Molecular Mechanism Of Astragaloside Ⅳ On Acute Kidney Injury Based On TLR4/NF-κB Signaling Pathway

Objective：1.Through the Traditional Chinese Medicine Inheritance support system software summarize the medication characteristics and laws of traditional Chinese medicine in the treatment of acute kidney injury reported in the existing literature,and obtain the common combination of traditional Chinese medicine in the clinical treatment of acute kidney injury.2.To study the main mechanism and potential target pathways of Astragalus in the treatment of acute kidney injury based on network pharmacology and molecular docking.3.Use animal experiments to verify the potential mechanism and target pathway of astragaloside Ⅳ in the treatment of acute kidney injury shown by network pharmacology and molecular docking.Methods：1.Data mining.The literature on the treatment of acute kidney injury with traditional Chinese medicine was searched by using China knowledge network,Wanfang Data,VIP,China biomedical literature service system and Pub Med database.The retrieval time was from the establishment of the database to January 10,2022.The traditional Chinese medicine prescriptions in the literature were screened and summarized.Through the analysis of traditional Chinese Medicine Inheritance auxiliary platform V2.5 software,the medication characteristics,prescription rules and new prescription combinations of traditional Chinese medicine prescriptions for the treatment of acute kidney injury were obtained.2.Network pharmacology and molecular docking analysisThe active ingredients of Astragalus were searched through TCMSP database,and the drug targets of Astragalus were searched by TCMSP and Swisstargetprediction.AKI-related disease targets were obtained using OMIM,Genecards,and Disgenet databases.Active ingredient-target network diagrams were constructed using Cytoscape 3.7.2 software.The STRING database was used to construct a protein-protein interaction network(PPI).GO enrichment and KEGG enrichment analysis were performed in R language to predict the potential mechanisms and target pathways of Astragalus in the treatment of acute kidney injury.The Auto Dock Tools-1.5.6 software was used to verify the molecular docking of the active ingredient astragaloside Ⅳ and TLR4,MYD88,NF-κB p65,Caspase-3 and Bcl-2 in the TLR4/NF-κB signaling pathway.3.Animal experimentThirty-six SPF SD rats were randomly divided into sham operation group(24,48hours),model group(24,48 hours),astragaloside Ⅳ group(24,48 hours),6 rats in each group.Barbital sodium 30 mg/ kg was anesthetized by intraperitoneal injection.In the sham-operated group,the abdominal cavity was opened,the bilateral renal arteries were dissociated,but renal artery clipping was not performed,and 1 ml of0.9% sodium chloride injection was injected into the tail vein after the abdomen was closed.In the model group and the astragaloside Ⅳ group,the ischemia-reperfusion animal model was established by bilateral renal artery occlusion.The model group was injected with 1 ml of 0.9 % sodium chloride injection through the tail vein after successful reperfusion,and the astragaloside Ⅳ group was injected with 1 ml of astragaloside Ⅳ solution through the tail vein after successful reperfusion.Blood urea nitrogen and creatinine in each group were detected by blood sampling from abdominal aorta after administration.Rat kidney tissue was collected for HE staining and Tunel staining.The levels of TLR4,MYD88,NF-κB p65,Caspase-3 and Bcl-2in renal tissue after ischemia-reperfusion injury were detected by Real-Time PCR and Western Blot.Results：1.Data mining:(1)There are 29 traditional Chinese medicines with a frequency of more than 10 times in the treatment of AKI,mainly cold and sweet.It mainly returns to the Liver,Spleen,Stomach,Heart,and Kidney meridians.(2)The most commonly used drug combination in AKI traditional Chinese medicine formulations is:Astragalus-Rhubarb,the core drugs are: Rhubarb,Astragalus,Salvia,Oyster,Poria,Dandelion,and the network pharmacology and molecular docking analysis of Astragalus were carried out.2.Network pharmacology and molecular docking analysisThrough network pharmacology,Through network pharmacology,88 active components and 961 drug targets of Astragalus were screened out.There are 761 disease targets related to AKI.After screening by R language,68 targets of Astragalus in the treatment of acute kidney injury were obtained.The three active ingredients with the highest degree value were obtained by analyzing the active ingredient-target network diagram: astragaloside Ⅳ,acetyl astragaloside and astragalus polysaccharide.Through PPI topology analysis,it was found that STAT3,CASP3,MAPK8,BCL2,etc.may be the key core targets of Astragalus in the treatment of acute kidney injury.GO and KEGG enrichment analysis of drug-disease common targets showed that the targets of astragalus in the treatment of acute kidney injury were mainly enriched in TLR/NF-κ B,PI3K-Akt,MAPK signaling pathways and other related pathways.Astragaloside Ⅳ was the active ingredient with the highest degree value from the analysis of the active ingredient-target network diagram,and the TLR/NF-κ B signaling pathway was the main signaling pathway for astragaloside Ⅳ in the treatment of acute kidney injury,and the molecular docking results showed that astragaloside Ⅳ Good binding ability to TLR4,MYD88,NF-κ B p65,Bcl-2,Caspase-3.3.Animal experimentA rat model of ischemia-reperfusion was established.At 24 hours and 48 hours after operation,the indexes of rats in the model group were higher than those in the sham operation group.At 24 and 48 hours after operation,the levels of blood urea nitrogen and creatinine in the astragaloside Ⅳ group decreased,with 48 hours after operation.The hour group was the most significant.It indicated that astragaloside Ⅳ could improve the renal function of the model group rats.At 24 and 48 hours after operation,the renal tissue injury scores of the rats in the model group were higher than those in the sham operation group,and the renal tissue injury scores of the rats in the astragaloside Ⅳ group were significantly lower than those in the model group at 2 4and 48 hours after the operation,indicating that astragaloside Ⅳ can reduce the risk of kidney damage.ischemia-reperfusion injury.TUNEL cell apoptosis test at 24 and 48 hours after operation showed that the percentage of apoptosis in the model group was higher than that in the sham operation group,and the percentage of apoptosis in the astragaloside Ⅳ group was lower than that in the model group at 24 and 48 hours after operation,suggesting that Astragaloside Ⅳ can reduce the apoptosis of renal tubular epithelial cells.At 24 hours and 48 hours after operation,the expression of TLR4,MYD88,NF-κ B p65 and Caspase-3 in the model group was increased,and the expression of Bcl-2 was decreased.Astragaloside Ⅳ could inhibit the expression of TLR4,MYD88,NF-κB p65 and Caspase-3.expression,increasing the expression of Bcl-2.Conclusion:Astragaloside Ⅳ has a protective effect on renal function after renal ischemia-reperfusion injury in rats,and this protective mechanism may be related to the regulation of TLR4/NF-κB signaling pathway.