| Backgroud and purposeWith hign mortality, acute lung injury (ALI) is a great threat to human health. To seek effective drugs for the repair of acute lung injury and lower the mortality has become a topic of intensive interest. ALI is a critical clinical syndrome with progressive dyspnea, refractory hypoxemia, which pathological features mainly include inflammatory reaction and alveolar-capillary membrane injury result from severe infection, trauma, shock, etc. Endotoxima is a key etiological factor for ALI. Toll-like receptors (TLRs) represent a conserved family of innate immune recognition receptors that play key roles in detecting microbes, initiating innate immune responses, and linking innate and adaptive immunity. TLR4signaling which is the major member of the TLR4is triggered by the interaction with lipopolysaccharide (LPS), the major cell-wall component of Gram-negative bacteria. All TLRs but TLR3utilize the intracellular adapter protein MyD88in order to induce NF-κB activation and subsequent inflammatory mediator expression, such as IL-1、 IL-6、IL-8、TNF-α, induce lung tissue damage characterized by extensive alveolar damage leading to the disruption of the alveolar-capillary barrier, pulmonary edema, and neutrophil infiltration. The lung is frequently affected both by systemic inflammatory reaction(SIR) and compensatory anti-inflammatory response (CAR). ALI/ARD canbe proportional repaired if keeping the SIR and CAR into balance. The development of a cute lung injury seems to be associated with local and systemic IL-10concentrations. Ecdysteroids (insect moulting hormones) display many pharmacological effects in mammals. Nowaday, as we know, its actions include:1)Promote nucleic acid and Protein synthesis;2)adjust glueose metabolism;3)modulate lipid metabolism;4)regnlate gene expression, Most researchers’interest in ecdysteroid-inducible gene expression systems is in the functional analysis of cloned genes, which can be put under the control of ecdysteroid-regulated promoter s and transfected into mammalian cells;5)improve immune function;6)Posses antioxidant activity;7)Promote angiogenesis and establishment of collateral circulation in ischemie area;8)promote proliferation of varied cells.The previous investigations of our study group reveal that Ecdysterone can improve the repair of acute lung injury, but the function mechanism is still unknown. Therefore, in this study we will observe the effects of ecdysteroids on the impact of ATII in the a model of LPS-induced ALI in vivo, and investigate the influence of EDS on LPS-activated TLR4signal transduction in vitro.The purposes of this study are to confirm whether there is anti-inflammatory effect of EDS for ALI, to elucidate the protective mechanism of EDS on LPS-induced ALI.Methods1、One hundred and twenty male adult Wister rats were randomized into5groups:normal control group (control); ALI group (LPS); ecdysterone-treated group (LPS+EDS20mg/kg), ecdysterone-treated group (LPS+EDS30mg/kg), ecdysterone-treated group (LPS+EDS40mg/kg), receiving intravenous injection of EDS at the dose of20mg/kg,30mg/kg,40mg/kg one hour after the administration of LPS respectively. All the rats were injected intraperitonealy with8mg/kg LPS except for the control group. The blood samples and lung tissue were collected at2,4and24h after LPS injection.2、PaO2, the levels of lung coefficient, lung penetrating index(LPI) and neutrophil counts in BALF were measured at the different time points. The mRNA expressions of IL-10in the lung tissues were detected by fluorescence quantitative real-time RT-PCR at the different time points. In addition, the protein and mRNA expressions of TLR4in the lung tissues were detected by Western blotting and fluorescence quantitative real-time RT-PCR, and the concentrations of TNF-a and IL-10in plasma by ELISA at the different time points. Meanwhile, the pathological changes in lung tissue were observed by light microscopy at6h after LPS injection, and the ultrastructural changes of lung tissue were observed by electron microscopy at24h after LPS injection.Result1、In addition to prohibiting the dropping of PaO2, ecdysterone could mitigate the acute lung injury which increased the levels of lung coefficient, LPI, neutrophil counts in BALF.2、The instructive injury induced by LPS observed under electron microscope and light microscope was decreased significantly in groups LPS+EDS compared to group LPS. In ALI group, there were alveolar septum widening, capillary congestion, interstitial and alveolar edema, intra-alveolar hemorrhage, and multiply inflammatory cells infiltration in the lungs of ALI rats. Disappearance of superficial microvilli of type II alveolar cells and atrophy of Osmiophilic lamellar body were also decreased. The pathological changes were lessened obviously in EDS treatment groups compare with that in ALI group.3、The lung IL-10mRNA expression levels of Groups LPS, LPS+EDS (20mg/kg), LPS+EDS (30mg/kg), and LPS+EDS (40mg/kg) were all significantly higher than that of Group control (all P<0.05). The lung IL-10mRNA expression levels of Groups LPS+EDS were all significantly higher than those of Group LPS (P<0.05) at the different time points. Meanwhile, the lung IL-10mRNA expression levels of Group LPS+EDS (40mg/kg) were all significantly higher than those of Group LPS+EDS (20mg/kg)(all P<0.05) at the different time points. However there were no significant differences between Groups LPS+EDS (20mg/kg) and LPS+EDS (30mg/kg)(P>0.05) at24hours.4、The lung TLR4protein expression levels of Groups LPS were all significantly higher than those of control group, and achieved the peak value at4h, then decreased gradually at24h. The lung TLR4protein expression levels were all significantly lower than those of Groups LPS (all P<0.05) at the different time points. Meanwhile, the lung TLR4protein expression levels of Groups LPS+EDS (40mg/kg) were all significantly lower than those of Groups LPS+EDS (20mg/kg)(all P<0.05) at the different time points. However there were no significant difierences between Groups LPS+EDS (20mg/kg) and Groups LPS+EDS (30mg/kg)(P>0.05) at24h, and beween Groups LPS+EDS (30mg/kg) and Groups LPS+EDS (40mg/kg) at4h,24h (all P>0.05).5、The lung TLR4mRNA expression levels of Groups LPS whose changes were similar to the lung TLR4protein were all significantly higher than those of control group, and achieved the peak value at4h, then decreased gradually at24h. The lung TLR4protein expression levels of EDS treatment groups were all significantly lower than those of Groups LPS (all P<0.05) at the different time points. Meanwhile, Groups LPS+EDS (40mg/kg) were all significantly lower than those of Groups LPS+EDS (20mg/kg)(all P<0.05) at the different time points. There were significant difierences between Groups LPS+EDS (20mg/kg) and Groups LPS+EDS (30mg/kg) at2h,4h(all P<0.05). 6、In EDS treatment groups, the concentration of IL-10in the serum and SOD in lung tissue was higher than those of ALI groups at2h,4h and24h obviously (all P<0.05vs ALI groups), meanwhile, the concentration of TNF-a in the serum was lower than those of ALI groups at2h,4h and24h obviously (all P<0.05vs ALI groups).Conclusion1、EDS therapy significantly protects lung from LPS-induced injury in rats by the suppression of pulmonary edema and pulmonary inflammation and alleviating the impair of AT-II. The mechanism may be increase of the expression of IL-10mRNA and inhibition of the overexpression of TLR4protein and mRNA, especially when give rats an dose40mg/kg of ecdysterone.2、Treatment with EDS could inhibit the production of TNF-a in the serum in LPS-induced ALI mice, and increase the production of IL-10in the serum, which are beneficial to the banlance of SIR and CIR. |
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