| Inflammation is a highly complex process to defend against foreign challenge or tissue injury, which involves various cell types (such as lymphocyte, macrophage, granulocyte, and endothelial cell, etc.) and multi-components (cytokines, vascular active substances, chemokines, adhesion molecules, inflammation-related enzymes). Inflammation seems like a double-edged sword:proper inflammatory response will help our body clearing the damage, fending off infection and promoting wound healing. However, excessive inflammatory response will in turn promote the occurrence and development of lots of inflammatory diseases, therefore affecting people’s health.Acute lung injury (ALI) is a life-threatening syndrome characterized by severe lung inflammation and increased microvascular permeability, and acute respiratory distress syndrome (ARDS) is the most severe form of lung injury. A variety of clinical disorders, such as pneumonia, aspiration of gastric contents, sepsis, major trauma, acute pancreatitis, can induce the occurrence of ALI. Studies suggest that the excessive, uncontrolled inflammatory response in the lung is the root cause of ALI. Clinical uses of corticosteroids, prostaglandins, prostacyclin, surfactant, lisofylline, ketoconazole and N-ace-tylcysteine have shown unsatisfactory significant improvements in patients’ mortality, regardless of the unwanted side effects of these drugs. Thus, a drug with better therapeutic effects and fewer side effects is urgently needed.The dry root of Peucedanum praeruptorum Dunn has been long used in traditional Chinese medicine for its remarkable effects on respiratory diseases. Praeruptorin A, B, C, D and E(PA, PB, PC, PD and PE), a class of pyranocoumarin, have been confirmed to be the main constituents in this herb. Previous studies showed the protective effects of total coumarins extracted from Peucedanum praeruptorum Dunn (TCP) on dimethylbenzene-induced ear swelling in mice and egg white-induced paw swelling in rats. In addition, TCP has been demonstrated to inhibit the airway hyper-responsiveness and inflammation in a mouse model of allergic airway disease. These data showed the anti-inflammatory activities of TCP in vivo. However, the pharmacological effect and the underlying mechanism of each Praeruptorin have not been investigated. In the present study, the anti-inflammatory activities of PC, PD and PE were investigated in LPS-stimulated murine macrophages. The effects of these Praerutorins on the expression of inflammatory mediators and cytokines, as well as activation of NF-kB, MAPK and STAT pathways were examined. In addition, the acute lung injury mouse models induced by LPS or HC1were established, and the effects of Praerutorins on inflammatory cells influx, cytokine release, protein leakage, MPO activity, as well as NF-kB activation were detected to investigate the protective effects and underlying mechanism of these compounds in ALI mice.The major research methods and results are divided into two parts:part I:Praeruptorin C, D and E (PC, PD and PE) inhibited inflammatory responses in LPS-activated macrophages1. Praeruptorin C, D and E (PC, PD and PE) inhibited NO production in LPS-activated macrophageRAW264.7cells were co-cultured with2,4,8and16μg/ml praeruptorins and1 μg/ml LPS for18h. NO production in the cell culture medium were detected by Griess reagent. Our results showed that stimulation with LPS resulted in a9-fold increase in NO production compared with control, and then treatment with praeruptorins inhibited NO production in a dose-dependent manner with the IC50values of7.6μg/ml (PC),5.6μg/ml(PD), and5.8μg/ml (PE).2. Cytotoxicity study of Praeruptorin C, D and E (PC, PD and PE) in macrophagesTo investigate whether the inhibitory effect of Praeruptorins on NO production is due to a cytotoxic effect in macrophages, the cytotoxicity of Praeruptorins was assessed. Cells were treated with Praeruptorins at a dose of2,4,8,16and32μg/ml for24h. The results showed that treatment with Praeruptorins did not affect the RAW264.7cell viability at doses ranging from2to16μg/ml.3. Praeruptorin C, D and E (PC, PD and PE) inhibited iNOS expression in LPS-activated macrophageRAW264.7cells were co-cultured with4,8and16μg/ml praeruptorins and1μg/ml LPS for18h. The total protein of RAW264.7cells was extracted, and the iNOS expression was then investigated by Western blot. The results showed that stimulation with LPS resulted in marked increase of iNOS expression compared with control cells. Treatment with praeruptorins inhibited iNOS expression in a dose-dependent manner.4. Praeruptorin C, D and E (PC, PD and PE) inhibited TNF-a and IL-6release in LPS-activated macrophageRAW264.7cells were co-cultured with2,4,8and16μg/ml praeruptorins and1μg/ml LPS for18h. TNF-a and IL-6production in the cell culture medium were detected by ELISA method. The results showed that stimulation with LPS resulted in marked increase of TNF-a and IL-6production compared with control cells. Treatment with praeruptorins inhibited cytokines release in a dose-dependent manner. The IC50values of PC, PD and PE for inhibition of TNF-a production were40.7,17.3, and18.5μg/ml, respectively. For inhibition of IL-6production, the IC50values of PC, PD and PE were11.9,4.9, and5.1μg/ml, respectively.5. Praeruptorin C, D and E (PC, PD and PE) inhibited TNF-a, IL-6and iNOS mRNA expression in LPS-activated macrophageRAW264.7cells were co-cultured with2,4,8and16μg/ml praeruptorins and1μg/ml LPS for6h. Total cellular RNA from the treated cells was extracted by using Trizol reagent. Then, the mRNA expressions of TNF-a, IL-6and iNOS were quantitated by Real-time-PCR methods. The results showed that praeruptorins inhibited TNF-a, IL-6and iNOS mRNA expression in a dose-dependent manner. Treatment with16μg/ml PC, PD or PE lead to35%,45%, and47%inhibition of TNF-a mRNA expression, respectively (Fig.3c). For inhibition of IL-6mRNA expression, same concentration of PC, PD, or PE caused55%,72%, and70%inhibition, respectively. For inhibition of iNOS mRNA expression, Treatment with16μg/ml PC, PD or PE lead to53%,75%, and78%inhibition.6. Praeruptorin C, D and E (PC, PD and PE) inhibited NF-kB activation in LPS-activated macrophageWestern blot and immunofluorescence analysis were performed to examine whether praeruptorins depresses NF-kB activation. The results showed that the p65subunit of NF-kB was translocated into the nucleus after LPS challenge for1h. However, treatment with praeruptorins markedly inhibited the translocation of p65. In the immunofluorescence assay, a low level of p65activity was observed in control macrophages. When stimulated by LPS for1h, p65was activated and translocated into nuclei, whereas treatment with praeruptorins inhibited the p65nuclear translocation.We then investigated the effect of praeruptorins on LPS-induced IκB-a degradation to examine the molecular mechanisms by which praeruptorins inhibit NF-kB transcriptional activity. Our results showed that LPS treatment induced a marked degradation of the proteins. In contrast, loss of cytoplasmic IkB-α was inhibited by praeruptorins. 7. Praeruptorin C, D and E (PC, PD and PE) inhibited STAT-3activation in LPS-activated macrophageThe results showed that LPS-induced tyrosine phosphorylation of STAT1and STAT3started at2-h post-LPS stimulation, and the significant phosphorylation levels lasted to6h. Thus, we investigated the effect of praeruptorins on STAT pathway at4h after LPS stimulation. Pretreatment of16μg/ml praeruptorins did not affect LPS-stimulated STAT1phosphorylation, but they markedly suppressed STAT3tyrosine phosphorylation without impairing total STAT3proteins. AG490, a positive control, markedly inhibited phosphorylation on both STAT1and STAT3.8. Praeruptorin C, D and E (PC, PD and PE) did not inhibited MAPKs activation in LPS-activated macrophageTo investigate the effects of pyranocoumarins on LPS-induced MAPK pathway, the phosphorylation levels of p38, ERK1/2, and JNK were examined. Our results showed that PC, PD, and PE did not inhibit LPS-induced phosphorylation of p38and JNK. On the contrary, these compounds significantly augmented the LPS-induced ERK1/2phosphorylation.part Ⅱ:Praeruptorin D and E attenuate lipopolysaccharide/hydrochloric acid induced acute lung injury in mice1. Effects of Praeruptorin A, C, D and E (PA, PC, PD and PE) on inflammatory cell counts and cytokine levels in Bronchoalveolar lavage fluid (BALF) of LPS-induced ALI miceTo examine the effects of Praeruptorins on LPS-induced pulmonary inflammation, PA, PC, PD or PE at a dose of80mg/kg were gavaged1h before intranasal administration of LPS. BALF was collected4h after LPS challenge. LPS markedly induced the increase of total cell and PMNs counts, as well as the release of TNF-a and IL-6in BALF. Pretreatment with PD and PE, but not PA and PC, significantly reduced these changes. There was no significant difference between PD and PE group.2. Dose response relationship of Praeruptorin D and E (PD and PE)A set of experiments were performed to study the dose response relationship of PD and PE. Our results showed that mice treated with80mg/kg PD or PE alone showed no significant difference in total cell and PMNs counts, when compared with control group. In LPS challenged mice, oral administration of both PD and PE led to a dose-dependent lowering of the total cell and PMNs counts in BALF. PD at doses of20,40and80mg/kg led to10%,33%and58%decrease of PMNs counts, respectively, whilst same doses of PE caused11%,43%and53%inhibition, respectively.Because vascular leakage is another hallmark of ALI, we also examined total protein concentrations in BALF. The results showed that PD and PE depressed LPS-induced protein contents in a dose-dependent manner.Both PD and PE dose-dependently reduced the LPS-induced increase of TNF-a and IL-6levels. PD at doses of80mg/kg led to51%and59%inhibitions of TNF-a and IL-6production, respectively. Similarly,80mg/kg of PE caused56%decrease of TNF-a level and51%inhibition of IL-6release, respectively.3. Pretreatment with Praeruptorin D or E (PD or PE) decreased MPO activity in LPS-induced ALI miceMPO activity in tissues is an indicator of PMNs infiltration. The results showed that instillation of LPS resulted in a significant increase in MPO activity in mouse lung tissues. In parallel with the number of PMNs, the level of MPO activity was partially inhibited by both PD and PE.4. Pretreatment with Praeruptorin D or E (PD or PE) attenuated lung histopathology changes in LPS-induced ALI mice.In the present study, no evident histological alteration was observed in lung specimens of normal mice. However, the instillation of LPS resulted in significant lung injury, evidenced by a large amount of neutrophil infiltration around the pulmonary vessel and interstitial spaces, a marked swelling of the alveolar walls, and severe hemorrhage as well as alveolar structural damage. These pathological changes were improved by pretreatment with80mg/kg of PD or PE.5. Pretreatment with Praeruptorin D or E (PD or PE) inhibited NF-kB activation in LPS-induced ALI miceWe evaluated IkB-α degradation and nuclear NF-kB p65expression by Western blot analysis to investigate the molecular mechanism whereby treatment with PD and PE attenuated the development of acute lung injury. In our study, mice treated with LPS showed significant decrease of IκB-α protein, when compared with control group. In contrast, loss of cytoplasmic IκB-a was markedly inhibited by pretreatment with PD or PE.On the other hand, mice challenged with LPS showed significant increase of NF-κB-p65subunits in the nucleus, as compared with control group. However, treatment with80mg/kg of PD or PE markedly inhibited the NF-icB-p65translocation.6. Pretreatment with Praeruptorin D or E (PD or PE) protected mice from HCl-inducedALITo evaluate the protective effects of PD and PE in the HCl-induced ALI mouse model, mice were gavaged with80mg/kg of PD or PE1h before HC1challenge, BALF and lung tissue samples were collected4h after HC1challenge. HC1instillation significantly induced increases of inflammatory cell counts, IL-6level and total protein concentration in BALF. However, HC1did not increase the release of TNF-a (data not shown). The HCl-induced PMNs influx, IL-6production and protein extravasation were dramatically reduced by oral administration of both PD and PE.The effects of PD and PE on lung histopathology in HCl-treated mice were also examined. The results showed that the pathological changes induced by HC1 instillation, such as severe hemorrhage, neutrophil infiltration and alveolar structural damage, were attenuated by both PD and PE.Based on the above results, the conclusions of this paper are as follows:1. Praeruptorin C, D and E (PC, PD and PE) inhibited LPS-induced macrophage inflammatory responseA:PC, PD and PE inhibited NO production and iNOS expression in LPS-activated macrophage.B:PC, PD and PE inhibited TNF-a and IL-6releases and mRNA expressions in LPS-activated macrophage.C:PC, PD and PE inhibited NF-kB and STAT-3activation in LPS-activated macrophage.2. Praeruptorin D and E (PD and PE) attenuate lipopolysaccharide/hydrochloric acid induced acute lung injury in mice.A:PD and PE protected mice from LPS-induced acute lung injury.a):PD and PE inhibited the inflammatory cells influx, cytokines release and protein leakage in BALF of LPS-challenged mice.b):PD and PE inhibited the MPO activity in the lungs in of LPS-challenged mice.c):PD and PE attenuated the pathological changes in the lung of LPS-challenged mice.B:PD and PE protected mice from HCl-induced acute lung injury.a):PD and PE inhibited the inflammatory cells influx, IL-6release and protein leakage in BALF of HCl-challenged mice.b):PD and PE attenuated the pathological changes in the lung of HCl-challenged mice. |
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