Analysis Of Total Saponins In Tribulus Terrestris And Study On The Mechanism Of Its Anti Cerebral Ischemia Effect Based On Multiomics

Objective:Characterization of the chemical composition of total saponins from Tribulus terrestris（GSTTF）based on mass spectrometry technology;Explore the protective effect of oral GSTTF on rats with middle cerebral artery occlusion（MCAO）;Transcriptomics and non targeted metabolomics studies were conducted to investigate the intervention effect of GSTTF on the gene and metabolic profiles of brain tissue in MCAO rats,and to explore the protective targets and network of GSTTF against MCAO rats;Western blotting（WB）was used to investigate the mechanism of GSTTF on calcium ions and TLR4/MYD88 signaling pathway in MCAO rats;Network pharmacology elucidates the active ingredients and targets of GSTTF in the treatment of ischemic stroke（IS）.Method:1.Based on liquid phase and mass spectrometry technology,the chemical composition was characterized by comparing with the retention time,molecular weight and secondary fragment information of the standard product and with the molecular weight and secondary fragment information of the existing compounds in the self-built library.2.30 male SD rats were randomly divided into a sham group,a model group,and a GSTTF group.14 days before surgery,rats in the GSTTF group were orally administered GSTTF200mg/kg daily,while rats in the Sham group and Model group were orally administered the same dose of physiological saline daily.On the 14th day,rats in different groups were orally administered with drugs or their respective test substances for one hour,and MCAO rat models were established using thread embolization method,23 h after the preparation of the model（1 h before treatment）,the rats were intragastric according to groups;2.Evaluate the neurological symptoms of rats using the Longa method,followed by intraperitoneal injection of 10%chloral hydrate at 3 m L/kg anesthesia.Collect blood from the abdominal aorta,centrifuge at 3000 r/min for 10 min to separate serum and store it in liquid nitrogen.Immediately cut off the head after blood collection,remove the olfactory bulb,cerebellum,and brainstem,wash with 4℃physiological saline,cut a brain slice of about 2mm in the coronal plane of the brain,and freeze the remaining part in liquid nitrogen.The brain slice is placed in a 2%2,3,5-triphenyltetrazolium（TTC）solution at37℃for 30 minutes in a dark manner.After staining,fix it in 4%paraformaldehyde for30 minutes,take photos,and calculate the ischemic area of the brain slice using Image-J software;3.Illumina Nova Seq 6000 sequencing was used to obtain the gene expression matrix of each group of samples,and DESeq2 software was used to analyze the significance of differentially expressed genes（DEGs）between the Model group and the Sham group,as well as between the GSTTF group and the Model group.The threshold for DEGs was set as follows:│Log2（FC）│≥1,P<0.05.Use R package"heatmap"（version 1.0.12）to perform hierarchical clustering on DEGs between different groups,use Venn diagram to identify overlapping DEGs,and use them as targets for GSTTF treatment of IS.Use DAVID database to perform gene ontology（GO）and Kyoto Encyclopedia of Genes and Genomes（KEGG）enrichment analysis on DEGs,and visualize them;4.Apply LC-MS technology for metabolomics analysis of brain tissue samples,and use principal component analysis（PCA）and partial least squares discriminant analysis（PLS-DA）methods for pattern recognition.Screen differential metabolites through projection value（VIP）,P-value,and fold change factor（FC）values.Based on precise molecular weight and MS/MS fragments,differential metabolites were identified in Human Metabolome Database（HMDB）,Metlin and other databases.The KEGG database was used to determine the metabolic pathways involved in differential metabolites.The Met Scape database was used to construct the regulation of GSTTF on the metabolism protein interaction network in the brain of MCAO rats;5.Measure the content of cyclic adenosine monophosphate（c AMP）,inositol triphosphate（IP3）,and diacylglycerol（DAG）in brain tissue using the reagent kit method.Western blotting was used to detect the protein expression levels of PLC,IP3R,PKC,PKA,RYR2,and Camk II in the calcium ion signaling pathway,as well as the protein expression level of My D88,IKK,and NF-κB in the TLR4 signaling pathway;6.Exploring the active ingredients and targets of GSTTF in the treatment of IS using targeted network pharmacology combined with molecular docking.Result:1.The HPLC analysis of GSTTF identified 9 peaks by comparing the retention time with the standard sample;The mass spectrometry analysis of GSTTF identified 59compounds through database comparison,addition ion and neutral loss inference;2.Compared with the Model group,the GSTTF group showed a significant decrease in cerebral ischemic area and neurological score,as well as plasma CRP,IL-6,and TNF-αlevels were significantly decreased in rats;GSTTF can significantly increase SOD activity,reduce MDA activity,significantly reduce NO content;The levels of c AMP in plasma and Ca²⁺in brain tissue of GSTTF group rats were significantly reduced,indicating that GSTTF can alleviate MCAO induced calcium overload in the rat brain.The mechanism may be related to the activation of calcium dependent kinases,release of inflammatory factors,and release of free radicals;3.GSTTF can significantly reverse the abnormal m RNA expression levels of IL-4,Grpr,Htr7,Cxcl10,and Pla2g5 in rat brain tissue caused by the MCAO model;GO and KEGG pathway enrichment analysis showed that GSTTF can regulate calcium ions and inflammatory responses,suggesting that GSTTF may exert anti IS effects through pathways related to inflammation and calcium regulation（Toll like receptor signaling pathway and calcium signaling pathway）;4.GSTTF exerts anti IS effects by regulating changes in metabolic products in rat brain tissue.Statistical analysis highlighted 39 differential metabolites that underwent significant changes during MCAO modeling.GSTTF has a significant regulatory effect on the content of these differential metabolites,mainly through metabolic pathways such as glycerophospholipid metabolism,sphingolipid metabolism,and arachidonic acid metabolism;5.GSTTF reduced the levels of c AMP,IP3,and DAG in cerebral ischemic rats,indicating that GSTTF has a regulatory effect on the calcium signaling pathway.By inhibiting the calcium signaling pathway（PLC/IP3R/PKC and PKA/RYR1/Camk II pathways）,GSTTF alleviates calcium overload in MCAO rat brain tissue and inhibits My D88/IKK/NF-κB signaling pathway significantly reduces the expression of My D88 and p-NF-κB,increases the expression of IKK and NF-κB exerts anti-inflammatory effects;6.Construct a chemical composition-target-IS-pathway network diagram,and use node connectivity to screen out a total of 22 active ingredients that may be GSTTF anti IS active substances.Molecular docking indicates that C16,C55,and C57 exhibit strong binding abilities with ptgs2,drd2,and htr1a,respectively,suggesting that these three compounds play an important role in the treatment of IS.Conclusion:GSTTF has a significant therapeutic effect on IS rats induced by MCAO surgery.During the treatment process,multiple metabolites and targets in the rat body are regulated,and multiple metabolic and signaling pathways are altered;GSTTF mainly regulates the calcium ion signaling pathway（PLC/IP3R/PKC and PKA/RYR1/Camk II pathways）and the My D88/IKK/NF-κB signaling pathway is used to treat IS rats with the characteristics of multi-component and multi-target effects.