration of inflammatory cytokines tumor necrosisfactor (TNF)-α, interleukin (IL)-1β and IL-8in collected supernatants were measuredrespectively by enzyme-linked immunosorbent assay (ELISA). The effect of shikonin inLPS-stimulated PBMCs was analyzed statistically.2. The animal model of ALI was established by intratracheal instillation of LPS inBALB/c mice. After LPS administration, the bronchoalveolar lavage fluid (BALF) andlung tissues were harvested at3h、6h、12h and24h. The concentration of TNF-α and IL-1βin BALF was measured by ELISA. Pulmonary histological changes were observedmicroscopically by hematoxylin-eosin stain and lung wet/dry weight ratios were evaluated.The myeloperoxidase (MPO) activity and nitric oxide (NO) concentration in lung tissuehomogenates were measured. The vadility and reliability of the model induced by LPSinstillation were conformed by clinical findings which met the histo-pathologicalcharacteristics of ALI for the next in vivo studies.3. Sixty male BALB/C mice were randomly devided into6groups (n=10, each):control group, shikonin group (50mg/kg), LPS group, and three different dosage (12.5,25and50mg/kg) for shikonin-treated groups. Shikonin or vehicle was given with annasogastric gavage1hour before intratracheal instillation of LPS (5mg/kg). The degree ofpulmonary injury was evaluated6hours after LPS challenge, including pulmonaryhistopathological changes, the productions of TNF-α and IL-1β and the concentration oftotal proteins in BALF, the concentrations of MPO and NO in lung tissues, and the lungwet/dry weight ratios. To see insight of the potential mechanism of shikoninanti-inflammatory effect, the cyclooxygenase-2(COX-2) and inducible nitric oxidesynthase (iNOS) and the NF-κB DNA binding activity in lung tissues.Results1. Compared with control and shikonin groups, the concentration of TNF-α, IL-1βand IL-8in supernatants in LPS group significantly increased, with values in199.3±21pg/ml,100.3±11.24pg/ml and101.0±11.6pg/ml respectively (P<0.05). However, the production of TNF-α, IL-1β and IL-8was efficiently reduced to126.3±10.0pg/ml,55.7±9.1pg/ml and60.7±12.1pg/ml respectively by pretreatment of shikonin..2. Compared with control group, the concentrations of TNF-α and IL-1β in LPSgroup was significantly increased (P<0.05). After intratracheal instillation of LPS3h, theconcentration of TNF-α and IL-1β in BALF peaked to3038±344.1pg/ml and1841.0±320.0pg/ml respectively. The peak activity of MPO and the concentrations of NOin lung tissues reached to5.09±1.04U/g and697.9±63.18μmol/g respectively after LPSinstillation. Compared with control group, the activity of MPO and the concentrations ofNO in LPS group was markedly increased (P<0.05). After intratracheal instillation of LPS24h, the lung wet/dry ratios was reached to5.32±0.38. Compared with control group, thelung wet/dry ratios in LPS group significantly increased (P<0.05). Lung tissues stainedwith hematoxylin-eosin in the control group showed a normal structure microscopically,while the evident histopathologic abnormalities, including infiltration of inflammatorycells into the interstitial and alveolar spaces, severe hemorrhage in the alveolus, alveoluscollapse, interstitial edema and widespread alveolar wall thickness in lung specimens wasdisplayed in LPS group.3. The productions of the pro-inflammatory cytokines TNF-α and IL-1β and theconcentration of total proteins in BALF was decreased by using shikonin decreased with adose-dependent pattern. Similarly the concentrations of myeloperoxidase (MPO) and nitricoxide (NO) in lung tissues demonstreated the same manner changes as thosepro-inflammatory cytokines. The lung wet/dry weight ratios, as the index of pulmonaryedema, were markedly decreased by shikonin pretreatment. Moreover, shikoninpretreatment could significantly attenuate LPS-induced pulmonary histopathologicalchanges, especially on inflammatory cells infiltration. In addition, LPS-induced activationof COX-2and iNOS in lung tissues could be significantly suppressed by shikoninpretreatment proved with western blotting. And the nuclear levels of NF-κB in lungtissues were also dose-dependently suppressed by shikonin pretreatment as compared withLPS group. Conclusions1. Shikonin could reduce the production of inflammatory cytokines TNF-α, IL-1βand IL-8in LPS-induced PBMCs. It indicated that the shikonin lays the role ofanti-inflammatory effect in vitro.2. The ALI murine experimental model was successfully induced by intratrachealinstillation of LPS and evidenced with the typical pulmonary histopahtological changes,the decreased production of TNF-α and IL-1β in BALF, reduced activity of MPO andincreased concentrations of NO as well as increased lung wet/dry ratios in lung tissues. Webelieved that the the ALI murine model was an ideal and reliable potent to perform furtherin vivo study..3. Shikonin pretreatment could significantly attenuate pulmonary histopathologicalchanges including weaking pulmonary edema, diminishing inflammatory cytokines andprotein in BALF, and reduceing the neutrophils infiltration in lung tissues.. The potentialmechanism of this action may involve the inhibitions of iNOS and COX-2expression byregulating NF-κB activation..Conclusively, this study revealed the evidences that shikonin plays the effecintanti-inflammatory and protective roles against LPS-induced ALI in vitro and in vivo andthe potential mechanism of these actions may be partly attributed to the inhibition of iNOSand COX-2expression by down-regulating NF-κB activation. These experimental resultsof this study suggest that shikonin may be a potential beneficial agent for ALI.