Protective Effect Of Emodin On Septic Acute Lung Injury In Rats

Objective:Animal experiment:SD rat trachea was injected with Lipopolysaccharide(LPS)to establish the model of sepsis acute lung injury.To explore the protective effect of emodin on sepsis ALI in rats,the pathogenesis of acute lung injury(ALI)was investigated from three aspects:systemic inflammatory response,pulmonary edema caused by pulmonary water metabolism disorder and lung tissue cell apoptosis.Cell experiment:ALI model was established by inducing HPAEpic with LPS.Based on the JNK/MAPKs pathway,emodin was explored to regulate the activation of Caspase-3 by apoptotic factors through death receptor pathway and mitochondrial pathway,and finally inhibit the apoptosis of HPAEpic after acute lung injury,so as to improve its anti-apoptotic mechanism.Methods:1.Animal experiment:80 SD rats were randomly divided into four groups:Blank control group(Blank group),normal saline negative control group(NS group),model group(LPS group),and emodin intervention group(EM+LPS group).The emodin intervention group was gavaged with 35mg/kg emodin sodium carboxymethyl cellulose suspension,and the other groups were gavaged with 0.5%sodium carboxymethyl cellulose solution.After 5 days of continuous administration,rats in the model group and emodin intervention group were treated with tracheal dripping LPS to establish the model of sepsis ALI.Rats in normal saline negative control group were given equal volume of NS(5ml/kg)by trachea dripping.The blank control group was not treated.Wet/Dry ratio(W/D)and lung permeability index(LPI)of lung tissues in each group were measured at 12h and 24h after modeling.The pathological sections of lung tissue were observed by light microscopy after HE staining.The ultrastructure of lung tissue was observed under transmission electron microscope.The inflammatory factors in lung tissue homogenate were determined by enzyme-linked immunosorbent assay.The Epithelial sodium channels(ENa C)ofα,βandγ;AQP1;AQP5,subunits were detected by western blot and Realtime PCR assay.Na~+-K~+-ATPase activity was determined.Cell apoptosis in lung tissue was detected by TUNEL labeling.2.Cell experiment:the same group of HPAEpic in the logarithmic phase of growth was randomly divided into seven groups,which were negative control group(NC group);Model group(LPS group);Emodin intervention group(EM+LPS group);Inhibitor intervention group(SP+LPS group);Emodin+inhibitor intervention group(EM+SP+LPS group);Emodin group(EM group);Inhibitor group(SP group).Before LPS modeling,the negative control group was given 0.1%DMSO;The emodin intervention group was pretreated and incubated with emodin40umol/L.Inhibitor intervention group was pretreated and incubated with JNK inhibitor SP600125 10umol/L.The emodin+inhibitor intervention group was pretreated and incubated with emodin 40umol/L and JNK inhibitor SP60012510umol/L.The emodin group was incubated with emodin 40umol/L.Inhibitor group was incubated with JNK inhibitor SP600125 10umol/L.LPS 10ug/ml was added to the other four groups except the negative control group,emodin group and inhibitor group after 30min incubation in the cell incubator.After modeling,the expressions of apoptotic factors Fas/Fas L in death receptor pathway,apoptotic factors Bax/Bcl-2 in mitochondrial pathway and Caspase-3 gene in late apoptotic participants were detected by RT-PCR.Meanwhile,the expression of Caspase-3 protein was detected by Western blot.To explore the potential mechanism of emodin inhibiting apoptosis of HPAEpic cells after acute lung injury.Results:1.Anti-inflammatory effect of emodin:12h after modeling,the interventional administration of emodin could inhibit the levels of early inflammatory factor 1L-6,IFN-γand TNF-αin lung tissue homogenate,and increase the level of anti-inflammatory factor 1L-10.The levels of 1L-6 and TNF-αinflammation in ALI lung tissue homogenate were also decreased 24h after modeling,and the inflammatory response was reduced(P<0.05).2.Emodin improves lung water metabolism:Through the detection of general state,the wetting/drying ratio and lung permeability index of the LPS model group were significantly increased at 12h and 24h after modeling,and the alveolar capillary barrier was damaged,leading to acute pulmonary edema.Emodin enhanced the active transport mediated by sodium ion channels,increased the fluid clearance rate in the alveolar cavity,decreased the lung tissue W/D and LPI,and improved acute pulmonary edema(P<0.05).2.1.Severe acute lung injury rat model group showed pathological changes of different degrees at 12h and 24h after modeling.Under light microscope,typical pulmonary edema and inflammation were observed,such as widening of alveolar wall,narrowing of alveolar cavity and exudation of interstitial red blood cells.Under electron microscope,the alveolar epithelial cells were damaged,the cell structure was blurred,and the lamellar body empty gun increased.The above pathological changes were alleviated after emodin intervention compared with model group.2.2.Emodin could reduce wet/dry ratio and lung permeability index of ALI model at12h and 24h after modeling(P<0.05).2.3.12h after modeling,emodin intervention could increase the m RNA expressions ofα,βandγ-ENa C and the protein expressions ofα,βandγ-ENa C in the lung tissues of ALI model,and also enhance the activity of Na~+-K~+-ATPase(P<0.05).2.4.24h after modeling,emodin could increase the m RNA expression of AQP1 and AQP5 and the protein expression of AQP1 and AQP5 in ALI lungs(P<0.05).3.Emodin alleviates lung tissue cell apoptosis:The intervention of emodin at 12h and 24h after modeling could reduce the apoptosis of ALI lung tissue cells and improve acute lung injury.4.The mechanism of emodin inhibiting apoptosis of HPAEpic after acute lung injury:4.1.At 6h and 12h after modeling,emodin decreased the expressions of pro-apoptotic factors Fas m RNA and Fas L m RNA through the death receptor pathway(P<0.05).4.2 After 12h and 24h modeling,emodin intervention increased the expression of anti-apoptotic factor Bcl-2 m RNA and inhibited the expression of pro-apoptotic gene Bax m RNA through mitochondrial pathway(P<0.05).4.3 Emodin at 6h,12h and 24h after modeling could reduce the expression of Caspase-3 protein and Caspase-3 m RNA through the JNK/MAPKs pathway,and inhibit the apoptosis of HPAEpic after acute lung injury(P<0.05).4.4 Emodin can reduce Fas L m RNA and Bax m RNA of apoptotic factors through JNK/MAPKs pathway 12 hours after modeling;24h after modeling,emodin inhibited the expression of Bax m RNA through the JNK/MAPKs pathway,and inhibited the apoptosis of HPAEpic(P<0.05).Conclusion:1.At 12h and 24h after modeling,emodin achieved anti-inflammatory effects through multi-target regulation of inflammatory factors.2.12h after modeling,emodin enhanced the activities of sodium channels and Na~+-K~+-ATPase,but 24h after modeling,emodin increased the expressions of AQP1 and AQP5 genes and proteins in active transport.At different time points,emodin intervention can be used in different stages of the active transport of alveolar edema fluid,so as to enhance the clearance rate of alveolar fluid,reduce the alveolar-capillary permeability and the wet/dry ratio of lung tissue,and improve pulmonary edema.3.At 12h and 24h after modeling,emodin played a protective role on LPS-induced sepsis ALI rats by reducing the apoptosis of lung tissue cells.4.At 6h and 12h after modeling,emodin inhibited the expression of apoptotic factor Fas/Fas L gene through death receptor pathway;At 12h and 24h after modeling,emodin could inhibit the apoptosis of HPAEpic cells after acute lung injury by regulating the expression of apoptotic factor Bax/Bcl-2 gene through mitochondrial pathway.5.Emodin regulates apoptosis factors in death receptor pathway and mitochondrial pathway through JNK/MAPKs signaling pathway,inhibits the activation of Caspase-3,and plays a role in inhibiting the apoptosis of HPAEpic after acute lung injury.