Tanshinone Ⅱa-induced Attenuation Of Lung Injury In Endotoxemic Mice Is Associated With Reduction Of Hif-1α Expression

Background:Acute lung injury (ALI)/acute respiratory distress syndrome (ARDS), a severe complication of stress situations such as trauma, burns and sepsis, is a frequent complication with high rates of morbidity and mortality. Even with recent success, there is still a tremendous need for continued efforts to explore the underlying pathophysiological mechanisms of ALI and to prevent and cure this disease. Our previous study showed that pharmacologic pretreatment with tanshinone IIA (TIIA) could significantly reduce lethality in lipopolysaccharide (LPS)-treated mice and attenuate LPS-induced lung injury both in vivo and in vitro. However, the underlying mechanisms responsible for the protective effects of TIIA are still not clear.LPS, a potent inflammatory factor that has been implicated in the pathogenesis of septic shock, has been reported to induce hypoxia-inducible factor 1α(HIF-1α) protein accumulation under nonhypoxic conditions in many cell types, especially in monocytes and macrophages. Different from hypoxia and many other stimuli, LPS induces HIF-1αaccumulation via increasing HIF-1αmRNA level and protein translation, and even decreasing its degradation. Currently, studies further show that HIF-1αplays pivotal roles in the development of myeloid cell-mediated inflammation and LPS-induced toll like receptor 4-mediated sepsis, and in controlling macrophage inflammatory response in skin and joints and promoting the phagocytic function of macrophages. Therefore, interventions that limit the HIF-1α-mediated inflammatory response in macrophages might benefit in the prevention of tissue damage associated with prolonged inflammation. However, whether HIF-1αparticipates in the development of LPS-induced ALI and whether the protective effect of TIIA against lung injury is related to HIF-1αhave not yet been reported.In the current study, therefore, we confirmed the protective effects of TIIA against LPS-induced lung injury and inflammatory response, investigated whether the protective effects of TIIA was related to its depression on HIF-1αexpression, and explored the possible mechanisms accounting for the inhibition of HIF-1αexpression by TIIA in LPS-stimulated macrophages.METHODSAnimal experimentMale BALB/c mice, weighing 18 - 22 g, were randomly divided into four groups, i.e., 1) saline control group (n = 8): mice received saline 0.5 h before the second saline administration; 2) TIIA control group (n = 8): mice received TIIA (10 mg/kg) 0.5 h before saline administration; 3) LPS group (n = 8): mice received saline 0.5 h before LPS (10 mg/kg) administration; and 4) TIIA/LPS administration. In all groups, administration was via intraperitoneal injection and measurements were made at 6 h after the second administration. Measurements included histological study, total and neutrophil counts, and lactate dehydrogenase (LDH) in bronchoalveolar lavage fluid (BALF), inflammatory cytokines (TNF-α, IL-1βand IL-6) in serum and BALF, and HIF-1αcontent in the lungs.Cell experimentExponentially growing NR8383 and RAW264.7 macrophages (about 80% confluence) were deprived of FCS for 16 to 20 h. For dose- and time-dependent effects of LPS on HIF-1αexpression, cells were treated with the indicated doses of LPS (0.5 - 10μg/ml) for the indicated periods (0 - 8 h). For dose-dependent effects of TIIA (1 - 20μg/ml) on LPS-induced HIF-1αexpression, relative regulatory factors and inflammatory cytokines, cells were pretreated with TIIA or different inhibitors for 30 min, followed by incubation with LPS for 6 h or 12 h.To investigate the role of HIF-1αin the production of inflammatory cytokines, RAW264.7 macrophages cells were transfected with HIF-1αsiRNA and a scramble sequence by using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions.Results1. Pretreatment with TIIA improved LPS-induced lung injuryCompared with saline control rats, LPS significantly increased LDH content (P < 0.01), indicating that LPS induced ALI in the treated rats. However, pretreatment with TIIA markedly reduced LDH content (P < 0.05).LPS instillation caused a marked lung injury, characterized by hemorrhage, edema, thickened alveolar wall and formation of hyaline membranes. However, with TIIA pretreatment in the TIIA/LPS group, these changes were less pronounced compared with the LPS group.2. Pretreatment with TIIA inhibited LPS-induced inflammatory response both in vivo and in vitroCompared with saline control rats, LPS significantly increased total cell and neutrophil counts in BALF (P < 0.01). However, pretreatment with TIIA markedly reduced the above mentioned parameters, indicating that TIIA significantly suppressed the infiltration of inflammatory cells into lungs (P < 0.05).We found that TNF-α, IL-6, and IL-1βwere only minimally expressed in serum and BALF from the control groups. Six hours after LPS administration, the expression of TNF-α, IL-6, and IL-1βwas markedly increased (P < 0.01), but these levels were significantly attenuated by pretreatment with TIIA (P < 0.05).We also detected TNF-α, IL-6, and IL-1βcontent in the cell supernatant from RAW264.7 cells. 1μg/ml LPS increased TNF-α, IL-6, and IL-1βcontent in the cell supernatant (P < 0.05 and P < 0.01, respectively). Compared with the LPS group, TIIA concentration-dependently reduced LPS-induced TNF-α, IL-6, and IL-1βcontent (P < 0.05 and P < 0.01, respectively).3. Role of HIF-1αin LPS-induced inflammatory responseWe stimulated macrophages in the presence of LPS for 6 h. Western blotting results showed that HIF-1αprotein levels in the two macrophage cell lines were induced by LPS and the induction of HIF-1αby LPS (0.5 - 10μg/ml) was dose-dependent. Time-course studies were then performed using 1μg/ml LPS stimulation. An increase was seen by LPS stimulation for 2 h and the maximal induction was obtained between 6 and 8 h in the presence of LPS.We used HIF-1αsiRNA to study if HIF-1αaffected the production of inflammatory cytokines. Results showed that HIF-1αsiRNA greatly suppressed TNF-α, IL-6 and IL-1βcontent in the supernatant from RAW264.7 cells treated with LPS for 12 h.4. Pretreatment with TIIA inhibited LPS-induced HIF-1αSimilar to the effects of TIIA on LPS-induced inflammatory cytokines, pretreatment with TIIA led to a dose-dependent reduction in HIF-1αprotein levels in macrophages. Meanwhile, pretreatment with TIIA decreased the up-regulation of HIF-1αin lungs induced by LPS.5. Pretreatment with TIIA did not affect LPS-induced HIF-1αmRNA expression in macrophagesLPS induced HIF-1αmRNA expression in NR8383 and RAW264.7 cells (P < 0.05). In TIIA-pretreated group, however, TIIA had no significant effect on LPS-induced HIF-1αmRNA expression compared to the LPS group.6. TIIA interfered with protein translational machinery for HIF-1αin macrophagesWestern blotting results showed that LPS greatly increased AKT and MAPK phosphorylation in NR8383 and RAW264.7 cell lines, but pretreatment with TIIA could significantly block LPS-induced activation of AKT and MAPK.Western blotting results showed that LPS induced the phosphorylation of p70S6K1, S6 ribosomal protein, 4E-BP1, and eIF4E in the two cell lines. However, TIIA pretreatment concentration-dependently reversed the phosphorylation of these proteins.7. Pretreatment with TIIA promoted LPS-induced HIF-1αprotein degradation via the proteasomal pathway In the presence of CHX, the half-lives of HIF-1αprotein in the cells pretreated with TIIA (10μg/ml) were 4.9 min and 5 min in NR8383 and RAW264.7 cells, respectively, which were significantly shorter than those in cells treated without TIIA (8.5 min and 9 min, respectively). Ttreatment of the cells with TIIA decreased HIF-1αprotein levels, but TIIA-induced HIF-1αinhibition was prevented completely by MG132.Conclusion:(1)TIIA could alleviate LPS-reduced lung injury and inflammatory response;(2)The protective effect of TIIA was correlated with the inhibition of LPS-induced HIF-1αaccumulation;(3)TIIA could not inhibit HIF-1αmRNA level induced by LPS;(4)TIIA could affect HIF-1αprotein synthesis by the inhibition of PI3K/AKT and MAPK pathways and related protein translational regulator, including p70S6K1, S6 ribosomal protein, 4E-BP1, and eIF4E.(5)TIIA could enhance the degradation of HIF-1αprotein via the proteasomal pathway.