Allergic rhinitis (AR) has become a tremendous disease burden worldwide. Only a few studies have explored the effects of environmental exposure on the prevalence of AR in children in China.2.5), air humidity, temperature, and passive smoking) with AR in adolescents aged 10–13 years in Chongqing. Data from 4,146 participants in urban and rural areas between March 2019 and May 2019 were collected.In the present study, we investigated the associations of environmental exposure  was marginally associated with a high risk of AR.The overall prevalence of AR was 17.50% in adolescents. After adjusting for other covariates, AR was positively correlated with the annual mean PM2.5 exposure, high air temperature, and low air humidity were associated with a high risk of AR in adolescents. Our findings have potential implications for public health strategies and interventions aimed at reducing the burden of AR in adolescents.High PM Allergic rhinitis (AR) affects 2–25% of children and has become a tremendous disease burden worldwide , 2. With2.5 pollution was the top concern in China in the last decade. According to the Environmental Quality Report (2015) released by the Ministry of Environmental Protection (MEP) (http://www.zhb.gov.cn/), the annual mean PM2.5 concentration in China was 50 μg/m3 (ranging from 11 to 125 μg/m3), which far exceeds the new National Ambient Air Quality Standard II  of 35 μg/m3. A cross-sectional study found that higher air pollution levels were significantly associated with a greater risk for respiratory diseases and decreased lung function in Chinese children .Anthropometric measurements were conducted by well-trained pediatric nurses. Well-trained nurses from the Children's Hospital of Chongqing Medical University carried out the physical examinations. After they were rigorously trained, an examination based on the operation manual was carried out, and the training was according to the anthropometric examination guidelines recommended by the World Health Organization (WHO) . All antThe diagnosis of current AR followed the clinical practice guideline of allergic rhinitis . The dia2.5 for each participant were assessed using satellite-based PM2.5 concentrations at 1-km spatial resolution. Monthly mean PM2.5 concentrations across China were estimated from 2018 to 2019 using machine learning approaches , and the Wilcoxon rank sum test was used for comparison of these variables between the two groups. The prevalence rates of AR were reported as numbers (n) and percentages of the total (%), and the χ2 test was used to test the difference between the two groups. Moreover, univariate logistic regression (model 1: crude model) was performed to investigate the association between PM2.5 and AR risk. Then, age, sex, BMI, PS, family income, parental marital status, paternal education level, and mother's education level were adjusted for multivariate logistic regression in model 2. All statistical analysis was performed by SAS 9.4 software . A significant difference was defined by an α level of 0.05 with a two-sided test.Differences in anthropometric variables between the two groups were assessed using Student's P = 0.0022).The general characteristics of the participants are shown in 2.5 concentration, monthly mean PM2.5 concentration, and air temperature in the AR group were significantly higher than those of in the non-rhinitis group, while the annual mean and monthly mean air humidity in the AR group were significantly lower than those in the non-rhinitis group , monthly mean PM2.5 concentration , annual mean air temperature , and monthly mean air temperature  but negatively related to annual mean air humidity , after adjusting for demographic and other covariates .Furthermore, the association between the PManalysis . Similar2.5 was positively associated with the risk of AR after adjusting for air temperature and humidity . PS was marginally associated with a high risk of AR . Also, a higher mother's education level  was associated with a high risk of AR  Phase Three found that current AR in the 13- to 14-year age group was 14.6%, which was consistent with our results . The pre2.5 concentration worldwide. In 2013, only 4.1% of Chinese cities achieved the annual mean standard of ≤ 35 μg/m3  and second-level criterion of the Chinese national air quality standard (35 μg/m3).China had the highest PM35 μg/m3 . The ann.2 μg/m3 . During hongqing . However2.5 and AR risk remains inconsistent. In a pooled analysis of six birth cohorts, point estimates for associations between nitrogen dioxide, PM2.5 mass, and PM2.5 absorbance with AR were positive, but only PM2.5 was statistically significant . PM components, in particular iron, copper, and zinc, reflect poorly regulated non-tailpipe road traffic emissions, which may increase the risk of AR in children. Future studies should analyze the effect of the concentration and composition of different PM2.5 on AR risk.The present study has several limitations. First, this study is a cross-sectional study. Longitudinal analysis is required to study the incidence of AR, and the association between risk factors and AR incidence in the four seasons. Second, information on allergic sensitization or inflammatory cytokine levels was undetected for further confirmation of the diagnosis of AR. Our study has focused on the combination of nasal and conjunctival symptoms (in the absence of intercurrent infection) and a pediatrician's physical examination as the most relevant definition of AR. This may lead to a few overdiagnosis of AR since we did not perform atopy tests, including SPTs and sIgE tests and NAC, to confirm the allergen of the children with rhinitis. Third, we only analyzed the concentrations of PM2.5 exposure, high air temperature, and low air humidity were associated with a high risk of AR in adolescents, and PS was associated with a higher risk of AR.In conclusion, this study partially filled in the gap in the epidemiological investigation of AR in Chinese adolescents. High PMThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Institutional Review Board at the Children's Hospital of Chongqing Medical University. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.XL conceived and designed the study. FLia, FLiu, and XA collected the data. XL, CQ, PZ, WF, and XT participated in the physical measurement. CQ wrote the manuscript. All authors critically reviewed and approved the final manuscript.This work was supported by the National Key Research and Development Project of the Ministry of Science and Technology of China (2017YFC0211705), the Major Health Project of Chongqing Science and Technology Bureau (No. CSTC2021jscx-gksb-N0001), CQMU Program for Youth Innovation in Future Medicine (No. W0088), Young and Middle-aged Medical Outstanding Expert Project of Chongqing Municipal Health Commission, Joint Medical Research Project of Chongqing Municipal Health Commission and Chongqing Science and Technology Bureau (2020MSXM062), Technology Foresight and Institutional Innovation Project of Chongqing Science and Technology Bureau (cstc2020jsyj-zzysbAX0016), Young Scientists Fund Program of the National Natural Science Foundation of China (81502826), and Young Scientists Fund Program of the Education Commission of Chongqing (KJQN201900443). The funders had no role in the study design, the data collection and analysis, the decision to publish, or the preparation of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Compared to the isotype control group, DOX concentrations in six tissues slightly decreased under AR pre-administration but only showed statistical significance (p < 0.05) in the liver. Using network analysis, we showed that five of the nine representative AR components were not localized to the vicinity of the DOX disposition-associated module. These findings suggest that AR may mitigate DOX-induced toxicity by affecting drug targets rather than drug disposition.Doxorubicin (DOX) is an essential component in chemotherapy, and Astragali Radix (AR) is a widely used tonic herbal medicine. The combination of DOX and AR offers widespread, well-documented advantages in treating cancer, e.g., reducing the risk of adverse effects. This study mainly aims to uncover the impact of AR on DOX disposition in vivo. Rats received a single intravenous dose of 5 mg/kg DOX following a single-dose co-treatment or multiple-dose pre-treatment of AR (10 g/kg × 1 or × 10). The concentrations of DOX in rat plasma and six tissues, including heart, liver, lung, kidney, spleen, and skeletal muscle, were determined by a fully validated LC-MS/MS method. A network-based approach was further employed to quantify the relationships between enzymes that metabolize and transport DOX and the targets of nine representative AR components in the human protein–protein interactome. We found that short-term (≤10 d) AR administration was ineffective in changing the plasma pharmacokinetics of DOX in terms of the area under the concentration–time curve (AUC, 1303.35 ± 271.74 μg/L*h versus 1208.74 ± 145.35 μg/L*h,  Doxorubicin (DOX), also known as Adriamycin, is widely used in clinics for the treatment of many different types of cancer, such as acute myeloid leukemia and breast cancer . DOX stoAstragali Radix (AR) is a common, traditional Chinese medicine that has long been used as an anti-oxidative and anti-inflammatory herbal prescription for multiple complex diseases, from diabetes to asthma and cancer . EvidencDOX administered as a conventional injection is widely distributed in the plasma and tissues but can hardly cross the blood–brain barrier . There aHere, we hypothesized that, in addition to acting on DOX targets, AR might mitigate the side effects by reducing DOX exposure in vivo. To test this hypothesis, we developed and validated a simple, specific, and sensitive liquid chromatography–tandem mass spectrometry (LC-MS/MS) method for the determination of DOX in rat plasma and six tissues , allowing us to analyze the effects of AR co-treatment and pre-treatment on DOX disposition. Moreover, enzymes that metabolize and transport DOX are not randomly scattered in the human protein–protein interactome (PPI), but tend to cluster in the same neighborhood, known as the DOX disposition-associated module . The tar2 > 0.995) in all biological samples with broad dynamic quantification ranges, from two to three orders of magnitude (The representative extracted ion chromatograms of DOX and IS in rat plasma and tissues are illustrated in agnitude . Intra-dagnitude . Recoveragnitude . The posagnitude . Moreoveagnitude . These rmax), the total area under the concentration–time curve (AUC0-t), and the half-life (t1/2) were in the range of 838.4 ng/mL to 2157.8 ng/mL, 1054.7 µg/L*h to 1940.1 µg/L*h, and 25.0 h to 39.3 h, respectively. These results were consistent with previously reported animal experiment data . TheIn summary, we reported the effects of AR on the pharmacokinetics and tissue distribution of DOX using a novel analytical method to determine DOX in rat plasma and six tissues. Specifically, we observed that the plasma pharmacokinetics of DOX was not affected by AR co- and pre-treatment, whereas DOX concentrations in six tissues were slightly decreased under AR pre-administration for 10 consecutive days. In addition, network-based measures revealed that representative AR components could hardly act on the enzymes involved in DOX metabolism and transport. This study provided valuable data for understanding the DOX–AR combination and might help uncover the mechanisms of AR in alleviating DOX-induced toxicity.


YU-Pingfeng San (YPFS) can regulate inflammatory response to alleviate the symptoms of nasal congestion and runny rose in allergic rhinitis (AR). However, the mechanism of action remains unclear. In this study, 30 active ingredients of three effective herbs included in YPFS and 140 AR/YPFS-related genes were identified by database analysis. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that the targets were mainly enriched in immune inflammatory-related biological processes and pathways. Finally, three hub gene targeting epidermal growth factor receptor (EGFR), mitogen-activated protein kinase 1 (MAPK1), and protein kinase B1 (AKT1) related to YPFS and AR were identified by network pharmacology analysis. YPFS treatment decreased the expression of EGFR, MAPK1, and AKT1 in ovalbumin (OVA)-induced AR mice and impaired the production of inflammatory factors interleukin (IL)-4, IL-5, and IL-13, thus alleviating immunoglobulin E (IgE) production and the symptoms of scratching nose in AR. Through molecular docking analysis, we found that the active ingredients decursin, anomalin, and wogonin of YPFS could bind to EGFR, MAPK1, and AKT1 proteins. Moreover, decursin treatment impaired the expression of IL-4 and IL-5 in human PBMCs. These results suggested that YPFS could alleviate the AR inflammatory responses by targeting EGFR, MAPK1, and AKT1, showing the mechanism of action of YPFS in AR treatment. Allergic rhinitis (AR) is an allergic disease of the nasal mucosa caused by a variety of factors. Clinical manifestations are characterized by nasal itching, sneezing, hypersecretion, and nasal mucosal swelling . AR is oAstragali Radix , Atractylodis Macrocephalae Rhizoma , and Saposhnikoviae Radix , at a content ratio of 3:1:1 . Those chemical compounds of YPFS with identifiable properties of absorption, distribution, metabolism, and excretion (ADME) and druggable properties, were selected as active components for further target prediction, with a cutoff of oral bioavailability of 30% and drug-likeness (DL) of 0.18  and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of AR/YPFS-related genes was performed to identify genes in the biological processes (BP), cellular components (CC), molecular functions (MF) categories, and pathways using the ClusterProfiler R Package . The dat8 .9 a protein interaction functional enrichment analysis website, to construct the Protein–protein interaction (PPI) network with a cutoff of high confidence of 0.7  2014-0007). All mice were raised in a specific pathogen-free level environment in the Research Building of Yuhuangding Hospital, Yantai. All experimental animals were fed with standardized sterile feed and water, which was sterilized by high-pressure steam and cooled to room temperature. All experimental animals were dissected after fasting for 12 h. All animal experiments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the Yantai Yuhuangding Animal Ethics Committee.Ovalbumin-induced AR mice were induced through two stages: The basic sensitization stage and the stimulation stage. In the basic sensitization stage, 200 μl of a mixture comprising 40 μg OVA  and 2 mg aluminum hydroxide  gel were injected intraperitoneally into the OVA-induced AR group seven times on days 1, 3, 5, 7, 9, 11, and 13. In the stimulation stage, mice received nasal drops comprising 20 μl of 5% OVA solution following atomization with 5% ovalbumin saline solution in a homemade simple atomizer for 30 min, from days 21 to 27 . The micYPFS comprises three kinds of herbs, HQ , BZ , and FF  at a content ratio of 3:1:1. The three herbs were mixed, soaked in water for 1 h, and then decocted two times using an automatic decocting machine for 1.5 h each time. The decoctions  were filtered through double gauze to concentrate the filtrate to the final concentration of 1 g/ml and stored at 4°C until use .YPFS was intragastrically administered to the AR mice in the YPFS group at a dose of 130 mg/20 g body weight for 14 days continuously . The samThe serum was separated from the upper layer of the blood after centrifugation for 20 min at 900 g. The concentrations of immunoglobulin E (IgE), interleukin (IL)-4, IL-5, IL-13, and interferon-gamma (IFN-γ) in mouse serum were detected using an Enzyme-linked immunosorbent assay (ELISA) kit  according to the manufacturer’s instructions.–ΔΔCT method  using the Trizol reagent  and reverse transcribed into cDNA using a reverse transcription kit . The qPCR step was performed using an SYBR Green qPCR Mix kit (SparkJade) in a StepOnePlus fluorescence quantitative PCR instrument . The expression levels of the genes encoding IL-4, IL-5, epidermal growth factor receptor (EGFR), mitogen-activated protein kinase 1 (MAPK1), and protein kinase B1 (AKT1) were detected. The relative expression level was calculated using the 2T method . The pri10 Ingredients involved in regulating immune inflammation were selected for molecular docking with the three hub genes decursin (11). The eutectic ligands isolated from the receptor and the active pocket of each target were identified using AutodockTools-1.5.6  was used to isolate Human PBMCs according to the manufacturer’s instructions. The Human PBMCs were cultured in 10% fetal bovine serum and 90% Roswell Park Memorial Institute-1640 medium for 48 h. Then, decursin  was added to PBMCs at concentrations of 0, 5, and 10 μM, respectively, and incubated for 48 h.t-tests, and p-value < 0.05 was considered statistically significant. The results are shown as the mean and standard deviation.The experimental data were analyzed using GraphPad Prism 8  using unpaired The workflow chart for network pharmacology analysis and experimental verification is shown in A total of 49 AR-associated target genes were obtained from the DrugBank database, 1,311 AR-associated target genes were obtained from the Genecards database, and 214 AR-associated target genes were obtained from the OMIM database. After combining the related target genes from the three databases and the removal of duplicates, a total of 945 AR-associated target genes were obtained . About 1Functional enrichment analysis was performed on the 140 AR/YPFS-related genes using GO, which showed that these genes were mainly enriched in processes involved in immune regulation, such as regulation of inflammatory response, leukocyte migration and chemotaxis, regulation of leukocyte migration, lymphocyte differentiation, and T cell activation . KEGG paTo understand the interaction regulation of the 140 AR/YPFS-related genes, we uploaded them to the String database to obtain a PPI network diagram . Using CCompared with those in the control group, serum IgE, IL-4, IL-5, and IL-13 levels were significantly higher in the OVA-induced AR group . The IFNCompared with those in the control group, the frequency and times of nasal scratching in the OVA-induced AR group mice increased significantly , whereasEgfr, Mapk1, and Akt1 in the nasal mucosa of the OVA-induced AR group were significantly increased, and no significant expression differences were found between the YPFS treatment group and the control group  and 2 types (Th2) immune responses and T cell cytokine production. Through + T cell differentiation to Th2 cells, which produce the cytokines IL-4, IL-5, and IL-13, thus inducing a classical type 2 inflammatory response  to produce the type 2 cytokines IL-5 and IL-13 and also acting on dendritic cells (DCs). DCs present antigens to resident lymph nodes to activate CD4response . In our AR is an allergic disease of the nasal mucosa caused by the imbalance of differentiation of T cells. YPFS is recommended for the management of AR by the Chinese medicine clinical practice guidelines and can relieve the clinical symptoms of AR. However, how YPFS functions is still not clear. YPFS could impair the inflammatory response by reducing thymic stromal lymphopoietin (TSLP), tumor necrosis factor-alpha (TNFα), and IL-6 production . YPFS alKEGG pathway enrichment analysis showed that 140 AR/YPFS-related genes were enriched in pathways involved in regulating AR, such as the PI3K/AKT pathway, the hypoxia-inducible factor 1 alpha (HIF1α) signaling pathway, and the MAPK, RAS, and JAK/STAT signaling pathways. The PI3K/AKT signaling pathway is involved in a variety of AR inflammatory responses, including Th2-mediated inflammation , the perThree hub genes, EGFR, MAPK1, and AKT1, were identified through PPI analysis and Cytoscape analysis. Accumulating studies have shown that EGFR, MAPK1, and AKT1 participate in AR regulation. EGFR was significantly elevated in patients with AR , and thein vivo by reducing the number of eosinophils, increasing eosinophil apoptosis, reducing airway mucus production, and reducing airway hyperresponsiveness. Studies have shown that wogonin might play a role in the treatment of allergic diseases by regulating Th1/Th2 cytokine imbalance and histamine release in mast cells (The compounds decursin, anomalin, and wogonin, screened from YPFS, can potentially bind to EGFR, MAPK, and AKT and inhibit their expression. Decursin has been reported to be associated with inflammatory immunity . A novelst cells . These sIn vivo experiments showed that YPFS reduced the IgE level in serum, decreased the production of Th2-type cytokines, and relieved AR symptoms. Meanwhile, EGFR, MAPK1, and AKT1 were found to be downregulated by YPFS in OVA-induced AR mice, and be targeted by active ingredients decursin, anomalin, and wogonin from YPFS through molecular docking prediction. These results showed that YPFS could relieve the AR inflammatory response and symptoms mainly via active ingredients decursin, anomalin, and wogonin targeting and inhibiting EGFR, MAPK1, and AKT1, which may provide novel ingredients for AR treatment. However, these results should be further verified using more detailed molecular mechanism studies, contributing to the in-depth research and application of traditional Chinese medicines.In summary, we identified the active ingredients in YPFS and 140 AR/YPFS-related genes through network pharmacology analysis. The original contributions presented in this study are included in the article/The studies involving human participants were reviewed and approved by Yantai Yuhuangding Animal Ethics Committee. The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by Yantai Yuhuangding Animal Ethics Committee.YY and YL designed and directed the study. ZL and QS organized the public data and wrote the manuscript. ZL, XL, XS, QS, and ZS performed the experimental work and analyzed the data. FS, CL, and QS took charge of data visualization. XS, YZ, and YL revised the manuscript. All authors contributed to the article and approved the submitted version.


It is controversial as to whether the withdrawal of antiresorptive (AR) agents is necessary while treating medication-related osteonecrosis of the jaw (MRONJ). In this study, we investigated whether a drug holiday promoted sequestrum separation and improved the surgical outcomes of MRONJ patients with malignant tumors, who were undergoing high-dose AR therapy. In total, we included 103 MRONJ patients with malignant tumors as their primary disease who underwent surgery at Nagasaki University Hospital or Kansai Medical University Hospital from January 2009 to December 2020. We recorded the patients’ age, sex, primary disease, MRONJ stage, type and administration period of the AR agent, presence of diabetes, corticosteroid use, drug holiday period, white blood cell count, serum albumin, serum creatinine, outcomes, and computed tomography findings. The relationships between a drug holiday and sequestrum separation, and between a drug holiday and outcome, were analyzed. Drug holidays of 60, 90, and 120 days were not significant factors of sequestrum separation and did not influence patients’ surgical outcomes as per the univariate and multivariate analyses. MRONJ patients with cancer as their primary disease should be operated upon immediately and without drug holidays if their general condition permits surgery. Medication-related osteonecrosis of the jaw (MRONJ) is known to be one of the severe side effects of antiresorptive (AR) agents, such as bisphosphonate (BP) and denosumab (Dmab) ,2. The pSome systematic reviews have shown that the healing rate of MRONJ patients who undergo surgery is higher than that of those who receive conservative therapy ,5,6. HayThus, it is unclear whether a drug holiday of an AR agent is necessary before surgical treatment. The position papers of both AAOMS and JSOMS suggested that AR therapy may need to be discontinued until the MRONJ treatment is complete, although a drug holiday is not recommended for patients with high fracture risk or progressive bone metastases ,3. HowevThis was a retrospective observational study. From January 2009 to December 2020, 70 out of 175 MRONJ patients with malignant tumors, who underwent treatment at Nagasaki University Hospital or Kansai Medical University Hospital, received only conservative therapy. Of the 105 patients who underwent surgery, 103 were included in the study, excluding two patients whose data could not be collected.We recorded the following patient data: age, sex, primary disease, MRONJ site, MRONJ stage, type of AR agent, administration period of AR agent, presence of diabetes, corticosteroid use, drug holiday period, white blood cell count, serum albumin, serum creatinine, surgical procedure, outcomes, and computed tomography (CT) findings. The primary disease category was divided into solid cancer and multiple myeloma. The MRONJ stage was determined at the time of initial diagnosis and according to the AAOMS 2014) [014 [2]. Postoperative follow-up was based on monthly medical examinations and CT images every three months. Patients were asked to visit the hospital as per their general condition. The treatment outcome was divided into healing and non-healing. Healing was defined as a lack of osteolysis progression on the CT images, and the disappearance of all clinical symptoms, including bone exposure, pain, and infection, and the date of healing, was registered. We examined the relationship between a preoperative drug holiday of AR agents and treatment outcome in terms of healing time, as well as the relationship between the drug holidays and sequestrum separation.p-value of ˂ 0.05 was considered significant. The correlation between each variable and sequestrum separation was analyzed using the one-way ANOVA for continuous variables and Fisher’s exact test for categorical variables. The correlation between each variable and treatment outcome was analyzed using univariate and multivariate cox regression analyses. The relationship between a drug holiday and the treatment outcome was illustrated using the Kaplan–Meier method.All statistical analyses were performed using SPSS software , and a two-tailed The study protocol conformed to the ethical guidelines of the Declaration of Helsinki and the Ethical Guidelines for Medical and Biological Research Involving Human Subjects by the Ministry of Health, Labor, and Welfare of Japan. This study was approved by the Institutional Review Board (#21021509) of Nagasaki University Hospital. The need for informed consent was waived by the IRB of Nagasaki University Hospital. This was a retrospective study; thus, the research plan was published on the homepage of the participating hospitals according to the instructions of the IRB, and in accordance with the guaranteed opt-out opportunity.The number of MRONJ patients whose primary disease was a malignancy and who underwent surgical treatment was 103 . Nine pap = 0.095), 90 days (p = 0.641), or 120 days (p = 0.246) did not become a significant factor of sequester separation (Sequestrum separation was found in 9 out of 103 patients (8.7%). There were no factors related to the separation. A drug holiday before CT for more than 60 days (paration .p = 0.043), lower serum creatinine (p = 0.003), and localized osteolytic area (p = 0.014) were significantly correlated with poor treatment outcome (p = 0.220), 90 days (p = 0.394), or 120 days (p = 0.178) did not improve treatment outcomes.A univariate analysis revealed no significant factors related to the treatment outcome. According to a multivariate analysis using stepwise selection, the use of steroid ( outcome . Figure This study showed that drug holidays of 60, 90 and 120 days were not significant factors of sequestrum separation, and did not influence surgical outcomes in cancer patients with MRONJ undergoing surgical treatment.It is unclear whether surgical treatment or conservative treatment is the better strategy for MRONJ. In a retrospective multicenter study, the authors reported the superiority of surgical therapy over conservative therapy . TherefoThe AAOMS position paper recommended a 3-month drug holiday of a low-dose AR agent before invasive dental surgery in 2009 , but theIt is often not appropriate to withdraw the AR agent therapeutically in cancer patients; therefore, the position papers of AAOMS and JSOMS suggested that invasive dental treatments should be avoided ,3. HowevBP is bound tightly to the bone matrix, and osteoclasts take up BP into the cytoplasm during bone resorption, subsequently leading to apoptosis. BP may remain in the bone for months or years, and the bone resorption inhibitory effect lasts for a long time. Dmab is a monoclonal antibody which selectively binds to RANKL. Dmab is not absorbed in the bone tissue, but circulates in the blood. Dmab prevents the formation, function, and survival of osteoclasts, and has a half-life of 26 days in the blood ,14. FromIt is not clear how long AR agents should be withdrawn to recover from osteoclast inhibition. Recently, we examined surgical materials of osteoporosis patients receiving oral BP immunohistologically and found that osteoclast morphology did not recover with a drug holiday of less than 12 months . AlthougThere are some limitations in the study. Firstly, this is a retrospective analysis using a small number of patients; therefore, it is unclear whether the results obtained can be generalized. Secondly, this study was a cross-sectional study and did not longitudinally observe the effects of a drug holiday. However, to the best of our knowledge, this is the first study to investigate how a preoperative drug holiday affects sequestrum separation and treatment outcomes in patients receiving high-dose AR agent therapy. In future, studies with larger sample sizes and more detailed examinations should be conducted to confirm our results.Drug holidays of 60, 90 and 120 days were not significant factors for sequestrum separation and did not influence the surgical outcomes of cancer patients with MRONJ undergoing surgical treatment. MRONJ patients with cancer as their primary disease should be operated upon immediately without drug holidays if their general condition permits surgery.


The use of tele-rehabilitation in children was limited before the COVID-19 pandemic, due to culture, technology access, regulatory and reimbursement barriers.The study was conducted according to the CHERRIES (Checklist for reporting results of internet E-surveys) guidelines in order to provide quantitative and qualitative data about experience of patients with disabilities and their caregivers during Phase 1 of the COVID-19 pandemic, and their level of satisfaction. An online survey was developed using Google Forms and sent via email. The outcome measures were rated using a 5-point Likert Scale. Two additional open-ended questions were used to collect qualitative data.One hundred twenty-eight out of 261 families responded to the survey: 80.5% of the caregivers reported they were satisfied with the tele-rehabilitation. More than a half (53%) of the families reported a high level of satisfaction with the involvement they received in defining and sharing of rehabilitation goals.The implementation of telehealth during the COVID-19 lockdown has allowed us to gain more information about the potential of tele-rehabilitation, and resulted in an excellent satisfaction of caregivers. With appropriate education and consistent models of care, an increased use of telehealth may provide advances in remote patient care.Not applicable.The online version contains supplementary material available at 10.1186/s12875-022-01747-2. Following the spread of the COVID-19 outbreak in Italy in February 2020, strict measures have been implemented by the Italian Government. As a primary intervention to contain the SARS-CoV-2 infection spread, the Italian Government aimed at limiting the virus transmission by imposing a severe lockdown with daily-life restrictions and closure of educational, social, and manufacturing activities . Conversely, 18 (14%) caregivers were partially satisfied and 7 (5.5%) dissatisfied. None of the caregivers reported that they were very dissatisfied."level of satisfaction of tele-rehabilitation" (Question 33) on the dichotomized answer to the question "caregiver’s participation in the therapy session" (Question 14) [likelihood ratio test: p = 0.1899], even though there was a statistically significant association between the dichotomized answer to Question number 33 and the dichotomized answer to Question number 14 (Fisher’s exact test: p = 0.048).Logistic regression showed no influence of the answer to the question More than a half (68.5%) of the families reported a high level of satisfaction with the involvement received in the definition and sharing of rehabilitation goals, 47 (37%) stated that they were satisfied, 9 (7%) partially satisfied, 4 (3%) dissatisfied, 0 very dissatisfied.Concerning the achievement of work objectives provided by the IRP, 42 (32%) families were very satisfied, 69 (53%) satisfied, 13 (10%) partially satisfied, 6 (5%) dissatisfied, 0 very dissatisfied.Satisfaction with tele-rehabilitation in relation to the child’s ability to participate was rated as excellent by 70.3% of respondents [95% Conf. Interval].Satisfaction degree of tele-rehabilitation in relation to the child’s ability to participate according to age groups and to type of rehabilitation program are reported in Table The qualitative remarks at the end of the survey (open-ended questions) showed that many participants described the availability of the therapists as a positive aspect (30 families), as well as the continuity of the rehabilitation projects (20 families). Three caregivers expressed their desire to continue using video calls for meetings or therapies.We obtained a total of 130 open-ended responses (79 responses to the first question on aspects most appreciated and 51 responses to the second question that addressed comments or suggestions). After removing irrelevant responses, 74 responses for the first question and 30 for the second were finally analyzed.availability and competence of the therapists, continuity of treatment, involvement of caregivers, organizational aspects such as hours, flexibility, etc., revelation about feasibility and effectiveness. The number of participants supporting each theme can be found in Table The analysis of the first question on aspects most appreciated generated 5 themes: technical aspects , propose tele-rehabilitation as a possible alternative, perhaps integrated with face-to-face therapy, claim that face-to-face therapy is irreplaceable. The number of participants supporting each theme can be found in Table The analysis of the second question that addressed comments or suggestions generated 3 themes: According to our experience and literature , childreA particularly useful tool for the assessment of caregivers’ satisfaction is a questionnaire-based survey, because caregivers can answer questions anonymously and do not have to fear negative consequences because of their opinion. Moreover, self-administered questionnaires are—in contrast to interviews –cheaper, faster, and regarded as more voluntary .Our study indicates that tele-rehabilitation of children with neurodevelopmental disabilities through tele-medicine is well accepted by the families. Indeed, 80.5% of them reported that they were satisfied with remote rehabilitation.The possibility to continue treatment during the pandemic and the availability of therapists from remote were considered the main advantages.These findings are in line with previous preliminary data –22, whic"level of satisfaction of remote rehabilitation" and the dichotomized answer to the question "caregiver’s participation in the therapy session" (Fisher’s exact test: p = 0.048); caregivers who had participated in the therapy session reported that they were satisfied with remote rehabilitation. Also, our experience seems to show that the parents’ role in facilitating the rehabilitation interventions is much more prominent using an online modality, as they can be more actively engaged by the therapists, thus increasing their self-esteem in parenting. In addition, according to our experience and literature, to be close to their own needs and those of their children, it seems to be useful to parents for both psychological and educational aspects [Even if not directly assessed by the study tool, our direct experience during the pandemic seems to shows that tele-medicine in child neuropsychiatry produces positive effects for both the children and the families. On the child-side, we promoted and enabled continuity of care, maintained social contacts with their therapists, and reduced the risk of disrupting daily and weekly routines. Moreover, as extrapolations of this work, we can think that involving the parents in rehabilitation programs may improve the overall degree of satisfaction. Statistical analysis of data shows a statistically significant association between the dichotomized answer to Question “ aspects .The sample included in the present survey was highly representative of the usual customers of Italian Childhood and Adolescence Neurology and Psychiatry Units, and all the features of in-person rehabilitation that are usually offered have been covered remotely, although through innovative tools.The development of specific rehabilitation tools created by the therapists has allowed to translate to remote also therapies where physical contact is very relevant. An example is that of Equine-Assisted Therapy, where the therapists made video calls from the Department and created specific videos to maintain goals about the ability to relate and on self-care, and realized interactive PowerPoint files dedicated to the horse theme was created to allow working on communication skills, attention, concentration, and alternation. Moreover, therapists of Equine-Assisted Therapy have implemented the use of Augmentative Alternative Communication (AAC). Indeed, customized materials have been developed, some horse-themed choosing activities, stories about horse’s daily routine and other materials have been translated into AAC, for children with special communicative needs. Based on the results of the survey, we can infer that the relationship with the horse combined with a careful reformulation of the proposals has allowed maintaining high motivation to treatment despite the distance. As a result, 84% of the families rated the Equine-Assisted Therapy positively with regard to the child’s ability to participate, and positive evaluations were recorded for other types of intervention as well.The tools available to the families and the channels that were used were investigated in order to evaluate the accessibility to the tele-rehabilitation proposal: on-line platforms, e.g. Skype, were indicated as the most adequate instrument for rehabilitation purposes.Considering that this was the first experience of tele-rehabilitation for almost the entire sample, we anticipated there would be some limits and critical issues. Therefore, we assessed data from Sect. In order to understand whether some treatments carry more issues or pitfalls than others and can therefore be more difficult to convert to tele-rehabilitation, we analyzed the data about the child’s ability to participate and the data about general satisfaction for each type of rehabilitation program. Some disciplines may be more challenging to be performed through tele-therapy  and may require teaching of facilitation techniques to caregivers. In this view, the possibility to share previously recorded videos of rehabilitation sessions may help the families by supplying useful examples for continuity of care.Moreover, children with behavioral or attention problems may face more difficulties following tele-rehabilitation sessions rather than face-to-face engagement.One limitation of tele-rehabilitation that emerged from our survey is the child’s age, especially age younger than 3 years, even though the children in this age group were too few to permit further interpretation. As a matter of fact, toddlers need face-to-face rehabilitation treatments because of the importance of physical and relational contact. Indeed, they need treatments like physiotherapy or psychomotor therapy, where physical and relational contact is crucial.Even though limited to the COVID-19-related lockdown, the findings of our survey indicate a positive response of caregivers of children with neurodevelopmental disabilities to tele-rehabilitation.This result may encourage policy makers to implement these services into future healthcare models, recognizing the reduced costs of care and high patient/caregivers’ level of satisfaction. Indeed, considering the first positive response of caregivers of children with neurodevelopmental disabilities to tele-rehabilitation, during the course of the pandemic, Niguarda Hospital has implemented some tools for remote rehabilitation. The devices quota was increased , Zoom platform was installed on all computers (chosen as preferred method of communication for a company policy decision), and specific softwares for speech therapy rehabilitation in learning disabilities were purchased. For the future it could prove useful as remote rehabilitation improvement, a stable platform for rehabilitation interventions, telerehabilitation policies and procedures.Furthermore, with regard to the reimbursement system, it is important to consider that despite the Italian Ministry of Health issued a document on 17 March 2014 to provide guidelines for Telehealth development , telemedDuring the emergency due to Covid-19, the Lombardy Region issued a circular to allow delivery of therapy remotely, by voice, video-call, video web platforms for all Childhood and Adolescence Neuropsychiatric services, for pediatric patients and their families, in accordance with the Decree of the General Director of the Welfare DG of Lombardy Region N. 2906 of the 8th of March and N. 3553 of the 15th of March 2020.With this circular, during the pandemic emergency, tele-rehabilitation has been included in the list of reimbursed services and tariffs for these clinical services became comparable to those for the available usual care in presence.For the purpose of regulating the methods used to carry out tele-rehabilitation, as a result of the pandemic, on 17 December 2020 the new National Guidelines for the Provision of Telemedicine Services were approved by the Permanent State Regions and Autonomous Provinces Conference. This document is playing a crucial role in the development of telemedicine in Italy because it aims to ensure consistent development of telemedicine throughout the country, going beyond the individual regional, hospital and services particularities. Thanks to this document entitled “National guidelines for the provision of telemedicine services,” drafted by the Ministry of Health, healthcare services provided remotely are officially a part of the National Healthcare Service and an updated version replacing the previous guidelines of 2014.Italian regions must implement the Guidelines in their respective regional healthcare services. The Guidelines specify that the fee for a telemedicine service, tele-rehabilitation included, shall be equivalent to the fee for the same service when provided in the traditional way within the Basic Level of Assistance , including any cost-sharing. In this context, regions implemented telemedicine services within the Regional Healthcare Service, permitting a new method for providing health care, starting from tele-visits and limiting them to cases that involve ongoing care of patients who need ambulatory services and do not need to be examined.Although the results of our study give some insights about the value of tele-rehabilitation for parents of children with neurodevelopmental disabilities, some limitations should be emphasized, concerning the heterogeneous nature of the types of treatments that can receive different feedback from the same family.We are aware that the items and the questions identified for the questionnaire have been selected during the time of global lockdown, where the problems and the challenges were new for anyone. We tried to focus on the parents’ satisfaction about tele-rehabilitation, but we were unable to collect children’s opinions. So that, we cannot also exclude the possibility of missing relevant information to the remote care of children with neurodevelopmental disabilities. In order to capture the patient's satisfaction, for future research a useful approach can be to include in the questionnaire simple questions directed towards the children.Another limitation of our study can be identified in the moment when the questionnaire was administered, 20 weeks after the beginning of the intervention, which can be considered a quite long period, but we preferred to obtain the parents’ assessment closed to the end of the tele-rehabilitation process, that usually lasts at least four-five months.Telerehabilitation seems to have an impact on financial and social stress factors for patients, and other different study projects could capture this data. New job perspectives can be assumed to reduce the dissatisfaction reported by some parents, it is important to consider new ways of tele-rehabilitation: for example using more performing platforms or mixed interventions "face to face"—remote. For future research, in order to fully assess the role of the parent in rehabilitation with and without a component of tele-rehabilitation, it would be better to have a study with two separate cohorts that compare levels of involvement and parental satisfaction.The usual care of children with special needs has become even more challenging during the COVID-19 lockdown, since parents found themselves involved throughout the whole day and often without the support of educational and rehabilitation services. To mitigate the feeling of being alone in direct caregiving of children with disabilities, we have developed potentially effective strategies to support the families of children with disabilities using tele-medicine approaches. This experience has allowed us to gain more information about the potentiality of tele-rehabilitation and resulted in an excellent level of satisfaction of the caregivers. With appropriate education and consistent models of care, an increased use of telehealth may provide advances in remote patient care.Additional file 1.Additional file 2.


To determine the effects of augmented reality (AR) interventions on the function of the upper extremity (UE) and balance in children with spastic hemiplegic cerebral palsy (SHCP).post-hoc analysis was used to compare the groups in terms of balance. The data were analyzed by using SPSS version 21 and the level of significance was set at p < 0.05. Paired sample t-test and Wilcoxon signed-rank test was used for analyzing the changes in the total DASH and PBS scores within the groups, respectively. One-way ANOVA was used to determine the differences between the groups in the total DASH and PBS scores, while the Kruskal Wallis test was used for the differences between the groups in the PBS items. The data were analyzed by using SPSS version 21.In total, 30 children with SHCP, aged 6 to 12 years, were randomly divided into three interventional groups. Each group received an AR game, i.e., Balance It, Bubble Pop, or Scoop'd . The UE function and balance were assessed at the baseline and after 8 weeks of intervention through the Disability of Arm, Shoulder, and Hand (DASH) questionnaire and Pediatric Balance Scale (PBS), respectively. The mixed ANOVA was used to determine the combined with-in and between-the-groups differences in the function of the upper extremity. The Wilcoxon sign ranked test was used for with-in group changes, while the Kruskal Wallis test with the bonferroni correction p = 0.03). The total score of PBS also showed a significant difference between Bubble Pop and Balance It groups (p = 0.02).All the groups improved significantly in the total DASH and PBS scores post-intervention. A significant difference was determined in standing with one foot in front between Bubble Pop and Balance It groups (The AR interventions used in this study were found to be effective in improving the UE function and balance of children with SHCP. The Balance It game showed more promising results in improving the balance as compared with the other games, however, no significant difference was determined between the three AR games in terms of the UE function of the participants. Cerebral palsy (CP) is a group of permanent disorders of movement and posture causing activity limitations which are attributed to non-progressive disturbances in the brain occurring early in the development . AssociaSpastic hemiplegic cerebral palsy (SHCP) is associated with motor impairments , including deficits in motor coordination and muscular weakness . This haConventional approaches for the management of SHCP include medical, surgical, and rehabilitative strategies. Rehabilitation options typically include physiotherapy and occupational therapy . The useIn AR, the user has a real-time visual perception of every movement of the body, which is based upon the concept of mirror therapy . Since AAugmented reality therapy is an emerging intervention for the rehabilitation of neurological conditions such as CP. Based on the literature, the effects of AR interventions on the function of UE and balance of children with SHCP are not well known. In the light of limited evidence to support AR games for children with CP, and the need to determine the impact on specific sub-groups, this study aimed to determine the effect of AR intervention on the function of UE and balance in children with SHCP and to compare different AR games for their effectiveness in improving these aspects.A randomized clinical trial  was conducted at the National Institute of Rehabilitation Medicine (NIRM), Islamabad, Pakistan, for a time duration of 5 months, i.e., from November 2019 to March 2020. The study was initiated post approval from the Institutional Review Board and Ethics Committee of NIRM . Written informed consent was taken from the participants' legal guardians/next of kin.A total of 45 children with SHCP were assessed for eligibility, who visited NIRM during the recruitment period. However, 30 children fulfilled the inclusion criteria and showed a willingness to participate in the study and were thus recruited through a non-probability convenient sampling technique. The treatment groups were named according to the game that each group played. The AR games used in this study were designed and developed by WonderTree, Pakistan.The inclusion criteria were children with SHCP, aged between 6 and 12 years , who werChildren with attention deficits or short attention span, having musculoskeletal abnormalities and contractures, who had received or planned to receive motor therapy such as botulinum toxin or constraint-induced movement therapy in the past 6 months or during the study, those with visual and hearing impairment or a history of seizures which could be provoked by TV lights (confirmed by the pediatric neurologist and physiotherapist) were excluded from the study.n = 10), Scoop'd (n = 10), and Balance It (n = 10)  . RandomiThe apparatus included a Microsoft Kinect v2 sensor , which cAll the participants received 24 sessions (three sessions in a week), for a time duration of 8 weeks at the National Institute of Rehabilitation Medicine (NIRM), Islamabad, Pakistan. Each session lasted for 15 min. Before the intervention, the AR games were explained to the children, and a five-minute-long trial session was given to all the participants for better understanding. The intervention sessions were conducted in a quiet room. The participants were instructed to stand 4 feet away from the projection screen on which they could see themselves in real-time. One participant played the AR game at a time and every session was supervised by the treating physiotherapist.The participants were given verbal cues during the sessions to perform the required task and were encouraged to actively use both of their upper extremities. In each new session, the participants started with the game level which they had left in the last session. Furthermore, the total levels of the games were different, so an attempt was made to avoid any such influence on the results by providing the intervention for the same time duration to each group.This AR-based therapeutic game  had 30 lThis game  also conBalance It  consistePediatric Balance Scale (PBS), which has established validity  and relipost-hoc analysis was used to compare the groups. The effect size  was also calculated, and the level of significance was set at p < 0.05. The SPSS version 21 was used for the statistical analysis.The total score of DASH met the assumption of the parametric test so the mixed ANOVA was used to determine the combined with-in and between-the-groups differences in the function of the upper extremity. The total PBS score was not distributed normally, and the responses of PBS were on an ordinal scale, so the non-parametric Wilcoxon sign ranked test was used for with-in group changes, while the Kruskal Wallis test with the bonferroni correction In total, 30 children with SHCP participated in this study, of which 18 were males and 12 were females. The mean age of the participants was 8.76 ± 2.32 years. A comparison of the demographic and baseline characteristics revealed that there were no statistically significant differences among the three study groups with respect to the age, gender, side of impairment, GMFCS levels, and MAS grades at the start of the study. Therefore, all the groups were similar, comparable and homogenous in nature at the onset of the study .p < 0.05) in all the groups  standing on one foot , and placing alternate foot on stool  components of PBS. Pre–post analysis of the Bubble-Pop group showed that standing with one foot in front , placing alternate foot on the stool , and reaching forward with an outstretched arm  improved significantly. A significant improvement was also determined in the Balance It group in standing with one foot in front .Within groups analysis (pre- and post-intervention) showed that the balance of the participants, assessed through PBS as the total score, improved significantly , Bubble Pop , and Balance It  throughout the intervention , p < 0.001, ηp2 = 0.69] with a large effect size in the DASH score.Similarly, a significant improvement was determined in the DASH score in all the groups, including Scoop d , p = 0.67 ηp2 = 0.03]. Moreover, the Kruskal–Wallis test showed that there was no statistically significant difference in the domains of PBS (p ≥ 0.05), except in standing with one foot in front  and in the total PBS score  with a large effect size post-intervention  vs. 4(1.25), p = 0.03, r = 0.65]. The total score of PBS also showed a significant difference between Bubble Pop and Balance It groups . However, there was no significant difference (p ≥ 0.05) between the Balance It and Scoop'd groups as well as between the Bubble Pop and Scoop'd group in standing with one front in front component and the total PBS score post-intervention.The pre- and post-analysis between the interventional groups showed no statistically significant difference in the total DASH score [rvention . Furthervia the game scores play an integral role in motor relearning and enhancing neuroplasticity which might have contributed to improving the balance  improved significantly in all the groups. Though lower-extremity exercises using AR have shown improved balance in various medical conditions previously as well , however balance .In the Scoop'd group, the components of PBS such as standing with one foot in front, standing on one foot, and placing an alternate foot on the stool improved significantly. The movement of the participant in various directions to catch the ice-cream scoops in this game might have caused this improvement. Also, AR delivers visual and auditory feedback on balance parameters which can provide higher specificity and continuity and lower delay than the feedback delivered by a therapist . This miIn the Bubble Pop group, standing with one foot in front, placing an alternate foot on the stool, and reaching forward with an outstretched arm significantly improved post-intervention. Just like Scoop'd, this game also involved the participant in multi-directional movements that might be the reason for this improvement. The participant also had to extend their UE to pop the bubbles which may have contributed to improving the forward-reaching score in PBS.Standing with one foot in front also improved significantly in the Balance It group, which just like the previous two games involved multi-directional motion. According to a systematic review, balance training with feedback can result in improved ability to shift weight, reduced postural sway, and reduced attentional demands while standing . SimilarA significant improvement with a large effect size has been determined in the DASH score in all the groups post-intervention. The possible reasons for this improvement might be AR-based cognitive engagement during play, enhanced problem-solving, neuroplasticity changes, and increased motivation. AR creates repetitive task-specific and task-oriented practices in a real environment, the flexibility of adjusting task difficulties, and the potential for interaction and social play . AR alsoThe comparison between the groups showed that there was no significant difference in the total DASH score and in the domains of PBS except in standing with one foot in front and the total PBS score. There was a significant difference determined in standing with one foot in front between Bubble Pop and Balance It groups. The total score of PBS also showed a significant difference between Bubble Pop and Balance It groups. Furthermore, the Balance It game showed a better improvement in balance as compared to the other games. Though all the games involved motion in different directions, however, the time spent in a specific posture and movement pattern differed greatly in each game and this might would have caused such differences among the groups.AR allows optimum exercise orientation toward objectives with high patient motivation and is also enjoyable to use . AR techAugmented reality is a promising intervention that could be used during rehabilitation for children with CP as an addition to other appropriate therapies . The resThe limitations of this study are that there was no control group and the total levels of the game played by each participant were not noted. These should be incorporated into future research along with including larger sample size and longer study duration to determine the comparative effectiveness of AR games and standard physiotherapy, and to attain further improvements. It is also suggested that these AR games may be improved by adding elements of variation and challenge to achieve greater and generalized improvement in the function of UE and body balance in children with SHCP.Augmented reality games used in this study were found to be effective in improving the function of the upper extremity and balance of children with SHCP. The Balance It game showed more promising results in improving the balance as compared to the other games, however, no significant difference was determined between the three AR games in terms of the UE function of the participants.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Institutional Review Board and Ethics Committee of NIRM. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin. Written informed consent was obtained from the minor(s)' legal guardian/next of kin for the publication of any potentially identifiable images or data included in this article.WM and MA: substantial contributions to the conception and design of the study. WM, RB, and QM: acquisition of data for the study. WM and WA: interpretation of data for the study. WA: analysis of the data for the study. WM and RB: drafted the work. WM, WA, MA, and QM: revised it critically for important intellectual content. WM, RB, WA, MA, and QM: final approval of the version to be published and agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


E. sinensis (APE) and determine whether AR-9 could improve many symptoms of a HL rat induced by a high-fat diet (HFD) by modulating gut flora and its metabolites. Above all, AR-9 was derived from APE using ion-exchange chromatography, and its structure was deconstructed by Fourier transform infrared spectrometer (FT-IR), circular dichroism (CD) spectroscopy, and UHPLC-Q-Exactive-Orbitrap MS. Then, an HFD-induced HL model in SD rats was established and used to clarify the regulatory effects of AR-9 (dose of 3 mg/kg) on HL. Normal diet–fed rats were taken as the control. The plasma samples and liver were harvested for biochemical and histopathological examinations. 16S rRNA gene sequencing and untargeted metabolomics were sequenced to assess changes in gut flora and its metabolites from rat fecal samples. Finally, Spearman’s correlation analysis was used to assess the relationship between lipid-related factors, gut flora, and its metabolites so as to evaluate the mechanism of AR-9 against HL. The results of the separation experiments showed that the amino acid sequence of AR-9 was AVFPSIVGR, which was a fragment of the actin protein from Blattaria insects. Moreover, HFD rats developed exaltation of index factors, liver lipid accumulation, and simple fibrosis for 8 weeks, and the profiles of gut flora and its metabolites were significantly altered. After treatment, AR-9 decreased the levels of lipid factors in plasma and the extent of liver damage. 16S rRNA gene sequencing results indicated that AR-9 significantly increased the relative abundance of beneficial bacteria Bacteroidetes and reduced the relative abundance of the obesity-associated bacteria Firmicutes. Furthermore, AR-9 changed gut microbiota composition and increased the relative abundance of beneficial bacteria: Lactobacillus, Clostridium, Dehalobacterium, and Candidatus arthromitus. Fecal metabolomics showed that the pathway regulated by AR-9 was “arginine biosynthesis”, in which the contents were citrulline and ornithine. Spearman’s correlation analysis revealed that two metabolites (ornithine and citrulline) showed significantly negative correlations with obesity-related parameters and positive correlations with the gut genera (Clostridium) enriched by AR-9. Overall, our results suggested interactions between gut microbial shifts and fecal amino acid/lipid metabolism and revealed the mechanisms underlying the anti-HL effect of AR-9. The abovementioned results not only reveal the initial anti-HL mechanism of AR-9 but also provide a theoretical basis for the continued development of AR-9.The dysbiosis of gut flora and its metabolites plays important roles in the progression of hyperlipidemia (HL), and some bioactive peptides are available for HL treatment. In this study, we aimed to isolate an active peptide (AR-9) from active peptides of  Hyperlipidemia, which is accompanied by an increase in triglycerides (TGs), total cholesterol (TC), and low-density lipoprotein cholesterol (LDL-C), is a common clinical metabolic disease . In addivia the changes in their composition and metabolites , can inhibit lipid production and accelerate lipid oxidation by mediating signaling pathways to achieve regulation of lipid factors  used in traditional Chinese medicine (TCM) was first recorded in Shennong’s Herbal Classic written during the Han dynasty . The specimens were preserved at the chemical laboratory of Binzhou Medical University. Pepsin  and trypsin  were all obtained from Shanghai Sinopharm Reagent Group Co., Ltd . The kits of total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) were purchased from Nanjing Jiancheng Bioengineering Institute .E. sinensis after pulverization was added into deionized water to the concentration of 10% (weight/volume). After being denatured for 10 min at 90°C and cooled to 40°C, the pH of the sample was adjusted to 2.0 with 1.0 M HCl. The solution was then supplemented with pepsin (1.0% of the drug) and subjected to enzymatic hydrolysis for 1.0 h. Thereafter, the pH of the primary solution was adjusted to 8.0 with 1.0 M NaOH before the addition of trypsin (1.0% of drug), followed by incubation for 3.0 h with stirring. Finally, the enzymatic solution was soaked in boiling water for 15 min to inactivate the enzymes. The mixture was then centrifuged at 5,000 rpm for 10 min.The resulting supernatants were separated into different constituents using an ultrafiltration cartridge of a 3,000 Da nominal molecular weight cut-offs (NMWC) membrane . For salt removal, the abovementioned solution was passed through 500-Da dialysis membranes and lyophilized. The lyophilized sample was redissolved with deionized water and further isolated using a DEAE-Sepharose Fast Flow anion-exchange column (2.6 × 40 cm). The column was equilibrated with 0.03 mM Tris-HCl buffer (pH 8.0). The sample was added to the protein chromatography system (AKTA explorer 10.0) and eluted with a stepwise gradient of NaCl aqueous solutions (from 0.1 to 2 M) prepared in Tris-HCl buffer. The fractions were combined according to the number of elution peaks to gather them. After dialyzing, freeze-drying, and concentrating, the fractions which include AR-9 were obtained .The collected components were analyzed by reversed phase-high-performance liquid chromatography (RP-HPLC) to obtain the highest purity compound of AR-9. The separation was achieved on Waters symmetry C18 column . The mobile phases consisted of (A) water containing 0.1% trifluoroacetic acid and (B) acetonitrile (ACE) containing 0.1% trifluoroacetic acid . The gra−1 at 30°C.As we know, CD spectroscopy of protein is highly sensitive toward the secondary structure and the same applies for peptides. Therefore, the secondary structure of AR-9 was analyzed by CD spectroscopy using the reported method . MoreoveTM peptide XB C18 column  was used as a separation medium, mobile phase A was deionized water , ACE with 0.1% FA was set as mobile phase B. Seven microliter AR-9 (10 mg dissolved by 100 ml 0.1% FA) was injected into the analysis platform and separated using a linear gradient elution: 0–3 min, 2–6% B; 3–42 min, 6–20% B; 42–47 min, 20–35% B; 47–48 min, 35–100% B; 48–60 min, 100% B. The flow rate was 300 nL/min.Finally, the complete amino acid sequence of AR-9 was determined by an Easy nLC1200-Q-Eaxtive plus mass spectrometer  which contained a binary pump, an autosampler, and a column oven. The bioZenm/z 350–1,800 in the positive mode with a resolution of 70,000, and the precursor ion of AR-9 (Charge state ≥+1) was fragmented with the high-energy collision of 28.0 NEC. The temperature of the capillary was 275°C, and the spray voltage was set as 2,200 V. The product ions were measured on an orbit with a resolution of 17,500 (AGC 1e5). For dynamic exclusion, the specific parameters were as follows: a repeat duration of 25 s and exclusion duration of 25 s.The ESI-MS/MS information of AR-9 was collected using a Q-Exactive Orbitrap mass spectrometer  with a data-dependent scan mode. The mass range of the MS scan was set to ad libitum for 1 week. Afterward, all rats were randomly divided into the control group  and the HL group (24 rats) according to body weight. The rats in the control group were fed normal rodent chow , and those in the HL group were fed with a HFD, containing a standard chow diet (65%), sucrose (20%), lard (15%), cholesterol (5%), sodium cholate (5%), and 5% yolk powder  for 8 weeks (n = 6), simvastatin group  at the dose of 5 mg/kg/d (drug weight/body weight/day), APE group  at the dose of 25 mg/kg/d, and AR-9 group  at the dose of 3 mg/kg/d. This dose was determined based on the percentage of AR-9 in APE  rats  were purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd 2019-0003). Before experimental interventions, all animals must be maintained under standard animal room conditions  with standard feed and water  8 weeks . Then, tAfter the final gavage, all rats were starved for 12 h, receiving only deionized water. Fresh feces from all rats were collected and stored at −80°C refrigerators for fecal metabolomics and intestinal microbial analysis. The rats of five groups were killed in parallel using 10% pentobarbital sodium by intraperitoneal injection. The blood samples were collected from the abdominal aorta and were centrifuged  to obtain plasma for biochemical analysis . The tissues of the liver were fixed in 4% paraformaldehyde fixation (PFA)  for subsequent analysis.The levels of TG, TC, LDL-C, and HDL-C in plasma samples from all rats were measured using a microplate reader . The hepatic tissues fixed in 4% PFA were dehydrated and embedded in paraffin, cross-sectioned into 4-µm-thick slices, and stained with hematoxylin–eosin (H&E). The sections of the remaining liver tissues were cleaned with PBS and cultured with 60% isopropanol for 5 min and then dyed in 0.5% Oil Red O staining liquid  for 20 min. After being cleaned by PBS, all sections were then stained with hematoxylin stain  for 2 min. The abovementioned indicators were all used to evaluate the anti-HL function of AR-9.The fecal pellets stored at −80°C were freeze-dried and crushed. 100 mg powders were ultrasonically extracted with 500 µl cold water and 500 µl cold ethanol for 30 min in succession. Then, the two extracts were combined and centrifuged ; the resulting supernatant was collected and evaporated by nitrogen at 4°C . These rChromatographic separation was performed on a Dionex Ultimate 3000 UHPLC system equipped with a WPS-3000 TRS autosampler, a TCC-3000RS column oven, and an HPG-3400RS binary pump . An ACQUITY UPLC BEH HILIC column  was operated at 40°C. The mobile phase comprised (A) 0.1% FA in water and (B) ACE with a flow rate of 0.3 ml/min. The elution parameters were 0.00–5.00 min, 5% A; 5.00–6.00 min, 5–20% A; 6.00–10.00 min, 20–25%; 10.00–13.00 min, 25–40%; 13.00–16.00 min, 40%; 16.00–16.10 min, 40–5%; 16.10–20.00 min, 5% A.m/z 70–1,050, auxiliary gas heater temperature was 350°C, and capillary temperature was 320°C.The ion spray voltages were set at 3.5 kV in the positive ion mode and 3.0 kV in the negative ion mode. The auxiliary gas flow was 10 arb, sheath gas flow was 30 arb, the mass range of the MS scan was m/z, peak intensity, and retention time.The LC-MS/MS data were processed with Compound Discoverer 3.0 software  for noise cancellation, baseline correction, and normalization to obtain reliable data sets with some information, including p-value (formed from relative intensity). The structures, molecular weights, and codes of differential metabolites were assigned according to the human metabolome database (https://hmdb.ca/).Afterward, the processed data sets were added to SIMCA-P 14.0 software  for performing the principal component analysis (PCA) and orthogonal to partial least squares-discriminate analysis (OPLS-DA). Among them, PCA was applied to discriminate the interesting separation trend of all groups. OPLS-DA was used to characterize metabolic perturbation of HL. In addition, S-plots were used to screen the differential metabolites of HL treated by AR-9 combined with other judgment methods, such as variable importance in projection (VIP) (generated in the OPLS-DA mode) and Bacterial DNA was isolated from fecal samples using the cetyltrimethylammonium bromide (CTAB) method . The DNAThe sequencing libraries were generated using the TruSeq® DNA PCR-Free Sample Preparation Kit  according to the manufacturer’s recommendations, and index codes were added. The library quality was assessed on the Qubit @ 2.0 Fluorometer (Thermo Scientific). At last, the library was sequenced on an Illumina NovaSeq platform.Appropriate methods including ANOVA and LEfSe were used to identify the bacteria with different abundance among samples and groups . Alpha dt-test were used to assess statistical significance for multiple comparisons among the different groups. p < 0.05 and p < 0.01 were defined as statistically significant. The statistical analyses and figures were performed using GraphPad Prism 8.0 software.All results were analyzed using statistical product and service solutions (SPSS) 22.0 software and were presented as Mean ± SD. One-way ANOVA and Student’s The Spearman correlation analysis was performed to determine the potential correlations of the intestinal microbial, differential metabolites, and biochemical indicators. The correlations of the abovementioned three variables were visually displayed using heat maps, separately. Finally, the intestinal microbes and biomarkers that reflect the anti-HL function of AR-9 were extracted.The result of APE separated by anion-exchange chromatography is shown in −1), amide B , amide I , and amide II . As shown in −1, which prompted the presence of αⅡ-helical structure in AR-9; the bands around 3,300 cm−1 and 3,100 cm−1 illustrated that they were generated by Fermi resonances between amide II and NH stretching vibrations. The amide I band mostly consists of C=O stretching vibrations and C-N groups, while the amide II band not only consists primarily of N–H bending but also C-N and C-C stretching vibrations , m/z 171.11 (b2), m/z 318.19 (b3), m/z 175.11 (y1), m/z 232.13 (y2), m/z 331.21 (y3), m/z 444.29 (y4), m/z 531.33 (y5), m/z 628.38 (y6), and m/z 775.45 (y7). The molecular weight of the entire peptide was 944.54280. The fragment ion at m/z 72.08 was produced due to the loss of hydroxyl groups (17 Da) of alanine. The fragment ions at m/z 775.41 ([M-171.11+2H]+) and m/z 628.38 ([M-318.19+2H]+) indicated the presence of valine ([b2-b1+H2O]) and phenylalanine ([b3-b2+H2O]). Moreover, the loss of proline ([y6-y5+H2O]), serine ([y5-y4+H2O]), isoleucine ([y4-y3+H2O]), valine ([y3-y2+H2O]), glycine ([y2-y1+H2O]), and y1-arginine ([M + H]+) was detected. Therefore, the amino acid sequence of AR-9 was speculated as alanine–valine–phenylalanine–proline–serine–isoleucine–valine–glycine–arginine (AVFPSIVGR). In order to determine the correctness of the inferred structure, AR-9 was synthesized by solid-phase peptide synthesis and re-analyzed using UPLC-MS/MS with the same method. DPIs and retention time of synthetic were consistent with those of the isolated compound.LC-MS/MS is an available tool for the identification, quantification, and analysis of peptides. In the large majority of cases, the identification of peptide sequences has been enabled from collision-induced dissociation (CID) MSch tools . The reshttp://pepdraw.com/) and the PEPFOLD3 database (https://bioserv.rpbs.univ-paris-diderot.fr/services/PEP-FOLD3/) (Blatta americana) using UniProt (https://www.uniprot.org/).The physical and chemical indicators of AR-9 were predicted using the PepDraw database (-FOLD3/) , and thep < 0.05) and the decrease of HDL-C compared with the levels in the normal diet–fed rats. After treatment, AR-9 and APE all mitigated these adverse changes, compared to the model group, and AR-9 and APE could reduce the levels of TG, TC, and LDL-C and increase the level of HDL-C. Interestingly, the ability of AR-9 to resist the lipid is stronger than that of APE . The results of the pathway analysis revealed that there were three primary disturbed pathways (p < 0.05) of feces between Con vs. Mod, involving 1) phenylalanine metabolism; 2) arginine biosynthesis; 3) histidine metabolism. Moreover, there were two primary disturbed pathways (p < 0.05) of feces between AR-9 vs. Mod, involving 1) arginine biosynthesis and 2) arginine and proline metabolism. The color and size of each circle in p-values and pathway impact values, respectively. The results suggested that the disturbed pathways in response to HL and AR-9 treatment were mainly arginine biosynthesis.To clarify the exact pathways of AR-9 resisting HL, differential metabolites with Con vs. Mod were imported into the MetaboAnalyst database  were conducted on the results of 16S rRNA sequencing based on the 97% similarity level. It could be found from The beta diversities were described by a principal component analysis  and a non-metric multidimensional scaling (NMDS) analysis. Both PCoA  and NMDSFirmicutes and Bacteroidetes were the most predominant phylum in gut bacteria of rats and account for the mainly relative abundance in all samples, as shown in Bacteroidetes in the Mod group decreased, while Firmicutes increased. In contrast, compared to the Mod group, Firmicutes decreased in AR-9 group, whereas Bacteroidetes increased. The differently abundant taxa among experimental samples were identified using linear discriminant analysis effect size (LEfSe) (p < 0.05). The relative abundance of selected taxa in the Mod group relative to the AR-9 group is shown in Lactobacillus, Clostridium, Dehalobacterium, and Candidatus Arthromitus. Especially, Clostridium was present in a significantly lower proportion in the Mod group than the AR-9 group (p < 0.01). (LEfSe) . The ana (LEfSe) . In termp < 0.05) correlations with TC, TG, and LDL-C levels. These results indicated that the abovementioned compounds might be pivotal metabolites of the beneficial function of AR-9. Hereafter, the correlations between gut flora and lipid factors were revealed  but also increase HDL amounts in plasma. In addition, AR-9 reduced the accumulation of lipids in the liver based on Oil Red O staining and alleviated liver damage, which was caused by HFD.The first-line medicine for the treatment of HL includes three categories: statins, fibrates, and niacin . The aboFirmicutes and decreased that of Bacteroidetes; however, AR-9 reversed this trend. At the genus level, AR-9 could upregulate the relative abundance of four bacteria: Lactobacillus, Clostridium, Dehalobacterium, and Candidatus Arthromitus. Among them, Lactobacillus and Clostridium were directly related to metabolic diseases, such as type 2 diabetes, HL, atherosclerosis, and hepatic steatosis (In order to find out the mechanism of AR-9 against HL, the profiles of gut flora and its metabolites were studied in detail by 16S rRNA sequencing and fecal metabolomics. In gut flora results, a HFD significantly increased the relative abundance of teatosis . The conde novo lipid biosynthesis (Many studies had shown a positive correlation between HL and amino acid metabolism, such as tryptophan metabolism and arginine and proline metabolism. . In the ynthesis . MeanwhiLactobacillus, Bacteroides, and Clostridium have been illustrated to have the abovementioned function (via the metabolic function of Clostridium to achieve a beneficial effect of treating HL (Clostridium has not been revealed and verified. Many studies have confirmed that active peptides can be more easily absorbed into the circulatory system by converting to small molecules (Citrulline and ornithine are produced under the fermentation of gut flora, proteins, and bioactive peptides. function . Coincidating HL . Howeverolecules . Moreovevia acting on Clostridium.In this study, the effects of AR-9 on lipid factors and gut flora in SD rats with HFD-induced HL were studied. AR-9 could regulate the levels of TG, TC, LDL-C, and HDL-C in plasma and repair the damaged liver function. For gut flora and metabolomics, AR-9 mostly regulated the arginine biosynthesis pathway Dyslipidemia, the disorder of lipid metabolism, and decreased diversity and abundance of gut flora were ubiquitous in HL. It was proven by metabolomics that the metabolic profile of the feces of the rats in the HL group changed significantly. At the same time, the effect of AR-9 on preventing HL was evaluated. The existence of a link between metabolic biomarkers and the gut microbial has an important influence on the host. AR-9 can regulate amino acid metabolism, increase the diversity and abundance of gut flora, regulate the structure of gut flora, and thus contribute to health. This study identified key functional bacteria and potential biomarkers of HL in rats and established a correlation between them and AR-9 treatment. All results in this study will provide support for the continued development of AR-9.Clostridium and anti-HL of AR-9 and its absorbed components urgently should be resolved. Accordingly, the findings of the current study will be further investigated for uncovering the metabolic and microbial basis of AR-9 in the treatment of HL.There are some limitations of the present study. First, the identified differences in gut microbials and metabolites for diagnostic and prognostic evaluations were not verified by further study. Second, AR-9 is administered in a single dose. Third, the interactive relationships between AR-9 and