| Gouty arthritis（GA）is a metabolic rheumatic disease with high incidence in Jiangxi province,China and even the world,which seriously affects the daily life and health of patients.Meanwhile,the progression of GA is relatively straightforward:hyperuricemia（HUA）-acute gouty arthritis（AGA）-intermittent onset（DIP）-chronic gouty arthritis（CGA）.At present,the evaluation of clinical diagnosis and treatment effect of GA mainly relies on synovial fluid polarized light examination,conventional inflammatory indicators,clinical experience judgment of doctors and subjective description of patients,lacking relatively sensitive endogenous small molecular biomarkers,and the evolution mechanism of the progression of GA is unclear,which is worthy of further study.Therefore,with the deepening of the research on the modernization of traditional Chinese medicine,it is an urgent need for the current clinical treatment of GA to find the active monomer with high safety and clear pharmacodynamic effect from natural products according to the"multi-level"characteristics of the progression of GA and to clarify its mechanism of action,which is also the core research task of this paper.At present,colchicine,non-steroidal anti-inflammatory drugs and glucocorticoids were the main clinical treatments for GA in clinical practice.The curative effect was positive,but it was usually accompanied by adverse reactions,such as drug resistance,endogenous hormone inhibition and digestive tract stimulation.Meanwhile,the clinical treatment of GA with traditional Chinese medicine mainly consists of single-medicinal slices,experienced prescription of traditional Chinese medicine and proprietary Chinese medicine.It has certain curative effect,but the course of treatment was long and was often used in combination with chemical medicine,which makes it difficult to elaborate and study its mechanism of action.Therefore,with the deepening of the research on the modernization of traditional Chinese medicine,it is an urgent need for the current GA clinical treatment to search for the active monomer with high safety and clear efficacy from natural products and clarify its mechanism of action according to the"multi-level"characteristics of GA,which is also the core research task of this paper.Our research group found that pulchinenoside b4（P-B4）has strong anti-inflammatory activity on the MSU-crystaled induced arthritis,therefore,this paper aims to identify relevant pharmacodynamic biomarkers and potential therapeutic targets by integrated analysis of metabonomics,combined with quantitative and pharmacological verification,to clarify the specific regulation of pulchinenoside b4 on the expression of"gene-protein-metabolite"in vivo,and to elucidate its anti-gouty arthritis mechanism at molecular and cellular levels,and try to infer the optimal intervention stage and treatment endpoint of GA clinical intervention by P-b4.The pharmacodynamic part of this paper showed that Pulchinenoside b4（p-b4）had definite therapeutic effect on acute gouty arthritis（AGA）and was non-toxic（chapter1）.Meanwhile,the study of serum metabolomics in GA rats after p-b4 intervention revealed an important practical problem related to the diagnostic criteria of GA:The 32endogenous"pharmacodynamic biomarkers"remained at the same level as the acute episode when the apparent symptomsof GA（joint swelling and pain threshold）subsided spontaneously（chapter 2）.This may be the underlying cause of the next acute episode,or even a sustained episode.Therefore,we believe that the regression of clinical symptoms or the decline of some biochemical indicators（such as serum uric acid）cannot be regarded as the end point of GA treatment,and the phenomenon like p-b4eliminating inflammatory response and callback of endogenous biomarkers is the marker of treatment effectiveness.However,the anti-GA mechanism of P-b4 is still undefined,which will largely impede the subsequent research.Therefore,it is extremely necessary to elucidate the mechanism of p-b4 to eliminate GA inflammatory response while also callback endogenous biomarkers from the overall level in vivo,and try to infer the optimal stage and treatment endpoint of p-b4 in clinical treatment of GA in the current study phase.Serum samples from 579 subjects were collected within three years in this study,and were divided into training set（n=379）and validation set（n=200）.All samples were collected using uniform standards and serum biochemical indicators were obtained（chapter 3）.After a series of multivariate statistical analysis and correlation analysis,a total of 12 serum biochemical indicators were selected as risk factors for GA at each stage,and then the serum biochemical profile was obtained,which could effectively distinguish different stages of GA.On this account,we suppose that the bias of the overall profile of the patient’s serum biochemical indicators to a certain stage may lead to the appearance of symptoms.Therefore,we believe that the previous single indicator diagnosis method should be improved,and turn to adopt multiple indicators for clinical comprehensive diagnosis and prediction.On the basis of the above,clinical diagnostic models of five GA stages were established and prospectively tested by means of multiple ordered logistic regression.Then,the validation set data was substituted into the above models,and the predicted results were outputted and compared with the original results.Total area under the curve（AUC）in receiver operating characteristic（ROC）curves of the clinical diagnostic model was 0.9534 while AUC of five models were 0.9814（Control）,0.9288（HUA）,0.9752（AGA）,0.9056（DIP）,0.9759（CGA）.In addition,Kappa coefficient of the clinical diagnostic model was 0.80,which indicated a substantial accuracy of this model.This clinical diagnostic model could be applied clinically and in research to improve accuracy of identification of these stages of GA patients.Meanwhile,the serum biochemical profile revealed by this study could be use to assist the clinical diagnosis and prediction of GA（chapter 4）.The method of non-targeted metabolomics using UPLC Qtof-MS/MS was adopted to identify and evaluate the 496 diagnostic biomarkers in five GA clinical stages.Among them,12 metabolites satisfied the following three conditions simultaneously,which could be regarded as potential biomarkers for the progression of GA（chapter 5）:（1）Differential expression was found in all five stages of GA;（2）Metabolites could be detected in all five stages of GA;（3）There was a continuous and stable upward or downward trend of the metabolites in the five stages of GA.At the same time,the key metabolic pathways related to the progression of GA were enriched and analyzed,and11 key pathways and related upstream proteins were obtained（chapter 5）.Furthermore,the method of targeted metabolomics using UHPLC-QE-MS was adopted to perform accurate quantitative validation of 12 potential biomarkers identified（chapter 6）.It was found that Indoleacetic acid、DL-2-aminoadipic acid、kynurenic acid and N1-Methyl-2-pyridone-5-carboxamide showed the most regular dynamic changes in the five stages of GA.Therefore,we believe that these four metabolites are the most promising potential biomarkers for the progression of GA.The human monocytic THP1 cells induced by MSU crystals were used for GA inflammation model,and a comparative study was conducted on the expression of the following proteins and inflammatory factors in the control group,model group and P-b4 group:Neutrophilic alkaline phosphatase-3（NALP3）,cysteinyl aspartate specific proteinase-1（caspase-1）,apoptosis associated speck like protein（ASC）,interleukin-1β（IL-1β）,prostaglandin E2（PGE2）,tumor necrosis factorα（TNF-α）（chapter 6）.The above synthesis elucidates that four metabolites could distinguish HUA,AGA,DIP and CGA patients from control group and serve as accurate diagnostic biomarkers for different stages of GA,moreover,combined with the analysis of identified pharmacodynamic biomarkers and metabolic pathways,it was also shown that p-b4could significantly reduce the m RNA expression of NALP3 inflammasome-related protein in MSU-induced THP-1 cells,thereby inhibiting the biosynthetic of NALP3,caspase-1 and ASC,thereby reducing the release of monocytes to TNF-α,IL-1βand PGE2,and finally exert its pharmacodynamic effect on GA.By comparing 496 GA"diagnostic biomarkers"and 32 P-b4"pharmacodynamic biomarkers",it was found that the GA"diagnostic biomarkers"included 23 P-b4"efficacy biomarkers",so the semi-quantitative results of the 23 biomarkers in the serum of patients of five stages of GA were further extracted and analyzed（chapter 7）.It was found that the expression of these 23 biomarkers at the AGA stage was the most significant difference with control group,and the regulation trend was consistent with the results of animal experiments.Therefore,we speculated that the optimal clinical intervention stage of P-b4 on GA was the AGA stage.After the completion of this study,further studies on other targets of P-b4 in vivo,quality standard studies,preparation studies and pharmacokinetics studies will be carried out to improve the efficacy and safety evaluation,and preclinical studies of new drugs will be carried out.At the same time,it is expected to develop a new clinical diagnosis method of GA similar to"GA diagnostic kit",which can help clinicians predict the progress of GA faster and more accurately and improve the prognosis of patients. |
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