| Objective The material basis and mechanism of Asarum were predicted by network pharmacology,and the pulmonary toxicity mechanism of Asarum was explored by toxicogenomics,metabolomics,routine biochemical and pathological methods and the predicted results of network pharmacology.Methods Network pharmacological methods were used to screen active ingredients,targets and diseases of Asarum.The material basis and mechanism of Asarum were constructed by constructing compounds-targets,targets-diseases,compounds-targets-diseases and target-related biological functions and pathway networks.Eighty male Sprauge-Dawley(SD)rats were randomly divided into four groups: blank control group,Asarum low-dose group(0.27 g · kg-1),Asarum medium-dose group(0.81 g · kg-1)and Asarum high-dose group(1.35g·kg-1),20 rats in each group.The administration period was four weeks(28 days).The control group was given distilled water of equal volume.During the administration,the dosage was adjusted weekly according to the weight changes of rats.During the experiment,the general conditions(skin,hair,secretion of five senses,fecal traits,activity and behavioral activities)of rats in each group were observed daily.Weight was measured weekly and daily intake was recorded.Four weeks later,the samples were collected,weighed before anesthesia,and abdominal aorta blood was taken to determine the arterial blood p H,arterial partial oxygen pressure(Pa O2),arterial partial pressure of carbon dioxide(Pa CO2),standard bicarbonate(SB),arterial oxygen saturation(Sa O2),alveolar-arterial partial oxygen pressure difference(A-ADO2).The organ coefficients were calculated by weighing the lungs of the high-dose group and the control group.The morphological changes were observed by routine HE staining.The differentially expressed genes in lung were detected by gene chips.Screening candidate genes based on the results of network pharmacology,the expression levels of Tmprss6,Prkag3,Antxr1,Klk11,Rag2,Olr77,Cd7,Il20,Loc69,C6,Loc68,Cd163 and Ccl20 in lung tissues were detected by quantitative PCR.The expression levels of Ampk,Bcl2,Caspase 3,Il1,Il20,Matriptase2,Nf?b,Nptx2 and Rag2 in lung tissues of different groups were detected by Western blot and immunohistochemistry.Metabolomics was used to detect the changes of metabolites in lung tissues of rats in different groups.Result Results of network pharmacology: Asarum contains 192 compounds and 234 related targets.Compound-related targets of Asarum include tumors,cardiovascular and cerebrovascular diseases,inflammation,respiratory diseases,psychosis,nervous system diseases,pain syndromes,digestive system diseases and so on.Enrichment analysis of KEGG pathway of Asarum target indicated that Asarum may play a role in the treatment of cancer-related diseases;Asarum may play a therapeutic role in inflammatory diseases or its mechanism may be related through the role of inflammatory-related pathways,including NF?B,AMPK,MAPK,TNF,Ras,Wnt,VEGF,p53,PI3K-Akt,Toll-like receptor,PPAR,c GMP-PKG,c AMP signaling pathway and so on.The mechanism of Asarum may be closely related to lipid metabolism,energy metabolism and other metabolic pathways.General Situation of Rats: During the whole experimental period,the hair of rats in the control group and the low-dose group was glossy,responsive,well-moving,and there was no obvious abnormality in their behavior.From the second week,the rats in the high-dose group began to show the effects of decreased gloss of hair,slow reaction and mental depression.The body weight of rats in each dose group increased gradually with the time of gastric administration,and there was significant difference compared with themselves(P < 0.05).There was no significant difference in the same period between each group and the first three weeks(P > 0.05).In the fourth week,compared with the control group,there was significant difference with the high-dose group(P<0.05),there was no significant change in the low-dose group and the middle-dose group.Compared with the control group,the lung organ coefficient increased in high-dose group(P<0.05),there was no significant change in the low dose group and the middle dose group(P >0.05).Arterial blood gas measurement: Compared with the control group,Pa O2 and Sa O2 in high-dose group were lower,A-ADO2 was higher,with significant difference(P<0.05).Compared with the control group,there was no significant difference in each index between the low-dose group and the middle-dose group(P >0.05).Morphological changes of rat lungs: Compared with the control group,inflammatory cell infiltration appeared in pulmonary interstitium of each group.The pathological changes of lung tissue in low-dose group were milder,and those in high-dose group were the most serious.The morphological changes of lung tissue were positively correlated with the increase of Asarum dosage.The alveolar wall was partly widened.A few alveolar tubes,alveolar sacs and alveolar dilatation were accompanied by alveolar septal rupture,and alveolar wall was congested to varying degrees.Gene chip results: Selection of differentially expressed genes showed that 344 genes were significantly altered in lung tissue of high-dose group,of which 259 were significantly up-regulated and 85 were significantly downregulated.The analysis of differentially expressed genes showed that the results mainly focused on metabolic pathways,including olfactory pathway,MAPK signaling pathway and calcium signaling pathway.Signal molecules interacting with most genes were related to neuroactive ligands.Cell processes were mainly transport and catabolism in receptor interaction.Tissue system was mainly involved in the immune system.The main diseases are cancer,immune system diseases,kidney degenerative diseases and so on.Differentially expressed genes are related to the expression of olfactory receptors and immune factors activating inflammation-related genes.Validation of differentially expressed genes: Compared with the control group,quantitative PCR results showed that the expression levels of Tmprss6,Prkag3,Antxr1,Klk11,Rag2,Olr77,Cd7,Il20,Loc69,C6,Loc68 and Cd163 were up-regulated,while the expression levels of Ccl20 were down-regulated in Asarum high-dose group,there was no significant difference between the middle-dose group and the low-dose group.Western blot results showed that,compared with the control group,the expression of proteins such as Ampk,Caspase3,Il1,Il20,Matriptase2,Nf?b and Rag2 was significantly increased,the expression of Bcl2 was significantly down-regulated and the expression of Nptx2 was insignificant in high-dose group,there was no significant difference between the middle-dose group and the low-dose group.Immunohistochemical results showed that,compared with the control group the expression of proteins such as Ampk,Caspase 3,Il1,Il20,Matriptase2,Nf?b and Rag2 was significantly increased,while the expression of Bcl2 was down-regulated and the expression of Nptx2 was insignificant in the high-dose group,there was no significant difference between the middle-dose group and the low-dose group.Metabonomic results: 14 metabolites of lung homogenate were identified as biomarkers,namely isoleucine,pyrimidine,citric acid,L-serine,iminodiacetic acid,taurine,phosphatidylethanolamine,D-aloxose,inositol,L-glutamine,heptanoic acid,adenosine and glycerol.The toxicity of Asarum to the lungs is closely related to the pathways of tricarboxylic acid cycle,glycolysis,oxidative stress,immune damage and lipid metabolism.Asarum pulmonary toxicity is closely related to the metabolic pathway of sphingolipid.Conclusion 1.Long-term high-dose Asarum has certain damage to lung function and tissue morphology.The toxicity of Asarum to the lungs is closely related to the dosage of Asarum,that is,the dosage of Asarum is small,and the degree of damage is slight.On the contrary,the degree of damage is deep.2.Asarum pulmonary toxicity changes in gene expression before target organ damage.The mechanism of Asarum pulmonary toxicity may be related to the influence of differentially expressed genes on Ampk-Nf?b pathway,Bcl2 pathway,inflammatory and immune-related complements,chemokines,interleukin,olfactory receptors,AMPK and other factors,leading to immune injury and activation of inflammatory response.3.The mechanism of Asarum on lung toxicity is closely related to the pathways of tricarboxylic acid cycle,glycolysis,oxidative stress,immune injury and lipid metabolism. |
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