| Ratinales:Kidney-Yang deficiency syndrome is a common syndrome pattern in traditional Chinese medicine(TCM),which was firstly recorded in Yellow Emperors Classic of Internal Medicine(Huangdi Neijing),the earliest systematic monograph in TCM.It is a classic syndrome associated with the deficiency syndrome of kidney yang and warm disorder,and the patients usually have chills,hypo sexuality and night urination.With the modernization of TCM,dysfunction of hypothalamic-pituitary-target glands(adrenal,thyroid and gonad)has been proved to be closely related to Kidney-Yang deficiency syndrome.Guilingji is a famous health preserving prescription from the Ming Dynasty,which consists of 28 herbs and is made by traditional method.It is famous for its efficacy in strengthening the brain,kidney and enhancing the appetite.Up to now,studies on Guilingji mainly focus on erectile dysfunction,aging etc.And there are some clinical studies on Kidney-Yang deficiency syndrome while little pharmacologic study.In this study,the pharmacological effects of Guilingji were evaluated and LC-MS metabolomics analyzed the regulation of Guilingji on biomarkers and associated metabolic pathways,which was for the further study and clinical application of Guilingji.Objective:This study aimed to reveal the pharmacological effects of Guilingji against Kidney-Yang deficiency syndrome in rats and discuss the regulation of Guilingji on metabolites and metabolic pathways based on LC-MS metabolomics,which hoped to provide scientific basis for the clinical application of Guilingji.Methods:1.Kidney-Yang deficiency rat model was constructed by high-dose injection of hydrocortisone.Rats were randomly divided into 6 groups:control group(CON),model group(MOD),positive drug group(P)and low-dose group(37.5 mg/kg),medium-dose group(75 mg/kg),and high-dose group(150 mg/kg)of Guilingji for continuous administration for 30 days.The pharmacological efficacy of Guilingji on Kidney-Yang deficiency rats was evaluated with traditional pharmacodynamic indicators(behavioral indicators,histological examination and biochemical parameters).2.The serum,adrenal and testicular metabolic profiles of Kidney-Yang deficiency syndrom rats were acquired by LC-MS metabonomics techniques.The effects of Guilingji were analyzed by the regulation on the metabolites associated with Kidney-Yang deficiency syndrome from the serum,adrenal gland and testisof rats3.The metabolic pathways of the biomarkers related to Kidney-Yang deficiency syndrome were also explored as the evaluation indicators of effects of Guilingji on Kidney-Yang deficiency syndrome ratsResults:1.Pharmacological effects:compared with the control group,rats in the model group showed emaciated,dim hair,depilate,languorous,sluggish,crouched and the weights,number of crossings and rearings and the levels of serum hormone of rats in model group were all significantly decreased,which indicated the success of Kidney-Yang deficiency model in rats.After the administration of Guilingji,the signs,weights,number of crossings and the levels of serum hormone were all obviously regulated2.Serum metabolomics results:PLS-DA model score plots showed that obvious separation occurred in the control group and model group,moreover,obvious separations were also observed among the groups treated by Guilingji and model group,which indicated that Guilingji had regulative effects on the serum metabolic profiles of Kidney-Yang deficiency rats.At last,22 differentiated metabolites related to Kidney-Yang Deficiency were identified,among them,21 metabolites were regulated by Guilingji and 7 metabolites,(LPC(20:4)、PC(0:0/18:1)、LPC(18:2)、LPC(20:0)、Platelet-activating factor、L-leucine、Oleamide)were significantly restored compared with those of the model group(P<0.05).Pathway analysis showed that leucine-isoleucine metabolism,ether ester metabolism,bile acid metabolism,etc.are the main pathways that improved by Guilingji3.Adrenal metabolomics results:PLS-DA model score plots showed that obvious separation occurred in the control group and model group and were also observed among the groups treated by Guilingji and model group,which indicated Guilingji had regulative effects on the adrenal metabolic profiles of Kidney-Yang deficiency rats.At last,19 differentiated metabolites related to Kidney-Yang deficiency were identified,among them,15 metabolites were significantly restored compared with those of the model group after the administration of Guilingji(P<0.05),which belong to lipids,nucleotides,and vitamins.Pathway analysis showed that sphingolipid metabolism,nicotinate and nicotinamide metabolism,arachidonic acid metabolism and purine metabolism are the main pathways that improved by Guilingji.4.Testicular metabolomics results:PLS-DA model score plots showed that obvious separation occurred in the control group and model group suggesting that the testicular metabolic profiles were altered by Kidney-Yang Deficiency.Moreover,obvious separations were observed among the groups treated by Guilingji and model group,which indicated Guilingji had regulative effects on the testicular metabolic profiles of Kidney-Yang deficiency rats.At last,31 differentiated metabolites related to Kidney-Yang Deficiency were identified,among them,26 metabolites were significantly restored(P<0.05)compared with those of the model group after the administration of Guilingji,which belong to lipids,nucleotides,vitamins and amino acids.Pathway analysis showed that glyoxylate and dicarboxylate metabolism,sphingolipid metabolism,glutathione metabolism,valine,leucine and isoleucine biosynthesis and ether lipid metabolism are the main pathways that improved by GuilingjiConclusin:Serum metabolomics and target tissue metabolomics are holistic and systemic,and complement each other.Guilingji had great effects on Kidney-Yang deficiency in rats by the regulation of lipids metabolism,ncleotide metabolism,aino acid metabolism,vtamin metabolism etc.,which might be associated with steroid hormone biosynthesis,oxidant-antioxidant balance and energy acquisition. |
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