Andrographolide Protects Anainst LPS-induced Acute Lung Injury By Inactivation Of NF-κB

BackgroundNF-κB is a central transcriptional factor and a pleiotropic regulator of manygenes involved in acute lung injury (ALI). Andrographolide is found in theplant of Andrographis paniculata and widely used in Traditional ChineseMedicine, exhibiting potently anti-inflammatory property by inhibitingNF-κB activity.Methods1. Forty male BALB/c mice were randomly divided into4groups (n=10),control group, LPS group, low dosage andrographolide (Sigma, St.Louis, MO, USA) treatment group (LPS+Andro-L group) and highdosage andrographolide treatment group (LPS+Andro-H group).2. Mice were anesthetized with pentobarbital sodium, followed by10μgLPS in50μL sterile saline intratracheal injection with a needle.Seventy-two hours later, the mice were exsanguinated afterpentobarbitone anesthesia.3. Total cellular counts, neutrophils, macrophage and lymphocytes in BALF were measured by Diff-Quik. The levels of TNF-α, IL-6andIL-1β in BALF were analyzed by ELISA. MPO activities in the lungtissues were detected. Wet to dry ratio (W/D ratio) was used to measurethe severity of pulmonary edema. HE staining was used to observe thepathological changes, and pulmonary injury scores were analyzed.Transmission Electron Microscope (TEM) was used to observe the typeII alveolar epithelial cells. The mRNA expression of VEGF andVCAM-1was measured by RT-PCR. The protein expression of VEGF,VCAM-1, IKKβ, phospho-IKKβ, IκBα, phospho-IκBα, NF-κB p65,phospho-NF-κB p65and β-actin were measured by western blot.NF-κB p65DNA binding activities were analyzed by TransAM NFκB p65Chemi Transcription Factor Assay Kit.4. Statistical analyses were performed with Sigmaplot software (SPSS).Each point corresponds to mean±SD Statistical differences weredetermined by one-way or two-way analysis of variance (ANOVA) andP <0.05was considered to be statistically significant.Results1. Three days after LPS injection, total cells, neutrophils and macrophageswere remarkably increased in BALF. However, andrographolidetreatments dose-dependently reduced total cells, neutrophils andmacrophages in BALF. However, there was no difference found inlymphocytes in BALF among groups. 2. Three days after LPS injection, TNF-α, IL-6and IL-1β in BALF in themice with LPS injection were markedly higher than the mice in controlgroup. However, TNF-α, IL-6and IL-1β in BALF in the mice withandrographolide injections were decreased.3. MPO activity in model group was markedly higher than control group.However, MPO activity was dose-dependently reduced byandrographolide.4. The mice received LPS injection showed higher W/D than in controls.Nevertheless, W/D significantly decreased after andrographolideadministrations. Additionally, the effect of andrographolide inpulmonary inflammation and pulmonary edema was in a dose dependentmanner.5. Histological evaluation of lung sections3day after the LPS injectionshowed evidence of notable inflammatory cells infiltration, interstitialedema, interalveolar septal thickening and intraalveolar and interstitialpatchy hemorrhage. However, after andrographolide interventions, thelung injury scores in the lung tissues were ameliorated in a dosedependent manner.6. After72h LPS stimulation, typical changes were observed in type IIalveolar epithelial cells in the lung tissues. However, the ultrastructurechanges were attenuated by andrographolide.7. Compared with control group, enhanced mRNA expression of VCAM-1 and VEGF were found after LPS stimulation. Meanwhile,LPS-stimulated enhanced VCAM-1and VEGF mRNA expression wasdose-dependently inhibited by andrographolide.8. Compared with control group, enhanced protein expression of VCAM-1and VEGF were found after LPS stimulation. Meanwhile,LPS-stimulated enhanced VCAM-1and VEGF protein expression wasdose-dependently inhibited by andrographolide.9. Our investigation found a markedly increased p65subunitphophorylation72h after LPS stimulation, which was attenuated byandrographolide administrations.10.Our results showed that NF-κB p65DNA-binding activities weresignificantly increased72h after LPS stimulation. However, theenhanced DNA-binding activities of NF-κB p65were dose-dependentlyreduced by andrographolide treatments.ConclusionsOur data indicate that andrographolide dose-dependently attenuated theseverity of LPS-induced ALI, more likely by means ofandrographolide-mediated NF-κB inhibition. These findings suggestandrographolide may be considered as an effective and safe drug for thepotential treatment of ALI/ARDS. However, further clinic studies should becarried out to identify the value of andrographolide in practice use.