The Protective Effect And Mechanism Of AEP On PM2.5-induced Lung Injury

Objectives:In recent years,haze weather caused by environmental pollution has frequently appeared in my country,which has a serious impact on people’s lives and health.Among them,high concentration of PM2.5 is the main reason for the formation of haze weather.PM2.5 has a small particle size and complex components.When breathing into the lungs,its insoluble components are mainly deposited in the bronchi and alveoli,inducing inflammation in the lungs,causing a series of lung diseases such as pneumonia and asthma.The damage to the lungs cannot be ignored.Therefore,it is very important to find reasonable and effective preventive and therapeutic drugs to relieve PM2.5-induced lung injury.Andrographis paniculata(Burm.f.)Nees is a traditional Chinese medicine with the special effects of clearing heat,detoxifying and cooling blood,and is It is widely used to prevent and treat acute respiratory infections,acute bacillary dysentery and other diseases.The previous study of our research group found that Andrographis diterpene lactone effective parts(AEP)and andrographolide(A)both have strong anti-inflammatory effects,which can reduce the degree of inflammatory infiltration in the bronchial and lung tissues and reduce the release of inflammatory factors in the rat asthma model To increase the level of immune factors.Andrographolide derivative andrographolide PDS,as a clinical drug,has a significant effect on the treatment of pneumonia and upper respiratory tract infections.Therefore,this subject establishes a rat lung injury model by way of tracheal infusion of PM2.5,and A and PDS were used as controls to study the protective effect of AEP on PM2.5-induced lung injury in rats,and human bronchial epithelium BEAS-2B cells are used as a model to explore the protective mechanism of AEP on PM2.5-induced BEAS-2B cell damage,to reveal the preventive effect and mechanism of AEP on PM2.5-induced lung injury,and to provide new ideas for prevention and treatment of PM2.5 induced and aggravated respiratory diseases.Methods and Results:1.The protective effect of AEP on PM2.5 induced lung injury in rats.56 male wistar rats were randomly divided into 7 groups with 8 rats in each group.The control group,PM2.5 model group,group A(200 mg/kg),AEP low,medium and high(100,200,400 mg/kg)group,PDS group(200 mg/kg),except PDS group abdominal cavity It was administered by injection,and the remaining groups were given by intragastric administration,once a day,for a period of 28 days.On the 24 th day,the control group was instilled with 0.9% saline,and the PM2.5 model group and each administration group were instilled with PM2.5 suspension(8 mg/kg)three times every other day.24 hours after the last exposure,the rats were sacrificed and the alveolar lavage fluid(BALF)was collected to detect the content of alkaline phosphatase(AKP),acid phosphatase(ACP)and total protein(TP)in BALF,HE staining observation of lung Pathological changes.The results showed that: Compared with the control group,the contents of AKP,ACP,and TP in BALF of the PM2.5 model group were significantly increased(P<0.01);compared with the model group,the content of related enzymes in the BALF of the rats in the administration group Are reduced.HE staining results showed that the alveolar interstitium in the PM2.5 model group was significantly thickened,the interstitial edema was severe,and there was a large amount of inflammatory cell infiltration;the rats in the AEP-administered group had reduced lung injury and inflammatory infiltration.improve.It shows that AEP can reduce the content of related enzymes induced by PM2.5 and has a protective effect on lung injury in rats,and the ability of AEP to regulate enzyme activity is stronger than that of A and PDS.2.The protective mechanism of AEP on PM2.5-induced BEAS-2B cell damage.Using MTT method to detect different concentrations of PM2.5 suspension(0,100,200,400,600,800 μg/m L),AEP(1,2,4,8 μg/m L),A(1,2,3,4,5 μg/m L),PDS(10,20,30,40 μg/m L)on the proliferation ability of BEAS-2B cells,the selected PM2.5 exposure condition is 400 μg/m L 48 h,A is given The drug concentration is in the range of 5 μg/m L,and the administration concentration of AEP is in the range of 8 μg/m L.Then the experiment was divided into control group,PM2.5 group(400 μg/m L),PM2.5+A(2 μg/m L)group,PM2.5+ AEP low,medium and high(1,2,4 μg/m L))Dose group and PM2.5+PDS(2 μg/m L)group,the drug-adding group was pretreated with drugs for 1 hour,then added 400 μg/m L of PM2.5,and co-cultured for 48 hours.MTT method was used to detect the vitality of BEAS-2B cells in each experimental group.The results showed that: compared with the control group,the PM2.5 group significantly inhibited the cell viability of BEAS-2B cells(P<0.01);compared with the PM2.5 group,each administration group could significantly increase cell viability(P<0.05).ELISA kit was used to detect the content of TNF-α and IL-6 in the cell culture fluid of each group,the colorimetric method was used to determine the LDH activity in the cell culture fluid of each group.the chemical fluorescence method was used to detect the intracellular ROS intensity,and the colorimetric method was used to detect the intracellular SOD activity.The results showed that: compared with the control group,the LDH activity,TNF-α and IL-6 contents in the cell culture fluid of the PM2.5 group were significantly increased(P<0.01);Compared with the PM2.5 group,each index in the cell culture fluid of each administration group were significantly reduced(P<0.05),and the AEP administration groups showed a dose-dependent.Compared with the control group,the ROS intensity of the PM2.5 group was significantly increased,and the SOD activity was significantly decreased(P<0.01);compared with the PM2.5 group,the SOD content in the cells of each administration group increased significantly(P<0.05),the ROS fluorescence intensity in cells of AEP high-dose administration groups was significantly reduced(P<0.05).It shows that AEP can protect PM2.5-induced cell damage by inhibiting the release of inflammatory factors and enhancing the activity of antioxidant enzymes.The Western blot method was used to detect the expression of the key proteins IκBα,p-IκBα,HO-1 and the transfer of p65 and Nrf2 into the nucleus in the NF-κB and Nrf2/HO-1 signaling pathways of each experimental group.Fluorescence quantitative PCR experiments were used to detect the effects of each experimental group on the expression levels of NF-κB,IL-6,Nrf2 and HO-1 m RNA.The results showed that: compared with the control group,the expression of p-IκBɑ and HO-1 protein in the PM2.5 group was significantly increased(P<0.01),and the expression of nucleus p65 was significantly increased(P<0.01),the expression of nuclear Nrf2 increased;Compared with the PM2.5 group,each administration group could up-regulate the expression of IκBɑ and cytoplasmic p65 protein,down-regulate p-IκBɑ and nucleus p65 protein expression,and up-regulate HO-1 and nucleus Nrf2 protein expression.The results of fluorescence quantitative PCR experiments showed that: the NF-κB,IL-6,Nrf2 and HO-1 m RNA c DNA of each experimental group were effectively amplified,and the specificity of the amplified products was good.Compared with the control group,the NF-κB,IL-6 m RNA expression of the cells in the PM2.5 group was significantly increased(P<0.01),and the expression of Nrf2 and HO-1 m RNA was increased significantly(P<0.05);compared with the PM2.5 group,each administration group could significantly reduce NF-κB and IL-6 m RNA expression(P<0.05),and the AEP administration group can significantly up-regulate the expression of Nrf2 and HO-1 m RNA(P<0.05).Conclusion:AEP has a protective effect on the lung injury induced by PM2.5 in rats,which can significantly reduce the content of ACP and AKP,reduce the TP exudation,relieve the thickening of the alveolar interstitium and the infiltration of inflammatory cells,reduce the inflammatory response,and thus play a role Lung protection.AEP can significantly reduce PM2.5-induced BEAS-2B cell damage,reduce LDH activity nd TNF-α,IL-6 content,down-regulate the expression of p-IκBɑ protein,inhibit the transfer of p65 to the nucleus and its transcription level,and then inhibit the NF-κB pathway activation,inhibit the transcription level of the inflammatory factor IL-6,reduce inflammation response,and increased the expression of Nrf2 at the transcription level and protein level,thereby activating the Nrf2/HO-1 pathway,up-regulating the expression of HO-1 at the transcription level and protein level,and enhancing antioxidant capacity,Protects against cell damage induced by PM2.5.