Investigation On Salidroside Alleviating PM2.5 Caused Lung Injury Via Inhibition Of Apoptosis And Pyroptosis Through Regulating NLRP3 Inflammasome Based On "WE" Medicine

BackgroundLung injury caused by acute exposure to ambient fine particulate matter with dp<2.5μm（PM2.5）is a new global public health problem with the development of industrialization and urbanization in recent years.Exposure to PM2.5 leads to the release of proinflammatory materials,contributing to lung inflammation and tissue damage,as well as increasing the morbidity and mortality from respiratory diseases.In PM2.5induced lung injury,apoptosis and pyroptosis play important roles.On the one hand,PM2.5 initiates the oxidative stress response,which generates reactive oxygen species（ROS）,followed by the activation of NLRP3 inflammasome and downstream proinflammatory cytokines release.Pyroptosis induced by NLRP3 inflammasome pathway enhances the tissue damage and triggers lung diseases.On the other hand,ROS can also induce apoptosis via either the mitochondria mediated intrinsic pathway or the NLRP3 downstream factor Caspase-1,further damaging the lung.“WE”medicine（Western and Eastern Medicine）is the current trend of medical development.The new challenges of global public health requires the integration of Western medicine and Eastern medicine to solve health problems in a complete and comprehensive way.In traditional Chinese medicine（TCM）,PM2.5 belongs to the“environmental toxin”category,and Qi deficiency is the main mechanism for PM2.5’s pathogenesis.Rhodiola can nourish qi and activate blood circulation,unblock blood vessels and relieve asthma with detoxification function,providing relevant theoretical basis for preventing and treating lung injury caused by acute exposure to PM2.5.Basic researches reveal that PM2.5 induced lung injury is due to the two different cell-deaths:apoptosis and pyroptosis.Recent studies show that salidroside can inhibit NLRP3pathway mediated pyroptosis and apoptosis,reduce inflammation,and prevent various diseases.Therefore,we hypothesize that“The mechanism of Salidroside alleviating PM2.5 caused lung injury is via the inhibition of apoptosis and pyroptosis through regulating NLRP3 inflammasome”.ObjectiveTo find the possible target proteins of Sal for PM2.5 induced lung injury,network pharmacology was conducted.Based on the results of network pharmacology,experiments were performed to focus on the protective mechanism of Sal precaution on PM2.5 induced lung injury,mainly investigating the regulation of Sal on NLRP3pathway dependent pyroptosis,apoptosis and lung inflammation by using NLRP3activator——Nigericin（Nig）.From this point,our research deepens the understanding of protective roles of Sal and provides a novel strategy and target for the development of anti-lung damage botanical drugs.Methods（1）Network pharmacology:we used the online databases,including the Encyclopedia of Traditional Chinese Medicine（ETCM）,Pharm Mapper,Swiss Target Prediction,Comparative Toxicogenomics database（CTD）,Therapeutic Targe database（TTD）,Uniprot,and The Binding Database（Binding DB）to predict Sal potential targets based on the publications,existing experimental results,and structure similarity.After excluding the low confidential candidates,we used the remains for online STRING and Gene Ontology analysis,to identify the involved pathways or protein complexes.Meanwhile,by extracting the lung disease related targets from CTD,we analyzed the overlap between Sal-Target and Lung disease-Target with the help of online Venn Plot tool.All the results were plotted by using the R software（including stringr,Bioc Manager,Cluster Profile and ggplot2 packages）.Finally,the NCBI BLASTP tool was used for analyzing the sequence identity,and the proteins with high sequence identity in mice were used to identify potential drug-targets.（2）To investigate the impact of Sal on PM2.5 induced lung injury and the associated mechanism,C57BL/6 mice were intraperitoneally injected with Sal for 3days before the respiratory systems were exposed to PM2.5.Experiment One:Fourty-two mice were divided into 6 groups randomly:1)PM_2.5 group;2)Control（Con）group;3)Sal control group（Sal-H,100 mg/kg）;4)Sal-H+PM_2.5 group;5)Sal-L+PM_2.5（Sal-L,50 mg/kg）group;6)Nig group（Sal-H+Nig+PM2.5）.To evaluate the effect of Sal precaution,each group was observed for up to 120 hours based on the physical health and survival.Finally,the survival rate was calculated and plotted.Experiment Two:Fourty-two mice were randomly divided into the same 6 groups as the Experiment One:1)PM2.5 group,2)Con group,3)Sal control group,4)Sal-H+PM2.5 group,5)Sal-L+PM2.5 group,6)Nig group.The mice were sacrificed 12 hours after PM2.5 treatment.The apoptosis and pyroptosis in lung cells were studied by observing the TUNEL and immunofluorescence,apoptotic index,and pyroptosis rae.The expressions of proteins involved in pyroptosis（e.g.,NLRP3,ACS,cleaved-caspase 1,Gasdrmin D）,the proteins involved in apoptosis（Bax,Bcl 2,caspase 3,cleaved PARP 1）,and inflammatory proteins（p65）in lung tissues were evaluated by western blotting.Meanwhile,the proinflammatory cytokines（IL-1β,IL-18 and TNF-α）in serum and bronchoalveolar lavage fluid（BALF）were quantified by ELISA,exploring the molecular details of Sal precaution on pyroptosis,apoptosis and inflammation.Additionally,we used the wet/dry lung weight ratio（W/D）,lung water content,HE stains,Transmission electron microscopy（TEM）,and lactate dehydrogenase（LDH）release to evaluate the overall lung structural damage,verifying the protection on lung structure provided by Sal precaution.ResultsBy using network pharmacology,we found that Sal potential targets were mainly involved in NLRP3 inflammasome mediated pyroptosis and apoptosis pathways.In experiment one,we found that（1）The survival rate of mice in the PM2.5 group was significantly reduced,and the survival status of the mice was poor;while Sal increased the survival rate dramatically,compared with the PM2.5 group,which confirmed the protection provided by of Sal precaution against mice death;（2）Even with high dosage Sal pre-treatment,in Nigericin co-treated group,mice still frequently died from PM2.5 exposure.In Experiment Two,（1）PM2.5 exposure increased the W/D ratio,lung water content,and damage scores in lung HE staining.Lung morphology by HE and Ultrastructure by TEM showed obvious pathological lesions in PM2.5 group.Salidroside pre-administration can significantly improve the above pathological indicators,thereby alleviating PM2.5-induced pathological damages of lung tissue.（2）The content of inflammatory factors（TNF-α,IL-1β,IL-18）in the BALF and serum of mice in the PM2.5 group was significantly increased.TUNEL/NLRP3 and Caspase-1 fluorescence signals were enhanced in the PM2.5 model group,and the pyroptosis pathway was activated;LDH release rate was increased in PM2.5 group,indicating cell membrane damages;electron microscope observation of the ultrastructure of alveolar macrophages and alveolar epithelial cells showed that the cell membranes in the PM2.5model group were swollen and ruptured,and the cells were severely damaged,showing a typical phenomenon of pyroptosis.Pre-administration of Sal can effectively improve the above indicators and reduce the lung injury caused by PM2.5 exposure,and the protective effect was positively correlated with the dosage of Sal.The expressions of NLRP3 inflammasome-related proteins（NLRP3,ASC,cleaved-caspase1）and pyroptosis-related proteins（GSDMD,IL-1β,IL-18）were significantly increased in lung tissue cells exposed to PM2.5,while Sal pre-administration can reduce the expression of the above proteins in lung tissue,inhibit cell pyroptosis,and reduce lung inflammation,thereby alleviating the lung injury caused by PM2.5 exposure.But Nigericin reversed the above effects of Sal.These results suggested that Sal might inhibit pyroptosis through the NLRP3/caspase-1 signaling pathway,reduce oxidative stress and inflammatory response in the lungs,and thereby alleviated lung injury caused by PM2.5 exposure.3)The results of immunofluorescence and western blotting showed that Sal reduced the apoptotic index,inhibited the Bcl2-Bax/caspase-3/PARP1apoptosis pathway,and inhibited the downstream NF-κB pathway,reduced lung inflammation,and protected PM2.5-induced acute lung injury.Likewise,Nig could reverse this effect,suggesting that the protective effect of Sal against apoptosis might also be associated with the NLRP3 inflammasome.（4）The translocation（cytosol to nucleus）of proinflammatory NF-κB pathway proteins p65 was significantly increased in PM2.5-exposed lung tissue cells,indicating the activation of this pathway,and Sal pre-administration could reduce the related protein levels to inhibit inflammation.ConclusionsPre-administration of Sal can inhibit NLRP3-dependent pyroptosis and apoptosis by regulating relevant proteins expressions,which reduced the inflammation to protect mice from the PM2.5 acute exposure induced lung inflammation and lung injury,which was the potential molecule details of the protective role of Sal in the lung injury caused by acute exposure to PM2.5 based on“WE”medicine.