Apreliminary Study On The Metabolomics Of Xuebijing Injection In Rats With Paraquat Poisoning

Objective: This study used a non-targeted metabolomics method based on gas chromatography-mass spectrometry(GC-MS)and ultra-high performance liquid chromatography-time-of-flight mass spectrometry(UPLC-QTOF-MS)to study the therapeutic effect of Xuebijing injection(XBJ)on paraquat(PQ)poisoning rats,searching for metabolites related to PQ poisoning by chemometrics,and exploring the effects of XBJ on potential metabolites,and preliminarily elucidating the mechanism of action of XBJ to provide a theoretical basis for rational application in clinical practice.Methods: According to the random number table method,the rats were divided into Control group,PQ group and XBJ group.At the same time,every group was subdivided into the first day,the third day,the seventh day and the twenty-first day subgroups that including six rats.The PQ group was intragastrically administered with 40 mg/kg PQ for one time,and the other two groups were administered with 1 mL of normal saline.Two hours later,XBJ group received 8mL/kg·d of XBJ injection by tail vein injection,and PQ group and control group received the same amount of normal saline by tail vein injection.Then,all groups were administered continuously for seven days.Finally,the rats were sacrificedon the first day,the third day,the seventh day,and the twenty-first day,respectively.Subsequently,the lung tissue and blood of the rats were collected.The upper lobe of right lung was detected for ELISA and the lower lobe of right lung was detected for pathology.In addition,different pretreatments were performed on plasma samples for GC-MS and UPLC-Q-TOF-MS analysis.The detected endogenous metabolites were extracted and converted using the instrumental software.Then the data was input to the SMICA-P 14.1 software for principal component analysis(PCA)and partial least squares discriminant analysis(PLS-DA),and meanwhile imported to MATLAB software for random forest analysis(RF).In order to uncover the potential differential metabolites,we used variable importance in the projection indicators(VIP)to screen the metabolites.Finally,t-test analysis was performed on the potential biomarkers to identify the suitable biomarkers,and the metabolic regulation pathway was analyzed by MetaboAnalyst 4.0 software.Results:(1)Pathological examination showed that pulmonary edema and pulmonary hemorrhage occurred in the PQ group at the first and third days compared with the control group.But the pulmonary interstitial fibroblast proliferation was occurred after the seventh day,and it still shown some fibroblast proliferation on the twenty-first day.These phenomenons indicated that the experiment of lung injury model was successful.(2)The pathological results of this experiment also showedthat the degree of lung injury in rats was significantly better than that in PQ group after intervention with XBJ.And the biochemical test results of this study showed that all biochemical indexes varied after XBJ intervention.Superoxide dismutase(SOD)and malondialdehyde(MDA)were statistically different(P<0.05)at the third day and the seventh day,while hydroxyproline(HYP)was statistically significant(P<0.05)at the third,the seventh,and the twenty-first days.These results clearly displayed that XBJ has an importantly therapeutic effect on PQ poisoning by weakening the early oxidative stress,reducing HYP content,and cutting back the process of lung injury.(3)The missing values in the data was removed using the “80% rules”,the repeatability and precision of the methods were investigated by QC samples.The relative standard deviation(RSD)of the retention time and the intensity of metabolites were all met the analytical requirements.The plasma endogenous metabolites were identified by standard library and NIST 14.0 database in GC-MS,and by local standard database,HMDB database,and online database of LC-MS method.Finally,seventeen potential biomarkers related to PQ poisoning were matched according the VIP and t-test.What’s the most important is the concentrations of following metabolites such as L-valine,succinic acid,D-galactose,linoleic acid,L-tryptophan,indoxylphenol,sphingosine 1-phosphate,oleic acid,lysophosphatidylcholine PC(22:4),9,10-epoxyoctadecenoic acid,8,11,14-eicosatrienoic acid and pantothenic acid were corrected after XBJ intervention.Then,several metabolic pathways were deduced,which including amino acid metabolism,sphingolipid and phospholipid metabolism,fatty acid metabolism,energy metabolism and pantothenic acid and CoA metabolism.Therefore,these metabolic pathways discovered in this study are clinically valuable for elucidating the therapeutic effects of XBJ.Conclusions:(1)In this study,our results showed that XBJ had a protective effect on PQ-induced lung injury in rats by using the pathological and biochemical examinations.(2)Additional,the results demonstrated that GC-MS and UPLC-QTOF-MS metabolomics methods are suitable to study the metabolic changes of acute lung injury induced by PQ poisoning on rats,which providing a potential protective mechanism for XBJ to alleviate PQ-induced acute lung injury.These pathways involve amino acid metabolism,sphingolipid and phospholipid metabolism,fatty acid metabolism,energy metabolism,pantothenic acid and CoA metabolism.