| 【Objective】To establish hierarchical quantitative diagnosis model for type 2 diabetes mellitus(T2DM)with syndrome of qi and yin deficiencies,and explore the medication rules of Traditional Chinese Medicine(TCM)formulas in treating T2DM with qi and yin deficiencies.To preliminarily explore the mechanisms of compound core herb combination in the intervention of T2DM and conduct experimental verification.【Methods】Study of quantitative diagnosis: Candidate diagnostic items for quantitative diagnosis of T2DM with qi and yin deficiencies were initially screened based on literature research and expert consultations.Clinical questionnaires were developed and cross-sectional investigations were carried out in the Hospital of Chengdu University of Traditional Chinese Medicine,Chengdu First People’s Hospital,Ningxia Chinese Medicine Research Center,and Mei Shan Shi Peng Shan Qu Chinese Medicine Hospital.Univariate logistic regression analysis,multivariate logistic regression analysis,ROC curve analysis,and quartile analysis were used to obtain the final diagnostic items,diagnostic equation,diagnostic threshold,and degree grading standard.The diagnostic model was evaluated by goodness of fit tests,areas under the ROC curve,and decision curve analyses in the verification group and modeling group,respectively.Finally,the diagnostic model was developed as a mobile application based on Java Script language.Study of data mining: Clinical researches of TCM intervention on T2DM with qiyin deficiencies or treating T2DM through qi-yin nourishing method were retrieved from CNKI,VIP,Sino Med,Wanfang,and Pubmed databases.The compound data in qualified literature were extracted and a database was established.SPSS modeler software and SPSS statistics software were adopted to analyze the herb dosage form,single-herb frequency,herb property,herb flavor,meridian tropism,dose,hierarchical clustering,and association rule,so as to deeply explore the medication rules of compound prescriptions.Study of network pharmacology: The active ingredients and targets of the core triangular herb "Huangqi-Shengdihuang-Shanyao(HSS)" obtained based on data mining were searched from TCMSP,BATMAN-TCM,and Drug Bank databases.The disease-related targets of T2DM were searched from Pharm Gkb,Gene Cards,OMIM,and TTD databases.The protein-protein interaction(PPI)analyses of the HSS-T2DM intersection targets were performed by using STRING online tool and Cytoscape software.The GO and KEGG signaling pathway enrichment analyses for intersection targets were performed through R software.Autodock Vina software was adopted to conduct the molecular docking of core proteins with its corresponding active ingredients.Study of in-vitro experiment: After continuous intragastric administration of HSS formula granules for 1 week,blood was collected from abdominal aorta of SD rats to obtain herb-containing serum.Insulin resistance-HepG2(IR-HepG2)cell model was induced by insulin solution combined with high glucose medium.The groups were divided into normal control group,blank group,model control group,HSS-low dose group,HSS-medium dose group,and HSS-high dose group.The cellular viability,glucose consumption,glycogen content,malondialdehyde(MDA),superoxide dismutase(SOD),and reactive oxygen species(ROS)were detected using the corresponding kits.The mRNA expressions of PI3 K,AKT,and GSK-3β were assayed through real-time PCR,and the protein expressions of PI3 K,AKT,p-AKT,GSK-3β,and p-GSK-3β were assayed through Western blot.【Results】Study of quantitative diagnosis:(1)Through literature research and expert consultations,28 diagnostic items for T2DM with qi-yin deficiencies such as "mental fatigue","shortness of breath and reluctance to talk","lassitude",and "spontaneous sweating" were preliminarily screened.(2)Based on the data of 326 T2DM cases in cross-sectional investigation,8 items including "lassitude","blurred vision","night sweating","feverishness in the palms and soles","scanty coating","low voice","thready pulse",and "deficient pulse" were finally included in the regression model after screening by univariate and multivariate logistic regression analyses.(3)Each diagnostic item in the regression model was assigned 0-3 points according to different degrees,and the diagnosis equation was calculated according to the regression coefficient β as follows: diagnostic score = "lassitude" + 1.204 × "blurred vision" +1.383 × "night sweating" + 1.961 × "feverishness in the palms and soles" + 3.989 × "scanty coating" + 1.600 × "low voice" + 1.598 × "thready pulse" + 2.548 × "deficient pulse".(4)The diagnostic threshold of the model is: slight qi-yin deficiencies(10.448≤ diagnostic score <15.653),midrange qi-yin deficiencies(15.653 ≤ diagnostic score≤ 30.014),serious qi-yin deficiencies(diagnostic score > 30.014).(5)The results of goodness of fit tests,area under ROC curve,and decision curve analyses indicate that a high calibration ability,differentiation ability,and clinical application value of the diagnostic model.(6)The diagnostic model was presented in the form of a mobile phone application named "Quantitative Diagnosis Calculator for Type 2 Diabetes with Qi-Yin Deficiencies".Study of data mining:(1)A total of 663 literatures,241 compound prescriptions,and 175 herbs were included in the data mining.(2)Blood-stasis syndrome is the most common accompanied syndrome in T2DM patients with qi-yin deficiencies.(3)There are various dosage forms for treating T2DM with qi-yin deficiencies,among which decoction,capsule,and granule are the most commonly reported.(4)Huangqi,Shengdihuang,and Shanyao are the most frequently used single-herbs in the treatment of T2DM with qi-yin deficiencies.(5)The overall dosages of Huangqi,Shanyao,and Danshen in all compound prescriptions are relatively large,while the dosages of Wuweizi,Huanglian,and Zhimu are small.(6)Danshen is the most commonly used herb for invigorating blood and removing stasis in blood-stasis accompanied syndrome;Huanglian and Zhimu are the most commonly used herbs for clearing heat in heat accompanied syndrome;Huanglian and Cangzhu are the most commonly used herbs for removing dampness in phlegm or dampness accompanied syndrome.(7)The application dosage of Shengdihuang in the accompanied syndrome of heat is relatively large while that in the accompanied syndrome of phlegm or dampness is relatively small;the application dosage of Huanglian in the accompanied syndrome of phlegm or dampness is relatively large while that in the main syndrome of qi-yin deficiencies is relatively small;The overall application dosage of Xuanshen in the accompanied syndrome of phlegm or dampness is relatively large.(8)Cold and neutral are the highfrequency herb properties of the compound prescriptions;sweet and bitter are the highfrequency herb tastes;lung meridian,spleen meridian,and kidney meridian are the high-frequency herb meridian tropism.(9)The couplet and triangular herbs with the highest support degree are "Huangqi-Shengdihuang" and "Huangqi-ShengdihuangShanyao",respectively.(10)There are 8 potential core prescriptions for the treatment of T2DM with qi-yin deficiencies,including "Mudanpi,Zexie,Fuling,Shanyao,Shanzhuyu,Shudihuang,Dangshen,Baizhu,Gancao","Tianhuafen,Zhimu,Maidong,Wuweizi,Beishashen","Chishao,Chuanxiong,Danggui,Honghua,Shuizhi","Taizishen,Huangjing,Xuanshen,Cangzhu,Danshen,Gegen,Huangqi","Shengdihuang,Huanglian,Renshen,Gouqizi,Wuweizi,Yuzhu,Shihu","Shengdihuang,Huangqi,Gegen,Maidong,Shanyao","Huangqi,Gegen,Shanyao,Shendihuang,Tianhuafen",and "Huangqi,Gouqizi,Danshen,Shengdihuang,Shanyao".Study of network pharmacology:(1)A total of 10903 disease-related targets of T2DM were obtained,and 29 active ingredients of "Huangqi-Shengdihuang-Shanyao" that may act on T2DM were screened out.These active ingredients were associated with 196 T2DM-related targets.(2)Quercetin,kaempferol,and formononetin were the active ingredients that act on the largest number of T2DM targets.(3)GO analysis of the intersection targets enriched 2396 biological processes,95 cellular components,and198 molecular functions.(4)KEGG analysis of the intersection targets enriched 175 pathways including PI3K-Akt signaling pathway,MAPK signaling pathway,and AGERAGE signaling pathway,among which the PI3K-Akt pathway was associated with the largest number of intersection targets.(5)A total of 17 core proteins were obtained through the topology analyses of PPI network.The results of molecular docking showed that these core proteins were well bound to the corresponding ingredient ligands.Study of in-vitro experiment:(1)Low,medium,and high doses of HSS-containing serum had no significant effect on the cellular viability of IR-HepG2 cells.(2)HSScontaining serum could effectively increase glucose consumption and glycogen content of IR-HepG2 cells.(3)HSS-containing serum could effectively reduce the MDA and ROS levels in IR-HepG2 cells,and increase the SOD activity in IR-HepG2 cells.(4)HSS-containing serum could significantly up-regulate PI3 K mRNA expression and down-regulate GSK-3β mRNA expression in IR-HepG2 cells,which present a certain dose gradient trend.(5)HSS-containing serum could significantly up-regulate the expression of PI3 K,p-Akt,and p-GSK-3β proteins in IR-HepG2 cells,and downregulate the expression of GSK-3β proteins,which present a certain dose gradient trend.【Conclusion】(1)The model construction of "Quantitative Diagnosis Calculator for Type 2Diabetes with Qi-Yin Deficiencies" provides an objective basis for the standardized syndrome differentiation of T2DM.Mobile application is a convenient and practical presentation of diagnosis model.(2)Data mining comprehensively reveals the herb dosage forms,high-frequency herbs,dose ranges,herb propertie and herb taste distributions,and herb association rules of the compound prescriptions for T2DM with qi-yin deficiencies,which provides references and ideas for clinical and scientific research work on T2DM with qi-yin deficiencies.(3)The mechanism of HSS acting on T2DM is a complex network with multitarget and multi-pathway.This network pharmacology study lays a foundation for further exploring the potential therapeutic mechanisms of "Huangqi-ShengdihuangShanyao",a core triangular herb in treating T2DM with qi-yin deficiencies,and also provides a reference for researchers in related fields to carry out further experimental works.(4)HSS-containing serum could effectively ameliorate insulin resistance of IRHepG2 cells,and its therapeutic mechanism may be related to the activation of PI3K/AKT/GSK-3β signaling pathway and the improvement of antioxidant stress resistance of model cells.This in-vitro experiment provides a reference for further exploring the potential scientific principles of treating T2DM with herbs of qi-yin nourishing. |
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