| This project comes from the key research and development project of Shaanxi Province,"Research and Application of quality Marker of Classical prescription Shaoyao Gancao decoction"(NO.2019SF-300).Neuropathic pain is pain caused by somatosensory nervous system disease or nerve injury.It lasts for a long time and is difficult to cure,which seriously affects the quality of life of patients.Shaoyao Gancao Decoction was first published in Treatise on Febrile and Miscellaneous Disease.It is composed of two traditional Chinese medicines,Radix Paeoniae Alba and Radix Glycyrrhizae.It is a classic prescription for relieving spasm and relieving pain.Many scholars have carried out chemical component analysis and pharmacological effects research on Shaoyao Gancao Decoction,but the material basis and mechanism of action of Shaoyao Gancao Decoction for the treatment of neuropathic pain have not been well explained,which has affected the clinical application and further drug development of Shaoyao Gancao Decoction.Therefore,it is urgent to use modern scientific and technological methods to reveal the pharmacodynamic material basis and relevant mechanism of Shaoyao Gancao Decoction in the treatment of neuropathic pain.This paper mainly studies the pharmacological material basis and mechanism of Shaoyaoglycyrhiza decoction,the compatibility of Shaoyao Gancao decoction and the quality markers of Shaoyao Gancao decoction,providing scientific basis for the precise clinical application of Shaoyao Gancao decoction and laying a foundation for the further drug development of Shaoyao Gancao decoction.This paper is divided into four chapters.The research contents and results of each chapter are as follows:Chapter 1.Literature research Section 1.Data analysis of TCM application of Paeoniae Radix and Glycyrrhizae Radix et RhizomaIn this section,the keyword mapping method is used to count the compatibility information of Paeoniae Radix and Glycyrrhizae Radix et Rhizoma in traditional Chinese medicine prescriptions,and the frequency statistics and network diagram are used to mine the application information of Paeoniae Radix and Glycyrrhizae Radix et Rhizoma.The results show that the common doses of Paeoniae Radix and Glycyrrhizae Radix et Rhizoma are 31.25 g and 15.53 g.The common compatibility ratio was 1:1,accounting for 37.06% of the total.The commonly used compatibility drugs of Paeoniae Radix-Glycyrrhizae Radix et Rhizoma were Angelica sinensis Radix,Chuanxiong Rhizoma,Ginseng Radix et Rhizoma,Poria and Rehmannia Radix,accounting for 16.63% of the total.The commonly used methods were water decocting,raw internal medicine,wine decocting,external uses,water alcohol decocting together,paste decocting,vinegar decocting.The main efficacy of the prescriptions were analgesia,tonic deficiency,detoxification,dispelling wind,detumescence and improving eyesight.Diseases were attributed to internal medicine,surgery,gynaecology,pediatrics,ophthalmology and otorhinolaryngology.In addition,the dosage,compatibility ratio and preparation usage were correlated with efficacy and disease attribution.Paeoniae Radix-Glycyrrhizae Radix et Rhizoma were most commonly used in internal medicine and surgery.The highest frequency of the combined dosage was 62.50 g,which was commonly used for analgesia.The highest frequency of compatibility ratio was 1:1,which was commonly used to relieve pain,detoxify,and dispel wind.Decocting in water has the highest frequency,and was often used to relieve pain,detoxify and tonify deficiency.Section 2.The research idea and design of this paperThe purpose of this section is to explain the research ideas,the design of the paper and the overall framework of the research content.Around the classic prescription Shaoyao Gancao decoction,taking neuropathic pain as the disease model,the following key problems are to be solved:(1)to clarify the functional material basis of Shaoyao Gancao decoction;(2)to reveal the action mechanism of Shaoyao Gancao decoction;(3)to clarify the quality markers of Shaoyao Gancao decoction.Finally,it provides a scientific basis for the establishment of the quality standard of Shaoyao Gancao decoction and the exploration of ideas and methods for the quality control of classical prescriptions.Chapter 2.Study on the effect and mechanism of compatibility of components in Shaoyao Gancao decoction Section 1.Study on the analgesic effect of Shaoyao Gancao decoctionIn this section,the rat model of neuropathic pain was established by selective injury of sciatic nerve branches(SNI).Pregabalin was given as a positive drug,and Shaoyao Gancao decoction was treated with low,middle and high doses of Shaoyao Gancao decoction.The mechanical pain threshold and biochemical indexes of rats were used as indexes to investigate the effect of Shaoyao Gancao decoction on neuropathic pain rats.Through the study,it was found that the mechanical pain threshold of rats was decreased by SNI model,and the pain-related indexes were regulated.After administration of Shaoyao Gancao decoction,it was found that the mechanical pain threshold and biochemical indexes of the three dose groups of Shaoyao Gancao decoction could be adjusted back,and the improvement effect of the middle dose group(the clinical equivalent dose of human)was the best.Section 2.Study on analgesic mechanism of Shayao Gancao decoction based on metabolomicsIn this section,the analgesic mechanism of Shaoyao Gancao decoction on SNI-induced neuropathic pain in rats was studied based on the metabonomic method of UPLC-Q-TOF/MS.The results showed that a total of 128 differential metabolites(84 from negative ion model and 44 from positive ion model)were identified between sham operation group and SNI group.A total of 40 differential metabolites were identified and identified as potential biomarkers(22 from negative ion model and 18 from positive ion model)from Shaoyao Gancao decoction group and SNI group.Five metabolic pathways of Shaoyao Gancao decoction in neuropathic pain were clarified,namely,glycerophospholipid metabolism,linoleic acid metabolism,α-linolenic acid metabolism,glycosylphosphatidylinositol(GPI)anchored biosynthesis and arachidonic acid metabolism.Shaoyao Gancao decoction can adjust the changes of endogenous markers in rats caused by neuropathic pain model,indicating that Shaoyao Gancao decoction may treat neuropathic pain by regulating the above pathway.The metabolic pathway also provides a reference for further elucidating the potential target of Shaoyao Gancao decoction in the treatment of neuropathic pain.Section 3.Study on material basis of analgesic effect of Shaoyao Gancao Decoction based on multi-index comprehensive index method and PLSR integration strategyIn this section,the blood components of Shaoyao Gancao decoction was obtained by UPLC-Q-TOF/MS technology,and Pearson correlation coefficient method was used to analyze the correlation between components and biomarkers based on metabonomics identification.The use of multi-index comprehensive index method and PLSR method to reveal the contribution of various components to the curative effect,and finally find the active components of Shaoyao Gancao decoction in the treatment of neuropathic pain,and then explain the material basis of the analgesic effect of Shaoyao Gancao decoction.The results showed that 44 components in Shaoyao Gancao decoction were blood components,and 14 kinds of analgesic components were found by Pearson correlation coefficient method.Paeonol,DL-arabinose,Benzoic acid,Hispaglabridin A and Paeoniflorin C were selected as the effective components of Shaoyao Gancao decoction in the treatment of neuropathic pain by the method of multi-index comprehensive index and PLSR integrated strategy.Chapter 3.Study on the effect and mechanism of the composition compatibility of Shaoyao Gancao decoction Section 1.Evaluation of analgesic effect of paeoniflorin-liquiritinIn this section,paeoniflorin and liquiritin,the representative components of Shaoyao Gancao decoction,were selected to evaluate its analgesic effect.The SNI model was established,and the sham operation group,SNI model group,paeoniflorin-liquiritin low,middle and high dose groups and positive drug pregabalin group were set up.The mechanical pain threshold of rats was used as the detection index to investigate the effect of paeoniflorin-liquiritin on neuropathic pain rats.The results showed that SNI model decreased the mechanical pain threshold of rats.After administration of paeoniflorin-liquiritin,it was found that all three dose groups could significantly increase the mechanical pain threshold of rats,and the improvement effect of the middle dose group was the best.Section 2.Study on analgesic mechanism of paeoniflorin-liquiritin based on lipidomicsIn this section,the analgesic mechanism of paeoniflorin-liquiritin on SNI-induced neuropathic pain in rats was studied based on lipidomics.The results showed that 18 differential metabolites were screened out in plasma samples by VIP >1 and P < 0.05.There were 9 phosphatidylcholine,5 triglycerides,3lysophosphatidylcholine and 1 choline ethanolamine,and 5 differential metabolites in spinal cord tissue were screened by VIP > 1 and P < 0.05.It contains 2phosphatidylcholine,1 lysophosphatidylcholine,1 lysophosphatidylethanolamine and1 ceramide.Paeoniflorin-liquiritin can reverse the changes of differential metabolites induced by neuropathic pain model in rats.The pathways of glycerolphospholipid metabolism,linoleic acid metabolism,α-linolenic acid metabolism,glycerol metabolism,steroid biosynthesis,sphingolipid metabolism and arachidonic acid metabolism were obtained from these differential metabolites.It is suggested that paeoniflorin-liquiritin may play a role in the treatment of neuropathic pain by regulating the above pathway.Section 3.Study on the analgesic mechanism of paeoniflorin-liquiritin based on transcriptomicsIn this section,the L3-L6 spinal cord tissues of rats in sham operation group,SNI model group and paeoniflorin-liquiritin middle dose group were sequenced by transcriptome technique,and the differentially expressed genes in SNI model group and paeoniflorin-liquiritin combination group were screened.Then the differentially expressed genes were analyzed by cluster analysis,GO analysis and KEGG pathway enrichment analysis.The results showed that there were 342 differentially expressed genes between SNI model group and paeoniflorin-liquiritin middle dose group,of which 64 genes were significantly up-regulated and 278 genes were significantly down-regulated.The enrichment of these differentially expressed genes is mainly related to c AMP signaling pathways,calcium signaling pathways,PPAR signaling pathways,glycerophospholipid metabolism and so on.This paper preliminarily elucidates the scientific basis of paeoniflorin-liquiritin on neuropathic pain,and provides a theoretical basis for popularizing paeoniflorin-liquiritin in the treatment of neuropathic pain.Section 4.Combined analysis and mechanism of analgesic effect of paeoniflorin-liquiritin based on lipidomics and transcriptomicsIn this section,the combination of liposome technique and transcriptome technique was used to explore the mechanism of paeoniflorin and liquiritin in the treatment of neuropathic pain,and q RT-PCR was used to further verify the corresponding gene changes in each pathway.The results showed that there were strong associations between metabolites in 7 pathways and differential genes,which were glycerolphospholipid metabolism,linoleic acid metabolism,α-linolenic acid metabolism,glycerol metabolism,sphingolipid metabolism,arachidonic acid metabolism and steroid biosynthesis.The corresponding gene changes in PLC-IP3R1-PKC-CREB pathway in the above glycerophospholipid metabolism were further verified by q RT-PCR,which proved that paeoniflorin-liquiritin could reverse the changes of differential metabolites induced by SNI in rats,that is,paeoniflorin-liquiritin could treat neuropathic pain by regulating PLC-IP3R-PKC-CREB pathways.Chapter 4.Quantitative identification and value transfer of quality markers of Shaoyao Gancao Decoction Section 1.Quantitative identification of quality markers of Shaoyao Gancao decoctionThis section constructs a comprehensive evaluation index system of the quality marks of traditional Chinese medicine in order to study the quality markers of Shaoyao Gancao decoction.Firstly,the analytic hierarchy process is used to analyze the effectiveness,testability and specificity of the primary index,and then the entropy weight method is used to analyze the secondary index supported by literature and experimental data under each primary index.After calculating the weight of each factor in the index layer,the comprehensive score of each component was calculated and the quality markers of Shaoyao Gancao decoction were obtained.The results showed that the validity weight of the first level index of Shaoyao Gancao decoction was 59%,the testability was 31%,and the specificity was 10%.The number of literatures and experimental data of the secondary index of effectiveness accounted for 67.60% and 32.40% respectively,and the weights of the number of literatures,the proportion of HPLC,the average content of medicinal materials,the variability of medicinal material content and the transfer rate of medicinal materials-prescriptions were 18.90%,14.17%,27.58%,14.48% and 24.88%,respectively.The weight of the number of plants of the secondary index of specificity is 100%.After comprehensive scoring,the top 10 components were paeoniflorin > quercetin > albiflorin >glycyrrhizic acid > naringenin > liquiritin > oxypaeoniflorin > benzoylpaeoniflorin >liquiritigenin > isoliquiritigenin.These components can be preliminarily determined as the quality markers of the analgesic effect of Shaoyao Gancao decoction Section 2.Quantity value transfer of quality markers of Shaoyao Gancao decoctionIn this section,the quantity transfer of medicinal materials-slices-material standards of Shaoyao Gancao decoction was studied by high performance liquid chromatography(HPLC).The inter-batch quality consistency and traceability of the substance benchmarks were ensured by paste yield,content determination and fingerprint.The results show that the data of gallic acid,oxypaeoniflorin,albiflorin,paeoniflorin,liquiritin,benzoic acid,isoliquiritin,quercetin,benzoylpaeoniflorin and glycyrrhizic acid fluctuated within 70%-130% of the average,and no discrete data appeared.Paeoniflorin,quercetin,albiflorin,glycyrrhizic acid,naringin,liquiritin,oxypaeoniflorin and benzoylpaeoniflorin can be detected in materials,slices and material standards,and the above components are finally confirmed as the quality markers of Shaoyao Gancao decoction. |
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