| Diabetes mellitus（DM）is a metabolic disease characterized by absolute or relative insulin deficiency and resulting hyperglycaemia,the incidence of which is increasing rapidly worldwide.Type 1 diabetes mellitus（T1DM）and type 2 diabetes mellitus（T2DM）are the main subtypes of diabetes mellitus and both are independent risk factors for kidney disease.Studies have shown that even with conventional glucose-lowering therapy,more than 30%of diabetic patients still suffer from chronic kidney damage.Therefore,the development of drugs that lower glucose and alleviate kidney damage is important for the prevention and treatment of diabetic kidney complications.Ginseng（Panax Ginseng C.A.Mey）is one of the classical Chinese medicines for the treatment of diabetes.Recent studies have shown that ginseng and its pharmacological components have significant effects on regulating blood glucose and pancreatic islet function in diabetic rats,but there is a lack of systematic and holistic studies on its pharmacological effects and mechanisms of action against diabetes induced kidney injury.Metabolomics and transcriptomics,the main methods in systems biology,can systematically analyze the overall changes of endogenous small molecule metabolites and gene expression in response to external stimuli,and have become a powerful tool for the elucidation of the pharmacological effects of Chinese medicine.Therefore,in the current study,integrated the metabolomics approach based on liquid-liquid coupling technology with the renal tissue transcriptomics approach based on RNA-seq analysis,the mechanism of action of ginseng aqueous extract against kidney injury in T1DM and T2DM rats were investigated at the metabolic and genetic levels.The scientific implications of ginseng for the prevention and treatment of diabetic kidney injury will be explored and elucidated in the current study.The main research includes the following aspects:1.Pharmacodynamics and metabolomics research on the prevention and treatment effect of ginseng aqueous extract against kidney injury in diabetic ratsIn this study,pharmacodynamic and metabolomic approaches were used to investigate the therapeutic effects and metabolic mechanisms of ginseng aqueous extract on kidney injury in diabetic rats.Firstly,to investigate the effects of ginseng aqueous extract on blood glucose,pancreatic islet function and renal function in T2DM rats,a HFD combined with low-dose STZ induced T2DM rat model and a high-dose STZ induced T1DM rat model were established,and subsequently administrated with ginseng aqueous extract for four weeks.The results showed that ginseng aqueous extract significantly alleviated the hyperglycemia and lipid disorders in diabetes rats,with significantly reduced the serum urea nitrogen and creatinine levels in diabetes rats.The morphological changes of kidney pathology were also improved.Secondly,the serum and urine metabolic profiles of diabetic rats were investigated by based on UPLC-Q-TOF/MS after four weeks of ginseng aqueous extract administration.The metabolic biomarkers affected by ginseng aqueous extract in diabetic rats were identified.Integrated with multivariate statistics and bioinformatics analysis,the biological meanings of the biomarkers affected by ginseng were illustrated.The results showed that ginseng aqueous extract could significantly improve the alteration of serum and urine metabolic profiles in rats with T2DM and T1DM.25 endogenous metabolites in serum and 23 endogenous metabolites were identified as the biomarkers for the therapeutic effects of ginseng aqueous extract against renal injury in diabetic rats,mainly including phenylalanine,histidine,citric acid and uracil,which significantly involved in tryptophan,phenylalanine,tyrosine,purine and pyrimidine metabolic pathways.In the meanwhile,ginseng aqueous extract could significantly regulated 30serum metabolic biomarkers（16 in serum and 14 in urine）in T1DM rats,including lactate,p-cresol sulfate,isocitrate,pyruvate and acetic acid,mainly involved in the regulation of metabolic pathways of amino acids（phenylalanine metabolism,tyrosine metabolism,tryptophan metabolism）and energy metabolism（TCA cycle,pyruvate cycle,glycolysis/gluconeogenesis）.The different pathogenesis of T1DM and T2DM may result in differences in metabolic regulatory effect of ginseng on T2DM and T1DM rats.It is showed that,ginseng aqueous extract present predominant regulatory effect on the amino acid metabolism pathways in T2DM rats than that in T1DM rats.However ginseng aqueous extract took advantages in the regulating energy metabolism disturbance in T1DM rats,mainly by inhibiting pyruvate metabolism in T1DM rats,while promoting downstream glycolysis and TCA cycle,and subsequently increasing the body’s metabolism of glucose.Moreover,the common features of the regulatory effect of ginseng on metabolic profiles of diabetic rats were analyzed.It is found that,amino acid metabolism（phenylalanine metabolic pathway,tryptophan metabolism,etc.）,glucose metabolism（TCA cycle and glycolysis/gluconeogenesis）,and linoleic acid metabolism were the common metabolic pathways regulated by ginseng aqueous extract in both types of diabetic rats,which were critically associated with gut microbiota dysbiosis,renal dysfunction and energy metabolism imbalance.It is also closely related to the drivers of diabetes and diabetic nephropathy such as intestinal flora dysregulation,renal dysfunction and energy metabolism imbalance.This suggests that ginseng aqueous extract may play a pharmacological role in prevention the progression of diabetic kidney injury,mainly by regulating amino acid metabolism disorders,maintaining energy metabolism homeostasis and modulating metabolic processes related to the structure of intestinal flora.2.Renal transcriptomic study the prevention and treatment effect of ginseng aqueous extract against kidney injury in diabetic ratsIn the current study,RNA-seq-based transcriptomics was applied to investigate the effects of ginseng aqueous extract on renal gene expression in T1DM and T2DM rats,and integrated metabolomic association analysis to illustrate the mechanism of action of ginseng aqueous extract in preventing renal injury in diabetic rats.First of all,the transcriptomic assay results showed that ginseng aqueous extract could significantly regulated the renal gene profile changes induced by T2DM and T1DM in rats.Combined with the bioinformatics analysis,a total of 1634 differential genes in the kidney of T2DM rats were regulated by ginseng aqueous extract,mainly involved in cell surface receptor signaling pathway,transmembrane transporter protein activity,PPAR signaling pathway,phenylalanine metabolism,citric acid metabolism and glycolysis/gluconeogenesis pathways,with Ppara,Pck1,Fbp2,G6pc and Hk2 were identified critically involved.In the meanwhile,325 differentially expressed genes for ginseng intervention in T1DM kidney was identified.Then,transcriptomic integrated metabolomic correlation analysis showed that,ginseng aqueous extract could significantly regulate the key genes and metabolic biomarkers involved in PPAR signaling pathway,amino acid metabolism and energy metabolism pathways in T2DM rats,thus exerting renal protective effects.While ginseng aqueous extract was mainly involved in the regulation of amino acid and lipid metabolism in T1DM kidneys.It was further verified by RT-q PCR method that ginseng could significant back-regulation effect on m RNA expression of metabolism-related differential expressed genes（B3galt2,Mogat2 and Pipox）and inflammation response-related differential expressed genes（Ilb,Il21r,Clec7a and Cd84）in T1DM renal.Moreover,the comprehensive transcriptomic results revealed that there were 68 differential expressed genes co-regulated by ginseng aqueous extract on T2DM and T1DM,mainly including Tlr7,Hk2,Clec5a and Mogat2.The core pathways co-regulated by ginseng were TLR signaling pathway,NF-κB signaling pathway and chemokine signaling pathway which closely related to the inflammatory responses in DN progression.It indicates that the prevention and treatment effect of ginseng aqueous extract against renal injury in diabetic rats was critically involved in improvement of renal inflammatory response in renal.In conclusion,this proportion of the study elucidates the mechanism of action of ginseng aqueous extract in preventing kidney injury in diabetic rats from the aspect of the genetic level,which clarifies that inflammatory signaling pathways such as TLR and NF-κB may be the common key mechanism of ginseng aqueous extract in intervening kidney injury in both T1DM and T2DM.3.Network pharmacology analysis of ginseng against diabetic kidney injuryIn the current study,a network pharmacology approach was used to explore the potential active ingredients and targets of ginseng to intervene in the progression of diabetic kidney injury.A total of 66 ginseng active ingredients and 132 ingredient-related targets were obtained based on the network pharmacology database screening of ginseng potential active ingredients and their targets.Functional enrichment analysis showed that most of these targets are membrane protein complexes and transport complex constituent proteins,and are involved in biological processes such as positive regulation of intercellular signaling and inflammatory factor response.These targets are associated with KEGG pathways such as TLR signaling pathway,NOD-like receptor signaling and insulin signaling.Meanwhile,integrating the results of network pharmacology and transcriptomics results,it is found that they were co-enriched in TLR signaling pathway and chemokine signaling pathway that tightly related to inflammatory response.The network pharmacology results were corroborated with the transcriptomic results,further revealing that ginseng and its active ingredients may exert diabetic nephroprotective effects by regulating TLR/NF-κB in the high glucose-induced renal inflammatory environment.On this basis,the interactions between the 11active ingredients and key targets such as TLRs,IL1B,TNF,PTGS2,IKBKB,JUN,PPARG and GSK3B in the network pharmacological analysis were validated using a molecular docking approach.The results showed that ginseng active ingredients panaxadiol,ginsenoside Rh4 and frutinone A could form a stable conformation with TLRs,possibly inhibiting their protein expression and activity and subsequently blocking the activation of TLR/NF-κB signaling pathway.The above results integrated transcriptomic and network pharmacological analyses to verify that ginseng and its active ingredients can improve the inflammatory environment in diabetic kidney injury through TLRs/NF-κB pathway-mediated inflammatory responses,and consequently exert a mechanism of action against diabetic kidney injury.4.Study of the regulatory effect of ginseng aqueous extract on TLR4/NF-κB/NLRP3pathway in diabetic kidney injuryBased on the results of metabolomics,transcriptomics and network pharmacology,western blot and other molecular biology methods were applied to verify the regulatory effects of ginseng aqueous extract on key differential genes in TLR family and TLR4/NF-κB/NLRP3 inflammatory pathway in the kidney of two diabetic rat models.It was demonstrated that ginseng aqueous extract significantly modulated TLR2,TLR4,TLR7 and TLR9 protein and m RNA expression changes in T1DM and T2DM-induced rat kidney tissues,and regulated NF-κB and NLRP3 inflammasome-related protein and m RNA expression and inhibited inflammatory factor release.Meanwhile,to further verify the regulatory effects of ginseng aqueous extract on renal inflammatory response,RAW264.7macrophages were treated with high glucose conditions to simulate the in vivo diabetic environment.Then the intervention effects of different concentrations of ginseng aqueous extract on macrophage proliferation and TLR4/NFκB/NLRP3signaling pathway were investigated.The results showed that both ginseng aqueous extracts showed no significant effects on macrophage viability under low and high glucose conditions;both low and high concentrations of ginseng aqueous extracts significantly inhibited TLR4 protein expression and overexpression of NLRP3inflammatory vesicle-related proteins NLRP3 and ASC in macrophages induced with high glucose,which in turn blocked NLRP3 inflammatory vesicle activation and reduced NLRP3 in macrophages The secretion of IL-18 and IL-1β,the downstream products of NLRP3 inflammasome,was significantly reduced in macrophages.In this part of the study,using in vivo diabetic animal models and high glucose-induced in vitro macrophage models,we further confirmed that ginseng aqueous extract could inhibit the activation of TLR4/NF-κB/NLRP3 pathway in diabetic kidney to block the release of inflammatory factors in diabetic kidney inflammation,therefore to improve the inflammatory infiltration of diabetic-induced kidney injury,alleviate the diabetic-induced renal inflammatory response,and subsequently prevent diabetic nephropathy injury.In summary,this study was conducted to clarify the protective effects of ginseng aqueous extract on renal injury in diabetic rats based on pharmacological methods.Then,integrate metabolomics,transcriptomics and network pharmacology analysis approaches were applied to systematically reveal the molecular mechanism of ginseng aqueous extract against diabetic renal injury from the aspect of metabolite-genes-active component.In addition,using in vivo diabetic models and an in vitro high glucose-induced macrophage model,the mechanism of ginseng aqueous extract to prevent the progression of diabetic kidney injury by inhibiting the TLR4/NF-κB/NLRP3 pathway-mediated inflammatory response were further elucidated.The current study combined with advanced analytical techniques to explore the mechanism of action of Chinese herbal medicines,providing a theoretical basis for the elucidation of the mechanism of action of ginseng aqueous extracts against diabetic kidney injury.It is also providing a new methodological route for the systematic study on the mechanism of action in Chinese herbal medicines. |
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