Study On The Mechanism Of Tribulus Terrestris Saponins Intervening Cerebral Ischemia Rats Based On Metabonomics

Objective: To explore the protective effect of total saponins of Tribulus terrestris(GSTTF)on middle cerebral artery occlusion(MCAO)rats;to study the effects of GSTTF on MCAO using non-targeted metabolomics methods of gas-phase mass spectrometry(GC-MS)and liquid-phase mass spectrometry(LC-MS)Intervention of rat serum,urine and brain tissue metabolic spectrum,explore potential biomarkers of MCAO rats,explore the regulation of GSTTF on the metabolic network of MCAO rats,and explaint the mechanism of GSTTF anti-ischemic stroke(IS)combined with network pharmacology.Method:1.Thirty male SD rats were randomly divided into MCAO group,GSTTF group and sham operation group.Four days before the operation,rats in the GSTTF group were injected with 10 m L/kg of GSTTF in the tail vein every day,and rats in the sham operation group and MCAO group were injected with the same dose of normal saline every day in the tail vein.On the fifth day,rats in each group were injected with their respective test substances in the tail vein.After 1 hours,the MCAO model was prepared by by the suture method;2.The neurological symptoms of the rats were evaluated according to the Longa method,and urine was collected,followed by intraperitoneal injection of 10% chloral hydrate 3m L/kg for anesthesia,blood was collected from the abdominal aorta,and centrifuged at3000 r/min for 10 min to separate serum and liquid nitrogen for storage.Take out the brain,remove the olfactory bulb,cerebellum,and brainstem,and wash with physiological saline at 4℃.Cut a slice of about 2mm in the middle of the coronal plane of the brain.The rest was cryopreserved in liquid nitrogen.The slice was placed at 2% 5-Triphenyltetrazolium(TTC)solution at 37 ℃,was stained for 30 minutes in the dark,fixed with 4%paraformaldehyde for 30 minutes after staining,then photographed,and the ischemic area of the brain slice was calculated by Image-J software;3.The UHPLC/GC-MS(UPLC-LTQ/Orbitrap MS)technology is used to analyze serum,urine and brain tissue samples,using principal component analysis(PCA)and partial least squares-discriminant analysis(PLS-DA)method for pattern recognition.Look for potential biomarkers through the importance of predictor variables(VIP value),correlation coefficient(P value),and fold change(FC value).Identify potential biomarkers in Human Metabolome Database(HMDB),Metlin and other databases based on precise molecular weight and MS/MS fragmentation patterns.Use the KEGG database to determine metabolic pathways involving potential biomarkers;4.Use network pharmacology to screen the active ingredients and targets of GSTTF in the treatment of IS,and explore the mechanism of GSTTF in the treatment of IS in combination with potential biomarkers.Result:1.GSTTF can significantly reduce the neurological symptom score of MCAO rats.TTC staining of brain tissue shows that GSTTF can significantly reduce the area of cerebral infarction after MCAO;2.Metabonomics analysis shows that there are disorders of serum,urine and brain metabolism in middle cerebral artery occlusion(MCAO)rats.GSTTF can regulate fatty acid metabolism,amino acid metabolism,carbohydrate metabolism,unsaturated fatty acid biosynthesis and α-linolenic acid metabolism multiple metabolic pathways to reverse the deviation of serum metabolism caused by MCAO.GSTTF can reverse deviation in urine metabolism through multiple metabolic pathways including valine,leucine and isoleucine biosynthesis,citric acid cycle,pantothenic acid and coenzyme A biosynthesis,β-alanine metabolism,glutathione metabolism,etc.The brain metabolism deviation caused by MCAO can be reversed through multiple metabolic pathways including complement and coagulation cascade,sphingolipid metabolism,glycerophospholipid metabolism,glutathione metabolism and platelet activation;3.Network pharmacology studies have shown that GSTTF treats IS through targets such as Soat1,Hcrtr2,etc,and may exert neuroprotective,anti-inflammatory and anti-apoptotic effects through the cyclic adenosine monophosphate(cAMP)signaling pathway.Conclusion: Tribulus terrestris fruit saponins have a significant therapeutic effect on rats with cerebral ischemic stroke caused by MCAO surgery.During the treatment process,many metabolic pathways in rats have changed.Tribulus terrestris fruit saponins can mainly regulate fatty acid metabolism,amino acid metabolism,citric acid cycle and many other metabolic pathways to treat rats with ischemic stroke.