The Effect And Mechanism Of Traditional Chinese Medicine Gushiling Capsule For Intervention Of Glucocorticoid-induced Osteonecrosis Of The Femoral Head Based On Lipid Metabolomics

Background:Glucocorticoid-induced osteonecrosis of the femoral head(GIONFH)is a difficult disease and multi morbidity with extremely high disability rate clinically.Glucocorticoids(GCs)are widely used and have significant effects,but there are increasing cases of osteonecrosis of the femoral head in patients requiring long-term or/and high-dose use of glucocorticoids due to the presence of glucocorticoid side effects.“Hormonal therapy” for “SARS” in 2003,although saving many patients’ lives,however,also left many sequelae in ill patients,with GIONFH incidence as high as 53.5%.Here,in the face of a novel coronavirus pneumonia(COVID-19)outbreak,glucocorticoids remain an important weapon in the fight against severe infection and in the salvage of critically ill patients,but we are at the same time at risk of developing GIONFH.Therefore,how to prevent the occurrence of GIONFH has received much attention from the medical community at home and abroad.It is of great significance to explore how to prevent the occurrence of side effects such as glucocorticoid induced avascular femoral head necrosis while effectively applying glucocorticoids to treat the disease,guided by the idea of “preventing disease” in traditional Chinese medicine(TCM),to exert the advantages of TCM to prevent the disease.Gushiling capsule(GSLC)is a traditional pure traditional Chinese medicine formulation that has been clinically used for many years,and its main components contain 21 Chinese herbs including Salvia miltiorrhiza,chuanxiong,and Panax notoginseng.There have been clinical studies showing that GSLC has significant efficacy for the treatment of GIONFH and its sequelae,with fewer side effects.The results of animal model experiments also showed that GSLC could improve the disorder of blood lipid metabolism and lower blood lipid at an early stage,thus reducing the incidence of GIONFH and delaying the progression of the disease.However,the results of the modernization study of traditional Chinese medicine(TCM)GSLC are quite limited,and there are few systematic studies on its effects and mechanism of action.Metabolomics is a method for the quantitative identification and qualitative analysis of cellular endogenous and exogenous metabolites to reveal the relative relationship between the metabolic pathways of biological metabolites and the observed physiological and/or pathological changes.Metabolomics has been widely used in the detection of biomarkers,monitoring of drug efficacy and pathogenesis,early detection and clinical diagnosis of many diseases,such as cancer,heart disease and diabetes.In particular,untargeted metabolomics based on ultra-high performance liquid chromatography-tandem mass spectrometry(UHPLC-MS/MS)can obtain comprehensive information of metabolic changes,which can then evaluate the efficacy of drugs and explore the related mechanisms by which they exert their effects.However,a metabolomics study on the effect and mechanism of GSLC in preventing GIONFH has not been reported.Objective:In this study,we aimed to establish the serum metabolite profiles of a rabbit model of GIONFH,explore the biomarker changes and their metabolic pathways based on lipid metabolomics technology for early serum metabolomics of GIONFH rabbits,and screen out “risk factors” of GIONFH for early monitoring,diagnosis and intervention of GIONFH,for new reference and basis.In addition,using the above methods,we explore the efficacy and its metabolic mechanism of traditional Chinese medicine(TCM)GSLC for the prevention and treatment of GIONFH in rabbits,screen out the relevant potential biomarkers for their function,and determine their metabolic pathways.Methods:In this study,a relevant experimental study was conducted by preparing an animal model of GIONFH,and serum samples of experimental rabbits were profiled for lipid metabolomics using an untargeted metabolomics based on UHPLC-MS/MS.Principal component analysis(PCA)was applied to give an overview of the differential metabolites of the different groups.In multivariate statistical analysis,ezinfo 3.0 software was used for ratio analysis,partial least squares-discriminant analysis(PLS-DA)and orthogonal partial least squares-discriminant analysis(OPLS-DA),and PLS-DA and OPLS-DA were used to screen potential biomarkers and preliminarily identify the differences between groups.The HEML 1.0 package was used to create heat map to display the relative numbers of differential metabolites,and to perform hierarchical clustering analysis.Metbra Analytist 3.0 was used to analyze the metabolic pathways of the identified key metabolite datasets.Magnetic resonance imaging(MRI)was used to detect the imaging manifestations of avascular necrosis of the femoral head.Transmission electron microscopy(TEM)was used to observe the ultrastructural changes such as chromatin,nucleolus,mitochondria,and lysosomes in the nucleus of osteocytes in the femoral head.HE staining was used to detect the histopathological changes of the femoral head samples in the control,model,and GSLC groups.Results:(1)The metabolic profile of the experimental rabbit serum samples collected at different time points by UHPLC-MS/MS was used to obtain metabolic differences between the different time points.Principal component analysis(PCA)results showed that there was a separation between the metabolic profiles at the starting point and 1 week,indicating an obvious metabolic difference between the two groups.This was also further confirmed by partial least squares-discriminant analysis(PLS-DA)and orthogonal partial least squares-discriminant analysis(OPLS-DA).we identified 39 metabolites at the starting point and 1 week,mainly including phospholipids and amino acids.Evaluated by receiver operating characteristic curve(ROC curve),the above model has good prediction performance and accuracy.Heat maps were made using Heml 1.0 software to comprehensively analyze differential metabolites.The results showed significant clustering and correlation of metabolites.Biological pathway analysis using Metabo Analyst 3.0revealed 8 main metabolic pathways.Finally,the imaging and pathological changes of the femoral head were detected by magnetic resonance imaging and transmission electron microscope.The results of magnetic resonance imaging showed that there was no obvious abnormal signal in the femoral head of experimental rabbits at the starting point.The abnormal MRI signal appeared after glucocorticoid intervention for 3 weeks.T2 weighted imaging showed spotted,thin line and uneven high signal.The results of transmission electron microscope showed that there was no obvious abnormality in the substructure of femoral head osteocytes at the starting point.After glucocorticoid intervention for 6 weeks,some cells showed cytoplasmic concentration,cell body reduction,deep staining of nucleus,irregular nuclear membrane,local fragmentation and formation of apoptotic bodies.(2)The scores of principal component analysis(PCA)and partial least squares discriminant analysis(PLS-DA)showed that there was an obvious separation trend between the metabolites of the control group and the model group and GSLC group(1,2 and 3 weeks),but the separation trend between the metabolites of the control group and GSLC group(4,5 and 6 weeks)decreased significantly.The OPLS-DA score plot showed that there were significant differences in metabolites between the model group and GSLC group(4,5 and 6 weeks),while the metabolomic characteristics of GSLC intervention for 6 weeks showed a similar trend to that of the control group.In rabbits serum samples,we identified 10 potential metabolite biomarkers,mainly phospholipids,and proved that they were related to GIONFH.HE staining and MRI showed that the pathological changes and imaging manifestations of femoral head necrosis in GSLC group were better than those in model group,which was consistent with the changes of serum metabolite spectrum.Conclusion:(1)Based on lipid metabolomics technology,this study identified the biomarkers and metabolic pathways of early serum metabolomics of GIONFH rabbits,screened out the relevant "risk factors" of GIONFH,and provided a new strategy for the early monitoring,diagnosis and intervention of GIONFH.(2)The study of lipid metabolomics in this paper shows that GSLC plays a role in the prevention and treatment of GIONFH by regulating the disordered lipid metabolism spectrum of GIONFH(mainly involving phospholipids)to the normal level.We also found that these metabolites are mainly related to glycerol phospholipid metabolism,sphingolipid metabolism,linoleic acid metabolism andα-linolenic acid metabolism,which fully shows the overall effect of GSLC in preventing and treating GIONFH through multiple pathways,levels and targets.(3)Our study used non-targeted metabonomics technology for the first time to clarify the pharmacological metabolic effect of GSLC in GIONFH rabbits,which provided a new perspective for the study of the preventive and therapeutic effect and metabolic mechanism of GSLC in GIONFH,and also provided a new scientific basis for its wide application in traditional Chinese medicine clinic.