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Zielsetzungen: | Ziel dieser Studie | DER | Ziel dieser Studie war es, die Verringerung der VL und der Infektiosität durch Phthalocyanin-Mundwasser und -Nasenspray bei Patienten mit COVID-19 zu untersuchen. | PMC10326526 |
Methoden: | Zeitpunkt der klinischen Diagnose | DER | Patienten mit leichter COVID-19-Erkrankung wurden für die Teilnahme an einer dreifachblinden, randomisierten und kontrollierten Studie rekrutiert. Die Teilnehmer wurden einer von drei Gruppen zugeteilt: Gruppe 1: nicht-aktive Mundspülung und Kochsalz-Nasenspray; Gruppe 2: Phthalocyanin-Mundspülung und Kochsalz-Nasenspray und Gruppe 3: Phthalocyanin-Mundspülung und Phthalocyanin-Nasenspray. VL wurde in nasopharyngealen und oropharyngealen Abstrichen untersucht, die zum Zeitpunkt der klinischen Diagnose zu Studienbeginn sowie 24 und 72 Stunden nach Beginn der Spülprotokolle entnommen wurden. | PMC10326526 |
Ergebnisse: | der | DER | In die Analyse wurden 46 Teilnehmer einbezogen: 15 in Gruppe 1, 16 in Gruppe 2 und 15 in Gruppe 3. Nach 72 Stunden war die Verringerung der VL in Gruppe 3 signifikant höher (mittlere Abnahme der Zyklusschwelle: 11,21) als in Gruppe 1 (mittlere Abnahme der Zyklusschwelle: 5,53). Außerdem war nur die mittlere VL in Gruppe 3 nach 72 Stunden auf ein nicht-ansteckendes Niveau gesunken. | PMC10326526 |
Wichtigste Schlussfolgerungen: | DER | Die Verwendung von Phthalocyanin-Mundspülungen und -Nasensprays ist wirksam bei der Verringerung der SARS-CoV-2-Infektiosität. | PMC10326526 |
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Introduction | coronavirus disease | DISEASE | The coronavirus disease (COVID-19) pandemic began in December 2019 in China and spread worldwide, causing serious consequences and prompting a race to search for drugs to treat and prevent the disease. The nasal and oral cavities contain a large number of angiotensin-converting enzyme [ | PMC10326526 |
Patients and methods | PMC10326526 |
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Study design | Patients were recruited from the COVID-19 outpatient clinic of the University Hospital of Londrina-Paraná, Brazil, from November 1, 2020, to February 1, 2021. The study was a prospective randomized triple-blinded randomized controlled trial. Participants were instructed to use nasal and oral rinsing protocols according to the following groups: Group 1, non-active mouthwash and saline nasal spray (SNS); Group 2, phthalocyanine mouthwash and SNS; and Group 3, phthalocyanine mouthwash and phthalocyanine nasal spray. They were instructed to gargle for 30 sec and rinse for 30 sec with 5 ml of the mouthwash solution and to spray two pumps of the nasal spray into each nostril five times a day for three consecutive days. Participants’ VLs were assessed using reverse-transcription polymerase chain reaction (RT-PCR) on the first and third days of using the mouthwash and nasal spray. Each participant’s adherence to the rinsing protocols was recorded.The inclusion criteria were patients aged 18 to 80 years who tested positive for SARS-CoV-2 and were diagnosed within 7 days of symptom onset. The exclusion criteria included patients with cycle threshold (Ct) values greater than 31 and patients who had contraindications to using mouthwash or nasal spray for medical reasons or because of an inability to gargle and spit. All participants received standard care (antibiotics, corticosteroids, and anticoagulants) according to the World Health Organization standard treatment guidelines [ | PMC10326526 |
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Randomization and masking | Sample randomization was performed as follows: the mouthwash bottles and nasal sprays were placed in consecutively numbered closed packages. An Excel (Microsoft, Redmond, WA, US) database was created from these numbered packages and used for randomization. After randomization, packages with mouthwash bottles, oral care kits (dental floss, toothbrush, and fluoride toothpaste, Rabbit Corp, Brazil), and nasal sprays were delivered to the participants. | PMC10326526 |
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RNA extraction and quantitative PCR for SARS-CoV-2 | SARS-CoV-2 infection | SARS-COV-2 INFECTION | Viral RNA was extracted from 100 µL of nasopharyngeal or oropharyngeal swab, using the automated extractor, EXTRACTA 32 (Loccus, Cotia, Brazil), and magnetic bead extraction kits (MVXA-P016 FAST), and following the manufacturer’s instructions (Loccus, Cotia, Brazil). A negative extraction control (UltraPure DNase/RNase-Free Distilled Water, Thermo Fisher Scientific, Waltham, MA, USA) was added to each extraction run.The diagnosis of SARS-CoV-2 infection and monitoring of VL were performed by real-time reverse transcription-polymerase chain reaction (RT-PCR) using fluorescent probes (Taqman) [ | PMC10326526 |
Statistical analysis | Initially, descriptive and exploratory analyses of all data were performed. Data were summarized as frequencies and percentages for categorical variables and as means and standard deviations or medians and interquartile ranges for continuous variables. Then, the three groups were compared using Fisher’s exact test for categorical variables and generalized linear models for continuous variables (age and onset of symptoms). The Ct values met the assumptions for parametric analysis and were analyzed using generalized linear models for repeated measures. They were also analyzed for variations in the Ct value in relation to the baseline by one-way analysis of variance and Tukey’s test. Finally, associations between groups and a classification of infectivity (high, intermediate, and low) were analyzed based on Ct values using Fisher’s exact test. All analyses were performed using the R Core Team program (R Foundation for Statistical Computing, Vienna, Austria) with a significance level of 5%. All tests were based on the null hypothesis of the three interventions having an equivalent effect on the Ct value. | PMC10326526 |
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Results | A total of seventy-five patients with mild COVID-19 were recruited for the study (Figure 1 As shown in Table 1 The Ct values decreased significantly in all three groups (Table 2 Figure 2 | PMC10326526 |
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Discussion | VIRUS, CAVITY | In the present study, all three groups experienced a decrease in Ct values over time, indicating a reduction in the VL. These results are in accordance with previous reports that gargling and rinsing with mouthwash as well as rinsing the nasal cavity are associated with a reduction in the SARS-CoV-2 VL [Considering that the highest VL can be found in the first week after symptom onset [This study has some limitations. The sample size was limited. Furthermore, Ct values are semi-quantitative and the RT-PCR evaluation only indicates the presence or absence of virus fragments. More precise results could be obtained if the evaluation measured the infective capacity of viable viruses after using the rinsing protocols. In the present study, we adopted the measure of infectivity as previously described by Jaafar et al. [ | PMC10326526 |
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Conclusion | Nasal and oral rinsing with phthalocyanine antiseptic solutions can reduce the SARS-CoV-2 VL and reduce infectivity to low or non-contagious levels after 3 days of use. Further studies with larger sample sizes are required to confirm these results. | PMC10326526 |
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Notes | PMC10326526 |
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Acknowledgements | INFECTIOUS DISEASES | We thank Dr. Lucas Marques da Costa Alves for his support regarding the topic of infectious diseases. | PMC10326526 |
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Patient consent | All participants provided written informed consent. | PMC10326526 |
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Ethics statement | The study protocol was approved by the Research Ethics Committees of the State University of Londrina, Paraná, Brazil (CAAE 37277420.0.0000.5231), and the followed procedures were in accordance with the Helsinki Declaration of 1975, as revised in 1983.The study was registered in the Brazilian Registry of Clinical Trials (ReBEC) (RBR-8tygcz7) and complied with the Consolidated Standards of Reporting Trials (CONSORT) 2010 checklist. | PMC10326526 |
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Authors’ contributions |
Study design, article writing, data analysis, and final review: ANCS, FVV, BFO, PSSSStudy design: AASGL, ZNTData collection: LMV, NPS, GLT, PLCSC, MFOHR, TMSS, MFOHRData analysis: MABL | PMC10326526 |
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Conflict of interests | Dr. Vilhena has a patent classification pending.Prof. Dr. da Silva Santos reports grants from CNPq process nº 309525/2018-7.The other authors declare that they have no competing interests. | PMC10326526 |
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Comparisons between groups (G1–3) regarding patient characteristics | PMC10326526 |
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Mean (standard deviation) of the VL estimated by the Ct value obtained in the PCR reaction as a function of the group and time. Different letters (upper case comparing horizontally between groups and lowercase comparing vertically between times) indicate statistically significant differences. Group 1 (G1): use of mouthwash containing placebo + placebo nasal spray; Group 2 (G2): use of mouthwash containing phthalocyanine + placebo nasal spray; Group 3 (G3): use of mouthwash containing phthalocyanine + nasal spray containing phthalocyanine | PMC10326526 |
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CONSORT flow diagram | PMC10326526 |
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Qualitative data of mean VL and infectivity estimated by the Ct value as a function of group and time | PMC10326526 |
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Subject terms | quadriceps muscle mass, critically ill | BLOOD, CRITICALLY ILL, INSULIN RESISTANCE, CRITICALLY ILL | Intermittent (or bolus) feeding regimens in critically ill patients have been of increasing interest to clinicians and scientists. Changes in amino acid, fat and carbohydrate metabolites over time might yet deliver other benefits (e.g. modulation of the circadian rhythm and sleep, and impacts on ghrelin secretion, insulin resistance and autophagy). We set out to characterise these changes in metabolite concentration. The Intermittent versus Continuous Feeding in Critically Ill paitents study (NCT02358512) was an eight-centre single-blinded randomised controlled trial. Patients were randomised to received a continuous (control arm) or intermittent (6x/day, intervention arm) enteral feeding regimen. Blood samples were taken on trial days 1, 7 and 10 immediately before and 30 min after intermittent feeds, and at equivalent timepoints in the control arm. A pre-planned targeted metabolomic analysis was performend using Nuclear Resonance Spectroscopy. Five hundred and ninety four samples were analysed from 75 patients. A total of 24 amino acid-, 19 lipid based-, and 44 small molecule metabolite features. Across the main two axes of variation (40–60% and 6–8% of variance), no broad patterns distinguished between intermittent or continuous feeding arms, across intra-day sampling times or over the 10 days from initial ICU admission. Logfold decreases in abundance were seen in metabolites related to amino acids (Glutamine − 0.682; Alanine − 0.594), ketone body metabolism (Acetone − 0.64; 3-Hydroxybutyric Acid − 0.632; Acetonacetic Acid − 0.586), fatty acid (carnitine − 0.509) and carbohydrate metabolism ( Maltose − 0.510; Citric Acid − 0.485). 2–3 Butanediol, a by-product of sugar-fermenting microbial metabolism also decreased (− 0.489). No correlation was seen with change in quadriceps muscle mass for any of the 20 metabolites varying with time (all | PMC10636009 |
Introduction | critically ill, ill | CRITICALLY ILL | Intermittent or bolus enteral feeding regimens in critically ill patients have been of increasing interest to clinicians and scientistsCritically ill patients lose 2–3% of muscle mass each day as a result of decreased muscle protein synthesis and unchecked protein breakdownHowever, it has been speculated that other less well-explored benefits of intermittent feeding might improve patient outcome or experienceWe hypothesise that intermittent enteral feeding may modulate amino acid, fat and carbohydrate metabolite profiles over time, when compared to continuous feeding. For example, alterations in amino acid trafficking may be advantageous as a result of addressing the muscle full effect, providing support for future trials combining intermittent feeding with a co-intervention addressing bioenergetics failure to improve physical function | PMC10636009 |
Methods | PMC10636009 |
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Study design and participants | critically ill | CRITICALLY ILL | The Intermittent versus Continuous Feeding in critically ill patients study (IVC study, NCT02358512) was a multicentre single-blinded randomised controlled trial performed on 8 UK intensive care units (ICU), in which adult (≥ 18 years of age) patients were randomised (1:1 ratio, concealed allocation) to receive either a continuous or intermittent enteral feeding regimen5.Chest. 158:183–194. The original study received ethics committee approval (National Research Ethics Service Committee London-Queens Square; REC reference 14/LO/1792; IRAS project ID 160,281) and was registered on clinicaltrials.gov before randomisation was commenced (05/11/15). All research was performed in accordance with the Declaration of Helsinki. Prospective informed assent was obtained in writing from a nominated personal consultee or professional consultee. Retrospective participant consent was obtained on return of participant’s mental capacity. Permission to use participants’ data if capacity did not return or they did not survive was included in the assent process. | PMC10636009 |
A pre-planned targeted metabolomic cohort ancillary study was performed | Sequential Organ Failure Assessment, multi-organ failure | MULTI-ORGAN FAILURE | In brief, patients were required to have been admitted to the ICU for ≤ 24 h prior to enrolment. Included patients were those anticipated to be mechanically ventilated for > 48 h, who required enteral feeding via a nasogastric tube, had multi-organ failure (Sequential Organ Failure Assessment (SOFA) score > 2 in ≥ 2 domains at admission), had a likely ICU stay of > 7 days, and who were considered likely to survive > 10 days. The characteristics and demographics of patients randomised to each limb were well balanced. | PMC10636009 |
Sample collection | BLOOD | Blood samples were collected in heparinised tubes and the supernatant was removed and frozen at − 80 | PMC10636009 |
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Measures of muscle mass | Rectus Femoris Cross-Sectional Area (RFCSA) was measured at randomisation (as baseline) and at day 10 (primary outcome of the original trial) | PMC10636009 |
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Metabolite analysis | BLOOD | Blood samples were analysed using a targeted metabolomics approach using Nuclear Magnetic Resonance Spectroscopy (NMR). For each sample, 300 μL of plasma was mixed with 300 μL of a 90/10 H | PMC10636009 |
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2D NMR spectra for metabolites identification | For metabolite identification, 2D NMR spectra were acquired on one of the samples, in particular [ | PMC10636009 |
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Sample size estimate | critically ill | CRITICALLY ILL | This was an exploratory study of the metabolome under different feeding regimes in critically ill patients. Without pilot data existing power calculation packages e.g. Metaboanalyst (( | PMC10636009 |
Statistical analysis | The statistical analyses were performed using R version 4.0.3 (Following NMR analyses, separate data matrices of metabolite abundances for lipid species, small molecule species and amino acids were generated and each analysed separately as targeted panels of metabolites. Each metabolite panel was first filtered to remove missing data points and/or those with negative abundance values, followed by log transformation, centering and scaling to bring metabolite abundances within the same dynamic range. Exploratory visualisations were then generated using principal component analyses to assess for any obvious effects of the feeding regimens on metabolite abundances. To determine differences in metabolite abundance between feeding regimens, across time and interactions between feed and time, analyses were performed using a moderated empirical Bayesian mixed effects model approach with the limma (version 3.18) R package, with the primary outcome being a difference in metabolite profiles between armsLinear mixed effect modelling was chosen over standard statistical approaches (e.g. repeated measures ANOVA) as it deals better with designs where the repeated measure is continuous (i.e. in this case, time), where the design may become unbalanced (e.g. due to attrition) and where non-independence of data due to clustering within individuals exists, as is likely to be the case here. In addition we used limma to generate empirical Bayes estimates of metabolite-wise variance | PMC10636009 |
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Ethical approval | The study received ethics committee approval (National Research Ethics Service Committee London-Queens Square; REC reference 14/LO/1792; IRAS project ID 160,281) and was registered on clinicaltrials.gov before randomisation was commenced. | PMC10636009 |
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Results | RENAL | Of the 121 patients randomised, 62 were in the intervention feeding (IF) group, and 59 in the control feeding (CF) group. Five hundred and ninety four samples were available for 75 patients (Table Characteristics of the 75 patients whose metabolome abundances are described. LOS = Length of Stay; APACHE II = Acute Physiology and Chronic Health Evaluation Score; SOFA = Sequentional Organ Failure Assessment; RRT = Renal Replacement Therapy; NMBA = Neuromuscular Blockade administration. Data are mean (95% confidence intervals), except for # indicating median with range.Plasma metabolomic analyses using NMR identified a total of twenty-four amino acid metabolite features, nineteen lipid based metabolite features and forty-four other small molecule metabolite features across five hundred and ninety observations/samples (Figs. | PMC10636009 |
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Exploratory visualisations | We first examined the metabolite panels for broad patterns of variability that might underpin physiologically relevant changes as a result of feeding regimen; time since admission or time of day. Although a few observations from each panel were clustering out with others, along the main two axes of variation (PC1—explaining between 40 and 60% of variance and PC2—explaining 6 and 8% of variance) no broad patterns distinguished between intermittent or continuous feeding arms, across intra-day sampling times or over the 10 days from initial ICU admission (Fig. ( | PMC10636009 |
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Specific metabolite alterations | We next addressed the question of specific metabolite alterations between the feeding arms using a mixed effect moderated empirical Bayesian approachSummary of all metabolites and associated Human Metabolite Database (HMDB) ID number and related biological pathway whose concentration was found to change significantly over the duration of ICU stay, following removal of a non-significant feeding arm effect by collapsing data into a single dataset together with their associated Human Metabolite Database (HMDB) ID number and related biological pathway. Where two peaks of the same metabolites are seen (representing different isomers) these are numbered separately. “Unknown” refers to metabolites not seen in the HMDB.( | PMC10636009 |
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Clinical correlates | REGRESSION, REGRESSION | Finally, to assess whether there was any relationship between these groups of metabolites and clinical outcome following ICU admission, we used linear regression analysis to examine the relationship between the 20 metabolites varying over time and the primary clinical endpoint of change in muscle mass during admission (restricted to only n = 35 for this analyses due to need for complete data sets for all at 0 and 10 days). Patients in this cohort had a mean RFSummary of Linear Regression Analyses of relationship between significantly changing metabolites and change in muscle cross sectional area over 10 day ICU stay. Where two peaks of the same metabolites are seen (representing different isomers) these are numbered separately. “Unknown” refers to metabolites not seen in the HMDB.Summary of Linear Regression Analyses of relationship between significantly changing metabolites and Sequential Organ Failure Assessment (SOFA) score over a 10 day ICU stay. Where two peaks of the same metabolites are seen (representing different isomers) these are numbered separately. “Unknown” refers to metabolites not seein the HMDB. | PMC10636009 |
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Discussion | critically ill, dysbiosis, critically ill patientThese | CRITICALLY ILL, COLLAPSE, CRITICAL ILLNESS, KLEBSIELLA, MULTI-ORGAN FAILURE | We performed a detailed metabolic analysis of amino acid, lipid and small molecule profiling in response to intermittent enteral feeding compared to continuous enteral feeding in critically ill patients, in the context of a multi-centre randomised controlled trial. We examined these three specific metabolite panels, composed of twenty four amino acid metabolites, nineteen lipid based metabolites and 44 small molecule metabolites over a four hour window within a day and longitudinally up to 10 days, in addition to between arms. No differences were seen between groups at each of the individual time points. Using a mixed effect moderated empirical Bayesian approach, we confirmed this finding, allowing us to collapse the study arms and examine changes over time of specific metabolites. Decreases in relative concentrations of small metabolites related to fatty acid, ketone acid and carbohydrate metabolism were observed over the first 10 days of critical illness.Metabolic profiling of these critically ill patients was surprisingly stable over time, with alterations only being seen at 10 days as opposed to variations at earlier timepoints. This may reflect the population chosen, as all patients had multi-organ failure, and reach an abnormal equilibrium of cellular metabolism early, and resolve slowly. This is in keeping with our previous observations in skeletal muscle and the limited clinical metabolomics data in critically ill patientsDecreases in circulating glutamine have been well described in the critical care literature, although supplementation is associated with an increase in mortality, perhaps through ammonia generation2,3-Butandiol is produced from sugar-fermenting microbes (e.g. Klebsiella sp, Entrobacter Sp, Serratia Sp, Bacillus Sp), and the decrease in metabolite levels are likely to reflect dysbiosis of critical illness, of which our understanding is still nascentTeasing apart the relationship between these processes across time and determining the true biological significance of the findings remains challenging, and will remain so until experimental and interventional studies can perturb these processes in the critically ill. Our data are the first to demonstrate these changes longitudinally as opposed to across cohorts, strengthening the case for focusing intervention development on these pathways. Metabolite profiling has been used in the past to determine effects of interventions such as Vitamin DWe were unable to demonstrate a difference in metabolite patterns between trial arms at individual timepoints. These data suggest that intermittent feeding is unable to sufficiently alter metabolism in critically ill patients, and therefore is unlikely to be better than continuous feeding in affecting end-organ metabolism. This does not, however, rule out the existence of other benefits of intermittent feeding regimens, such as increased nutrition delivery by overcoming difficulties in the process of nutritional delivery to the critically ill patientThese data represent one of the the largest matrices of metabolomics data in critical illness, as a result of the repeated sampling within days and across 7–10 days which, to our knowledge, has not been performed previously. The multi-centre nature of the trial suggests that these data have significant external validity. Most of the results presented are independent of feeding regimen across length of ICU admission | PMC10636009 |
Conclusions | critically ill | CRITICALLY ILL | A 6-times per day intermittent feeding regimen did not alter metabolite patterns across time compared to continuous feeding in critically ill patients, within a 24 h period, nor across a 10-day intervention. Future research on intermittent feeding regimens should focus on clinical process benefits, or extended gut rest and fasting. | PMC10636009 |
Supplementary Information | The online version contains supplementary material available at 10.1038/s41598-023-46490-5. | PMC10636009 |
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Acknowledgements | HEART | We acknowledge the Centre for Biomolecular Spectroscopy at King’s College London funded by the Wellcome Trust and British Heart Foundation (ref. 202,767/Z/16/Z and IG/16/2/32,273). We are grateful to the staff and patients (and families) at all recruiting centres for their time and support. | PMC10636009 |
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Author contributions | D.W., P.A., Z.P., S.H. conceived and designed the manuscript. A.M., D.B., H.M., N.H., Z.P. conceived, designed and delivered the original trial. T.F., M.C. A.G., I.G., D.W. performed the analysis. D.W., Z.P., A.M., P.A. wrote the main manuscript. All authors reviewed the manuscript. | PMC10636009 |
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Funding | LCRN, A. S. | MAY | HM is supported by the National Institute of Health Research’s Comprehensive Biomedical Research Centre at University College London Hospitals (UCLH NIHR BRC, BRC202 rev/CM/AM/101320; RCF236/AMcN/2015); J P Moulton Charitable Foundation (JM29/04/ 14; JM02/06/15); Intensive Care Foundation (New Investigator Award, A. S. McN.); London South Local Clinical Research Network (LCRN) (D. E. B.: November 2014- May 2015); North Thames LCRN (A. S. McN.: January 2017-March 2017); American Society of Parenteral and Enteral Nutrition Rhoads Research Foundation (Z. A. P.: January 2018-January 2020). | PMC10636009 |
Data availability | Datasets generated are available on reasonable request to the corresponding author ([email protected]). | PMC10636009 |
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Code availability | Code used for analyses is available at: | PMC10636009 |
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Competing interests | Thomas’ NHS, P. | MUSCLE WASTING | This work was supported (in part) by the ASPEN Rhoads Research Foundation. D. E. B. reports speaker fees from, Baxter Healthcare; advisory board fees from Baxter Healthcare;. N. H. reports unrestricted grants from Philips and ResMed outside the direct area of work commented on here with the funds held and managed by Guy’s and St Thomas’ NHS Foundation Trust; financial support from Philips for the development of MYOTRACE technology that has a patent filed in Europe (US pending) outside the area of work commented on here; personal fees for lecturing from Philips-Respironics, Philips, ResMed, and Fisher-Paykel both within and outside the area of work commented on here; N. H. is on the Pulmonary Research Advisory Board for Philips outside the area of work commented on here with the funds for this role held by Guy’s and St Thomas’ NHS Foundation Trust. H. E. M. has a patent, “The Use of Inhibitors of the Renin-Angiotensin System,” which relates in part to the prevention of muscle wasting, issued. Z. A. P. reports personal fees from Faraday Pharmaceuticals, Lyric Pharmaceuticals,Bioage, Fresenius Kabi, Nestlé, Orion, and GlaxoSmithKline, outside the submitted work.
None declared (DW, IJG, AM,PJA, ALG, MRC, TF, SDR). | PMC10636009 |
References | PMC10636009 |
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1. Introduction | rheumatoid arthritis, Rheumatoid arthritis, RA, multiple joints | RHEUMATOID ARTHRITIS, RHEUMATOID ARTHRITIS, DISEASE | The study aimed to elucidate the effective chemical composition and molecular mechanism of rheumatoid arthritis (RA) treatment with Jishe Qushi capsules (JSQS) and perform clinical validation. The effective chemical components were screened by a database. We used Cytoscape software to construct the key target-RA composite target network of JSQS. Gene Ontology biofunctional analysis and Kyoto encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed for the key targets, followed by molecular docking validation of core key targets. Ninety-nine patients chosen were divided into 49 cases in the treatment group and 50 cases in the control group according to the random number table method. The control group was treated with the combination of methotrexate (MTX) plus Glucosidorum Tripterygll Totorum. The treatment group was treated with MTX plus JSQS. The treatment effects of the 2 groups were evaluated. A total of 118 key anti-RA targets were obtained for JSQS. Quercetin in Rheumatoid arthritis (RA) is a common immune disease characterized by the symmetrical destruction of multiple joints.Chinese traditional medicine has a curative effect in the treatment of RA. These medicines—such as Glucosidorum Tripterygll Totorum and total glucosides of | PMC10470707 |
2. Materials and methods | PMC10470707 |
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2.1. Network pharmacology | PMC10470707 |
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2.1.1. Acquisition and screening of the chemical composition of JSQS. | The Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) database was used to retrieve all chemical components of the Chinese herbal medicine composition of | PMC10470707 |
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2.1.2. Collection of disease target information and screening of cross-targets. | RA | RHEUMATOID ARTHRITIS, DISEASES | Using “rheumatoid arthritis” as the keyword, we searched the GeneCards and Online Mendelian Inheritance in Man human gene platforms for confirmed genes related to RA, downloaded the database into Excel, and de-duplicated the genes. The cross-targets of diseases and drugs were obtained by using Weishengxin. | PMC10470707 |
2.1.3. Construction of protein interactions. | RA | DISEASE | The targets of RA intersection with JSQS were imported into the String database, and the species was set as “homo sapiens.” Additionally, the minimum interaction score correlation was set as “highest confidence” (≥0.400). The drug–disease protein–protein interaction (PPI) network map was initially obtained by downloading the TSV file and processing it with Cytoscape 3.90 software. The core targets were selected by analyzing the network degree and other parameters with the help of the plug-in Network Analyzer. | PMC10470707 |
2.1.4. Construction of network model. | RA | DISEASES | The compounds and related targets obtained from JSQS were categorized, as well as the targets that intersected with the diseases. The active ingredient-target network of the capsules for RA treatment was constructed by Cytoscape 3.90 software. The network topology parameters, such as degree and betweenness, were used to screen the key active ingredients of the drug using the built-in tools of the software. | PMC10470707 |
2.1.5. GO enrichment analysis and KEGG pathway enrichment analysis. | RA | The drug-disease intersection targets were imported into the Database for Annotation, Visualization and Integrated Discovery (DAVID), and the species was set as human species. The kyoto encyclopedia of genes and genomes (KEGG) pathway and Gene Ontology (GO) enrichment analyses were submitted to establish a “core target-biological pathway” network to screen out the major pathways. The KEGG enrichment bubble chart and GO term enrichment were created online using the results of bioinformatics analysis to reflect the interactions between drug targets and RA target-related pathways. | PMC10470707 |
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2.2. Analysis of molecular docking technology | We obtained the 3D crystal structure of the target protein from the RCSB PDB ( | PMC10470707 |
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2.3. Clinical controlled trials | PMC10470707 |
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2.3.1. General clinical information | Rheumatoid Arthritis, X-ray stage, X-ray stage I, ±, malignancy, ≤, proteinuria, allergy, RA | INFECTIOUS DISEASES, RHEUMATOID ARTHRITIS, DISEASE, GASTROINTESTINAL ULCER, ALLERGY, INTERSTITIAL PULMONARY FIBROSIS, PERIPHERAL NEUROPATHY, DISEASE, DISEASES | Ninety-nine patients with RA treated at the Second Affiliated Hospital of Guizhou University of Traditional Chinese Medicine from January 2021 to 2022 were selected for the study. Among them, 18 cases were men, and 81 cases were women. Their age ranged from 24 to 75 years, with an average age of 56.34 ± 12.80 years. Disease duration ranged from 7 months to 11 years, with an average disease duration of 5.21 ± 2.74 years. Inclusion criteria comprised: patients aged 18 to 60 years; patients that met the diagnostic criteria of active RA according to Chinese Guidelines for Rheumatoid Arthritis Treatment (2018 edition); patients diagnosed for the first time, patients who did not receive formal drug treatment after diagnosis, or patients who received formal treatment but stopped taking drugs for more than 3 months; and patients with 2.6 ≤ Disease Activity Score using 28 joint counts <5.1, joint function classification of I to III, and X-ray stage I to III of both hands.Exclusion criteria were: patients aged <18 years or >60 years; patients with grade IV joint function and/or X-ray stage IV of both hands; patients with a history of hormone and/or biological treatment within 1 month; patients with severe cardiac, hepatic, renal, or another important organ insufficiency; patients with other serious diseases, such as malignancy, active gastrointestinal ulcer, and infectious diseases; patients with severe extra-articular manifestations, such as peripheral neuropathy, proteinuria, and interstitial pulmonary fibrosis; and patients with allergy to the drug of our study. | PMC10470707 |
2.3.2. Drug | ’ major symptoms, pain | The control group received MTX (16 tablets/bottle, Shanghai Xinyi Pharmaceutical Co., Ltd., Lot no. 036180503) 10 mg once a week and Glucosidorum Tripterygll Totorum (100 tablets/bottle, Guizhou Hanfang Pharmaceutical Co., Ltd., lot no. 1459001) 20 mg thrice daily for 4 consecutive weeks. The treatment group received MTX, 10 mg once a week, combined with JSQS (0.45 g, 36 capsules/bottle, from Department of Pharmacy, Second Affiliated Hospital of Guizhou University of Traditional Chinese Medicine, lot no. 20180101) 4 capsules each time, thrice daily for 4 consecutive weeks. Both groups took folic acid tablets 10 mg (100 tablets/bottle, Changzhou Pharmaceutical Factory Co., Ltd., lot no. 18082511) on the next day during the MTX administration. Patients of both groups were allowed to take celecoxib capsules (6 capsules/box, Pfizer Pharmaceutical Co., Ltd., lot no. W64713) orally at a maximum dose of 2 capsules per day during the study period due to joint pain, and the number of taken capsules and the withdrawal time were recorded separately for both groups.The criteria for determining efficacy were formulated with reference to the Guiding Principles for Clinical Research on New Chinese Medicines (Trial) 2020. Efficacy was defined as the overall improvement rate of patients’ main symptoms and signs of ≥75%, while erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) were normal or significantly improved. Progress was defined as the overall improvement rate of patients’ main symptoms and signs of ≥50%, while ESR and CRP improved. Effectiveness was defined as the overall improvement rate of patients’ major symptoms and signs of ≥30%, while ESR and CRP were improved or not improved. Ineffectiveness was defined as the overall improvement rate of patients’ main symptoms and signs of <30%, while ESR and CRP did not improve. The total effective rate was calculated as follows:Total effective rate = (significant + improvement + effective)/ total number of cases. | PMC10470707 |
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2.3.3. Observed indicators. | PMC10470707 |
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2.3.3.1. Comparing the main symptoms and signs of the 2 groups. | morning stiffness, pain | The joint pressure pain, swollen joint count, time of morning stiffness, and visual analog scale (VAS) of the 2 groups were compared before and after treatment. As VAS, a 10-cm scale was selected, with 0 representing a completely pain-free state and 10 representing intolerably severe pain. Patients chose the corresponding scale according to their pain level, and the physician in charge chose the corresponding score. | PMC10470707 |
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2.3.3.2. Comparing the level of changes in key clinical indicators. | Rheumatoid, fasting venous blood | BLOOD | First, 5 mL of fasting venous blood was collected from patients in both groups before and after treatment. After centrifugation at 3000 r/min for 10 minutes, the supernatant was pretreated and stored at low temperature and used to detect the relevant indexes of patients. Blood ESR was measured by the Westergren methods. CRP was measured by immunodiffusion. Rheumatoid factor (RF) was measured by latex agglutination test. Anti-cyclic citrullinated peptide (anti-CCP) was measured by enzyme-linked immunosorbent assay. The reagents were purchased from Shanghai Chemical Biotechnology Co., and the operation procedure was performed strictly according to the manufacturer instructions. | PMC10470707 |
2.3.3.3. Comparing the levels of inflammatory factor changes in the 2 groups. | fasting venous blood, tumor necrosis, TNF-α | TUMOR NECROSIS | Five mL of fasting venous blood was collected from patients in both groups before and after treatment. After centrifugation at 3000 r/min for 10 minutes, the supernatant was pretreated and stored at 4°C and used to detect the relevant indexes of patients. The levels of serum interleukin-6 (IL-6), serum interleukin-17 (IL-17), and tumor necrosis factor (TNF-α) in the 2 groups were measured by enzyme-linked immunosorbent assay. The respective kits were purchased from Shanghai Enzyme Link Biotechnology Co., and the operation procedure was performed strictly according to the manufacturer instructions. | PMC10470707 |
2.3.3.4. Comparing the level of changes in immune function indexes in the 2 groups. | fasting venous blood | First, 5 mL of fasting venous blood was collected from patients in both groups before and after treatment. After centrifugation at 3000 r/min for 10 minutes, the supernatant was taken for pre-treatment. The changes in the expression levels of helper T cells 17 (Th17) and regulatory T cells (Treg) were detected using a flow cytometer (Serena [China] Medical Technology Co., Ltd, model Sparrow), and the operation procedure was performed strictly according to the manufacturer instructions. | PMC10470707 |
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2.4. Observation of adverse reactions | ADVERSE REACTIONS | We observed the adverse reactions during treatment in both groups. No obvious clinical signs and symptoms of discomfort were found. | PMC10470707 |
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2.5. Statistical methods | Statistical differences were considered as significant if the | PMC10470707 |
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3. Results | PMC10470707 |
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3.1. Acquisition and screening of the chemical composition of Chinese herbal medicines in JSQS | A total of 33 active compounds were collected from Potential active ingredients of JSQS (TCMSP).CLR = Cholesterol, DFV = 4',7-Dihydroxyflavanone, DL = drug similarity, OB = oral bioavailability.Potential active ingredients of JSQS (ChemMapper). | PMC10470707 |
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3.2. Collection of disease target information and screening of cross-targets | RA | RHEUMATOID ARTHRITIS, DISEASE | A total of 4881 and 28 RA-related targets were obtained from the GeneCards and Online Mendelian Inheritance in Man databases, respectively. The RA targets collected from the 2 databases were integrated and de-duplicated, providing a total of 4791 targets. The disease and drug targets were imported into Weishengxin to obtain the Venn diagram, as shown in Figure Common targets of Jishe Qushi capsules in RA.Cross-targeting of disease and drug. Note: pink represents the number of targets for RA, blue represents the number of targets for active constituents of Jishe Qushi capsules, and the middle part represents the intersection targets of the 2. RA = rheumatoid arthritis. | PMC10470707 |
3.3. Construction of protein interactions | RA | RHEUMATOID ARTHRITIS, DISEASE | A total of 118 targets based on the intersection of JSQS and RA were imported into the String platform. The PPI network map was initially obtained after setting the corresponding parameters, and the downloaded TSV file was opened in Cytoscape 3.90 software to draw the PPI network map. The larger the value of Degree and Betweenness Centrality, the larger the shape of the node, and the larger the value of Betweenness Centrality, the closer the color of the node was to dark green, as shown in Figure Jishe Qushi capsules and RA have a common target PPI network. The circle represents the disease target, and the line represents the relationship between the targets. PPI = protein–protein interaction, RA = rheumatoid arthritis. | PMC10470707 |
3.4. Compound-intersection targets-disease network of JSQS | RA | RHEUMATOID ARTHRITIS | One hundred eighteen common targets were used to construct a “Chinese herbal medicine-active ingredient-target” network containing 166 nodes and 423 edges, using Cytoscape software, as shown in Figure Basic information on key compounds.The orange rectangle represents the target point where the JSQS and RA intersect, and the other triangular nodes represent the active ingredients of the drug, with the same color attributed to the same herbal medicine. JSQS = Jishe Qushi capsules, RA = rheumatoid arthritis. | PMC10470707 |
3.5. GO enrichment analysis and KEGG pathway enrichment analysis | Human cytomegalovirus infection, cancer, Pancreatic cancer, Kaposi sarcoma-associated herpesvirus infection, Hepatitis B, Hepatitis C | LEGIONELLOSIS, HUMAN PAPILLOMAVIRUS INFECTION, SMALL CELL LUNG CANCER, PROLIFERATION, CANCER, MEASLES, SALMONELLA INFECTION, PANCREATIC CANCER, EPSTEIN-BARR VIRUS INFECTION, BLADDER CANCER, PROSTATE CANCER, ALCOHOLIC LIVER DISEASE, HEPATITIS B, ATHEROSCLEROSIS, HEPATITIS C | The targets were imported into the DAVID data platform for GO annotation analysis using “homo sapiens” as the study context. A total of 210 entries were obtained for GO enrichment analysis, including 159 entries for biological process (BP), 12 entries for cell composition (CC), and 39 entries for molecular function (MF). The top 10 entries were visualized (Q < 0.05) and ranked by gene size. BP was mainly involved in the positive regulation of gene expression, positive regulation of transcription from RNA polymerase II promoter, positive regulation of cell proliferation, positive regulation of transcription, DNA-templated, negative regulation of transcription from RNA polymerase II promoter, response to drug, negative regulation of apoptotic process, regulation of transcription from RNA polymerase II promoter, positive regulation of mitogen-activated protein kinase (MAPK) cascade, and response to estradiol. CC was mainly involved in cytoplasm, nucleoplasm, cytosol, nucleus, chromatin, macromolecular complex, receptor complex, transcription factor complex, perinuclear region of cytoplasm, and death-inducing signaling complex. MF was mainly involved in protein binding, identical protein binding, transcription factor binding, enzyme binding, macromolecular complex binding, transcription factor activity, sequence-specific DNA binding, RNA polymerase II core promoter proximal region sequence-specific DNA binding, RNA polymerase II transcription factor activity, sequence-specific DNA binding, DNA binding, and ubiquitin protein ligase binding. All 3 processes with respective involvement are shown in Figure GO enrichment analysis of key targets. Note: red represents biological processes, blue represents cellular component, and green represents molecular function. GO = gene ontology.98 pathways were obtained by KEGG enrichment analysis through the DAVID data platform, and the top 20 pathways were screened for visualization, which showed that the key targets were mainly involved in Pathways in cancer, phosphatidylinositol-3-kinaseRAC–serine/threonine-protein kinase signaling pathway, Kaposi sarcoma-associated herpesvirus infection, Human cytomegalovirus infection, Measles, Hepatitis C, Hepatitis B, Epstein-Barr virus infection, Proteoglycans in cancer, Lipid and atherosclerosis, MAPK signaling pathway, Prostate cancer, aryl hydrocarbon receptor nuclear translocator signaling pathway, Alcoholic liver disease, Salmonella infection, Human papillomavirus infection, Bladder cancer, Legionellosis, Pancreatic cancer, Small cell lung cancer as shown in Figure Signaling pathways.HIF-1 = aryl hydrocarbon receptor nuclear translocator, MAPK = mitogen-activated protein kinase, PI3K-Akt = phosphatidylinositol-3-kinaseRAC–serine/threonine-protein kinase.KEGG enrichment analysis of key targets Note: the x-axis represents the gene ratio, the y-axis represents the enriched pathways, the size of the dots indicates the gene number, and the color of the dots represents the | PMC10470707 |
3.6. Results of molecular docking | INS | TP53, INS, IL6, VEGFA, MYC, CASP3, ESR1, EGF, CCND1, PPARG, ERBB2, NFKBIA, TLR4, RELA, and CASP8 were selected to find the corresponding effective chemical components for molecular docking, and the docking scores were used to determine the affinity. The docking results are shown in Figure Results of molecular docking.Core compound-core target molecular docking diagram. (A) Molecular docking diagram of VEGFA and Vanillic acid; (B) molecular docking diagram of PPARG and Protocatechuic aldehyde; (C) molecular docking diagram of PPARG and Vanillic acid; (D) molecular docking diagram of PPARG and 3,5-dimethyl-4-hydroxybenzaldehyde. | PMC10470707 |
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3.12. Comparison of adverse reactions between the 2 groups | thrombocytopenia, nausea, vomiting, RA | ADVERSE EVENTS, THROMBOCYTOPENIA, ADVERSE REACTION, ADVERSE REACTIONS | There were no adverse events that seriously affected the treatment of patients in both groups. Two patients had a mild elevation of glutamate transaminase, 1 patient had nausea, and 1 patient had thrombocytopenia during the treatment in the treatment group, and the incidence of adverse reactions was 8.2%. In the control group, 2 patients had a mild elevation of glutamate transaminase, and 1 patient had vomiting during the treatment period, with an adverse reaction incidence of 6.0%. All of them received symptomatic treatment, which did not affect RA treatment, and there was no significant difference in the incidence of adverse reactions between the 2 groups. | PMC10470707 |
4. Discussion | cancer, vasculitis, pain, RA synovitis, morning stiffness, Synovitis, pannus, joints, RA | INS, CANCER, VASCULITIS, DISEASE, NEOVASCULARIZATION, SYNOVITIS, PANNUS, PATHOLOGY | The clinical drugs used for the treatment of RA can be broadly classified into 2 categories according to their mode of action: those for the improvement of symptoms and those for the improvement of cartilage metabolism. However, symptom improvement drugs cannot fundamentally reverse the development of RA pathology. With the development of Chinese medicine extraction technology, the application of Chinese medicine preparation extracts for the treatment of RA has been increasingly reported and supported by relevant basic experimental studies. JSQS can effectively protect chondrocytes and maintain the integrity of cartilage, reduce the level of inflammatory cytokines, and play a certain therapeutic role in RA.In this study, 141 chiropteran capsule targets were mapped to 4791 RA target genes, 118 anti-RA key targets of JSQS were obtained, and a JSQS-key target-RA composite target network map was constructed. The quercetin in Quercetin has an anti-inflammatory effect on RA by reducing the ratio of MMP-13/TIMP-1, promoting the inhibition of cartilage extracellular matrix degradation, and protecting articular cartilage.The results of KEGG enrichment analysis showed that JSQS were mainly used to treat RA through the pathways in cancer, phosphatidylinositol-3-kinaseRAC–serine/threonine-protein kinase signaling pathway, MAPK signaling pathway, and aryl hydrocarbon receptor nuclear translocator signaling pathway, among others. The topological analysis of this study showed that TP53, INS, IL6, VEGFA, MYC, CASP3, ESR1, EGF, CCND1, PPARG, ERBB2, NFKBIA, TLR4, RELA, and CASP8 are the core key targets. Molecular docking was performed according to the corresponding active chemical components, suggesting binding fractions of >70 between PPARG and VEGFA and 3,5-dimethyl-4-hydroxybenzaldehyde, protocatechuic-aldehyde, vanillic acid, which had good binding energy. It indicated that these active chemical components might play an important role in RA treatment. However, further in vivo and in vitro studies are needed to confirm the possible existence of a therapeutic axis.PPAR, a member of the nuclear receptor superfamily that regulates lipid, glucose, and amino acid metabolism,Synovitis and vasculitis of joints are the basic pathological factors of RA. Neovascularization is considered to be one of the main factors in the formation and maintenance of pannus in RA, and some studies have shown that VEGF can increase vascular permeability and play a very critical role in the formation and development of pannus in RA synovitis.This study identified the possible material basis and molecular mechanism of JSQS in RA treatment. Thus, our team conducted a controlled clinical trial to verify the therapeutic effect of JSQS in RA treatment. The results showed that the capsules could effectively improve morning stiffness, joint pain, VAS score, other indicators of RA, clinical symptoms, and patient quality of life. The results of laboratory tests, such as ESR, CRP, RF, and anti-CCP, also indicated effective disease control, reduced levels of inflammation-related factors, such as TNF-α, IL-6, and IL-17, and significantly improved Th17/Treg balance. In conclusion, the multicomponent, multipathway, and multitarget synergistic interventions of JSQS can significantly improve clinical symptoms and quality of life of RA patients and delay the progression of the disease. Insufficient sample size is a regret of this study due to time and financial constraints. After obtaining the preliminary results, we will carry out a multicenter study, and at the same time, we will optimize the design of the study protocol on the line, in order to provide a theoretical basis and guidance for the clinical application of Chinese medicine. | PMC10470707 |
Abbreviations: | rheumatoid arthritis, TD No., necrosis | RHEUMATOID ARTHRITIS, NECROSIS | anti-cyclic citrullinated peptideC-reactive proteinDatabase for Annotation, Visualization and Integrated Discoverydrug similarityerythrocyte sedimentation rateGene Ontologyinterleukin-17interleukin-6Jishe Qushi capsuleskyoto encyclopedia of genes and genomesmitogen-activated protein kinasemethotrexateprotein–protein interactionrheumatoid arthritisrheumatoid factortraditional Chinese medicineTraditional Chinese Medicine Systems Pharmacology Database and Analysis Platformhelper T cells 17tumor necrosis factorregulatory T cellsvisual analog scaleThe datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.The authors have no conflicts of interest to disclose.This research was supported by the Science and Technology Project of Traditional Chinese Medicine and Ethnic Medicine in Guizhou Province (QZYY-2020-076) and Guizhou University of Traditional Chinese Medicine National and Provincial Science and Technology Innovation Talent Team Cultivation Programs: GZUTCM TD No. [2022]004.All the patients signed the informed consent form, and the study was approved by Ethics Committee of The Second Affiliated Hospital of Guizhou University of Traditional Chinese Medicine (RYW2020012).How to cite this article: Li Y, Zhang N, Peng X, Ma W, Qin Y, Yao X, Huang C, Zhang X. Network pharmacology analysis and clinical verification of Jishe Qushi capsules in rheumatoid arthritis treatment. Medicine 2023;102:34(e34883) | PMC10470707 |
References | PMC10470707 |