The number of patients undergoing joint arthroplasty is increasing worldwide. An Enhanced Recovery After Surgery (ERAS) pathway for hip and knee arthroplasty was introduced in an Italian high-volume research hospital in March 2018.The aim of this mixed methods observational study is to perform a health technology assessment (HTA) of the ERAS pathway, considering 938 procedures performed after its implementation, by means of a hospital-based approach derived from the EUnetHTA  Core Model. The assessment process is based on dimensions of general relevance, safety, efficacy, effectiveness, economic and financial impact, equity, legal aspects, social and ethical impact, and organizational impact. A narrative review of the literature helped to identify general relevance, safety and efficacy factors, and a set of relevant sub-dimensions submitted to the evaluation of the professionals who use the technology through a 7-item Likert Scale. The economic and financial impact of the ERAS pathway on the hospital budget was supported by quantitative data collected from internal or national registries, employing economic modelling strategies to identify the amount of resources required to implement it.The relevance of technology under assessment is recognized worldwide. A number of studies show accelerated pathways to dominate conventional approaches on pain reduction, functional recovery, prevention of complications, improvements in tolerability and quality of life, including fragile or vulnerable patients. Qualitative surveys on clinical and functional outcomes confirm most of these benefits. The ERAS pathway is associated with a reduced length of stay in comparison with the Italian hospitalization average for the same procedures, despite the poor spread of the pathway within the country may generate postcode inequalities. The economic analyses show how the resources invested in training activities are largely depreciated by benefits once the technology is permanently introduced, which may generate hospital cost savings of up to 2054,123.44 € per year.Galeazzi Hospital’s ERAS pathway for hip and knee arthroplasty results preferable to traditional approaches following most of the HTA dimensions, and offers room for further improvement. The more comparable practices are shared, the before this potential improvement can be identified and addressed. The spread of chronic musculoskeletal disorders prompted the United Nations, the World Health Organization and 37 countries to proclaim that we are living in the Bone and Joint Decade . The neeImproving the value of healthcare became a global priority . At thisThese efforts are particularly clear in joint arthroplasty (JA), due to high number of procedures delivered worldwide . A numbeAn ERAS pathway for hip and knee arthroplasty was introduced in a high-volume research hospital, IRCCS Orthopedic Institute Galeazzi , in March 2018. The aim of this study is to perform a multidimensional evaluation of the pathway 9 months after its implementation, in order to i) compare benefits and disadvantages with conventional JA, ii) identify subsequent challenges and room for improvement, iii) offer a potential comparison with similar practices worldwide.An HTA is conducted considering 938 hip and knee JAs performed within the ERAS pathway from March to October 2018. The assessment is based on a hospital version  of the EQualitative, quantitative, or mixed assessments of the technology are performed according to the type of evidence investigated in the single domain. Qualitative assessments of the ERAS pathway are performed through the administration of a 7-item Likert scale validated questionnaire  to the 3Quantitative assessment is performed by gathering comparable data from 1) internal and/or national registries, such as length of hospital stay (LOS) in acute ward or in rehabilitation setting, and 2) economic analysis tools recommended by the literature. Any comment on the results is postponed to the Focusing on statistical analysis, data were analysed considering descriptive statistics, frequencies, and distributions. After having tested the normal distribution of all the variables investigated (− 1 < Skewness< 1 and − 1 < Kurtosis< 1), independent sample t-tests were used to describe the existence of statistically significant differences between the presence or the absence of ERAS pathway, within the qualitative perceptions.All analyses were conducted with a significance level of 0.05. Results are expressed as average value ± standard error.A healthcare technology is relevant when comparable with another existing procedure to treat a significant epidemiological need. JA is considered the best solution against osteoarthritis and rheumatoid arthritis when conservative treatment is not effective, with increasing worldwide patients undergoing surgery primarily due to aging, overweight and trauma , 16.In Italy, a dedicated National Register  was introduced in 2006 in order to collect epidemiological and demographic information associated with patient characteristics from the entire country. According to the last report , joint aA high variety in patient characteristics, surgeon preferences, logistical arrangements, and regional healthcare financing makes it difficult to compare the approaches adopted by different providers, both in Italy and other European countries . MoreoveThe contents of conventional treatment and ERAS pathway as currently provided by Galeazzi Hospital are reported in Table Faster recovery must not preclude safe recovery. Up to 52% of reduced hospital LOS was obtained without a significant increase in readmissions –27, exceThe 938 procedures under assessment included hip and knee, primary and revision, unilateral and bilateral, total and unicompartmental, simultaneous and staged JAs, offering an exhaustive sample of surgical procedures. Eight different surgical teams accepted to adopt the ERAS pathway for all the patients undergoing hip or knee arthroplasty in their unit, without selecting for patient characteristics or type of surgery. Follow-up visits are generally performed after 6 weeks, 3 months, 6 months and 1 year, depending on the surgeon’s decision, the evolution of recovery and patient characteristics. Since all the procedures were performed between March and October 2018, at the moment of writing it is impossible to collect data on long-run complications, readmissions, and patient experiences of pain.The lack of comparative data required preliminary estimations of safety to be carried out by means of a qualitative questionnaire, completed by the 27 healthcare professionals currently employed in the ERAS pathway. According to the EUnetHTA Core Model, they evaluated 9 safety indicators which were largely retrieved from the before-mentioned literature review Fig. .Fig. 1SERAS pathway dominates conventional treatment on patient safety, reporting a slight improvement in the mean rate (0.29 ± 0.011 vs 0.07 ± 0.006). General security, tolerability, pain, and risk of readmission are the items in which the technology dominates with a difference of more than 0.50 units, calculated by the mean preferences expressed by the professionals through the 7-item Likert Scale. On the contrary, the technology seems not to be equally safe in terms of errors within the procedure, which may trigger risks to the healthcare providers and caregivers which do not comply with a certain innovation introduced within the pathway. However, the mean difference perceived in comparison with the conventional procedure is under 0.50 units.Efficacy is different from effectiveness. Efficacy is retrieved from the literature as a parameter of how a certain treatment performs in different settings and/or different patients. Effectiveness is a real-world indicator of how the same treatment performs in a specific setting on specific patients. Then, the efficacy of a certain intervention (“can it work?”) is a preliminary condition to test the effectiveness of the same intervention under usual clinical circumstances (“does it work in practice”?) , 39.A number of studies support the efficacy of accelerated pathways. A retrospective comparative study between conventional and accelerated total knee revisions (TKRs) resulted in faster postoperative recovery for those patients who underwent the latter . AccordiAccelerated perioperative care and rehabilitation after hip and knee arthroplasty demonstrated good results under different settings and circumstances , which pSpecific clinical and logistical indicators to assess the effectiveness of accelerated pathways have been identified in maximum 3 days of acute ward LOS, improved patient autonomy and function, positive PROMS, and survivorship after surgery , 38.Comparing the average patient LOS in Galeazzi acute and rehabilitation wards  with the average LOS in Italian hospitals , to perform a hip or knee arthroplasty Table .Administering a qualitative questionnaire on 5 indicators of effectiveness  retrieveReporting the number of postoperative deaths based on legal complaints.In the absence of long-term data on patients who underwent surgery in the ERAS pathway, a preliminary estimation of the effectiveness of the technology was performed in three steps.The average LOS associated with the ERAS pathway is reduced in comparison to Italian hospitals. Patients undergoing surgery within the pathway are slightly younger and more likely to be males Fig. .Fig. 2EAccording to the qualitative survey, ERAS dominates the conventional treatment in each of the five indicators, maintaining a difference of more than 0.5 units of preference (1.90 ± 0.076 vs 0.60 ± 0.095). No patients died during hospitalization or after discharge.Value is given by healthcare outcomes per dollar spent . AccelerAn Activity Based Costing (ABC) , in ordeA Cost-Effective Analysis (CEA), by means of an Incremental Cost-Effectiveness Ratio (ICER), in order to define the cost-effectiveness ratios generated by conventional treatment and ERAS technology, based on the hospital internal data .A Budget Impact Analysis (BIA), calculated from the number of patients treated within the ERAS pathway, multiplied by the cost of the average process, assuming the hospital perspective , and conThe economic and financial impact of the ERAS pathway was evaluated by adopting the following approaches –48.An AAll costs are expressed in Euros.The cost of the technology is calculated by the sum of the human and technical resources employed along the process, assuming the hospital perspective Table .Table 4Assuming the hospital perspective, the ERAS pathway costs € 34,19 more than the conventional pathway, where the human resources employed in the preoperative phase represent the main difference.Qualitative indicators of effectiveness do not allow rigorous comparisons between similar pathways, and postoperative survivorship is a short-term indicator of effectiveness. Then, an evidence-based comparison between ERAS pathway and conventional treatment can be performed by adopting hospital LOS as a preliminary indicator of the latter Table .Table 5The CEA resulted in −56.98 ICER score, which underlines the greater effectiveness of the ERAS pathway against a moderate economic investment. According to the above, considering an average economic value of a single hospitalization day in Orthopaedic surgery equal to € 218.14, the reduction in LOS will be translated in an economic saving per patient equal to € 130.88 for the hospitalization in Orthopaedic surgery.Guaranteed the effectiveness and safety of each treatment, the ERAS pathway is associated with reduced patient LOS and increased discharge directly at home. Considered the average reimbursement of € 2244.77 per patient rehabilitation charged by the hospital to the SSN, the technology under assessment generates significant savings in terms of public healthcare expenditure.Table the cost of admission to orthopaedic surgery ;the cost of the surgical activity ;the cost of the rehabilitation activities.Assuming the public funder perspective, the analysis shows how the introduction of the new technology saved € 688,738.93 to the SSN in less than 1 year, following from reduced LOS and no need for inpatient rehabilitation. However, this evaluation is performed by comparing 12 months of conventional treatment against 9 months of accelerated pathway. In order to uniform the evaluation, consider the scenario in which the ERAS pathway entirely replaced conventional treatment, 1) assuming the same number of patients (1271) from 1 year to the next; 2) including in detail the following items of healthcare expenditure  in comparison to the baseline scenario. Thus, the ERAS accelerated pathway is more affordable than the conventional treatment.The dimension of equity can be distinguished on a macro and a micro level , 50.On the macro level, equity is a matter of allocative justice. Achieving the same outcomes with fewer resources  allows the public funder to free up financial resources which can be reinvested in treating other morbidities, or a higher number of arthritic patients, with a potential reduction in hospital waiting lists, providing better access to free and quality health care . In this sense, the economic and financial assessments support equity of the ERAS pathway on a macro level.On the micro level, equity is a matter of healthcare accessibility to vulnerable categories of patients. Caution must be kept in case of patients who find it difficult to bear early mobilization and intensive rehabilitation, be them psychiatric, old, frail, or affected by other clinical morbidities (i.e. chronic) or conditions of risk. Despite accelerated pathways show improved accessibility to protected categories of patients , 51, 52,A qualitative investigation of the equity domain is conducted through 9 indicators retrieved from EUnetHTA Core Model  accessi.Fig. 3EThe conventional treatment  seemed slightly preferable than the ERAS pathway  when analysed in light of the equity domain.The impact of the ERAS pathway from the perspective of society is here declined in terms of i) the human and economic burden removed from the patient and/or his/her family or caregivers, ii) the estimated productivity loss caused by the attendance of the healthcare environment, iii) the need to support the spread and safety of the technology with adequate legal procedures and market regulation.Points i) and iii) had been evaluated by the administration of a 7-item Likert Scale qualitative questionnaire to the healthcare professionals answering the questionnaire Figs.  and 5; pERAS pathway dominated the conventional treatment  in light of the burden removed by the technology, with the exception of two indicators: economic accessibility to the patient (ability to pay), and social determinants of compliance and technology understanding .The perceived reduction of time spent by the patient in the healthcare environment Fig.  is confiMultiplying the average days spent outside the healthcare environment by the number of patients who underwent surgery under the ERAS pathway, in 9 months, the technology saved 4115 days of hospital attendance in comparison to the conventional treatment.Figure The percentage of professionals (right column) agreeing or not to perform a certain investment (left column), including potential specific suggestions Table ;The cost of 30-h specific training to all the professionals who employ the technology Table .The sustainability of innovation is given not only from a profitable ratio between costs and benefits, but also from the amount of investments required to replace the previous technology. The potential investments required to implement the ERAS pathway are here described in terms of:The benefits of telemedicine , permaneIntroducing a 30-h training activity would require to the hospital an additional investment of € 34,480.50. Considering the € 2054,123.44 estimated savings generated by the introduction of the ERAS pathway in 1 year, the economic investment in training activities should be largely outweighed by the economic benefits induced by the technology.JA procedures are increasingly performed to treat hip and knee arthritis each year, supported by a number of international studies designed to improve their clinical tolerability and financial sustainability. The ERAS pathway introduced in Galeazzi Hospital, which aims to improve the treatment by investing on preoperative planning and perioperative integration of the single medical and surgical performance, can therefore be considered relevant.According to the perception of the professionals involved in the ERAS pathway, the new technology dominates the conventional treatment in terms of general clinical security, tolerability by the patient, reduced pain, and risk of adverse events or readmissions. Improved scores could be explained by the multidisciplinary commitment required by the technology, better communication among the professionals, progress in pain management, and accurate evaluation of the single patient , 60, 61.Increasing literature shows accelerated pathways to dominate more conventional approaches in terms of reduced pain scores, earlier functional recovery, improved quality of life, and better PROMs. This dimension was not supported by a systematic review of the evidence. However, the primary interest of the manuscript is to perform a validated HTA on a specific hospital-based preliminary experience, then the dimension of effectiveness is expected to provide more valuable and substantial information - in comparison to the dimension of efficacy - to share with the clinical and academic community.The technology reduced LOS in comparison to the conventional treatment performed in Italian hospitals. Improved clinical and functional outcomes were reported by the professionals employed within the pathway. The limited time available for data collection and the subsequent impossibility to include PROMs suggest caution in the interpretation of these results. However, longer-lasting clinical studies based on similar outcomes, PROMs included, from 90 days to 10 years after surgery, support positive expectations towards the technology , 62–64. The ABC analysis has estimated that the ERAS pathway costs to the hospital € 34.19 more than the previous, conventional treatment. The incremented cost is mainly due to more investments in human resources at the preoperative phase, in order to provide patient assessment and education. Despite the benefits of pre-operative education for hip and knee replacement are controversial , 66, curA moderate additional investment at the preoperative phase is outweighed by a reduction in hospital LOS, which - once the safety of the procedures is maintained - reduces in turn the time spent by the patient in the healthcare environment, generates significant cost savings to the funder, and potentially reduce waiting lists in order to provide a higher number of treatments. The cost-effectiveness of the ERAS pathway is therefore supported by a CEA. It may be argued that reduced hospital LOS is a poor indicator of the effectiveness of a medical treatment, for increased risk of readmissions and increased cost-shifting to the patient may follow. With regard to cost-shifting, ERAS pathway outpatient rehabilitation is provided by Galeazzi Hospital and funded by SSN, without any additional cost to the patient. With regard to readmissions, the lack of long-term data about patient recovery is actually a limit of the present assessment; however, waiting for those data to be available, evidence from longer-lasting studies and positive impressions by the involved professionals support the cost-effectiveness of the ERAS pathway.Assuming the hospital perspective, projections of one-year implementation of the ERAS pathway on the same number of patients treated in 2017 under the conventional pathway (as if the technology entirely replaced the previous treatment), generated an estimated saving of € 2054,123.44 per year. Based on these analytical tools, the ERAS pathway is expected to generate positive economic and financial impact. Although the estimations are based on projections on the same number of months and patients, they offer a perspective of the significant economic benefits that the systematic adoption of the ERAS pathway should generate on the long term.According to the professionals involved in the ERAS pathway, the conventional treatment dominates the new technology on 6 out of 9 indicators. Lower outcomes are reported in relation to the accessibility of the technology in the area, the accessibility to post-discharge facilities, and the possibility to choose post-discharge facility together with patient. These results are probably due to the poor spread of the technology outside the hospital: if outpatient rehabilitation requires a specific approach which is compatible with an accelerated pathway, at least in terms of perioperative coordination among professionals, patients who live far from the hospital will find it difficult to obtain adequate support, even more if they are elderly, disabled or frail , 49, 50.The functional impairment caused by joint pain is a human and economic burden, since quality of life, autonomy and productivity are critically threatened. JA is considered the best solution against osteoarthritis and rheumatoid arthritis when a conservative treatment is not effective. Accelerated pathways present the additional advantage to reduce the time spent within the healthcare environment. However, the new technology seems to present some issues in terms of economic accessibility to the patient and social determinants of compliance. The problem of economic accessibility may occur to patients who need to undergo outpatient rehabilitation compatible with the ERAS pathway. Since such a specific approach may not be provided or funded by the SSN throughout the entire country, the patients who live far from Galeazzi Hospital are likely to pay the service out-of-pocket. The inability of a patient to access equal healthcare based on the area of the country in which he/she was born, or lives, is known in the literature as post-code lottery . SimilarWith regard to the social determinants of compliance, patients respond to clinical instructions when they are able to understand them, and believe that they will be helpful in solving their problems . MultidiWith regard to the subdomain of productivity loss, the professionals perceive the ERAS pathway to reduce the time spent in the healthcare environment, as a consequence of reduced hospital LOS and increased discharge directly at home. With regard to the subdomain of legal procedures and regulation, both the conventional treatment and the ERAS pathway need improvements in terms of care providers’ compliance to guidelines and patients’ data protection. The spread of the new technology, supported by a permanent audit, a multidisciplinary commitment between professionals, clinical feedback, and the share of best practice among researchers, are likely to support this need , 46, 48.The economic resources invested in support of professional training activities were estimated to be largely outweighed by benefits once the technology is permanently introduced. Most of the expenditure is likely to occur during the phase of implementation. The more cooperation between the professionals involved in the pathway, hospital governance and administration, the better identification of the organizational issues to be addressed, included the enrollment of dedicated staff. Therefore, the organizational impact of the ERAS pathway is probably sustainable and cost-effective in the long-term.This HTA involved the commitment of a researcher (not on the payroll) and the support from a few administrative employees  to access to the data. Therefore, it did not require any additional cost to the hospital. Conversely, clinical, organizational and economic benefits are expected from the improvement of the technology.The aim of an HTA is to choose the technology associated to the best “added value” among different alternatives. Value can be conceived in several ways, which include different outcomes, various time ranges and several perspectives. The ERAS pathway under assessment meets most of the considered outcomes and presents considerable room for further improvement. The more experiences are shared and compared, the before this room for improvement can be addressed.


Objective: The technology, network pharmacology and molecular docking technology of the ultra performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (UPLC-Q-TOF-MS/MS) were used to explore the potential molecular mechanism of Platycodon grandiflorum (PG) in the treatment of lung cancer (LC).Methods: UPLC-Q-TOF-MS/MS technology was used to analyze the ingredients of PG and the potential LC targets were obtained from the Traditional Chinese Medicine Systems Pharmacology database, and the Analysis Platform (TCMSP), GeneCards and other databases. The interaction network of the drug-disease targets was constructed with the additional use of STRING 11.0. The pathway enrichment analysis was carried out using Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) in Metascape, and then the “Drug-Ingredients-Targets-Pathways-Disease” (D-I-T-P-D) network was constructed using Cytoscape v3.7.1. Finally, the Discovery Studio 2016 (DS) software was used to evaluate the molecular docking.Results: Forty-seven compounds in PG, including triterpenoid saponins, steroidal saponins and flavonoids, were identified and nine main bioactive components including platycodin D were screened. According to the method of data mining, 545 potential drug targets and 2,664 disease-related targets were collected. The results of topological analysis revealed 20 core targets including caspase 3 (CASP3) and prostaglandin-endoperoxide synthase 2 (PTGS2) suggesting that the potential signaling pathway potentially involved in the treatment of LC included MAPK signaling pathway and P13K-AKT signaling pathway. The results of molecular docking proved that the bound of the ingredients with potential key targets was excellent.Conclusion: The results in this study provided a novel insight in the exploration of the mechanism of action of PG against LC. Lung cancer (LC) is a malignant tumor with the highest morbidity and mortality worldwide, mostly in male than in female, indeed known as “the king of cancer” . At presPlatycodon grandiflorum (PG) is a plant belonging to the family of campanulaceae, and the dried root is used in TCM to regulate the lung meridian. PG exerts the effect of smoothing lung, dispelling the phlegm, and expelling the pus, and represents the main treatment to cure sore throat, vomiting due to a purulent carbuncle infection in the lung, hypochondriac pain in the chest and other syndromes  A. DC. Campanulaceae from Professor Fu Xiaomei. PG decoction pieces were processed by Jiangxi Jiangzhong TCM Decoction Co., Ltd. according to the processing method of the Chinese Pharmacopoeia 2015 edition. Next, dried PG pieces were crushed into a 40 mesh powder and stored in the laboratory of the Jiangxi University of TCM.Methanol, acetonitrile, and formic acid used for high performance liquid chromatography (HPLC) were purchased from ACS . Methanol for herb extraction was purchased from Xilong Scientific Co., Ltd . Ultrapure water was obtained from a Milli-QB system . PG root pieces (simply defined as PG pieces according to the Chinese Pharmacopoeia 2015 edition that assumes that PG is the root) were purchased from Jiangxi Jiangzhong Herbal Pieces Co., Ltd . The original PG medicinal material was purchased from Yiyuan, Shandong province, and was identified as the dried root of A total of 15 pure compounds were used as reference standards (purity≥98%). Among them, deapio-platycodin D, platycodin D, polygonatoside C1, rubinin, luteolin, kaempferol, apigenin, caffeic acid, ferulic acid, adenosine, and lobetyolin were purchased from Chengdu Chroma-Biotechnology Co., Ltd . 3-O-β-D-glucopyranosyl platycodigenin, rutin, chlorogenic acid, and linoleic acid were purchased from Sichuan Vicky Biotechnology Co., Ltd .Ten milligrams of each reference compound  were weighed and transferred into a 10-ml volumetric flask. Methanol was added to reach the volumetric mark, and the solution was shaken well, stored at 4 °C and used as a stock solution. Then, the appropriate amount of stock solution was transferred into a 5 ml volumetric flask, and methanol was added to reach the volumetric mark. The solutions were filtered using a 0.22 μm microporous membrane to obtain the standard solutions.A total of 2.0 g PG powder was accurately weighed and transferred into a round bottom flask with 50 ml 50% methanol. The solution was mixed well, incubated for 0.5 h at room temperature, and ultrasonically treated for 30 min using an ultrasonic cleaning instrument . The extracted solution was centrifuged at 14,000 rpm for 15 min at room temperature, and filtered using a 0.22 μm microporous membrane before qualitative analysis.+, AB Sciex, Forster City, CA, United States) with an electrospray ionization source (ESI). Acquity UPLC BEH C18 column (2.1 × 100 mm × 1.7 µm) was used to perform chromatographic separation with a flow rate of 0.25 ml/min at 40 °C. The linear gradient program used in this work was composed of a mobile phase system including solvent A  and solvent B  according to this detailed composition: solvent A at 5–23% for 10 min, 23–25% for 6 min, 25% for 4 min, 25–29% for 3 min, 29–95% for 7 min, 95–5% for 2.1 min, isocratic eluted at 5% for 2.9 min.The chemical analysis was performed on a connected UPLC system  -hybrid triple quadruple time-of-flight mass spectrometer (Triple TOF™ 5600The settings of Q-TOF-MS/MS parameters were as follows: ion source gas 1 (GSI) and gas 2 (GS2) were both set at 50 psi, curtain gas (CUR) was set at 40 psi, ion spray voltage floating (ISVF) was set at 5500 V in the positive mode while 4500 V was set in the negative mode, ion source temperature (TEM) was set at 500 °C, collision energy (CE) was set at 60 V, collision energy spread (CES) was set at 15 V, declustering potential (DP) was set at 100 V, and nitrogen was used as a nebulizer and auxiliary gas. Samples were analyzed in both positive and negative ionization modes with a scanning mas-to-charge (m/z) range from 100 to 1,250. Data were collected in the information-dependent acquisition (IDA) mode and analyzed by PeakView®1.2 software .https://scifinder.cas.org), the Traditional Chinese Medicine Systems Pharmacology database, and the Analysis Platform  database. Then, a PG ingredients database was established, containing the basic information, such as ingredient name and molecular formula. A total of 161 ingredients in PG were collected, and the specific information is listed in the ® 1.2 to perform the ingredient analysis. Chemicals were identified according to the reference standards, chromatographic elution behavior, mass fragment pattern, and mass spectral library .The chemical PG ingredients were collected from existing databases, such as SciFinder (http://www.swisstargetprediction.ch/) (http://www.lilab-ecust.cn/pharmmapper/) (https://pubchem.ncbi.nlm.nih.gov/) (http://lsp.nwu.edu.cn/tcmsp.php) (http://www.uniprot.org/), and the library of the targets of the active components was obtained. The known disease targets were searched in the databases of GeneCards (https://www.genecards.org/) (http://www.disgenet.org/) (The prediction targets of the active PG components were obtained using the following databases: Swiss Target Prediction (ion.ch/) , Pharmmamapper/) , Pubchemih.gov/)  and TCMSmsp.php) . The prods.org/)  and DisGet.org/)  using “Lhttps://string-db.org/). The protein type was set to “Homo sapiens” and the minimum interaction score was 0.4. The PPI network diagram was obtained, imported into Cytoscape v3.7.1 software, and CentiScape was used to calculate the degree centrality (DC) to filter PPI network core targets.R software was used to intersect PG related targets and LC related targets, and the overlapping targets were uploaded into STRING 11.0 (http://www.metascape.org/) is a gene annotation tool that integrates many authoritative databases such as GO, KEGG, UniProt and DrugBank. It allows the completion of pathway enrichment analysis and biological process annotation and performs gene-related protein network analysis and drug analysis, providing comprehensive and detailed information regarding each gene  and PathwayBuilderTool_2.0 software to further explain the role of the core targets in gene function and signaling pathway.The gene symbols of the core targets were introduced into Metascape, “The files related to “drug-core ingredient,” “core ingredient-core target,” “pathway-core target,” and “disease-core target” were established, and the D-I-T-P-D network was constructed using Cytoscape v 3.7.1 to explain the multi-effect synergistic mechanism of PG.https://www.rcsb.org/). Then the molecular docking function of DS software is used to perform component-target molecular docking in the LibDock module.Discovery Studio 2016 software (DS) is a commonly used molecular modeling and simulation software widely used in the field of drug design and optimization, protein structure and function . The PDB®1.2, and the composition was screened by “XIC manager”. The structural formula of the target compound was matched with the secondary fragment ion, and the compounds were further identified according to the matching degree and the law of ion bond breaking. The compounds with a matching degree greater than 80% and in accordance with the law of bond breaking were considered. According to the reference  properties of the chemical constituents were predicted by DS, and apigenin, caffeic acid, kaempferol, linoleic acid, methyl linolenic acid, and ferulic acid were selected. Although some components do not meet the DS screening criteria, they are still considered as active components in order to select the active components of PG more comprehensively. For example, luteolin and robinin were obtained from TCMSP, with oral bioavailability greater than 30% and drug-likeness greater than 0.18 despite they did not meet the DS screening criteria, so they are considered as active ingredients. Recent studies showed that platycodin D is one of the main components of PG, and has a certain preventive and therapeutic effect on LC , thus plA total of 545 targets of the selected nine active components in PG were obtained using the Swiss Target Prediction, Pubchem, TCMSP and pharmmapper databases. A total of 2664 LC related targets were collected using GeneCards and DisGenet databases using “Lung cancer” as the keyword .The Venn diagram of drug-disease overlapping targets was obtained using the R software to intersect PG related targets and LC related targets , revealip< 0.01, minimum count 3, and enrichment factor>1.5. A total of 849 GO biological functions and 116 KEGG enrichment items were obtained. The GO functions related to the treatment of LC included oxidative stress response (GO:0006979), active regulation of cell migration (GO:0030335), regulation of DNA binding transcription factor activity (GO:0051090), and regulation of cytokine-mediated signaling pathway (GO:0019221)  , mostly 0019221) . The fir0019221) . This anIn order to explain the complex mechanism of multi-ingredient-multi-target-multi-pathway of PG in the treatment of LC, this study selected the top 10 pathways and used Ctyoscape v 3.7.1 to construct a D-I-T-P-D network, as shown in p < 0.01)  .The analysis of the D-I-T-P-D network showed that each core component interacted with multiple targets. Thus, the DS software was used to dock the core components with the core target molecules in order to verify the interaction among them. It is generally accepted that the higher the score of the ligand binding to the receptor, the greater the possibility of interaction. The docking results of luteolin and protein PTGS2 docking are shown in .The cluster analysis revealed that GAPDH, VEGFA, EGFR, JUN, CCND1, MAPK8, FOS and CASP3 could be included into one group, while EP300, AKT1, HSP90AA1, MAPK3, TNF, SIRT1, ESR1, PTGS2, TP53, FN1, MAPK1 and IL6 could be included in one category. SIRT1, PTGS2, CASP3, VEGFA, MAPK8 and GAPDH were the targets with strong binding ability according to the molecular docking. Linoleic acid, methyl linolenate, luteolin, apigenin and kaempferol were the core components with strong binding ability according to the molecular docking, and the docking scores of these five components with SIRT1 were greater than 120, followed by methyl linolenate, kaempferol, linoleic acid, luteolin and apigenin. This result suggested that these five components have a good binding activity with SIRT1 and might play a key role in the treatment of LC using PGThe etiology of LC is complex and so far, no final conclusion was obtained. It is related to smoking, air pollution, dietary factors, decreased immune function and genetic factors , while TIn this study, 47 non-volatile ingredients were identified from the alcohol extract of PG by the UPLC-Q-TOF-MS/MS technique. According to the ADMET parameters and other basic properties of the ingredients, and combined with the results in the literature, nine main active ingredients including platycodin D and apigenin were selected. The results showed that they play an important role by affecting 285 overlapping genes involved in the treatment of LC. The PPI network showed that the DC values of GAPDH, AKT1 and TP53 were greater than 175, and they were predicted as the most relevant targets. The enrichment analysis of the GO pathway and KEGG pathway in the 20 core targets showed that most of the functional enrichment results of GO were apoptosis, oxidative stress and energy metabolism. Our hypothesis was that oxidative stress response might be the most important biological process managed by PG in the treatment of LC. The results of the KEGG pathway enrichment analysis showed that the top 10 pathways included cancer pathway, TNF signaling pathway, MAPK signaling pathway, P13K-AKT signaling pathway and FoxO signaling pathway. Among them, MAPK signaling pathway, PI3K-AKT signaling pathway, FoxO signaling pathway and HIF-1 signaling pathway were related to cell proliferation and apoptosis, oxidative stress and inflammation. MAPK signaling pathway is one of the most important pathways regulated by PG in the treatment of LC. Related studies showed that the MAPK signaling pathway participates in the regulation of cell cycle, apoptosis and proliferation of NSCLC cells, and inhibits the expression of P-glycoprotein (P-gp), multidrug resistance gene 1 (MDR1), TP53 and other proteins, thus inhibiting the growth of NSCLC cells, blocking cell cycle and inducing apoptosis . PI3K-AKThe D-I-T-P-D network showed that the same target could interact with many ingredients. For example, PTGS2 could bind ferulic acid, luteolin, linoleic acid, apigenin and sophorin, while CASP3 can bind methyl linolenic acid, apigenin, ferulic acid and robinin, indicating that multiple active ingredients in PG might act on the same target. In addition, our results showed that apigenin was related to CASP3, AKT1, FOS, JUN, MAPK8, TNF, VEGFA, and CCND1, and linoleic acid was related to PTGS2, TP53, ESR1, MAPK1, MAPK8 and IL6, which was consistent with the results obtained by molecular docking, suggesting that PG could act on multiple targets through the same active component. This result also explained the characteristics of multi-ingredient and multi-target synergism of PG, providing a basis in the mechanism of PG to treat LC. Our hypothesis was that PG was closely related to cell proliferation and apoptosis in the treatment of LC according to the GO pathway and KEGG pathway enrichment analysis. Several core components of PG could interact with PTGS2 and CASP3, potentially promoting cancer cell apoptosis through specific signaling pathways such as MAPK signaling pathway and P13K-AKT signaling pathway in order to cure LC.The survival analysis results showed that the top five core targets  in the PPI network were closely related to the survival of LC patients. The overall survival of LC patients with a high expression of these genes was significantly lower than that of patients with their low expression, suggesting that its overexpression is related to a poor prognosis in LC patients. Thus, they could be used as biomarkers to evaluate LC prognosis.α (ERRα), thus providing a strong support in the study of the anti-cancer mechanism of kaempferol (The results of molecular docking showed that apigenin, luteolin, linoleic acid, kaempferol and methyl linolenate might be the potential active ingredients. Some studies showed that apigenin has a cytotoxic effect on human LC cisplatin-resistant cell line A549/DDP, and indeed it effectively inhibits its growth and reverse its drug resistance . Zhou Liempferol . Howeverempferol . Apigeniempferol  and peonempferol  also conPG is one of the most commonly used TCM in clinical practice, since it has a significant therapeutic effect on lung-related diseases and has been used in China for thousands of years. In this study, the chemical constituents of PG were analyzed and identified. A total of nine active PG components and 285 overlapping targets of PG and LC were screened. PPI network, GO and KEGG enrichment analysis showed that the mechanism of PG in the treatment of LC might be related to its involvement in cancer cell apoptosis, inflammation and oxidative stress through the MAPK signaling pathway and P13K-AKT signaling pathway. The network pharmacological method developed in this study provided another strategy for a comprehensive understanding of the mechanism of PG in the treatment of LC.


Biomarkers may contribute to our understanding of the pathophysiology of various diseases. Type 2 diabetes (T2D) and coronary heart disease (CHD) share many clinical and lifestyle risk factors and several biomarkers are associated with both diseases. The current analysis aims to assess the relevance of biomarkers combined to pathway groups for the development of T2D and CHD in the same cohort.2PM coefficient of determination to assess the explained disease risk.Forty-seven serum biomarkers were measured in the MONICA/KORA case-cohort study using clinical chemistry assays and ultrasensitive molecular counting technology. The T2D (CHD) analyses included 689 (568) incident cases and 1850 (2004) non-cases from three population-based surveys. At baseline, the study participants were 35–74 years old. The median follow-up was 14 years. We computed Cox regression models for each biomarker, adjusted for age, sex, and survey. Additionally, we assigned the biomarkers to 19 etiological pathways based on information from literature. One age-, sex-, and survey-controlled average variable was built for each pathway. We used the RThe associations of many biomarkers, such as several cytokines or the iron marker soluble transferrin receptor (sTfR), were similar in strength for T2D and CHD, but we also observed important differences. Lipoprotein (a) (Lp(a)) and N-terminal pro B-type natriuretic peptide (NT-proBNP) even demonstrated opposite effect directions. All pathway variables together explained 49% of the T2D risk and 21% of the CHD risk. The insulin-like growth factor binding protein 2  best explained the T2D risk . For CHD, the myocardial-injury- and lipid-related-pathways were most important and both explained about 4% of the CHD risk.The biomarker-derived pathway variables explained a higher proportion of the T2D risk compared to CHD. The ranking of the pathways differed between the two diseases, with the IGF/IGFBP-system-pathway being most strongly associated with T2D and the myocardial-injury- and lipid-related-pathways with CHD. Our results help to better understand the pathophysiology of the two diseases, with the ultimate goal of pointing out targets for lifestyle intervention and drug development to ideally prevent both T2D and CHD development. Type 2 diabetes (T2D) and coronary heart disease (CHD) are among the most common chronic diseases in Western industrialized countries and cardiovascular diseases (CVD) are the main cause of death . AlthougOur study examines the common soil hypothesis by investigation of biomarkers, which reflect diverse etiologic pathways. Serum and plasma biomarkers are increasingly used to elucidate pathophysiological changes leading to T2D and CHD. Biomarkers are objectively measurable and are not subject to recall bias. Numerous examples show the benefit of biomarkers for etiologic research , 11. HowThe current investigation was embedded within the Monitoring of Trends and Determinants in Cardiovascular Disease (MONICA) Augsburg study, which was initiated in the early 1980s by the World Health Organization to study risk factors of premature CVD in regionally defined communities by a standardized protocol. In previous analyses, several biomarkers have already been investigated with respect to their association with either T2D e.g. –16) or C or C16])The design and all procedures of this prospective case-cohort study within the population-based MONICA/KORA Augsburg cohort have been described in detail before . Brieflyn = 141 developed incident T2D) plus all 548 additional incident T2D cases.The incidence of T2D was assessed using a written follow-up questionnaire sent to all participants of the three baseline surveys in 1997/98, 2002/03, and 2008/09. Furthermore, all S1 participants were invited to a follow-up examination in 1987/88. Self-reported incident T2D status and the date of diagnosis were validated by a questionnaire mailed to the treating physician or medical chart review. Only participants for whom the treating physician clearly reported a diagnosis of T2D or for whom a diagnosis of T2D was mentioned in the medical records or who were taking antidiabetic medication were classified as cases. The mean follow-up time ± standard deviation (SD) was 12.0 ± 3.5 years, the median was 14.0 years. The T2D case-cohort study included only participants without prevalent diabetes at baseline. It comprised a randomly drawn subcohort of 1991 individuals (of whom th Revision (ICD 9): 410–414 and 798). Until December 2000, the diagnosis of a major, non-fatal MI and coronary death was based on the MONICA algorithm [n = 159 developed incident CHD) plus all 409 additional incident CHD cases.CHD was defined as non-fatal MI as well as coronary death and sudden death  for measurement of serum high density lipoprotein (HDL) cholesterol, total cholesterol, and uric acid. All other 44 biomarkers were measured from serum samples stored at − 80 °C.On average, 16% of the biomarker data were missing, with a large variability between the biomarkers D), 30%). In order to enable unbiased analyses and to use the available data most efficiently, we replaced missing biomarker values in each of the two case-cohort datasets, using 20-fold multiple imputation by chained equations (MICE) (R version 3.2.3 and R package MICE version 2.25) –29.Biomarker measurements with a right-skewed distribution were ln-transformed and all biomarkers were 0,1)-standardized. The measurement methods, coefficients of variation, proportions of missing values pre-imputation and decisions on ln-transformations are summarized in Additional file -standardThe biomarkers were selected with regard to their potential importance for either T2D or CHD pathophysiology based on prior knowledge from experimental and epidemiological studies. The current study used all available biomarker data from the two case-cohort studies for simultaneous analysis of both diseases in the form of a secondary data analysis. For this purpose, the biomarkers were grouped according to pathophysiological aspects; these biomarker groups are called ‘pathway variables’ in the following. Each biomarker was assigned to one pathway based on information from literature Fig. . This apDescriptive analyses of baseline characteristics were computed for cases and non-cases using the SAS procedure SURVEYMEANS . To account for the case-cohort design, weighting was performed using the survey- and sex-specific sampling weights. Medians and interquartile ranges were calculated as median of percentiles over multiple results of 20 imputations; proportions were calculated as arithmetic means over the 20 imputation results.Correlations between the age, sex, and survey adjusted biomarker residuals and between the pathway variables were investigated separately in the subcohorts of the T2D and CHD case-cohort studies by Pearson’s coefficients. Fisher’s Z-transformation and re-transformation was used to compute average correlation coefficients over the 20 imputed datasets , 31.To assess the relation of the -standardized biomarker variables with incident T2D and incident CHD, Cox proportional hazards regression models adjusted for age, sex, and survey were built. Regression coefficients were calculated using Barlow’s weighting method to account for the case-cohort design . Robust 2 determines the ratio of the outcome variability explained by the model to the total variability. The variability explained by the model was estimated separately in each imputed dataset and combined by calculating the arithmetic mean. In order to determine the T2D and CHD outcome variabilities explained by all pathway variables, p value < 0.05 were considered statistically significant.For calculation of the explained time-to-event outcome variability , the coefficient of determination  account , 36. In mentclass2pt{minimTable The unadjusted baseline values of all 47 biomarkers are shown in Additional file The age, sex, and survey adjusted associations of the single biomarkers with incident T2D and incident CHD, sorted according to the assigned pathways, are shown in Fig. Pearson’s correlations were lower than 0.8 between the single age, sex, and survey controlled biomarker variables  compared to CHD (21%). We also showed that the pathway variables that independently explained most of the disease risk differed between incident T2D and incident CHD. Moreover, the age, sex, and survey adjusted associations of some single biomarkers differed strongly for T2D and CHD, with Lp(a) and NT-proBNP even demonstrating opposite effect directions. However, the associations of many single biomarkers, such as several cytokines or the iron biomarker soluble transferrin receptor (sTfR), were quite similar for T2D and CHD.The IGF/IGFBP system pathway was most strongly associated with T2D development in the univariate analyses, followed by the adipose-derived hormones and the hormone regulation pathways. For CHD, the lipid related, the stress and antioxidants, and the myocardial injury pathways individually were most important. Nonetheless, the associations of some single biomarkers were similar for T2D and CHD and the lipid related pathway  and endothelial dysfunction pathway  both strongly explained the T2D as well as the CHD development when the pathways were individually investigated.LPA gene region [While the two biomarkers sE-selectin and sICAM-1 that represent the endothelial dysfunction pathway were positively associated with both T2D and CHD, more caution is required when interpreting the results from the lipid related pathway. Total cholesterol and sPLA2-IIA were both positively and HDL-cholesterol was inversely associated with incident T2D and CHD. Lp(a), however, was inversely associated with T2D and positively with CHD development. Accordingly, we inverted the age, sex, and survey adjusted Lp(a) residuals when we built the lipid related pathway variable for the T2D analysis, but not when we built this variable for the CHD analysis. This needs to be considered when interpreting the results. While it has been known for a long time that elevated Lp(a) increases the CHD risk, several prospective studies have shown more recently that the association with T2D is inverse –39. The e region  and lowee region . The rese region , 39. Sime region , 44, clie region . The effe region , 46, 47.In our investigation, not only Lp(a), but also NT-proBNP, a marker of vascular function and neurohumoral activity, showed an inverse association with incident T2D and a positive association with incident CHD, which is consistent to the literature –50.The analysis of single pathways revealed the strongest individual associations. However, these associations might partly be due to correlations to other pathway variables. We have therefore also investigated the independent contribution of each pathway by excluding one pathway variable at a time from a full model including all 19 pathway variables. None of the pathway variables independently and strongly explained both the development of T2D and CHD.For T2D, only the IGF/IGFBP pathway independently explained the development of the disease strongly. This pathway, which was represented by IGFBP-2, had already shown the most pronounced association in the univariate assessment; increased IGFBP-2 serum levels conferred a decreased T2D risk. In recent years, evidence has accumulated that insulin-like growth factor 1 (IGF-1) and at least part of its binding proteins (IGFBPs) play an important role in glucose homeostasis . The IGFThe independent contribution of all other pathways was substantially weaker in the T2D analysis. Interestingly, the liver marker pathway, which was represented by fetuin-A , ranked on the second last place in the univariate assessment but ranked second best in the analysis of the independent contribution. The reason was that the estimated proportion of explained risk was only slightly attenuated when the other pathway variables were included in the model (from 2.4 to 1.8% explained risk), because the liver marker pathway was only weakly correlated with other important pathways. In our study, higher levels of fetuin-A were associated with an increased risk of T2D development, which a recent meta-analysis has also convincingly illustrated . In thisConcerning CHD, the effect of the stress and antioxidants pathway seen in the univariate assessment was strongly attenuated by the inclusion of the other pathway variables in the model (from 7.0 to 0.6%). In contrast, the myocardial injury and lipid related pathways were both independently strongly related to CHD development. The myocardial injury pathway was represented by ultra-sensitive troponin I, a marker of myocardial injury. Troponin I is used to diagnose acute MI in clinical practice and has also been shown to be predictive for incident CHD in individuals of the general population without prior CHD , 61.So far, only few studies have compared to what extent biomarkers that reflect different pathophysiological processes can explain the development of T2D or CHD relative to each other and even less, if any, have directly compared the relevance for both diseases. In their study, Montonen et al. used an effect decomposition method to compare how well the four most strongly associated biomarkers from four selected pathways explain the development of T2D . Our appOur study has shown that some of the investigated biomarkers are associated with both T2D and CHD while some are only associated with one of the two diseases. In two instances, the effect direction even differed between the two studies. Moreover, the pathway variables that explained most of the disease risk differed between incident T2D and incident CHD. While there are a number of genetic studies that have examined comprehensively the common grounds of T2D and CHD , 64, we We would like to point out that no conclusions on causality may be drawn from observational studies like our own. Thoroughly conducted Mendelian Randomization studies and eventually randomized clinical trials are necessary to prove the causality of the observed relationships. However, observational biomarker studies may contribute importantly to the development of pathophysiological hypotheses .The main strength of our study is the prospective design, where all biomarkers were measured at baseline in non-diseased study participants. All cases developed during the long follow-up of 14 years. In order to minimize the risk of including T2D cases who were undiagnosed at baseline in the incident T2D analyses, all type 2 diabetic cases who were diagnosed during the first year after the baseline survey were excluded. A second strength is that we investigated many biomarkers from different metabolic pathways simultaneously. Noteworthy is also that we investigated T2D and CHD according to the same analysis approach in the same study population, which allows a direct comparison between the pathophysiological processes which play a role for these two frequent and highly related diseases. Another strength is the large sample size of our study, which includes more than 500 incident cases for each of the two investigated diseases. Last but not least, we developed a novel approach to investigate biomarker data from various pathways to get etiologic clues.There are also some limitations that should be considered. This study has re-examined available biomarker data in their totality. This means that the biomarkers were not necessarily selected for the reason that they are representative for a specific pathway. It also means that the pathway variables were defined based on different numbers of biomarkers, which we have statistically taken into account by calculation of an average biomarker variable per pathway. Nevertheless, metabolic pathways with more biomarkers tend to be more representatively covered than pathways with only one or two biomarkers. For example, inclusion of additional biomarkers from the IGF/IGFBP system pathway such as IGF-1, IGF-2, IGFBP-1, and IGFBP-3 would have been helpful to better estimate the contribution of this system to the T2D and CVD risk. Moreover, the assignment to the pathophysiologic pathways was ambiguous for some biomarkers, and several biomarkers might also have fitted to other pathway groups. We are also aware that the investigation of pathway variables is a simplifying approach. Therefore, we analyzed the data not only on the level of the pathway but also on the level of the single biomarkers. Finally, the opposite inclusion direction of some biomarkers in the formation of the pathway variables for T2D and CHD limits the comparability between the two diseases for these pathways. In order to avoid erroneous conclusions, these differences are described and discussed in the manuscript.In our study, the biomarker-derived pathway variables collectively explained a substantially larger proportion of the risk of T2D compared to that of CHD. The ranking of the pathway variables differed between the two diseases: The IGF/IGFBP system pathway was by far most strongly associated with T2D . For CHD, the myocardial injury and lipid related pathways were most strongly associated and both independently explained about 4% of the CHD risk. Our analysis of the single biomarkers showed that the age, sex, and survey adjusted associations of many biomarkers were similar in strength for T2D and CHD, but there were also important differences. Lipoprotein (a) and NT-proBNP even demonstrated opposite effect directions. Our study thus adds to the evidence that there exists a common soil for both diseases, but also clearly shows that the common soil hypothesis has its limits. Our results help to better understand the pathophysiology of the two diseases, with the ultimate goal of pointing out targets for lifestyle intervention and drug development to ideally prevent both T2D and CHD development.Additional file 1: Text S1. Comments on analytical approach. Table S1. Measurement methods, coefficients of variation (CV), missing values pre-imputation in the T2D and CHD case-cohort studies, and decisions on ln-transformations. Table S2. Baseline biomarker values of cases and non-cases in the T2D and CHD case-cohort studies. Table S3. Age, sex, and survey adjusted hazard ratios (HR) with 95% confidence intervals per standard deviation (SD) increase in biomarker concentration for incident T2D and incident CHD. Figure S1. Flowchart showing sample sizes and reasons for exclusions. Figure S2. Pearson’s correlations between the pathway variables, calculated in the subcohort of the T2D case-cohort study. Figure S3. Pearson’s correlations between the pathway variables, calculated in the subcohort of the CHD case-cohort study.Additional file 2: Figure S4 (a). Pearson’s correlation coefficients r between the age, sex, and survey adjusted biomarker residuals, calculated in the subcohort of the T2D case-cohort study. Figure S4 (b). Pearson’s correlation coefficients r between the age, sex, and survey adjusted biomarker residuals, calculated in the subcohort of the CHD case-cohort study.


Hemerocallis fulva L. is a common Traditional Chinese Medicine (TCM). Its flower buds are known to have ability to clear away heat and dampness, detoxify, and relieve depression. Ancient TCM literature shows that its roots have a beneficial effect in calming the spirit and even the temper in order to reduce the feeling of melancholy. Therefore, it is inferred that the root of Hemerocallis fulva L. can be used as a therapeutic medicine for depression. This study aims to uncover the pharmacological mechanism of the antidepressant effect of Hemerocallis Radix (HR) through network pharmacology method. During the analysis, 11 active components were obtained and screened using ADME—absorption, distribution, metabolism, and excretion— method. Furthermore, 267 HR targets and 740 depressive disorder (DD) targets were gathered from various databases. Then protein–protein interaction (PPI) network of HR and DD targets were constructed and cluster analysis was applied to further explore the connection between the targets. In addition, gene ontology (GO) enrichment and pathway analysis was applied to further verify that the biological process related to the target protein is associated with the occurrence of depression disorder. In conclusion, the most important bioactive components—anthraquinone, kaempferol, and vanillic acid—can alleviate depression symptoms by regulating MAOA, MAOB, and ESR1. The proposed network pharmacology strategy provides an integrating method to explore the therapeutic mechanism of multi-component drugs on a systematic level.The global depression population is showing a significant increase.  The burden of depression and other mental health conditions is on the rise globally. Depression alone accounts for 4.3% of the global burden of disease with more than 300 million people affected. People with mental health disorders often have higher rates of mortality, 40% to 60% greater than the general population. Therefore, the World Health Organization (WHO) ranked depression as the second most important disease to be considered in 2020 .Mental disorders often affect, and are affected by, other diseases. If worse comes to worst, suicide is the case. Depression and suicide are significant public health concerns, with over 40,000 Americans dying by suicide each year . Beyond Guidelines on the duration of antidepressant prescriptions differ in detail, but in general, about 6 to 12 months as WHO and NICE  recommended. However, patients with long-term antidepressant treatment have trade-offs in adherence that might lead to discontinuation of these drugs. Taiwanese, especially elders, has a special place in their heart for Traditional Chinese Medicine (TCM). Many of us believe that TCM has benefits toward human health. In addition, more than 6.4 million people went to a Chinese medication clinic, 3% of them have been diagnosed with mental or behavioral disorders [Hemerocallis fulva L.—Orange daylily or Nepenthe—is a common TCM. A species of perennial flowering daylily in genus Hemerocallis . It is native to Himalaya, East Europe, China, Japan, and Korea [Hemerocallis fulva are considered to have sweet, cool, and non-toxic properties and to be associated with the spleen, liver, and bladder meridians [Hemerocallis Radix (HR)—the root of Hemerocallis fulva L.—can be used as a therapeutic medicine for depression or as an auxiliary medication.nd Korea ,6,7,8, aeridians . Its floeridians ,11. TherSo far, several analytical methods, such as HPLC and LC-MS/MS has been reported to evaluate the effective compounds . NeverthBased on the network pharmacology model we constructed, the therapeutic mechanisms of HR toward DD were clarified. HR components were collected from databases. Next, numerous ADME—absorption, distribution, metabolism, and excretion—methods were used to screen for potential active components. Then the obtained data were used for gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. All PPI networks will be further studied through cluster analysis. Finally, Component-Target-Pathway network is calculated and analyzed by the network visualization tool Cystoscape. Although every TCM herb contains multiple components, only a couple of them lives up to the standard of pharmacokinetic property. In this study, four ADME-related models were used to screen out the active components in HR, including oral bioavailability (OB), Caco-2 permeability (Caco-2), drug-likeness (DL), and GI absorption. Beyond that, all screen-out components should follow Lipinski’s rule of five. After this process, a few components that did not meet the ADME criteria were added back into the database because of their high bioactive or curative effect proved in previous studies. Therefore, a total of 28 components were collected through databases. All identified components were subjected to ADME screening, and 11 of the 28 passed the ADME criteria. That is to say, these are the active component of HR. The detail information is shown in According to the pharmacokinetic evaluation of HR, aloe-emodin, α-boswellic acid, anthraquinone, β-boswellic acid, chrysophanol, colchicine, hemerocallone, kaempferol, puerarin, rhein, and vanillic acid were selected to be the active components of this herb, which were chosen in the following network pharmacology investigation. Thus, a network pharmacology approach was established to uncover the treatment mechanism of depression.Three databases were engaged to assemble component-related targets. Total of 357 identified targets were collected through PubChem. 178 remain after deleting those overlapping targets. Among these components, chrysophanol, kaempferol, and aloe-emodin have the highest number of targets, where α-Boswellic acid and Hemerocallone have no identified target at all. For predicted targets, two databases were put into use. Total of 22 targets were predicted by HitPick under the condition of precision ≥ 50%. Twenty remains after deleting those overlapping the targets with colchicine having the highest number of targets, and hemerocallone has 0 predicted target. A total of 138 targets were predicted by SEA under the condition of MaxTc ≥ 0.5 which stands for the maximum tanimoto similarity between compounds. Total of 92 remain after deleting those overlapping the targets. Among these components, vanillic acid, kaempferol, and anthraquinone have the highest number of targets, whereas β-Boswellic acid, α-Boswellic acid, and Hemerocallone have less number of predicted targets.To explore the therapeutic mechanism of HR in the treatment of DD, 267 targets and 11 components were used to construct the component-target (C-T) network . All of http://www.cytoscape.org/) [Based on the result from gene database DisGeNET, there were a total of 740 candidate targets relevant to depressive disorder. Gene IDs of these targets were input into String, species were limited to “Homo sapiens,” and confidence score was set at 0.7. Then, it was imported into Cytoscape3.7.1 (pe.org/)  to constThere are 709 nodes and 5506 edges in the DD target PPI network . The cloSeven clusters were found after DD target network was analyzed through MCODE (K-core = 5). This demonstrates that these clusters may be the most relevant to DD in studies at present. The details are described in Cluster 1 contains 63 nodes and 1042 edges with a score of 33.613. The seed node of this cluster is PDYN  which compete with and mimic the effects of opiate drugs. It plays a role in a number of physiologic functions, including pain perception and responses to stress. PDYN is also involved in the regulation of chemical synaptic transmission and neuropeptide signaling pathway.Cluster 2 contains 25 nodes and 200 edges with a score of 16.667. The seed node of this cluster is HTR4 (5-hydroxytryptamine receptor 4) which is one of the several different receptors for serotonin, a biogenic hormone that functions as a neurotransmitter, a hormone, and a mitogen. The activity of this receptor is mediated by G proteins that stimulate adenylate cyclase. It is involved in the regulation of chemical synaptic transmission, G protein-coupled receptor signaling pathway, and the regulation of appetite [Cluster 3 contains 35 nodes and 187 edges with a score of 11. The seed node of this cluster is IL6 (Interleukin 6) which is a potent inducer of the acute phase response. It plays an essential role in the final differentiation of B-cells into Ig-secreting cells and induces nerve cell differentiation. IL6 is also involved in the regulation of neuroinflammatory response and negative regulation of neurogenesis. With meta-analysis results and many individual studies approved, depression is associated with the activation of the immune system, including increased expression of proinflammatory cytokines such as TNF-α and IL-6 . Cluster 4 contains 38 nodes and 147 edges with a score of 7.946. The seed node of this cluster is NR1D1 (Nuclear receptor subfamily 1 group D member 1) which is related to metabolic, inflammatory and cardiovascular processes. It can also increase the hepatic expression of CYP7A1 via repression of NR0B2 and NFIL3 which are negative regulators of CYP7A1. Cluster 5 contains 11 nodes and 37 edges with a score of 7.4. The seed node of this cluster is PMS1 (PMS1 protein homolog 1). This protein is thought to be involved in the repair of DNA mismatches and the gene ontology (GO) annotations related to this gene include ATPase activity and mismatched DNA binding .Cluster 6 contains 26 nodes and 84 edges with a score of 6.72. The seed node of this cluster is MAPK8 (Mitogen-activated protein kinase 8). Diseases associated with MAPK8 include fatty liver disease and renal fibrosis and the GO annotations related to this gene include transferase activity, transferring phosphorus-containing groups, and protein tyrosine kinase activity. Cluster 7 contains 37 nodes and 89 edges with a score of 4.944. There is no seed node presented in this cluster.After the intersection process, we found there is an overlap between HR and DD targets. As shown in Three clusters were obtained after conducting clustering analysis for HR-DD PPI network (K-core = 2). The details are described in p-value cutoff set at 0.05 and the p-value were adjusted using the method introduced by Benjamini and Hochberg [The major targets could be categorized into various functional modules by Gene Ontology enrichment analysis. GO enrichment analysis based on the topGO package —availablHochberg . It contHochberg ,23,24, sThe GO enrichment analysis of the aforementioned network showed that total of 390 GO entries were obtained and top20 of each category were selected. Biology process (BP) is shown in p-value cutoff = 0.05) were screened and the top10 pathways were selected using Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis  pathway exhibits the highest number of target connections (degree = 12), which includes consequential target genes such as MAOA, GRIN2A, GRIN2B, and CALM1. The results show that HR can down-regulate MAOA, MAOB, and simultaneously up-regulate CALM1 . MAOA an2+) is one of the most significant intracellular messengers; the appropriate concentration of Ca2+ is necessary for neuronal excitability. When the Ca2+ concentration increases, Ca2+, calmodulin (CaM), and CaM kinase IIα (CaM KIIα) combine together to form the Ca2+-CaM-CaM KIIα signaling pathway, which is important in the plasticity of the central nervous system, learning and memory, mind, behavior, and other types of cognitive activities. In the previous Additionally, calcium  plays an important role in the regulation in neurotransmission and calmodulin-related gene expression is altered in lateral habenula and frontal cortex of Major depressive disorder patients . TherefoThe details of other related pathways are described in TCM might simultaneously target multiple physiological processes to arouse the human body’s potentiality to recover from unhealthiness. Unfortunately, the potential targets of Chinese herbs are difficult to be identified. The research strategy of network pharmacology provides a unique and innovative path for the study of TCM, including the mechanism of action of multi-component and multi-target. In the present study, we employed network pharmacology approaches to find potential bioactive ingredients from the herb HR associated with the important DD-related targets. By combining the advantages of data mining, machine learning, and neural network together, we predicted that multiple active components would target several proteins related to depressive disorder. Through the aforementioned analysis—pharmacokinetic analysis, C-T network, PPI network, GO enrichment and pathway analysis, T-P network, and cluster analysis—we found that the herb HR contained important candidate bioactive components for depression disorder treatment. To more intuitively determine the relation among component, target, and pathway, Hemerocallis Radix has a beneficial effect in calming the spirit and even the temper, in order to reduce the feeling of melancholy. Pharmacological analysis has indicated that the active components in HR —aloe-emodin, α-boswellic acid, anthraquinone, β-Boswellic acid, chrysophanol, colchicine, hemerocallone, kaempferol, puerarin, rhein, and vanillic acid—impart beneficial effects on DD and related complications through the action of target proteins in various metabolic pathways. EGFR, GRIN2B, HDAC2, MAOB, MAOA, CYP2E1, GRIN2A, CYP2D6, ESR1, and GRIN1 as hub genes and cluster analysis showed that GRIN2D, HDAC5, and CYP2B6 are the center genes from three major clusters. PPI network unveiled the interaction between HR and DD-related targets, and found possibly significant targets in a more circumstantial point of view through the clustering method. Third, GO enrichment analysis indicated that all the targets interacting with the bioactive components of HR have a total of 390 GO entries and top20 of each category  were selected. Most of these play important parts in the central nervous system that affect different steps of the synthesis or transportation of neurotransmitters. Finally, the pathway analysis proved that bioactive components of HR exert a synergistic effect on the treatment of depressive disorder through numerous pathways such as alcoholism pathway, amphetamine addiction pathway, Rap1 signaling pathway, Ras signaling pathway, drug metabolism—cytochrome P450 pathway, metabolism of xenobiotics by cytochrome P450 pathway, and so on. These pathways mostly down-regulate MAOA, MAOB, EGFR, and CYP2E1, while simultaneously up-regulating CALM1. This matches with the hub genes from PPI network, in which EGFR has been proved to be a significant depression-related gene [MAOA and MAOB are crucial target genes of various mental diseases, and MAOA is particularly important in depression and anxiety [In this study, we try to interpret the synergistic effect of HR on DD from four aspects. First, C-T network showed that the average number of targets per component is 24.3, and the mean degree of components per target is 1.9. This clearly established that HR fits one of the most important characteristics of TCM—multi-component and multi-target. Second, PPI network recognized ted gene , MAOA an anxiety ,26. More anxiety . Althoug anxiety .14H8O2. Previous studies showed that anthraquinone has only moderate inhibition effect or is even inactive against mouse Monoamine Oxidase (MAO) enzyme [8H8O4, found in some forms of vanilla and many other plant extracts. It is reported to possess strong antioxidant, anti-inflammatory, antinociceptive, and neuroprotective effects [Furthermore, the top3 key components and their related genes are presented in ) enzyme ,32. Howe) enzyme . Consist) enzyme ,35. In s) enzyme . Vanilli effects ,38,39,40 effects . Interes effects ,43,44 an effects .http://lsp.nwu.edu.cn/tcmsp.php) [http://mesh.tcm.microbioinformatics.org) [http://www.swissadme.ch/index.php) [All components of HR were obtained from Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform   and TCM-ics.org) . TCMSP ddex.php) .ADME—absorption, distribution, metabolism, and excretion—is used in drug discovery to optimize the balance of properties necessary to convert leads into good medicines. The proper ADME screening can assure those candidates have better pharmacokinetic properties and hopefully, minimize drug–drug interactions in the future.In this study, four ADME-related models were used to screen out the active components in HR, including OB, Caco-2, DL, and GI absorption. Beyond that, all screen-out components should follow Lipinski’s rule of five.Lipinski’s rule of five is a rule of thumb to evaluate if a chemical compound with certain pharmacological or biological activities could be a likely orally active drug in humans. In this rule, compounds who met the requirements of MW, Hdon, Hacc, AlogP, and RBN seemed to be more possible to become a drug ,50. OB is defined as “the rate and extent to which the active ingredient or active moiety is absorbed from a drug product and becomes available at the site of action” by the Food and Drug Administration (FDA) . It portHuman intestinal cell line Caco-2 is a most common tool to study the passive diffusion of drugs across the intestinal epithelium. The Caco-2 cell model is a human-cloned colonic adenocarcinoma cell that is structurally and functionally similar to intestinal epithelial cells. The transport rates of components (nm/s) in Caco-2 monolayers represents the intestinal epithelial permeability . Those chttp://www.drugbank.ca/) [DL is an established concept for drug design that is used to estimate which compounds have the “drug-like” properties. The DL values of these components were calculated by the database-dependent DL evaluation approach based on Tanimoto coefficient, which equation is showing below. In this equation, A represents the molecular descriptor of herbal components, and B is the average molecular property of all components in DrugBank (ank.ca/) . The thrank.ca/) . Any comGastrointestinal (GI) absorption is a pharmacokinetic behavior crucial to estimate the various stages of the drug discovery processes, which can be calculated by an accurate predictive model, the Brain Or IntestinaL EstimateD permeation method (BOILED-Egg) . This moLastly, there are some screen-out components we add back into the database. Vanillic acid, puerarin, α-Boswellic acid, β-Boswellic acid and Chrysophanol were added back into the list because numerous studies proved that they are beneficial for hepatoprotection ,55,57,58To obtain the target of active components in HR, the commonly used databases, i.e., HitPick , similarhttp://www.disgenet.org/) [The DD targets were gathered from the DisGeNET database (et.org/) , which oet.org/)  in Cytoshttp://bioinformatics.psb.ugent.be/webtools/Venn/) was applied to calculate the intersection between HR and DD targets. Artificial neural network was constructed through the network visualization software Cytoscape, which is used to visualize and analyze the molecular interactions and biological pathway. Draw Venn Diagram  was adopted to analyze the representative biological processes and pathways associated with HR against DD. All obtained targets from previous preparation were imported. Total of 390 GO entries were found and top20 entries of each category, namely BP, CC, and MF, were obtained with the p-values were set at 0.05 as the cut-off criterion. A total of 40 pathways were screened and top10 pathways were selected. The results of the analysis were annotated by Pathview [https://www.bioconductor.org/).The latest pathway data were obtained from the KEGG database  for KEGGPathview  in the RIn this research, the network model of "component-target-pathway" interaction was established through Cytoscape3.7.1. In network interactions, nodes represent components, targets, and pathways, whereas edges represent the interaction of each other. Based on this neural network model, the pathway of active components and targets in depressive disorder is initially explored to provide a preliminary theoretical basis for the design of subsequently targeted drugs. The aforementioned results highlight that HR has intervention effects on DD through multiple bioactive compounds and targets. In conclusion, anthraquinone, kaempferol, and vanillic acid as the main bioactive components can alleviate depression symptoms by down-regulated MAOs. While there is no literature that shows the effect of anthraquinone and vanillic acid on ESR1, studies showed that it acts as an important factor of depression especially in women ,69,70. EHemerocallis Radix on depressive disorder. It will provide new ideas for further research on ethnopharmacology, Traditional Chinese Medicinal herbs, and ethnic compounds. The targets, clusters, biological processes, and pathways associated with HR were discovered through this study. HR target-DD target network exhibited the effective bioactive components, potential pharmacology, and molecular mechanism of HR for treating DD. This study provides a suitable combination strategy to uncover the therapeutic mechanism of multi-component drugs on a systematic level, which lays the foundation for the clinical application.In the present study, a network-based computational strategy was established to uncover the pharmacological mechanism of the compounds of The alcoholism pathway is regulated at different levels by various mechanism and can crosstalk with other signaling pathways. The results of our study support the role of anthraquinone, kaempferol, and vanillic acid on MAOA, MAOB, and ESR1 in the etiology of depressive disorder. Many studies focusing medicinal herbs were carried out with animal models. However, several researches suggested that it shows different results in humans, indicating the importance of assessing the effect of the abovementioned active components on specific target toward the treatment of depressive disorder in human. While there is no ample evidence of the effect of anthraquinone and vanillic acid on ESR1, further research in this area may reveal details in assigning causative relationships.